Natural killer cells expressing dual-targeting chimeric antigen receptors for CD19 and CD22, and uses thereof

Abstract

The present invention relates to NK cells expressing dual-targeting chimeric antigen receptors for CD19 and CD22, and uses thereof. In the present invention, it was found that the delivery efficiency of a CAR expression vector and the production efficiency of CAR-NK cells are excellent when, among the cell surface proteins of PBMCs for producing CAR-NK cells (UCI-101), CD16 is expressed at 70% or less, natural killer group 2D (NKG2D) is expressed at less than 10%, CD57 is expressed at 30% or less, low-density lipoprotein receptor (LDLR) is expressed at 0.1% or more, and natural cytotoxicity triggering receptor 3 (NKp30) is expressed at less than 10%. In addition, optimal conditions for inducing NK cell differentiation and optimal conditions for transduction, under which the delivery efficiency of a CAR expression vector and the production efficiency of CAR-NK cells are excellent, were established, and CAR-NK cells produced by the method according to the present invention were found to exhibit an excellent antitumor effect in an animal model, and thus can be effectively used as a composition for preventing or treating diseases related to CD22 (or CD19) expression or diseases related to B cells.

Description

Natural Killer Cells Expressing Dual-Targeting Chimeric Antigen Receptors for CD19 and CD22 and Uses Thereof

[0001]

[0002] The present invention relates to NK cells expressing dual-target chimeric antigen receptors for CD19 and CD22 and their uses, and more specifically, to a method for producing CAR-NK cells targeting CD19 and CD22, and a pharmaceutical composition for the prevention or treatment of B cell-mediated diseases comprising CAR-NK cells produced by the said method.

[0003]

[0004] CD22 is expressed in most B-cell leukemias and lymphomas, including NHL, acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (B-CLL), and especially acute non-lymphocytic leukemia (ANLL).

[0005] The development of CD22-specific antibodies is underway for the treatment or diagnosis of diseases associated with such CD22 expression. International Patent Publication WO1998-041641 contains V H 44 and V L A recombinant anti-CD22 antibody having a cysteine ​​residue at the 100 position is disclosed, and International Patent Publication WO 1998-042378 discloses an anti-CD22 antibody for the treatment of B-cell malignancies.

[0006] As mentioned above, monoclonal antibodies are primarily produced using mice for therapeutic purposes. However, non-human antibodies, such as mouse-derived monoclonal antibodies, are considered foreign antigens within the human body and trigger an immune response; furthermore, their short half-life limits their therapeutic efficacy.

[0007] To solve the above problem, humanized antibodies have been developed in which the portion of the antibody excluding only the antigen-binding site is replaced with a human antibody. The currently used method for replacing mouse antibodies with humanized antibodies involves selecting the human antibody gene most similar to the antibody to be replaced and replacing only the CDR region of the mouse antibody with the human antibody CDR location using a method called CDR transplantation. Since most of the gene of such humanized antibodies has been humanized, they have the advantage of reducing the immune response within the human body.

[0008]

[0009] Meanwhile, antibodies specific to the various other B cell surface markers (antigens) mentioned above are being developed for the treatment of B cell disorders, diseases, autoimmune diseases, and transplant rejection. In addition to the CD22 antigen, CD19 is a commonly used antigen target, and many clinical trials for CAR-T cells targeting it are also underway. However, there are differences in the expression levels of target antigens depending on the cell, such as in leukemia cells that do not express CD19, and single CAR or single CAR-T cell therapies targeting only one antigen may result in problems such as the loss of the target antigen due to the immune evasion strategies of tumor cells. In fact, CD19-negative relapses in which CD19 is not expressed have been observed in patients with B-cell ALL (acute lymphocytic leukemia) (up to 25% of B-cell ALL patients who initially responded to CD19 CAR-T therapy), and this phenomenon has been identified as a mechanism of tumor cell resistance to CAR-T cell therapy (Maude, SL, et al., N. Engl. J. Med., 378:439-448, 2018).

[0010] To address this, CAR-immune effector cells targeting dual or multiple antigens are being studied, and targeting two antigens simultaneously can reduce the possibility of antigen-losing variants.

[0011] In particular, NK cells are cytotoxic lymphocytes that constitute an important component of the innate immune system. These cells have various functions, and in particular, they have the ability to kill tumor cells, virus-infected cells, cells undergoing oncogenic transformation, and other abnormal cells in vivo. Since NK cells secrete IL-3 and Granulocyte-Macrophage Colony-stimulating Factor, they are less likely to cause CRS, and the PD-1 levels secreted by NK cells are substantially low and cause almost no immunosuppression. Furthermore, they are known to enhance anti-PD-1 immunotherapy by migrating dendritic cells to tumors, thus proving the superiority of CAR-NK development. In addition, preclinical and clinical trial results for CAR-NK cell therapy have confirmed that it can effectively eliminate hematological and solid tumor cells, highlighting its potential to be developed as an anticancer immunotherapy for a wide range of cancers, including solid tumors (EL Siegler, et al., Cell Stem Cell, 23:160-161, 2018).

[0012]

[0013] Accordingly, the present invention has selected antibodies that bind to CD22 to reduce immune responses within the human body, and has manufactured bispecific chimeric antigen receptors (Bivalent CAR or Bispecific CAR) and bispecific CAR-T cells targeting CD22 and CD19 using these antibodies (Korean Registered Patent No. 10-2393776).

[0014] In order to establish more effective CAR-immune effector cells, CAR-NK cells that are dual-specific to CD22 and CD19 were prepared, and a final culture process to improve the CAR expression rate in NK cells was established, thereby completing the present invention.

[0015]

[0016] Accordingly, the objective of the present invention is to provide a method for producing CAR-NK cells targeting CD19 and CD22 and CAR-NK cells targeting CD19 and CD22 produced by said method.

[0017] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a disease mediated by B cells comprising CAR-NK cells targeting CD19 and CD22 prepared by the above method.

[0018]

[0019] In order to achieve the aforementioned purpose,

[0020] The present invention relates to a method for differentiating peripheral blood mononuclear cells (PBMCs) that express CD16 at 70% or less, NKG2D (natural killer group 2D) at less than 10%, CD57 at 30% or less, LDLR (low-density lipoprotein receptor) at 0.1% or more, and NKp30 (Natural cytotoxicity triggering receptor 3) at less than 10% into NK cells by treating them with dexamethasone, and then

[0021] The method comprises the step of transfecting the NK cells with a vector containing a polynucleotide encoding a bispecific chimeric antigen receptor (CAR) targeting CD19 and CD22, and

[0022] The present invention provides a method for producing CAR-NK cells targeting CD19 and CD22, characterized in that the above-mentioned bispecific chimeric antigen receptor comprises a CD19-binding domain and a CD22-binding domain; a transmembrane domain; a costimulatory domain; and an intracellular signal transduction domain.

[0023]

[0024] In a preferred embodiment of the present invention, the dexamethasone is administered at the beginning of NK cell differentiation induction (day 0), and differentiation induction can be performed for 5 to 10 days in a medium containing dexamethasone.

[0025] In another preferred embodiment of the present invention, to induce NK cell differentiation, the cells may be co-cultured with feeder cells expressing one or more proteins selected from the group consisting of IL-2, IL-15, IL-21, OX40L (CD134 ligand), and lunasin, and the feeder cells may be K562 cell lines, preferably irradiated K562 cell lines.

[0026] In another preferred embodiment of the present invention, the vector may be a viral vector, preferably a lentiviral vector, and may be treated with 3 to 7 MOI (Multiplicity of infection).

[0027] In another preferred embodiment of the present invention, prostaglandin E2 (PGE2) and a polyoxyethylene-polyoxypropylene block copolymer may be additionally included to increase the expression rate of the chimeric antigen receptor (CAR) during the transduction process.

[0028] In another preferred embodiment of the present invention, the CD19-binding domain and the CD22-binding domain may be connected in the order of the light chain variable region of an antibody that specifically binds to CD19 - the heavy chain variable region of an antibody that specifically binds to CD22 - the light chain variable region of an antibody that specifically binds to CD22 - the heavy chain variable region of an antibody that specifically binds to CD19.

[0029] In another preferred embodiment of the present invention, the heavy chain variable region of the antibody specifically binding to CD22 comprises a CDR1 region represented by the amino acid of SEQ ID NO. 1, a CDR2 region represented by the amino acid of SEQ ID NO. 2, and a CDR3 region represented by the amino acid of SEQ ID NO. 3.

[0030] The light chain variable region of the antibody specifically binding to the above CD22 includes a CDR1 region represented by the amino acid of SEQ ID NO. 4, a CDR2 region represented by the amino acid of SEQ ID NO. 5, and a CDR3 region represented by the amino acid of SEQ ID NO. 6, and

[0031] The heavy chain variable region of the antibody specifically binding to the above CD19 comprises a CDR1 region represented by the amino acid of SEQ ID NO. 11, a CDR2 region represented by the amino acid of SEQ ID NO. 12, and a CDR3 region represented by the amino acid of SEQ ID NO. 13, and

[0032] The light chain variable region of the antibody that specifically binds to the CD19 may include a CDR1 region represented by the amino acid of SEQ ID NO. 14, a CDR2 region represented by the amino acid of SEQ ID NO. 15, and a CDR3 region represented by the amino acid of SEQ ID NO. 16.

[0033]

[0034] In another preferred embodiment of the present invention, the transmembrane domain is a protein derived from any one selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1, the co-stimulation domain is a protein derived from any one selected from the group consisting of CD28, 4-1BB, OX-40 and ICOS, and the signal transduction domain may be a protein derived from CD3ζ.

[0035] In another preferred embodiment of the present invention, a hinge region may be additionally included between the C end of the binding domain and the N end of the transmembrane domain.

[0036]

[0037] In order to achieve other purposes,

[0038] The present invention provides CAR-NK cells targeting CD19 and CD22 prepared by the above method.

[0039] In a preferred embodiment of the present invention, the CAR-NK cells may be characterized by having low expression of CD16, NKG2D, LDLR, and NKp30, and a high expression rate of CD57.

[0040]

[0041] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of a B cell-mediated disease comprising CAR-NK cells targeting CD19 and CD22 prepared by the above method.

[0042] In a preferred embodiment of the present invention, the disease mediated by the B cell may be selected from the group consisting of tumors, lymphomas, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indeterminate NHL, refractory NHL, refractory indeterminate NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt lymphoma, and mantle cell lymphoma.

[0043]

[0044] In the present invention, it was confirmed that among the cell surface proteins of PBMCs for producing CAR-NK cells (UCI-101), CD16 is expressed at 70% or less, NKG2D (natural killer group 2D) at less than 10%, CD57 at 30% or less, LDLR (low-density lipoprotein receptor) at 0.1% or more, and NKp30 (Natural cytotoxicity triggering receptor 3) at less than 10%, resulting in excellent delivery efficiency of the CAR expression vector and CAR-NK cell production efficiency. Furthermore, optimal conditions for NK cell differentiation induction and transduction were established that resulted in excellent delivery efficiency of the CAR expression vector and CAR-NK cell production efficiency. Since it was confirmed that CAR-NK cells produced by the method of the present invention exhibit excellent anti-tumor effects in animal models, they can be usefully utilized as a composition for the prevention or treatment of diseases related to CD22 (or CD19) expression or diseases related to B cells.

[0045]

[0046] Figure 1 is a schematic diagram showing a bispecific chimeric antigen receptor (Bispecific CAR) targeting CD19 and CD22.

[0047] Figure 2 is a schematic diagram showing a method for producing CD19xCD22-CAR-NK cells using peripheral blood mononuclear cells (PBMC).

[0048] Figure 3 is a schematic diagram for establishing optimal treatment conditions for dexamethasone. The method in Figure 3a involved treating NK cells with dexamethasone at a concentration of 100 nM or 1000 nM at the beginning of differentiation induction (Day 0), followed by culture under conditions of medium exchange after 1 hour of exposure, medium exchange after 24 hours of exposure, and exposure for 7 days without medium exchange.

[0049] In addition, the method in Fig. 3b involved treating NK cells with dexamethasone at a concentration of 100 nM or 1000 nM after 7 days of differentiation induction, followed by culture under conditions of medium exchange after 1 hour of exposure, medium exchange after 24 hours of exposure, and exposure for 7 days without medium exchange.

[0050] Figure 4 shows data when dexamethasone was administered during the early stages of NK differentiation induction using the method of Figure 3a. Figure 4a shows the NK cell growth curve according to dexamethasone treatment concentration and exposure time, Figure 4b shows the PBMC population, and Figure 4c shows the NK cell expansion fold.

[0051] Figure 5 shows the data when dexamethasone was administered 7 days after NK differentiation induction using the method of Figure 3b. Figure 5a shows the NK cell growth curve according to dexamethasone treatment concentration and exposure time, Figure 5b shows the degree of PBMC growth, and Figure 5c shows the NK cell expansion fold.

[0052] Figure 6 shows data on the optimized timing of transduction to improve the CAR expression rate of CD19xCD22-CAR-NK cells. Figure 6a is a schematic diagram of the experiment according to the timing of CD19xCD22-CAR lentivirus vector transduction, and Figure 6b is data confirming the CAR expression rate according to the timing of CD19xCD22-CAR lentivirus vector transduction.

[0053] Figure 7 shows data on the optimization of additives during transduction to enhance the CAR expression rate of CD19xCD22-CAR-NK cells. Figure 7a shows data confirming the CAR expression rate according to the type of additive during CD19xCD22-CAR lentivirus vector transduction, and Figure 7b shows the CAR expression rate expressed as a percentage. Figure 7c shows data on the CD19 and CD22 expression rates on the final day of culture (top left), CD19 and CD22 expression rates according to culture days (top right), and CD19xCD22-CAR-NK cell proliferation rate (bottom) of CD19xCD22-CAR-NK cells prepared by transduction under PGE2 + Lentiboost conditions.

[0054] Figure 8 shows data obtained by optimizing the MOI of the CD19xCD22-CAR lentivirus vector to improve the CAR expression rate of CD19xCD22-CAR-NK cells, and (a) data confirming the CAR expression rate and (b) cell viability 7 days after transduction.

[0055] Figure 9 shows data confirming (a) the growth rate of NK cells, (b) the growth rate of CD19xCD22-CAR-NK cells, and (c) the CAR expression rate according to the type of donated PBMC when the final culture process established to improve the CAR expression rate in NK cells was applied.

[0056] Figure 10 shows the surface markers of CD19xCD22-CAR-NK cells according to the type of donated PBMC when the final culture process established to improve CAR expression rate in NK cells was applied.

[0057] Figure 11 is data analyzing the marker expression patterns of PBMCs by donor to establish selection criteria for donated PBMCs.

[0058] Figure 12 is data analyzing the phenotype of CD19xCD22-CAR-NK cells (UCI-101) prepared by the optimal manufacturing method of the present invention. Figure 12a is the result of exogenous and endogenous CD3ζ Western blotting analysis in PBNK (periopheral blood NK cell) and UCI-101, and Figure 12b is data confirming aging markers of PBNK and UCI-101.

[0059] Figure 13a shows data confirming the CAR expression rate and cytotoxicity against target cells (Raji cells and Daudi cells) of CD19xCD22-CAR-NK cells (UCI-101) prepared by the optimal manufacturing method of the present invention, and Figure 13b shows data confirming the degree of cytokine secretion when CD19xCD22-CAR-NK cells are co-cultured with target cells (Raji cells).

[0060] Figure 14 is data showing the luminescence values ​​of U2932-Luc cells to evaluate the efficacy of UCI-101 when UCI-101 was administered once to a U2932 blood cancer animal model as shown in (a) to confirm the antitumor effect of CD19xCD22-CAR-NK cells (UCI-101).

[0061] Figure 15 shows the antitumor effect of CD19xCD22-CAR-NK cells (UCI-101) according to administration concentration, to confirm the antitumor effect in a U2932 blood cancer animal model as shown in the schematic diagram of (a) by administering UCI-101 at a low concentration (2.5 x 10⁻⁶). 6 Cells), medium concentration (5 x 10 6 Cells), high concentration (1 x 10 7 (b) Data showing the luminescence values ​​of U2932-Luc cells to evaluate the efficacy of UCI-101 when administered repeatedly to cells.

[0062] Figure 16 shows the antitumor effect of CD19xCD22-CAR-NK cells (UCI-101) according to administration concentration in a U2932 blood cancer animal model as shown in the schematic diagram of (a), by administering UCI-101 at a medium concentration (5 x 10⁻⁶). 6 (b) Data showing the luminescence values ​​of U2932-Luc cells to evaluate the efficacy of UCI-101 when administered at 7-day intervals.

[0063] Figure 17 shows (a) CD19 to confirm the antigen-specific cytotoxic activity of UCI-101. - / - , CD22 - / - , CD19 - / - CD22 - / - (b) Data on the expression levels of CD19 and CD22 confirmed by constructing KO cell lines and CD19xCD22-CAR-NK cells (UCI-101) and CAR-T cells and CD19xCD22-CAR-NK cells (UCI-101) and CD19xCD22-CAR-T cells.

[0064] Fig. 18 is the CD19 manufactured in Fig. 17 - / - , CD22 - / - , CD19 - / - CD22 - / - This is data confirming (a) antigen-specific cytotoxicity and (b) the degree of cytokine secretion by CD19xCD22-CAR-NK cells and CD19xCD22-CAR-T cells when KO cell line (U2932) and CD19xCD22-CAR-NK cells and CD19xCD22-CAR-T cells were cultured, respectively.

[0065]

[0066] The present invention will be described in detail below.

[0067]

[0068] The present invention relates to a method for differentiating peripheral blood mononuclear cells (PBMCs) that express CD16 at 70% or less, NKG2D (natural killer group 2D) at less than 10%, CD57 at 30% or less, LDLR (low-density lipoprotein receptor) at 0.1% or more, and NKp30 (Natural cytotoxicity triggering receptor 3) at less than 10% into NK cells by treating them with dexamethasone, and then

[0069] The method includes the step of introducing a vector comprising a polynucleotide encoding a bispecific chimeric antigen receptor (CAR) targeting CD19 and CD22 into the NK cells.

[0070] The present invention relates to a method for producing CAR-NK cells targeting CD19 and CD22, characterized in that the above-mentioned bispecific chimeric antigen receptor comprises a CD19-binding domain and a CD22-binding domain; a transmembrane domain; a costimulatory domain; and an intracellular signal transduction domain.

[0071]

[0072] More specifically, the above manufacturing method comprises: (a) a step of treating peripheral blood mononuclear cells (PBMCs) isolated from blood with dexamethasone to induce differentiation into NK cells, and isolating the activated NK cells; and

[0073] (b) a step of transducing the activated NK cells into a vector comprising a polynucleotide encoding a bispecific chimeric antigen receptor (CAR) targeting CD19 and CD22, and

[0074] The PBMC of step (a) above expresses CD16 at 70% or less, NKG2D (natural killer group 2D) at less than 10%, CD57 at 30% or less, LDLR (low-density lipoprotein receptor) at 0.1% or more, and NKp30 (Natural cytotoxicity triggering receptor 3) at less than 10%, and

[0075] The present invention relates to a method for producing CAR-NK cells targeting CD19 and CD22, characterized in that the above-mentioned bispecific chimeric antigen receptor comprises a CD19-binding domain and a CD22-binding domain; a transmembrane domain; a costimulatory domain; and an intracellular signal transduction domain.

[0076]

[0077] In the present invention, the dexamethasone is administered at the beginning of NK cell differentiation induction (day 0), and differentiation induction can be performed for 5 to 10 days in a medium containing dexamethasone.

[0078] In the present invention, to induce differentiation of the NK cells, the cells may be co-cultured with a feeder cell expressing one or more proteins selected from the group consisting of IL-2, IL-15, IL-21, OX40L (CD134 ligand), and lunasin, and the feeder cell may be a K562 cell line, preferably an irradiated K562 cell line.

[0079] In the present invention, the vector may be a viral vector, preferably a lentivirus vector, and may be treated with 3 to 7 MOI (Multiplicity of infection).

[0080] In the present invention, prostaglandin E2 (PGE2) and a polyoxyethylene-polyoxypropylene block copolymer may be additionally included to increase the expression rate of the chimeric antigen receptor (CAR) during the transduction process. In the present invention, Poloxamer synperonic F108 (Non-ionic, Amphiphilic poloxamer synperonic F108, LentiBOOST) was preferably used as the polyoxyethylene-polyoxypropylene block copolymer, but is not limited thereto.

[0081]

[0082] In a specific embodiment of the present invention, as a result of establishing optimal conditions for improving the CAR expression rate of CD19xCD22-CAR-NK cells, (1) dexamethasone is treated at the beginning of NK differentiation induction (day 0), differentiation induction is performed for 5 to 10 days (preferably 7 days) in a medium containing dexamethasone, (2) CD19xCD22-CAR lentivirus vector is treated at 3 to 7 MOI (preferably 5 MOI) when transfecting the NK cells differentiated in (1), and (3) PGE2 and LentiBOOST are additionally included to increase the expression rate of the chimeric antigen receptor (CAR) when transfecting.

[0083] In addition, it was confirmed that among the cell surface proteins of PBMCs for producing CAR-NK cells (UCI-101), CD16 is expressed at 70% or less, NKG2D (natural killer group 2D) at less than 10%, CD57 at 30% or less, LDLR (low-density lipoprotein receptor) at 0.1% or more, and NKp30 (Natural cytotoxicity triggering receptor 3) at less than 10%, the delivery efficiency of the CAR expression vector and the efficiency of producing CAR-NK cells are excellent, and it was confirmed that CD19xCD22-CAR-NK cells produced by the method of the present invention show excellent anti-tumor effects in animal models.

[0084]

[0085] In the present invention, the CD19-binding domain and the CD22-binding domain may be connected in the order of the light chain variable region of an antibody that specifically binds to CD19 - the heavy chain variable region of an antibody that specifically binds to CD22 - the light chain variable region of an antibody that specifically binds to CD22 - the heavy chain variable region of an antibody that specifically binds to CD19.

[0086] Specifically, the heavy chain variable region of the antibody specifically binding to CD19 comprises a CDR1 region represented by the amino acid of SEQ ID NO. 11, a CDR2 region represented by the amino acid of SEQ ID NO. 12, and a CDR3 region represented by the amino acid of SEQ ID NO. 13.

[0087] The light chain variable region of the antibody that specifically binds to the CD19 may include a CDR1 region represented by the amino acid of SEQ ID NO. 14, a CDR2 region represented by the amino acid of SEQ ID NO. 15, and a CDR3 region represented by the amino acid of SEQ ID NO. 16.

[0088]

[0089] In the present invention, the bispecific or bivalent chimeric antigen receptor is a CAR capable of simultaneously binding two different types of antigens. In the present invention, a bispecific chimeric antigen receptor targeting both CD19 and CD22 is preferably prepared, and the CD19-binding domain may use any known anti-CD19 antibody sequence without limitation.

[0090] In the present invention, a signal peptide may be additionally included at the N-terminus of the CD19-binding domain and the CD22-binding domain, and the "signal peptide" generally refers to a peptide chain for guiding protein delivery. The signal peptide may be a short peptide having a length of 5 to 30 amino acids, and in the present invention, the amino acid sequence of SEQ ID NO. 33 was preferably used.

[0091] In the present invention, a hinge region may be further included between the C-terminus of the CD19-binding domain and the CD22-binding domain and the N-terminus of the transmembrane domain, said hinge region may be derived from CD8α and preferably represented by the amino acid sequence of SEQ ID NO. 34. The "hinge region" generally refers to a connection region between the antigen-binding region and the immune cell Fc receptor (FcR)-binding region.

[0092] In the present invention, the "transmembrane domain" generally refers to a domain of a CAR that passes through a cell membrane and connects to an intracellular signaling domain to perform signaling. The transmembrane domain may be derived from a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1, and preferably may be represented by the amino acid sequence of SEQ ID NO. 35.

[0093] In the present invention, the "costimulatory domain" generally refers to an intracellular domain capable of providing an immunostimulatory molecule, which is a cell surface molecule necessary for an effective response of lymphocytes to antigens. The costimulatory domain described above may include a costimulatory domain of CD28, and may include a costimulatory domain of the TNF receptor family such as the costimulatory domains of OX40 and 4-1BB, and preferably may be 4-1BB represented by the amino acid sequence of SEQ ID NO. 36.

[0094] In the present invention, "intracellular signal transduction domain" generally refers to a domain located inside a cell and capable of transmitting a signal. In the present invention, the intracellular signal transduction domain is the intracellular signal transduction domain of a chimeric antigen receptor. For example, the intracellular signal transduction domain may be selected from the CD3ζ intracellular domain, CD28 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain, and OX40 intracellular domain, and preferably may be CD3ζ represented by the amino acid sequence of SEQ ID NO. 37.

[0095] In the present invention, the vector is a recombinant viral vector, preferably a lentiviral vector, comprising an operablely linked EF1α promoter; a polynucleotide encoding a signal peptide; a polynucleotide encoding a CD19-binding domain and a CD22-binding domain; a polynucleotide encoding a transmembrane domain; and a polynucleotide encoding an intracellular signaling domain, and may additionally comprise a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) to increase protein expression.

[0096] The above EF1α promoter may be represented by the nucleotide sequence of SEQ ID NO. 26 and, if necessary, may include a sequence that is 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the nucleotide sequence of SEQ ID NO. 27.

[0097]

[0098] In another aspect, the present invention relates to CAR-NK cells targeting CD19 and CD22 produced by the above method.

[0099] In the present invention, the CAR-NK cells may be characterized by having low expression of surface markers CD16, NKG2D, LDLR, and NKp30, and a high expression rate of CD57.

[0100]

[0101] In another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of a disease mediated by B cells comprising CAR-NK cells targeting CD19 and CD22 prepared by the above method.

[0102] In the present invention, the disease mediated by the B cell may be selected from the group consisting of tumors, lymphomas, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indeterminate NHL, refractory NHL, refractory indeterminate NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt lymphoma, and mantle cell lymphoma.

[0103]

[0104] In the present invention, the composition may include a therapeutic agent for a disease mediated by B cells, and the therapeutic agent may exist in a covalently bound state to an antibody that specifically binds to CD19 or CD22, or may be administered in combination with the CD22-CAR immune effector cells or CD19xCD22-CAR immune effector cells of the present invention.

[0105] The above therapeutic agents include small molecule drugs, peptide drugs, toxins (e.g., cytotoxins), etc.

[0106] The above-mentioned low-molecular-weight drug exhibits pharmaceutical activity of interest and may generally be a compound with a molecular weight of about 800 Da or less or 2000 Da or less. Inorganic low-molecular-weight refers to a molecule that does not contain any carbon atoms, whereas organic low-molecular-weight refers to a compound that contains at least one carbon atom.

[0107] The above-mentioned peptide drug refers to an amino acid containing a polymeric compound, which includes naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimics. The above-mentioned peptide drug may be obtained by chemical synthesis or generated from a genetically encoded source (e.g., a recombinant source). The molecular weight of the peptide drug may range from 200 Da to 10 kDa or greater.

[0108] The above toxin is preferably a cytotoxin, and cytotoxins include, but are not limited to, ricin, abrine, diphtheria toxin, Pseudomonas extracellular toxin (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, American pokeweed antiviral protein (PAP), botulinum toxin, briodine, momordine, and buganin.

[0109] In addition, the therapeutic agent may be an anticancer agent. Anticancer agents reduce the proliferation of cancer cells and include non-peptide (i.e., non-protein) compounds, encompassing cytotoxic agents and cell proliferation inhibitors. Non-limiting examples of anticancer agents include alkylating agents, nitrosores, anmetatals, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds may also be used.

[0110]

[0111] In the above pharmaceutical composition, CD19xCD22-CAR NK cells may be the only active ingredient in the therapeutic or diagnostic composition, or may be used together with other active ingredients including other antibody components such as anti-T cells, anti-IFNγ or anti-LPS antibodies, or non-antibody components such as xanthine.

[0112] Preferably, the pharmaceutical composition comprises a therapeutically effective amount of the antibody of the present invention. As used herein, the term "therapeutically effective amount" refers to the amount of therapeutic agent required to treat, improve, or prevent a target disease or condition, or the amount of therapeutic agent required to produce a detectable therapeutic or preventive effect. For any antibody, the therapeutically effective dose may be initially determined by cell culture assays or by animal models, such as rodents, rabbits, dogs, pigs, or primates. Animal models may also be used to determine appropriate concentration ranges and routes of administration. This information may be used to determine useful doses and routes for human administration.

[0113] The precise effective dose for human patients may vary depending on the severity of the disease state, the patient's general health condition, the patient's age, weight and sex, diet, time of administration, frequency of administration, drug composition, response sensitivity, and tolerance / response to treatment. The above dose may be determined by conventional experiments and is within the scope of the clinician's judgment. Generally, the effective dose is 0.01 to 50 mg / kg, preferably 0.1 to 20 mg / kg, and more preferably about 15 mg / kg.

[0114] The composition may be administered to the patient individually or in combination with other preparations, drugs, or hormones.

[0115] The dosage of the antibody of the present invention administered depends on the nature of the condition to be treated, the grade of malignant lymphoma or leukemia, and whether the antibody is used for disease prevention or to treat an existing condition.

[0116] The frequency of administration depends on the half-life of the antibody molecule and the duration of the drug's effect. If the antibody molecule has a short half-life (e.g., 2 to 10 hours), it is necessary to provide a dose once a day or more. Or, if the antibody molecule has a long half-life (e.g., 2 to 15 days), it is necessary to provide a dose once a day, once a week, or once every 1 or 2 months.

[0117] Additionally, the pharmaceutical composition may contain a pharmaceutically acceptable carrier for the administration of antibodies. The carrier itself must not induce the production of antibodies harmful to the individual receiving the composition and must be non-toxic. Suitable carriers may be slowly metabolized macromolecules, such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, and inactive viral particles.

[0118] Pharmaceutically acceptable salts may be used, for example, mineral salts such as hydrochloride, hydrobromide, phosphate, and sulfate, or salts of organic acids such as acetic acid, propionic acid, malonic acid, and benzoic acid.

[0119] Pharmaceutically acceptable carriers in the therapeutic composition may additionally include liquids such as water, saline solution, glycerol, and ethanol. Additionally, auxiliary substances such as humectants, emulsifiers, or pH buffers may be present in the composition. The carriers may be formulated as tablets, pills, coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions for ingestion of the pharmaceutical composition by a patient.

[0120] Preferred forms for administration include forms suitable for parenteral administration by, for example, injection or infusion (e.g., bolus injection or serial infusion). When the product is for injection or injectable use, it may take the form of a suspension, solution, or emulsion in an oil or water-soluble excipient, which may include formulation agents such as a suspending agent, preservative, stabilizer, and / or dispersant. Alternatively, the antibody molecule may be in an anhydrous form and may be reconstituted with a suitable sterile solution before use.

[0121] Once formulated, the composition of the present invention may be administered directly to a patient. The patients to be treated may be animals. However, it is preferable to adapt the composition for administration to human patients.

[0122] The pharmaceutical composition of the present invention may be administered by any route, including but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intravertebral, intraventricular, transdermal, transcutaneous (e.g., see WO 98 / 20734), subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, vaginal, or rectal routes. A hypospray may be used to administer the pharmaceutical composition of the present invention. Typically, the therapeutic composition may be prepared as an injectable substance as a liquid solution or suspension. Additionally, a solid form suitable for a liquid excipient solution or suspension may be prepared prior to injection.

[0123] Direct delivery of the composition may generally be achieved by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, or intramuscular injection, or it may be delivered into the interstitial space of the tissue. Additionally, the composition may be administered to a wound site. Dosage administration may be a single-dose schedule or a multiple-dose schedule.

[0124] The active ingredient in the composition may be an antibody molecule. It may be susceptible to degradation within the gastrointestinal tract. Therefore, if the composition is administered via a route involving the gastrointestinal tract, it will be necessary to contain a mechanism that protects the antibody from degradation but releases the antibody once it has been absorbed from the gastrointestinal tract.

[0125] A complete discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ, 1991).

[0126]

[0127] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0128]

[0129] Example 1: Preparation of a bispecific chimeric antigen receptor expression vector targeting CD19 / CD22

[0130] In the present invention, antibodies that bind to CD22 were selected, and bispecific chimeric antigen receptors (Bivalent CAR or Bispecific CAR) and bispecific CAR-T cells targeting CD22 and CD19 were manufactured using these antibodies (Korean Registered Patent No. 10-2393776). Based on this, a lentivirus vector (CD19xCD22-CAR lentivirus) expressing a bispecific chimeric antigen receptor targeting CD19 and CD22 was manufactured.

[0131] As shown in the schematic diagram of FIG. 1,

[0132] EF1α promoter (sequence number 26);

[0133] Polynucleotide encoding a signal peptide (Sequence No. 27);

[0134] Polynucleotide encoding a CD19 / CD22-binding domain;

[0135] Polynucleotide encoding the CD8 hinge region (Sequence No. 28);

[0136] Polynucleotide encoding a transmembrane domain (Sequence No. 29);

[0137] Polynucleotide encoding 4-1BB (co-stimulation domain) (Sequence No. 30);

[0138] Polynucleotide encoding CD3ζ (intracellular signaling domain) (SEQ No. 31); and

[0139] CAR DNA composed of a polynucleotide encoding WPRE (SEQ No. 32) was synthesized in vitro and inserted into a third-generation lentivirus vector.

[0140]

[0141] In the present invention, the CD19-binding domain used was a known anti-CD19 antibody (FMC63), and the CD22-binding domain used was the 2G1(V4) antibody of Table 1 below.

[0142]

[0143] Sequence Information of Anti-CD22 Antibody-2G1(V4) 2G1-V4 Sequence Information Sequence Number Heavy Chain Variable Region CDR1GFSLTSYDI Sequence Number 1 Heavy Chain Variable Region CDR2IWTGGGT Sequence Number 2 Heavy Chain Variable Region CDR3VPHYYGYAMDYW Sequence Number 3 Light Chain Variable Region CDR1QDINKY Sequence Number 4 Light Chain Variable Region CDR2YTS Sequence Number 5 Light Chain Variable Region CDR3LQYDNLLT Sequence Number 6 Heavy Chain Variable Region Amino Acid Sequence EVQLQESGPGLVKPSQTLSLTCTVSGFSLTSYDISWIRQPPGKGLEWLGVIWTGGGTNYNSALKSRVTISKDNSKSQVSLKLSSVTAADTAVYYCVPHYYGYAMDYWGQGTTVTVSS Sequence Number 7 Light Chain Variable Region Amino Acid Sequence EIVLTQSPATLSLSPGERATLSCRASQDINKYIAWYQQKPGQAPRLLIHYTSTRQTGIPARFSGSGSGRDYTLTISSLEPEDFAVYYCLQYDNLLTFGGGTKLEIK Sequence Number 8 Heavy chain variable region nucleotide sequence GAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGAAGCCGAGCCAGACTCTTTCTCTGACCTGCACCGTGTCCGGCTTCTCTCTTACGAGCTACGACATCTCGTGGATCCGGCAGCCGCCTGGGAAAGGCTTAGAGTGGCTAGGGGTGATTTGGACCGGCGGGGGTACCA ACTACAACTCCGCGCTCAAATCCCGCGTCACTATTTCTAAGGACAATTCCAAGAGCCAGGTCTCGCTGAAGCTCTCGTCCGTGACCGCCGCGGACACCGCAGTTTATTACTGCGTGCCTCATTACTACGGCTACGCCATGGATTATTGGGGCCAGGGCACCACAGTAACAGTCAGCTCCSEQ ID NO.9경쇄가변부위염기서열GAGATCGTGCTGACTCAGAGCCCGGCCACCCTTAGCCTGAGTCCAGGCGAGCGCGCTACGTTGTCATGCCGAGCTTCCCAGGACATTAACAAGTACATCGCGTGGTACCAGCAGAAGCCCGGACAGGCCCCCCGCCTGCTCATCCACTACACCTCCACCCGCCAGACTGGCATCCCTGCCAGGTTTTCAGGCTCCGGTTCTGGCCGTGACTACACCCTGACCATCTCTAGTTTGGAGCCCGAAGATTTCGCCGTGTACTACTGTCTGCAATATGACAACCTGCTGACCTTCGGAGGGGGTACCAAGCTGGAGATCAAG서열번호 10

[0144] Sequence Information of Anti-CD19 Antibody (FMC63) CD19 Sequence Information Sequence Number Heavy Chain Variable Region CDR1GVSLPDYG Sequence Number 11 Heavy Chain Variable Region CDR2IWGSETT Sequence Number 12 Heavy Chain Variable Region CDR3AKHYYYGGSYAMDY Sequence Number 13 Light Chain Variable Region CDR1QDISKY Sequence Number 14 Light Chain Variable Region CDR2HTS Sequence Number 15 Light Chain Variable Region CDR3QQGNTLPYT Sequence Number 16 Heavy Chain Variable Region Amino Acid Sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVS Sequence Number 17 Light Chain Variable Region Amino Acid Sequence MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT Sequence Number 18Heavy chain variable region sequenceTCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATA CTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCSEQ ID NO.19Light chain variable region base sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGG AACTGTTAAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACASEQ ID NO. 20

[0145] The above CD19xCD22-binding domain is formed by connecting the light chain variable region (CD19VL) of an antibody specifically binding to CD19 - the heavy chain variable region (CD22VH) of an antibody specifically binding to CD22 - the light chain variable region (CD22VL) of an antibody specifically binding to CD22 - the heavy chain variable region (CD19VH) of an antibody specifically binding to CD19 in sequence (LoopCAR), and the sequence information for the polynucleotides encoding them is as follows:

[0146] CD19x2G1(V4): CD19VL represented by the nucleotide sequence of SEQ ID NO. 20 - Linker represented by the nucleotide sequence of SEQ ID NO. 21 (linker 1 in Fig. 1) - CD22VH represented by the nucleotide sequence of SEQ ID NO. 9 - Linker represented by the nucleotide sequence of SEQ ID NO. 24 (linker 6 in Fig. 1) - CD22VL represented by the nucleotide sequence of SEQ ID NO. 140 - Linker represented by the nucleotide sequence of SEQ ID NO. 53 (linker 1 in Fig. 1) - CD19VH represented by the nucleotide sequence of SEQ ID NO. 19.

[0147]

[0148] HEK293FT cells were co-transfected with three lentivirus vectors: pMDLg / pRRE (Addgene, cat# #12251), pMD2.G (Addgene, cat##12259), and pRSV-Rev (Addgene, cat##12253), to produce CD19 / CD22-CAR lentiviruses. For co-infection, HEK293FT cells were cultured with the three vectors for 4 hours using Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) and Opti-MEM+GlutaMAX (gibco, cat# 51985-034) medium.

[0149] As a result of confirming whether CD19xCD22 specific CARs are expressed in HEK293FT transfected with a lentivirus vector, as shown in Figures 13 and 14, it was confirmed that anti-CD19 / anti-CD22 antibody expression occurs normally.

[0150]

[0151] Example 2: Establishment of Optimal Dexamethasone Treatment Conditions for Promoting NK Cell Differentiation Induction

[0152] In the present invention, dexamethasone was used to promote the induction of differentiation of PBMCs into NK cells, and to establish optimal treatment conditions, NK cell growth curves were examined according to the treatment concentration, exposure time, and treatment time of dexamethasone.

[0153] Dexamethasone was administered according to the method shown in the schematic diagram of Fig. 3; specifically, the timing of dexamethasone treatment was arbitrarily set to Day 0 (the first) and Day 7 (the last) of the initial NK activation stage, and the treatment concentration was arbitrarily set to 100 nM and 1000 nM within the range of 50 to 1000 nM. Three exposure times were arbitrarily set: 1 hour, 24 hours, and 7 days of incubation without washing. The initial culture condition was 1.5 x 10⁶ PBMCs 6 cells / mL, K562 5 x 10 5 Co-culture was performed at a cell / mL ratio, and the medium used was serum-free medium.

[0154]

[0155] As a result, as shown in Figures 4a and 5b, it was confirmed that the final NK cell concentration on day 14 of culture was highest when 1000 nM dexamethasone was administered on day 0 of culture and cultured for 7 days without washing.

[0156]

[0157] The PBMC cell population was determined using a flow cytometer on day 14 of culture. As shown in Figures 4b and 5b, it was confirmed that the number of NK cells was generally higher when dexamethasone was treated on day 0 of culture compared to when it was treated on day 7. In particular, when comparing the groups showing high cell growth in the growth curve, the conditions exposed on day 7 of culture showed an NK cell count of approximately 50%, whereas the conditions exposed on day 0 of culture showed a higher NK cell count of approximately 90%.

[0158] The NK cell expansion fold was also found to be the highest at over 700 times under the condition where dexamethasone was administered on day 0 of culture and cultured for 7 days without washing.

[0159] Overall, it was confirmed that the earlier dexamethasone is administered during culture and the longer the exposure time, the higher the number of NK cells and the lower the proportion of other T cells, and it can also help with cell growth.

[0160]

[0161] Example 3: Establishment of Optimal Conditions for Enhancing CAR Expression Rate in CD19xCD22-CAR-NK Cells - Confirmation of Cell Phenotype and CAR Expression

[0162] PBMCs were isolated from leukaphersis blood aliquots of healthy donors using Ficoll-Paque density gradient centrifugation and then frozen. The frozen PBMCs were thawed and co-cultured at a 3:1 ratio with K562 feeder cells irradiated at 100 Gy to induce differentiation into NK cells. After co-culture, cells were collected for transduction on days 3, 5, 7, and 10 of NK differentiation induction. The cells were centrifuged at 450 xg for 5 minutes at RT, and then treated with 5 MOI of the CD19xCD22-CAR lentivirus vector prepared in <Example 1>. After 24 hours, the NK cells were harvested, replaced with NK culture medium supplemented with IL-2 and IL-15, and cultured for a total of 14 additional days. On the final culture day, NK cells were treated with CD56 (APC), CD3 (Pacific blue), and CD19 (PE) recombinant proteins, and cell phenotype and CAR expression were analyzed using a flow cytometer (Cytek).

[0163]

[0164] As a result, as shown in Figure 6, it was confirmed that the CAR expression rate was best when the CD19xCD22-CAR lentivirus vector was transduced on days 7 and 10 of NK differentiation induction.

[0165]

[0166] Example 4: Establishment of Optimal Conditions for Enhancing CAR Expression Rate in CD19xCD22-CAR-NK Cells - Confirmation of CAR Expression According to Additives During Transduction

[0167] PBMCs were isolated from leukaphersis blood aliquots of healthy donors using Ficoll-Paque density gradient centrifugation and subsequently frozen. The frozen PBMCs were thawed and co-cultured with K562 feeder cells irradiated at 100 Gy at a 3:1 ratio to induce differentiation into NK cells. Co-culture was performed for a total of 7 days, with 200 mL of culture medium added on days 3 and 5 for a total culture volume of 600 mL. After centrifuging the cells co-cultured for 7 days at 1,500 rpm and 18°C, CD3 T cells were removed using a positive selection method (MACS, CD3 depletion kit). After obtaining activated NK cells, 2 x 10 7 Cells were placed in T75 flasks and divided into (1) a group treated with 1:100 Lentiboost® (Poloxamer synperonic F108; Non-ionic, Amphiphilic poloxamer synperonic F108, Sirion Bioteck) alone, (2) a group treated with 10 μg / mL Vectofusin (Miltenyi Biotec) alone, (3) a group treated with 10 μM PGE2 (Sigma) alone, (4) a group treated with 10 μM PGE2 + 10 μg / mL Vectofusin combination, and (5) a group treated with 10 μM PGE2 + 1:100 Lentiboost® combination.

[0168] The CD19xCD22-CAR lentivirus vector was diluted to the same culture volume as the NK cells and treated at 5 MOI. After 24 hours, the NK cells were harvested and replaced with NK cell culture medium supplemented with IL-2 and IL-15, and CD19xCD22-CAR-NK cells were produced by additionally culturing for a total of 7 days.

[0169] The cell concentration on culture days 3, 5, and the final culture day (7) is 5 x 10 5 After diluting with NK culture medium to cells / mL, CAR-positive cells were analyzed using a flow cytometer (Cytek) after treatment with CD19-FITC and CD22-PE (AcroBio systems) recombinant proteins. Statistical analysis was performed using the Student-t Test in GraphPad Prism software (P-value, 0.001 > ***).

[0170] The results of the analysis of CAR-positive cells and the analysis of the percentage of CAR-positive cells on day 7 after transduction are shown in Figures 7a and 7b, and it was confirmed that the CAR expression rate was highest in the PGE2 + Lentiboost® combined treatment group.

[0171]

[0172] CD19xCD22-CAR-NK cells were prepared from PBMCs of three healthy donors using the same PGE2 + Lentiboost® combined treatment conditions established above. On the final day of culture, the CAR expression rate and proliferation rate were checked, confirming that CAR expression and CD19xCD22-CAR-NK cell proliferation had occurred (Fig. 7c). Untransfected normal NK cells were used as a negative control.

[0173]

[0174] Example 5: Establishment of Optimal Conditions for Enhancing CAR Expression Rate in CD19xCD22-CAR-NK Cells - Establishment of MOI

[0175] To enhance the CAR expression rate of CD19xCD22-CAR-NK cells and to confirm CAR expression according to MOI of the CD19xCD22-CAR lentiviral vector, CD19xCD22-CAR lentiviral vectors were transduced at 0 MOI, 0.5 MOI, 1 MOI, 2 MOI, 5 MOI, and 10 MOI, respectively, on day 7 of NK differentiation induction. 24 hours after transduction, NK cells were harvested, replaced with NK cell culture medium supplemented with IL-2 and IL-15, and cultured for an additional 7 days. 7 days after transduction, CD19xCD22-CAR-NK cells transduced at each MOI were collected and treated with CD56 (APC), CD3 (Pacific blue), CD19 (PE) recombinant protein, and CD22 (FITC) recombinant protein, and CAR expression was analyzed using a flow cytometer (Cytek). The cell viability was verified using a cell counting device.

[0176]

[0177] As a result, as shown in Figure 8, it was found that the transduction efficiency approached the maximum value under 5 MOI conditions.

[0178]

[0179] Example 6: Preparation of CD19xCD22-CAR-NK cells using the final culture process established in the present invention

[0180] In the present invention, based on the experimental results of the above examples,

[0181] (1) Dexamethasone is administered at the beginning of NK differentiation induction (day 0), and differentiation induction is performed in a medium containing dexamethasone for 5 to 10 days (preferably 7 days), and

[0182] (2) When transfecting the CD19xCD22-CAR lentivirus vector into the NK cells differentiated in (1), the cells are treated at 3 to 7 MOI (preferably 5 MOI), and

[0183] (3) A culture process was established that additionally includes PGE2 and LentiBOOST to increase the expression rate of the chimeric antigen receptor (CAR) during transduction.

[0184]

[0185] In the present invention, the final culture process established above was applied to PBMCs (donor A to F) isolated from 6 healthy donors to induce differentiation of NK cells, and the growth rate of NK cells is shown in Fig. 9a.

[0186] CD19xCD22-CAR-NK cells were prepared by transfecting the NK cells cultured above with a CD19xCD22-CAR lentivirus vector, and then inoculated into a 10 L bioreactor (Eppendorf) on the 8th day after transfection and cultured in large quantities for a total of 8 days. After inoculating the bioreactor, 1.6 L of NK culture medium was added at 2-day intervals for a total of 3 times. Culture conditions were maintained at 37°C, 5% CO2, and 100 rpm, and the pH was maintained between 6.5 and 7.2.

[0187] During the culture period, 3 mL of sample was taken daily, and the cell count and viability were measured using an automated cytometer. On the final day of culture, a final volume of 350 mL of cell culture medium was recovered through a continuous centrifugation process using a UniFuge instrument. The recovered culture bags were washed a total of five times with PlasmaLyte A 148 strains, and then frozen after adding CS10 and Human Serum Albumin. During mass culture, CAR expression levels were determined by measuring fluorescence signals using a flow cytometer after treating cells with CD19-FITC and CD22-PE (AcroBio systems) recombinant proteins.

[0188] The growth rate and CAR expression rate of CD19xCD22-CAR-NK cells are shown in Figures 9b and 9c, respectively.

[0189] In addition, from day 0 to day 15 of culture, the samples were stained using surface markers CD16-FITC (Biolegend), NKG2D-APC (BD Pharmingen), CD57-BV785 (Biolegend), LDLR-PE (Biolegend), and NKp30-APC / Cy7 (BD Pharmingen), and analyzed using a flow cytometer. The results are shown in Figure 10.

[0190] Looking at the results in Figures 9 and 10, it was found that when CD19xCD22-CAR-NK cells were produced by applying the culture process established in the present invention, there were significant differences in the CAR expression rate and the expression rate of surface markers depending on the type of donated PBMC. Accordingly, the present invention aimed to establish PBMC conditions suitable for producing CD19xCD22-CAR-NK cells.

[0191]

[0192] Example 7: Establishment of PBMC Selection Criteria Suitable for CD19xCD22-CAR-NK Cell Production

[0193] In the present invention, based on the results of <Example 6> above, we aimed to establish criteria for selecting PBMC suitable for manufacturing CD19xCD22-CAR-NK cells.

[0194] First, PBMCs isolated from each donor were stained using CD16-FITC (Biolegend), NKG2D-APC (BD Pharmingen), CD57-BV785 (Biolegend), LDLR-PE (Biolegend), and NKp30-APC / Cy7 (BD Pharmingen), analyzed using a flow cytometer, and the characteristics of the donor were analyzed by analyzing the expression patterns of each gene.

[0195]

[0196] As a result, it was confirmed that the PBMC isolated from Donor B in the process of <Example 6> above had a higher CAR gene delivery efficiency and a higher NK cell proliferation rate than the PBMC isolated from other donors (Figs. 9 and 10), and unlike other donors, showed a unique gene expression pattern as shown in Fig. 11 and Table 3 below.

[0197]

[0198] Donor B Surface Marker Analysis Results Selection Marker Selection Criteria (%) Symbol CD16 70% or less 70% ≥ CD16 NKG2D Less than 10% 10% > NKG2 DCD5 7 30% or less 30% ≥ CD5 LDLR 0.1% or more 0.1% ≤ LDLR NKp 30 Less than 10% 10% > NKp 30

[0199]

[0200] Based on Table 3 above, the selection criteria for donors with high CAR gene delivery efficiency were established as follows: CD16: 70% or less, NKG2D: less than 10%, CD57: 30% or less, LDLR: 0.1% or more, and NKp30: less than 10%.

[0201]

[0202] Surface marker analysis and verification of screening criteria satisfaction for each donor %Donor ADonor BDonor CDDonor DDonor EDonor F Item Expression Rate Suitability Expression Rate Suitability Expression Rate Suitability Expression Rate Suitability Expression Rate Suitability CD16 50.6 Suitable 59.3 Suitable 59.7 Suitable 79.5 Unsuitable 80.8 Unsuitable 69.1 Suitable NKG 2D 10.3 Unsuitable 7.55 Suitable 8.54 Suitable 7.74 Suitable 10.1 Unsuitable 9.8 Suitable CD5 77.87 Suitable 24.3 Suitable 36.3 Unsuitable 35.5 Unsuitable 0.18 Suitable 28.4 Suitable LDLR 0 Unsuitable 0.5 Suitable 0 Unsuitable 0 Unsuitable 0.21 Suitable 0 Unsuitable NKG 3018 Unsuitable 2.77 Suitable 1.3 Suitable 35 Unsuitable 40.1 Unsuitable 16.6 Unsuitable Sampling Conditions Fulfilled XOXXXXNK Manufactured Results Donor A Donor B Donor CD Donor D Donor E Donor FCD19 Expression (TD 10) 6.95 % 51.0 % 10.3 % 12.9 % 9.76 % 13.4 % CD22 Expression (TD 10) 6.67 % 57.0 % 28.3 % 9.63 % 13.1 % 10.0 % NK Cell Count (Culture 15) 4.392 X 10⁶ 10 6.023X10 10 3.134X10 10 3.299X10 10 4.353X10 10 2.532X10 10

[0203]

[0204] When the selection criteria for the donor established above were applied to the PBMCs of each donor to check if they met the selection conditions, it was confirmed that only Donor B was suitable, and in the case of other donors, CAR expression and the number of CAR-NK cells were very low.

[0205]

[0206] Example 8: Analysis of CD19xCD22-CAR-NK cells (UCI-101) prepared under optimal conditions

[0207] In the present invention, CD19xCD22-CAR-NK cells were prepared as in <Example 6> using Donor B PBMC selected under the conditions of <Example 7>, and were named "UCI-101".

[0208] To confirm the characteristics of UCI-101, the surface type of PBNK (periopheral blood NK cell, control) and UCI-101 (CAR-NK) was analyzed.

[0209] First, Western blotting was performed to measure the expression levels of exogenous and endogenous CD3ζ. Western blotting was carried out according to standard procedures and transferred to a PVDF (Bio-rad) membrane. The primary antibodies used were anti-CD3ζ (Cell Signalling Technology, 1:1000) and anti-β-actin-HRP (Santa Cruz, 1:1000). As a secondary antibody, an anti-rabbit IgG (Santa Cruz, 1:5000) HRP-conjugated antibody was used, and detection was performed using the ChemiDoc Imaging System (Biorad, BR17001401) with ECL reagent (Super Signal West Pico, Thermo Scientific). The bar graphs represent the quantification results of the overlapping Western blot gel images as mean ± standard deviation using the Prism program. The gel images were quantified using the ImageJ program and normalized based on the β-actin quantification values. Subsequently, the PBNK result value was set to 1 to compare and analyze the expression levels of CAR-NK.

[0210]

[0211] As a result, as shown in Figure 12a, exogenous CD3ζ expression increased in UCI-101, which means that CAR was normally expressed on the surface of NK cells.

[0212]

[0213] Next, to determine the degree of aging of UCI-101 with and without transduction, PBNK without lentivirus vector treatment was prepared using the same culture method with PBMC from a healthy donor identical to UCI-101. Subsequently, UCI-101 and PBNK were stained with the fluorescent antibodies CD57-BV785 (Biolegend), CD56-BV421 (Biolegend), CD158a.h-APC (BD Pharmingen), and CD158e1.e2 (BD Pharmingen), and then analyzed using a flow cytometer.

[0214] As a result, as shown in Figure 12b, it was confirmed that the aging of UCI-101 due to transduction was not accelerated.

[0215]

[0216] Example 9: Confirmation of the apoptotic effect of CD19xCD22-CAR-NK cells (UCI-101) on CD22 or CD19-expressing cells

[0217] The CAR expression rate and cytotoxicity against target cells (Raji cells and Daudi cells) of CD19xCD22-CAR-NK cells (UCI-101) prepared by the optimal manufacturing method of the present invention were confirmed.

[0218] Raji cells, derived from Burkkit lymphoma, and Daudi cells, human B-cell lymphoma cells, are known as representative B cell lines that express CD19 and CD22 antigens.

[0219] First, to confirm the CAR expression rate of UCI-101 prepared in <Example 8> above, cells were treated with CD19-FITC and CD22-PE (AcroBio systems) recombinant proteins, and the fluorescence signal was measured using a flow cytometer. The target cells, Raji and Daudi cells, were cultured for at least one week to allow for a stabilization period before use. The target cells were 1 x 10⁶ 7After CTV labeling (100 nM) at a concentration of cells / mL for 20 minutes in a 37℃ incubator, the thawed PBNK or UCI-101 was washed at least twice and co-cultured for 4 hours.

[0220] In this case, the number of cells is calculated based on the total number of cells, Ratio 5 x 10 5 Based on cells, the cells were cultured in 24-well plates with an E:T ratio of UCI-101 to target cells of 9:1, 3:1, and 1:1. After 4 hours, the cells were harvested, stained with PI, and subjected to flow cytometry analysis. The degree of cytotoxicity of the target cells was measured using the following Equation 1 by analyzing the level of PI expression within the CTV-positive gate.

[0221]

[0222] [Mathematical Formula 1]

[0223] % Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)]

[0224]

[0225] Experimental: Luminescence values ​​derived from the culture medium of target cells and CAR-NK cells combined

[0226] Effector Spontaneous: Luminescence value derived from the medium of CAR-NK cells alone

[0227] Target Spontaneous: Luminescence value derived from the medium of target cells only

[0228] Target Maximum: Luminescence value derived from 100% lysis of target cells (using lysis reagent)

[0229]

[0230] As a result, as shown in Figure 13a, it was confirmed that UCI-101 specifically kills target cells, namely Raji cells and Daudi cells.

[0231]

[0232] Next, the secretion of TNF-alpha and IFN-gamma cytokines from the supernatant obtained upon cell recovery was measured using ELISA (Enzyme Linked Immunosorbent assay). ELISA was performed according to standard procedures (R&D systems), and the final absorbance was analyzed using a Multiplate Reader (Molecular Devices, spectramax id3 system).

[0233] As a result, as shown in Figure 13b, it was confirmed that UCI-101 secretes cytokines that play an important role in cell killing ability and anticancer activity.

[0234]

[0235] Example 10: Confirmation of antitumor effects of CD19xCD22-CAR-NK cells (UCI-101) in an animal model

[0236] In this invention, to confirm the antitumor effect of CD19xCD22-CAR-NK cells (UCI-101), the antitumor effect of UCI-101 was confirmed in a U2932 blood cancer animal model.

[0237] Male NOG mice (NOD.Cg-PrkdcscidIl2rgtm1Sug / Jic) were supplied by CIEM Japan and used in animal studies after acclimatization for one week. Animal studies were conducted with the approval of the Animal Ethics Committee, and the testing was commissioned to Samda Bio Co., Ltd. Acclimatized mice were fed U2932-Luc cell line at a rate of 4 x 10⁶ per mouse. 6Cells were intravenously injected. The time of infusion with the U2932-Luc cell line was set as Day 0, and 4 days later, CD19xCD22-CAR-NK cells (UCI-101) were administered at a rate of 5 x 10⁶ CAR-positive cells per animal. 6 Cells were injected intravenously (Fig. 14a). Images were taken on days 12, 16, 19, 23, and 27 using IVIS (Perkin Elmer) imaging equipment, and the substrate used was measured 5 minutes after intraperitoneal injection of Luciferin (Perkin Elmer). For each measurement, 3 to 5 mice were placed under respiratory anesthesia before measurement.

[0238] IVIS imaging results were examined on days 12, 16, 19, 23, and 27 after U2932-Luc cell administration. Emission values ​​were imaged using pseudocolor. Color bars indicated relative luciferase activity in units of p / sec / cm2 / sr, with a minimum of 1 x 10 6 , maximum 1 x 10 7 Represents the value.

[0239] Next, in order to establish the administration concentration and administration cycle of UCI-101 in the present invention, UCI-101 was administered as shown in the schematic diagrams of FIG. 15a and FIG. 16a.

[0240] Specifically, to establish the administration concentration of UCI-101, CD19xCD22-CAR-NK cells were injected into mice with the U2932-Luc cell line at low concentrations (2.5 x 10⁶) based on CAR-positive cells per animal at 4, 7, 12, and 19 days later. 6 Cells), medium concentration (5 x 10 6 Cells), high concentration (1 x 10 7 Cells were intravenously injected four times per group (Fig. 15a).

[0241] In addition, CD19xCD22-CAR-NK cells were injected into mice at medium concentrations (5 x 10⁶) based on CAR-positive cells per animal 4, 11, and 18 days after injection of the U2932-Luc cell line.6 Cells were intravenously injected three times per group (Fig. 16a).

[0242]

[0243] As a result, as shown in Figures 14b, 15b, and 16b, it was confirmed that tumor cells were killed by the administration of UCI-101.

[0244]

[0245] Example 11: Confirmation of Antigen-Specific Cytotoxicity of CD19xCD22-CAR-NK Cells (UCI-101)

[0246] 11-1: Preparation of CD19- / -, CD22- / -, CD19- / -CD22- / - KO U2932 cell lines

[0247] U2932 cells, the target cells, are cells that express both CD19 and CD22. To confirm the antigen-specific cytotoxic effect of UCI-101, CD19- / -, CD22- / -, and CD19- / -CD22- / - KO U2932 cell lines were constructed. The KO cell lines were constructed by electroporation using sgRNA (Alt-R®CRISPR-Cas9 sgRNA (IDT)) and Cas9 genes (Atl-R®Sp Cas9 Nuclease V3 (IDT) Cat#. 1081059) under conditions of 1450 V, 10 ms, and 3 pulses.

[0248] The sequence information of the sgRNA used in the experiment is as follows:

[0249] CD19: 5'-CTGTGCTGCAGTGCCTCAA-3' (Sequence No. 38)

[0250] CD22: 5'-TCCTAGAGGGGGTTCCAATG-3' (Sequence No. 39)

[0251]

[0252] Subsequently, KO cell lines were selected using positive selection or negative selection methods (MACS) with anti-CD19 microbeads (Miltenyi, #130-050-301) and anti-CD22 microbeads (Miltenyi, #130-046-401).

[0253] Selected cell lines were treated with Anti-CD19 (PE) and Anti-CD22 (BV650) antibodies, and the expression levels of CD19 and CD22 were confirmed using a flow cytometer (Fig. 17).

[0254]

[0255] 11-2: CD19xCD22-CAR-T cell manufacturing

[0256] In addition, as a comparative example, CD19xCD22-CAR-T cells were prepared by the following method.

[0257] PBMCs were isolated from leukaphersis blood aliquots of healthy donors using Ficoll-Paque density gradient centrifugation and then frozen. The frozen PBMCs were thawed, and T cells were isolated using a negative selection method (stem cell non-T cell Target kit). The isolated T cells were cultured for 2 days under 200 U / mL IL-2 conditions. On day 2, the cells were harvested and 1 x 10⁶ 7 After suspending to a cell / mL concentration, the CD19xCD22-CAR lentivirus vector was treated at 0.5 MOI for 10 minutes. After 10 minutes, the culture medium was replaced with one containing 200 U / mL IL-2, and on days 3 and 5, the cell concentration was set to 5 x 10 5Culture medium was added to achieve a cell / mL level. After additional culture until day 7, the phenotypes of CD19xCD22-CAR-NK cells and CD19xCD22-CAR-T cells were analyzed via flow cytometry. As shown in Figure 17b, this represents the percentage of CAR-positive cells within CD56-positive and CD3-negative gates, and the percentage of CAR-positive cells within CD3-positive gates.

[0258]

[0259] 11-3: Confirmation of Antigen-Specific Cytotoxicity

[0260] Target cells, the WT-U2932 cell line and CD19 and CD22 antigens, KO U2932 cell line: 1 x 10 7 CTV labeling (100 nM) was performed at a concentration of cells / mL for 20 minutes in a 37°C incubator. After washing at least twice, CD19xCD22-CAR-NK cells and CD19xCD22-CAR-T cells were co-cultured for 4 hours. At this time, the cell count was calculated based on the total cell count using the ratio 5 x 10 5 Based on cells, effector cells (CAR-NK cells and CAR-T cells) and target cells (E:T Ratio) were cultured in 24-well plates at 9:1, 3:1, and 1:1. After 4 hours, cells were harvested, stained with PI, and flow cytometrically analyzed. The degree of cytotoxicity of target cells was measured by analyzing the level of PI expression within a CTV-positive gate.

[0261]

[0262] As a result, as shown in Figure 18a, it was found that CD19xCD22-CAR-NK cells exhibited superior cytotoxicity against target cells compared to CD19xCD22-CAR-T cells, and in particular, high cytotoxicity was confirmed against the WT-U2932 cell line, which expresses both CD19 antigen and CD22 antigen.

[0263]

[0264] 11-4: Confirmation of Cytokine Secretion by CAR-NK and CAR-T Cells

[0265] The amount of IFN-gamma cytokine secreted from the supernatant obtained upon cell recovery was measured using ELISA (Enzyme Linked Immunosorbent assay). The ELISA was performed according to standard procedures (R&D systems), and the final absorbance was analyzed using a Multiplate Reader (Molecular Devices, spectramax id3 system). For the measurement sample, the amount of IFN-gamma was measured from the supernatant obtained after co-culturing in the E:T = 5:1 solution used in the aforementioned cytotoxicity confirmation test.

[0266] As a result, as shown in Figure 18b, it was confirmed that the cytokine secretion ability of CD19xCD22-CAR-NK cells was superior to that of CD19xCD22-CAR-T cells.

[0267]

[0268] In the present invention, optimal conditions for inducing NK cell differentiation and optimal conditions for transduction were established, which have excellent delivery efficiency of the CAR expression vector and CAR-NK cell production efficiency. Since it was confirmed that CAR-NK cells produced by the method of the present invention exhibit excellent anti-tumor effects in animal models, they can be usefully utilized as a composition for the prevention or treatment of diseases related to CD22 (or CD19) expression or diseases related to B cells.

Claims

1. Peripheral blood mononuclear cells (PBMCs) expressing CD16 at 70% or less, NKG2D (natural killer group 2D) at less than 10%, CD57 at 30% or less, LDLR (low-density lipoprotein receptor) at 0.1% or more, and NKp30 (Natural cytotoxicity triggering receptor 3) at less than 10% are treated with dexamethasone and differentiated into NK cells, and then The method comprises the step of transfecting the NK cells with a vector containing a polynucleotide encoding a bispecific chimeric antigen receptor (CAR) targeting CD19 and CD22, and A method for producing CAR-NK cells targeting CD19 and CD22, characterized in that the above-mentioned bispecific chimeric antigen receptor comprises a CD19-binding domain and a CD22-binding domain; a transmembrane domain; a costimulatory domain; and an intracellular signal transduction domain.

2. In Paragraph 1, A method of preparation characterized by treating the above dexamethasone at the beginning of NK cell differentiation induction (day 0) and performing differentiation induction in a medium containing dexamethasone for 5 to 10 days.

3. In Paragraph 1, A method for manufacturing, characterized by co-culturing with a feeder cell expressing one or more proteins selected from the group consisting of IL-2, IL-15, IL-21, OX40L (CD134 ligand), and lunasin to induce differentiation of the above NK cells.

4. In Paragraph 1, The above vector is a virus vector, and A manufacturing method characterized by treating NK cells with 3 to 7 MOI (Multiplicity of infection).

5. In Paragraph 1, A manufacturing method characterized by additionally including prostaglandin E2 (PGE2) and a polyoxyethylene-polyoxypropylene block copolymer to increase the expression rate of a chimeric antigen receptor (CAR) in the above transduction process.

6. In Paragraph 1, A method for manufacturing, characterized in that the above CD19-binding domain and CD22-binding domain are connected in the order of the light chain variable region of an antibody that specifically binds to CD19 - the heavy chain variable region of an antibody that specifically binds to CD22 - the light chain variable region of an antibody that specifically binds to CD22 - the heavy chain variable region of an antibody that specifically binds to CD19.

7. In Paragraph 6, The heavy chain variable region of the antibody specifically binding to CD22 comprises a CDR1 region represented by the amino acid of SEQ ID NO. 1, a CDR2 region represented by the amino acid of SEQ ID NO. 2, and a CDR3 region represented by the amino acid of SEQ ID NO. 3, and The light chain variable region of the antibody specifically binding to the above CD22 includes a CDR1 region represented by the amino acid of SEQ ID NO. 4, a CDR2 region represented by the amino acid of SEQ ID NO. 5, and a CDR3 region represented by the amino acid of SEQ ID NO. 6, and The heavy chain variable region of the antibody specifically binding to the above CD19 comprises a CDR1 region represented by the amino acid of SEQ ID NO. 11, a CDR2 region represented by the amino acid of SEQ ID NO. 12, and a CDR3 region represented by the amino acid of SEQ ID NO. 13, and A method for manufacturing, characterized in that the light chain variable region of the antibody specifically binding to CD19 comprises a CDR1 region represented by the amino acid of SEQ ID NO. 14, a CDR2 region represented by the amino acid of SEQ ID NO. 15, and a CDR3 region represented by the amino acid of SEQ ID NO.

16.

8. In Paragraph 1, The above-mentioned transmembrane domain is a protein derived from any one selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1, and The co-stimulation domain is a protein derived from any one selected from the group consisting of CD28, 4-1BB, OX-40, and ICOS, and A method for manufacturing, characterized in that the above signal transduction domain is a protein derived from CD3ζ.

9. In Paragraph 1, A manufacturing method characterized by additionally including a hinge region between the C-terminus of the CD19-binding domain and the CD22-binding domain and the N-terminus of the transmembrane domain.

10. A CAR-NK cell targeting CD19 and CD22, prepared by the method of any one of claims 1 to 9.

11. The CAR-NK cell according to claim 10, characterized in that the CAR-NK cell has low expression of CD16, NKG2D, LDLR, and NKp30, and a high expression rate of CD57.

12. A pharmaceutical composition for the prevention or treatment of a B cell-mediated disease comprising CAR-NK cells targeting CD19 and CD22 prepared by the method of any one of claims 1 to 9.

13. In Paragraph 12, A pharmaceutical composition for the prevention or treatment of a B cell-mediated disease, characterized in that the B cell-mediated disease is selected from the group consisting of tumors, lymphomas, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indeterminate NHL, refractory NHL, refractory indeterminate NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt lymphoma, and mantle cell lymphoma.

Images