Novel car-t cells and monoclonal antibodies

Abstract

The present invention addresses the problem of providing anti-CD47 CAR-T cells that can be prepared easily and efficiently.　The problem can be solved by anti-CD47 CAR-γδ T cells that express a chimeric antigen receptor including an antigen binding domain that recognizes CD47 as an antigen, a transmembrane domain, and an intracellular signaling domain.

Description

Novel CAR-T cells and monoclonal antibodies

[0001] This invention relates to anti-CD47 CAR-γδT cells, anti-CD47 monoclonal antibodies, and the use thereof.

[0002] CD47 (Cluster of Difference 47) is a transmembrane protein expressed on the cell surface. CD47 functions as a "don't eat me" signal to macrophages, potentially suppressing their phagocytic activity. Furthermore, hematological malignancies such as myeloid leukemia, lymphoma, and myeloma are often CD47-positive.

[0003] 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).

[0004] As mentioned above, since hematological malignant tumor cells are often CD47-positive, CD47 could be a candidate for a cancer cell surface antigen in CAR-T therapy.

[0005] Special Publication No. 2020-517244

[0006] In the preparation of anti-CD47 CAR-T cells, if the T cells into which the anti-CD47 CAR gene is introduced express CD47, a problem may arise where fructuring occurs, resulting in reduced gene transfer efficiency (in other words, a lower yield of anti-CD47 CAR-T cells).

[0007] The above-mentioned problems with fractorides can be solved, for example, by suppressing CD47 expression in T cells into which genes have been introduced by RNA interference, genome editing, etc., or by co-administering anti-CD47 CAR-T cells with anti-CD47 antibodies, but these methods require complicated procedures and high costs. Therefore, the main objective of the present invention is to provide anti-CD47 CAR-T cells that can be prepared simply and efficiently.

[0008] Furthermore, conventional CAR-T cells are prepared using non-human antibodies, such as those from mice. Therefore, when conventional CAR-T cells are administered to humans, they may be recognized as antigens, resulting in antibody production and potentially leading to rejection reactions or other problems. Thus, a further objective of the present invention is to provide anti-CD47 CAR-T cells with reduced immunogenicity in humans.

[0009] [1] According to one aspect of the present invention, anti-CD47 CAR-γδ T cells are provided that express a chimeric antigen receptor that recognizes CD47 as an antigen. [2] In the anti-CD47 CAR-γδ T cells described in [1] above, the chimeric antigen receptor may recognize human CD47 as an antigen. [3] In the anti-CD47 CAR-γδ T cells described in [1] or [2] above, the chimeric antigen receptor may include an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the antigen-binding domain may include a light chain variable region and a heavy chain variable region derived from a human antibody. [4] In the anti-CD47 CAR-γδT cells described in [3] above, the antigen-binding domain may include a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2. [5] According to another aspect of the present invention, a cell population comprising 80% or more of the anti-CD47 CAR-γδT cells described in any of [1] to [4] above is provided. [6] According to another aspect of the present invention, an anti-CD47 monoclonal antibody or its antigen-binding fragment is provided, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2. [7] In yet another aspect of the present invention, a pharmaceutical composition for treating or preventing a disease related to CD47 expression is provided, comprising anti-CD47 CAR-γδT cells as described in any of [1] to [4] above, or an anti-CD47 monoclonal antibody or antigen-binding fragment thereof as described in [6] above. [8] In the pharmaceutical composition according to [7] above, the disease related to CD47 expression may be cancer or arteriosclerosis. [9] In the pharmaceutical composition according to [8] above, the cancer may be a hematological malignancy.

[0010] According to embodiments of the present invention, by using γδ T cells, the above-mentioned problem of fructoliside can be suppressed, and anti-CD47 CAR-T cells can be provided simply and efficiently. Furthermore, by using light chain variable regions and heavy chain variable regions derived from human antibodies screened from a human antibody library, anti-CD47 CAR-T cells with reduced immunogenicity to humans can be provided.

[0011] This is a schematic diagram illustrating an example of a DNA construct containing the gene encoding anti-CD47 CAR. This figure shows the results of detecting anti-CD47 CAR expression in cell populations before and after transduction by flow cytometry. This figure shows the results of detecting CD47 expression in cell populations before and after transduction by flow cytometry. This figure shows the results of an in vitro cytotoxicity assay of anti-CD47 CAR-γδT cells against KMM1 cells. This figure shows the results of an in vitro cytotoxicity assay of anti-CD47 CAR-γδT cells against BV-173 cells. This figure shows the results of an in vitro cytotoxicity assay of anti-CD47 CAR-γδT cells against THP-1 cells. This figure shows the results of detecting CD47 expression in cell populations before transduction by flow cytometry. This figure shows the results of detecting anti-CD47 CAR expression in cell populations before and after transduction by flow cytometry.

[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-CD47 CAR-γδ T cells According to one aspect of the present invention, anti-CD47 CAR-γδ T cells are provided that express a chimeric antigen receptor (anti-CD47 CAR) that recognizes CD47 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 make up the majority of T cells and are relatively easy to proliferate, so generally, CAR-T cells are prepared by introducing a gene encoding CAR into αβ T cells proliferated from peripheral blood mononuclear cells. However, according to newly acquired findings by the present inventors, αβ T cells after proliferation stimulation for gene transfer express CD47, and when anti-CD47 CAR is expressed, fructidine may be produced. In contrast, γδ T cells after proliferation stimulation express CD47, but do not produce potent fructidine, and anti-CD47 CAR can be expressed with high gene transfer efficiency. Therefore, by expressing anti-CD47 CAR in γδ T cells, fructidine can be avoided or suppressed without requiring additional operations such as RNA interference or genome editing, thereby enabling the simple and efficient production of anti-CD47 CAR-T cells (more specifically, anti-CD47 CAR-γδ T cells). Furthermore, the cytotoxic effect of γδ T cells is HLA-independent. Therefore, the above anti-CD47 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-CD47 CAR is not particularly limited in the embodiments of the present invention. For example, γδ T cells expressing anti-CD47 CAR are V γ９ V δ２ It can contain subtypes.

[0014] The anti-CD47 CAR described above includes an antigen-binding domain that recognizes CD47, preferably human CD47, as an antigen, a transmembrane domain, and an intracellular signaling domain. Preferably, the anti-CD47 CAR includes an antigen-binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. These domains may be bound in tandem in this order. A leader sequence may also be positioned at the tip of the antigen-binding domain. The amino acid sequence and nucleotide sequence of human CD47 are registered under GenBank accession number Z25521.

[0015] The antigen-binding domain described above may include, for example, the light chain variable region (VL) and the heavy chain variable region (VH) contained in the antibody. In one embodiment, the antigen-binding domain includes a single-chain Fv (scFv). The scFv may be a fusion protein in which the VL and VH of the antibody are linked by a short linker peptide of, for example, 10 to 25 amino acids. The scFv may contain the VL and VH in this order toward the transmembrane domain, or in this order from the transmembrane domain.

[0016] The antibodies derived from VL and VH mentioned above may be non-human antibodies such as mouse antibodies or rat antibodies, or humanized antibodies thereof, or they may be human antibodies.

[0017] In one embodiment, the antigen-binding domain includes VL and VH derived from human antibodies. By including VL and VH derived from human antibodies in the antigen-binding domain, anti-CD47 CAR-γδ T cells with reduced, preferably avoided, immunogenicity to humans can be obtained. In this specification, VL or VH derived from human antibodies may include not only VL or VH directly derived from humans (hereinafter sometimes referred to as fully human VL or VH), but also fully human VL or VH into which amino acid mutations (e.g., substitution, deletion, insertion, addition) have been introduced. Fully human VL or VH is VL or VH identified based on human raw materials (including human gene libraries, etc.), and may be, for example, VL or VH contained in human antibodies isolated from humans or VL or VH encoded by nucleic acids isolated from humans. The immunogenicity of the above human antibody-derived VL and VH to humans can be confirmed by immunoassays such as enzyme-linked immunosorbent assay (ELISA). The immunogenicity of the above-mentioned human antibody-derived VL and VH in humans can be evaluated, for example, by immobilizing an antibody sample containing human antibody-derived VL and VH and the constant region (Fc) of the human antibody on an immunoplate, reacting it with the subject's serum, and then detecting the antibody bound to the antibody sample using an anti-human Fc antibody. In human antibody-derived VL and VH, the amino acid mutations that can be introduced into fully human VL and VH may be, for example, within a range that shows reduced immunogenicity in humans compared to non-human antibody-derived VL and VH, or within a range where the introduction of amino acid mutations does not significantly increase immunogenicity in humans.

[0018] In one embodiment, the antigen-binding domain includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 1 or its homologous amino acid sequence, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2 or its homologous amino acid sequence. The amino acid sequences of SEQ ID NO: 1 and 2 are amino acid sequences that may be included in fully human type VL and fully human type VH, respectively. Therefore, by using an antigen-binding domain that includes VL and VH and binds to CD47, anti-CD47 CAR-T cells with suitably reduced immunogenicity to humans, preferably anti-CD47 CAR-T cells that do not exhibit immunogenicity to humans, can be obtained.

[0019] In this specification, "homologous amino acid 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 original amino acid sequence (for example, the amino acid sequence of SEQ ID NO: 1 or 2). The homologous amino acid sequence may be an amino acid sequence that includes 1 to 25, 20 or less, 15 or less, 12 or less, 10 or less, 8 or less, 5 or less, or 3 or less amino acid mutations relative to the original amino acid sequence.

[0020] The above amino acid mutations may be substitutions, deletions, insertions, and / or additions. Amino acid substitutions are preferably conservative substitutions. Examples of conservative substitutions include substitutions between acidic amino acids, basic amino acids, polar, uncharged amino acids, aromatic amino acids, and aliphatic amino acids.

[0021] 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).

[0022] Preferably, the light chain and heavy chain complementarity-determining regions (CDRs) 1, 2, and 3 of the homologous amino acid sequence have a sufficiently high degree of sequence identity with the light chain and heavy chain CDRs 1, 2, and 3 of the original amino acid sequence. The number of amino acid mutations that can be introduced into the light chain and heavy chain CDRs 1, 2, and 3 of the homologous amino acid sequence is, for example, 0 to 3, preferably 0 to 2, and more preferably 0 or 1.

[0023] Various structures are known for the hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signaling domain that constitute the CAR. 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.

[0024] The hinge domain connects the antigen-binding domain and the transmembrane domain. The hinge domain is designed, for example, to provide space (length) and flexibility for the antigen-binding domain to access the target epitope. The transmembrane domain can anchor the CAR to the T cell membrane. Both the hinge domain and the transmembrane domain may contain domains derived from, for example, CD8, CD28, etc.

[0025] The co-stimulatory domain can enhance the proliferation and persistence of T cells. The co-stimulatory domain may contain one or more domains derived from, for example, 4-1BB, CD28, CD27, ICOS, etc.

[0026] Intracellular signaling domains can activate immune responses by T cells. These intracellular signaling domains may include, for example, domains derived from CD3ζ.

[0027] The leader sequence can efficiently express CAR on the cell surface. The leader sequence may include, for example, a CD8α signal peptide or a GM-CSF receptor sequence.

[0028] The anti-CD47 CAR-γδ T cells described above can be prepared by any suitable method. A method for preparing anti-CD47 CAR-γδ T cells may include, for example, preparing γδ T cells (Step I) and introducing a gene encoding anti-CD47 CAR (anti-CD47 CAR gene) into the γδ T cells (Step II).

[0029] [Step I] In Step I, γδ T cells are prepared. The γδ T cells to be prepared may be prepared using pluripotent stem cells (e.g., iPS cells, ES cells), hematopoietic stem cells, or non-stem cells. γδ T cells prepared using non-stem cells may have advantages in that they have a lower risk of becoming cancerous and their preparation costs are reduced.

[0030] For example, γδ T cells can be obtained by isolating them from blood (e.g., peripheral blood) collected from an individual. Since the proportion of γδ T cells in blood cells is small, it is preferable to culture and proliferate the γδ T cells isolated from the blood. For example, peripheral blood mononuclear cells can be isolated from the peripheral blood of an individual by gravity centrifugation or the like, and γδ T cells can be proliferated from these peripheral blood mononuclear cells. The individual is preferably a human or a non-human mammal (mouse, rat, rabbit, dog, pig, goat, cattle, horse, monkey, etc.).

[0031] Any suitable method can be used to proliferate γδ T cells from peripheral blood mononuclear cells. For example, one method is to culture peripheral blood mononuclear cells in a culture medium containing growth factors. As the culture medium, commercially available animal cell culture media, such as CTS Optmizer, can be used. ＴＭ You can use T cell culture media such as the Series or suspension cell culture media such as RPMI 1640 medium. The culture medium may be changed as needed during the culture period.

[0032] Growth factors include bisphosphonates, alkylamines, and recombinant human fibronectin (e.g., retronectin). （Ｒ）Examples include cytokines, anti-CD3 antibodies, anti-CD28 antibodies, etc. Cytokines may be IL-2, IL-7, IL-15, etc. Growth factors may be used individually 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 multiple growth factors, or culture in the presence of a single growth factor may be combined with culture in the presence of one or more growth factors. Preferably, the growth factors include bisphosphonates, and more preferably further include cytokines. As cytokines, for example, one or more selected from IL-2, IL-7, and IL-15 may be used. In one embodiment, two or more including bisphosphonates and IL-2 or IL-15 may be used in combination. By incubating peripheral blood mononuclear cells in the presence of growth factors (especially bisphosphonates), γδTCRs are activated in the peripheral blood mononuclear cells, and γδT cells can be selectively activated and proliferated.

[0033] 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.

[0034] 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 1 μM to about 10 μM. In one embodiment, the concentrations of cytokines such as IL-2, IL-7, and IL-15 may each be about 200 IU / mL to about 1000 IU / mL.

[0035] In step I, γδ T cells may be prepared as a cell population containing γδ T cells and other cells (e.g., αβ T cells). For example, the cell population prepared in step I may be a lymphocyte population or a T cell population. The proportion (purity) of γδ T cells in the cell population prepared in step I is preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, for example, 90% or more, 95% or more, or 98% to 100%. The proportion of cells having predetermined characteristics in the cell population can be evaluated by analytical methods such as flow cytometry or PCR. For example, the proportion of γδ T cells in the cell population can be evaluated by measuring the γδ TCR positivity rate by flow cytometry.

[0036] 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 bisphosphonates 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.

[0037] The proportion of CD47-positive cells (in other words, the CD47 positivity rate) in the cell population prepared in step I (more specifically, the cell population including γδ T cells) may be, for example, 80% or more, 85% or more, 90% or more, or 95% or more. One of the features of the present invention is that even when the anti-CD47 CAR gene is introduced into a cell population with a high proportion of CD47-positive cells, a high gene transfer efficiency can be achieved, in other words, anti-CD47 CAR-T cells can be obtained in high yield.

[0038] [Step II] In Step II, the anti-CD47 CAR gene is introduced into the γδ T cells prepared in Step I. The anti-CD47 CAR gene typically includes a gene encoding an antigen binding domain, a gene encoding a transmembrane domain, and a gene encoding an intracellular signaling domain.

[0039] Figure 1 is a schematic diagram illustrating an example of a DNA construct containing the anti-CD47 CAR gene that can be used in the present invention. The DNA construct 1 containing the anti-CD47 CAR gene has a configuration in which, for example, a base sequence (a) encoding a leader sequence, a base sequence (b) encoding the VL of an anti-CD47 antibody (e.g., the amino acid sequence represented by SEQ ID NO: 1), a base sequence (c) encoding a linker, a base sequence (d) encoding the VH of an anti-CD47 antibody (e.g., the amino acid sequence represented by SEQ ID NO: 2), a base sequence (e) encoding a hinge domain and a transmembrane domain, a base sequence (f) encoding a co-stimulatory domain, and a base sequence (g) encoding an intracellular signaling domain are sequentially linked.

[0040] Any appropriate method can be used as the method for introducing and expressing the anti-CD47 CAR gene into γδ T cells. Examples include biological gene transfer methods using viral vectors and the like, physical gene transfer methods such as electroporation, and chemical gene transfer methods such as lipofection and the calcium phosphate method. Among these, a gene transfer method using a viral vector can be preferably used.

[0041] The viral vector is not particularly limited as long as the produced viral particles infect γδ T cells and the γδ T cells express the anti-CD47 CAR. Specific examples of the viral vector include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors. Among these, retroviral vectors can be preferably used because the transgene is integrated into the chromosome of the host cell with high efficiency, and high expression of the above gene can be maintained.

[0042] Gene transfer into γδ T cells using a retroviral vector can be performed, for example, by co-transfecting a vector plasmid comprising the target gene sequence, LTR, and packaging signal, and a packaging plasmid comprising the gag sequence, pol sequence, and env sequence but no packaging signal into packaging cells (e.g., Cos cells, 293T cells), and collecting the produced viral particles (virions) to infect the γδ T cells. Alternatively, a packaging cell that expresses gag, pol, and env and produces empty viral particles may be transfected with a vector plasmid comprising the target gene sequence to produce viral particles (virions). As the retroviral vector plasmid, various commercially available products can be used, and the target gene can be inserted into a multiple cloning site (MCS) using a standard cloning method.

[0043] As the retroviral vector plasmid, various commercially available products can be used, and the target gene can be inserted into a multiple cloning site (MCS) using a standard cloning method. In one embodiment, a retroviral vector plasmid having a marker gene is used from the viewpoint of selection, sorting, etc. of transduced cells. As the marker, for example, fluorescent proteins such as GFP and EGFP can be used.

[0044] As the packaging cells and the method for transfecting the packaging cells with a retroviral vector plasmid or the like, those commonly used in gene transfer using a retroviral vector can be applied.

[0045] Viral particles produced from the packaging cells can be obtained as a virus solution, for example, by filtering the culture supernatant through a filter.

[0046] 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.

[0047] After the above infection, cells expressing anti-CD47 CAR can be easily selected and isolated by flow cytometry analysis using, for example, a labeled antibody that binds to anti-CD47 CAR (e.g., a fluorescent anti-G4S linker antibody).

[0048] In one embodiment, the proportion of cells expressing anti-CD47 CAR in the cell population after the gene transfer treatment may be, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and even more preferably 95% or more. Also, the proportion of γδ T cells in the cell population after the gene transfer treatment may be, for example, 70% or more, more preferably 80% or more, even more preferably 85% or more, for example 90% or more, 95% or more, or 98% to 100%. Therefore, the proportion of anti-CD47 CAR-γδ T cells in the cell population after the gene transfer treatment may be, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and even more preferably 95% or more. Also, the proportion of CD47-positive cells in the cell population after the gene transfer treatment may be, for example, 55% or more, 60% or more, 65% or more, or 70% or more. The proportion of each cell type in the above cell population may be the proportion of the cell population including all cells (excluding dead cells) after the gene transfer treatment, or the proportion of the lymphocyte population or T cell population. Furthermore, the proportion of each cell type in the above cell population may be the proportion of the cell population after, for example, 48 hours or more, 72 hours or more, or 168 hours or more have passed since the gene transfer treatment.

[0049] The anti-CD47 CAR-γδ T cells obtained in step II can be maintained or expanded over a long period after the introduction of the anti-CD47 CAR gene. For example, from 10 mL of peripheral blood, for example, 5 × 10⁶ cells can be cultured. ６ One or more, preferably 1 x 10 ７ One or more anti-CD47 CAR-γδ T cells can be obtained. Furthermore, these anti-CD47 CAR-γδ T cells can maintain their cytotoxic effect against CD47-positive cells even after freeze-thawing. Therefore, these anti-CD47 CAR-γδ T cells can be cryopreserved, for example, in liquid nitrogen and used after thawing.

[0050] B. Anti-CD47 Monoclonal Antibody According to one aspect of the present invention, an anti-CD47 monoclonal antibody or its antigen-binding fragment that binds to CD47, preferably human CD47, is provided. The anti-CD47 monoclonal antibody comprises a VL containing the amino acid sequence of SEQ ID NO: 1 or its homologous amino acid sequence, and a VH containing the amino acid sequence of SEQ ID NO: 2 or its homologous amino acid sequence. The amino acid sequence of SEQ ID NO: 1 is an amino acid sequence that may be included in fully human VL, and the amino acid sequence of SEQ ID NO: 2 is an amino acid sequence that may be included in fully human VH. Therefore, the anti-CD47 monoclonal antibody or its antigen-binding fragment has suitably reduced immunogenicity to humans, and preferably does not exhibit immunogenicity to humans.

[0051] The above-mentioned anti-CD47 monoclonal antibody is typically an immunoglobulin and has a unit structure comprising two light chains and two heavy chains. Each light chain comprises the above-mentioned VL and a light chain constant region (CL). Each heavy chain comprises the above-mentioned VH and a heavy chain constant region (CH). CL and CH may be human CL and human CH, respectively. The anti-CD47 monoclonal antibody may be, for example, IgG, IgM, IgA, IgD, or IgE.

[0052] The antigen-binding fragment of the anti-CD47 monoclonal antibody includes the above-mentioned VL and VH and can bind to CD47, preferably human CD47. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, etc.

[0053] The above-mentioned anti-CD47 monoclonal antibody or its antigen-binding fragment can be obtained by any suitable method. For example, the anti-CD47 monoclonal antibody or its antigen-binding fragment can be expressed in host cells by incorporating the gene encoding the anti-CD47 monoclonal antibody or its antigen-binding fragment into an expression vector and introducing it into any suitable host cell. The gene encoding the anti-CD47 monoclonal antibody or its antigen-binding fragment can be artificially synthesized based on the amino acid sequences of VL and VH, the composition of the desired anti-CD47 monoclonal antibody or its antigen-binding fragment, etc. Examples of host cells include animal cells, plant cells, insect cells, yeast, bacteria, etc. The antigen-binding fragment can also be obtained by enzymatic treatment of immunoglobulin.

[0054] C. Pharmaceutical Compositions According to one aspect of the present invention, a pharmaceutical composition is provided comprising anti-CD47 CAR-γδ T cells as described in Section A and / or an anti-CD47 monoclonal antibody or its antigen-binding fragment as described in Section B. The pharmaceutical composition may be used to treat or prevent diseases associated with CD47 expression. For example, a pharmaceutical composition comprising anti-CD47 CAR-γδ T cells may be suitably applied to CAR-T therapy for diseases associated with CD47 expression. The anti-CD47 monoclonal antibody or its antigen-binding fragment may also be included in the pharmaceutical composition as a conjugate with a cytotoxic agent, for example, an antibody-drug conjugate (ADC).

[0055] Diseases associated with CD47 expression include cancer and arteriosclerosis. Cancer can be hematological malignancies or solid tumors. Hematological malignancies include leukemia (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL)), lymphoma (e.g., follicular lymphoma (FL), marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma / Waldenström macroglobulinemia (LPL / WM), mantle cell lymphoma (MCL), and diffuse large B cell lymphoma). Examples of cancers include cellular lymphomas (DLBCL, NOS), Burkitt lymphoma / high-grade B-cell lymphoma (BL / HGBL), peripheral T-cell lymphoma (PTCL), extranodal NK / T-cell lymphoma nasal type (ENKL), adult T-cell leukemia / lymphoma (ATL), and Hodgkin lymphoma (HL), and myeloma (e.g., multiple myeloma (MM), solitary plasmacytoma, AL amyloidosis, and POEMS syndrome). Examples of solid tumors include lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain tumors, head and neck cancers, esophageal cancer, skin cancer, melanoma, retinoblastoma, thymic cancer, gastric cancer, colorectal cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, bile duct cancer, and pancreatic cancer.

[0056] The amount of anti-CD47 CAR-γδ T cells and / or anti-CD47 monoclonal antibodies or their antigen-binding fragments in the pharmaceutical composition should be such that it can exert the desired pharmacological effect.

[0057] The method of administering the pharmaceutical composition may be oral or parenteral administration, and parenteral administration is preferred. Parenteral administration methods include intravenous administration, subcutaneous administration, intratumoral administration, intraperitoneal administration, intraventricular administration, and intramuscular administration.

[0058] The pharmaceutical composition is prepared into an appropriate dosage form according to the method of administration. Depending on the dosage form of the pharmaceutical composition, any suitable pharmaceutically acceptable carrier and / or additives may be added as appropriate.

[0059] The dosage, administration rate, administration interval, and number of administrations of the pharmaceutical composition may be appropriately determined according to the conditions of the recipient (weight, age, sex, etc.) and the patient's condition.

[0060] The target of administration is preferably a human or non-human mammal, and more preferably a human. As described above, the VL and VH contained in the anti-CD47 CAR-γδ T cells and the anti-CD47 monoclonal antibody or its antigen-binding fragment may be of human origin. Therefore, the efficacy and safety of the pharmaceutical composition containing these may be maintained even when administered multiple times to a human. For example, the pharmaceutical composition may be applied to patients with relapsed or refractory hematological malignancies.

[0061] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0062] [Cells] Lymphoprep ＴＭ PBMCs were isolated from the peripheral blood of healthy subjects using a Serumwerk Bernburg microscope. The isolated PBMCs were cultured in a CTS OpTMizer T Cell Expansion SFM (Thermo Fisher Scientific) supplemented with zoledronic acid (Novartis) and IL-2 (PeproTech). Retrovirus packaging cell line PG13, myeloma-derived cell line KMM1, chronic myeloma-derived cell line BV-173, and acute leukemia-derived cell line THP-1 were purchased from ATCC. 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) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 U / mL penicillin, and 100 μg / mL streptomycin (PS) (FUJI FILM Wako Pure Chemical Corporation) at 5% CO2. ２were cultured below. KMM1 cells, BV-173 cells, and THP-1 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS and PS. [Flow Cytometry Analysis] For cell analysis, a BD Accuri C6 Plus Flow Cytometer (Becton, Dickinson and Company) was used. The measurement data were analyzed using BD Accuri C6 Plus software and FlowJo (Becton, Dickinson and Company). 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 Laboratories) staining. For cell staining, two types of antibodies, anti-G4SLinker-Alexa647 (E7O2V, Cell Signaling Technology) and anti-CD47-APC (B6H12, abcam), were used.

[0063] [Experimental Example 1: Acquisition of Fully Human Anti-CD47 scFv] <Screening of Fully Human Anti-CD47 scFv> Referring to JP-A-2005-185281, WO2008 / 007648, WO2006 / 090750, and the like, a phage (hereinafter referred to as antibody phage) library displaying human scFv was prepared based on nucleic acids derived from lymphocytes of healthy humans and humans suffering from tonsillitis. Fully human anti-CD47 scFv was screened from the above antibody phage library by the panning method. hCD47-His recombinant protein was added to the wells of a Maxisorp Immunoplate (Thermo Fisher Scientific), and the plate was allowed to stand overnight at 4°C. After blocking with 3% BSA, the antibody phage library (1.0 × 10 １３CFU was added and the mixture was reacted at 37°C for 1 hour. After washing the wells with PBS, the antigen-bound antibody phages were eluted with 0.1 M Glycine-HCl (pH 2.5) and recovered, and used to infect E. coli (DH12S). E. coli with inserted phagemid DNA was selected in 2×YT liquid medium containing 100 μg / mL ampicillin and 1% glucose. The selected E. coli were infected with M13KO7 helper phage, and antibody phages were grown in the culture supernatant. These were recovered and concentrated by PEG precipitation and used for the next panning. This cycle was repeated from the second time onward, with the blocking solution changed to 1% skim milk, for a total of three pannings. The antibody phages recovered in the third panning were used to infect E. coli (DH12S), and cultured in LB plates containing 100 μg / mL ampicillin and 0.2% glucose to form E. coli colonies. Colonies on plates were inoculated into 2×YT medium containing 100 μg / mL ampicillin, 0.05% glucose, and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Antibody phages were secreted into the E. coli culture supernatant by shaking culture overnight at 30°C. The scFv presented by the above antibody phage has the pIII protein, a coat protein of M13-phage, fused to its C-terminus. The following ELISA was performed using this culture supernatant.

[0064] <Selection of fully human anti-CD47 scFv> Anti-CD47 antibody-positive clones were selected by ELISA. hCD47-His recombinant protein was added to wells of Maxisorp Immunoplate (Thermofisher Scientific) and left to stand overnight at 4°C. After blocking the immobilized wells with 5% BSA, the above E. coli culture supernatant was added to each well and incubated at 37°C. After washing the wells with PBS-T containing 0.05% Tween, rabbit anti-cp3 antibody (MBL) was added as a secondary antibody and incubated at 37°C. After washing the wells with PBS-T containing 0.05% Tween, peroxidase-conjugated goat anti-rabbit IgG (H+L chain) (MBL) was added as a tertiary antibody and incubated at 37°C. TMB (Thermo Fisher Scientific) was added to each well and allowed to develop color at room temperature. Then, 2N sulfuric acid was added to stop the peroxidase reaction, and the absorbance of the sample at a wavelength of 450 nm was measured. Positive clones were selected from the measurement results.

[0065] <Sequencing Analysis> DNA was extracted from positive clones (No. 6-11). The nucleotide sequence of scFv presented by the antibody phage was determined by sequencing analysis. The nucleotide sequence analysis was performed by Eurofins Genomics using the sequencing primer T7ETZ (5'-TAATACGACTCACTTATAGGG-3': SEQ ID NO: 3). The above nucleotide sequence was converted to an amino acid sequence to determine the amino acid sequence of scFv presented by the antibody phage (SEQ ID NO: 4). The determined amino acid sequence is shown in Table 1. In the amino acid sequence shown in Table 1, the underlined sequence (GGGGGSGGGGGSGGGGGS (SEQ ID NO: 5)) is the linker sequence, and the amino acid sequence from Glu1 to Arg122 on the N-terminus (SEQ ID NO: 1) is VL, and the amino acid sequence from Met138 to Ser355 on the C-terminus (SEQ ID NO: 2) is VH.

[0066]

[0067] [Experimental Example 2: Preparation of Nucleic Acid Encoding Anti-CD47 CAR] <Preparation of Retroviral Vector> A nucleic acid encoding anti-CD47 CAR (see Figure 1) containing the following in order: CD8α signal peptide (a), anti-CD47 scFv of Sequence ID No. 4 (b-d), CD8α hinge domain and transmembrane domain (e), 4-1BB intracellular domain (f), and CD3ζ domain (g) was inserted into the retroviral vector pDON5 (TaKaRa). The amino acid sequences of the CD8α signal peptide, CD8α hinge domain and transmembrane domain, 4-1BB intracellular domain, and CD3ζ domain are shown in Table 2.

[0068]

[0069] <Preparation of Virus Solution> G3T-hi cells were seeded in a 6-well plate 24 hours before transfection. The above G3T-hi cells were co-transfected with a retroviral vector containing nucleic acid encoding anti-CD47 CAR using TransIT-293 reagent (Mirus) with gag-pol and env expression vectors. After 48 hours, the supernatant containing the ecotropic retrovirus produced 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 polyblen 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 polyblen were added. The above PG13 cell infection procedure was performed a total of 5 times. After the final infection, the supernatant of PG13 cell cultures was collected and filtered through a 0.45 μm filter to obtain an anti-CD47 CAR retrovirus solution.

[0070] [Experimental Example 3: Preparation of Anti-CD47 CAR-γδ T Cells] <Preparation of γδ T Cells> Human peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors using Lymphoprep gradient centrifugation. PBMCs were stimulated to promote γδ T cell proliferation by culturing them for 4 days in T cell medium containing 200 IU / mL IL-2 and 5 μM zoledronic acid solution (ZOL) (OpTmizer T cell expansion supplement, 10% fetal bovine serum, 1% penicillin / streptomycin, and OpTmizer containing 2 mM L-glutamine).

[0071] <Transduction> Transduction of γδ T cells was performed on Day 4 of PBMC stimulation with ZOL and IL-2. Specifically, a cell suspension containing the proliferated γδ T cells and an anti-CD47 CAR retrovirus solution were added in a 1:1 ratio to a polypropylene tube coated with retronectin (TaKaRa), and the tube was centrifuged at 1200 g for 1.5 hours. After 24 hours, the γδ T cells were reinfected with the retrovirus using the same procedure. This yielded anti-CD47 CAR-γδ T cells. Furthermore, when the proportion of γδ T cells in the cell suspension used for transduction was evaluated by flow cytometry using an anti-γδ TCR antibody, the proportion of γδ T cells was over 80%.

[0072] Cells before transduction (Day 4) and after transduction (48 hours after final infection) were stained with anti-G4SLinker-Alexa 647, and the CAR positivity rate was detected by flow cytometry. The results are shown in Figure 2. Cells before transduction (Day 4) and after transduction (48 hours after final infection) were also stained with anti-CD47-APC, and the CD47 positivity rate was detected by flow cytometry. The results are shown in Figure 3. The CAR positivity rate in Figure 2 and the CD47 positivity rate in Figure 3 represent the positivity rates in the lymphocyte population (lymphocyte population) of the lymphocyte region, which was gated based on SSCs and FSCs, respectively.

[0073] As shown in Figure 2, the CAR positivity rate (effectively the proportion of anti-CD47 CAR-γδ T cells) in the transduction-treated cell population was 99.3%. On the other hand, as shown in Figure 3, the CD47 positivity rate in the cell population before transduction was 99.7%, but it decreased slightly to 72.4% in the transduction-treated cell population. Furthermore, these results indicate that although the majority of γδ T cells activated for transduction were CD47-positive, strong fracturing occurred between anti-CD47 CAR-γδ T cells. In addition, a CAR positivity rate of over 95% was maintained even one week after transduction. In the preparation of the anti-CD47 CAR-γδ T cells described above, 5 × 10⁶ cells were produced from 10 mL of peripheral blood. ６ One or more anti-CD47 CAR-γδ T cells were obtained.

[0074] [Experimental Example 4: Cytotoxicity Assay] Target cells (KMM1 cells, BV-173 cells, and THP-1 cells) and Effector cells (anti-CD47 CAR-γδT cells or untransduced γδT cells) were co-cultured in 96-well plates. To demonstrate cytotoxic activity dependent on the number of effector cells, cells were seeded so that the effector-to-target ratio (E:T) was in the range of 1:10 to 1:2. The number of surviving target cells 24 hours after seeding was evaluated by flow cytometry.

[0075] As shown in Figures 4-6, anti-CD47 CAR-γδT cells showed superior antitumor effects against KMM1 cells, BV-173 cells, and THP-1 cells in an effector cell number-dependent manner. In the graphs shown in Figures 4-6, the vertical axis represents the ratio of surviving target cells to the number of surviving target cells when non-transduced γδT cells (Mock γδT cells) are used as effector cells.

[0076] [Experimental Example 5: Preparation of Anti-CD47 CAR-αβT Cells] A cell suspension containing high-purity αβT cells was prepared in the same manner as in Experimental Example 3, except that 20 IU / mL IL-2, anti-CD3 antibody, and anti-CD28 antibody were used as growth factors instead of 200 IU / mL IL-2 and 5 μM ZOL, and the cells were infected with anti-CD47 CAR retrovirus. Cells before transduction were stained with anti-CD47-APC, and the CD47 positivity rate was detected by flow cytometry. The results are shown in Figure 7. Cells before transduction and after transduction (one week after final infection) were stained with anti-G4SLinker-Alexa 647, and the CAR positivity rate was detected by flow cytometry. The results are shown in Figure 8. The E1 region in the left panel of Figure 7 and the E3 region in the left panel of Figure 8 are lymphocyte regions gated based on SSCs and FSCs. The right panel of Figure 7 shows the detection results of the CD47 positivity rate in the cell population (lymphocyte population) of the E1 region, and the right panel of Figure 8 shows the detection results of the CAR positivity rate in the cell population (lymphocyte population) of the E3 region.

[0077] As shown in Figure 7, the CD47 positivity rate in the lymphocyte population before transduction was 100%. On the other hand, as shown in Figure 8, one week after transduction, the proportion of lymphocytes in the entire cell suspension had decreased significantly, and the CD47 positivity rate in the lymphocyte population was 49.7%. From these results, it is suggested that αβ T cells activated by proliferation stimulation are CD47 positive, and that fractorides are produced by the expression of anti-CD47 CAR.

[0078] Anti-CD47 CAR-γδT cells, etc., according to embodiments of the present invention can be suitably used in the pharmaceutical field.

Claims

1. Anti-CD47 CAR-γδ T cells that express a chimeric antigen receptor that recognizes CD47 as an antigen.

2. The anti-CD47 CAR-γδ T cell according to claim 1, wherein the chimeric antigen receptor recognizes human CD47 as an antigen.

3. The anti-CD47 CAR-γδ T cell according to claim 1, wherein the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the antigen-binding domain comprises a light chain variable region and a heavy chain variable region derived from a human antibody.

4. The anti-CD47 CAR-γδ T cell according to claim 3, wherein the antigen-binding domain comprises a light chain variable region containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:

2.

5. A cell population comprising 80% or more of the anti-CD47 CAR-γδT cells described in claim 1.

6. An anti-CD47 monoclonal antibody or its antigen-binding fragment, comprising: a light chain variable region containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1; and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:

2.

7. A pharmaceutical composition for treating or preventing a disease related to CD47 expression, comprising anti-CD47 CAR-γδT cells as described in claim 1 or an anti-CD47 monoclonal antibody or its antigen-binding fragment as described in claim 6.

8. The pharmaceutical composition according to claim 7, wherein the disease associated with the expression of CD47 is cancer or arteriosclerosis.

9. The pharmaceutical composition according to claim 8, wherein the cancer is a hematological malignancy.

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