Anti-CD38 car-γδ t cells

Abstract

The present invention provides anti-CD38 CAR-T cells that can be easily produced. According to the present invention, there are provided anti-CD38 CAR-γδ T cells expressing a chimeric antigen receptor that recognizes CD38 as an antigen, a pharmaceutical composition comprising the anti-CD38 CAR-γδ T cells, and a method for producing the anti-CD38 CAR-γδ T cells.

Description

Anti-CD38 CAR-γδT cells 【0001】 The present invention relates to anti-CD38 CAR-γδT cells, a method for producing the same, and a pharmaceutical composition containing the above anti-CD38 CAR-γδT cells. 【0002】 CD38 (Cluster of Difference 38) is a transmembrane glycoprotein with a molecular weight of approximately 45 kDa and is a surface marker for plasma cells, PreB cells, and other cells. CD38 is involved in cell differentiation, activation, regulation of immune responses, and apoptosis. For example, in T cells and B cells, it is expressed in the early stages of development, disappears with maturation, but is known to be expressed again upon activation. 【0003】 As mentioned above, CD38 is expressed in normal cells, but it is known to be expressed at a higher level on the surface of hematological malignancies such as myeloma cells (e.g., multiple myeloma), malignant lymphoma cells, and leukemia cells compared to normal cells (e.g., Non-Patent Document 1). 【0004】 Furthermore, in recent years, research and application of CAR-T therapy, which uses CAR-T cells—T cells collected from patients into which genes encoding chimeric antigen receptors (CARs) that recognize cancer cell surface antigens—have been introduced and expressed, has been rapidly progressing as a type of cancer immunotherapy (for example, Patent Document 1). 【0005】 Special Publication No. 2020-517244 【0006】 Cytometry, 46:23-27, 2001 【0007】As mentioned above, CD38 is often highly expressed in hematological malignancies, making it a potential candidate for cancer cell surface antigens in CAR gene therapies such as CAR-T therapy. On the other hand, in the preparation of CAR-T cells, T cells for gene transfer are usually prepared by activating (and consequently proliferating) T cells, and it is known that T cells express CD38 during this activation and proliferation. Therefore, when anti-CD38 CAR-T cells are produced by introducing the gene encoding anti-CD38 CAR (anti-CD38 CAR gene) into T cells, self-killing, also known as fracturing, occurs, resulting in a reduction in their antitumor activity. To address this problem, for example, techniques such as RNA interference using siRNA or DNA editing using Crispr-Cas9 are employed to suppress CD38 expression in T cells into which the anti-CD38 CAR gene has been introduced, or anti-CD38 CAR-T cells are coexisted with anti-CD38 antibodies. 【0008】 In view of the above circumstances, the primary objective of the present invention is to provide anti-CD38 CAR-T cells that can be easily produced. The present invention also aims to provide anti-CD38 antibodies and anti-CD38 CARs that have no or low immunogenicity in humans. 【0009】[1] According to one aspect of the present invention, anti-CD38 CAR-γδ T cells are provided that express a chimeric antigen receptor that recognizes CD38 as an antigen. [2] In the anti-CD38 CAR-γδ T cells described in [1] above, the chimeric antigen receptor may recognize human CD38 as an antigen. [3] In the anti-CD38 CAR-γδ T cells described in [1] or [2] above, the chimeric antigen receptor may include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain linked in this order. [4] In the anti-CD38 CAR-γδ T cells described in [3] above, the extracellular antigen-binding domain may include a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 1 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 2 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2. [5] In the anti-CD38 CAR-γδT cell described in [3] above, the extracellular antigen-binding domain may include a light chain variable region comprising LCDR1, LCDR2, and LCDR3, and a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein LCDR3 may include the amino acid sequence represented by SEQ ID NO: 4, and HCDR3 may include the amino acid sequence represented by SEQ ID NO: 7. [6] In the anti-CD38 CAR-γδT cell described in [3] or [5] above, the extracellular antigen-binding domain may include a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 8 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 9. [7] In another aspect of the present invention, a pharmaceutical composition for treating or preventing a disease associated with CD38 expression is provided, comprising anti-CD38 CAR-γδT cells as described in any of [1] to [6] above. [8] In the pharmaceutical composition according to [7] above, the disease associated with CD38 expression may be selected from leukemia, malignant lymphoma, and multiple myeloma.[9] The pharmaceutical compositions described in [7] or [8] above may be used in combination with an anti-CD38 antibody drug.

[10] According to another aspect of the present invention, a method for producing anti-CD38 CAR-γδT cells is provided, comprising preparing γδT cells and introducing a gene encoding a chimeric antigen receptor that recognizes CD38 as an antigen into the γδT cells.

[11] In the production method described in

[10] above, the γδT cells may be prepared from non-stem cells.

[12] In the production method described in

[10] or

[11] above, preparing the γδT cells may involve incubating peripheral blood mononuclear cells derived from peripheral blood in the presence of at least one growth factor selected from bisphosphonates, alkylamines, recombinant human fibronectin, cytokines, anti-CD3 antibodies, and anti-CD28 antibodies.

[13] According to another aspect of the present invention, a chimeric antigen receptor that recognizes CD38 as an antigen is provided, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain linked in this order, wherein the extracellular antigen-binding domain comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, and a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 7.

[14] In the chimeric antigen receptor described in

[13] above, the extracellular antigen-binding domain may comprise a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 8 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 9.

[15] According to another aspect of the present invention, an anti-CD38 antibody or a functional fragment thereof is provided, comprising a light chain variable region comprising LCDR1, LCDR2, and LCDR3, and a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 7.

[16] The anti-CD38 antibody or functional fragment described in

[15] above may include a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 8 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 9 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 9.

[17] The anti-CD38 antibody or functional fragment described in

[15] or

[16] above may be an scFv. 【0010】 According to embodiments of the present invention, anti-CD38 CAR-T cells can be easily and efficiently obtained by introducing the anti-CD38 CAR gene into γδ T cells, thereby avoiding or suppressing fructoliside. 【0011】This is a schematic diagram illustrating an example of a DNA construct containing the anti-CD38 CAR gene that can be used in the present invention. This is a schematic diagram illustrating an example of the structure of a retroviral vector containing the anti-CD38 CAR gene. This is a diagram showing the results of evaluating the expression of CD38 in γδT cells before introduction of the anti-CD38 CAR gene by flow cytometry analysis. This is a diagram showing the results of evaluating the expression of anti-CD38 CAR in γδT cells after introduction of the anti-CD38 CAR gene by flow cytometry analysis. This is a diagram showing the results of evaluating the cytotoxicity of effector cells against KMM1 cells when co-cultured in vitro at various E:T ratios. This is a diagram showing the results of evaluating the cytotoxicity of effector cells against MM1.S cells when co-cultured in vitro at various E:T ratios. This is a diagram showing the results of evaluating the cytotoxicity of effector cells against KMM1 cells over time in vitro. This figure shows the results of evaluating the cytotoxicity of anti-CD38 CAR-γδ T cells against KMM1 cells when used in combination with an anti-CD38 antibody drug. This figure shows the bioluminescence intensity over time in mice transplanted with KMM1 cells. This image shows the bioluminescence of mice 29 days after KMM1 cell transplantation. This figure shows the survival curve of mice transplanted with KMM1 cells. This figure shows the results of evaluating the binding activity of antibodies to hCD38 by enzyme-linked immunosorbent assay (ELISA). (a) to (c) are figures showing the results of evaluating the reactivity of antibodies against RPMI8226 cells by flow cytometry analysis, respectively. (a) and (b) are figures showing the results of evaluating the cytotoxicity of Mock γδ T cells and human anti-CD38 CAR-T cells against KMM1 cells by flow cytometry analysis, respectively. This figure shows the results of evaluating the cytotoxicity of effector cells against KMM1 cells when co-cultured in vitro at various E:T ratios. This figure shows the results of evaluating the cytotoxicity of effector cells against MM1.S cells when co-cultured in vitro at various E:T ratios. This figure shows the bioluminescence intensity over time in mice transplanted with KMM1 cells. This image shows the bioluminescence of mice 28 days after KMM1 cell transplantation. 【0012】Preferred embodiments of the present invention will be described below, but the present invention is not limited to these embodiments. Unless otherwise appropriate in context, each embodiment can be combined as appropriate. In this specification, “~” representing a numerical range includes its upper and lower limits. Also in this specification, unless otherwise appropriate in context, “cell” includes “cell population.” A cell population may consist of one type of cell or two or more types of cells. 【0013】 A. Anti-CD38 CAR-γδ T cells According to one aspect of the present invention, anti-CD38 CAR-γδ T cells are provided that express a chimeric antigen receptor (anti-CD38 CAR) that recognizes CD38 as an antigen. T cells express an antigen receptor called a T cell receptor (TCR) on their surface and are broadly classified into αβ T cells, which express αβ type TCRs and are mainly responsible for adaptive immunity, and γδ T cells, which express γδ type TCRs and are mainly responsible for innate immunity. αβ T cells, which make up the majority of T cells, usually express CD38 during activation and proliferation. In contrast, according to newly acquired findings by the present inventors, γδ T cells do not express CD38 or express it only weakly during activation and proliferation. Therefore, by introducing the anti-CD38 CAR gene into γδ T cells, fracturides can be avoided or suppressed without requiring additional operations such as RNA interference or DNA editing, thereby enabling the efficient production of anti-CD38 CAR-T cells. Furthermore, the cytotoxic effect of γδ T cells is HLA-independent. Moreover, the above anti-CD38 CAR-γδ T cells can maintain their cytotoxic effect against CD38-positive cells even after freeze-thawing. Therefore, the above anti-CD38 CAR-γδ T cells can be suitably applied to off-the-shelf use through banking using allogeneic peripheral blood. Although there are multiple subtypes of γδ T cells, the subtype of γδ T cells expressing anti-CD38 CAR is not particularly limited in the embodiments of the present invention. For example, γδ T cells expressing anti-CD38 CAR are V γ９ V δ２ It can include subtypes. 【0014】The anti-CD38 CAR described above typically includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain linked in this order. Preferably, the anti-CD38 CAR includes an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain, and these regions are linked in tandem in this order. 【0015】 The extracellular antigen-binding domain contains an anti-CD38 antibody or a functional fragment thereof that recognizes CD38 as an antigen. As the anti-CD38 antibody, any suitable antibody capable of recognizing and specifically binding to CD38, preferably an IgG antibody, can be used. The anti-CD38 antibody may be, for example, a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody. The CD38 recognized as an antigen by the anti-CD38 CAR is preferably mammalian CD38, and includes human CD38. 【0016】 A functional fragment is a fragment of an antibody that retains some or all of the binding activity of the antibody from which it is derived. A single-chain Fv (scFv) is preferably used as the functional fragment. scFv may be a fusion protein in which the light chain variable region (VL region) and heavy chain variable region (VH region) of IgG are linked by a short linker peptide of, for example, 10 to 25 amino acids. The extracellular antigen-binding domain (e.g., scFv) may contain the VL region and VH region in this order toward the transmembrane domain, or in this order from the transmembrane domain. 【0017】 The VL region, also known as the complementarity-determining region (CDR), typically contains three highly variable regions (LCDR1, LFR2, LFR3) that determine antigen specificity, and four highly conserved regions (LFR1, LFR2, LFR3, LFR4) also known as the framework region (FR). These regions are arranged in the order LFR1, LFR1, LFR2, LFR2, LFR3, LFR3, and LFR4, from the amino terminus to the carboxyl terminus. 【0018】The VH region typically includes three complementarity-determining regions (HCDR1, HCDR2, HCDR3) and four framework regions (HFR1, HFR2, HFR3, HFR4). These regions are arranged in the order HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4, from the amino terminus to the carboxyl terminus. 【0019】 In one embodiment, the extracellular antigen-binding domain includes a VL region comprising the amino acid sequence represented by SEQ ID NO: 1 or its homologous sequence. 【0020】 In one embodiment, the extracellular antigen-binding domain includes a VH region comprising the amino acid sequence represented by Sequence ID No. 2 or its homologous sequence. 【0021】 In a preferred embodiment, the extracellular antigen-binding domain includes a VL region containing the amino acid sequence represented by SEQ ID NO: 1 or its homologous sequence, and a VH region containing the amino acid sequence represented by SEQ ID NO: 2 or its homologous sequence. For an anti-CD38 antibody containing the VL region containing the amino acid sequence represented by SEQ ID NO: 1 or its homologous sequence, and the VH region containing the amino acid sequence represented by SEQ ID NO: 2 or its homologous sequence, see, for example, WO2007 / 142241. 【0022】In one embodiment, the extracellular antigen-binding domain includes a VL region comprising an LCDR3 containing the amino acid sequence represented by SEQ ID NO: 4, for example, an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 4. The LCDR1 included in the VL region may be the amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence in which, for example, 1 to 3, preferably 1 or 2, amino acid mutations (substitutions, deletions, insertions, and / or additions) have been introduced to the above amino acid sequence. The LCDR2 included in the VL region may be the amino acid sequence of a DVS, or an amino acid sequence in which, for example, 1 or 2, preferably 1, amino acid mutations have been introduced to the above amino acid sequence. For example, the extracellular antigen-binding domain may include a VL region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 3, an LCDR2 consisting of the amino acid sequence of a DVS, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 4. In the VL region, it is preferable that each of the four FRs is derived from a human antibody. The amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence of the DVS, and the amino acid sequence represented by SEQ ID NO: 4 correspond to LCDR1-3 derived from human antibodies, respectively. Therefore, a fully human type VL region can be obtained by using a FR derived from human antibodies. Examples of FRs derived from human antibodies include FRs of human antibodies registered in known sequence databases such as GenBank. A fully human type VL region containing the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence of the DVS, and the amino acid sequence represented by SEQ ID NO: 4 is an example of a VL region containing the amino acid sequence represented by SEQ ID NO: 8. A preferred example of a VL region containing the extracellular antigen-binding domain is a VL region containing the amino acid sequence represented by SEQ ID NO: 8 or its homologous sequence. 【0023】In one embodiment, the extracellular antigen-binding domain includes a VH region containing HCDR3, which comprises the amino acid sequence represented by SEQ ID NO: 7, for example, HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 7. HCDR1 included in the VH region may be the amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence in which, for example, 1 to 3, preferably 1 or 2, amino acid mutations have been introduced into the above amino acid sequence. HCDR2 included in the VH region may be the amino acid sequence represented by SEQ ID NO: 6, or an amino acid sequence in which, for example, 1 to 3, preferably 1 or 2, amino acid mutations have been introduced into the above amino acid sequence. For example, the extracellular antigen-binding domain may include a VH region containing HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 5, HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 6, and HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 7. In the VH region, it is preferable that each of the four FRs is derived from a human antibody. The amino acid sequences represented by SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 correspond to HCDRs 1-3 derived from human antibodies, respectively. Therefore, by using FR derived from human antibodies, a fully human type VH region can be obtained. An example of a fully human type VH region containing the amino acid sequences represented by SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 is a VH region containing the amino acid sequence represented by SEQ ID NO: 9. A preferred example of a VH region containing the extracellular antigen-binding domain is a VH region containing the amino acid sequence represented by SEQ ID NO: 9 or its homologous sequence. 【0024】In a preferred embodiment, the extracellular antigen-binding domain comprises a light chain variable region including LCDR1, LCDR2, and LCDR3, and a heavy chain variable region including HCDR1, HCDR2, and HCDR3, wherein LCDR3 includes the amino acid sequence represented by SEQ ID NO: 4, and HCDR3 includes the amino acid sequence represented by SEQ ID NO: 7. For example, the extracellular antigen-binding domain may include a VL region comprising LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 3, LCDR2 consisting of the DVS amino acid sequence, and LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 4, and a VH region comprising HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 5, HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 6, and HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 7. Alternatively, for example, the extracellular antigen-binding domain may include a VL region comprising the amino acid sequence represented by SEQ ID NO: 8 or its homologous sequence, and a VH region comprising the amino acid sequence represented by SEQ ID NO: 9 or its homologous sequence. 【0025】 For example, the extracellular antigen-binding domain may include the amino acid sequence of the VL region or its homologous sequence and the amino acid sequence of the VH region or its homologous sequence of a known anti-CD38 antibody such as daratumumab or isatuximab. 【0026】In this specification, "homologous sequence" can have a sequence identity of 80% or more, for example 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more, with a reference amino acid sequence (for example, the amino acid sequence represented by SEQ ID NOs: 1, 2, 8, or 9). The homologous sequence may be an amino acid sequence that includes substitutions, deletions, insertions, or additions of 1 to 15, 12 or less, 10 or less, 8 or less, 5 or less, or 3 or less amino acids with respect to the reference amino acid sequence. With respect to the VL and VH regions, the light and heavy chains CDR1, 2, and 3 of the homologous sequence may have a high degree of sequence identity with the light and heavy chains CDR1, 2, and 3 of the reference amino acid sequence (for example, substitutions, deletions, insertions, or additions of 3 or less, 2 or less, 1 or less, or 0 amino acids). In particular, since LCDR3 and HCDR3 tend to contribute significantly to antigen specificity, it is preferable that they have a sufficiently high degree of sequence identity (for example, substitution, deletion, insertion, or addition of 0 or 1, preferably 0, amino acids) with the reference amino acid sequences of LCDR3 and HCDR3. 【0027】 The sequence identity of the above amino acid sequence can be calculated using known homology search software or homology search program. For example, the sequence identity of the amino acid sequence can be calculated using the default parameters in the BLAST search of the National Center for Biotechnology Information (NCBI). 【0028】 Various structures are known for the hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signaling domain in CARs. In embodiments of the present invention, any suitable structure can be adopted for these domains, as long as the effects of the present invention are obtained. 【0029】The hinge domain connects the extracellular antigen-binding domain and the transmembrane domain. The hinge domain is designed, for example, to provide the space (length) and flexibility for the extracellular antigen-binding domain to access the target epitope. The transmembrane domain anchors the CAR to the cell membrane of T cells. The hinge domain and the transmembrane domain can each contain domains derived from, for example, CD8, CD28, etc. 【0030】 The co-stimulatory domain can enhance the proliferation and persistence of T cells. The co-stimulatory domain can contain one or more domains derived from, for example, 4-1BB, CD28, CD27, ICOS, etc. 【0031】 The intracellular signaling domain can activate the immune response by T cells. The intracellular signaling domain can contain a domain derived from, for example, the CD3ζ domain. 【0032】 B. Method for producing anti-CD38 CAR-γδ T cells The anti-CD38 CAR-γδ T cells described in section A can be produced by any suitable production method. In one embodiment, the method for producing the anti-CD38 CAR-γδ T cells comprises: preparing γδ T cells (step I); introducing a gene encoding a chimeric antigen receptor (anti-CD38 CAR) that recognizes CD38 as an antigen into the γδ T cells (step II). 【0033】 [Step I] In step I, γδ T cells are prepared. The γδ T cells to be prepared may be prepared from stem cells or from non-stem cells. γδ T cells derived from non-stem cells may be advantageous in that they have no risk of carcinogenesis and the preparation cost is suppressed. 【0034】 The γδ T cells can be obtained, for example, by separating them from peripheral blood collected from an individual. γδ T cells obtained by maintaining or expanding (proliferating) the γδ T cells separated from peripheral blood may be used. The separation of γδ T cells from peripheral blood can be performed, for example, using a commercially available reagent for separating mononuclear cells (for example, the product name "Lymphoprep" ＴＭIt can be carried out by (”). The above-mentioned individual is preferably a human or a non-human mammal (such as mouse, rat, rabbit, dog, pig, goat, cow, horse, monkey, etc.). 【0035】 For example, γδ T cells can be obtained by separating peripheral blood mononuclear cells from the peripheral blood of the above-mentioned individual by density gradient centrifugation or the like, and proliferating γδ T cells from the peripheral blood mononuclear cells. 【0036】 As a method for proliferating γδ T cells from peripheral blood mononuclear cells, any appropriate method can be adopted. For example, a method of culturing peripheral blood mononuclear cells in a medium containing a growth factor can be mentioned. As the medium, a commercially available animal cell culture medium (for example, CTS Optimizer ＴＭ series or other culture media for T cells) or a suspension cell culture medium such as RPMI 1640 medium can be used. During the culture period, the medium may be replaced as necessary. 【0037】 Examples of the growth factor include bisphosphonate, alkylamine, recombinant human fibronectin (for example, Retronectin （Ｒ） ), cytokine, anti-CD3 antibody, anti-CD28 antibody, etc. The cytokine can be IL-2, IL-7, IL-15, etc. The growth factor may be used alone or in combination of two or more. When two or more growth factors are used in combination, peripheral blood mononuclear cells may be cultured in the presence of a plurality of growth factors, or a culture in the presence of a single growth factor and a culture in the presence of a single or a plurality of growth factors may be combined. Preferably, the growth factor contains bisphosphonate, and more preferably further contains cytokine. As the cytokine, for example, one or more selected from IL-2, IL-7, and IL-15 can be used. In one embodiment, two or more growth factors containing bisphosphonate and IL-2 or IL-15 can be used in combination. By incubating peripheral blood mononuclear cells in the presence of a growth factor (especially bisphosphonate), γδ TCR is activated in peripheral blood mononuclear cells, and γδ T cells can be selectively activated and proliferated. 【0038】Examples of bisphosphonates include pamidronic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, or salts or hydrates thereof. Among these, bisphosphonates having a nitrogen atom, such as pamidronic acid, alendronic acid, zoledronic acid, or salts or hydrates thereof (so-called aminobisphosphonates), can be preferably used. 【0039】 The concentration of growth factors in the culture medium should be within a range that can activate and proliferate γδ T cells. In one embodiment, the concentration of bisphosphonates such as zoledronic acid may be about 5 μM. In one embodiment, the concentrations of cytokines such as IL-2, IL-7, and IL-15 may each be about 200 to about 1000 IU / mL. 【0040】 In step I, γδ T cells may be prepared as a cell population including γδ T cells and other cells (e.g., αβ T cells). The cell population prepared in step I may contain γδ T cells in a proportion of, for example, 90% or more, preferably 95% or more, and more preferably 98% to 100%. 【0041】 In one embodiment, peripheral blood mononuclear cells are isolated from peripheral blood and cultured for several days (e.g., 3 to 5 days) in the presence of a bisphosphonate (e.g., zoledronic acid) and a cytokine (e.g., one or more selected from IL-2, IL-7, and IL-15). This can yield a cell population with a sufficiently high proportion of γδ T cells. Thus, the resulting cell population containing γδ T cells can be used directly in step II, in other words, without the removal of cells other than γδ T cells. If necessary, the cells may be cultured for a further few days (e.g., 2 to 4 days) in the absence of a bisphosphonate (e.g., zoledronic acid) and in the presence of cytokines. This can reduce the number of cells other than γδ T cells and yield a purer γδ T cell population. Alternatively, step II may be performed after removing cells other than γδ T cells from the cell population containing γδ T cells or after isolating only γδ T cells from the cell population containing γδ T cells. 【0042】The proportion of CD38-positive cells in the γδ T cells (more specifically, the cell population including γδ T cells) prepared in step I may be, for example, 25% or less, preferably 20% or less, and more preferably 10% or less. The γδ T cells (more specifically, the cell population including γδ T cells) prepared in step I can be used directly in step II without removing CD38-positive cells. If necessary, the cells may be used in step II after removing CD38-positive cells. The proportion of CD38-positive cells in the above cell population can be evaluated, for example, by flow cytometry analysis. Note that even among CD38-positive cells, cells with weak expression levels may not be involved in fracturing. Therefore, the proportion of CD38-positive cells may not directly reflect the efficiency of anti-CD38 CAR gene introduction (in other words, the proportion of cells expressing anti-CD38 CAR in the cell population after gene introduction). 【0043】 [Step II] In Step II, a gene encoding a chimeric antigen receptor that recognizes CD38 as an antigen (anti-CD38 CAR gene) is introduced into the γδ T cells prepared in Step I. The anti-CD38 CAR gene typically includes a gene encoding an extracellular antigen-binding domain, a gene encoding a transmembrane domain, and a gene encoding an intracellular signal transduction domain. 【0044】Figure 1 is a schematic diagram illustrating an example of a DNA construct containing the anti-CD38 CAR gene that may be used in the present invention. DNA construct 1 containing the anti-CD38 CAR gene has a structure in which the following are sequentially linked: a nucleotide sequence encoding the signal peptide of CD8α (a), a nucleotide sequence encoding the VL region of the anti-CD38 antibody (e.g., the amino acid sequence represented by SEQ ID NO: 1 or 8) (b), a nucleotide sequence encoding the linker (c), a nucleotide sequence encoding the VH region of the anti-CD38 antibody (e.g., the amino acid sequence represented by SEQ ID NO: 2 or 9) (d), a nucleotide sequence encoding the hinge domain and transmembrane domain of CD8α (e), a nucleotide sequence encoding the 4-1BB intracellular domain (f), and a nucleotide sequence encoding the CD3ζ chain (g). For details of anti-CD38 CAR and DNA constructs containing the anti-CD38 CAR gene, see, for example, WO2007 / 142241. Furthermore, the DNA construct containing the anti-CD38 CAR gene can be prepared, for example, by following the method of Imai et al. (C. Imai, K. Mihara et al., Leukemia, 2004, 18:676-684). 【0045】 Any suitable method can be used to introduce and express the anti-CD38 CAR gene in γδT cells. Examples include biological gene transfer methods using viral vectors, physical gene transfer methods such as electroporation, and chemical gene transfer methods such as lipofection and calcium phosphate methods. Among these, gene transfer methods using viral vectors are preferably used. 【0046】 The viral vector is not particularly limited, as long as the produced viral particles infect γδ T cells and the γδ T cells express anti-CD38 CAR. Retroviral vectors can be preferably used from the viewpoint that the introduced gene is incorporated into the chromosome of the host cell with high efficiency and that high expression of the gene can be maintained. Here, retroviral vector means so-called oncoretroviral vectors (hereinafter simply referred to as retroviral vectors), and includes lentiviral vectors, etc. 【0047】Gene transfer into γδ T cells using retroviral vectors can be performed, for example, by co-transfecting packaging cells (e.g., Cos cells, 293 T cells) with a vector plasmid containing the target gene sequence, LTR, and packaging signal, and a packaging plasmid containing the gag sequence, pol sequence, and env sequence, but without the packaging signal, and then recovering the produced viral particles (virions) and infecting γδ T cells with them. Alternatively, packaging cells expressing gag, pol, and env and producing empty viral particles may be transfected with a vector plasmid containing the target gene sequence to induce the production of viral particles (virions). 【0048】 Preferred examples of retroviral vector plasmids include MSCV (Murine Stern Cell Virus) vector plasmids and lentiviral vector plasmids. Various commercially available retroviral vector plasmids can be used; for example, the MSCV Retroviral Expression System (manufactured by Clontech) can be used. The target gene sequence can be inserted into the multi-cloning site (MCS) of the retroviral vector plasmid using standard cloning methods. 【0049】 In one embodiment, a retroviral vector plasmid containing an IRES (Internal ribosome entry site) sequence is used. The IRES sequence is a sequence located inside the mRNA chain that codes for a structure to which ribosomes directly bind, and is used in the mechanism by which translation is initiated by the direct binding of ribosomes, the so-called internal initiation mechanism. With a retroviral vector plasmid containing an IRES sequence, translation of eukaryotic cell mRNA can be made cap-structure independent. 【0050】 In one embodiment, a retroviral vector plasmid containing a marker gene is used from the viewpoint of selecting and sorting the gene-transferred cells. As the marker, fluorescent proteins such as GFP and EGFP may be used. 【0051】 For the packaging cells and the transfection method for retroviral vector plasmids into the packaging cells, the methods commonly used in gene transfer using retroviral vectors can be applied. 【0052】 Virus particles produced from packaging cells can be obtained as a virus solution, for example, by filtering the culture supernatant. 【0053】 Any suitable method can be used to infect γδ T cells with the obtained virus particles. For example, a method can be used in which the above virus solution is mixed with a positively charged gene transfer aid, and the resulting mixture is added to γδ T cells for culture (e.g., the polyblen method, the protamine method). The retronectin method can also be used. 【0054】 After the above infection, cells expressing anti-CD38 CAR can be easily selected and isolated by flow cytometry analysis using, for example, a labeled antibody that binds to anti-CD38 CAR (e.g., a fluorescent anti-G4S linker antibody, a fluorescent anti-mouse IgG antibody, etc.). 【0055】 In one embodiment, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more of the cells (cell population) after gene transfer treatment are anti-CD38 CAR-γδ T cells expressing anti-CD38 CAR. In other words, by step II, a cell population containing anti-CD38 CAR-γδ T cells at a rate of, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more can be obtained. The proportion of anti-CD38 CAR-γδ T cells in the cell population can be evaluated, for example, by flow cytometry analysis. 【0056】The anti-CD38 CAR-γδ T cells obtained as described above can be maintained or expanded for a long period of 30 days or more after the introduction of the anti-CD38 CAR gene. Furthermore, these anti-CD38 CAR-γδ T cells can maintain their cytotoxic effect against CD38-positive cells even after freeze-thawing. Therefore, these anti-CD38 CAR-γδ T cells can be cryopreserved, for example, in liquid nitrogen and used after thawing. 【0057】 C. Pharmaceutical Compositions According to another aspect of the present invention, a pharmaceutical composition for treating or preventing a disease related to CD38 expression is provided, comprising the anti-CD38 CAR-γδT cells described in Section A. The anti-CD38 CAR-γδT cells have cytotoxic effects on cells that express CD38, preferably cells that highly express CD38. Therefore, the pharmaceutical compositions according to embodiments of the present invention can be suitably applied to CAR-T therapy for diseases related to CD38 expression. 【0058】 Diseases associated with CD38 expression include malignant tumors such as cancer. Cancer includes hematological malignancies such as leukemia, malignant lymphoma, and multiple myeloma. Specific examples of hematological malignancies include multiple myeloma (MM), non-Hodgkin lymphomas (NHL) such as Burkitt lymphoma (BL), chronic B-lymphocytic leukemia (B-CLL), acute B and T lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, chronic myeloid leukemia (CML), hairy cell leukemia (HCL), follicular lymphoma, Waldenström macroglobulinemia, mantle cell lymphoma, Hodgkin lymphoma (HL), plasmacytoma, precursor B-cell lymphoblastic leukemia / lymphoma, plasmacytoma, giant cell myeloma, plasmacytoma, heavy chain myeloma, light chain or Bence Jones myeloma, and lymphomatoid granulomatosis. 【0059】The above pharmaceutical composition may be used in combination with an anti-CD38 antibody drug. By combining the cytotoxic effect of the anti-CD38 CAR-γδT cells with the action of the antibody drug (e.g., CDC activity, ADCC activity, ADCP activity, etc.), a more favorable therapeutic or preventive effect against the above disease can be obtained. Furthermore, since γδT cells have Fc receptors, a synergistic effect can be obtained when used in combination with an antibody drug. 【0060】 The above-mentioned pharmaceutical composition may be a combination of the above-mentioned anti-CD38 CAR-γδ T cells and an anti-CD38 antibody drug. Alternatively, the above-mentioned pharmaceutical composition may not contain an anti-CD38 antibody drug and may be administered in combination with a separately prepared anti-CD38 antibody drug. In this case, the pharmaceutical composition and the anti-CD38 antibody drug may be administered simultaneously, or each may be administered individually at an appropriate time. 【0061】 It is preferable that the anti-CD38 CAR-γδ T cells described above bind to an epitope at a site different from the epitope to which the anti-CD38 antibody drug binds. 【0062】 Examples of anti-CD38 antibody drugs used clinically include daratumumab and isatuximab. 【0063】 The above pharmaceutical composition may contain anti-CD38 CAR-γδ T cells and any anti-CD38 antibody drug, as well as any suitable pharmaceutically acceptable excipients depending on the dosage form. It may also further contain other drugs. The dosage form of the pharmaceutical composition is preferably parenteral, and may be, for example, an injection (including intravenous infusion). 【0064】 The dosage, administration rate, administration interval, and number of administrations of the above-mentioned pharmaceutical composition may be appropriately determined according to the conditions of the patient (height, weight, age, sex, etc.) and the patient's condition. 【0065】D. Anti-CD38 Antibody According to another aspect of the present invention, an anti-CD38 antibody or functional fragment thereof is provided, comprising a VL region including LCDR1, LCDR2, and LCDR3, and a heavy chain variable region including HCDR1, HCDR2, and HCDR3, wherein the LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 7. The anti-CD38 antibody specifically binds to CD38, preferably human CD38. The LCDR3 may consist of, for example, the amino acid sequence represented by SEQ ID NO: 4. The HCDR3 may consist of, for example, the amino acid sequence represented by SEQ ID NO: 7. Examples of LCDR1 included in the VL region include the amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence in which, for example, 1 to 3, preferably 1 or 2 amino acid mutations (substitutions, deletions, insertions, and / or additions) are introduced into the above amino acid sequence. Examples of the LCDR2 contained in the VL region include the amino acid sequence of DVS or an amino acid sequence in which, for example, one or two, preferably one amino acid mutations have been introduced into the above amino acid sequence. Examples of the HCDR1 contained in the VH region include the amino acid sequence represented by SEQ ID NO: 5 or an amino acid sequence in which, for example, one to three, preferably one or two amino acid mutations have been introduced into the above amino acid sequence. Examples of the HCDR2 contained in the VH region include the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence in which, for example, one to three, preferably one or two amino acid mutations have been introduced into the above amino acid sequence. In one embodiment, the antibody or its functional fragment may include a light chain variable region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 3, an LCDR2 consisting of the amino acid sequence of DVS, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 4, and a heavy chain variable region comprising an HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 5, an HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 6, and an HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 7. 【0066】In one embodiment, the anti-CD38 antibody comprises a VL region containing the amino acid sequence represented by SEQ ID NO: 8 or its homologous sequence, and a VH region containing the amino acid sequence represented by SEQ ID NO: 9 or its homologous sequence. 【0067】 The antibody described above is preferably an isolated antibody, and may be, for example, a monoclonal antibody. The antibody may be of any type IgM, IgD, IgG, IgA, or IgE. The constant region of the antibody is preferably the human constant region. 【0068】 The functional fragment of the above antibody comprises a fragment of the above antibody and can specifically bind to CD38, preferably human CD38. The functional fragment is F(ab'). ２ Examples include Fab', Fab, scFv, etc. 【0069】 The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. 【0070】 [Cells and Cultures] "Lymphoprep ＴＭPeripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy subjects using the Serumwerk Bernburg (Bernburg, Germany). The isolated PBMCs were cultured for 4 days in a CTS OpTmizer T Cell Expansion SFM (Thermo Fisher Scientific, Waltham, Massachusetts) supplemented with 5 μM zoledronic acid (Novartis, Basel, Switzerland) and 200 IU / mL IL-2 (PeproTech, Cranberry, New Jersey) to obtain γδ T cells. The retroviral packaging cell line "PG13 cells," myeloma-derived cell lines "KMM1 cells," "MM1.S cells," and "RPMI8226 cells" were purchased from ATCC (Manassas, Virginia). The lymphoma-derived cell line "Hut78 cells" were obtained from the Cell Bank of the Medical Cell Resource Center, Institute of Development, Aging and Cancer, Tohoku University. "G3T-hi cells" and "PG13 cells" were incubated at 37°C in Dulbecco's modified Eagle medium (D-MEM) (FUJI FILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, Massachusetts), 100 U / mL penicillin, and 100 μg / mL streptomycin (PS) (FUJI FILM Wako Pure Chemical Corporation, Osaka, Japan) at 5% CO2. ２ The cells were cultured as follows: "KMM1 cells" and "MM1.S cells" were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, Massachusetts) supplemented with 10% FBS and PS. "RPMI8226 cells" and "Hut78 cells" were cultured in RPMI 1640 medium supplemented with 10% FBS. 【0071】 [Statistical Analysis] Statistical analysis was performed using R ver 4.4.2. Two-group comparisons were performed using the Student T-test, and three-group comparisons were performed using the Tukey-Kramer's HSD test. A p-value of 0.05 or less was defined as a statistically significant difference. 【0072】[Experimental Example 1: Composition of a Retroviral Vector Containing the Anti-CD38 CAR Sequence] From a lentiviral vector constructed in the same manner as in the example of WO2007 / 142241, the gene sequence encoding anti-CD38 CAR, including the signal peptide of CD8α, anti-CD38scFV, the hinge domain and transmembrane domain of CD8α, the intracellular domain of 4-1BB, and the CD3ζ domain, was excised using restriction enzymes and inserted into the retroviral vector pDON5 (TaKaRa, Kyoto, Japan). The anti-CD38scFV sequence includes a VL region containing the amino acid sequence represented by SEQ ID NO: 1 and a VH region containing the amino acid sequence represented by SEQ ID NO: 2. The schematic composition of the obtained retroviral vector is shown in Figure 2. 【0073】[Experimental Example 2: Introduction of the anti-CD38 CAR gene into γδ T cells] G3T-hi cells were seeded in a 6-well plate 24 hours before transfection. Using TransIT-293 reagent (Mirus, Madison, Wisconsin), a retroviral vector containing the anti-CD38 CAR gene and gag-pol and env expression vectors were transfected into G3T-hi cells. After 48 hours, the supernatant containing the ecotropic retrovirus generated from the G3T-hi cells was collected, filtered through a 0.45 μm filter, and stored at -80°C. PG13 cells were seeded in a 6-well plate, and after 24 hours, the ecotropic virus supernatant diluted 4-fold in D-MEM medium and polybren adjusted to a final concentration of 8 μg / mL were added. After more than 4 hours, the supernatant was discarded, and fresh ecotropic virus supernatant and polybren were added. The above PG13 cell infection procedure was performed a total of five times. After the final infection, the PG13 cell culture supernatant was collected and filtered through a 0.45 μm filter to obtain a retrovirus solution. As described above, peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from healthy adults and stimulated for 4 days in the presence of zoledronic acid and IL-2 to promote the proliferation of γδ T cells, obtaining a nearly pure activated γδ T cell population in preparation for retrovirus transduction. The activated γδ T cell suspension and the above retrovirus solution were added in a 1:1 ratio to polypropylene tubes coated with retronectin (TaKaRa, Kyoto, Japan), and the tubes were centrifuged at 1200 g for 1.5 hours. After 24 hours, anti-CD38 CAR-γδ T cells were obtained by reinfecting the cells with the retrovirus using the same procedure. 【0074】 CD38 expression in γδ T cells before retroviral infection and anti-CD38 CAR expression in γδ T cells after retroviral infection (approximately 48 hours after the final infection) were evaluated by flow cytometry analysis as described below. The results are shown in Figures 3 and 4. 【0075】<Flow cytometry analysis> For cell analysis, a BD Accuri C6 Plus flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ) was used. Measurement data were analyzed using BD Accuri C6 Plus software and FlowJo (Becton, Dickinson and Company, Franklin Lakes, NJ). A negative gate was set using unstained samples, and a positive gate was set using stained target cells. The dead cell population was excluded using propidium iodide (PI) (Dojindo Molecular Technologies, Kumamoto, Japan) staining. For cell staining, anti-G4S Linker-Alexa647 (E7O2V, Cell Signaling Technology, Danvers, MA) and anti-CD38-APC (HIT2, Becton, Dickinson and Company, Franklin Lakes, NJ) were used. 【0076】 As shown in FIG. 3, the percentage of CD38-positive cells in γδ T cells before retroviral infection was approximately 20%, and the CD38 expression in γδ T cells before retroviral infection was weakly positive. On the other hand, as shown in FIG. 4, in γδ T cells after retroviral infection, the anti-CD38 CAR gene was introduced with an introduction efficiency exceeding 90%. Currently, the introduction efficiency of the CAR gene during the production of anti-CD19 CAR-αβ T cells currently in clinical use is about 25%. Therefore, according to the embodiments of the present invention, it can be seen that anti-CD38 CAR-γδ T cells were obtained with very high efficiency. Here, "the anti-CD38 CAR gene was introduced with an introduction efficiency exceeding 90%" means that the percentage of cells expressing anti-CD38 CAR in the cell population after infection exceeds 90%. 【0077】 The anti-CD38 CAR-γδ T cells obtained as described above can be cultured for a long term of 30 days or more, and 10 ７More than [number] anti-CD38-CAR-γδT cells were obtained. Furthermore, the following experiments 3 to 6 were performed using the anti-CD38-CAR-γδT cells obtained as described above. 【0078】 [Experimental Example 3: In Vitro Cytotoxicity Evaluation 1] The cytotoxic activity dependent on the number of effector cells was investigated. Specifically, target cells (KMM1 cells or MM1.S cells) and effector cells (anti-CD38 CAR-γδT cells or anti-CD38 CAR-non-transduced γδT cells (Mock-γδT cells)) were co-cultured in 96-well plates with an effector-to-target ratio (E:T) of 1:8 to 1:2. The number of surviving target cells (number of CD38-positive cells) was evaluated by flow cytometry analysis 24 hours after seeding. The results are shown in Figures 5 and 6. In the figures, the vertical axis shows the ratio of surviving target cells to the number of surviving target cells when Mock-γδT cells were used as effector cells. 【0079】 [Experimental Example 4: In vitro Cytotoxicity Evaluation 2] We investigated cytotoxic activity that depends on the co-culture period. Specifically, target cells (KMM1 cells) were cultured for 1 × 10⁻⁶ days. ５ Effector cells (anti-CD38 CAR-γδT cells or Mock-γδT cells) are placed at a density of 2 × 10⁻¹⁶ cells / well. ４ Cells were seeded at a density of cells / well, and the number of viable target cells (CD38-positive cells) was measured by flow cytometry analysis between 0 and 48 hours. The results are shown in Figure 7. In the figure, the vertical axis shows the ratio of the measured number of viable target cells to the number of target cells at seeding. "No add" indicates an evaluation system in which effector cells were not seeded. 【0080】 As shown in Figures 5-7, anti-CD38 CAR-γδT cells exhibited selective, cell-number-dependent, and time-dependent cytotoxicity against KMM1 cells and MM1.S cells. 【0081】 [Experimental Example 5: In vitro cytotoxicity evaluation 3] A competitive assay was performed using an anti-CD38 antibody drug. Specifically, target cells (KMM1 cells) were subjected to 1 × 10⁶ ５Seeds were seeded at a density of cells / well, and daratumumab (Janssen Pharmaceutica, Basel, Belgium) or isatuximab (Sanofi, Paris, France) was added to a final concentration of 0 μg / mL, 100 μg / mL, or 1000 μg / mL. One hour after addition, effector cells (anti-CD38 CAR-γδ T cells) were divided into 1 × 10⁶ cells. ４ Cells were seeded at a density of cells / well. The number of viable target cells (CD38-positive cells) was evaluated by flow cytometry 24 hours after seeding of effector cells. The results are shown in Figure 8. In the figure, the vertical axis represents the ratio of viable target cells to the number of viable target cells when effector cells were not seeded, at each antibody drug concentration. 【0082】 As shown in Figure 8, the treatment exhibited high cytotoxicity to target cells regardless of the presence or absence of anti-CD38 antibody drugs. This indicates that CAR-T cell therapy using anti-CD38 CAR-γδ T cells does not compete with treatment using anti-CD38 antibody drugs, and that these can be used in combination. 【0083】 [Experimental Example 6: In vivo Antitumor Activity Evaluation 1] Five-week-old male NOD / Shi-scid, IL-2RγKO Jic (NOG) mice were purchased from CLEA Japan (Tokyo, Japan). They were reared at the Fujita Health University's experimental animal facility in a pathogen-free animal facility at 23±3°C with a 12-hour light-dark cycle. After a one-week acclimatization period, KMM1 cells (5 × 10) into which luciferase had been introduced were introduced. ６KMM1 cells were transplanted into the tail vein of each mouse. Effector cells (anti-CD38 CAR-γδT cells or Mock-γδT cells) were administered via tail vein 4 days after KMM1 cell transplantation (E:T = 1:5). After KMM1 cell transplantation, VivoGlo luciferin, in vivo grade (Promega, Madison, Wisconsin) was administered intraperitoneally on a regular basis to monitor tumor cell proliferation. Bioluminescence was measured using LagoX (Spectral Instruments Imaging, Tucson, Arizona). The bioluminescence intensity of mice up to 29 days after KMM1 cell transplantation and the bioluminescence image at 29 days are shown in Figures 9 and 10. The survival rate of transplanted mice is shown in Figure 11. In the figure, "NT" or "w / o γδTcells" represents the untreated group. Each group consisted of seven mice. 【0084】 As shown in Figures 9-11, anti-CD38 CAR-γδT cells showed effective antitumor effects against NOG mice transplanted with KMM1 cells. 【0085】[Experimental Example 7: Acquisition of Human Anti-CD38 Antibody] <Preparation of scFv> Referring to Japanese Patent Publication No. 2005-185281, WO2008 / 007648, WO2006 / 090750, etc., a library of phages (hereinafter referred to as antibody phages) that present human scFv was prepared based on nucleic acids derived from lymphocytes of healthy humans and humans with tonsillitis. Human anti-CD38 antibodies were screened from this antibody phage library by the panning method. Screening was performed by the panning method using hCD38 recombinant protein (ECD, His Tag) (Sinobiological Corporation, Cat: 10818-H08H) as the antigen. The eluted phages were collected, infected with Escherichia coli (DH12S), and cultured in LB plates containing 100 mg / ml ampicillin and 0.2% glucose to form colonies. E. coli colonies were inoculated into 2×YT medium containing 100 mg / ml ampicillin and 0.05% glucose, and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added. The cells were incubated overnight at 30°C to induce the secretion of antibody phages into the medium. These antibody phages displayed scFv-cp3 type antibodies, in which the pIII protein, a coat protein of the M13 phage, was fused to the C-terminus of scFv. This E. coli culture supernatant was used in a binding assay to the hCD38 antigen. 【0086】<Selection of anti-CD38 antibodies> (1) Evaluation of binding activity to hCD38 recombinant protein hCD38 recombinant protein (ECD, His Tag) (Sinobiological Japan, Cat: 10818-H08H) was immobilized in wells of Maxisorp Immunoplate (Thermo Fisher Scientific). After blocking with BSA, scFv-cp3 antibody expressed in E. coli culture supernatant was reacted. The above reaction was performed using E. coli culture supernatant concentrated 0.5-fold, 1-fold, or 2-fold. The samples were washed with PBST, reacted with mouse anti-pIII antibody, washed again, and then reacted with peroxidase-conjugated goat anti-mouse IgG (H+L chain) (MBL). After PBST washing, TMB (Thermo Fisher Scientific) was added and color development was performed. After stopping the reaction with 2N sulfuric acid, the absorbance was measured at a wavelength of 450 nm. From the measurement results, two positive clones (A10-335 and A10-343) that had binding activity to the hCD38 recombinant protein were selected as anti-CD38 antibodies. The measurement results for the two positive clones and an example of a negative clone (F081-007) are shown in Figure 12. In the figure, the bars on the left show the measurement results in wells where CD38 was solidified, and the bars on the right show the measurement results in wells where CD38 was not solidified. 【0087】 (2) Evaluation of reactivity to RPMI8226 cells The reactivity of the positive clones selected in (1) above to RPMI8226 cells was confirmed by flow cytometry analysis as follows. RPMI8226 cells express hCD38 on the cell membrane. RPMI8226 cells were cultured and harvested. The culture supernatant of E. coli was concentrated to a 10-fold concentration by ammonium sulfate precipitation. The scFv-cp3 antibody in the concentrated culture supernatant was reacted with the harvested cells. 1% BSA and 0.05% NaN ３ After washing with PBS containing [a specific substance], the sample was reacted with mouse anti-pIII antibody. After further washing, the sample was reacted with anti-mouse Alexa-647 (Jackson ImmunoResearch Laboratories). After washing, 0.05% NaN [a specific substance] was added. ３The antibodies were suspended in PBS containing the specified antibody and analyzed using a BD Accuri C6 Plus flow cytometer (BD Biosciences). From the results of this analysis, the antibody clone (A10-335) that showed stronger reactivity was selected. Figures 13(a) and (b) show graphs plotting the fluorescence intensity and cell number of RPMI8226 cells when reacted with the scFv-cp3 antibody in the concentrated culture supernatant in the above flow cytometry analysis. In the figures, "Control" indicates the results when an anti-G4S linker antibody was used instead of the scFv-cp3 antibody. Furthermore, Figure 13(c) shows the results of evaluating the reactivity to RPMI8226 cells in the same manner as above, using a PE-labeled mouse anti-CD38 antibody (BD Pharmaingen, product name "PE monoclonal anti-human CD38 #555460") as a positive control instead of the scFv-cp3 antibody. In the figure, "Isotype control" shows the results when a PE-labeled mouse isotype control antibody (BD Pharmaingen, product name "PE-conjugated mouse IgG1 Isotype control #556029") was used instead of the scFv-cp3 antibody. 【0088】 As can be seen from Figures 12 and 13, antibody clone (A10-335) showed lower reactivity to hCD38 than antibody clone (A10-343) in ELISA, but showed higher reactivity to RPMI8226 cells than antibody clone (A10-343) in flow cytometry analysis. 【0089】 (3) Evaluation of reactivity to KMM1 cells and Hut78 cells The reactivity of the antibody clone (A10-335) selected in (2) above to KMM1 cells and Hut78 cells was evaluated. KMM1 cells express CD38 on their cell membrane, while Hut78 cells do not express CD38 on their cell membrane. Each cell type was cultured and harvested. The harvested cells were reacted with scFv-cp3 antibody secreted into the culture supernatant of E. coli. The cells were then treated with 1% BSA and 0.05% NaN ３After washing with PBS containing [a specific substance], it was reacted with rabbit anti-G4S linker Alexa 647 (cell signaling). After washing, 0.05% NaN ３ The antibody clone (A10-335) was suspended in PBS containing the specified substance and analyzed using a BD Accuri C6 Plus flow cytometer (BD Biosciences). The analysis results confirmed that the antibody clone (A10-335) reacted with KMM1 cells but not with Hut78 cells. Based on these results, the antibody clone (A10-335) was selected as the antibody for CAR-T cell production. 【0090】 <Sequencing Analysis of Anti-CD38 Antibody> DNA was extracted from the antibody clone (A10-335). The nucleotide sequence of the scFv presented on the antibody phage was determined by sequencing analysis. The sequencing primer T7ETZ (5'-TAATACGACTCACTTATAGGG-3' (SEQ ID NO: 10)) was used for the sequencing analysis. The analysis was outsourced to Eurofins Genomics. The determined nucleotide sequence is shown in Table 1. In the table, the underlined portion indicates the nucleotide sequence of the linker. The obtained nucleotide sequence was converted to an amino acid sequence to determine the amino acid sequence of the scFv of the antibody clone (A10-335). The determined amino acid sequence is shown in Table 2. In the table, the underlined portion indicates the amino acid sequence of the linker. Database search using the Blast program confirmed that this antibody is a novel antibody. 【0091】 【0092】 【0093】 By applying NCBI's IgBlast-based CDR sequence delimiter, the CDR sequences of the VL region (positions 1-110) and VH region (positions 126-249) contained in scFv, represented by sequence number 12, were identified. The results are shown in Table 3. 【0094】 【0095】[Experimental Example 8: Preparation of Human Anti-CD38 CAR-T Cells] <Preparation of Nucleic Acid Encoding Anti-CD38 CAR> A nucleic acid encoding human anti-CD38 CAR (see Figure 1) containing the CD8α signal peptide (a), the scFv of Sequence ID No. 12 (b-d), the hinge domain and transmembrane domain of CD8α (e), the intracellular domain of 4-1BB (f), and the CD3ζ domain (g) in this order was inserted into the retroviral vector pDON5 (TaKaRa, Kyoto, Japan). The amino acid sequences of the CD8α signal peptide, the hinge domain and transmembrane domain of CD8α, the intracellular domain of 4-1BB, and the CD3ζ domain are shown in Table 4. 【0096】 【0097】 Human anti-CD38 CAR-γδ T cells were obtained in the same manner as in Experimental Example 2, except that a retroviral vector containing nucleic acid encoding human anti-CD38 CAR obtained as described above was used. 【0098】 [Experimental Example 9: In Vitro Cytotoxicity Evaluation 4] The cytotoxic activity of human anti-CD38 CAR-γδT cells against KMM1 cells was investigated. Specifically, target cells (KMM1 cells) and effector cells (human anti-CD38 CAR-γδT cells or human anti-CD38 CAR-non-transduced γδT cells (Mock-γδT cells)) were seeded in a 96-well plate with an effector-to-target ratio (E:T) of 1:2 (target cell count: 1 × 10⁶). ５ The cells were cultured in a well. After 24 hours from seeding, the number of viable target cells (number of CD38-positive cells) was evaluated by flow cytometry. The results are shown in Figure 14. In the figure, the circled area represents the region where CD38-positive cells were gated, and corresponds to viable KMM1 cells. 【0099】 As shown in Figure 14, the proportion of surviving KMM1 cells to total flow cytometry events after co-culture with human anti-CD38 CAR-γδ T cells was 4.0%, and the proportion of surviving KMM1 cells to total flow cytometry events after co-culture with Mock-γδ T cells was 30.2%. 【0100】[Experimental Example 10: In Vitro Cytotoxicity Evaluation 5] The cytotoxic activity of anti-CD38 CAR-γδT cells obtained in Experimental Example 2 (hereinafter sometimes referred to as "mouse anti-CD38 CAR-γδT cells") and human anti-CD38 CAR-γδT cells obtained in Experimental Example 8 against KMM1 cells and MM1.S cells was verified. Specifically, target cells (KMM1 cells or MM1.S cells) and effector cells (mouse anti-CD38 CAR-γδT cells, human anti-CD38 CAR-γδT cells, or anti-CD38 CAR-free γδT cells (Mock-γδT cells)) were seeded in 96-well plates so that the effector-to-target ratio (E:T) was 1:10 to 1:2 (target cell count: 1 × 10⁶). ５ The cells were cultured in a well. After 24 hours from seeding, the number of viable target cells (CD38-positive cells) was evaluated by flow cytometry. The results are shown in Figures 15 and 16. In the figures, the vertical axis shows the ratio of the number of viable target cells to the number of viable target cells when Mock-γδT cells were used as effector cells. 【0101】 As shown in Figures 15 and 16, both mouse anti-CD38 CAR-γδT cells and human anti-CD38 CAR-γδT cells exhibited cell-number-dependent cytotoxicity against KMM1 cells and MM1.S cells. Human anti-CD38 CAR-γδT cells showed higher cytotoxicity against KMM1 cells and MM1.S cells than mouse anti-CD38 CAR-γδT cells. 【0102】 [Experimental Example 11: In vivo antitumor activity evaluation 2] Five-week-old male NOD / Shi-scid, IL-2RγKO Jic (NOG) mice were purchased from CLEA Japan (Tokyo, Japan). They were reared at the experimental animal facility of Fujita Health University in a pathogen-free animal facility at 23±3°C with a 12-hour light-dark cycle. After a one-week acclimatization period, KMM1 cells (2.0 × 10⁶) into which luciferase had been introduced were introduced. ６ Cells (mouse cells) were transplanted into the tail vein of each mouse. Effector cells (human anti-CD38 CAR-γδT cells or Mock-γδT cells) were administered via the tail vein 4 days after KMM1 cell transplantation (effector cell dosage: 1.0 × 10⁻¹⁶). ７Cells / mouse). After transplantation of KMM1 cells, VivoGlo luciferin, in vivo grade (Promega, Madison, Wisconsin) was administered intraperitoneally on a regular basis, and tumor cell proliferation was monitored. Bioluminescence was measured using LagoX (Spectral Instruments Imaging, Tucson, Arizona). The bioluminescence intensity of mice up to 28 days (Day 28) after KMM1 cell transplantation and the bioluminescence image at Day 28 are shown in Figures 17 and 18. In the figures, "NT" represents the untreated group. There were three mice in each group. 【0103】 As shown in Figures 17 and 18, human anti-CD38 CAR-γδT cells showed effective antitumor effects against NOG mice transplanted with KMM1 cells. 【0104】 Anti-CD38 CAR-γδ T cells according to embodiments of the present invention can be suitably used in medical fields such as CAR-T therapy.

Claims

1. Anti-CD38 CAR-γδ T cells that express a chimeric antigen receptor that recognizes CD38 as an antigen.

2. The anti-CD38 CAR-γδ T cell according to claim 1, wherein the chimeric antigen receptor recognizes human CD38 as an antigen.

3. The anti-CD38 CAR-γδ T cell according to claim 1, wherein the chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain linked in this order.

4. The anti-CD38 CAR-γδ T cell according to claim 3, wherein the extracellular antigen-binding domain comprises a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 1 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 2 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

2.

5. The anti-CD38 CAR-γδ T cell according to claim 3, wherein the extracellular antigen-binding domain comprises a light chain variable region including LCDR1, LCDR2, and LCDR3, and a heavy chain variable region including HCDR1, HCDR2, and HCDR3, wherein LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence represented by SEQ ID NO:

7.

6. The anti-CD38 CAR-γδ T cell according to claim 3, wherein the extracellular antigen-binding domain comprises a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 8 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 9 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

9.

7. A pharmaceutical composition for treating or preventing a disease related to CD38 expression, comprising the anti-CD38 CAR-γδT cells described in claim 1.

8. The pharmaceutical composition according to claim 7, wherein the disease associated with the expression of CD38 is selected from leukemia, malignant lymphoma, and multiple myeloma.

9. The pharmaceutical composition according to claim 7, which is used in combination with an anti-CD38 antibody drug.

10. A method for producing anti-CD38 CAR-γδT cells, comprising preparing γδT cells and introducing a gene encoding a chimeric antigen receptor that recognizes CD38 as an antigen into the γδT cells.

11. The method for producing γδ T cells according to claim 10, wherein the γδ T cells are prepared from non-stem cells.

12. The method for producing the γδ T cells according to claim 10, comprising incubating peripheral blood mononuclear cells derived from peripheral blood in the presence of at least one growth factor selected from bisphosphonates, alkylamines, recombinant human fibronectin, cytokines, anti-CD3 antibodies, and anti-CD28 antibodies.

13. A chimeric antigen receptor that recognizes CD38 as an antigen, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain linked in this order, wherein the extracellular antigen-binding domain comprises a light chain variable region including LCDR1, LCDR2, and LCDR3, and a heavy chain variable region including HCDR1, HCDR2, and HCDR3, wherein LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence represented by SEQ ID NO:

7.

14. The chimeric antigen receptor according to claim 13, wherein the extracellular antigen-binding domain comprises a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 8 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 9 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

9.

15. An anti-CD38 antibody or functional fragment thereof, comprising a light chain variable region including LCDR1, LCDR2, and LCDR3, and a heavy chain variable region including HCDR1, HCDR2, and HCDR3, wherein LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence represented by SEQ ID NO:

7.

16. The anti-CD38 antibody or functional fragment according to claim 15, comprising: a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 8 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8; and a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 9 or a homologous sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

9.

17. The anti-CD38 antibody or functional fragment thereof according to claim 15, which is scFv.

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