A technology for preparing universal humanized CAR19-dnt cells and application thereof

Abstract

Provided are a technology for preparing universal humanized CAR19-DNT cells and an application thereof. Specifically, provided is a universal CAR-T cell targeting CD19, which expresses an exogenous CAR construct having a structure as shown in Formula I, L-scFv-H-TM-C-CD3ζ (Formula I), wherein L is nothing or a signal peptide sequence; scFv is an antibody single-chain variable region sequence targeting CD19; H is nothing or a hinge region; TM is a transmembrane domain; C is a costimulatory signal molecule; and CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ. The universal humanized CAR19-DNT cell has the advantages of low immunogenicity, no need for gene editing to avoid GvHD, high safety, high specificity, and significant tumor killing effect, and the construction method can realize large-scale production and has low production cost.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy technology, specifically relating to a technical method for preparing universal humanized chimeric antigen receptor CART-19 cells and its application. Background Technology

[0002] Chimeric antigen receptor T (CAR) therapy is one of the most promising cancer immunotherapies. Two CAR cell products approved by the FDA in 2017 are derived from peripheral blood mononuclear cells (PBMCs) isolated from cancer patients and genetically engineered to incorporate specific antigen receptors (CARs). This enhances the targeting, killing activity, and persistence of T cells in PBMCs, and their recognition of tumor cell surface antigens is not MHC-restricted. Common CARs consist of an extracellular antigen-binding region, a transmembrane region, and an intracellular T cell receptor signal transduction region (such as CD3ζ and CD28). The extracellular antigen-binding region consists of the light chain (VL) and heavy chain (VH) of a monoclonal antibody, linked by a hinge to form a single-chain fragment variable (scFv), capable of recognizing specific tumor antigens. CAR-T cell technology has evolved to its third generation. First-generation CARs consist of a single-chain antibody (scFv) that recognizes tumor surface antigens, an antigen-binding site, and an immune receptor tyrosine activation motif. Extracellularly, the scFv recognizes tumor marker molecules, but lacks co-stimulatory signals, resulting in poor clinical outcomes, manifested as short T-cell survival time and low cytokine secretion. Second-generation CARs introduced co-stimulatory signal sequences, enhancing T-cell cytotoxicity, proliferation, and survival time, and promoting cytokine release, achieving success in the clinical treatment of acute lymphoblastic leukemia. Third-generation CARs incorporate tandem CD28 and 4-1BB co-stimulatory molecules and CD3ζ in the cytoplasmic region, resulting in even better efficacy with this dual co-stimulatory signal sequence. A schematic diagram of the structure of the third-generation CARs is shown below. Figure 1 As shown.

[0003] Currently, autologous CAR-T cell therapy has achieved a complete remission rate of over 90% in treating CD19-targeted B-cell acute lymphoblastic leukemia (B-ALL); in lymphoma treatment, the complete remission rate is also as high as 64%. The efficacy of CAR-T technology in treating these two diseases is unmatched by any other drug. In my country, acute lymphoblastic leukemia accounts for 80% of childhood acute leukemia, with an incidence rate of 1 in 100,000, and accounts for 20% of all adult leukemia cases. Immunotherapy is an important treatment for many serious diseases, including cancer, autoimmune diseases, and even severe viral infections, with successful cases of immunotherapy.

[0004] While CD19-targeted CAR-T therapy has shown remarkable efficacy in clinical trials, it also has several drawbacks. Besides causing severe side effects such as cytokine storms, there are four major problems: First, some late-stage patients with low lymphocyte counts or poor lymphocyte quality lose the opportunity for CAR-T treatment due to a lack of sufficient autologous immune cells. Second, there have been reports of viral genes being transduced into residual tumor cells in peripheral blood when using CAR-T products prepared from the patient's own cells, leading to patient death. Third, most currently developing universal CAR-T cell products use gene editing technology to knock out genes that can cause graft-versus-host disease, such as the TCR receptor; however, knocking out these genes requires a complex construction process and large-scale sequencing, and has a high off-target rate. Finally, because preparing autologous CAR-T cell products is a one-to-one personalized treatment, the preparation cost is high, and the preparation process is complex, resulting in expensive products that most patients cannot afford, increasing the social medical burden.

[0005] Therefore, there is an urgent need in this field to develop a universal (ready-to-use) CART cell and its construction method that is derived from peripheral blood of healthy donors, does not require complex gene editing methods to knock out the TCR gene that causes graft-versus-host disease, has low immunogenicity (does not induce host anti-graft immune response, has a long retention time in vivo), high safety, significant killing effect, and low production cost. Summary of the Invention

[0006] In view of the series of problems of existing related technologies, the present invention provides a technical method for preparing universal antigen chimeric receptor CAR19-DNT cells and its application.

[0007] The purpose of this invention is to provide a universal (ready-to-use) CART cell and its construction method that are derived from peripheral blood of healthy donors, do not require complex gene editing methods to knock out the TCR gene that causes graft-versus-host disease, have low immunogenicity (do not induce host anti-graft immune response, have long retention time in vivo), high safety, significant killing effect and low production cost.

[0008] In a first aspect of the invention, a universal CAR-T cell targeting CD19 is provided, said universal CAR-T cell expressing an exogenous CAR construct having a structure as shown in Formula I.

[0009] L-scFv-H-TM-C-CD3ζ (Formula I)

[0010] In the formula,

[0011] L represents the absence of a signal peptide sequence;

[0012] scFv is the single-chain variable region sequence of an antibody targeting CD19;

[0013] H represents the area with no hinge or no connection.

[0014] TM represents a transmembrane domain;

[0015] C is a co-stimulatory signaling molecule;

[0016] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ;

[0017] Each "-" independently represents a linking peptide or peptide bond that connects the above elements.

[0018] In another preferred embodiment, the CD19-targeting universal CAR-T cell is a CD19-targeting universal CAR-double-negative T cell (CAR-DNT cell).

[0019] In another preferred embodiment, the TCR in the universal CAR-T cells is either knocked out or not knocked out.

[0020] In another preferred embodiment, the TCR in the universal CAR-T cells was not knocked out.

[0021] In another preferred embodiment, L is a signal peptide selected from the following histones: CD8, GM-CSF, CD4, CD137, or a combination thereof.

[0022] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0023] In another preferred embodiment, the scFv comprises a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO:6 and a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:5.

[0024] In another preferred embodiment, the scFv also contains a linker peptide sequence between VH and VL.

[0025] In another preferred embodiment, the linker peptide sequence has an amino acid sequence as shown in SEQ ID NO:7.

[0026] In another preferred embodiment, the scFv has an amino acid sequence as shown in SEQ ID NO:4.

[0027] In another preferred embodiment, H is a hinge region selected from the following histones: CD8, CD28, CD137, or a combination thereof.

[0028] In another preferred embodiment, H is the hinge region derived from CD8.

[0029] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0030] In another preferred embodiment, TM includes a transmembrane region derived from CD8 and / or a transmembrane region derived from CD28.

[0031] In another preferred embodiment, C is a co-stimulatory signaling molecule selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0032] In another preferred embodiment, C includes co-stimulatory signaling molecules derived from 4-1BB and / or co-stimulatory signaling molecules derived from CD28.

[0033] In another preferred embodiment, the CAR construct has an amino acid sequence as shown in SEQ ID NO:14.

[0034] In a second aspect of the invention, a method for preparing universal CAR-DNT cells targeting CD19 as described in the first aspect of the invention is provided, comprising the steps of:

[0035] (i) Provide an expression vector containing a nucleotide sequence encoding a CAR construct as shown in Formula I;

[0036] L-scFv-H-TM-C-CD3ζ (Formula I)

[0037] In the formula,

[0038] L represents the absence of a signal peptide sequence;

[0039] scFv is the single-chain variable region sequence of an antibody targeting CD19;

[0040] H represents the area with no hinge or no connection.

[0041] TM represents a transmembrane domain;

[0042] C is a co-stimulatory signaling molecule;

[0043] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ;

[0044] Each "-" independently represents a linking peptide or peptide bond that connects the above elements;

[0045] (ii) Providing a DNT cell culture medium containing at least one DNT cell; transducing the expression vector from step (i) into the DNT cell to obtain a universal CAR-DNT cell targeting CD19 as described in the first aspect of the invention; and

[0046] (iii) Optional detection steps (ii) to obtain universal CAR-DNT cells.

[0047] In another preferred embodiment, the expression vector is selected from DNA, RNA, or a combination thereof.

[0048] In another preferred embodiment, the expression vector is selected from the group consisting of plasmids, viral expression vectors, transposons, or combinations thereof.

[0049] In another preferred embodiment, the viral expression vector is selected from the group consisting of lentiviral vectors, adenoviral vectors, retroviral vectors, or combinations thereof.

[0050] In another preferred embodiment, the expression vector is a viral expression vector, and the method (ii) further includes the step of: viral packaging of the viral expression vector of step (i).

[0051] In another preferred embodiment, the viral expression vector is a retroviral expression vector or a lentiviral expression vector, preferably a lentiviral expression vector.

[0052] In another preferred embodiment, the polynucleotide sequence integrated into the viral expression vector of step (i) is shown in SEQ ID NO:16.

[0053] In another preferred embodiment, step (ii) includes co-transfecting the viral expression vector from step (i) with the packaging plasmids psPAX2 and pMD2.0G into the viral packaging host cell.

[0054] In another preferred embodiment, in step (i), the polynucleotide sequence contains the nucleotide sequence of scFv as shown in formula (I) of SEQ ID NO:15.

[0055] In another preferred embodiment, in step (i), the polynucleotide sequence contains a nucleotide sequence of L as shown in formula (I) of SEQ ID NO:8.

[0056] In another preferred embodiment, in step (i), the polynucleotide sequence contains a nucleotide sequence of H as shown in formula (I) of SEQ ID NO:9.

[0057] In another preferred embodiment, in step (i), the polynucleotide sequence contains a nucleotide sequence of TM as shown in formula (I) of SEQ ID NO:10.

[0058] In another preferred embodiment, in step (i), the polynucleotide sequence contains a nucleotide sequence of C in the coding formula (I) as shown in SEQ ID NO:11.

[0059] In another preferred embodiment, in step (i), the polynucleotide sequence contains the nucleotide sequence of CD3ζ as shown in formula (I) of SEQ ID NO:12.

[0060] In another preferred embodiment, the virus packaging host cell is a mammalian cell, preferably a 293T cell.

[0061] In another preferred embodiment, before step (ii), the following step is further included:

[0062] (iia) Collect peripheral blood samples from healthy donors;

[0063] (iib) Remove CD4 from the peripheral blood sample + CD8 + T cells, thereby obtaining DNT cells;

[0064] (iic) In a culture flask coated with CD3 monoclonal antibody, the DNT cells obtained in (iib) are cultured and amplified in a suitable culture medium to obtain the culture system containing DNT cells required in step (ii).

[0065] In another preferred embodiment, the culture medium in step (iic) contains a substance selected from the group consisting of: gentamicin, recombinant human interleukin-2, recombinant human interleukin-7, recombinant human interleukin-12, recombinant human interleukin-15, autologous plasma, AB serum, or a combination thereof.

[0066] In another preferred embodiment, the culture medium in step (iic) does not contain recombinant or wild-type human interleukin-4.

[0067] In another preferred embodiment, the culture medium in step (iic) does not contain AB serum.

[0068] In another preferred embodiment, in the culture medium of step (iic), the concentration of gentamicin is 40-80 units / ml (preferably 60 units / ml), the concentration of recombinant human interleukin-2 is 150-1000 IU / ml (preferably 200-250 IU / ml), the concentration of recombinant human interleukin-15 is 5-20 ng / ml (preferably 10 ng / ml), the concentration of recombinant human interleukin-7 is 1-5 ng / ml (preferably 2 ng / ml), the concentration of recombinant human interleukin-12 is 5-20 ng / ml (preferably 10 ng / ml), the concentration of autologous plasma is 3-25 v% (preferably 10-20 v%), and / or the concentration of AB serum is 4-8 v% (preferably 6 v%).

[0069] In another preferred embodiment, in step (ii), the concentration of DNT cells in the DNT cell culture medium is 1 × 10⁻⁶. 6 Cells / ml to 4×10 6 Cells / ml, preferably 1×10⁻⁶. 6 Cells / ml to 2×10 6 Cells / ml, more preferably 0.5 × 10⁻⁶. 6 Cells / ml to 1×10 6 Cells / ml

[0070] In another preferred embodiment, in step (ii), the MOI of the virus is 0.1 to 10, more preferably 0.1 to 8, and most preferably 5.

[0071] In another preferred embodiment, the detection in step (iii) is performed 7-14 days after the transduction in step (ii), preferably 8 days or 13 days later.

[0072] In a third aspect of the invention, a pharmaceutical composition is provided, comprising:

[0073] (a) Universal CAR-T cells targeting CD19 as described in the first aspect of the invention; and

[0074] (b) Pharmaceutically acceptable carriers, diluents or excipients.

[0075] In another preferred embodiment, the pharmaceutical composition is a liquid pharmaceutical composition.

[0076] In another preferred embodiment, the pharmaceutical composition is an injectable preparation.

[0077] In another preferred embodiment, the concentration of the CD19-targeting universal CAR-T cells in the pharmaceutical composition is 1 × 10⁻⁶. 6 -5×10 6 Cells / ml, preferably 1×10⁻⁶. 6 -2×10 6 Cells / ml

[0078] In a fourth aspect of the invention, there is provided the use of a universal CAR-T cell targeting CD19 as described in the first aspect of the invention for the preparation of pharmaceutical compositions or formulations for the prevention and / or treatment of cancer.

[0079] In another preferred embodiment, the tumor is selected from the group consisting of hematologic malignancies, solid tumors, or combinations thereof.

[0080] In another preferred embodiment, the tumor is a hematologic tumor.

[0081] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

[0082] In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, squamous cell carcinoma of the lung, anal cancer, head and neck tumor, or combinations thereof.

[0083] In a fifth aspect of the invention, a method for preventing and / or treating a disease is provided, comprising the steps of administering, to a desired subject, a universal CAR-T cell targeting CD19 as described in the first aspect of the invention, or a pharmaceutical composition as described in the third aspect of the invention.

[0084] In another preferred embodiment, the disease is cancer or a tumor.

[0085] In another preferred embodiment, the desired object is a human or a non-human mammal.

[0086] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0087] Figure 1 The structure of the three generations of CART is shown.

[0088] Figure 2 A schematic diagram of the humanized CD19 CAR construct is shown.

[0089] Figure 3 A three-dimensional model of humanized CD19 with a mutation in the CDR1 domain (V27G) is shown.

[0090] Figure 4 The results show that on days 8 and 13 of culture, the proportion of DNT cells in the culture system was identified by flow cytometry using CD3, CD4, and CD8 antibody labeling, and the efficiency of DNT cells infected with antibody-carrying lentiviruses was detected by specific anti-human CD19 variable region antibody labeling, thereby calculating the percentage of CART cells in the cell product.

[0091] Figure 5 shows the comparison of the in vitro specific tumor-killing activities of humanized CAR19-DNT against HeLa and HeLa-CD19 target cells on days 8 and 13 of culture, respectively; where 5-A shows the real-time killing effect monitored by RTCA (Real-Time Cytotoxicity Assay) at an effector-to-target ratio of 2:1; 5-B shows the real-time quantitative killing effect monitored by RTCA (Real-Time Cytotoxicity Assay) 18 hours after the addition of effector cells at an effector-to-target ratio of 2:1. Detailed Implementation

[0092] Through extensive and in-depth research and screening, the inventors have developed, for the first time, a method for constructing universal chimeric antigen receptor CAR19-DNT cells. Experiments have demonstrated that the universal chimeric antigen receptor CAR19-DNT cells provided by this invention are derived from peripheral blood of healthy donors, eliminating the need for complex gene editing methods to knock out the TCR gene that causes graft-versus-host disease. They exhibit low immunogenicity (does not induce a host anti-graft immune response, has a long in vivo retention time), high safety, and specifically target the human CD19 antigen, demonstrating significant tumor-killing effects. Furthermore, the construction method provided by this invention allows for large-scale production at low cost. This invention was completed based on these findings.

[0093] the term

[0094] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0095] As used herein, the term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0096] As used herein, the terms “give” and “administer” are used interchangeably to mean the physical introduction of the product of the invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0097] As used in this article, the term "DNT cells" refers to double-negative T cells, a type of TCRαβ with a unique phenotype. + and / or TCRγδ + T lymphocytes express CD3 molecules but not CD4, CD8, NK, iNKT, or other characteristic surface molecules (markers) of cells, and play unique and diverse physiological roles in the human immune system.

[0098] Double negative T (DNT) cells refer to CD3 cells that are normally present in peripheral blood. + CD4 - CD8 - Mature T lymphocyte subsets comprise approximately 1-3% of peripheral blood mononuclear cells. DNT cells express CD3 molecules and αβ- or γδ-T cell receptors (TCRs) on their surface, but do not express CD4 and CD8 molecules, and are unresponsive to invariant natural killer T (iNKT) cell-specific αGalCer, thus differing from conventional T cells, NK cells, and NKT cells. DNT cells exert cytotoxicity and antigen-presenting functions through various natural mechanisms, while simultaneously releasing cytokines / chemokines to activate a broader immune response, making them promising clinical therapeutic candidates.

[0099] The advantages of DNT cells as a candidate drug for cell immunotherapy include:

[0100] DNT cells can be used for various tumor indications. They selectively target corresponding ligands expressed on various hematologic or solid tumors, such as AML, lymphoma, cervical cancer, and lung cancer, through receptors such as NKG2D and DNAM-1 on their cell surface. They also secrete TNF-α, IFN-γ, Grazyme B, Peforin, IL-2, and IL-4 to exert direct and indirect tumor-killing activities. Furthermore, they can be used in combination with immune checkpoint inhibitors such as PD-1 and CTLA-4, as well as chemotherapy drugs, exhibiting synergistic therapeutic effects.

[0101] DNT cells are not restricted by MHC (histocompatibility complex) molecules when killing tumor cells. Allogeneic DNT cells donated by healthy donors are infused into patients without killing or toxicizing normal cells in the patient's body, and do not affect the further differentiation of hematopoietic stem cells. They do not cause graft-versus-host disease (GvHD) or host-versus-graft reaction. The infused allogeneic DNT cells can remain in the patient's body for a long time to exert tumor-killing activity.

[0102] DNT cells from healthy donors can be efficiently expanded to 5 x 10^6 cells per ml of peripheral blood. 8 The above-mentioned quantities allow for the collection and cryopreservation of cells for the treatment of multiple patients, enabling clinical patients to receive treatment immediately upon diagnosis. This eliminates the product preparation waiting period required for autologous CAR-T products, making them truly off-the-shelf universal (ready-to-use) cell products. Combining the aforementioned broad-spectrum antitumor activity, high safety, versatility, and ability to be used in combination with other immunotherapies, this invention designs and constructs a universal humanized chimeric antigen receptor CART-19 product targeting CD19, and further develops a series of gene-edit-free universal CAR-T cell products based on the DNT cell platform.

[0103] Chimeric antigen receptor (CAR)

[0104] Chimeric antigen receptors (CARs) consist of an extracellular antigen recognition domain, typically a single-chain variable fragment (scFv), a transmembrane region, and an intracellular co-stimulatory signaling domain. CAR design has evolved as follows: First-generation CARs contained only one intracellular signaling component, CD3ζ or FcγRI. Because they had only one activation domain, they could only induce transient T cell proliferation and limited cytokine secretion, failing to provide long-term T cell proliferation signals and sustained in vivo anti-tumor effects, thus failing to achieve satisfactory clinical efficacy. Second-generation CARs introduced a co-stimulatory molecule, such as CD28, 4-1BB, OX40, or ICOS, significantly improving function compared to first-generation CARs and further enhancing the persistence of CAR-T cells and their ability to kill tumor cells. Third- and fourth-generation CARs were developed by tandem with new immune co-stimulatory molecules such as CD27 and CD134.

[0105] The extracellular domain of CARs can recognize one or more specific antigenic determinants (epitopes), and then transduce this signal through intracellular domains, causing cell activation and proliferation, cytotoxicity, and cytokine secretion, thereby eliminating target cells. First, autologous or allogeneic (healthy donor) PBMCs are isolated, activated, and genetically modified to produce CAR-containing immune cells, which are then injected into the patient. These cells specifically kill tumor cells by directly recognizing tumor cell surface antigens in a non-MHC-restricted manner.

[0106] Specifically, the chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and / or a ζ-chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than an antigen receptor or its ligands.

[0107] Connectors can be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR.

[0108] As used herein, the terms "linker" and "hinge region" are used interchangeably and generally refer to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular or cytoplasmic domain of a polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2-100 amino acids, and most preferably 3-50 amino acids.

[0109] When expressed in immune cells, the CAR of this invention enables antigen recognition based on antigen-binding specificity. When it binds to associated antigens on tumor cells, it leads to tumor cell death, resulting in a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with an intracellular domain derived from one or more co-stimulatory molecules and / or the ζ chain. Preferably, the antigen-binding domain is fused with an intracellular domain combining a CD28 co-stimulatory signaling molecule, a 4-1BB co-stimulatory signaling molecule, and a CD3ζ signaling domain.

[0110] As used herein, the basic structure of the chimeric antigen receptor of this invention includes: a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The selection of the tumor-associated antigen directly affects its therapeutic effect on tumors. In this invention, the chimeric antigen receptor of this invention targets CD19.

[0111] CD19 is a transmembrane glycoprotein of approximately 95 kDa, primarily expressed in early B cells. It is expressed in both normal and malignant B lymphocytes and is considered one of the most reliable surface markers covering a relatively long stage of B cell development.

[0112] In a preferred embodiment of the present invention, a single-chain antibody against CD19 is selected as the antigen-binding domain targeting CD19 in the CAR of the present invention.

[0113] In a preferred embodiment, the amino acid sequence of the scFv is as shown in SEQ ID NO:4, which can bind efficiently to the CD19 molecule.

[0114] In this invention, the antigen-binding domain targeting CD19 also includes a conserved variant of the scFv, meaning that compared to the amino acid sequence of the scFv of this invention, at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or analogous properties to form a polypeptide. In this invention, the number of added, deleted, modified, and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%. In this invention, the number of added, deleted, modified, and / or substituted amino acids is typically 1, 2, 3, 4, or 5, preferably 1-3, more preferably 1-2, and most preferably 1.

[0115] For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one implementation, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected, or modified by amino acid substitution, to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0116] Preferably, the CAR construct of the present invention has the following structure: CD8 leader sequence - CD19 scFv (VL-connector-VH) - CD8 hinge region - CD8TM-41BB-CD3-ζ.

[0117] In a preferred embodiment, the amino acid sequence of the CAR of the present invention is shown as positions 1-489 of SEQ ID NO:14. It is particularly noteworthy that in the amino acid sequence of the CD19-targeting CAR of the present invention, position 173 corresponds to glycine (Gly).

[0118] Universal CAR-T cells targeting CD19

[0119] As used herein, the terms "CAR19-DNT cell", "universal CAR-DNT cell targeting CD19", "CAR-T cell of the present invention", and "universal CAR-T cell of the present invention" are used interchangeably and all refer to the universal double-negative T cell of the first aspect of the present invention that expresses a chimeric antigen receptor having the structure of Formula I that specifically targets the human CD19 molecule.

[0120] In this invention, the CAR construct of this invention is expressed in CD19-targeting universal CAR-DNT cells.

[0121] Preferably, the TCR in the CD19-targeting universal CAR-DNT cells is not knocked out.

[0122] Preparation method

[0123] This invention also provides a method for preparing the universal CAR-DNT cells targeting CD19 of this invention, comprising the steps of:

[0124] (i) Provide an expression vector containing a nucleotide sequence for expressing the CAR construct of the present invention;

[0125] (ii) Providing a DNT cell culture medium containing at least one DNT cell; transducing the expression vector from step (i) into the DNT cell to obtain the universal CAR-DNT cell targeting CD19 of the present invention; and

[0126] (iii) Optional detection steps (ii) to obtain universal CAR-DNT cells.

[0127] Preferably, the expression vector in the preparation method of the present invention is a viral expression vector, and more preferably a lentiviral expression vector.

[0128] It is worth noting that obtaining the DNT cell culture medium in step (ii) of the method requires the following steps:

[0129] (iia) Collect peripheral blood samples from healthy donors;

[0130] (iib) Remove CD4 from the peripheral blood sample + CD8 + T cells, thereby obtaining DNT cells;

[0131] (iic) In a culture flask coated with CD3 monoclonal antibody, the DNT cells obtained in (iib) are cultured and amplified in a suitable culture medium to obtain the culture system containing DNT cells required in step (ii).

[0132] Preferably, the culture medium in step (iic) contains a substance selected from the group consisting of: gentamicin, recombinant human interleukin-2, recombinant human interleukin-7, recombinant human interleukin-12, recombinant human interleukin-15, autologous plasma, AB serum, or a combination thereof.

[0133] In another preferred embodiment, in the culture medium of step (iic), the concentration of gentamicin is 40-80 units / ml (preferably 60 units / ml), the concentration of recombinant human interleukin-2 is 150-100 IU / ml (preferably 200-250 IU / ml), the concentration of recombinant human interleukin-15 is 5-20 ng / ml (preferably 10 ng / ml), the concentration of recombinant human interleukin-7 is 1-5 ng / ml (preferably 2 ng / ml), the concentration of recombinant human interleukin-12 is 5-20 ng / ml (preferably 10 ng / ml), the concentration of autologous plasma is 3-25 v% (preferably 10-20 v%), and / or the concentration of AB serum is 4-8 v% (preferably 6 v%).

[0134] In a preferred embodiment of the invention, the culture medium in step (iic) does not contain recombinant or wild-type human interleukin-4, and does not contain AB serum.

[0135] Pharmaceutical Composition

[0136] This invention provides a universal CAR-T cell formulation containing CD19-targeting cells as described in the first aspect of this invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the pharmaceutical composition is a liquid formulation. Preferably, the formulation is an injectable preparation. Preferably, the concentration of the CAR-T cells in the formulation is 1 × 10⁻⁶. 5 -1×10 8 Cells / ml, more optimal 1×10 5 -1×10 6 Cells / ml

[0137] In one embodiment, the formulation may include buffer solutions such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0138] Therapeutic applications

[0139] The present invention also provides the use of CD19-targeting universal CAR-T cells of the first aspect of the present invention and the pharmaceutical composition of the third aspect of the present invention for the preparation of pharmaceutical compositions or formulations for the prevention and / or treatment of cancer, and for the treatment and / or prevention of cancer.

[0140] The cancers described include tumors at various stages of development, including clinically stage I to IV tumors. Cancers can include non-solid tumors (such as hematologic malignancies, such as leukemia and lymphoma) or solid tumors. Types of cancer treated with the CAR of this invention include, but are not limited to, carcinomas, germ cell tumors, and sarcomas, and certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0141] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-borne) cancers include leukemia, including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal dysplasia.

[0142] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0143] The universal CAR-T cells of this invention can also be used as a vaccine type for in vitro immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0144] For the preparation of ex vivo immune cells, at least one of the following occurs in vitro before the cells are administered into mammals: i) cell expansion, ii) introduction of nucleic acid encoding CAR into the cells, and / or iii) cryopreservation of the cells.

[0145] In vitro cell processing procedures are well known in the art and are discussed more fully below. In short, cells are isolated from mammals (preferably humans) and genetically modified (i.e., transduced or transfected in vitro) using a vector expressing the CAR disclosed herein. CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous, allogeneic, or syngeneic relative to the recipient.

[0146] In addition to the use of cell-based vaccines for use with respect to ex vivo immune cells, the present invention also provides compositions and methods for use in vivo to enhance immune responses against targeted antigens in patients.

[0147] The present invention provides a method for treating tumors, comprising administering an effective amount of the universal CAR-T cells of the present invention to a subject in need.

[0148] The universal CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-15, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may comprise target cells as described herein, combined with one or more pharmaceutically or clinically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0149] The pharmaceutical compositions of the present invention can be administered in a manner suitable for treating (or preventing) a disease. The amount and frequency of administration will be determined by factors such as the characteristics of the patient's condition, the type and severity of the disease—although the appropriate dosage can be determined through clinical trials.

[0150] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that a pharmaceutical composition including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 7Administered at a dose of cells per kg body weight (including all integer values ​​within those ranges). The T-cell composition may also be administered at these doses multiple times. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be determined by a physician monitoring the patient's disease progress.

[0151] The composition can be administered in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intraperitoneally or intrapleurally. In another embodiment, the T-cell composition of the present invention is preferably administered via intravenous (iv) injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection. (CART cell products are primarily administered via intravenous infusion, but can be injected directly into the tumor, lymph node, or site of infection.)

[0152] In some embodiments of the invention, cells are activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels, and administered to a patient in combination with any number of relevant therapeutic modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir, interleukin-2, IFN-γ, cytarabine (also known as ARA-C), other cytotoxic chemotherapeutic agents, checkpoint inhibitors such as PD-1 antibodies, anti-CTLA-4 antibodies, and agents that inhibit cytokine storms, such as tocilizumab antibodies against the IL-6 receptor, and other treatments. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and antibodies or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0153] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment.6 One to 1×10 10 The universal CAR-DNT cells of the present invention are administered to a patient, for example, via intravenous infusion.

[0154] The main advantages of this invention include:

[0155] 1) The CD19 CAR in this invention is a humanized chimeric antigen receptor, which has the advantages of low immunogenicity and low likelihood of causing immune rejection compared with the whole mouse chimeric antigen receptor.

[0156] 2) The CAR19-DNT cells in this invention are universal immune cell products that can be prepared without gene editing. The process is simple and the safety is better. DNT cells do not require the participation of TCR molecules to kill tumors, do not have MHC restriction, and will not cause GvHD and HvG. They can be used as a universal immune cell product, avoiding the cumbersome construction process required to knock out genes such as TCR, as well as the problems of large-scale sequencing and high off-target efficiency.

[0157] 3) The method for constructing and expanding CAR-DNT provided in this invention can achieve large-scale production and reduce production costs. Compared with one-to-one individualized treatment, this universal CAR-DNT cell product greatly reduces production costs and alleviates the burden on patients.

[0158] 4) The CAR-DNT cells provided in this invention are not limited by the source of patients and can be expanded on a large scale from healthy donors. The cells have better activity and can provide immediate treatment to patients, solving the problems of low number, poor quality and insufficient quantity of lymphocytes in patients with advanced tumors in the current autologous CAR-T cell therapy.

[0159] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0160] This invention uses a second-generation CD19-CAR, which contains a humanized CD19scFv (clone 11) with a mutant V27G in the CDR1 region of VH. Figure 2 A schematic diagram of the humanized CD19 CAR construct is shown. The invention is further illustrated below by means of embodiments, but the invention is not limited to these specific embodiments.

[0161] Example 1: Construction of humanized anti-CD19FMC63scFv-41BB-CD3ζ

[0162] This invention inserts a humanized CD19-ScFv-CAR structure between the XbaI and EcoRI sites of a lentiviral vector. This structure contains an insert fragment of humanized CD19 ScFv-41BB-CD3ζ between the XbaI and EcoRI cloning sites.

[0163] 1.1 Humanized CD19 antibody: sequences of VH, VL, and scFv

[0164] This invention obtained a humanized CD19 scFv from the mouse CD19FMC63scFv clone, and selected its CDR1 mutation to create a humanized scFv (clone 11). The structure of the humanized CD19 scFv is: VL-linker peptide-VH. The linker peptide sequence is GSTGSSGKPGSGEGSTKG (SEQ ID NO.:7).

[0165] The nucleotide sequences in bold are the sequence of humanized CD19VL (SEQ ID NO: 1); the nucleotide sequences in regular font are VH (SEQ ID NO: 2, bolded to indicate the mutation encoding G (ggc)); and the nucleotide sequences in italics in the middle are (SEQ ID NO: 3) encoding the linker peptide sequence GSTGSGSGKGPGSGEGSTKG (SEQ ID NO: 7).

[0166] Humanized CD19 scFv (SEQ ID NO: 4): bold text is VL, regular text is VH, underlined text is the CDR region, the mutated amino acid position is the 27th position of VH (SEQ ID NO: 6), displayed as large italic G (V27G of VH).

[0167] In the amino acid sequences, the amino acid sequence in bold is VL (SEQ ID NO: 5); the amino acid sequence in regular font is VH (SEQ ID NO: 6); and the amino acid sequence in italics is the linker peptide (SEQ ID NO: 7).

[0168] This invention uses 3D models ( Figure 3 In antigen-antibody binding experiments, it was found that mutations in the CDR1 region of the humanized CD19 antibody VH can improve the binding ability of the antibody to the CD19 antigen.

[0169]

[0170] 1.2 Humanized CD19-CAR sequence

[0171] The humanized CD19-CAR structure is shown in Figure 2 below. The humanized scFv CAR sequence is cloned into a lentiviral vector using the EF1a promoter.

[0172] The following nucleotide sequence is the CD8 leader sequence of humanized CD19ScFv-CD8 hinge-TM8-41BB-CD3ζ. The CAR structure comprises a human CD8 signal peptide, humanized CD19 scFv (VL-linker–VH), CD8 hinge, CD8 transmembrane domain, 41BB co-stimulatory domain and CD3ζ activation domain ( Figure 2 ). CD8 leader sequence-CD19 scFv(V L -Linker–V H )-CD8 hinge-CD8 TM-41BB-CD3-zeta:

[0173] <CD8 leader>

[0174]

[0175] <Humanized CD19 (VL-linker-VH), clone 11 scFv>(the mutated sequence ggc encoding V27G is marked in bold)

[0176]

[0177]

[0178] <CD8 hinge>

[0179]

[0180] <cd8tm>

[0181]

[0182] <4-1BB costimulatory domain>

[0183]

[0184] <CD3ζ>

[0185]

[0186] <EcoRI restriction site>

[0187]

[0188] The amino acid sequence of the humanized CD19-4-1BB-CD3-CAR protein is as follows (for construct structure, please refer to [link]). Figure 3 Mutations in CDR1 VH are indicated by bold underline:

[0189]

[0190] The nucleotide sequence of the humanized CD19-4-1BB-CD3-CAR protein is as follows:

[0191]

[0192] Example 2: Lentiviral Packaging

[0193] 293T cells were cultured at 37°C in a 5% CO2 incubator in DMEM + 10% FBS medium. On day 2, when the cells reached 90% confluence, they were co-transformed with the expression plasmid and packaging plasmids psPAX2 and pMD2.0G. The plasmids were mixed at a suitable molar ratio and added to the culture dish, gently shaken and mixed, and then placed in the incubator. After 48-72 hours, the virus could be harvested, and the floating dead 293T cells were removed by centrifugation. The virus-containing medium was then filtered, concentrated, purified, aliquoted, frozen at -80°C, and the titer was determined.

[0194] Example 3: Preparation of CAR-19DNT cells

[0195] 3.1 Collection of human peripheral blood samples

[0196] Collect 30-400 ml of peripheral blood from a healthy donor into a tube containing sodium heparin.

[0197] 3.2 Preparation and Detection of CAR19-DNT Cells

[0198] 3.2.1 Preparation of CAR19-DNT cells

[0199] Method 1:

[0200] On day 0, following the manufacturer's instructions, using... The kit (Stem Cell Technologies Inc.) removes CD4 by using RBC rosetting. + CD8 + T cells. Blood samples were labeled with anti-human CD4 and CD8 removal reagents and incubated at room temperature for 20 minutes. The blood was then separated into layers in 50 ml centrifuge tubes with an equal volume of Ficoll-Hypaque density gradient. After centrifugation at 2500 rpm for 25 minutes, CD4-removed PBMCs were collected at the Ficoll-plasma interface. + and CD8 + The cells, namely DNT cells, were washed once with 0.9% physiological saline. The obtained DNT cells were then incubated at 75 cm⁻¹. 2 Culture flasks with 1-6×10 6 Cells per ml were cultured in AIM-V medium at 37°C and 5% CO2. The culture flasks were coated with anti-human CD3 monoclonal antibody (clone OKT3) (5-20 μg / ml). The AIM-V medium contained gentamicin (60 units / ml), recombinant human interleukin-2 (250 IU / ml), recombinant human interleukin-15 (10 ng / ml), recombinant human interleukin-7 (2 ng / ml), recombinant human interleukin-12 (10 ng / ml), and autologous plasma (20 v%).

[0201] On day 1, after culturing the prepared DNT cells overnight, they were infected with lentivirus with an MOI of 5 overnight. Subsequently, AIM-V medium was added daily or every other day depending on the cell status.

[0202] On day 5, DNT cells were fully activated and proliferating vigorously. They were then adjusted to a size of 1-3 × 10⁶ cells using fresh AIM-V proliferation medium (containing 60 units / ml gentamicin, 500 IU / ml recombinant human interleukin-2 and recombinant human interleukin-15 (10 ng / ml), recombinant human interleukin-7 (2 ng / ml), recombinant human interleukin-12 (10 ng / ml), and autologous plasma (10 v%)). 6 Continue to expand and culture at a concentration of cells / ml;

[0203] On the 7th day, the height reached 175cm. 2 Culture flasks, with 1-3×10 6 Cells per ml were cultured for 3 days in AIM-V medium containing gentamicin (60 units / ml), recombinant human interleukin-2 (250 IU / ml), recombinant human interleukin-15 (10 ng / ml), recombinant human interleukin-12 (10 ng / ml) and 50 ng / ml anti-human CD3 monoclonal antibody.

[0204] On the 10th day, 175cm 2 Cells from culture flasks were transferred into culture bags at a concentration of 1-3 × 10⁻⁶. 6 Cells per ml were cultured in AIM-V medium containing gentamicin (60 units / ml), recombinant human interleukin-2 (500 IU / ml), recombinant human interleukin-15 (10 ng / ml), recombinant human interleukin-12 (10 ng / ml) and 100 ng / ml anti-human CD3 monoclonal antibody.

[0205] On day 14, CAR-19 DNT cells were harvested. Cells were collected in 250ml conical-bottom centrifuge flasks, centrifuged at 900×g for 10 minutes, and then washed with physiological saline containing 2.5% human serum albumin (also known as "solvent"). The collected CAR-19-DNT cells were then centrifuged at 0.1-1×10⁻⁶ cells / mL. 7 The CAR19-DNT cell preparation is frozen in liquid nitrogen at a concentration of 1 cell / mL. After passing quality inspection, it can be used clinically.

[0206] Method 2:

[0207] On day 0, following the manufacturer's instructions, using... The kit (Stem Cell Technologies Inc.) removes CD4 by using RBC rosetting. + CD8 + T cells. Blood samples were labeled with anti-human CD4 and CD8 removal reagents and incubated at room temperature for 20 minutes. The blood was then separated into layers in 50 ml centrifuge tubes with an equal volume of Ficoll-Hypaque density gradient. After centrifugation at 2500 rpm for 25 minutes, CD4-removed PBMCs were collected at the Ficoll-plasma interface. + and CD8 + The cells, namely DNT cells, were washed once with 0.9% physiological saline. The isolated and purified DNT cells (magnetic beads:DNT cells = 1:1) were mixed with CD3 / CD28 magnetic beads and incubated at 75 cm⁻¹. 2 Culture flasks with 1-6×10 6 Cells per ml were cultured in AIM-V medium at 37°C and 5% CO2, wherein the AIM-V medium contained gentamicin (60 units / ml), recombinant human interleukin-2 (500 IU / ml), recombinant human interleukin-15 (2 ng / ml), recombinant human interleukin-7 (2 ng / ml), recombinant human interleukin-12 (10 ng / ml) and autologous plasma (20 v%).

[0208] On day 1, after culturing the prepared DNT cells overnight, they were infected with lentivirus with an MOI of 5 overnight. Subsequently, AIM-V medium was added daily or every other day depending on the cell status.

[0209] On day 5, after the DNT cells in the culture flask were thoroughly agitated, the CD3 / CD28 magnetic beads were removed using a magnetic rack. The DNT cells were then adjusted to a size of 1-3 × 10⁶ cells using fresh AIM-V proliferation medium (containing 60 IU / ml gentamicin, 500 IU / ml recombinant human interleukin-2 and recombinant human interleukin-15 (10 ng / ml), recombinant human interleukin-7 (2 ng / ml), recombinant human interleukin-12 (10 ng / ml), and autologous plasma (10 v%)). 6 Continue to expand and culture at a concentration of cells / ml;

[0210] On day 7, the cells were transferred to 175 cm² culture flasks and incubated at a rate of 1–3 × 10⁻⁶. 6 Cells per ml were cultured for 3 days in AIM-V medium containing gentamicin (60 units / ml), recombinant human interleukin-2 (500 IU / ml), recombinant human interleukin-15 (10 ng / ml), and recombinant human interleukin-12 (10 ng / ml).

[0211] On the 10th day, 175cm 2 Cells from culture flasks were transferred into culture bags at a concentration of 1-3 × 10⁻⁶. 6 Cells per ml were cultured in AIM-V medium for up to day 14, which contained gentamicin (60 units / ml), recombinant human interleukin-2 (500 IU / ml), recombinant human interleukin-15 (10 ng / ml), and recombinant human interleukin-12 (10 ng / ml).

[0212] On day 14, CAR-19 DNT cells were harvested. Cells were collected in 250ml conical-bottom centrifuge flasks, centrifuged at 900×g for 10 minutes, and then washed with physiological saline containing 2.5% human serum albumin (also known as "solvent"). The collected CAR19-DNT cells were then centrifuged at 0.1-1×10⁻⁶ cells / mL. 7 The CAR19-DNT cell preparation is frozen in liquid nitrogen at a concentration of 1 cell / mL. After passing quality inspection, it can be used clinically.

[0213] 3.2.2 Detection of CAR-19 DNT cell phenotype and positivity rate

[0214] DNT phenotype and CAR19-DNT positivity rate were detected on days 8 and 13, respectively. At this point, high-purity DNT cells (purity exceeding 85%) were obtained. Figure 4 ) and CAR19-DNT cells with high hCD19-CAR positivity were identified in the culture system using flow cytometry by labeling with anti-human CD3, CD4, and CD8 antibodies. Figure 4 Simultaneously, the transduction positivity rate was detected using FMC63-CD19 antibody labeling against humanized CD19. Figure 4 ).

[0215] Example 4: Cell killing experiment

[0216] HeLa cells expressing CD19 (a stable HeLa-CD19 cell line) were established using lentiviral infection as target cells, while HeLa cells not expressing CD19 were used as target cell controls. Untransduced CAR-CD19 DNT cells and humanized CAR-CD19 CD19CAR-DNT cells were collected on days 8 and 13 of culture, respectively, as effector cells with an effector-to-target ratio of 2:1. Real-Time Cytotoxicity Assay (RTCA) was used to monitor the killing effect in real time.

[0217] The results are shown in Figure 5: Compared with DNT cells without CD19-CAR transduction, CAR19-DNT cells transduced with humanized CD19CAR specifically killed HeLa-CD19 cells. There was no significant difference in the killing of CD19-negative HeLa cells between the two cell types. This indicates that humanized CAR19-DNT cells specifically target HeLa-CD19 cells expressing CD19 antigen (Figure 5). Figure 5-A shows the real-time dynamic killing behavior monitored by RTCA at an effector-to-target ratio of 2:1; the real-time quantitative killing results monitored by RTCA at the maximum killing time (18 hours) are shown below. Figure 5-B As shown. Quantitative killing results showed that CAR19-DNT cells on days 8 and 13 of culture exhibited specific killing effects against HeLa-CD19 as high as 98.52% and 98.10%, respectively. Figure 5-B Furthermore, the maximum lethal effect can be maintained over time as the lethality increases. Figure 5-A ).

[0218] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Zhejiang Ruijiamei Biotechnology Co., Ltd. <120> A technique for preparing universal humanized CAR19-DNT cells and its application <130> P2022-2200 <150> CN202010314336.0 <151> 2020-04-20 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 321 <212> DNA <213> Artificial Sequence <400> 1 gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60 attacctgcc gcgcgagcca ggatattagc aaatatctga actggtatca gcagaaaccg 120 ggcaaagcgc cgaaactgct gatttatcat accagccgcc tgcatagcgg cgtgccgagc 180 cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240 gaagattttg cgacctatta ttgccagcag ggcaacaccc tgccgtatac ctttggcggc 300 ggcaccaaag tggaaattaa a 321 <210> 2 <211> 360 <212> DNA <213> Artificial Sequence <400> 2 caggtgcagc tgcaggaaag cggcccgggc ctggtgaaac cgagcgaaac cctgagcctg 60 acctgcaccg tgagcggcgg cagcctgccg gattatggcg tgagctggat tcgccagccg 120 ccgggcaaag gcctggaatg gattggcgtg atttggggca gcgaaaccac ctattataac 180 agcgcgctga aaagccgcgt gaccattagc gtggatacca gcaaaaacca gtttagcctg 240 aaactgagca gcgtgaccgc ggcggatacc gcggtgtatt attgcgcgaa acattattat 300 tatggcggca gctatgcgat ggattattgg ggccagggca ccctggtgac cgtgagcagc 360 <210> 3 <211> 54 <212> DNA <213> Artificial Sequence <400> 3 ggctccacct ctggatccgg caagcccgga tctggcgagg gatccaccaa gggc 54 <210> 4 <211> 245 <212> PRT <213> Artificial Sequence <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln 115 120 125 Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser 130 135 140 Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Pro Asp Tyr Gly Val Ser 145 150 155 160 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile 165 170 175 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Val 180 185 190 Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser 195 200 205 Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr 210 215 220 Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 225 230 235 240 Val Thr Val Ser Ser 245 <210> 5 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 6 <211> 120 <212> PRT <213> Artificial Sequence <400> 6 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <400> 7 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly <210> 8 <211> 63 <212> DNA <213> Artificial Sequence <400> 8 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 9 <211> 135 <212> DNA <213> Artificial Sequence <400> 9 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 10 <211> 72 <212> DNA <213> Artificial Sequence <400> 10 atctacatct gggcgcccct ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gc 72 <210> 11 <211> 126 <212> DNA <213> Artificial Sequence <400> 11 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 12 <211> 342 <212> DNA <213> Artificial Sequence <400> 12 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaat ag 342 <210> 13 <211> 6 <212> DNA <213> Artificial Sequence <400> 13 gaattc 6 <210> 14 <211> 489 <212> PRT <213> Artificial Sequence <400> 14 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 130 135 140 Lys Gly Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 145 150 155 160 Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Pro 165 170 175 Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 180 185 190 Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala 195 200 205 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 210 215 220 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro 260 265 270 Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 275 280 285 Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 290 295 300 Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 305 310 315 320 Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys 325 330 335 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 340 345 350 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 355 360 365 Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 370 375 380 Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu 385 390 395 400 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 405 410 415 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 420 425 430 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 435 440 445 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 450 455 460 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 465 470 475 480 Leu His Met Gln Ala Leu Pro Pro Arg 485 <210> 15 <211> 735 <212> DNA <213> Artificial Sequence <400> 15 gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60 attacctgcc gcgcgagcca ggatattagc aaatatctga actggtatca gcagaaaccg 120 ggcaaagcgc cgaaactgct gatttatcat accagccgcc tgcatagcgg cgtgccgagc 180 cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240 gaagattttg cgacctatta ttgccagcag ggcaacaccc tgccgtatac ctttggcggc 300 ggcaccaaag tggaaattaa aggctccacc tctggatccg gcaagcccgg atctggcgag 360 ggatccacca agggccaggt gcagctgcag gaaagcggcc cgggcctggt gaaaccgagc 420 gaaaccctga gcctgacctg caccgtgagc ggcggcagcc tgccggatta tggcgtgagc 480 tggattcgcc agccgccggg caaaggcctg gaatggattg gcgtgatttg gggcagcgaa 540 accacctatt ataacagcgc gctgaaaagc cgcgtgacca ttagcgtgga taccagcaaa 600 aaccagttta gcctgaaact gagcagcgtg accgcggcgg ataccgcggt gtattattgc 660 gcgaaacatt attattatgg cggcagctat gcgatggatt attggggcca gggcaccctg 720 gtgaccgtga gcagc 735 <210> 16 <211> 1473 <212> DNA <213> Artificial Sequence <400> 16 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggatattc agatgaccca gagcccgagc agcctgagcg cgagcgtggg cgatcgcgtg 120 accattacct gccgcgcgag ccaggatatt agcaaatatc tgaactggta tcagcagaaa 180 ccgggcaaag cgccgaaact gctgattat cataccagcc gcctgcatag cggcgtgccg 240 agccgcttta gcggcagcgg cagcggcacc gattttaccc tgaccattag cagcctgcag 300 ccggaagatt ttgcgaccta ttattgccag cagggcaaca ccctgccgta taccttttggc 360 ggcggcacca aagtggaaat taaaggctcc acctctggat ccggcaagcc cggatctggc 420 gagggatcca ccaagggcca ggtgcagctg caggaaagcg gcccgggcct ggtgaaaccg 480 agcgaaaccc tgagcctgac ctgcaccgtg agcggcggca gcctgccgga ttatggcgtg 540 agctggattc gccagccgcc gggcaaaggc ctggaatgga ttggcgtgat ttggggcagc 600 gaaaccacct attataacag cgcgctgaaa agccgcgtga ccattagcgt ggataccagc 660 aaaaaccagt ttagcctgaa actgagcagc gtgaccgcgg cggataccgc ggtgtattat 720 tgcgcgaaac attattatta tggcggcagc tatgcgatgg attattgggg ccagggcacc 780 ctggtgaccg tgagcagcac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840 atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 900 gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cctggccggg 960 acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020 aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagagaa 1080 gatggctgta gctgccgatt tccagaagaa gaaggag gatgtgaact gagagtgaag 1140 ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1200 ctcaatctag your hand ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260 gagatggggg gaaagccgag aaagaac cctcaggaag gcctgtacaa tgaactgcag 1320 aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380 aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440 cttcacatgc aggccctgcc ccctcgctaa go to 1473

Claims

1. A universal CAR-double-negative T cell targeting CD19, characterized in that, The universal CAR-double-negative T cells express an exogenous CAR construct having the structure shown in Formula I. L-scFv-H-TM-C-CD3ζ (Formula I) In the formula, L is the CD8 signal peptide sequence, and its amino acid sequence is shown as positions 1 to 21 of SEQ ID NO: 14; scFv is a single-chain variable region sequence of an antibody targeting CD19, wherein the scFv contains a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO:6, and a light chain variable region (VL) with an amino acid sequence as shown in SEQ ID NO:5; H is the hinge region derived from CD8, and its amino acid sequence is shown as positions 267 to 311 of SEQ ID NO: 14; TM is a transmembrane domain derived from CD8, and its amino acid sequence is shown as positions 312 to 335 of SEQ ID NO: 14; C is a co-stimulatory signaling molecule derived from 4-1BB, and its amino acid sequence is shown at positions 336 to 377 of SEQ ID NO: 14; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ, and its amino acid sequence is shown in positions 378 to 489 of SEQ ID NO: 14; Each "-" independently represents a linking peptide or peptide bond connecting the above elements; Furthermore, the CAR construct has an amino acid sequence as shown in SEQ ID NO:

14.

2. The universal CAR-double-negative T cells targeting CD19 as described in claim 1, characterized in that, The TCR in the universal CAR-double-negative T cells was either knocked out or not knocked out.

3. The universal CAR-double-negative T cells targeting CD19 as described in claim 1, characterized in that, The TCR in the universal CAR-double-negative T cells was not knocked out.

4. The universal CAR-double-negative T cells targeting CD19 as described in claim 1, characterized in that, The amino acid sequence of the scFv is shown in SEQ ID NO:

4.

5. A method for preparing universal CAR-double-negative T cells targeting CD19 as described in claim 1, characterized in that, Including the following steps: (i) Provide an expression vector containing a polynucleotide sequence encoding a CAR construct as shown in Formula I; L-scFv-H-TM-C-CD3ζ (Formula I) In the formula, L is the CD8 signal peptide sequence, and its amino acid sequence is shown as positions 1 to 21 of SEQ ID NO: 14; scFv is a single-chain variable region sequence of an antibody targeting CD19, wherein the scFv contains a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO:6, and a light chain variable region (VL) with an amino acid sequence as shown in SEQ ID NO:5; H is the hinge region derived from CD8, and its amino acid sequence is shown as positions 267 to 311 of SEQ ID NO: 14; TM is a transmembrane domain derived from CD8, and its amino acid sequence is shown as positions 312 to 335 of SEQ ID NO: 14; C is a co-stimulatory signaling molecule derived from 4-1BB, and its amino acid sequence is shown at positions 336 to 377 of SEQ ID NO: 14; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ, and its amino acid sequence is shown in positions 378 to 489 of SEQ ID NO: 14; Each "-" independently represents a linking peptide or peptide bond connecting the above elements; Furthermore, the CAR construct has the amino acid sequence shown in SEQ ID NO: 14; (ii) Providing a DNT cell culture medium containing at least one double-negative T cell; transducing the expression vector from step (i) into the double-negative T cell to obtain the universal CAR-double-negative T cell targeting CD19 as described in claim 1, wherein in the double-negative T cell culture medium, the concentration of gentamicin is 40-80 units / ml, the concentration of recombinant human interleukin-2 is 150-1000 IU / ml, the concentration of recombinant human interleukin-15 is 5-20 ng / ml, the concentration of recombinant human interleukin-7 is 1-5 ng / ml, the concentration of recombinant human interleukin-12 is 5-20 ng / ml, the concentration of autologous plasma is 3-25 v%, and / or the concentration of AB serum is 4-8 v%; and (iii) Detect the universal CAR-double-negative T cells obtained in step (ii).

6. The method as described in claim 5, characterized in that, The expression vector is a lentiviral expression vector.

7. The method as described in claim 5, characterized in that, The polynucleotide sequence integrated into the expression vector in step (i) is shown in SEQ ID NO:

16.

8. The method as described in claim 5, characterized in that, In step (i), the polynucleotide sequence contains the nucleotide sequence of scFv as shown in formula (I) of SEQ ID NO:

15.

9. A pharmaceutical composition, characterized in that, include: (a) Universal CAR-double-negative T cells targeting CD19 as described in any one of claims 1-4; and (b) Pharmaceutically acceptable carriers.

10. Use of a universal CAR-double-negative T cell targeting CD19 as described in any one of claims 1-4, characterized in that, Used to prepare a pharmaceutical composition or formulation for treating cancer, wherein the cancer is diffuse large B-cell lymphoma (DLBCL).

Images