JP2015092865A - Humanized anti-CD20 chimeric antigen receptor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel treatment strategy against lymphoma.SOLUTION: The invention provides a humanized anti-CD20 chimeric antigen receptor comprising an extracellular domain comprising an anti-CD20 humanized monoclonal antibody scFv fragment, a membrane penetrating domain, and an intracellular signal domain for an immunocyte effector function. The invention also provides a CD20 specific immunocyte which expresses the humanized anti-CD20 chimeric antigen receptor on a cell surface and is produced by genetic engineering.

Description

  The present invention relates to a humanized CD20 chimeric antigen receptor, immune cells expressing the same, a method for producing the same, use thereof, and the like.

  Anti-CD20 humanized chimeric monoclonal antibodies (CD20mAb: rituximab, ofatumumab, etc.) have already been administered to many patients as pharmaceuticals and their clinical effects have been confirmed. However, because the half-life of antibody drugs is as short as 2 to 3 weeks, it requires repeated administration, and repeated administration causes the emergence of CD20 low-expressing strains, resulting in treatment resistance Has been reported. The mechanism of action of antibody drugs is broadly divided into complement-dependent cytotoxic activity (CDC) and antibody-dependent cell-dependent cytotoxic activity (ADCC), and rituximab is said to be heavily involved in ADCC.

  A chimeric antigen receptor (hereinafter also referred to as “CAR”) is typically a cell of a molecule that carries a single-chain variable region of an antibody as an extracellular domain, transmembrane region, CD3ζ, and a costimulatory signal. It has a structure that connects inner domains. By binding to the antigen according to the specificity of the antibody, a signal is transmitted to T cells, and T cell division occurs after target cell injury, cytokine release, and stimulation. It is possible to give specificity to T cells by gene transfer, and the preparation of cells is generally easier than the case of culturing and amplifying endogenous tumor-specific T cell receptor (TCR) positive T cells. In addition, the treatment method using CAR (CAR-T therapy) shows higher cytotoxic activity than antibody therapy, and multiple treatments are required because it is amplified autonomously by antigen stimulation in one infusion. There is no point. Since the introduction of the TCR gene targets a complex of HLA and the peptide presented on it, it can be treated only when the patient has a specific HLA (HLA-restricted). It is also advantageous that there is no restraint. That is, if the target antigen is positive for the tumor cells, it is possible to treat all patients who are positive for the target antigen. On the other hand, since all cells are targeted if the antigen is positive, normal tissues are also targeted when the antigen is expressed in addition to the target cells (on-target side reaction). For this reason, it is difficult to select a target antigen for CAR-T therapy, and this point is the most problematic for enhancing versatility and clinical application.

  CAR uses the intracellular domain of co-stimulatory molecules (CD28, 4-1BB, etc.) so that immune cells can be stimulated just by signals from CAR, even if co-stimulation is not transmitted from the target cell. Can be activated. Normally, tumor cells do not express ligands such as CD80 / 86, so even if endogenous tumor-specific T cells are present, only incomplete stimuli can be transmitted, resulting in energy. CAR can provide costimulation with only one signal. This is another advantage over TCR gene transfer therapy.

  In CAR-T therapy, as described above, if the target antigen is expressed in any amount other than the tumor, an on-target side reaction occurs, resulting in serious side effects, and sometimes fatal. Therefore, as described above, selection of a target antigen is most important, and B-cell differentiation antigens such as CD20 / CD19 are the most advanced in the clinical trials of CAR-T therapy that have been conducted so far. A research group at the University of Pennsylvania created a CAR by connecting a mouse-derived CD19 single-chain antibody and the intracellular domain of 4-1BB as described above, and introduced it into peripheral blood T cells of chemoresistant chronic lymphocytic leukemia. . An amazing result that remission is obtained in 3 out of 3 cases by administering this to a patient has been reported (Non-patent Document 1). The effectiveness of CD19CAR-T therapy is also reported in Patent Document 1 and Non-Patent Document 2. On the other hand, with regard to CAR-T therapy targeting CD20, an example using mouse antibody scFv has been reported (Non-patent Document 3). There is also a report of CD20 CAR-T therapy using CD28 intracellular domain (Non-patent Document 2).

Japanese Patent No. 5312721

N Engl J Med. 2011; 365: 725-733. Cancer Res 2006; 66: 10995-11004. MOLECULAR THERAPY Vol. 9, No. 4, April 2004 577-586

  As described above, the therapeutic method using the anti-CD20 humanized chimeric monoclonal antibody (CD20mAb) shows a certain effect. However, repeated administration is necessary due to its half-life. In addition, since the main action is expressed by the action of T cells and NK cells in the patient, administration of the antibody alone is inefficient. Furthermore, if the antigen expression in the target cells is weak, good therapeutic results cannot be expected. That is, in order for the humanized CD20 antibody to exert its effect, the CD20 expression on the target cell surface needs to be relatively high.

  On the other hand, the extracellular domain of the chimeric antigen receptor takes the structure of an antibody, but most of those developed so far utilize mouse-derived antibodies and have a problem of immune rejection when applied to humans. Although an effect can be expected in a patient with reduced immunity, an effect cannot be expected in a patient with an autoimmune disease or a patient with no immunity reduction.

  Therefore, the main object of the present invention is to provide a novel therapeutic strategy for lymphoma, particularly a case showing resistance to treatment with an anti-CD20 humanized chimeric monoclonal antibody and a highly malignant lymphoma with decreased CD20 expression.

  In the course of research in view of the above problems, the present inventors focused on CAR-T therapy and focused on the importance of CD20 as a target antigen, and the humanized anti-CD20 monoclonal antibody scFv fragment was extracellular domain. We designed a new CAR structure and verified its effectiveness. Specifically, the effectiveness of immune cell therapy using a novel CAR structure was examined in relation to the expression level of CD20 on the target cell surface. As a result, it was found that the newly-structured CAR recognizes CD20 expression that is considerably lower than that of CD20 mAb and exerts its function. This fact means that the newly-structured CAR is also effective against cases showing resistance to treatment with CD20 mAb and tumor cells with reduced CD20 expression. On the other hand, as a result of detailed examination, important knowledge was obtained about the characteristic of CAR necessary for enhancing the therapeutic effect.

  Here, it is known that CD20 is expressed not only in tumor cells but also in normal plasma cells, and CD20 mAb is applied to treatment targeting antibody-producing CD20 positive B cells even in non-neoplastic diseases . As an alternative therapy, a therapy using a novel structure of CAR can be used. Specifically, autoimmune diseases such as treatment-resistant systemic lupus erythematosus, treatment-resistant idiopathic thrombocytopenic purpura, and poor regenerative disease where anti-HLA antibodies were acquired by frequent blood transfusions, resulting in ineffective transfusion. It is expected that T-cell therapy using a novel structure of CAR will exert a high therapeutic effect on diseases and conditions such as anemia and hemophilia that have acquired inhibitors by supplementation of coagulation factors.

The invention shown below is mainly based on the above-mentioned results and considerations.
[1] A humanized anti-CD20 chimeric antigen receptor comprising an extracellular domain containing an scFv fragment of an anti-CD20 humanized monoclonal antibody, a transmembrane domain, and an intracellular signal domain for effector functions of immune cells.
[2] The humanized anti-CD20 chimeric antigen receptor according to [1], wherein the anti-CD20 humanized monoclonal antibody has a dissociation constant (Kd value) for human CD20 antigen of 7 to 10 nM.
[3] The humanized anti-CD20 chimeric antigen receptor according to [1] or [2], wherein the intracellular signal domain includes CD3ζ and an intracellular domain of a costimulatory molecule.
[4] The humanized anti-CD20 chimeric antigen receptor according to [3], wherein the costimulatory molecule is CD28 or 4-1BB.
[5] The humanized anti-CD20 chimeric antigen receptor according to any one of [1] to [4], wherein the transmembrane domain is a transmembrane domain of CD28.
[6] The humanized anti-CD20 chimeric antigen receptor according to any one of [1] to [5], which contains a spacer domain between the extracellular domain and the transmembrane domain.
[7] The humanized anti-CD20 chimeric antigen receptor according to [6], wherein the spacer domain consists of an Fc fragment of human IgG.
[8] The humanized anti-CD20 chimeric antigen receptor according to [7], wherein the human IgG is IgG4.
[9] The humanized anti-CD20 chimeric antigen receptor according to any one of [1] to [8], wherein the linker constituting the scFv fragment is a peptide linker having 8 to 25 amino acid residues.
[10] The humanized anti-CD20 chimeric antigen receptor according to any one of [1] to [9], wherein the dissociation constant of the anti-CD20 monoclonal antibody for human CD20 antigen is about 7.2.
[11] The amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) constituting the scFv fragment are a combination of the following (1) to (3): [1] to [9] The humanized anti-CD20 chimeric antigen receptor according to any one of
(1) VL is the amino acid sequence of SEQ ID NO: 1, and VH is the amino sequence of SEQ ID NO: 2;
(2) VL is the amino acid sequence of SEQ ID NO: 3, and VH is the amino sequence of SEQ ID NO: 4;
(3) VL is the amino acid sequence of SEQ ID NO: 5, and VH is the amino sequence of SEQ ID NO: 6.
[12] DNA encoding the humanized anti-CD20 chimeric antigen receptor according to any one of [1] to [11].
[13] A recombinant expression vector that retains the DNA of [12] in an expressible manner.
[14] A genetically engineered CD20-specific immune cell that expresses the humanized anti-CD20 chimeric antigen receptor according to any one of [1] to [11] on the cell surface.
[15] The CD20-specific immune cell according to [14], wherein the immune cell is a T cell.
[16] A cell preparation comprising a therapeutically effective amount of the CD20-specific T cell according to [14] or [15].
[17] Selected from the group consisting of B-cell malignant lymphoma, treatment-resistant chronic lymphocytic leukemia, treatment-resistant idiopathic thrombocytopenic purpura, treatment-resistant systemic lupus erythematosus, and hemophilia with coagulation inhibitors The cell preparation according to [16], which is used for treatment, prevention, or improvement of a disease or a patient who cannot obtain a blood transfusion effect due to an anti-HLA antibody.
[18] The cell preparation according to [17], which is applied to a case resistant to an anti-CD20 humanized chimeric monoclonal antibody.

New CD20 CAR design. The cell surface state when CAR is introduced and expressed (top) and the nucleic acid construct for CAR introduction (bottom) are shown. CD20 CAR gene introduction scheme (top) and measurement results (bottom). CD20 expression level on the cell surface in various cell lines. Complement dependent cytotoxicity (CDC) by rituximab against CD20-CEM (left). After incubation for 30 minutes in the presence of rituximab (10 μg / ml) and 20% rabbit complement, Annexin-V / DAPI staining was performed. Cytotoxic activity (right) by CD20 CAR-T cells against CD20-CEM. CD20 CAR T-cells were proliferated for 1 course with irradiated LCL to select CD8 positive cells, and a ⁵¹ Cr release assay was performed. CD20 expression level of CD20-CEM with typical CD20 expression. Production of IFN-γ in CD20CAR-T cells after stimulation with CD20-CEM with representative CD20 expression. CD20 CAR-T cells (reactive cells) and CD20-CEM (stimulating cells) were mixed 1: 1 and stimulated (incubation for 4 hours), then fixed and stained. CD20CAR-T cell division by stimulation of CD20-CEM with typical CD20 expression. CD20 CAR-T cells (reacting cells) and CD20-CEM (stimulating cells) were mixed by 1: 1 and stimulated (incubation for 96 hours), and then subjected to analysis. Intracellular signaling by stimulation of CD20-CEM with typical CD20 expression. CD20 CAR-T cells (reacting cells) and CD20-CEM (stimulating cells) were mixed at 5: 1 for stimulation (incubation for 10 minutes), and then fixed and stained. Effect of CD20 CAR-T cells on CD20 low-expressing cell lines and clinically isolated cells. Relationship between extracellular domain (scFv) affinity and IFN-γ production. Relationship between extracellular domain (scFv) affinity and cytotoxic activity. Relationship between extracellular domain (scFv) affinity and CAR-T cell proliferation after stimulation of irradiated LCL. Relationship between linker length and percentage of IFN-γ producing cells. Examination of cytotoxic activity against clinically isolated cells with reduced CD20 expression.

1. Humanized anti-CD20 chimeric antigen receptor The first aspect of the invention relates to a humanized anti-CD20 chimeric antigen receptor (CAR). The humanized anti-CD20 chimeric antigen receptor is a structure comprising an extracellular domain derived from a humanized antibody specific for CD20, and a transmembrane domain and an intracellular signal domain that have different origins from the extracellular domain. The CAR of the present invention comprises an extracellular domain containing an scFv fragment of an anti-CD20 humanized monoclonal antibody, a transmembrane domain, and an intracellular signal domain for effector functions of immune cells. Hereinafter, each domain will be described.

(1) Extracellular domain In the present invention, the extracellular domain exhibits CD20-specific binding properties. The extracellular domain contains an scFv fragment of an anti-CD20 humanized monoclonal antibody. The anti-CD20 humanized monoclonal antibody is a humanized monoclonal antibody using CD20 as an antigen. A humanized monoclonal antibody is an antibody in which the structure of a monoclonal antibody of another animal species (eg, mouse or rat) is similar to that of a human antibody, and only the constant region of the antibody is replaced with that of a human antibody. A chimeric antibody and a human-type CDR-grafted antibody (PTJohons et al., Nature 321,522 (1986) in which parts other than CDRs (complementarity determining regions) present in constant regions and variable regions are substituted with those of human antibodies. ))including. In order to increase the antigen-binding activity of human CDR-grafted antibodies, a method for selecting a human antibody framework (FR) highly homologous to a mouse antibody, a method for producing a humanized antibody having a high homology, a mouse CDR for a human antibody Techniques for further substitution of amino acids in the FR region after transplantation have already been developed (US Patent No. 5585089, US Patent No. 5693761, US Patent No. 5693762, US Patent No. 6180370, European Patent No. 451216) , European Patent No. 682040, Patent No. 2828340, etc.) and can also be used for the production of humanized antibodies.

  As the anti-CD20 humanized monoclonal antibody in the present invention, one having high affinity for CD20 may be adopted. Preferably, an anti-CD20 humanized monoclonal antibody having a dissociation constant (Kd value) of 7 to 10 nM for human CD20 antigen is used so as to increase the effect expected of the CAR of the present invention, that is, the damaging activity against tumor cells and the like. . In a particularly preferred embodiment, an anti-CD20 humanized monoclonal antibody having a dissociation constant (Kd value) for human CD20 antigen of about 7.2 nM is utilized. The dissociation constant can be measured by the measurement method shown in the examples of JP 2010-189402 A. However, as long as the same measurement result can be obtained, other measurement methods capable of measuring the Kd value for the antigen expressed on the cell surface may be employed.

  As antibodies that satisfy the above dissociation constant conditions (Kd value of 7 to 10 nM), antibody vv, antibody fv, antibody fa disclosed in JP2010-189402A (Table 5 of JP2010-189402A) Reference). Antibody vv has the amino acid sequence of SEQ ID NO: 1 in the light chain variable region (VL) and the amino sequence of SEQ ID NO: 2 in the heavy chain variable region (VH). Similarly, antibody fv has an amino acid sequence of VL in SEQ ID NO: 3 and an amino sequence of VH in SEQ ID NO: 4. Antibody fa has an amino acid sequence of SEQ ID NO: 5 in VL and an amino sequence of SEQ ID NO: 6 in VH. The dissociation constants (Kd value, nM) of these antibodies for human CD20 antigen are 7.02 (antibody vv), 7.21 (antibody fv), and 10.09 (antibody fa) (Table 5 of JP 2010-189402 A). See).

  In the present invention, the scFv fragment of the anti-CD20 humanized monoclonal antibody is used for the extracellular domain. An scFv fragment is a structure in which an immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) are linked via a linker, and retains the ability to bind to an antigen. As the linker, for example, a peptide linker can be used. A peptide linker is a linker consisting of a peptide in which amino acids are linked in a straight chain. A typical example of a peptide linker is a linker composed of glycine and serine (GGS linker or GS linker). The GGS linker and glycine and serine, which are amino acids constituting the GS linker, have a small size per se and are difficult to form higher-order structures in the linker. The length of the linker is not particularly limited. For example, a linker having 5 to 25 amino acid residues can be used. As shown in the Examples described later, it has been clarified that a longer linker is more advantageous for the CAR function than a short linker. Therefore, the length of the linker is preferably 8-25, more preferably 15-20. Specific examples of the linker are shown in the examples described later.

(2) Transmembrane domain The transmembrane domain is interposed between the extracellular domain and the intracellular signal domain. As the transmembrane domain, a transmembrane domain such as CD28, CD3ε, CD8α, CD3, CD4 or 4-1BB can be used. You may decide to use the transmembrane domain which consists of the polypeptide constructed artificially. Specific examples of the transmembrane domain are shown in Examples described later. Functionally equivalent to those shown in the embodiments can also be used.

(3) Intracellular signal domain The intracellular signal domain transmits a signal necessary for exerting an effector function of an immune cell. That is, an intracellular signal domain that can transmit a signal necessary for activation of immune cells when the extracellular domain binds to CD20 is used. The intracellular signal domain includes a domain for transmitting a signal via the TCR complex (for convenience, referred to as “first domain”) and a domain for transmitting a costimulatory signal (for convenience, “second domain”). Called). In addition to CD3ζ, an intracellular domain such as FcεRIγ can be used as the first domain. CD3ζ is preferably used. As the second domain, an intracellular domain of a costimulatory molecule is used. Examples of costimulatory molecules include CD28, 4-1BB (CD137), CD2, CD4, CD5, CD134, OX-40, or ICOS. Preferably, the intracellular domain of CD28 or 4-1BB is employed.

  The connection mode of the first domain and the second domain is not particularly limited, but preferably, it is known that costimulation was strongly transmitted when CD3ζ was connected distally in the past case. The second domain is arranged on the side. A plurality of identical or different intracellular domains may be linked in tandem to form the first domain. The same applies to the second domain.

  The first domain and the second domain may be linked directly or a linker may be interposed between them. As the linker, for example, a peptide linker can be used. A peptide linker is a linker consisting of a peptide in which amino acids are linked in a straight chain. The structure, characteristics, etc. of the peptide linker are as described above. However, as a linker here, you may use what consists only of glycine. The length of the linker is not particularly limited. For example, a linker having 2 to 15 amino acid residues can be used. Specific examples of the intracellular signal domain are shown in the examples described later. Functionally equivalent to those shown in the embodiments can also be used.

(4) Other elements A structure in which an extracellular domain and a transmembrane domain are linked via a spacer domain is preferable. That is, the CAR of a preferred embodiment includes a spacer domain between the extracellular domain and the transmembrane domain. The spacer domain is used to promote binding between CAR and CD20 antigen. For example, an Fc fragment of human IgG (preferably human IgG4) can be used as a spacer domain. In addition, a part of the extracellular domain of CD28, a part of the extracellular domain of CD8α, and the like can be used as the spacer domain. A spacer domain can also be provided between the transmembrane domain and the intracellular signal domain.

2. Nucleic acid encoding humanized anti-CD20 chimeric antigen receptor The second aspect of the present invention relates to a nucleic acid encoding the CAR of the present invention. The nucleic acid encoding the CAR of the present invention refers to a nucleic acid from which the CAR is obtained when it is expressed, that is, a nucleic acid (typically DNA) having a base sequence corresponding to the amino acid sequence of the CAR. Say. Therefore, as long as it has a sequence corresponding to the amino acid sequence, it may be a nucleic acid having a sequence portion that does not correspond to the amino acid sequence. Of course, codon degeneracy is also considered. The nucleic acid of the present invention can be used for the preparation of immune cells expressing the CAR of the present invention (the third aspect of the present invention). Typically, for the preparation of the immune cells, a vector (recombinant expression vector) that holds the nucleic acid of the present invention in an expressible manner is constructed. As used herein, the term “vector” refers to a nucleic acid molecule that can transport a nucleic acid molecule inserted therein into a target (ie, an immune cell or a progenitor cell thereof), and its form, origin, etc. are particularly limited. It is not a thing. Various types of vectors are available. For example, plasmid vectors, phage vectors, cosmid vectors, YAC vectors, BAC vectors, virus vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad Sci., 84: 7413-7417, 1987), HVJ (Hemagglutinating virus of Japan) -liposomes (Dzau, VJ, Mann, M., Morishita, R. et al., Proc. Natl. Acad. Sci., 93) : 11421-11425, 1996, Kaneda, Y., Saeki, Y. & Morishita, R., Molecular Med. Today, 5: 298-303, 1999).

  The gene transfer method using a virus vector skillfully utilizes the phenomenon that a virus infects cells, and high gene transfer efficiency can be obtained. Adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, Sendai virus vectors and the like have been developed as virus vectors. Among these, in adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors, a foreign gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected. Each viral vector can be prepared according to a previously reported method or using a commercially available dedicated kit. For example, a retroviral vector can be prepared by the following procedure. First, a virus genome (gag, pol, env gene) other than the packaging signal sequence between LTRs (Long Terminal Repeat) existing at both ends of the virus genome is removed, and a target gene is inserted therein. The viral DNA thus constructed is introduced into a packaging cell that constitutively expresses the gag, pol, and env genes. Thereby, only the vector RNA having the packaging signal sequence is incorporated into the viral particle, and a retroviral vector is produced.

  Usually, a promoter is incorporated in the recombinant expression vector of the present invention. Within the recombinant expression vector, the promoter is operably linked to the nucleic acid of the invention. In the recombinant expression vector having such a configuration, the nucleic acid of the present invention can be forcibly expressed in the target cell by the action of the promoter. Here, “the promoter is operably linked to the nucleic acid of the present invention” is synonymous with “the nucleic acid of the present invention is placed under the control of the promoter” and is usually 3 ′ of the promoter. The nucleic acid of the present invention is linked to the terminal side directly or via another sequence. As the promoter, CMV-IE (cytomegalovirus early gene-derived promoter), SV40ori, retrovirus LTP, SRα, EF1α, β-actin promoter and the like can be used.

  A poly A addition signal sequence, poly A sequence, enhancer sequence, selectable marker sequence, tag sequence and the like can also be arranged in the recombinant expression vector. The use of a poly A addition signal sequence or poly A sequence improves the stability of mRNA resulting from the recombinant expression vector. The poly A addition signal sequence or poly A sequence is linked to the nucleic acid of the present invention on the downstream side. On the other hand, the use of an enhancer sequence can improve the expression efficiency. If a recombinant expression vector containing a selectable marker sequence is used, the presence or absence (and introduction rate) of the recombinant expression vector can be confirmed using the selectable marker.

  For recombination operations (insertion of the nucleic acid, promoter, selection marker gene, etc. of the present invention into a vector, etc.), standard recombinant DNA techniques (for example, Molecular Cloning, Third, etc.) such as a method using a restriction enzyme and DNA ligase. Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).

3. CD20-specific immune cells expressing humanized anti-CD20 chimeric antigen receptor A third aspect of the present invention is an immune cell that expresses the CAR of the present invention on the cell surface (hereinafter referred to as "cell of the present invention"). provide. In the cells of the present invention, CARs present on the cell surface recognize and bind to the CD20 antigen, whereby signals are transmitted into the cells and activated. The cell of the present invention is produced by introducing the nucleic acid of the present invention into a target cell. Usually, target cells are transformed with a recombinant expression vector containing the nucleic acid of the invention. T cells, NK cells, or progenitor cells thereof (hematopoietic stem cells, lymphocyte progenitor cells, etc.) can be used as target cells. As T cells here, CD4 positive CD8 negative T cells, CD4 negative CD8 positive T cells, CD4 negative CD8 negative T cells, CD4 positive CD8 positive T cells, T cells prepared from iPS cells, αβ-T cells, γδ -T cells can be mentioned. Various cell populations can be used as long as they contain T cells or progenitor cells as described above. PBMC (peripheral blood mononuclear cells) collected from peripheral blood is one of the preferred target cells. A cell population obtained by passage and / or purification of a cell population collected from a living body can also be used as a target cell.

  Transformation of the target cell may be performed by a conventional method. For example, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al., Proc. Natl. Acad. Sci. USA 84,7413-7417 (1987)), infection (when using a viral vector), etc., can be used for transformation. As is clear from the operation, the cells of the present invention are prepared ex vivo.

  The cells of the present invention include B cell lymphoma (follicular malignant lymphoma, diffuse malignant lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B cell lymphoma, CD20 positive Hodgkin lymphoma, etc.), treatment-resistant chronic lymphocytic leukemia, treatment For the treatment, prevention, or improvement of patients with resistant idiopathic thrombocytopenic purpura, treatment-resistant systemic lupus erythematosus, hemophilia with coagulation inhibitors, or aplastic anemia patients whose anti-HLA antibodies have failed to transfuse. Can be used. “Treatment” includes alleviation of symptoms or concomitant symptoms characteristic of the target disease (mildness), prevention or delay of deterioration of symptoms, and the like. “Prevention” refers to preventing or delaying the onset / onset of a disease (disorder) or its symptoms, or reducing the risk of onset / onset. On the other hand, “improvement” means that a disease (disorder) or a symptom thereof is alleviated (lightened), improved, ameliorated, or cured (including partial healing).

  The cells of the present invention are particularly useful for cases in which the expression of CD20 is reduced. Specific examples of such cases include those that are resistant to treatment with CD20 mAb (such as rituximab, ofatumumab) (Alvaro-Naranjo T, Jaen-Martinez J, Guma-Padro J, Bosch-Princep R, Salvado-Usach MT. CD20- negative DLBCL transformation after rituximab treatment in follicular lymphoma: a new case report and review of the literature.Ann Hematol. Sep 2003; 82 (9): 585-588 .; Yasuhito Terui TS, Yuji Mishima, Yuko Mishima, Natsuhiko Sugimura, Kiyotsugu Kojima, Masahiro Yokoyama, Kiyohiko Hatake. Identification of CD20 Mutations in Malignant Lymphoma: Can They Be Predictors of Response to Rituximab? Paper presented at: 47th American Society Of Hematology Annual Meeting; December 12, 2005, 2005; Atlanta .; Davis TA, Czerwinski DK, Levy R. Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression.Clin Cancer Res. Mar 1999; 5 (3): 611-615 .; Kinoshita T, Nagai H, Murate T, Saito H. CD20-negative relapse in B-cell lymphoma after t reatment with Rituximab. J Clin Oncol. Dec 1998; 16 (12): 3916.), CD20 negatively converted B-cell malignant lymphoma, patients with treatment-resistant CD20 low expression chronic lymphocytic leukemia, and the like.

  Here, it is known that CD20 is expressed not only in tumor cells but also in normal plasma cells, and CD20 mAb is applied to treatment targeting antibody-producing CD20 positive B cells even in non-neoplastic diseases . As an alternative therapy for this, the treatment strategy according to the present invention is expected to be effective. Specifically, autoimmune diseases such as treatment-resistant systemic lupus erythematosus, treatment-resistant idiopathic thrombocytopenic purpura, and aplastic anemia in which anti-HLA antibodies have been acquired by frequent blood transfusions, resulting in ineffective transfusion. The use of CAR-T cell therapy using the present invention is envisaged for diseases and medical conditions such as hemophilia that have acquired inhibitors by coagulation factor supplementation.

The cells of the present invention can also be provided in the form of cell preparations. The cell preparation of the present invention contains a therapeutically effective amount of the cells of the present invention. For example, for single administration, 10 ⁴ to 10 ¹⁰ cells are contained. Various components (vitamins) for the purpose of cell activation, proliferation or differentiation induction, such as dimethyl sulfoxide (DMSO) and serum albumin for the purpose of cell protection, such as antibiotics for the purpose of blocking bacterial contamination , Cytokines, growth factors, steroids, etc.) may be included in the cell preparation.

  The administration route of the cell or cell preparation of the present invention is not particularly limited. For example, administration is by intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Local administration may be used instead of systemic administration. Examples of the local administration include direct injection into a target tissue / organ / organ. The administration schedule may be prepared in consideration of the subject's (patient) sex, age, weight, disease state, and the like. In addition to single administration, multiple administration may be performed continuously or periodically.

1. New CD20 CAR design and examination of effects (1) CD20 CAR design (Figure 1)
The light chain variable region and the heavy chain variable region of the humanized CD20 antibody were linked by a linker (SEQ ID NO: 11) to form a single chain variable region (scFv). Human IgG4-derived Fc (hinge, CH2, CH3) (SEQ ID NO: 12), CD28 transmembrane region (SEQ ID NO: 13), CD28 intracellular domain (SEQ ID NO: 14), glycine linker (GGG), CD3ζ (SEQ ID NO: 15) A CAR to which a T2A sequence (SEQ ID NO: 16) and tEGF (truncated EGF) (SEQ ID NO: 17) were bound was designed. For comparison, five types of CARs with different scFv (20vv-a, 20fv-a, 20fa-a, 20sv-a, 20sa-a) were prepared. The VL and VH constituting the scFv of each CAR are as follows. In addition, DNA encoding each CAR (with a GM-CSF leader sequence added to the 5 'end of the sequence encoding VL and a HindIII site added to the 5' end and a NotI site added to the 3 'end) is 20vv. -a is the sequence of SEQ ID NO: 18, 20fv-a is the sequence of SEQ ID NO: 19, 20fa-a is the sequence of SEQ ID NO: 20, 20sv-a is the sequence of SEQ ID NO: 21, and 20sa-a is the sequence of SEQ ID NO: 22. .
20vv-a: VL (SEQ ID NO: 1): VH (SEQ ID NO: 2)
20fv-a: VL (SEQ ID NO: 3): VH (SEQ ID NO: 4)
20fa-a: VL (SEQ ID NO: 5): VH (SEQ ID NO: 6)
20sv-a: VL (SEQ ID NO: 7): VH (SEQ ID NO: 8)
20sa-a: VL (SEQ ID NO: 9): VH (SEQ ID NO: 10)

  For each CAR, the DNA encoding it was incorporated into a retrovirus packaging vector (LZRS-pBMN-Z) using HindIII / NotI sites created at both ends (LZRS-CD20 CAR). The LZRS-CD20 CAR vector was introduced into Phoenix-Ampho cells, which are retrovirus packaging cells, and the culture supernatant was collected to obtain a retrovirus.

(2) CD20 CAR gene introduction On the 0th day, peripheral blood was collected from a donor, and mononuclear cells (PBMC) were separated by specific gravity centrifugation. PBMC were purified to CD3 positive / CD8 positive using CD3 or CD8 immunomagnetic beads. Stimulation was then performed using CD3 / 28 beads (beads: T cells = 1: 3).

  On the third day, seed the stimulated cells in a 24-well cell culture dish coated with retronectin, and then add the retrovirus that had been cryopreserved to a MOI (multiplicity of infection) of about 3 and spinfer Spinfection (32 ° C., 2100 rpm, 45 minutes). When the gene transfer efficiency was measured from the 7th day to the 9th day, gene expression was observed in about 50 to 80% of cells (FIG. 2 left). When the cells were stimulated with CD20 gene-introduced K562 cells, CD20-specific production of interferon γ was observed (right side of FIG. 2).

(3) Expression of CD20 on the cell surface in various cell lines Dr. ACM Martens et al. Provided cell lines in which CD20 was expressed at various intensities in CEM cells, which were originally T cell lines that did not express CD20. . CD20 expression on the cell surface of CD20-CEM and various B cell tumor cell lines was quantified (ABC: antibody binding capacity DAKO QIFI kit).
. In normal tumor cell lines, more than 100,000 molecules are often expressed, but RRBL1 (Hiraga J, Tomita A, Sugimoto T, Shimada K, Ito M, Nakamura S, Kiyoi H , Kinoshita T, Naoe T. Blood. 2009 May 14; 113 (20): 4885-93; WO 2008/038587 pamphlet) and about 240 to 500,000 molecules in CEM-CD20 cells. The range is covered (FIG. 3).

(4) Complement-dependent cytotoxicity (CDC) by rituximab and cytotoxic activity by CD20 CAR-T cells against CD20-CEM
The CDC with rituximab for CD20-CEM is shown on the left of FIG. CDC activity is significantly reduced in cells with low CD20 expression. The cytotoxic activity of CD20 CAR-T cells against CD20-CEM is shown on the right side of FIG. The CD20 low expression strain of about MFI 1000 to 10,000 showed cytotoxic activity almost the same as that of the high expression strain. This result means that CAR-T therapy exhibits significant cytotoxic activity even if the target antigen CD20 is low expressed compared to antibody therapy.

(5) IFN-γ production ability CD20 CAR-T cells were stimulated using CD20-CEM (Fig. 5) having typical CD20 expression, and the IFN-γ production ratio was examined. Strain with very low CD20 expression (CEM-extremely low) did not show significant IFN-γ production, but stimulation with other CD20-CEM strains produced comparable IFN-γ production (FIG. 6).

(7) Evaluation of cell division CD20 CAR-T cells were stimulated using CD20-CEM (Fig. 5) having typical CD20 expression, and cell division after stimulation was evaluated. No significant division / proliferation was observed in strains with very low CD20 expression (CEM-extremely low), but similar division / proliferation was observed with other CD20-CEM strains (FIG. 7). .

(8) Examination of intracellular signaling CD20 CAR-T cells were stimulated using the previous 4 cell lines, and intracellular signaling was examined. No significant intracellular signaling was observed in strains with very low CD20 expression (CEM-extremely low), but stimulation with other strains of CD20-CEM showed similar levels of intracellular signaling. (FIG. 8). When these results and the above results (IFN-γ production ability, evaluation of cell division) are combined, in order to give significant stimulation to CD20 CAR-T cells, several thousand molecules or more of CD20 are required per cell. It was thought that there was.

(9) Effects of CD20 CAR-T cells on CD20 low-expressing cell lines and clinically isolated cells
RRBL1 is a cell line established from relapsed cells in patients who have been treated with rituximab for recurrent follicular lymphoma and reported that CD20 expression is reduced (Hiraga J, Tomita A, Sugimoto T, Shimada K, Ito M, Nakamura S, Kiyoi H, Kinoshita T, Naoe T. Blood. 2009 May 14; 113 (20): 4885-93; International Publication No. 2008/038587 Pamphlet). CD20 CAR-T cells also showed significant cytotoxic activity against RRBL1 (FIG. 9 upper). On the other hand, as a result of examining the effect on cells derived from patients with malignant lymphoma that recurred in pleural effusion (Pt-1), the CD20 positive fraction at the time of recurrence was about 13%, but the cytotoxic activity exceeded that ( FIG. 9 bottom). From the above results, it was found that CD20 CAR-T cells have a significant cytotoxic activity even after rituximab administration even when CD20 expression is decreased.

(10) Summary
It was revealed that CD20 CAR-T cells can also recognize CD20 low-expressing target cells. The threshold of recognition was fairly low, with hundreds of molecules per cell in the cytotoxic activity test. On the other hand, in order to induce division or the like in stimulated T cells, it was necessary to express several thousand molecules of CD20 per cell. In comparison with rituximab, it was clearly shown to be highly effective even for low-expressing strains. This fact suggests that CD20 CAR-T cell therapy is effective even for target cells with decreased CD20 expression, and has clinical significance.

2. 2. Examination of the relationship between the structure and effects of CD20 CAR 2-1. Affinity for antigen (1) Relationship between affinity of extracellular domain (scFv) and IFN-γ production Light chain variable region (VL) of five types of humanized anti-CD20 antibodies (see JP 2010-189402 A) having different affinity ) And a scFv designed based on the heavy chain variable region (VH) (20vv-a, 20fv-a, 20fa-a, 20sv-a, 20sa-a). The dissociation constant (Kd value (nM)) for CD20 is vv 7.07, fv 7.21, fa is 10.09, sv is 12.58, sa is 14.66, and the affinity is vv>fv>fa>sv> sa (special (See Table 5 of Kai 2010-189402).

  After each CAR was transfected into CD8-positive T cells, the introduced cells were purified with a gene transfer marker (EGFR) (FIG. 10 left), and the ratio of IFN-γ producing cells was evaluated. CAR-T cells with affinity greater than fa specifically recognized CD20 and produced IFN-γ, whereas CAR-T cells with affinity less than sv did not produce IFN-γ. In addition, among the antibody groups used, it was shown that the higher the affinity, the higher the ratio of IFN-γ producing cells (FIG. 10 right).

(2) Relationship between extracellular domain (scFv) affinity and cytotoxic activity After introducing the above five types of CAR into CD8-positive T cells, the introduced cells are purified, and 1-course stimulation / proliferation with γ-irradiated LCL after the CD20 transgenic K562 was evaluated by a ⁵¹ Cr release assay the cytotoxic activity as targets ⁽⁵¹ Cr releasing assay). CAR-T cells having an affinity of fa or higher showed good cytotoxic activity, but CAR-T cells having an affinity of sv or lower showed almost no cytotoxic activity (FIG. 11). In addition, among the antibody groups used, it was shown that fv having the second highest affinity shows the highest cytotoxic activity.

(3) Relationship between extracellular domain (scFv) affinity and CAR-T cell proliferation after stimulation with irradiated LCL After introducing the above five types of CAR into CD8-positive T cells, the introduced cells were purified. On day 0, CAR-T cells (0.5 × 10 ⁶ cells) were stimulated with γ-irradiated LCL (3.5 × 10 ⁶ cells) and cultured in the presence of IL-2 (50 IU / ml). Up to 20fv-a, the second highest affinity, growth efficiency correlated with affinity, but 20vv-a, which had the highest affinity, was rather less efficient and similar to 20fa-a (Figure 12).

(4) Summary (extracellular domain (scFv) affinity optimal for designing CD20-CAR)
The above results show that the proportion of IFN-γ-producing cells increases as the affinity increases (FIG. 10), but the cytotoxic activity and the cell proliferation efficiency by LCL stimulation are rather decreased at 20vv-a, which has the highest affinity. (FIGS. 11 and 12). Affinities that are too high will reduce activity, possibly due to activation-induced cell death (AICD), etc. due to overstimulation. In CAR-T cell therapy, CAR-T cells meet target antigen-positive cells and cause cytotoxic activity, and when they proliferate, antitumor effects are exerted and enhanced. Also, in clinical practice, it is assumed that there are cases where administration is carried out after stimulated proliferation such as irradiated LCL outside the body at the stage of cell preparation, and 20vv-a, 20fv-a and 20fa-a are excellent in cytotoxic activity and proliferation efficiency. (Dissociation constant (Kd value) range of about 7 to about 10 nM) is effective, especially 20 fv-a (dissociation constant (Kd value) of about 7.2 nM), which showed the most excellent characteristics, is optimal. I can say that.

2-2. Relationship between the length of the linker and the proportion of IFN-γ producing cells The VL and VH constituting the Fv region of the humanized anti-CD20 antibody are linked by a long linker (18AA; SEQ ID NO: 11) and a short linker (7AA: SEQ ID NO: 23). Two types of CAR were designed. Genes were introduced into CD3-positive T cells (FIG. 13 left), and the proportion of IFN-γ producing cells was evaluated. Both recognized CD20 specifically and produced IFN-γ, but CAR-T cells with long linkers had a higher proportion of IFN-γ producing cells than CAR-T cells with short linkers (right in FIG. 13). .

2-3. Cytotoxic activity on clinically isolated cells with reduced CD20 expression
After gene transfer of CD20 CAR (20fa-a) into CD8 positive T cells, the introduced cells are purified, and after 1-course stimulation growth with γ-irradiated LCL, the clinically isolated cells are targeted for ⁵¹ Cr release assay And evaluated. For patients with chronic lymphocytic leukemia (CLL) who had decreased CD20 expression after repeated chemotherapy and antibody therapy and became refractory to rituximab, the remaining CLL cells after treatment with ofatumumab were isolated from the patient's peripheral blood, Used as a specimen (target). The CD20 expression of CLL cells in the specimen was extremely reduced (FIG. 14 left). CD20 CAR-T cells also showed cytotoxic activity against such antibody therapy-refractory CD20 very low expressing cells (FIG. 14 right). Thus, it was shown that the newly established CD20 CAR-T cells are also effective for refractory cases of rituximab and ofatumumab antibody therapy targeting a common antigen (CD20).

  The present invention provides a new therapeutic strategy targeting CD20. The CD20 CAR-T cell therapy using the present invention is also effective against CD20 low-expressing target cells. In CD19CAR-T cell therapy, normal plasma cells were not targeted because they did not express CD19, but CD20 was also expressed in plasma cells. Can suggest treatment. CD20 CAR-T cell therapy using the present invention is expected to show a higher effect than CD20 antibody therapy in autoimmune diseases and pathologies where autoantibodies are problematic. Specifically, autoimmune diseases such as treatment-resistant systemic lupus erythematosus and treatment-resistant idiopathic thrombocytopenic purpura, and poor regenerative disease where anti-HLA antibodies were acquired by frequent blood transfusions, resulting in ineffective transfusion. It is expected that antibody-producing cells will be removed to exert therapeutic effects against diseases and conditions such as anemia and hemophilia that have acquired inhibitors by supplementation of coagulation factors.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (18)

  A humanized anti-CD20 chimeric antigen receptor comprising an extracellular domain comprising an scFv fragment of an anti-CD20 humanized monoclonal antibody, a transmembrane domain, and an intracellular signal domain for effector functions of immune cells.   The humanized anti-CD20 chimeric antigen receptor according to claim 1, wherein the anti-CD20 humanized monoclonal antibody has a dissociation constant (Kd value) for human CD20 antigen of 7 to 10 nM.   The humanized anti-CD20 chimeric antigen receptor according to claim 1 or 2, wherein the intracellular signal domain comprises CD3ζ and an intracellular domain of a costimulatory molecule.   4. The humanized anti-CD20 chimeric antigen receptor of claim 3, wherein the costimulatory molecule is CD28 or 4-1BB.   The humanized anti-CD20 chimeric antigen receptor according to any one of claims 1 to 4, wherein the transmembrane domain is a transmembrane domain of CD28.   The humanized anti-CD20 chimeric antigen receptor according to any one of claims 1 to 5, comprising a spacer domain between the extracellular domain and the transmembrane domain.   The humanized anti-CD20 chimeric antigen receptor according to claim 6, wherein the spacer domain consists of an Fc fragment of human IgG.   8. The humanized anti-CD20 chimeric antigen receptor of claim 7, wherein the human IgG is IgG4.   The humanized anti-CD20 chimeric antigen receptor according to any one of claims 1 to 8, wherein the linker constituting the scFv fragment is a peptide linker having 8 to 25 amino acid residues.   10. The humanized anti-CD20 chimeric antigen receptor of any one of claims 1-9, wherein the anti-CD20 monoclonal antibody has a dissociation constant for human CD20 antigen of about 7.2. The amino acid sequence of a light chain variable region (VL) and a heavy chain variable region (VH) constituting the scFv fragment is any one of the following combinations (1) to (3): The humanized anti-CD20 chimeric antigen receptor described in:
(1) VL is the amino acid sequence of SEQ ID NO: 1, and VH is the amino sequence of SEQ ID NO: 2;
(2) VL is the amino acid sequence of SEQ ID NO: 3, and VH is the amino sequence of SEQ ID NO: 4;
(3) VL is the amino acid sequence of SEQ ID NO: 5, and VH is the amino sequence of SEQ ID NO: 6.   DNA encoding the humanized anti-CD20 chimeric antigen receptor according to any one of claims 1-11.   A recombinant expression vector that retains the DNA of claim 12 in an expressible manner.   A genetically engineered CD20-specific immune cell that expresses the humanized anti-CD20 chimeric antigen receptor according to any one of claims 1 to 11 on the cell surface.   The CD20-specific immune cell according to claim 14, wherein the immune cell is a T cell.   A cell preparation comprising a therapeutically effective amount of the CD20-specific T cell according to claim 14 or 15.   A disease selected from the group consisting of B-cell malignant lymphoma, treatment-resistant chronic lymphocytic leukemia, treatment-resistant idiopathic thrombocytopenic purpura, treatment-resistant systemic lupus erythematosus, and hemophilia with coagulation inhibitors, or The cell preparation according to claim 16, which is used for treatment, prevention, or improvement of a patient whose blood transfusion effect cannot be obtained by an anti-HLA antibody.   The cell preparation according to claim 17, which is applied to a case resistant to an anti-CD20 humanized chimeric monoclonal antibody.

Images