Anti-tumor combination therapy comprising Anti-CD19 antibody and gamma delta t-cells

Abstract

To provide an improved method or therapeutic agent for treating many types of cancers including CD19-expressing tumors.SOLUTION: The invention provides a composition comprising an anti-CD19 antibody for use in the treatment of hematologic cancer, where the anti-CD19 antibody is administered in combination with γδ T cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 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. [Background technology]

[0002] CD19 is a 95-kDa transmembrane glycoprotein of the immunoglobulin superfamily containing two extracellular immunoglobulin-like domains and an extensive cytoplasmic tail. This protein is a pan-B lymphocyte surface receptor and is ubiquitously expressed from early stages of pre-B cell development onward until downregulation during terminal differentiation into plasma cells. It is B lymphocyte lineage-specific and is not expressed on hematopoietic stem cells or 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 the development of humoral immune responses. It functions as a costimulatory molecule in conjunction with CD21 and CD81, and is important for B cell responses to T cell-dependent antigens. The cytoplasmic tail of CD19 physically associates with a family of tyrosine kinases that trigger downstream signaling pathways via the SRC family of protein tyrosine kinases. CD19 is an attractive target in cancers of lymphoid origin because it is highly expressed in almost all chronic lymphocytic leukemias (CLL) and non-Hodgkin's lymphomas (NHL), as well as many other different types of leukemia, including acute lymphocytic leukemia (ALL) and hairy cell leukemia (HCL).

[0003] Tafasitamab (formerly MOR00208 and XmAb® 5574) is a humanized monoclonal antibody targeting the antigen CD19, a transmembrane protein involved in B-cell receptor signaling. Tafasitamab is engineered with an IgG Fc region to enhance antibody-dependent cellular cytotoxicity (ADCC), improving a key mechanism for tumor cell killing and potentially increasing efficacy compared with conventional, non-enhanced antibodies. Tafasitamab has been or is currently being studied in several clinical trials, including for CLL, ALL, and NHL. In some of these trials, tafasitamab is used in combination with idelalisib, lenalidomide, or venetoclax.

[0004] Despite the recent discovery and development of several anticancer drugs, the poor prognosis of many types of cancer, including CD19-expressing tumors, means that there remains a need for improved methods or therapeutic agents for treating such types of cancer. Therefore, the present inventors have confirmed that the combined administration of γδ T cells and a CD19-specific antibody or antibody fragment has excellent effects on the treatment of B cell-derived malignant lymphoma, thereby completing the present invention. Summary of the Invention

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

[0006] Since their discovery in the 1980s, γδ T cells have simultaneously been recognized to play an important role in infectious diseases and malignant tumors such as cancer. Activated γδ T cells possess potent cytotoxicity and broad tumor recognition capabilities, independent of the 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). To date, it has been shown that the antitumor effects of γδ T cells can be substantially enhanced by anti-CD20 antibodies (Tokuyama et al. 2008; Hoeres et al. 2018). Furthermore, the Fc-enhanced anti-CD20 antibody obinutuzumab, when combined with γδ T cells, exhibits increased tumor cell killing 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. Therefore, it is an object of the present disclosure to provide alternative combination therapies comprising antibodies and γδ T cells.

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

[0009] In this disclosure, we combined γδ T cells with the CD19-targeting antibodies tafasitamab (Fc-enhanced) and Xmab5603 (non-Fc-enhanced) and evaluated their antitumor activity in patient-derived CLL, MCL, and B-ALL samples, as well as various lymphoma and leukemia cell lines, in ADCC assays. Overall, we observed an increased rate of cell lysis when γδ T cells were combined with the Fc-enhanced anti-CD19 antibody tafasitamab compared to the non-Fc-enhanced Xmab5603 or a 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 in this study for the Fc-enhanced CD19-targeting antibody tafasitamab. Tafasitamab exhibited potent γδ T cell-mediated antitumor activity against several lymphoma and leukemia cell lines, as well as primary patient-derived CLL, MCL, and BALL cells, and may hold a promising approach for lymphoma and leukemia therapy.

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

[0012] In other embodiments, suitable γδ T cells may be derived from blood (e.g., peripheral blood). Methods for isolating and expanding Vδ1 cells from blood include, for example, those described in U.S. Pat. No. 9,499,788 and International Patent Publication No. WO 2016 / 198480, each of which is incorporated herein by reference in its entirety. Vγ9Vδ2 T cells may also be isolated from peripheral blood and further cultured ex vivo. Culture of Vγ9Vδ2 T cells may 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, those 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 heterologous targeting constructs 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, coreceptor agonists, and / or cytokines, e.g., IL-4, IL-1 5, and / or IFN-γ) in gas-permeable bioreactor bags for up to 21 days or more. Variations of this method, as well as 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 the methods described, for example, in International Patent Publications WO2017 / 197347 and WO2016 / 081518 (U.S. Patent Application Publication No. 2016 / 0175338), which are incorporated by reference in their entireties.

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

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

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

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

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

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

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

[0022] In yet another embodiment, the antibody specific for CD19 comprises the following heavy chain constant region: EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) and the following light chain constant region: DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[0023] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma delta T cells (γδ T cells) for use in treating cancer, wherein the γδ T cells comprise an enriched γδ T cell population. In one embodiment, the enriched γδ T cell population comprises unengineered or engineered γδ T 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 γδ T cell population for use in treating cancer.

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

[0025] In one aspect, the present disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma delta T cells (γδ T cells) for use in treating cancer, wherein the γδ T cells are isolated from peripheral blood, tumor tissue, or non-hematopoietic tissue. In one embodiment, the γδ T cells are isolated from peripheral blood. In another embodiment, the γδ T cells are a population of γδ T cells isolated from peripheral blood. In another embodiment, the γδ T cells are a population of Vy9V52 T cells isolated from peripheral blood. In a further embodiment, the γδ T cells are a population of Vy9V52 T cells isolated from peripheral blood, wherein the Vy9V52 T cells have been 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 (γδ T cells) for use in treating cancer, which is a hematological cancer. In one embodiment, the hematological cancer is chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL), or acute lymphoblastic leukemia (ALL). In another embodiment, the hematological cancer is non-Hodgkin's lymphoma (NHL). In a further embodiment, the non-Hodgkin's 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's 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 (γδ T cells) for use in the treatment of cancer, wherein the antibody specific for CD19 and the γδ T cells are administered in separate doses.

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

[0029] In one aspect, the present disclosure provides a kit for use in treating cancer, comprising an antibody specific for CD19 and gamma delta T cells (γδ T cells). [Brief explanation of the drawings]

[0030] [Figure 1] Representative ADCC assay of MOR00208 at increasing effector-to-target cell (E:T) ratios. Specific cell killing results, expressed as the % of dead target cells mediated by MOR00208 (black) or IgG1 negative control (white) versus γδ T cells from a healthy donor, are shown for two target cell lines, Mino and Jeko. Four different E:T ratios between 0.7:1 and 20:1 were tested. [Figure 2] Representative ADCC assay of MOR00208 at increasing effector-to-target cell (E:T) ratios. Specific cell killing results, expressed as % of dead target cells mediated by MOR00208 (black) or IgG1 control (white) versus γδ T cells from a healthy donor, are shown for three target cell lines: U2932, REH, and Daudi. Four different E:T ratios between 0.7:1 and 20:1 were tested. [Figure 3]Representative ADCC assay of MOR00208 at increasing effector-to-target cell (E:T) ratios using primary tumor cells from two CLL patients and one B-ALL patient as target cells. Results of specific cell killing, expressed as the % of dead target cells mediated by MOR00208 (black) or IgG1 control (white) versus γδ T cells from a healthy donor, are shown for three experiments using primary tumor cells as target cells. Four different E:T ratios between 0.7:1 and 20:1 were tested. [Figure 4] Representative ADCC assay of MOR00208 at increasing effector-to-target cell (E:T) ratios using primary tumor cells from two MCL patients as target cells. Results of specific cell killing, expressed as % of dead target cells mediated by MOR00208 (black) or IgG1 control (white) versus γδ T cells from a healthy donor, are shown for two experiments using primary tumor cells as target cells. Four different E:T ratios between 0.7:1 and 20:1 were tested. DETAILED DESCRIPTION OF THE INVENTION

[0031] definition The term "CD19" refers to the protein known as CD19, which has 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: (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 sequence of MOR00208 / tafasitamab. The MOR00208 antibody is described in U.S. Patent Application Serial No. 12 / 377,251, which is incorporated 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 designated 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 regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies may 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 phrase "antibody fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, or stabilization of spatial distribution). Examples of binding fragments include, but are not limited to, Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragments, i.e., bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single antibody arm; dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker, allowing them to be produced as a single protein chain using recombinant methods; the VL and VH regions pair to form a monovalent molecule (known as a 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 are obtained using conventional techniques known to those skilled 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-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136).Antibody fragments may be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments may also be incorporated into single-chain molecules comprising 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. Pat. No. 5,641,870).

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

[0037] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the 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 cellular phagocytosis (ADCP); down-regulation 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 antibodies coupled to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells using cytotoxins. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas 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 classical 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 cellular 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 "hematological 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's lymphoma (NHL) is a heterogeneous malignant tumor originating from lymphocytes. In the United States (US), the incidence rate is estimated at 65,000 per year, with a mortality rate of approximately 20,000 (American Cancer Society, 2006; and SEER Cancer Statistics Review). The disease can occur at any age, with onset typically occurring in adults over 40 years of age, and incidence increases with age. NHL is characterized by the clonal proliferation of lymphocytes that accumulate in lymph nodes, blood, bone marrow, and spleen, although any major organ may be involved. The current classification system used by pathologists and clinicians is the World Health Organization (WHO) Classification of Tumors, which categorizes NHL into precursor and mature B-cell or T-cell neoplasms. PDQ currently categorizes NHL as indolent (low-grade) or aggressive (aggressive) for clinical trial participation. The indolent NHL group consists primarily of follicular subtypes, small lymphocytic lymphoma, mucosa-associated lymphoid tissue (MALT), and marginal zone lymphoma. Indolent NHL comprises approximately 50% of newly diagnosed B-cell NHL patients. Aggressive NHL primarily includes patients with histological diagnoses of diffuse large B-cell (DLBL, "DLBCL," or DLCL) (40% of all newly diagnosed patients are of the diffuse large cell subtype), Burkitt lymphoma, and mantle cell (MCL) lymphoma. The clinical course of NHL is highly variable. The primary determinant of clinical course is the histological subtype. Most indolent NHLs are considered incurable. Patients initially respond to either chemotherapy or antibody therapy, and most relapse. Previous studies have not demonstrated improved survival with early intervention. In asymptomatic patients, "watch and wait" is acceptable until the patient becomes symptomatic or the pace of disease appears to be accelerating. Over time, the disease can progress to more aggressive histology. Median survival is 8 to 10 years, and indolent patients often receive three or more lines of therapy during the course of their disease. Initial treatment for symptomatic indolent NHL has historically been combination chemotherapy.The most commonly used drugs include cyclophosphamide, vincristine, and prednisone (CVP) or cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP). Approximately 70% to 80% of patients respond to initial chemotherapy, with remissions lasting 2 to 3 years. Ultimately, the majority of patients relapse. The discovery and clinical use of the anti-CD20 antibody rituximab has significantly improved response and survival rates. The current standard of care for most patients is rituximab plus CHOP (R-CHOP) or rituximab plus 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 (diffuse large B-cell lymphoma) NHL. However, anti-CD20 monoclonal antibodies (mAbs) have significant limitations, including primary resistance (50% response in relapsed indolent patients), acquired resistance (50% response rate upon retreatment), rare complete responses (2% complete responses in the relapsed population), and a persistent pattern of relapse. Finally, many B-cell disorders cannot be treated using anti-CD20 antibody therapy because many B cells do not express CD20.

[0043] In addition to NHL, there are several types of leukemia resulting from dysregulation of B cells. 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 fight infection. CLL is the most common form of leukemia in adults. Men are twice as likely as women to develop CLL. However, age is a major risk factor. More than 75% of new cases are diagnosed in patients over the age of 50. More than 10,000 cases are diagnosed each year, with a mortality rate of nearly 5,000 per year (American Cancer Society, 2006; and SEER Cancer Statistics Review). CLL is incurable, but in most cases it progresses slowly. Many people with CLL lead normal, active lives for many years. Because of the slow onset of the disease, early intervention is not believed to improve survival or quality of life, so early-stage CLL is generally not treated. Instead, the condition is monitored over time. Initial CLL treatment depends on the exact diagnosis and disease progression. 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 first-line treatment for CLL due to its risks.

[0044] Another type of leukemia is small lymphocytic lymphoma ("SLL"), which is considered a CLL variant that lacks the clonal lymphocytosis required for a CLL diagnosis but otherwise shares pathologic and immunophenotypic features (Campo et al., 2011). The definition of SLL requires the presence of lymphadenopathy and / or splenomegaly. Additionally, 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 lymph node biopsies whenever possible (Hallek et al., 2008). The incidence of SLL is approximately 25% of CLL cases 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 "acute" refers to the undifferentiated, immature state of circulating lymphocytes ("blasts"), which, if left untreated, rapidly progresses with a life expectancy of weeks to months. ALL is most common in childhood, with peak incidence between the ages of 4 and 5. Children aged 12 to 16 are more likely to die than other children. Currently, at least 80% of childhood ALL cases are considered curable. Fewer than 4,000 cases are diagnosed each year, with approximately 1,500 deaths annually (American Cancer Society, 2006; and SEER Cancer Statistics Review).

[0046] As used in this context, a "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, 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, antibodies or antibody fragments according to the present disclosure are engineered or modified to improve effector function 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 comprises two constant domains, a CH2 domain and a CH3 domain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as 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 in accordance with the present disclosure are administered to patients in therapeutically effective amounts. A "therapeutically effective amount" refers to an amount sufficient to alleviate to some extent the clinical symptoms of a given disease or disorder. The amount effective for a particular therapeutic purpose will depend on the weight and general condition of the subject, as well as the severity of the disease or injury. It will be understood that determining appropriate dosages can be accomplished using routine experimentation, constructing a matrix of values, and testing different points within the matrix, all of which is 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, "in combination" includes simultaneous (e.g., contemporaneous) and consecutive administration in any order. As a non-limiting example, a first therapeutic agent (e.g., an agent such as an anti-CD19 antibody) may be administered to a patient at least 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, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours The compound may be administered at any time during the administration of the compound (e.g., 1 week, 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 or more weeks after).

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

[0053] As used herein, the term "TCR" or "T cell receptor" refers to dimeric heterologous cell surface signaling proteins that form alpha-beta or gamma-delta receptors. αβ TCRs recognize antigens presented by MHC molecules, whereas γδ TCRs recognize antigens independently of MHC presentation.

[0054] As used herein, the term "cell population" refers to a large number of cells.The cell population can be, for example, a mixed cell population derived from peripheral blood samples, umbilical cord blood samples, tumors, stem cell precursors, tumor biopsies, tissues, lymphatic fluid, or derived from the epithelial site of a subject that directly contacts the external environment, or derived from stem progenitor cells.Alternatively, the mixed cell population can be derived from mammalian cell in vitro culture, or established from peripheral blood samples, umbilical cord blood samples, tumors, stem cell precursors, tumor biopsies, tissues, lymphatic fluid, or derived from the epithelial site of a subject that directly contacts the external environment, or derived from stem progenitor cells.

[0055] An "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, the starting mixed cell population can be enriched for a particular γδ T cell population. In all embodiments, the enriched γδ T cell population contains a smaller percentage of an αβ T cell population.

[0056] As used herein, "expanded" means that the number of desired or target cell types (e.g. δ1, δ2 T cells and / or Vy9V52 T cells) in the enriched preparation is greater than the number in the initial or starting cell population.

[0057] Detailed Description of the Invention

[0058] Anti-CD19 antibody The use of CD19 antibodies in non-specific B-cell lymphoma is discussed in WO2007076950 (U.S. 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 disclosed in WO2005012493 (U.S. Patent No. 7,109,304), WO2010053716 (US 12 / 266,999) (Immunomedics); WO2007002223 (US 8097703) (Medarex); WO2008022152 (US 12 / 377,251) and WO2008150494 (Xencor), WO2008031056 (US 11 / 852,106) (Medimmune); WO 2007076950 (US 11 / 648,505) (Merck Patent GmbH); WO 2009 / 052431 (US 12 / 253,895) (Seattle), all of which are incorporated by reference in their entireties. Genetics); and WO2010095031(12 / 710,442) (Glenmark Pharmaceuticals), WO2012010562 and WO2012010561 (International Drug Development), WO2011147834 (Roche Glycart), and WO2012156455 (Sanofi).

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

[0061] The dose of an antibody or antibody fragment contained in a 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. Doses are usually calculated based on body weight, body surface area, age, or individual. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective dosages and schedules for administering pharmaceutical compositions containing antibodies or antibody fragments specific for CD19 may be determined empirically. For example, the patient's progress may be monitored by periodic evaluation, and the dosage may be adjusted accordingly. Furthermore, interspecies scaling of dosages may be performed 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 may be prepared by known methods. For example, the injectable preparation may be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Exemplary pharmaceutical compositions containing an antibody or antibody fragment specific to CD19 that can be used in the context of the present disclosure are disclosed, for example, in WO2008 / 022152 or WO2018 / 002031.

[0063] In certain administration methods, such as intravenous administration, it is preferable to administer the drug according to the patient's weight.In other administration methods, such as subcutaneous administration, it is preferable to administer the drug at a fixed dose.Those skilled in the art know which dose in one administration method is equivalent to another dose in another administration method.The rational decision to administer the drug as needed at the required dose from the required effective dose usually takes into account the pharmacodynamics of a specific drug.

[0064] The antibodies administered according to the present disclosure are administered to patients 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, i.e., 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 yet other embodiments, the antibodies of the present disclosure are administered at 15 mg / kg or greater.

[0065] The antibody of the present disclosure may be administered at different times, and the treatment cycle may have different lengths. The antibody may be administered daily, every other day, three times a week, weekly, or every other week. The antibody 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 a specific embodiment of the present disclosure, the antibody is 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 (e.g., peripheral blood) of a subject. 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. Vγ9Vδ2 T cells can be isolated from peripheral blood and further cultured ex vivo. 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 peripheral blood-derived Vγ9Vδ2 T cells: Peripheral blood is collected from donors. PBMCs are immediately isolated by density gradient centrifugation using Lymphoprep™ (Axis Shield, Norway) according to the manufacturer's instructions. PBMCs are cultured at a concentration of 1 × 10 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), and 100 IU / mL recombinant human interleukin-2 (rhIL-2) (BD Pharmingen, USA). 6 The cells were resuspended in 1000 x 10 cells / mL, activated with 5 µM ZOL, and seeded into 6-well plates at a cell culture density of 1–2 x 10 cells / mL. 6 Maintain cells at 0.5x100 cells / mL and replenish with fresh medium containing 100 IU / mL rhIL-2 only (no ZOL) every 2-3 days. After 7-8 days of culture, cells were collected and concentrated as described below.

[0068] Enrichment of Vγ9Vδ2 T cells: Ex vivo expanded Vy9V52 T cells were enriched using negative selection MACS with a TCRγ / δ+ T cell isolation kit (human) (Miltenyi Biotec, Germany). Cell viability and total cell number after enrichment were assessed using trypan blue exclusion. The percentage of Vy9V52 T cells was determined by flow cytometry using PeCy5-conjugated anti-CD3 (clone UCHT1) (eBioscience, San Diego, CA, USA) and FITC-conjugated anti-Vy9TCR from BD Biosciences (San Jose, CA, USA). The percentage of Vy9V52 T cells was identified by gating on the lymphocyte population using forward / side scatter and then gating on Vy9+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 in the presence of an aminobisphosphonate (e.g., zoledronic acid), synthetic phosphoantigen (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1PP, or 2-methyl-3-butenyl-1-pyrophosphate in the presence of IL-2 for 1-2 weeks to generate an enriched population of V52 cells. Alternatively, immobilized anti-TCRγδ (e.g., pan-TCRγδ) may induce preferential expansion of V52 cells from a population of PBMCs in the presence of IL-2 for, for example, about 14 days. In some embodiments, preferential expansion of V52 cells from PBMCs can be achieved upon culture with immobilized anti-CD3 antibodies (e.g., OKT3) in the presence of IL-2 and IL-4. In some embodiments, the aforementioned cultures are maintained for about 7 days before passaging in soluble anti-CD3, IL-2, and IL-4. Alternatively, artificial antigen-presenting cells may be used to promote preferential expansion of γδ T cells, such as V52 cells. For example, γδ T cells from PBMCs cultured in the presence of irradiated aAPCs, IL-2, and / or IL-21 may be expanded to generate a population of γδ T cells containing a high proportion of V52 cells, a moderate proportion of V51 cells, and some double-negative cells. In some embodiments of the foregoing methods, PBMCs may be pre-enriched or post-enriched (e.g., by positive selection with a TCRyd-specific agent or negative selection with a TCRa-specific agent). Such methods and other suitable methods for expanding γδ T cells, such as V52 cells, are described in detail in Deniger et al., Frontiers in Immunology 2014, 5, 636:1-10, which is incorporated herein by reference in its entirety. Furthermore, Almeida et al. (Clinical Cancer Research 2016, 22, 23; 5795-5805), which is incorporated herein by reference in its entirety, provides suitable methods for obtaining populations of V51 T cells that can be engineered to express the heterologous targeting constructs described herein.For example, in some embodiments, PBMCs are pre-enriched using magnetic bead sorting, which can yield greater than 90% γδ T cells.

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

[0071] γδ T cells present in non-hematopoietic tissues (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) may be isolated from any human or non-human animal non-hematopoietic tissue that can be obtained from a patient to provide cells suitable for manipulation by the methods of the invention. In some embodiments, the non-hematopoietic tissue (from which γδ T cells are obtained and expanded) is skin (e.g., human skin), which can be obtained by methods known in the art. In some embodiments, skin is obtained by punch biopsy. Alternatively, the methods for isolating and expanding 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, γδ T cells can be derived from human cancer tissues (e.g., solid tumor tissue). In other embodiments, γδ T cells can be derived from human non-hematopoietic tissues other than cancer tissues (e.g., tissues that do not have a substantial number of tumor cells). For example, γδ T cells can be derived from an area of ​​skin (e.g., healthy skin) away from nearby or adjacent cancer tissue.

[0072] The predominant γδ T cells in blood are primarily Vδ2 T cells, whereas the predominant γδ T cells in non-hematopoietic tissues are primarily Vδ1 T cells, with Vδ1 T cells comprising approximately 70–80% of the γδ T cell population present in non-hematopoietic tissues. However, some Vδ2 T cells are also found in non-hematopoietic tissues, such as the intestine, where they may comprise approximately 10–20% of γδ T cells. Some γδ T cells present in non-hematopoietic tissues express neither Vδ1 nor Vδ2 TCRs, and the inventors have termed them double-negative (DN) γδ T cells. These DN γδ T cells may be mostly Vδ3-expressing T cells, with a minority expressing Vδ5. Therefore, γδ T cells normally present in non-hematopoietic tissues and expanded by the methods of the present invention are preferably non-Vδ2 T cells, such as Vδ1 T cells, containing a small number of DN γδ T cells.

[0073] Generally, γδ T cells present in non-hematopoietic tissues can spontaneously proliferate when removed from physical contact with stromal cells (e.g., dermal fibroblasts). Therefore, the scaffold-based culture methods described above may be used to induce such detachment, resulting in de-inhibition of γδ T cells and subsequent proliferation. Thus, in some embodiments, there is no substantial TCR pathway activation during the expansion step (e.g., no exogenous TCR pathway activators are included in the culture). Furthermore, the present invention provides methods for expanding γδ T cells present in non-hematopoietic tissues, which methods do not involve contact with feeder cells, tumor cells, and / or antigen-presenting cells.

[0074] Treatment method As described herein, pharmaceutical compositions comprising unmanipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof can be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the compositions can be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. Unmanipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof can also be administered to reduce the likelihood of developing, acquiring, or worsening a condition. The effective amount of unmanipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof for therapeutic use can vary based on the severity and course of the disease or condition, previous treatments, the subject's health, weight, and / or response to drugs, and / or the judgment of the treating physician.

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

[0076] Methods for treating a condition in a subject using an enriched γδ T cell population and an antibody or antibody fragment specific for CD19 of the present disclosure can include administering a therapeutically effective amount of a non-manipulated enriched γδ T cell population, an engineered enriched γδ T cell population, and / or a mixture thereof to a subject. The enriched γδ T cell population of the present disclosure and / or a mixture thereof can be administered in various regimens (e.g., timing, concentration, dosage, interval between treatments, and / or formulation). The subject can also be pretreated, for example, with chemotherapy, radiation, or a combination of both, before receiving the enriched γδ T cell population and / or a mixture thereof of the present disclosure. As part of the treatment, a non-manipulated enriched γδ T cell population, an engineered enriched 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 with the first regimen meets a predetermined level of therapeutic benefit.

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

[0078] Administration method One or more non-manipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof of the present invention may be administered to a subject in any order or simultaneously. Multiple non-manipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof of the present invention may be administered simultaneously in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions, subcutaneous injections, or pills. Non-manipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof of the present invention may be packaged together or separately in a single package or multiple packages. One or all of the non-manipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof of the present invention may be administered in multiple doses. If not simultaneous, the timing between multiple administrations may vary by about one week, one month, two months, three months, four months, five months, six months, or even about one year. In some cases, the non-manipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof of the present invention may be expanded in vivo within the subject's body after administration to the subject. The non-manipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof may be frozen to provide cells for multiple treatments with the same cell preparation. The non-manipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof of the present disclosure, as well as pharmaceutical compositions comprising the same, may be packaged as kits. The kits may include instructions (e.g., written instructions) for using the non-manipulated, enriched γδ T cell populations, engineered, enriched γδ T cell populations, and / or mixtures thereof, as well as compositions comprising them.

[0079] In some cases, a method of treating cancer comprises administering a therapeutically effective amount of a non-engineered, enriched γδ T cell population, an engineered, enriched γδ T cell population, and / or a mixture thereof to a subject, wherein the administration treats the cancer. In some embodiments, the therapeutically effective amount of a non-engineered, enriched γδ T cell population, an engineered, enriched γδ T cell population, and / or a 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 a non-engineered, enriched γδ T cell population, an engineered, enriched γδ T cell population, and / or a mixture thereof is administered for at least 1 week. In some embodiments, a therapeutically effective amount of a non-engineered enriched γδ T cell population, an engineered enriched γδ T cell population, and / or a mixture thereof is administered for at least two weeks.

[0080] Embodiment The present disclosure provides a pharmaceutical combination comprising an antibody or antibody fragment specific for CD19 and gamma delta T cells (γδ T 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 (γδ T cells), wherein the γδ T cells comprise an enriched γδ T cell population, for use in the treatment of cancer. In one embodiment, the enriched γδ T cell population comprises unengineered or engineered γδ T 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 γδ T 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 (γδ T cells) for use in the treatment of cancer, wherein the γδ T cells comprise unengineered or engineered γδ T cells and / or a mixture thereof. In another embodiment, the γδ T cells are a population of unengineered γδ T cells. In yet another embodiment, the γδ T cells are a population of engineered γδ T cells.

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

[0084] In one aspect, the disclosure provides a pharmaceutical combination comprising an antibody specific for CD19 and gamma delta T cells (γδ T cells) for use in treating a cancer that is a hematological cancer. In one embodiment, the hematological cancer is chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL), or acute lymphoblastic leukemia (ALL). In another embodiment, the hematological cancer is non-Hodgkin's lymphoma (NHL). In a further embodiment, the non-Hodgkin's 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's lymphoma, and mantle cell lymphoma.

[0085] In certain embodiments, the present disclosure provides a pharmaceutical combination for use in treating cancer, comprising an antibody or antibody fragment specific for CD19 and gamma delta T cells (γδ T 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 yet other embodiments, it is 15 mg / kg or greater.

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

[0087] In certain embodiments, the present disclosure provides a pharmaceutical combination comprising a CD19-specific antibody or antibody fragment and gamma delta T cells (γδ T cells) for use in the treatment of cancer, wherein the components of the combination, the antibody CD19-specific antibody or antibody fragment and the γδ T cells, are administered separately. In certain embodiments, the γδ T cells are administered prior to the administration of the CD19-specific antibody or antibody fragment. In certain embodiments, the CD19-specific antibody or antibody fragment is administered prior to the administration of the γδ T cells. In embodiments, the components of the combination are administered at times when both components (drugs) are active in the patient at the same time. By "synergistic effect," it is implied that both drugs are effective in the patient at the same time. In embodiments, the components of the combination are administered together, simultaneously, separately, or sequentially, either physically or temporally. In embodiments, the components of the combination are administered simultaneously.

[0088] In certain embodiments, the present disclosure provides a pharmaceutical combination comprising an antibody or antibody fragment specific for CD19 and γδ T cells for use in the treatment of cancer, wherein the anti-CD19 antibody is administered weekly, biweekly, 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 said 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 comprising an antibody or antibody fragment specific for CD19 and γδ T cells for use in the treatment of cancer, wherein the antibody or antibody fragment specific for CD19 is administered weekly, biweekly, or monthly following an initial administration on day 1, and the BCL-2 inhibitor is first administered on day 8. In a further embodiment, after an initial administration on day 1, the anti-CD19 antibody or antibody fragment thereof is administered weekly for the first 3 months and biweekly for at least the next 3 months.

[0091] In one aspect, the present disclosure provides an anti-CD19 antibody or antibody fragment thereof for use in treating a patient with a hematological cancer, wherein said hematological cancer patient has non-Hodgkin's lymphoma, and wherein said anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T cells.

[0092] In one aspect, the present disclosure provides an anti-CD19 antibody or antibody fragment thereof for use in treating a patient with a hematological cancer, wherein the patient has non-Hodgkin's lymphoma, and wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T cells. In one embodiment, the patient has a hematological cancer with a non-Hodgkin's lymphoma, the non-Hodgkin's 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's lymphoma, and mantle cell lymphoma.

[0093] In one embodiment, the anti-CD19 antibody or antibody fragment thereof for use in treating a patient with hematological cancer 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 treating patients with hematological cancer 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: Contains DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 8).

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

[0096] In a further embodiment, the anti-CD19 antibody for use in treating patients with hematological cancers in combination with γδ T cells comprises a heavy chain having the sequence: EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) and a light chain having the sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[0097] In one embodiment, the anti-CD19 antibody or antibody fragment thereof for use in treating a patient with hematological cancer 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 that have 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 one embodiment, the anti-CD19 antibody or antibody fragment thereof for use in treating a patient with hematological cancer 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, 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 Fc numbering is according to the EU index as in Kabat.

[0099] In a further embodiment, the anti-CD19 antibody or antibody fragment thereof for use in treating a patient with hematological cancer in combination with γδ T cells comprises a heavy chain having the sequence: EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) and a light chain having the 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.

[0100] In a further embodiment, the anti-CD19 antibody or antibody fragment thereof for use in treating a patient with hematological cancer in combination with γδ T cells comprises a heavy chain having the sequence: EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) and a light chain having the 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, 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 Fc numbering is according to the EU index as in Kabat.

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

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

[0103] The present disclosure provides an antibody or antibody fragment specific for CD19 for use in treating cancer, wherein the antibody or antibody fragment specific for CD19 is administered in combination with γδ T cells. In one embodiment, the γδ T cells are isolated from peripheral blood, tumor tissue, or non-hematopoietic tissue. In one embodiment, the γδ T cells are isolated from peripheral blood. In another embodiment, the γδ T cells are a population of γδ T cells isolated from peripheral blood. In another embodiment, the γδ T cells are a population of γδ T cells isolated from peripheral blood and cultured in the presence of IL-2 and ZOL. In another example, the γδ T cells are a population of blood-derived Vγ9Vδ2 T cells.

[0104] The present disclosure provides an antibody or antibody fragment specific for CD19 for use in the treatment of cancer, wherein said antibody or antibody fragment specific for CD19 is administered in combination with γδ T cells, wherein said step of administering is performed simultaneously, sequentially, or in reverse order with the CD19-specific antibody and γδ T cells in combination.

[0105] In another embodiment, the present disclosure provides the use of a pharmaceutical combination comprising an antibody or antibody fragment specific for CD19 and γδ T 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 administering to a subject a combination of a CD19-specific antibody and γδ T cells. In another embodiment, the present disclosure provides a method for use in the treatment of cancer, the method comprising administering to a subject a combination of a CD19-specific antibody and γδ T cells, the administering being carried out by administering the CD19-specific antibody and γδ T cells in combination 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 (γδ T cells) for use in treating cancer, wherein the γδ T cells are administered in a therapeutically effective amount. In some embodiments, a therapeutically effective amount of lymphocytes (e.g., γδ T cells) obtained by any of the above methods may be administered to a subject (e.g., for the treatment of cancer, such as a hematological cancer) in a therapeutically effective amount. In some cases, a therapeutically effective amount of lymphocytes (e.g., γδ T cells) may be greater than 10×10 per dose. 12 Less than 9 x 10 cells (e.g., per dose) 12 Less than 8 × 10 cells per dose 12 Less than 7 × 10 cells per dose 12 Less than 6 × 10 cells per dose 12 Less than 5 × 10 cells per dose 12 Less than 4 × 10 cells per dose 12 Less than 3 x 10 cells per dose 12 Less than 2 x 10 cells per dose 12 Less than 1 x 10 cells per dose 12 Less than 9 × 10 cells per dose 11 Less than 8 × 10 cells per dose 11 Less than 7 × 10 cells per dose 11 Less than 6 × 10 cells per dose 11 Less than 5 × 10 cells per dose 11 Less than 4 × 10 cells per dose 11 Less than 3 x 10 cells per dose 11 Less than 2 x 10 cells per dose 11 Less than 1 x 10 cells per dose 11 Less than 9 × 10 cells per dose 10 Less than 7.5 × 10 cells per dose 10 Less than 5 × 10 cells per dose 10 Less than 2.5 × 10 cells per dose 10 Less than 1 x 10 cells per dose 10 Less than 7.5 × 10 cells per dose 9 Less than 5 × 10 cells per dose 9 Less than 2.5 × 10 cells per dose 9 Less than 1 x 10 cells per dose9 Less than 7.5 × 10 cells per dose 8 Less than 5 × 10 cells per dose 8 Less than 2.5 × 10 cells per dose 8 Less than 1 x 10 cells per dose 8 Less than 7.5 × 10 cells per dose 7 Less than 5 × 10 cells per dose 7 Less than 2.5 × 10 cells 7 Less than 1 x 10 cells per dose 7 Less than 7.5 × 10 cells per dose 6 Less than 5 × 10 cells per dose 6 Less than 2.5 × 10 cells per dose 6 Less than 1 x 10 cells per dose 6 Less than 7.5 × 10 cells per dose 5 Less than 5 × 10 cells per dose 5 Less than 2.5 × 10 cells per dose 5 Less than 1 x 10 cells or 1 x 10 per dose 5 (less than a cell).

[0108] In some embodiments, a therapeutically effective amount of γδ T cells (e.g., skin-derived γδ T cells, blood-derived γδ T cells) is greater than or equal to 10×10 over the course of treatment. 12 Less than 9 x 10 cells (e.g., over the course of treatment) 12 Less than 8 × 10 cells 12 Less than 7 × 10 cells 12 Less than 6 × 10 cells 12 Less than 5 × 10 cells 12 Less than 4 × 10 cells 12 Less than 3 × 10 cells 12 Less than 2 × 10 cells 12 Less than 1 x 10 cells 12 Less than 9 × 10 cells 11 Less than 8 × 10 cells 11 Less than 7 × 10 cells 11 Less than 6 × 10 cells 11 Less than 5 x 10 cells 11 Less than 4 × 10 cells 11 Less than 3 × 10 cells 11 Less than 2 × 10 cells 11 Less than 1 x 10 cells11 Less than 9 × 10 cells 10 Less than 7.5 × 10 cells 10 Less than 5 × 10 cells 10 Less than 2.5 × 10 cells 10 Less than 1 x 10 cells 10 Less than 7.5 × 10 cells 9 Less than 5 × 10 cells 9 Less than 2.5 × 10 cells 9 Less than 1 x 10 cells 9 Less than 7.5 × 10 cells 8 Less than 5 × 10 cells 8 Less than 2.5 × 10 cells 8 Less than 1 x 10 cells 8 Less than 7.5 × 10 cells 7 Less than 5 × 10 cells 7 Less than 2.5 × 10 cells 7 Less than 1 x 10 cells 7 Less than 7.5 × 10 cells 6 Less than 5 × 10 cells 6 Less than 2.5 × 10 cells 6 Less than 1 x 10 cells 6 Less than 7.5 × 10 cells 5 Less than 5 × 10 cells 5 Less than 2.5 × 10 cells 5 Less than 1 x 10 cells 5 (less than a cell).

[0109] In some embodiments, the dose of γδ T cells described herein includes 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 In some embodiments, the dose of γδ T cells described herein includes at least 1×10 cells / kg. 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 In some embodiments, the dose of γδ T cells described herein includes up to 1×10 cells / kg. 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.

[0110] combination The present disclosure provides an anti-CD19 antibody or antibody fragment thereof in combination with γδ T cells for use in treating hematological cancers, wherein the anti-CD19 antibody or antibody fragment thereof and the γδ T cells are administered in combination with one or more pharmaceutical agents. In one embodiment of the present disclosure, the anti-CD19 antibody or antibody fragment thereof and the γδ T cells are administered in combination with a pharmaceutical agent. In another embodiment of the present disclosure, the anti-CD19 antibody or antibody fragment thereof and the γδ T cells are administered in combination with one or more additional agents or pharmaceutical agents. In one aspect, the pharmaceutical agent is an additional pharmaceutical agent. In one embodiment of the present disclosure, the pharmaceutical agent is a biological or chemotherapeutic agent. In another embodiment of the present disclosure, the pharmaceutical agent is a therapeutic antibody or antibody fragment, a nitrogen mustard, a purine analog, a thalidomide analog, a phosphoinositide 3-kinase inhibitor, a BCL-2 inhibitor, or a Bruton's tyrosine kinase (BTK) inhibitor. In further embodiments, the pharmaceutical agent is rituximab, R-CHOP, cyclophosphamide, chlorambucil, uramustine, 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 antibody fragment thereof and γδ T cells for use in treating hematological cancer, wherein the anti-CD19 antibody or antibody fragment thereof and γδ T cells are administered in combination with rituximab, R-CHOP, cyclophosphamide, chlorambucil, uramustine, 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 on 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 and primary patient-derived tumor material from CLL (chronic lymphocytic leukemia), MCL (mantle cell lymphoma), and B-ALL (acute lymphoblastic leukemia). Furthermore, the antibody-dependent cell-mediated cytotoxicity activity of tafasitamab in the presence of γδ T cells was evaluated.

[0114] Various lymphoma and leukemia cell lines, as well as primary tumor cells from lymphoma and leukemia patients, were evaluated in antibody-dependent cellular cytotoxicity (ADCC) assays 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 of 0.7:1, 2.2:1, 6.7:1, and 20:1).

[0115] Materials, methods, and data analysis γδT 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 unlimited and high combinatorial diversity, the Vδ2 chain is preferentially paired with the Vγ9 chain. Vγ9Vδ2 T cells account for approximately 5% of peripheral blood T cells and represent the major γδ T cell subpopulation of this compartment. γδ T cells from seven different donors were isolated, stimulated, and stimulated, as listed below. [Table 2] As listed in the table, the stimulated cell population used in the described experiments consisted of three major populations. Only a small proportion of CD56+ / CD3- NK effector cells were present (<5%), and although TCRγδneg / CD3+ cells represented a larger proportion in some cases, these cells did not cause target cell lysis. Therefore, γδ T cells were primarily responsible for antibody-mediated cell killing.

[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] result Dose titration with lymphoma cell lines Initial dose titration experiments to obtain a valid assay configuration were performed using MOR00208 at concentrations ranging from 0.001 to 10 μg / ml in the presence of four E:T ratios of γδ T cells (0.7:1, 2.2:1, 6.7:1, and 20:1). A dose-dependent increase in the percentage of lysed Jeko and U2932 target cells was observed. As expected, the killing activity of the tested antibodies increased with higher E:T ratios, with the most pronounced effect observed at the highest E:T ratios tested, 6.7:1 and 20:1. Maximum cell lysis was achieved at MOR00208 concentrations of 0.1 μg / ml, 1 μg / ml, and 10 μg / ml for the two tested cell lines. Cell lysis showed γδ T cell-dependent donor variation, with maximum lysis ranging from 34.0% to 49.3% for Jeko and 22.5% for 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 effects of antibody-mediated cell killing by γδ T 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 potential of γδ T cells in combination with a CD19 antibody, tafasitamab was tested at 1 μg / ml using Mino, Daudi, Jeko, U2932, and REH cells as target cells (Figures 1 and 2). As before, four different E:T ratios between 0.7:1 and 20:1 were tested. 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 dose titration experiments, the killing activity of the tested antibodies increased, with the most pronounced effect observed at the highest E:T ratios tested, 6.7:1 and 20:1. Notably, the two cell lines with the lowest nonspecific killing (Mino and Jeko) exhibited the highest specific contribution of the antibody to cell-killing activity. In both cell lines, MOR00208 activity was statistically significantly different from the IgG1 control (Figure 1). In the three cell lines with higher nonspecific killing, the activity profile was similar (MOR00208 > IgG1 control), but the specific effect was limited (Figure 2).

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

[0120] conclusion In summary, γδ T cells have been identified as a potential effector cell population in antibody-based tumor therapy, as demonstrated in this study with the Fc-enhanced CD19-targeting antibody MOR00208. MOR00208 exhibited potent antitumor activity mediated by the presence of γδ T cells against several lymphoma and leukemia cell lines, as well as primary patient-derived CLL, MCL, and B-ALL cells, providing a reasonable rationale for combining MOR00208 with γδ T cells as a promising approach for lymphoma and leukemia therapy.

Claims

1. 1. An anti-CD19 antibody or antibody fragment thereof for use in treating a hematological cancer, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T cells.

2. 2. The anti-CD19 antibody or antibody fragment thereof of claim 1 for use as described in claim 1, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T cells, and the γδ T cells comprise an enriched γδ T cell population.

3. 10. The anti-CD19 antibody or antibody fragment thereof according to any one of the preceding claims for use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T cells, wherein the γδ T cells comprise non-engineered or engineered γδ T cells and / or mixtures thereof.

4. 10. The anti-CD19 antibody or antibody fragment thereof according to any one of the preceding claims for use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T-cells, the γδ T-cells being isolated from peripheral blood, tumor tissue or non-hematopoietic tissue for use in the treatment of cancer.

5. 10. The anti-CD19 antibody or antibody fragment thereof according to any one of the preceding claims for the use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T cells, the γδ T cells being isolated from peripheral blood and cultured in the presence of IL-2 and ZOL.

6. 10. The anti-CD19 antibody or antibody fragment thereof according to any one of the preceding claims for the use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T cells, and the γδ T cells are blood-derived Vγ9Vδ2 T cells.

7. 10. The anti-CD19 antibody or antibody fragment thereof according to any one of the preceding claims for use in the treatment of a hematological cancer, wherein the hematological cancer is chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL), or acute lymphoblastic leukemia (ALL), and wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with a γδ T-cell according to any one of the preceding claims.

8. 10. The anti-CD19 antibody or antibody fragment thereof according to any one of the preceding claims for the use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T-cells according to any one of the preceding claims, wherein the anti-CD19 specific antibody or antibody fragment thereof and the γδ T-cells are administered in separate ways.

9. 10. The anti-CD19 antibody or antibody fragment thereof according to any one of the preceding claims for the use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T-cells according to any one of the preceding claims, wherein the anti-CD19 specific antibody or antibody fragment thereof and the γδ T-cells are administered in a simultaneous manner.

10. 10. The anti-CD19 antibody or antibody fragment thereof according to any one of the preceding claims for use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with a γδ T-cell according to any one of the preceding claims, and wherein the anti-CD19 antibody or antibody fragment thereof 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).

11. 11. The anti-CD19 antibody or antibody fragment thereof according to claim 10 for use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with a γδ T-cell according to any one of the preceding claims, and wherein the anti-CD19 antibody or antibody fragment thereof comprises a heavy chain variable region: EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS (SEQ ID NO: 7) and the following light chain variable region: The anti-CD19 antibody or antibody fragment thereof, comprising: DIVMTQSPATHLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 8).

12. 12. The anti-CD19 antibody according to claim 11 for the use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T-cells according to any one of the preceding claims, and wherein the anti-CD19 antibody has a heavy chain EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTK YNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPE LLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

13. 12. The anti-CD19 antibody or antibody fragment thereof according to claim 11 for the use according to any one of the preceding claims, wherein the anti-CD19 antibody or antibody fragment thereof is administered in combination with γδ T-cells according to any one of the preceding claims, and wherein the anti-CD19 antibody has a light chain The anti-CD19 antibody or antibody fragment thereof comprises DIVMTQSPATHLSPGERATTLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

14. 10. A kit for treatment according to any one of the preceding claims, comprising an anti-CD19 antibody or antibody fragment thereof according to any one of the preceding claims and instructions for administering said anti-CD19 antibody or antibody fragment thereof in combination with γδ T-cells according to any one of the preceding claims.

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