Antitumor combination therapy comprising an anti-CD19 antibody and gamma delta T cells

Abstract

The present disclosure relates to combination therapies comprising an anti-CD19 antibody or antibody fragment thereof and gamma delta T cells (γδ T cells) for use in the treatment of leukemia or lymphoma.

Description

Technical Field

[0001] The present disclosure relates to a combination therapy comprising an anti-CD19 antibody or an antibody fragment thereof and gamma delta T cells (γδT cells) for use in the treatment of leukemia or lymphoma.

Background Art

[0002] CD19 is a 95 kDa transmembrane glycoprotein of the immunoglobulin superfamily that contains two extracellular immunoglobulin-like domains and an extensive cytoplasmic tail. This protein is a ubiquitous B-lymphocyte surface receptor that is expressed ubiquitously until it is downregulated during the final differentiation from pre-B cells to plasma cells from the early stages after the development of pre-B cells. It is specific to the B-lymphocyte lineage and is not expressed in hematopoietic stem cells and other immune cells, except for some follicular dendritic cells. CD19 functions as a positive regulator of B-cell receptor (BCR) signaling and is important for B-cell activation and proliferation, as well as for the generation of the humoral immune response. It functions as a co-stimulatory molecule in cooperation with CD21 and CD81 and is important for the B-cell response to T-cell-dependent antigens. The cytoplasmic tail of CD19 is physically associated with a family of tyrosine kinases that induce downstream signaling pathways via the src family of protein tyrosine kinases. Since CD19 is highly expressed in almost all chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL), as well as many other different types of leukemia, including acute lymphocytic leukemia (ALL) and hairy cell leukemia (HCL), it is an attractive target for cancers of lymphoid origin.

[0003] Tafasitamab (former names: MOR00208 and XmAb® 5574) is a humanized monoclonal antibody that targets the antigen CD19, a transmembrane protein involved in B cell receptor signaling. Tafasitamab is engineered in the IgG Fc region to enhance antibody-dependent cell cytotoxicity (ADCC), thereby improving an important mechanism for killing tumor cells and potentially leading to improved efficacy compared to conventional antibodies, i.e., non-enhanced antibodies. Tafasitamab has been studied or is currently being studied in several clinical trials, such as those for CLL, ALL, and NHL. In some of these trials, tafasitamab is used in combination with idelalisib, lenalidomide, or venetoclax.

[0004] Despite the discovery and development of several anticancer agents in recent years, there is still a need for improved methods or therapeutic agents for treating many types of cancers, including CD19-expressing tumors, due to the poor prognosis of such cancers. Accordingly, the inventors have confirmed that the combined administration of γδ T cells and an antibody or antibody fragment specific for CD19 has excellent effects on the treatment of B cell-derived malignant lymphoma, and have thus completed the present invention.

Summary of the Invention

[0005] The present disclosure provides a novel combination for use in the treatment of cancer, comprising an antibody or antibody fragment specific for CD19 and γδ T cells.

[0006] Since their discovery in the 1980s, γδ T cells have been recognized as playing important roles in malignancies such as infectious diseases and cancer. Activated γδ T cells possess potent cytotoxicity and broad tumor recognition capabilities independent of major histocompatibility complex (MHC) molecules present on target cells. Furthermore, γδ T cells have been shown to be potent mediators of antibody-dependent cell-mediated cytotoxicity (ADCC). At present, it has been shown that the antitumor effect of γδ T cells can be substantially enhanced by anti-CD20 antibodies (Tokuyama et al. 2008; Hoeres et al. 2018). Additionally, the Fc-enhanced anti-CD20 antibody obinutuzumab shows increased tumor cell death when combined with γδ T cells compared to non-Fc-enhanced antibodies such as rituximab.

[0007] However, the tumor cell killing activity of antibodies specific for surface antigens other than CD20 in the presence of γδ T cells has not yet been evaluated. Accordingly, the object of the present disclosure is to provide an alternative combination therapy comprising an antibody and γδ T cells.

[0008] To achieve the above object, the present disclosure provides a combination for use in the treatment of cancer, comprising an antibody or antibody fragment specific for CD19 and γδ T cells.

[0009] In the present disclosure, the inventors combined γδ T cells with the CD19-targeting antibodies tafasitamab (Fc-enhanced) and Xmab5603 (non-Fc-enhanced) and evaluated their antitumor activities in ADCC assays using patient-derived CLL, MCL, and B-ALL samples, as well as various lymphoma and leukemia cell lines. Overall, when γδ T cells were combined with the Fc-enhanced anti-CD19 antibody tafasitamab, an increase in the rate of cell lysis was observed compared to non-Fc-enhanced Xmab5603 or the negative control IgG1 antibody.

[0010] In summary, γδ T cells have been shown to be a potential effector cell population in antibody-based tumor therapy as demonstrated for the Fc-enhanced CD19-targeting antibody tafasitamab in this study. Tafasitamab exhibits potent antitumor activity mediated by γδ T cells against several lymphoma and leukemia cell lines, as well as CLL, MCL, and BALL cells derived from primary patients, and may hold promise as an approach for lymphoma and leukemia treatment.

[0011] The γδ T cells can be derived from any suitable autologous or allogeneic γδ T cells or populations thereof. In some embodiments, suitable γδ T cells for use as a source of the currently described γδ T cells include Vδ1 cells, Vδ2 cells, Vδ3 cells, Vδ5 cells, and Vδ8 cells. For example, provided herein is a method for isolating and expanding Vδ1 cells from non-hematopoietic tissues such as skin or intestine. For example, Vδ1 cells may be isolated from a human skin biopsy as described in US2018 / 0312808, which is hereby incorporated by reference in its entirety, particularly for the method of isolating Vδ1 cells from tissues.

[0012] In other embodiments, suitable γδ T cells can be derived from blood (e.g., peripheral blood). Methods for isolating and expanding Vδ1 cells from blood include, for example, the methods described in U.S. Patent No. 9,499,788 and International Patent Publication No. WO2016 / 198480, each of which is hereby incorporated by reference in its entirety. Also, Vγ9Vδ2 T cells can be isolated from peripheral blood and further cultured ex vivo. The culture of Vγ9Vδ2 T cells can be optimized in the presence of IL-2 and zoledronic acid (ZOL). Methods for isolating and expanding Vγ9Vδ2 T cells from blood include, for example, the method described in Hoeres et al. 2018.

[0013] In some embodiments, suitable γδ T cells can be derived from tumor tissue (e.g., tumor-infiltrating γδ T cells). Alternatively, suitable γδ T cells that can be engineered to express a heterologous target construct can be derived from non-hematopoietic tissue according to the methods described below. These cells can be cultured in the presence of one or more factors (e.g., TCR agonists, co-receptor agonists, and / or cytokines, such as IL-4, IL-15, and / or IFN-γ) in a gas-permeable bioreactor bag for up to 21 days or more. Modifications of this method, and other methods of obtaining Vδ1 T cells, are suitable as part of the present invention. For example, blood-derived Vδ1 T cells can alternatively be obtained using, for example, the methods described in International Patent Publications WO2017 / 197347 and WO2016 / 081518 (U.S. Patent Application Publication No. 2016 / 0175338), which are incorporated herein by reference in their entireties.

[0014] The present disclosure provides a pharmaceutical combination for use in the treatment of cancer, comprising an antibody or antibody fragment specific for CD19 and gamma delta T cells (γδ T cells).

[0015] In one aspect, the present disclosure provides a pharmaceutical combination for use in the treatment of cancer, comprising an antibody or antibody fragment specific for CD19 and gamma delta T cells (γδ T cells), wherein the antibody comprises a heavy chain variable region comprising an HCDR1 region comprising the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region comprising the sequence NPYNDG (SEQ ID NO: 2), and an HCDR3 region comprising the sequence GTYYYGTRVFDY (SEQ ID NO: 3), and a light chain variable region comprising an LCDR1 region comprising the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region comprising the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region comprising the sequence MQHLEYPIT (SEQ ID NO: 6) for use in the treatment of cancer.

[0016] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody or antibody fragment specific for CD19 for use in the treatment of cancer and gamma delta T cells (γδT cells), wherein the antibody comprises a heavy chain variable region comprising the HCDR1 region of SYVMH (SEQ ID NO: 1), the HCDR2 region of NPYNDG (SEQ ID NO: 2), and the HCDR3 region of GTYYYGTRVFDY (SEQ ID NO: 3) for use in the treatment of cancer, and a light chain variable region comprising the LCDR1 region of RSSKSLQNVNGNTYLY (SEQ ID NO: 4), the LCDR2 region of RMSNLNS (SEQ ID NO: 5), and the LCDR3 region of MQHLEYPIT (SEQ ID NO: 6).

[0017] In another aspect, the antibody or antibody fragment specific for CD19 comprises the following heavy chain variable region EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS (SEQ ID NO: 7) and the following light chain variable region DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 8).

[0018] In another aspect, the antibody or antibody fragment specific for CD19 has effector function. In another aspect, the antibody or antibody fragment specific for CD19 has enhanced effector function. In one embodiment, the effector function is ADCC. In one embodiment, the antibody or antibody fragment specific for CD19 has enhanced ADCC activity. In a further embodiment, the antibody or antibody fragment specific for CD19 comprises an Fc domain comprising an amino acid substitution at position S239 and / or I332, numbering according to the EU index as in the case of Kabat.

[0019] In yet another aspect, the antibody or antibody fragment specific for CD19 has the following heavy chain constant region contains ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9).

[0020] In a further aspect, the antibody specific for CD19 has the following light chain constant region contains RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

[0021] In yet another aspect, the antibody specific for CD19 has the following heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:9) and the following light chain constant region including RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:10).

[0022] In yet another aspect, the antibody specific for CD19 has the following heavy chain constant region EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) and the following light chain constant region comprising DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[0023] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 for use in the treatment of cancer and gamma delta T cells (γδT cells), wherein the γδT cells comprise an enriched γδT cell population. In one embodiment, the enriched γδT cell population comprises unmanipulated or manipulated γδT cells and / or mixtures thereof. In a further embodiment, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and an enriched γδT cell population for use in the treatment of cancer.

[0024] In another aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma-delta T cells (γδT cells) for use in the treatment of cancer, wherein the γδT cells are either unmanipulated or comprise manipulated γδT cells and / or mixtures thereof. In another embodiment, the γδT cells are a population of unmanipulated γδT cells. In another embodiment, the γδT cells are a population of manipulated γδT cells.

[0025] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma-delta T cells (γδT cells) for use in the treatment of cancer, wherein the γδT cells are isolated from peripheral blood, tumor tissue or non-hematopoietic tissue. In one embodiment, the γδT cells are isolated from peripheral blood. In another embodiment, the γδT cells are a population of γδT cells isolated from peripheral blood. In another embodiment, the γδT cells are a population of Vγ9Vδ2 T cells isolated from peripheral blood. In a further embodiment, the γδT cells are a population of Vγ9Vδ2 T cells isolated from peripheral blood, and the Vγ9Vδ2 T cells are cultured ex vivo in the presence of IL-1 and zoledronic acid (ZOL).

[0026] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma-delta T cells (γδT cells) for use in the treatment of cancer, which is a blood cancer. In one embodiment, the blood cancer is chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), small lymphocytic lymphoma (SLL), or acute lymphoblastic leukemia (ALL). In another embodiment, the blood cancer is non-Hodgkin lymphoma (NHL). In a further embodiment, the non-Hodgkin lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, and mantle cell lymphoma.

[0027] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma-delta T cells (γδT cells) for use in the treatment of cancer, wherein the antibody specific for CD19 and the γδT cells are administered in separate manners.

[0028] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma-delta T cells (γδT cells) for use in the treatment of cancer, wherein the antibody specific for CD19 and the γδT cells are administered in a simultaneous manner.

[0029] In one aspect, the present disclosure provides a kit comprising an antibody specific for CD19 and gamma-delta T cells (γδT cells) for use in the treatment of cancer.

Brief Description of the Drawings

[0030]

Figure 1

Figure 2

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Mode for Carrying Out the Invention

[0031] Definitions The term "CD19" refers to a protein known as CD19 having the following synonyms: B4, B lymphocyte antigen CD19, B lymphocyte surface antigen B4, CVID3, differentiation antigen CD19, MGC12802, and T cell surface antigen Leu-12.

[0032] The amino acid sequence of human CD19 is as follows: MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR (SEQ ID NO: 13).

[0033] "MOR00208", "XmAb5574", and "tafasitamab" are used as synonyms for anti-CD19 antibodies according to Table 1. Table 1 shows the amino acid sequences of MOR00208 / tafasitamab. The MOR00208 antibody is described in U.S. Patent Application Serial No. 12 / 377,251, which is incorporated herein by reference in its entirety. U.S. Patent Application Serial No. 12 / 377,251 describes an antibody designated 4G7H1.52 hybrid S239D / I332E / 4G7L1.155 (later named MOR00208 and tafasitamab).

[0034] As used herein, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds that interact with an antigen. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) that are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. The antibody can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0035] As used herein, the term "antibody fragment" refers to one or more portions of an antibody that retain the ability to specifically interact (e.g., by binding, steric hindrance, stabilization of spatial distribution) with an antigen. Examples of binding fragments include, but are not limited to, Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragment, i.e., a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; Fd fragment consisting of the VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single arm of an antibody; dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and isolated complementarity determining regions (CDRs). Further, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be joined by a synthetic linker using recombinant methods that enable them to be made as a single protein chain, and the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv)); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antibody fragment". These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments may also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136).The antibody fragment may be grafted onto a scaffold based on a polypeptide such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies). The antibody fragment may be incorporated into a single-chain molecule containing a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0036] "Administered" or "administration" includes, but is not limited to, delivery of a drug by an injectable form, e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes, or by a mucosal route, e.g., as a nasal spray or aerosol for inhalation, or as an ingestible solution, capsule, or tablet. Preferably, administration is by an injectable form.

[0037] The term "effector function" refers to biological activities that can be attributed to the Fc region of an antibody and that vary with antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0038] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which an antibody bound to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to an antigen-bearing target cell and subsequently kill the target cell using cytotoxins. While NK cells, which are primary cells mediating ADCC, express only FcγRIII, monocytes express FcγRI, FcγRII, and FcγRIII.

[0039] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the conventional complement pathway begins with the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) of the present disclosure, which are bound to their cognate antigens.

[0040] "Antibody-dependent cell phagocytosis" or "ADCP" refers to the mechanism of elimination of antibody-coated target cells by internalization by phagocytic cells such as macrophages or dendritic cells.

[0041] The term "blood cancer" includes blood-borne tumors and diseases or disorders involving abnormal cell proliferation and / or growth in tissues of hematopoietic origin, such as lymphoma, leukemia, and myeloma.

[0042] Non-Hodgkin lymphoma (「NHL」) is a heterogeneous malignant tumor originating from lymphocytes. In the United States (US), the incidence is estimated at 65,000 / year, and the mortality rate is approximately 20,000 (American Cancer Society, 2006; and SEER Cancer Statistics Review). This disease can occur at all ages, with the usual onset starting in adults over 40 years old, and the incidence increasing with age. NHL is characterized by the clonal proliferation of lymphocytes that accumulate in lymph nodes, blood, bone marrow, and the spleen, but any major organ may be involved. The current classification system used by pathologists and clinicians is the World Health Organization (WHO) tumor classification, which classifies NHL into precursor and mature B-cell or T-cell neoplasms. PDQ currently divides NHL into those eligible for participation in clinical trials as indolent (low-grade) or aggressive (aggressive). The indolent NHL group mainly consists of follicular subtypes, small lymphocytic lymphoma, MALT (mucosa-associated lymphoid tissue), and marginal zone. Indolent accounts for approximately 50% of newly diagnosed B-cell NHL patients. Aggressive NHL mainly includes patients who have received a histological diagnosis of diffuse large cell type B-cell (DLBL, 「DLBCL」, or DLCL) (40% of all newly diagnosed patients are diffuse large cell type), Burkitt lymphoma, and mantle cell (「MCL」). The clinical course of NHL is very diverse. The main determinant of the clinical course is the histological subtype. Most indolent types of NHL are considered incurable diseases. Patients initially respond to either chemotherapy or antibody therapy, and most relapse. Previous studies have not demonstrated an improvement in survival rate with early intervention. In asymptomatic patients, 「watchful waiting」 is allowed until the patient becomes symptomatic or the disease appears to be accelerating in pace. Over time, this disease can change to a more aggressive (aggressive) histology. The median survival period is 8 to 10 years, and indolent (low-grade) patients often receive more than three treatments during the treatment stage of the disease. The initial treatment for symptomatic indolent NHL patients has historically been combination chemotherapy.The most commonly used drugs include cyclophosphamide, vincristine, prednisone (CVP); or cyclophosphamide, adriamycin, vincristine, prednisone (CHOP). Approximately 70% to 80% of patients respond to the initial chemotherapy, and the remission period lasts about 2 to 3 years. Eventually, the majority of patients relapse. The discovery and clinical use of rituximab, an anti-CD20 antibody, have significantly improved response and survival rates. The current standard treatment for most patients is rituximab + CHOP (R-CHOP) or rituximab + CVP (R-CVP). Rituximab therapy has been shown to be effective in several types of NHL and is currently approved as first-line treatment for both indolent (follicular lymphoma) and aggressive NHL (diffuse large B-cell lymphoma). However, anti-CD20 monoclonal antibodies (mAbs) have significant limitations, including primary resistance (50% response in relapsed indolent patients), acquired resistance (50% response rate during retreatment), rare complete responses (2% complete response in the relapsed population), and a continuous pattern of relapse. Finally, many B cells do not express CD20, so many B-cell disorders cannot be treated using anti-CD20 antibody therapy.

[0043] In addition to NHL, there are several types of leukemia resulting from B-cell dysregulation. Chronic lymphocytic leukemia (also known as "chronic lymphocytic leukemia" or "CLL") is a type of adult leukemia caused by the abnormal accumulation of B lymphocytes. In CLL, malignant lymphocytes may appear normal and mature, but are unable to effectively combat infections. CLL is the most common form of adult leukemia. Men are twice as likely to develop CLL as women. However, the major risk factor is age. More than 75% of new cases are diagnosed in patients over 50 years old. More than 10,000 cases are diagnosed each year, and the mortality rate is nearly 5,000 per year (American Cancer Society, 2006; and SEER Cancer Statistics Review). CLL is an incurable disease, but in most cases it progresses slowly. Many people with CLL lead normal, active lives for years. Because the onset is slow, early intervention in CLL is not thought to improve survival or quality of life, so early CLL is generally not treated. Instead, the condition is monitored over time. The initial CLL treatment varies depending on the exact diagnosis and the progression of the disease. There are dozens of drugs used in CLL therapy. Combination chemotherapy regimens such as FCR (fludarabine, cyclophosphamide, and rituximab), and BR (ibrutinib, and rituximab) are effective for both newly diagnosed and relapsed CLL. Allogeneic bone marrow (stem cell) transplantation is rarely used as a first-line treatment for CLL because of the risks involved.

[0044] Another type of leukemia is small lymphocytic lymphoma ("SLL"), which lacks the clonal lymphocytosis required for a CLL diagnosis but otherwise shares the pathological and immunophenotypic features of CLL (Campo et al., 2011). The definition of SLL requires the presence of lymph node swelling and / or splenomegaly. Furthermore, the number of B lymphocytes in the peripheral blood should not exceed 5 × 109 / L. In SLL, the diagnosis should be confirmed by histopathological evaluation of a lymph node biopsy whenever possible (Hallek et al., 2008). The incidence of SLL is approximately 25% of CLL in the United States (Dores et al., 2007).

[0045] Another type of leukemia is acute lymphoblastic leukemia (ALL), also known as acute lymphocytic leukemia. ALL is characterized by the overproduction and continuous proliferation of malignant and immature white blood cells (also known as lymphoblasts) in the bone marrow. The term "acute" refers to the undifferentiated and immature state of circulating lymphocytes ("blasts"), which, if left untreated, progresses rapidly with a mean survival of weeks to months. ALL is most common in childhood, with the peak incidence at 4 - 5 years of age. Children aged 12 - 16 years are more likely to die than other children. Currently, at least 80% of pediatric ALL is considered curable. Fewer than 4,000 cases are diagnosed each year, and the number of deaths is approximately 1,500 per year (American Cancer Society, 2006; and SEER Cancer Statistics Review).

[0046] As used in this context, "subject" or "patient" refers to any mammal, including rodents such as mice or rats, and primates such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), or humans (Homo sapiens). Preferably, the subject or patient is a primate, most preferably a human patient, and even more preferably an adult human patient.

[0047] As used herein, the terms "engineered" or "modified" include the manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or some combination of these techniques). Preferably, the antibodies or antibody fragments according to the present disclosure are engineered or modified to improve one or more properties such as antigen binding, stability, half-life, effector function, immunogenicity, safety, etc. Preferably, the antibodies or antibody fragments according to the present disclosure are engineered or modified to improve effector functions such as ADCC.

[0048] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally includes two constant domains, namely the CH2 domain and the CH3 domain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region follows the EU numbering system, also known as the EU index, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0049] Antibodies administered according to the present disclosure are administered to a patient in a therapeutically effective amount. A "therapeutically effective amount" refers to an amount sufficient to effect some improvement in the clinical symptoms of a given disease or disorder. The amount effective for a particular therapeutic purpose depends not only on the body weight and general condition of the subject, but also on the severity of the disease or injury. It will be understood that the determination of an appropriate dosage can be achieved by using routine experimentation to construct a matrix of values and testing different points within the matrix, all of which are within the ordinary skill of a trained physician or clinical scientist.

[0050] The term "combination" or "pharmaceutical combination" refers to the administration of one treatment in addition to another treatment. Thus, "combined" includes administration simultaneously (e.g., at the same time) and sequentially in any order. By way of non-limiting example, a first therapeutic agent (e.g., an agent such as an anti-CD19 antibody) may be administered before (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks before), simultaneously with, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more after) the administration of a second treatment (e.g., a pharmaceutical such as γδT cells) to a patient.

[0051] As used herein, the term "γδT cells (gamma delta T cells)" refers to a subset of T cells that express a distinct T cell receptor (TCR), γδTCR, on their surface and are composed of one γ chain and one δ chain. The term "γδT cells" specifically includes, but is not limited to, all subsets of γδT cells, including vδ1, vδ2, vδ3, and Vγ9Vδ2 T cells, as well as naive, effector memory, central memory, and terminally differentiated γδT cells. By way of further example, the term "γδT cells" includes vδ4, vδ5, vδ7, and vδ8 Τ cells.

[0052] As used herein, the term "T lymphocyte" or "T cell" refers to an immune cell that expresses CD3 (CD3+) and a T cell receptor (TCR+). T cells play a central role in cellular immunity.

[0053] As used herein, the terms "TCR" or "T cell receptor" refer to heterodimeric cell surface signaling proteins that form alpha-beta or gamma-delta receptors. Alpha-beta TCRs recognize antigens presented by MHC molecules, while gamma-delta TCRs recognize antigens independent of MHC presentation.

[0054] As used herein, the term "cell population" refers to a plurality of cells. A cell population can be, for example, a mixed cell population derived from a peripheral blood sample, umbilical cord blood sample, tumor, stem cell precursor, tumor biopsy, tissue, lymph, or an epithelial site of a subject in direct contact with the external environment, or derived from stem progenitor cells. Alternatively, the mixed cell population can be derived from a mammalian cell in vitro culture, established from a peripheral blood sample, umbilical cord blood sample, tumor, stem cell precursor, tumor biopsy, tissue, lymph, or an epithelial site of a subject in direct contact with the external environment, or derived from stem progenitor cells.

[0055] A "concentrated, enriched" cell population or preparation refers to a cell population derived from a starting mixed cell population that contains a higher percentage of a particular cell type than the percentage of that cell type in the starting population. For example, a starting mixed cell population can be concentrated for a particular gamma-delta T cell population. In all embodiments, the concentrated gamma-delta T cell population contains a lower percentage of the alpha-beta T cell population.

[0056] As used herein, "expanded" means that the number of the desired or target cell type (e.g., delta1, delta2 T cells and / or Vgamma9Vdelta2 T cells) in a concentrated preparation is greater than the number of the cell population at the initial or starting time.

[0057] Detailed Description of the Invention

[0058] Anti-CD19 antibody The use of CD19 antibodies in non-specific B cell lymphoma has been discussed in WO2007076950 (US Patent Application Publication No. 2007154473), both of which are incorporated by reference. The use of CD19 antibodies in CLL, NHL and ALL is described in Scheuermann et al., CD19 Antigen in Leukemia and Lymphoma Diagnosis and Immunotherapy, Leukemia and Lymphoma, Vol. 18, 385-397 (1995), which is incorporated by reference in its entirety.

[0059] Additional antibodies specific for CD19 are described in WO2005012493 (US Patent No. 7109304), WO2010053716 (US12 / 266,999) (Immunomedics); WO2007002223 (US US8097703) (Medarex); WO2008022152 (12 / 377,251) and WO2008150494 (Xencor), WO2008031056 (US11 / 852,106) (Medimmune); WO 2007076950 (US11 / 648,505) (Merck Patent GmbH); WO 2009 / 052431 (US12 / 253,895) (Seattle Genetics); and WO2010095031 (12 / 710,442) (Glenmark Pharmaceuticals), WO2012010562 and WO2012010561 (International Drug Development), WO2011147834 (Roche Glycart), and WO2012156455 (Sanofi), all of which are incorporated by reference in their entirety.

[0060] The pharmaceutical composition contains an active agent, for example, an antibody for therapeutic use in humans. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier or excipient.

[0061] The dosage of the antibody or antibody fragment contained in the pharmaceutical composition according to the present disclosure administered to a patient may vary depending on the patient's age and size, symptoms, condition, route of administration, etc. The dosage is usually calculated based on body weight, body surface area, age, or per individual. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. An effective dosage and schedule for administering a pharmaceutical composition containing an antibody or antibody fragment specific for CD19 can be determined empirically. For example, the progression of the patient can be monitored by regular evaluation and the dosage adjusted accordingly. Furthermore, interspecies scaling of the dosage can be carried out using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut.Res. 8:1351).

[0062] The pharmaceutical composition may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, etc. These injectable preparations can be prepared by known methods. For example, an injectable preparation can be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous medium or an oily medium conventionally used for injection. Exemplary pharmaceutical compositions containing an antibody or antibody fragment specific for CD19 that can be used in the context of the present disclosure are disclosed, for example, in WO2008 / 022152 or WO2018 / 002031.

[0063] For certain administration methods such as intravenous administration, it is preferable to administer the drug according to the patient's body weight. For other administration methods, such as subcutaneous administration, it is preferable to administer the drug at a certain fixed dosage. Those skilled in the art know which dosage is equivalent to another dosage in another administration method. In making a reasonable decision to administer the drug at the required dosage from the required effective dosage, the pharmacodynamics of the specific drug is usually considered.

[0064] The antibodies administered in accordance with the present disclosure are administered to a patient in a therapeutically effective amount. A "therapeutically effective amount" refers to an amount sufficient to cure, alleviate, or partially prevent the clinical symptoms of a given disease or disorder, namely NHL and its complications. In certain embodiments, the antibodies of the present disclosure are administered at 9 mg / kg. In alternative embodiments, the antibodies of the present disclosure are administered at 12 mg / kg. In still other embodiments, the antibodies of the present disclosure are administered at 15 mg / kg or more.

[0065] The antibodies of the present disclosure may be administered at different times, and the treatment cycles may have different lengths. The antibodies may be administered daily, every other day, three times a week, weekly, or biweekly. The antibodies may also be administered for at least 4 weeks or more, at least 5 weeks or more, at least 6 weeks or more, at least 7 weeks or more, at least 8 weeks or more, at least 9 weeks or more, at least 10 weeks or more, at least 11 weeks or more, or at least 12 weeks or more. In certain embodiments of the present disclosure, the antibodies are administered at least once a week for at least 8 weeks.

[0066] Isolation and expansion of γδ T cells from blood In some embodiments, the γδ T cells of the present disclosure are derived from the blood of a subject (e.g., peripheral blood). For example, the γδ T cells can be derived from blood-derived Vδ2 cells or blood-derived Vδ1 cells. In another example, the γδ T cells can be derived from blood-derived Vγ9Vδ2 T cells. The Vγ9Vδ2 T cells can be isolated from peripheral blood and further cultured ex vivo. The culture of Vγ9Vδ2 T cells can be optimized in the presence of IL-2 and zoledronic acid (ZOL). Methods for isolating and expanding Vγ9Vδ2 T cells from blood include, for example, the methods described in Hoeres et al. 2018, or the following procedure:

[0067] Ex vivo expansion of Vγ9Vδ2 T cells derived from peripheral blood: Peripheral blood is collected from the donor. PBMCs are immediately separated by density gradient centrifugation using Lymphoprep™ (Axis Shield, Norway) according to the manufacturer's instructions. PBMCs are resuspended in CTS™ OpTmizer™ T Cell Expansion SFM (Life Technologies, Australia) supplemented with OpTmizer™ T Cell Expansion Supplement (1:38 dilution) (Life Technologies, Australia), 10% heat-inactivated FBS (HI-FBS), 100 IU / mL penicillin, 100 μg / mL streptomycin, 2 mmol L-glutamine (Life Technologies, Australia), 25 mM HEPES, 0.1% β-mercaptoethanol (Sigma-Aldrich, USA), 100 IU / mL of recombinant human interleukin 2 (rhIL-2) (BD Pharmingen, USA) to 1×10 6 / mL, activated with 5 μM ZOL, and seeded into 6-well plates. The cell culture density is maintained at 1 - 2×10 6 cells / mL and fresh medium containing only 100 IU / mL rhIL-2 (without ZOL) is replenished every 2 - 3 days. After 7 - 8 days of culture, the cells are collected and concentrated as described below.

[0068] Enrichment of Vγ9Vδ2 T cells: Vγ9Vδ2 T cells grown ex vivo are enriched using negative selection MACS with a TCRγ / δ+ T cell isolation kit (human) (Miltenyi Biotec, Germany). The cell viability and total cell number after enrichment are evaluated using trypan blue exclusion. The percentage of Vγ9Vδ2 T cells is determined by flow cytometry using a PeCy5-conjugated anti-CD3 (clone UCHT1) (eBioscience, San Diego, CA, USA) and an FITC-conjugated anti-Vγ9 TCR from BD Biosciences (San Jose, CA, USA). The percentage of Vγ9Vδ2 T cells is identified by gating on the lymphocyte population using forward scatter / side scatter and then gating on Vγ9+CD3+ double-positive cells.

[0069] In some embodiments, peripheral blood mononuclear cells (PBMCs) may be obtained from a subject according to any suitable method known in the art. PBMCs may be cultured for 1 to 2 weeks in the presence of IL-2 in the presence of an aminobisphosphonate (e.g., zoledronic acid), a synthetic phosphorylated antigen (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1PP, or 2-methyl-3-butenyl-1-pyrophosphate to generate a population enriched in Vδ2 cells. Alternatively, immobilized anti-TCRγδ (e.g., pan-TCRγδ) may induce preferential proliferation of Vδ2 cells from a population of PBMCs in the presence of IL-2, for example, for about 14 days. In some embodiments, preferential proliferation of Vδ2 cells from PBMCs may be achieved upon culturing with an anti-CD3 antibody (e.g., OKT3) immobilized in the presence of IL-2 and IL-4. In some embodiments, the aforementioned cultures are maintained for about 7 days prior to subculture in soluble anti-CD3, IL-2, and IL-4. Alternatively, artificial antigen-presenting cells may be used to promote preferential proliferation of γδ T cells such as Vδ2 cells. For example, γδ T cells derived from PBMCs cultured in the presence of irradiated aAPC, IL-2, and / or IL-21 may be expanded to generate a population of γδ T cells containing a high percentage of Vδ2 cells, a moderate percentage of Vδ1 cells, and some double-negative cells. In some embodiments of the aforementioned methods, PBMCs may be pre-concentrated or concentrated later (e.g., by positive selection with a TCRγδ-specific agent or negative selection with a TCRα-specific agent). Such methods and other suitable methods for the expansion of γδ T cells such as Vδ2 cells are described in detail in Deniger et al., Frontiers in Immunology 2014, 5, 636:1-10, which is hereby incorporated by reference in its entirety. Further, Almeida et al. (Clinical Cancer Research 2016, 22, 23; 5795-5805), which is hereby incorporated by reference in its entirety, provides suitable methods for obtaining a population of Vδ1 T cells that can be engineered to express the heterologous target constructs described herein.For example, in some embodiments, PBMCs are pre - enriched using magnetic bead sorting that can produce more than 90% γδ T cells.

[0070] Isolation and expansion of γδ T cells present in non - hematopoietic tissues from non - hematopoietic tissues The γδ T cells present in non - hematopoietic tissues obtained as described herein exhibit good tumor infiltration and retention capabilities. More detailed methods for the isolation and expansion of γδ T cells present in non - hematopoietic tissues can be found, for example, in GB application number 1707048.3 (WO2018 / 202808) and international patent publication number WO2017 / 072367 (U.S. Patent Application Publication No. 2018 / 0312808), which are incorporated herein by reference in their entireties.

[0071] γδ T cells present in non - hematopoietic tissues (e.g., skin - derived γδ T cells and / or non - Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) may be isolated from non - hematopoietic tissues of any human or non - human animal (which can be obtained from a patient to obtain cells suitable for manipulation by the methods of the present invention). In some embodiments, the non - hematopoietic tissue (from which γδ T cells are removed and expanded) is skin (e.g., human skin), which can be obtained by methods known in the art. In some embodiments, the skin is obtained by punch biopsy. Alternatively, the methods for isolation and expansion of γδ T cells provided herein can be applied to the gastrointestinal tract (e.g., colon), breast, lung, prostate, liver, spleen, and pancreas. γδ T cells can also be present in human cancer tissues, e.g., breast or prostate tumors. In some embodiments, the γδ T cells can be derived from human cancer tissues (e.g., solid tumor tissues). In other embodiments, the γδ T cells can be derived from non - hematopoietic tissues other than human cancer tissues (e.g., tissues having no substantial number of tumor cells). For example, the γδ T cells can be derived from areas of skin near or adjacent to, but distant from, cancer tissues (e.g., healthy skin).

[0072] The γδT cells that are dominant in the blood are mainly Vδ2T cells, and the γδT cells that are dominant in non-hematopoietic tissues are mainly Vδ1T cells. Vδ1T cells include about 70-80% of the γδT cell population present in non-hematopoietic tissues. However, some Vδ2T cells can also be found in non-hematopoietic tissues, such as the intestine, where they can include about 10-20% of the γδT cells. Some of the γδT cells present in non-hematopoietic tissues do not express either Vδ1TCR or Vδ2TCR, and the inventors named them double-negative (DN) γδT cells. Most of these DNγδT cells may be T cells that express Vδ3 and a small number express Vδ5. Therefore, the γδT cells that are normally present in non-hematopoietic tissues and are expanded by the method of the present invention are preferably non-Vδ2T cells, such as Vδ1T cells, that contain a small amount of DNγδT cells.

[0073] Generally, γδT cells present in non-hematopoietic tissues can spontaneously proliferate when physical contact with stromal cells (e.g., skin fibroblasts) is removed. Therefore, the above-described scaffold-based culture method can be used to induce such separation, and as a result, the γδT cells are released from suppression and proliferation is induced. Therefore, in some embodiments, there is no substantial TCR pathway activation during the proliferation step (e.g., exogenous TCR pathway activators are not included in the culture). Furthermore, the present invention provides a method for expanding γδT cells present in non-hematopoietic tissues, the method not including contact with feeder cells, tumor cells, and / or antigen-presenting cells.

[0074] Method of treatment As described herein, pharmaceutical compositions comprising an unmanipulated, enriched γδ T cell population, a manipulated, enriched γδ T cell population, and / or mixtures thereof can be administered for prophylactic treatment and / or therapeutic treatment. In therapeutic use, the composition can be administered to a subject already suffering from the disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. An unmanipulated, enriched γδ T cell population, a manipulated, enriched γδ T cell population, and / or mixtures thereof can also be administered to reduce the likelihood of developing, contracting, or worsening a condition. The effective amount of an unmanipulated, enriched γδ T cell population, a manipulated, enriched γδ T cell population, and / or mixtures thereof for therapeutic use can vary depending on the severity and course of the disease or condition, previous treatment, the health status, weight, and / or response to the drug of the subject, and / or the judgment of the physician treating the subject.

[0075] The unmanipulated, enriched γδ T cell populations, manipulated, enriched γδ T cell populations, and / or mixtures thereof of the present disclosure can be used to treat subjects in need of treatment of a condition.

[0076] Methods of treating a subject's condition using a concentrated γδ T cell population and an antibody or antibody fragment specific for CD19 of the present disclosure can include administering to the subject a therapeutically effective amount of an unmanipulated concentrated γδ T cell population, a manipulated and concentrated γδ T cell population, and / or a mixture thereof. The concentrated γδ T cell populations of the present disclosure, and / or mixtures thereof, can be administered in various regimens (e.g., timing, concentration, dosage, interval between treatments, and / or formulation). The subject can also be pre-treated, for example, with chemotherapy, radiation, or a combination of both, prior to receiving the concentrated γδ T cell populations of the present disclosure and / or mixtures thereof. As part of the treatment, an unmanipulated concentrated γδ T cell population, a manipulated concentrated γδ T cell population, and / or a mixture thereof can be administered to the subject in a first regimen, and the subject can be monitored to determine whether treatment in the first regimen meets a predetermined level of therapeutic effect.

[0077] The concentrated γδ T cell populations of the present disclosure, i.e., unmanipulated or manipulated, and / or mixtures thereof, can be used to treat a variety of conditions. In some cases, the unmanipulated, concentrated γδ T cell populations, the manipulated, concentrated γδ T cell populations, and / or mixtures thereof of the present disclosure can be used to treat cancers including solid tumors and blood cancers.

[0078] Route of Administration One or more non-manipulated, enriched γδT cell populations, manipulated, enriched γδT cell populations, and / or mixtures thereof of the present invention may be administered to a subject in any order or simultaneously. Simultaneously, multiple non-manipulated, enriched γδT cell populations, manipulated, enriched γδT cell populations, and / or mixtures thereof of the present invention may be provided in a single unified form, such as intravenous injection, or in multiple forms, such as multiple intravenous infusions, s.c., injections or pills. The non-manipulated, enriched γδT cell populations, manipulated, enriched γδT cell populations, and / or mixtures thereof of the present invention may be packed together or separately in a single package or multiple packages. One or all of the non-manipulated, enriched γδT cell populations, manipulated, enriched γδT cell populations, and / or mixtures thereof of the present invention may be given in multiple doses. If not simultaneous, the timing between multiple administrations may vary from about one week, one month, two months, three months, four months, five months, six months, or up to about one year. In some cases, the non-manipulated, enriched γδT cell populations, manipulated, enriched γδT cell populations, and / or mixtures thereof of the present invention may grow in vivo, in the subject's body, after administration to the subject. The non-manipulated, enriched γδT cell populations, manipulated, enriched γδT cell populations, and / or mixtures thereof may be frozen to provide cells for multiple treatments with the same cell preparation. The non-manipulated, enriched γδT cell populations, manipulated, enriched γδT cell populations, and / or mixtures thereof of the present disclosure, and pharmaceutical compositions containing the same, may be packaged as a kit. The kit may comprise instructions (e.g., written instructions) regarding the use of the non-manipulated enriched γδT cell populations, manipulated enriched γδT cell populations, and / or mixtures thereof, and compositions containing them.

[0079] In some cases, a method of treating cancer comprises administering to a subject a therapeutically effective amount of an unmanipulated, enriched γδT cell population, a manipulated, enriched γδT cell population, and / or a mixture thereof, wherein the administering comprises treating the cancer. In some embodiments, the therapeutically effective amount of the unmanipulated, enriched γδT cell population, the manipulated, enriched γδT cell population, and / or the mixture thereof is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the therapeutically effective amount of the unmanipulated, enriched γδT cell population, the manipulated, enriched γδT cell population, and / or the mixture thereof is administered for at least 1 week. In some embodiments, the therapeutically effective amount of the unmanipulated, enriched γδT cell population, the manipulated, enriched γδT cell population, and / or the mixture thereof is administered for at least 2 weeks.

[0080] Embodiment The present disclosure provides a pharmaceutical combination comprising an antibody or antibody fragment specific for CD19 and gamma delta T cells (γδT cells) for use in the treatment of cancer.

[0081] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma delta T cells (γδT cells) for use in the treatment of cancer, wherein the γδT cells comprise an enriched γδT cell population. In one embodiment, the enriched γδT cell population comprises unmanipulated or manipulated γδT cells and / or a mixture thereof. In further embodiments, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and an enriched γδT cell population for use in the treatment of cancer.

[0082] In another aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma delta T cells (γδT cells) for use in the treatment of cancer, wherein the γδT cells are unmanipulated or a combination comprising manipulated γδT cells and / or mixtures thereof. In another embodiment, the γδT cells are a population of unmanipulated γδT cells. In yet another embodiment, the γδT cells are a population of manipulated γδT cells.

[0083] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma delta T cells (γδT cells) for use in the treatment of cancer, wherein the γδT cells are isolated from peripheral blood, tumor tissue or non-hematopoietic tissue. In one embodiment, the γδT cells are isolated from peripheral blood. In another embodiment, the γδT cells are a population of γδT cells isolated from peripheral blood.

[0084] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma delta T cells (γδT cells) for use in the treatment of cancer which is a blood cancer. In one embodiment, the blood cancer is chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), small lymphocytic lymphoma (SLL), or acute lymphoblastic leukemia (ALL). In another embodiment, the blood cancer is non-Hodgkin lymphoma (NHL). In a further embodiment, the non-Hodgkin lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and mantle cell lymphoma.

[0085] In certain embodiments, the present disclosure provides a pharmaceutical combination for use in the treatment of cancer, comprising an antibody or antibody fragment specific for CD19 and gamma delta T cells (γδT cells), wherein the antibody or antibody fragment specific for CD19 is administered at 9 mg / kg. In alternative embodiments, the antibody or antibody fragment specific for CD19 is administered at 12 mg / kg. In still other embodiments, it is 15 mg / kg or more.

[0086] In embodiments, the antibody or antibody fragment specific for CD19 has cytotoxic activity. In embodiments, the antibody or antibody fragment specific for CD19 comprises a constant region having ADCC-inducing activity. In embodiments, the antibody specific for CD19 induces ADCC.

[0087] In certain embodiments, the present disclosure is a pharmaceutical combination for use in the treatment of cancer, comprising an antibody or antibody fragment specific for CD19 and gamma delta T cells (γδT cells), wherein the components of the combination, the antibody or antibody fragment specific for CD19 and the γδT cells, are administered separately. In certain embodiments, the γδT cells are administered prior to the administration of the antibody or antibody fragment specific for CD19. In certain embodiments, the antibody or antibody fragment specific for CD19 is administered prior to the administration of the γδT cells. In embodiments, the components of the combination are administered such that none of the components (drugs) are administered to the patient at the same time when they are active. By "synergistic effect", it is implied that none of the drugs are effective in the patient at the same time. In embodiments, the components of the combination are administered physically or temporally together, simultaneously, separately, or sequentially. In embodiments, the components of the combination are administered simultaneously.

[0088] In certain embodiments, the present disclosure provides a pharmaceutical combination for use in the treatment of cancer, comprising an antibody or antibody fragment specific for CD19 and γδT cells, wherein the anti-CD19 antibody is administered weekly, bi-weekly or monthly.

[0089] In certain embodiments, the present disclosure provides a pharmaceutical combination for use in the treatment of cancer, comprising an antibody or antibody fragment specific for CD19 and γδ T cells, wherein the antibody or antibody fragment specific for CD19 is administered at a concentration of 12 mg / kg.

[0090] In certain embodiments, the present disclosure provides a pharmaceutical combination for use in the treatment of cancer, comprising an antibody or antibody fragment specific for CD19 and γδ T cells, wherein the antibody or antibody fragment specific for CD19 is administered weekly, bi-weekly or monthly after the first administration on day 1, and the BCL-2 inhibitor is administered for the first time on day 8. In a further embodiment, the anti-CD19 antibody or its antibody fragment after the first administration on day 1 is administered weekly for the first three months and bi-weekly for at least the next three months.

[0091] In one aspect, the present disclosure provides an anti-CD19 antibody or an antibody fragment thereof for use in the treatment of a patient with a blood cancer, wherein the patient with a blood cancer has non-Hodgkin lymphoma, and wherein the anti-CD19 antibody or an antibody fragment thereof is administered in combination with γδ T cells.

[0092] In one aspect, the present disclosure provides an anti-CD19 antibody or an antibody fragment thereof for use in the treatment of a patient with a blood cancer, wherein the patient with a blood cancer has non-Hodgkin lymphoma, and wherein the anti-CD19 antibody or an antibody fragment thereof is administered in combination with γδ T cells. In one embodiment where the patient with a blood cancer has non-Hodgkin lymphoma, the non-Hodgkin lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and mantle cell lymphoma.

[0093] In one embodiment, the anti-CD19 antibody or antibody fragment thereof for use in the treatment of blood cancer patients in combination with γδ T cells comprises an HCDR1 region comprising the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region comprising the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region comprising the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region comprising the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region comprising the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region comprising the sequence MQHLEYPIT (SEQ ID NO: 6).

[0094] In a further embodiment, the anti-CD19 antibody or antibody fragment thereof for use in the treatment of blood cancer patients in combination with γδ T cells has a variable heavy chain of the following sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS (SEQ ID NO: 7) and a variable light chain of the following sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 8).

[0095] In another embodiment of the present disclosure, the anti-CD19 antibody or an antibody fragment thereof is a human antibody, a humanized antibody, a chimeric antibody, or an antibody fragment. In another embodiment of the present disclosure, the anti-CD19 antibody or an antibody fragment thereof is of the IgG isotype. In another embodiment, the antibody or antibody fragment is IgG1, IgG2, or an IgG1 / IgG2 chimera. In another embodiment of the present disclosure, the isotype of the anti-CD19 antibody is engineered to enhance antibody-dependent cell-mediated cytotoxicity. In another embodiment, the heavy chain constant region of the anti-CD19 antibody contains amino acids 239D and 332E, where the Fc numbering follows the EU index as in the case of Kabat. In another embodiment, the antibody is IgG1, IgG2, or IgG1 / IgG2, and the chimeric heavy chain constant region of the anti-CD19 antibody contains amino acids 239D and 332E, where the Fc numbering follows the EU index as in the case of Kabat.

[0096] In a further embodiment, the anti-CD19 antibody for use in the treatment of a blood cancer patient in combination with γδ T cells has a heavy chain having the following sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) and a light chain having the following sequence comprises DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[0097] In certain embodiments, an anti-CD19 antibody or antibody fragment thereof for use in the treatment of a blood cancer patient in combination with γδ T cells has a variable heavy chain of the following sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS (SEQ ID NO: 7) and a variable light chain of the following sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 8) or a variable heavy chain and a variable light chain having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the variable heavy chain of SEQ ID NO: 7 and to the variable light chain of SEQ ID NO: 8.

[0098] In certain embodiments, an anti-CD19 antibody or antibody fragment thereof for use in the treatment of a blood cancer patient in combination with γδ T cells has a variable heavy chain of the following sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS (SEQ ID NO: 7) and a variable light chain of the following sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 8) or a variable heavy chain having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the variable heavy chain of SEQ ID NO: 7 and a variable light chain having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the variable light chain of SEQ ID NO: 8, wherein the anti-CD19 antibody comprises an HCDR1 region comprising the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region comprising the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region comprising the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region comprising the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region comprising the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region comprising the sequence MQHLEYPIT (SEQ ID NO: 6). In another embodiment, the heavy chain region of the anti-CD19 antibody comprises amino acids 239D and 332E, where Fc numbering follows the EU index as in the case of Kabat.

[0099] In a further embodiment, an anti-CD19 antibody or an antibody fragment thereof for use in the treatment of a blood cancer patient in combination with γδ T cells has a heavy chain having the following sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) and a light chain having the following sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12) or a heavy chain and a light chain having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the heavy chain of SEQ ID NO: 7 and to the light chain of SEQ ID NO: 8, respectively.

[0100] In a further embodiment, an anti-CD19 antibody or an antibody fragment thereof for use in the treatment of a blood cancer patient in combination with γδ T cells has a heavy chain having the following sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 11) and a light chain having the following sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 12) Or heavy chains having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the heavy chain of SEQ ID NO: 7, and light chains having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the light chain of SEQ ID NO: 8, wherein the anti-CD19 antibody comprises an HCDR1 region comprising the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region comprising the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region comprising the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region comprising the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region comprising the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region comprising the sequence MQHLEYPIT (SEQ ID NO: 6). In another embodiment, the heavy chain region of the anti-CD19 antibody comprises amino acids 239D and 332E, wherein the Fc numbering follows the EU index as in the case of Kabat.

[0101] In one embodiment, the present disclosure provides an anti-CD19 antibody or an antibody fragment thereof, wherein the anti-CD19 antibody or an antibody fragment thereof is administered at a concentration of 12 mg / kg.

[0102] In a further embodiment, the anti-CD19 antibody or an antibody fragment thereof is administered weekly, bi-weekly, or monthly. In a further embodiment, the anti-CD19 antibody or an antibody fragment thereof is administered weekly for the first 3 months and bi-weekly for at least the next 3 months. In a further embodiment, the anti-CD19 antibody or an antibody fragment thereof is administered weekly for the first 3 months. In a further embodiment, the anti-CD19 antibody or an antibody fragment thereof is administered weekly for the first 3 months and bi-weekly for at least the next 3 months. In a further embodiment, the anti-CD19 antibody or an antibody fragment thereof is administered weekly for the first 3 months, bi-weekly for the next 3 months, and then monthly. In yet another further embodiment, the anti-CD19 antibody or an antibody fragment thereof is administered weekly for the first 3 months, bi-weekly for the next 3 months, and then monthly.

[0103] The present disclosure provides an antibody or antibody fragment specific for CD19 for use in the treatment of cancer, wherein the antibody or antibody fragment specific for CD19 is administered in combination with γδT cells. In one embodiment, the γδT cells are isolated from peripheral blood, tumor tissue, or non-hematopoietic tissue. In one embodiment, the γδT cells are isolated from peripheral blood. In another embodiment, the γδT cells are a population of γδT cells isolated from peripheral blood. In another embodiment, the γδT cells are a population of γδT cells isolated from peripheral blood and cultured in the presence of IL-2 and ZOL. In another example, the γδT cells are a population of blood-derived Vγ9Vδ2T cells.

[0104] The present disclosure provides an antibody or antibody fragment specific for CD19 for use in the treatment of cancer, wherein the antibody or antibody fragment specific for CD19 is administered in combination with γδT cells, and wherein the step of administration is performed by combining the antibody specific for CD19 and γδT cells simultaneously, sequentially, or in reverse order.

[0105] In another embodiment, the present disclosure provides the use of a combination of a pharmaceutical composition comprising an antibody or antibody fragment specific for CD19 and γδT cells for the preparation of a medicament for the treatment of cancer.

[0106] In another embodiment, the present disclosure provides a method for use in the treatment of cancer, the method comprising the step of administering to a subject a combination of an antibody specific for CD19 and γδT cells. In another embodiment, the present disclosure provides a method for use in the treatment of cancer, the method comprising the step of administering to a subject a combination of an antibody specific for CD19 and γδT cells, wherein the step of administration is performed by administering the antibody specific for CD19 and γδT cells simultaneously, sequentially, or in reverse order.

[0107] The present disclosure provides a pharmaceutical combination comprising an antibody or antibody fragment specific for CD19 and gamma-delta T cells (γδT cells) for use in the treatment of cancer, wherein the γδT cells are administered in a therapeutically effective amount. In some embodiments, a therapeutically effective amount of lymphocytes (e.g., γδT cells) obtained by any of the above methods can be administered to a subject (e.g., for the treatment of cancer such as blood cancer) in a therapeutically effective amount. In some cases, the therapeutically effective amount of lymphocytes (e.g., γδT cells) is less than 10×10 12 cells per dose (e.g., less than 9×10 12 cells per dose, less than 8×10 12 cells per dose, less than 7×10 12 cells per dose, less than 6×10 12 cells per dose, less than 5×10 12 cells per dose, less than 4×10 12 cells per dose, less than 3×10 12 cells per dose, less than 2×10 12 cells per dose, less than 1×10 12 cells per dose, less than 9×10 11 cells per dose, less than 8×10 11 cells per dose, less than 7×10 11 cells per dose, less than 6×10 11 cells per dose, less than 5×10 11 cells per dose, less than 4×10 11 cells per dose, less than 3×10 11 cells per dose, less than 2×10 11 cells per dose, less than 1×10 11 cells per dose, less than 9×10 10 cells per dose, less than 7.5×10 10 cells per dose, less than 5×10 10 cells per dose, less than 2.5×10 10 cells per dose, less than 1×10 10 cells per dose, less than 7.5×10 9 cells per dose, less than 5×10 9 cells per dose, less than 2.5×10 9 cells per dose, less than 1×109 less than cells, 7.5×10 per dose 8 less than cells, 5×10 per dose 8 less than cells, 2.5×10 per dose 8 less than cells, 1×10 per dose 8 less than cells, 7.5×10 per dose 7 less than cells, 5×10 per dose 7 less than cells, 2.5×10 7 less than cells, 1×10 per dose 7 less than cells, 7.5×10 per dose 6 less than cells, 5×10 per dose 6 less than cells, 2.5×10 per dose 6 less than cells, 1×10 per dose 6 less than cells, 7.5×10 per dose 5 less than cells, 5×10 per dose 5 less than cells, 2.5×10 per dose 5 less than cells, or 1×10 per dose 5 less than cells).

[0108] In some embodiments, the therapeutically effective amount of γδ T cells (e.g., skin-derived γδ T cells, blood-derived γδ T cells) is 10×10 over the course of treatment 12 less than cells (e.g., 9×10 over the course of treatment 12 less than cells, 8×10 12 less than cells, 7×10 12 less than cells, 6×10 12 less than cells, 5×10 12 less than cells, 4×10 12 less than cells, 3×10 12 less than cells, 2×10 12 less than cells, 1×10 12 less than cells, 9×10 11 less than cells, 8×10 11 less than cells, 7×10 11 less than cells, 6×10 11 less than cells, less than 5×10 11 less than cells, 4×10 11 less than cells, 3×10 11 less than cells, 2×10 11 less than cells, 1×1011 less than cells, 9×10 10 less than cells, 7.5×10 10 less than cells, 5×10 10 less than cells, 2.5×10 10 less than cells, 1×10 10 less than cells, 7.5×10 9 less than cells, 5×10 9 less than cells, 2.5×10 9 less than cells, 1×10 9 less than cells, 7.5×10 8 less than cells, 5×10 8 less than cells, 2.5×10 8 less than cells, 1×10 8 less than cells, 7.5×10 7 less than cells, 5×10 7 less than cells, 2.5×10 7 less than cells, 1×10 7 less than cells, 7.5×10 6 less than cells, 5×10 6 less than cells, 2.5×10 6 less than cells, 1×10 6 less than cells, 7.5×10 5 less than cells, 5×10 5 less than cells, 2.5×10 5 less than cells, or 1×10 5 less than cells).

[0109] In some embodiments, the dosage of γδ T cells described herein is about 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5×10 8 cells / kg. In some embodiments, the dosage of γδ T cells described herein is at least 1×10 6 , 1.1×10 6 , 2×10 6, 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5×10 8 cells / kg may be mentioned. In some embodiments, the dosage of γδ T cells described herein is at most 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5×10 8 cells / kg may be mentioned.

[0110] combination The present disclosure provides an anti-CD19 antibody or an antibody fragment thereof in combination with γδT cells for use in the treatment of blood cancer, wherein the anti-CD19 antibody or an antibody fragment thereof and γδT cells are administered in combination with one or more pharmaceuticals. In one embodiment of the present disclosure, the anti-CD19 antibody or an antibody fragment thereof and γδT cells are administered in combination with a pharmaceutical. In another embodiment of the present disclosure, the anti-CD19 antibody or an antibody fragment thereof and γδT cells are administered in combination with one or more additional agents or additional pharmaceuticals. In one aspect, the pharmaceutical is an additional pharmaceutical. In one embodiment of the present disclosure, the pharmaceutical is a biological or chemotherapeutic agent. In another embodiment of the present disclosure, the pharmaceutical is a therapeutic antibody or antibody fragment, nitrogen mustard, purine analog, thalidomide analog, phosphoinositide 3-kinase inhibitor, BCL-2 inhibitor or Bruton's tyrosine kinase (BTK) inhibitor. In a further embodiment, the pharmaceutical is rituximab, R-CHOP, cyclophosphamide, chlorambucil, uracil mustard, ifosfamide, melphalan, bendamustine, mercaptopurine, azathioprine, thioguanine, fludarabine, thalidomide, lenalidomide, pomalidomide, idelalisib, duvelisib, copanlisib, ibrutinib or venetoclax.

[0111] In another embodiment, the present disclosure provides an anti-CD19 antibody or an antibody fragment thereof and γδT cells for use in the treatment of blood cancer, wherein the anti-CD19 antibody or an antibody fragment thereof and γδT cells are administered in combination with rituximab, R-CHOP, cyclophosphamide, chlorambucil, uracil mustard, ifosfamide, melphalan, bendamustine, mercaptopurine, azathioprine, thioguanine, fludarabine, thalidomide, lenalidomide, pomalidomide, idelalisib, duvelisib, copanlisib, ibrutinib or venetoclax.

[0112] Antibody sequence [Table 1-1]

Table 1-2

Example

[0113] Example 1: Characterization of CD19 and CD20 expression in tested cell lines This study was conducted to evaluate the cytotoxic activity of the Fc-enhanced anti-CD19 antibody tafasitamab (MOR00208) mediated by γδ T cells from different donors against lymphoma and leukemia cell lines as well as primary patient-derived tumor materials from CLL (chronic lymphocytic leukemia), MCL (mantle cell lymphoma), and B-ALL (acute lymphoblastic leukemia). Furthermore, the antibody-dependent cell-mediated cytotoxic activity of tafasitamab in the presence of γδ T cells was evaluated.

[0114] Various lymphoma and leukemia cell lines, as well as primary patient-derived tumor cells from lymphoma and leukemia patients, were evaluated in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay using various concentrations of tafasitamab and an IgG1 negative control antibody. γδ T cells were isolated from seven different donors and used as effector cells at different effector-to-target cell ratios (E:T ratios 0.7:1, 2.2:1, 6.7:1, and 20:1).

[0115] Materials, methods, and data analysis γδ T cells: γδ T cells express Vγ and Vδ variable chains as part of a T cell receptor (TCR) complex that is structurally and functionally distinct from the major histocompatibility complex (MHC) that binds to the TCR of αβ T cells. Despite an unrestricted high combinatorial diversity, the Vδ2 chain preferentially pairs with the Vγ9 chain. Vγ9Vδ2 T cells account for approximately 5% of peripheral blood T cells and represent the major γδ T cell subset of this compartment. As listed below, γδ T cells from seven different donors were isolated, stimulated, and applied. [Table 2] As listed in the table, the stimulated cell populations used in the described experiments consisted of three major populations. Only a very small fraction (<5%) of CD56+ / CD3− NK effector cells were present, and TCRγδneg / CD3+ cells showed a larger population in some cases, but these cells did not cause lysis of target cells. Thus, γδT cells were mainly responsible for antibody-mediated cell death.

[0116] Cell lines and patient samples Cell lines (obtained from DSMZ): Mino (mantle cell lymphoma), Daudi (Burkitt lymphoma), Jeko-1 (mantle cell lymphoma), U2932 (DLBCL), REH (B-ALL) Patient samples (peripheral blood): 2×CLL (chronic lymphocytic leukemia), 2×MCL (mantle cell lymphoma), 1×B-ALL (acute lymphoblastic leukemia)

[0117] Results Dose titration with lymphoma cell lines Initial dose titration experiments to obtain a valid assay setup were performed using MOR00208 at concentrations of 0.001–10 μg / ml in the presence of four E:T ratios of γδT cells (0.7:1, 2.2:1, 6.7:1, and 20:1), and a dose-dependent increase in the percentage of lysed Jeko and U2932 target cells was observed. As expected, the killing activity of the tested antibody increased at higher E:T ratios, and the most distinct effects were observed at the highest E:T ratios tested, 6.7:1 and 20:1. The maximum effect of cell lysis was achieved at concentrations of 0.1 μg / ml, 1 μg / ml, and 10 μg / ml of MOR00208 in the two test cell lines. Cell lysis showed γδT cell-dependent donor variability, with maximum lysis of 34.0%–49.3% in Jeko and 22.5% in U2392 cells, respectively. A concentration of 1 μg / ml of MOR00208 (Fc-enhanced) was selected as the optimal concentration for a more comprehensive analysis of the Fc-dependent effect of antibody-mediated cell killing by γδT cells against leukemia and lymphoma cell lines and patient-derived tumor cells.

[0118] Cytotoxicity assay using lymphoma / leukemia cell lines To determine the tumor cell killing ability of γδ T cells in combination with CD19 antibodies, tafasitamab was tested at 1 μg / ml using Mino, Daudi, Jeko, U2932, and REH cells as target cells (Figures 1 and 2). As described above, four different E:T ratios between 0.7:1 and 20:1 were tested. And for all cell lines, the effect of MOR00208 in combination with γδ T cells was superior to that of the IgG1 control in combination with γδ T cells (MOR00208 > IgG1 control). As observed during the dose titration experiment, the killing activity of the tested antibodies increased, and the most distinct effect was found at the highest E:T ratios tested, 6.7:1 and 20:1. In particular, in the two cell lines with the lowest non-specific killing (Mino and Jeko), the specific contribution of the antibody to cell killing activity was observed to be the highest. In both cell lines, the MOR00208 activity was statistically significantly different from the IgG1 control (Figure 1). In the three cell lines with higher non-specific killing, the activity profiles were similar (MOR00208 > IgG1 control), but the specific effects were limited (Figure 2).

[0119] Cytotoxicity assay using primary patient cells Primary tumor cells from two CLL, two MCL, and one B-ALL patients were isolated as performed in cell lines and incubated with single donor γδ T cells at various E:T ratios (0.7:1 - 20:1) and 1 μg / ml of the CD19-targeting antibody. (Figures 3 and 4). Consistent with the observations in cell lines, MOR00208 showed a clear specific contribution to the killing activity of γδ T cells. This contribution increased with the E:T ratio for all primary cells and was most prominent at 20:1, the highest E:T ratio tested. The primary patient cells were tested in only one experiment.

[0120] Conclusion In summary, γδT cells have been found to be a potential effector cell population in antibody-based cancer therapy, as demonstrated by the Fc-enhanced CD19-targeting antibody MOR00208 in this study. MOR00208 exhibits potent anti-tumor activity mediated by the presence of γδT cells against several lymphoma and leukemia cell lines, as well as primary patient-derived CLL, MCL, and B-ALL cells, providing a reasonable basis for combining MOR00208 and γδT cells as a promising approach for lymphoma and leukemia treatment.

Claims

1. A composition comprising an anti-CD19 antibody for use in the treatment of blood cancer, wherein the anti-CD19 antibody is administered in combination with γδ T cells, the anti-CD19 antibody consists of two heavy chains and two light chains, each heavy chain comprises an HCDR1 region containing the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region containing the sequence NPYNDG (SEQ ID NO: 2), and an HCDR3 region containing the sequence GTYYYGTRVF DY (SEQ ID NO: 3) and comprises a heavy chain variable region, and each light chain comprises an LCDR1 region containing the sequence RSSKS LQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region containing the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region containing the sequence MQHLEYPI T (SEQ ID NO: 6) and comprises a light chain variable region, said composition.

2. The composition according to claim 1 for use according to claim 1, wherein the γδ T cells comprise a population of γδ T cells enriched with, said composition.

3. The composition according to claim 1 or 2 for use according to claim 1 or 2, wherein the γδ T cells comprise unmanipulated γδ T cells, said composition.

4. The composition according to claim 1 or 2 for use according to claim 1 or 2, wherein the γδ T cells comprise manipulated γδ T cells, said composition.

5. The composition according to any one of claims 1 to 4 for use according to any one of claims 1 to 4, wherein the γδ T cells are isolated from peripheral blood, said composition.

6. The composition according to any one of claims 1 to 4 for use according to any one of claims 1 to 4, wherein the γδ T cells are isolated from tumor tissue, said composition.

7. The composition according to any one of claims 1 to 4 for use according to any one of claims 1 to 4, wherein the γδ T cells are isolated from non-hematopoietic tissue, said composition.

8. The composition according to any one of claims 1 to 4 for use according to any one of claims 1 to 4, wherein the γδ T cells are isolated from peripheral blood and cultured in the presence of IL-2 and zoledronic acid (ZOL), said composition.

9. The composition according to any one of claims 1 to 4 for use according to any one of claims 1 to 4, wherein the γδ T cells are blood-derived Vγ9Vδ2 T cells, said composition. **Claim 10**: A composition according to any one of claims 1 to 9 for use according to any one of claims 1 to 9, wherein the anti-CD19 antibody and the γδ T cells are administered by separate methods, said composition. **Claim 11**: A composition according to any one of claims 1 to 9 for use according to any one of claims 1 to 9, wherein the anti-CD19 antibody and the γδ T cells are administered by a simultaneous method, said composition. **Claim 12**: A composition according to any one of claims 1 to 11 for use according to any one of claims 1 to 11, wherein the anti-CD19 antibody has enhanced antibody-dependent cell-mediated cytotoxicity activity, said composition. **Claim 13**: A composition according to any one of claims 1 to 12 for use according to any one of claims 1 to 12, wherein the anti-CD19 antibody comprises an Fc domain comprising an amino acid substitution at position S239 and / or I332, numbering according to the EU index as in the case of Kabat, said composition. **Claim 14**: A composition according to any one of claims 1 to 13 for use according to any one of claims 1 to 13, wherein the heavy chain constant region of the anti-CD19 antibody comprises amino acids 239D and 332E, where Fc numbering follows the EU index as in the case of Kabat, said composition. **Claim 15**: A composition according to any one of claims 1 to 14 for use according to any one of claims 1 to 14, wherein the amino acid sequence of each of the heavy chain variable regions is shown in SEQ ID NO: 7, and the amino acid sequence of each of the light chain variable regions is shown in SEQ ID NO: 8, said composition. **Claim 16**: A composition according to any one of claims 1 to 15 for use according to any one of claims 1 to 15, wherein the amino acid sequence of each of the heavy chains is shown in SEQ ID NO: 11, said composition. **Claim 17**: A composition according to any one of claims 1 to 16 for use according to any one of claims 1 to 16, wherein the amino acid sequence of each of the heavy chains is shown in SEQ ID NO: 11, and the amino acid sequence of each of the light chains is shown in SEQ ID NO: 12, said composition. **Claim 18**: A composition according to any one of claims 1 to 17 for use according to any one of claims 1 to 17, wherein the blood cancer is chronic lymphocytic leukemia, said composition.

19. The composition according to any one of claims 1 to 17 for use according to any one of claims 1 to 17, wherein the blood cancer is small lymphocytic lymphoma, said composition.

20. The composition according to any one of claims 1 to 17 for use according to any one of claims 1 to 17, wherein the blood cancer is acute lymphoblastic leukemia, said composition.

21. The composition according to any one of claims 1 to 17 for use according to any one of claims 1 to 17, wherein the blood cancer is non-Hodgkin lymphoma, said composition.

22. The composition according to claim 21 for use according to claim 21, wherein the non-Hodgkin lymphoma is follicular lymphoma, said composition.

23. The composition according to claim 21 for use according to claim 21, wherein the non-Hodgkin lymphoma is mucosa-associated lymphoid tissue lymphoma, said composition.

24. The composition according to claim 21 for use according to claim 21, wherein the non-Hodgkin lymphoma is marginal zone lymphoma, said composition.

25. The composition according to claim 21 for use according to claim 21, wherein the non-Hodgkin lymphoma is diffuse large B-cell lymphoma, said composition.

26. The composition according to claim 21 for use according to claim 21, wherein the non-Hodgkin lymphoma is Burkitt lymphoma, said composition.

27. The composition according to claim 21 for use according to claim 21, wherein the non-Hodgkin lymphoma is mantle cell lymphoma, said composition.

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