CAR T cells for the treatment of CD1a-positive cancer

Abstract

To provide therapeutic methods for the treatment of CD1a-positive T-ALL.SOLUTION: CD1a is exclusively expressed in cortical T-ALLs, a major subset of T-ALL. The expression of CD1a is restricted to cortical thymocytes and neither CD34+ progenitors nor T-cells express CD1a during ontogeny, confining the risk of on-target / off-tumor toxicity. The present invention provides CARs comprising a CD1a-targeting moiety which may be transduced or transformed into T cells. The resultant CARTs are suitable for the treatment of cortical T-ALLS.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a therapeutic method for treating CD1a-positive cancers such as T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. In particular, the present invention provides chimeric antigen receptor (CAR) T cells that can target CD1a. [Background technology]

[0002] T-cell lineage acute lymphoblastic leukemia (T-ALL) is a malignant disorder caused by leukemic transformation of thymic T-cell precursors. 1 T-ALL is phenotypic and genetically heterogeneous and is generally associated with genetic alterations / mutations of transcription factors involved in hematopoietic stem / progenitor cell (HSPC) homeostasis and major regulators of T cell development. 2 T-ALL accounts for 10%–15% and 20%–25% of all acute leukemias diagnosed in children and adults, respectively. 3、4 The median age at diagnosis is 9 years. 5~7 Intensive chemotherapy regimens have led to improved survival in T-ALL patients. However, event-free survival (EFS) and overall survival (OS) remain below 70%, and relapsed / refractory (R / R) T-ALL, in particular, has a poor outcome. Currently, there are no more viable treatment options than hematopoietic stem cell transplantation and conventional chemotherapy, which are associated with significant toxicity trade-offs. 4、8 Therefore, there is a growing need for new targeted therapies. T-cell lymphoblastic lymphoma (TCL) is etiologically and pathogenically different from T-ALL, but phenotypically very similar. The main difference is that TCL is found extramedullary, while T-ALL is a bone marrow disease.

[0003] Immunotherapy has generated unprecedented expectations in cancer treatment, relying on the immune system as a powerful weapon against cancer. In recent years, adoptive cell immunotherapy based on chimeric antigen receptors (CARs) has shown great potential. In CAR therapy, genetically modified T cells are redirected to specifically recognize and eliminate tumor cells expressing specific antigens, independently of the major histocompatibility complex.9、10 The success of redirecting chimeric antigen receptor (CAR) T cells (CART) against CD19 or CD22 is now beyond debate for B cell malignancies, mainly B-ALL. 11~14 However, strategies targeting T cell malignancies with CART remain difficult due to the co-expression of target antigens between CART and T lineage tumor cells. In this regard, CART against pan-T cell antigens has two major drawbacks: i) self-targeting / fratricide of CART and ii) T cell aplasia leading to lethal immunodeficiency. 15~17 。

[0004] Recent accurate studies have demonstrated that T cells transduced with any of the most highly expressed pan-T cell antigens, CD7, CD3, CD5, or TCR CAR, can efficiently eliminate T-ALL blasts in vitro and control the disease in vivo. 15~20 Nevertheless, approaches such as CRISPR / Cas9 genome editing or protein expression blockers are still far from clinical use and require the destruction of target antigens in T cells prior to CAR transduction to avoid fratricide induced by broad self-antigens. 15~17、19 。

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, there is still a need for a therapy that can successfully treat T-ALL. The present invention aims to provide a therapy for treating CD1a-positive T-ALL.

Means for Solving the Problems

[0006] The selection of antigens for which T cell redirection is desired is between normal T cells and malignant T cells Shows a major advancement in solving problems related to the co-expression of T cell markers. The lipid presenting molecule CD1a was identified as a suitable target for the treatment of a large subset of T-ALL, namely cortical T-ALL.

[0007] Developed and functionally characterized CD1a-specific CARTs that show strong cytotoxicity against T-ALL cell lines and primary cortical CD1a+ T-ALL cells in both in vitro and in vivo xenograft models. CD1a CARTs were demonstrated to expand continuously up to 200-fold, similar to mock T cells, and that redirection of CARTs against the CD1a antigen does not induce T cell fratricide. Also, the use of CD1a CARTs for cortical T-ALL eliminates the need for sophisticated genome editing to disrupt the target antigen in T cells prior to CAR transduction as a strategy to avoid fratricide induced by self-antigens. 15~17、19 . In steady-state hematopoiesis, CD1a is expressed only in a subset of cortical CD34+CD7+ thymic T precursors, and more primitive CD34 high CD7 high T precursors were further demonstrated to lack CD1a. Additionally, normal CD34+ HSPCs and mature T cells from multiple tissues do not express CD1a during ontogeny, minimizing the risk of on-target / off-tumor toxicity. Indeed, exposure of CD7+ thymocytes from human fetal thymus to CD1a CARTs results in the elimination of only CD1a+ cortical thymocytes, and the more primitive and later thymic T lineage populations (CD34+ and CD34-) are not targeted, limiting the on-target / off-tumor effect to the developmentally transient thymic population of cortical thymocytes, further confirming the fratricide resistance of CD1a CARTs.

[0008] The exclusive thymic localization of cortical thymocytes and the fact that a thymic subpopulation of CD34+CD7+CD1a-T cell precursors that physiologically / constantly mature into functional T cells reside upstream of CD1a+ cortical thymocytes provide an additional level of safety for the use of CD1a CART in R / R T-ALL patients. Irreversible toxicity or severe T cell dysplasia attributable to CD1a CART is not expected for the following reasons: i) the CD1a+ thymocyte population is a transient thymic T cell fraction that is ultimately regenerated by the upstream CD1a-T cell precursors; ii) CD1a CART itself normally responds to viral antigens and therefore may prevent the pathogen; iii) clinical use of specific antibodies against CD5 or CD7. 42 iv) There are several studies demonstrating extrathymic maturation of T cells and a balance between the innate and adaptive immune systems that can at least partially ensure immunological protection in patients who have undergone partial or complete thymectomy. 45~47 .

[0009] Therefore, in one embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular domain containing a CD1a targeting moiety, a transmembrane domain, and an intracellular signaling domain.

[0010] The present invention also provides nucleic acids encoding the CAR of the present invention. Furthermore, the present invention provides cells comprising the nucleic acids and / or CAR of the present invention. The present invention also provides a pharmaceutical composition comprising a plurality of cells according to the present invention and a pharmaceutically acceptable carrier or diluent.

[0011] The present invention provides cells or pharmaceutical compositions used as pharmaceuticals. In particular, the present invention provides a method for treating CD1a-positive cancer, comprising administering the cells or pharmaceutical compositions of the present invention to a patient in need thereof. [Brief explanation of the drawing]

[0012] [Figure 1]This figure shows CD1a expression in T-ALL, as well as normal hematopoiesis and thymocyte proliferation. (A) Immunophenotypes of de novo T-ALL samples (n=38) against specified markers. The upper and middle curly braces identify CD1a+ / ++ and CD1alow / + coT-ALL patients, respectively. The black dots at the bottom represent non-coT-ALL patients. (B) Representative FACS dot plots of coT-ALL patients. CD7+CD1a+ cells are coT-ALL blasts, and CD3+CD7+CD1a- (either CD4+ or CD8+) are normal mature T cells present in diagnostic samples. (C) CD1a is retained at relapse (n=5 diagnostic-relapsed coT-ALL pairs). Data are shown as CD1a expression in relapsed samples compared to diagnostic-fit samples (diagnosis is shown as 100% expression). (D) T cells and CD34+HSPCs do not express CD1a throughout ontogeny. (E) A scheme showing the phenotype of a developing thymic T cell population. (F) Representative FACS for pre-cortical (CD34-high CD7++ CD1a-) and cortical (CD34+ CD7++ CD1a+) thymocytes. DX: Diagnosis. RX: Relapse. [Figure 2]This figure shows that CD1a CART specifically targets and eliminates CD1a+ T-ALL cell lines in vitro. (A) Scheme of the CD1aCAR construct used. (B) CAR detection in 293 T cells using anti-scFv MoAb and GFP. (C) Representative CAR transduction and detection in CD4+ and CD8+ T cells (n=6). (D) Appropriate T cell activation (n=3). (E) Strong increase in activated T cells transduced with either mock or CD1a CAR without signs of flutorides (n=4). (F) Surface expression of CD1a (black line) in Jurkat, MOLT4 and NALM6 cell lines. (G) CD1a antigen density in cell lines, primary coT-ALL samples and primografts. (H) Cytotoxicity of CD1a CART and mock T cells against coT-ALL and B-ALL cell lines at specified E:T ratios in a 16-hour assay (n=4). (I) Absolute number of viable eFluor+ target cells measured by FACS in a 72-hour cytotoxicity assay with a 1:1 E:T ratio. (J) Representative FACS analysis of cytotoxicity by target cells labeled with eFluor670. (K) ELISA (n=4) showing high levels of production of inflammatory cytokines IL-2, TNFα, and IFNγ by CD1a CART exposed to Jurkat and NALM6 (negative control) cells in a 16-hour assay with a 1:1 E:T ratio. *p<0.05, **p<0.01, ***p<0.001. [Figure 3]This figure shows that CD1a CART specifically targets and eliminates CD1a+ T-ALL blasts derived from primary samples or PDX models in vitro. (A) CD1a vs. CD7 expression in coT-ALL blasts derived from primary patient / primograft. Shows % of CD1a+ blasts. (B) Cytotoxicity (in absolute number of eFluor+ cells) measured by FACS in a 48-hour cytotoxicity assay at a 4:1 E:T ratio (n=3). (C) Representative FACS analysis of CD1a in eFluor-labeled target cells at the end of the cytotoxicity assay to reveal the specificity of CD1a CART (n=3). (D) High levels of pro-inflammatory cytokine production by CD1a CART analyzed by ELISA in a 16-hour assay at a 4:1 E:T ratio (n=3 independent supernatants). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4] This figure shows that CD1a CART completely controls the progression of coT-ALL cells in a mouse xenograft setting. (A) Scheme of the xenograft model. NSG mice (n=6 mice / group) were intravenously injected with 3 × 10⁶ Luc-GFP-expressing Jurkat cells, followed by a single intravenous injection of 5 × 10⁶ mock cells or CD1a CART 3 days later. Tumor burden was monitored every 4 to 6 days by bioluminescence (BLI) using IVIS imaging. When mock-treated animals developed complete leukemia, half of the CD1a CART-treated animals were sacrificed, and leukemia burden and CART persistence were analyzed by FACS (BM, PB, and spleen). The remaining animals were rechallenged with 1.5 × 10⁶ Luc-Jurkat cells 6 weeks later and followed in the same manner. (B) IVIS imaging of tumor burden monitored by BLI at specified time. (C) Total radiance quantification (p / sec / cm² / sr) at specified time. †: Sacrifice. (D) Circulating Jurkat cells in the spleen 17 days after CART infusion. (E) T cell persistence in the spleen and mb at day 17, as well as at sacrifice. Data are shown as mean ± SD (n=6 mice / group). *p<0.05, **p<0.01, ***p<0.001. [Figure 5]This figure shows that CD1a CART completely eliminates the progression of primary CD1a+coT-ALL blasts in a PDX (Patient-Developmental X-ray) situation. (A) Scheme of the PDX model. NSG mice (n=5 or 6 mice / group) were intravenously injected with 1 × 10⁶ primary coT-ALL cells, followed by a single intravenous injection of 1 × 10⁶ mock or CD1a CART cells 3 days later. Tumor burden was monitored by FACS every two weeks, and by blood sampling and BM aspiration at 6 and 9 weeks. (B, C) Frequency of leukemia mice, and the level of leukemia in BM (B) and PB (C) 6 and 9 weeks after CART infusion. The left panel shows representative FACS plots. Primary CD1a+T-ALL blasts are shown in the box (gray). Effector T cells are shown outside the box in gray. Mouse cells are shown in black. (D) 9-week OS of coT-ALL primografts given either CD1a CART or mock T cells. (E) Time course of effector T cell persistence in PB (weeks 2-9) and BM (weeks 6 and 9). Each dot represents an independent mouse. **p<0.01, Malcolm-Cox test. [Figure 6]This figure shows that CD1a CART retains the ability to control the progression of CD1a+ cell lines and coT-ALL primary samples in rechallenge PDX situations. (A) IVIS imaging of Jurkat cell loading in rechallenged mice. (B) Quantification of total radiance over time (p / sec / cm2 / sr) in mice rechallenged with Jurkat cells. (C) Circulating Jurkat cells in PB 16 days after rechallenge. (D) Strong effector T cell persistence in PB, BM and spleen at sacrifice of rechallenged animals. (E) Scheme of a rechallenge PDX experiment using coT-ALL primary samples. CART-carrying PDX mice were rechallenged with 1 × 10⁶ primary CD1a+ T-ALL cells 7 weeks after initial CART infusion. (F) Secondary coT-ALL loading in engrafted PB (left panel) and BM (right panel) 6 weeks after leukemia rechallenge. Time course of effector T cell persistence in PB (weeks 2, 4, and 6) derived from PDX re-challenged with (G)coT-ALL primary samples. Each dot represents an independent mouse. *p<0.05, **p<0.01, ***p<0.001, ****<0.0001. [Figure 7] This figure shows that CD1a CART derived from a coT-ALL patient at the time of onset specifically lyses autologous CD1a+T-ALL blasts. (A) Scheme showing the experimental design of the autologous cytotoxicity assay. Mature (normal) CD3+CD1a-T cells were FACS purified from PB of coT-ALL patients, infected with CD1a CART, augmented, and exposed to autologous whole PBMCs. (B) FACS analysis of 48-hour autologous cytotoxicity assays in 1:1 and 4:1 E:T. The eFluor670-labeled whole PBMC target population contains CD1a+T-ALL blasts (top box) and mature CD3+CD1a-T cells (bottom box). (C) Quantification of CD1a CART-mediated specific lysis for coT-ALL blasts (top panel) and CD3+CD1a-mature T cells (bottom panel). (D) ELISpot showing the number of IFNγ SFCs from mock vs CD1a CART stimulated with a pool of peptides derived from CMV, EBV, and influenza (CEF). Staphylococcal enterotoxin B (SEB) was used as a positive control. [Figure 8] This figure shows the immunophenotypes of each individual CD1a++coT-ALL patient presented in this study. (A) Gating strategies to distinguish between mature normal T cells (CD3++CD1a-, either CD4+ or CD8+) and coT-ALL blasts (CD7+CD1a+). Note that coT-ALL blasts generally have abnormal expression of CD3 and / or CD4 / CD8. (B) FACS dot plots of CD7 / CD3 vs. CD1a for n=16 available CD1a++coT-ALL patients, showing the percentage of mature normal T cells (left quadrant) and coT-ALL blasts (right quadrant). [Figure 9] This figure shows the in vitro specificity of CD1a CART. (A) Scheme of CD1aCAR, CD22CAR, and mock constructs used in this study. (B) CD1a CART lyses the T-ALL cell line Jurkat, but CD22 CART does not lyse Jurkat. CD22 CART lyses the B-ALL cell line NALM6, but CD1a CART does not lyse NALM6. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 10] This figure shows the dose-dependent in vivo cytotoxicity of CD1a CART. (A) Tumor burden monitored by BLI at the start of the experiment (scale: 3 × 10⁴ p / sec / cm² / sr ~ 1 × 10⁵ p / sec / cm² / sr), confirming early and efficient T-ALL engraftment. (B) IVIS imaging of tumor burden monitored by BLI at specified time points for CART doses of 2 × 10⁶ p / sec / cm² / sr and 5 × 10⁶ p / sec / cm² / sr. (C) Total radiance quantification (p / sec / cm² / sr) at specified time points for CART doses of 2 × 10⁶ and 5 × 10⁶. N = 3 or 4 mice / group. †: sacrifice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. [Figure 11]This figure shows that CD1a CART does not target CD7+CD1a- thymocytes. Cytotoxicity assays against fetal thymocytes were performed on CD1a CART and mock T cells in a 4:1 E:T ratio at 16 and 72 hours (n=2). [Figure 12] This figure shows that the absolute number of CD1a-primary coT-ALL cells remained the same after exposure to either mock or CD1a CART. This confirms that CD1a expression was not lost or downregulated by immunosuppression. [Modes for carrying out the invention]

[0013] definition "Administering" a drug to a patient or "administering" a drug to a patient (and grammatically equivalent expressions) refers to direct administration, which may be administration by a medical professional to the patient or self-administration, and / or indirect administration, which may be the act of prescribing a drug. For example, instructing a patient to self-administer a drug, or a doctor prescribing a drug to a patient administering the drug to the patient.

[0014] The term "affibody" is derived from the Z domain of protein A and refers to a protein that has been modified to bind to a specific target (see Frejd & Kim, 2017. Exp Mol Med. 49(3): e306).

[0015] The term "antibody" refers to a molecule that contains at least one immunoglobulin domain that binds to a specific target or is immunologically reactive with a specific target. This term includes all antibodies and any antigen-binding portion or single-chain thereof, as well as combinations thereof. For example, the term "antibody" specifically includes bivalent antibodies and bivalent bispecific antibodies.

[0016] A typical antibody consists of at least two heavy chains ("HC") and two light chains ("LC") interconnected by disulfide bonds.

[0017] Each "heavy chain" includes a "heavy chain variable domain" (abbreviated as "VH" herein) and a "heavy chain steady domain" (abbreviated as "CH" herein). The heavy chain steady domain typically contains three steady domains CH1, CH2, and CH3.

[0018] Each "light chain" includes a "light chain variable domain" (abbreviated herein as "VL") and a "light chain steady domain" ("CL"). The light chain steady domain (CL) may be κ-type or λ-type. The VH domain and VL domain can be further subdivided into hypervariable regions called complementarity-determining regions ("CDR"), which contain more conserved regions called "framework regions" ("FW").

[0019] VH and VL each consist of three CDRs and four FWs arranged in the following order from the amino terminus to the carboxyl terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, and FW4. This disclosure specifically presents the VH sequence and the VL sequence, as well as the subsequences corresponding to CDR1, CDR2, and CDR3.

[0020] The precise amino acid sequence boundaries of a given CDR are described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme) and Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme). This can be determined using one of a number of known schemes, including the one mentioned above.

[0021] Therefore, it will be understood by those skilled in the art that the sequences of FW1, FW2, FW3, and FW4 are equally disclosed. For a given VH, FW1 is a subsequence between the N-terminus of VH and the N-terminus of H-CDR1, FW2 is a subsequence between the C-terminus of H-CDR1 and the N-terminus of H-CDR2, FW3 is a subsequence between the C-terminus of H-CDR2 and the N-terminus of H-CDR3, and FW4 is a subsequence between the C-terminus of H-CDR3 and the C-terminus of VH. Similarly, for a given VL, FW1 is a subsequence between the N-terminus of VL and the N-terminus of L-CDR1, FW2 is a subsequence between the C-terminus of L-CDR1 and the N-terminus of L-CDR2, FW3 is a subsequence between the C-terminus of L-CDR2 and the N-terminus of L-CDR3, and FW4 is a subsequence between the C-terminus of L-CDR3 and the C-terminus of VL.

[0022] The variable domains of the heavy and light chains contain regions that interact with binding targets, which are also referred to herein as "antigen-binding sites" or "antigen binding sites." The constant domains of the antibody can mediate the binding of the antibody to various cells of the immune system (e.g., effector cells) and to host tissues or host factors, including the first component (C1q) of the classical complement system. Exemplary antibodies of this disclosure include not only typical antibodies but also their bivalent fragments and variants, such as F(ab')2.

[0023] As used herein, the term “antibody” includes intact polyclonal antibodies, intact monoclonal antibodies, bivalent antibody fragments (such as F(ab')2), multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecules containing antigen-binding sites.

[0024] Antibodies can be any of the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, which are respectively designated as α, δ, ε, γ, and μ. Different classes of immunoglobulins have different known subunit structures and three-dimensional arrangements. Antibodies may be naked or conjugated with other molecules, such as therapeutic or diagnostic agents, to form immune complexes.

[0025] The term "anticalin" is derived from lipocalin and refers to a specific target. This refers to a protein that has been modified to conform to (Skerra, 2008. FEBS J. 275(11):2677-83). (See reference).

[0026] The term "antigen-binding fragment" or "Fab" refers to an antibody fragment containing one constant domain and one variable domain in both the heavy and light chains. Fab fragments can be obtained by digesting intact monoclonal antibodies with papain.

[0027] The term "cancer" refers to a state of uncontrolled, usually rapid, cell proliferation that lacks physiological function. This refers to a group of diseases that can be defined as any abnormal, benign or malignant neoplasm of tissue that arises and may invade or metastasize to other parts of the body.

[0028] The term "CD1a" refers to a non-polymorphic MHC class 1-associated cell surface glycoprotein expressed in association with β2-microglobulin. CD1a is expressed by cortical thymocytes, Langerhans cells, and finger-shaped incarcerated cells. CD1a is also expressed in certain T-cell lineage malignancies and Langerhans cell histiocytosis. CD1a is expressed in cortical thymocytes, epidermal Langerhans cells, dendritic cells, certain T-cell leukemias, and various other tissues. CD1a is structurally related to major histocompatibility complex (MHC) proteins and forms a heterodimer with β2-microglobulin. Exemplary sequences and data for human CD1a are deposited in the UniProtKB database under ID P06126.

[0029] "CD1a-positive" cancers, including "CD1a-positive" cancerous diseases, are cancers that include cells on which CD1a is present on the cell surface. The term "CD1a-positive" also refers to cancers in which cells containing the CAR of the present invention produce sufficient levels of CD1a on the cell surface to have a therapeutic effect mediated by the binding of the CAR to CD1a. In some embodiments, CD1a-positive cancers are cortical T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma or Langerhans cell histiocytosis (LCH).

[0030] The term "CD1a targeting moiety" refers to a substance capable of binding to CD1a. In relation to CARs, the CD1a targeting moiety targets T cells to CD1a-positive cells, preferably cancer cells. In relation to CARs, it should be understood that the CD1a targeting moiety is genetically encoded.

[0031] The term "chimeric antigen receptor" or "CAR" refers to a synthetic receptor that targets T cells to a selected antigen and reprograms the function, metabolism, and persistence of T cells (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124). Similarly, "CART" The term refers to T cells that contain CARs.

[0032] When used herein, “combination therapy,” “in combination with,” or “in combination with” refers to any form of combination, parallel, simultaneous, sequential, or intermittent therapy involving at least two different therapeutic modalities (i.e., compounds, components, targeting agents, or therapeutic agents). Therefore, these terms refer to the administration of the other therapeutic modality before, during, or after the administration of one therapeutic modality to the subject. Modalities in combination may be administered in any order. Therapeutic modalities are administered together (e.g., simultaneously in the same or distinct compositions, formulations, or unit dosage forms) or separately (e.g., on the same or different days, in any order according to an appropriate administration protocol for distinct compositions, formulations, or unit dosage forms) in accordance with the methods and administration plans prescribed by healthcare professionals or regulated by regulatory bodies. Generally, each therapeutic modality is administered at the dose and / or schedule determined for that therapeutic modality. Optionally, three or more modalities may be used in combination therapy. Furthermore, the combination therapies provided herein may be used in combination with other types of therapy. For example, other anti-cancer treatments may be selected from a group of therapies that are relevant to the current standard of care for the subject, including chemotherapy, surgery, radiotherapy (radiation), and / or hormone therapy.

[0033] "Complete response," "complete remission," or "CR" refers to the disappearance of all target lesions as defined in the RECIST v1.1 guidelines. This does not necessarily mean that the cancer is cured.

[0034] The term "co-stimulus signaling domain" refers to, for example, the CD3 of the TCR / CD3 complex. The term refers to the signaling region that provides signals to T cells that mediate T cell responses, including but not limited to activation, proliferation, differentiation, and cytokine secretion, in addition to the primary signal provided by the ζ chain. The costimulatory domain may include, but is not limited to, all or some ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, the costimulatory signaling domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate cellular responses, including activation, proliferation, differentiation, and cytokine secretion.

[0035] The terms "designed ankyrin repeat proteins" or "DARPin" refer to proteins derived from ankyrin repeats that have been modified to bind to specific targets (see Plueckthun, 2015. Annu Rev Pharmacol Toxicol. 55:489-511).

[0036] "Disease-free survival" (DFS) refers to the period during and after treatment in which a patient remains disease-free.

[0037] As used herein, the term “effective dose” of an active substance, such as a therapeutic agent like CART, is a sufficient amount to produce a beneficial or desired result, such as a clinical outcome, and therefore the “effective dose” depends on the context in which it is applied. For example, in the context of administering a therapeutic agent to treat T-ALL, an effective dose may reduce the number of cancer cells, reduce tumor size or burden, inhibit (i.e., delay, and in certain embodiments, halt) the invasion of cancer cells into peripheral organs, inhibit (i.e., delay, and in certain embodiments, halt) tumor metastasis, inhibit tumor growth to some extent, alleviate one or more cancer-related symptoms to some extent, and / or produce a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or possibly stable disease (SD), reduced progressive disease (PD), shortened time to progression (TTP), or any combination thereof. The term "effective dose" can be used interchangeably with "effective dosage," "therapeutic effective dose," or "therapeutic effective dose."

[0038] The term "fynomer" refers to the SH3 domain of human Fyn kinase. This refers to proteins that have been modified to bind to specific targets (Bertschinger et al., See 2007. Protein Eng Des Sel. 20(2):57-68.

[0039] The terms “individual,” “patient,” or “subject” are used interchangeably in this application to designate a human being and are not intended to be limiting. “Individual,” “patient,” or “subject” may be of any age, sex, and physical condition. The term “patient requiring it” typically refers to a patient with CD1a-positive cancer.

[0040] "Injection" or "to inject" refers to the introduction of a solution containing a therapeutic agent into the body via a vein for therapeutic purposes. Generally, this is achieved using an intravenous bag.

[0041] As used herein, "intracellular signaling domain" refers to all or part of one or more domains of a molecule (here, a chimeric receptor molecule) that results in lymphocyte activation. The intracellular domain of such a molecule mediates signaling by interacting with cellular mediators, resulting in proliferation, differentiation, activation, and other effector functions. Examples of intracellular signaling domains used in the CAR of the present invention include intracellular sequences of the CD3ζ chain and / or co-receptors that act in coordination to initiate signaling after CAR ligation, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same function. This includes the following. T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation and provide T cell receptor-like signals (primary cytoplasmic signaling sequences) and sequences that act antigen-independently and provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). Stimulatory primary cytoplasmic signaling sequences may contain signaling motifs known as receptor tyrosine activation motifs or ITAMs. Examples of primary cytoplasmic signaling sequences containing ITAMs include those derived from CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0042] The term "monobody" is derived from the fibronectin type III domain and refers to a protein that has been modified to bind to a specific target (see Koide et al., 2013. J Mol Biol. 415(2):393-405).

[0043] The term "nanobody" refers to a protein containing a soluble single antigen-binding V domain of a heavy chain antibody, preferably a camelid heavy chain antibody (see Bannas et al., 2017. Front Immunol. 8:1603).

[0044] Overall survival (OS) refers to the period from patient registration to death or termination at the last known survival date. OS includes an extension of life expectancy compared to untreated or unadministered individuals or patients. Overall survival refers to the situation in which a patient continues to live for a specified period, such as one year or five years from the time of diagnosis or treatment.

[0045] "Partial response" or "PR" refers to a treatment-responsive reduction in the sum of target lesion diameters by at least 30% of the baseline sum of diameters, as defined in the RECIST v1.1 guidelines.

[0046] The term "peptide aptamer" refers to a short sequence of 5-20 amino acid residues that can bind to a specific target. Peptide aptamers are typically inserted into the loop region of a stable protein scaffold (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12):1082-101).

[0047] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable diluent” means any solvent, dispersion, coating agent, antibacterial and antifungal agent, isotonic agent and absorption retarder that is compatible with the administration of a pharmaceutical product. The use of such media and active ingredients for pharmaceutical active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and do not limit the scope of the present invention, but include additional buffers, preservatives, cosolvents, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA, metal complexes (e.g., Zn-protein complexes), biodegradable polymers such as polyesters, salt-forming counterions such as sodium and polyhydric sugar alcohols, amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid and threonine, lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols ( Examples include organic sugars or sugar alcohols such as inositol and polyethylene glycol, sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate, low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins, and hydrophilic polymers such as polyvinylpyrrolidone. Other pharmaceutically acceptable substances include those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Acceptable carriers, excipients, or stabilizers may be included in the pharmaceutical compositions described herein, provided that they do not adversely affect the desired properties of the pharmaceutical composition.

[0048] "Progressive disease" or "advanced disease" refers to the appearance of another new lesion or tumor and / or the apparent progression of an existing non-target lesion, as defined in the RECIST v1.1 guidelines. Progressive disease or advanced disease may also refer to tumor growth of more than 20 percent since the start of treatment due to an increase in either the volume or extent of the tumor.

[0049] Progression-free survival (PFS) refers to the time from registration to disease progression or death. PFS is generally measured using the Kaplan-Meier method and the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 standard. Generally, progression-free survival refers to the state in which a patient continues to live without their cancer progressing.

[0050] The term "RECIST" refers to the Criteria for Assessing the Response of Solid Tumors. The RECIST guidelines, criteria, or standards describe a standard approach to the measurement and definition of solid tumors for objective assessment of tumor size changes used in clinical trials of adult and pediatric cancers. RECIST v1.1 refers to version 1.1 of the revised RECIST guidelines, and the European It is published in Journal of Cancers 45 (2009) 228-247.

[0051] The term "repebody" is derived from the leucine-rich repeat module and refers to a protein that has been modified to bind to a specific target (see Lee et al., 2012. PNAS. 109(9): 3299-3304).

[0052] The term "favorable response" generally refers to producing a beneficial state in a subject. In the context of cancer treatment, this term refers to producing a therapeutic effect in the subject. Favorable therapeutic effects in cancer can be measured in numerous ways (see Weber, 2009. J Nucl Med. 50 Suppl 1:1S-10S). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess the therapeutic efficacy of anti-cancer therapies. Regarding tumor growth inhibition, according to NCI standards, T / C ≤ 42% is the minimum level of antitumor activity. T / C < 10% is considered a high level of antitumor activity, and T / C (%) = median tumor volume in treated / median tumor volume in control × 100. A favorable response can be assessed, for example, by increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a reduction in progressive disease (PD), a shorter time to progression (TTP), or any combination thereof.

[0053] The term "sequence identity" refers to the percentage value obtained when comparing two sequences using a pairwise sequence alignment tool. In this case, sequence identity was obtained using the global alignment tool "EMBOSS Needle" with default settings (Rice et al., 2000. Trends Genet. 16(6):276-7, Li et al., 2015. Nucleic Acids Res. 43(W1):W580-4). The global alignment tool is available at https: / / www.ebi.ac.uk / Tools / psa / .

[0054] The terms "single-chain antigen-binding fragment" or "scFab" refer to a fusion protein comprising one variable domain and one constant domain of an antibody light chain attached to one variable domain and one constant domain of an antibody heavy chain, wherein the heavy and light chains are linked by a short peptide.

[0055] The term "single-stranded variable fragment" or "scFv" refers to a fusion protein containing variable domains of the heavy and light chains of an antibody linked together by a peptide linker. The term also includes disulfide-stabilized Fv (dsFv). Methods for stabilizing scFv via disulfide bonds are disclosed in Reiter et al., 1996. Nat Biotechnol. 14(10):1239-45. It is being done.

[0056] "Stable disease" refers to a disease that does not progress or recur, as defined in the RECIST v1.1 guidelines. In a stable disease state, there is neither sufficient tumor reduction to be considered a partial response nor sufficient tumor growth to be considered a progressive disease.

[0057] Time to tumor progression (TTP) is defined as the time from registration to disease progression. TTP is generally measured using the RECIST v1.1 criteria.

[0058] As used in this application, the terms “treatment” and “therapy” refer to a set of sanitary, pharmacological, surgical, and / or physical means used with the aim of improving a health problem, as well as for the purpose of curing and / or alleviating a disease and / or symptom. The terms “treatment” and “therapy” include preventive and curative methods, for both are directed toward maintaining and / or restoring the health of an individual or animal. Regardless of the cause of the symptoms, disease, and disability, the administration of a medicine suitable for alleviating and / or curing a health problem should be interpreted as a form of treatment or therapy in the context of this application.

[0059] Chimeric antigen receptor In one embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular domain containing a CD1a targeting moiety, a transmembrane domain, and an intracellular signaling domain.

[0060] CD1a targeting moiety In some embodiments, the CD1a-targeting moiety is an antibody, antikalin, lipid body, monobody, scFv, Fab, scFab, affibody, finomer, DARPin, nanobody, or a peptide aptamer that specifically binds to CD1a.

[0061] Binding molecules that specifically bind to CD1a can be very useful in the diagnosis and treatment of the aforementioned disorders. Several mouse monoclonal antibodies against CD1a are known in this field (Kelly (1994), Amiot et al. (1986), Furue et al. (1992)). However, mouse antibodies are limited for in vivo use due to problems associated with administering mouse antibodies to humans, such as a short serum half-life, inability to induce certain human effector functions, and the occurrence of undesirable immune responses to mouse antibodies (Van Kroonenburgh and Pauwels (1988)). Novel human antibodies that overcome these aforementioned shortcomings have been developed in recent years. (Bechan (2012) and Gitanjali (2005)). In addition to NA1 / 34.HLK, other Hybridomas, such as SIGMA ALDRICH's OKT6 (IgG1 isotype), are commercially available. It is.

[0062] Please see below: Amiot M., Bernard A., Raynal B., Knapp W., Deschildre C. and Boumsell L. (1986), J. Immunol. 136:1752-1757. Furue M., Nindl M., Kawabe K., Nakamura K., Ishibashi Y. and Sagawa K. (1992), J. Am. Acad. Dermatol. 27:419-42 Kelly KM, Beverly PC, Chu AC, Davenport V., Gordon I., Smith M. and Pritchard J. (1994), J. Pediatr. 125:717-722 Van Kroonenburgh MJ and Pauwels EK (1988), Nucl. Med. Commun. 9:919-930. Gitanjali Bechan, David W. Lee, R. Maarten Egeler and Robert J. Arceci Blood 2005 106:4815 Bechan, GI, Lee, DW, Zajonc, DM, Heckel, D., Xian, R., Throsby, M. , Meijer, M., Germeraad, WT, Kruisbeek, AM, Maarten Egeler, R. and Arceci, RJ (2012), Br J Haematol, 159: 299-310.

[0063] Phage display and combinatorial methods for generating antibodies are known in the art (for example, Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication No. 92 / 18619; Dower et al., International Publication No. 91 / 17271; Winter et al., International Publication No. 92 / 20791; Markland et al., International Publication No. 92 / 15679; Breitling et al., International Publication No. 93 / 01288; McCafferty et al., International Publication No. International Publication No. 92 / 01047, Garrard et al., and Ladner et al., International Publication No. 92 / 09690. International Publication No. 90 / 02809, Fuchs et al. (1991) Bio / Technology 9:1370-1372, Hay et al. (1992) Hum Antibod Hybridomas 3:81-85, Huse et al. (1989) Science 246:1275-1281, Griffiths et al. (1993) EMBO J 12:725-734, Hawkins et al. (1992) J Mol Biol 226:889-896, Clackson et al. (1991) Nature 352:624-628, Gram et al. (1992) PNAS 89:3576-3580, Garrad et al. (1991) Bio / Technology 9:1373-1377, Hoogenboom (This information is described in et al. (1991) Nuc Acid Res 19:4133-4137 and Barbas et al. (1991) PNAS 88:7978-7982, and is incorporated herein by reference.)

[0064] Furthermore, methods for generating and selecting non-immunoglobulin scaffolds that bind to specific targets are known in the art (see, for example, Skrlec, et al., 2015. Trends Biotechnol. 33(7):408-18).

[0065] In some embodiments, the CD1a targeting moiety is an antibody, scFv, Fab, or scFab comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides, where LCDR1 consists only of [QDINKY] (SEQ ID NO: 1), LCDR2 consists only of [YTS], LCDR3 consists only of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists only of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists only of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists only of [ARGFYTMDY] (SEQ ID NO: 6).

[0066] In some embodiments, the CD1a targeting portion is an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2, and HCDR3 polypeptides, where LCDR1 consists only of [QDINKY] (SEQ ID NO: 1), LCDR2 consists only of [YTS], LCDR3 consists only of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists only of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists only of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists only of [ARGFYTMDY] (SEQ ID NO: 6).

[0067] In some embodiments, the CD1a targeting moiety is an antibody, scFv, Fab, or scFab containing a VL domain and a VH domain, wherein the VL domain consists solely of SEQ ID NO: 7 and the VH domain consists solely of SEQ ID NO: 8.

[0068] In some embodiments, the CD1a targeting portion is an scFv containing a VL domain and a VH domain, where the VL domain consists only of sequence number 7 and the VH domain consists only of sequence number 8.

[0069] VL domain (Sequence ID 7) [RDIQMTQSPSSLSASLGGKVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYSFSISNLEPEDIATYYCLHYDNLPWTFGGGTKLEIKRA]

[0070] VH domain (SEQ ID NO: 8) [QVQLQQSGAELARPGASVKMSCKASGYAFSTYTMHWVKQRPRQGLEWIGYINPNSASTSYNENFKDKATLTADKSSNTAYMHLSSLTSEDSAVYYCARGFYTMDYWGQGTSVTVSS]

[0071] In some embodiments, the CD1a targeting portion is an scFv that includes or consists solely of sequence number 9.

[0072] scFv (sequence number 9) derived from clone NA1 / 34.HLK [QVQLQQSGAELARPGASVKMSCKASGYAFSTYTMHWVKQRPRQGLEWIGYINPNSASTSYNENFKDKATLTADKSSNTAYMHLSSLTSEDSAVYYCARGFYTMDYWGQGTSVTVSSGGGGGSGG GGSGGGGSGGGGSRDIQMTQSPSSLSASLGGKVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYSFSISNLEPEDIATYYCLHYDNLPWTFGGGTKLEIKRA]

[0073] transmembrane domain The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-binding or transmembrane protein. The transmembrane region may include at least one transmembrane region of the α, β, or ζ chain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

[0074] The transmembrane domain may be synthetic or a variant of a naturally occurring transmembrane domain. In some embodiments, the synthetic or variant transmembrane domain mainly contains hydrophobic residues such as leucine and valine.

[0075] In some embodiments, the transmembrane domains include the transmembrane domains of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or their variants, which have 95% sequence identity.

[0076] In some embodiments, the transmembrane domains include the transmembrane domains of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or their variants, which have 98% sequence identity.

[0077] In some embodiments, the transmembrane domain includes the transmembrane domains of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

[0078] In some embodiments, the transmembrane domain comprises the CD8 transmembrane domain or a variant thereof, the variant having 95% sequence identity.

[0079] In some embodiments, the transmembrane domain comprises the CD8 transmembrane domain or a variant thereof, the variant having 98% sequence identity.

[0080] In some embodiments, the transmembrane domain includes the transmembrane domain of CD8.

[0081] In some embodiments, the transmembrane domain includes sequence number 10 or a sequence having 95% sequence identity with sequence number 10.

[0082] In some embodiments, the transmembrane domain includes sequence number 10 or a sequence having 98% sequence identity with sequence number 10.

[0083] In some embodiments, the transmembrane domain includes SEQ ID NO: 10. In some embodiments, the transmembrane domain consists solely of SEQ ID NO: 10.

[0084] Transmembrane domain derived from CD8 (SEQ ID NO: 10) [TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC]

[0085] Intracellular signal transduction domains The intracellular signaling domain results in the activation of at least one function of a cell expressing the CAR after binding to a ligand expressed on tumor cells. In some embodiments, the intracellular signaling domain comprises one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain is a subset and / or variant of an intracellular signaling domain that results in the activation of at least one function of a cell containing the CAR.

[0086] In some embodiments, the intracellular signaling domain includes the intracellular domains of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66b, or their variants, which have 95% sequence identity.

[0087] In some embodiments, the intracellular signaling domain includes the intracellular domains of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66b, or their variants, which have 98% sequence identity.

[0088] In some embodiments, the intracellular signaling domain includes the intracellular domains of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66b.

[0089] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ or a variant thereof, the variant having 95% sequence identity.

[0090] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ or a variant thereof, the variant having 98% sequence identity.

[0091] In some embodiments, the intracellular signaling domain includes the intracellular domain of CD3ζ.

[0092] In some embodiments, the intracellular signaling domain includes SEQ ID NO: 11 or a sequence having 95% sequence identity with SEQ ID NO: 11.

[0093] In some embodiments, the intracellular signaling domain includes SEQ ID NO: 11 or a sequence having 98% sequence identity with SEQ ID NO: 11.

[0094] In some embodiments, the intracellular signaling domain includes SEQ ID NO: 11 or a sequence having 99% sequence identity with SEQ ID NO: 11.

[0095] In some embodiments, the intracellular signaling domain includes SEQ ID NO: 11. In some embodiments, the intracellular signaling domain consists solely of SEQ ID NO: 11.

[0096] Intracellular signal transduction domain derived from CD3ζ (SEQ ID NO: 11) [RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR]

[0097] Co-stimulatory signaling domain In some embodiments, CAR may further include a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain includes the intracellular domains of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, or variants thereof, the variants having 95% sequence identity.

[0098] In some embodiments, the co-stimulatory signaling domain includes intracellular domains of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, or their variants, the variants having 98% sequence identity.

[0099] In some embodiments, the co-stimulatory signaling domain includes the intracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or CD276.

[0100] In some embodiments, the co-stimulatory signaling domain comprises the intracellular domain of CD137 or a variant thereof, the variant having 95% sequence identity.

[0101] In some embodiments, the co-stimulatory signaling domain comprises the intracellular domain of CD137 or a variant thereof, the variant having 98% sequence identity.

[0102] In some embodiments, the co-stimulatory signaling domain includes the intracellular domain of CD137.

[0103] In some embodiments, the co-stimulatory signaling domain includes sequence number 12 or a sequence having 95% sequence identity with sequence number 12.

[0104] In some embodiments, the co-stimulatory signaling domain includes SEQ ID NO: 12 or a sequence having 98% sequence identity with SEQ ID NO: 12.

[0105] In some embodiments, the co-stimulatory signaling domain includes sequence number 12 or a sequence having 99% sequence identity with sequence number 12.

[0106] In some embodiments, the co-stimulus signaling domain includes SEQ ID NO: 12. In some embodiments, the co-stimulus signaling domain consists solely of SEQ ID NO: 12.

[0107] Co-stimulatory signaling domain derived from CD137 (SEQ ID NO: 12) [KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL]

[0108] Complete array CAR according to the present invention In some embodiments, CAR is (i) scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1 and HC scFv containing DR2 and HCDR3 polypeptides, wherein LCDR1 consists only of [QDINKY] (SEQ ID NO: 1), LCDR2 consists only of [YTS], LCDR3 consists only of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists only of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists only of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists only of [ARGFYTMDY] (SEQ ID NO: 6), (ii) A transmembrane domain comprising a sequence having 95% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 10, (iii) an intracellular signaling domain comprising sequence number 11 or a sequence having 95% sequence identity with sequence number 11, (iv) A co-stimulatory signaling domain comprising sequence number 12 or a sequence having 95% sequence identity with sequence number 12, Includes.

[0109] In some embodiments, CAR is (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, where LCDR1 consists only of [QDINKY] (SEQ ID NO: 1), LCDR2 consists only of [YTS], LCDR3 consists only of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists only of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists only of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists only of [ARGFYTMDY] (SEQ ID NO: 6), (ii) A transmembrane domain comprising a sequence having 98% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 10, (iii) an intracellular signaling domain comprising sequence number 11 or a sequence having 98% sequence identity with sequence number 11, (iv) A co-stimulatory signaling domain comprising sequence number 12 or a sequence having 98% sequence identity with sequence number 12, Includes.

[0110] In some embodiments, CAR is (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, wherein LCDR1 consists only of [QDINKY] (SEQ ID NO: 1), LCDR2 consists only of [YTS], LCDR3 consists only of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists only of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists only of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists only of [ARGFYTMDY] (SEQ ID NO: 6), (ii) A transmembrane domain comprising a sequence having 98% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 10, (iii) an intracellular signaling domain comprising sequence number 11 or a sequence having 99% sequence identity with sequence number 11, (iv) A co-stimulatory signaling domain comprising sequence number 12 or a sequence having 99% sequence identity with sequence number 12, Includes.

[0111] In some embodiments, CAR is (i) scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, LCDR1 consists only of [QDINKY] (SEQ ID NO: 1), LCDR2 consists only of [YTS], and LCDR3 consists of [LHYDNLP scFv consisting only of WT (sequence number 3), HCDR1 consisting only of [GYAFSTYT] (sequence number 4), HCDR2 consisting only of [INPNSAST] (sequence number 5), and HCDR3 consisting only of [ARGFYTMDY] (sequence number 6), (ii) A transmembrane domain containing Sequence ID No. 10, (iii) an intracellular signaling domain including sequence number 11, (iv) Co-stimulatory signaling domain including Sequence ID No. 12, Includes.

[0112] In some embodiments, CAR is (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, and the VH domain comprises HCDR1, HCDR2 and HCDR3 polypeptides, wherein LCDR1 consists only of [QDINKY] (SEQ ID NO: 1), LCDR2 consists only of [YTS], LCDR3 consists only of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists only of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists only of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists only of [ARGFYTMDY] (SEQ ID NO: 6), (ii) A transmembrane domain consisting of Sequence ID No. 10, (iii) an intracellular signaling domain consisting of Sequence ID No. 11, (iv) Co-stimulatory signaling domain consisting of Sequence ID No. 12, Includes.

[0113] In some embodiments, CAR is (i) an scFv containing a VL domain and a VH domain, wherein the VL domain consists only of sequence number 7 and the VH domain consists only of sequence number 8, (ii) A transmembrane domain comprising a sequence having 95% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 10, (iii) an intracellular signaling domain comprising sequence number 11 or a sequence having 95% sequence identity with sequence number 11, (iv) A co-stimulatory signaling domain comprising sequence number 12 or a sequence having 95% sequence identity with sequence number 12, Includes.

[0114] In some embodiments, CAR is (i) an scFv containing a VL domain and a VH domain, wherein the VL domain consists only of sequence number 7 and the VH domain consists only of sequence number 8, (ii) A transmembrane domain comprising a sequence having 98% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 10, (iii) an intracellular signaling domain comprising sequence number 11 or a sequence having 98% sequence identity with sequence number 11, (iv) A co-stimulatory signaling domain comprising sequence number 12 or a sequence having 98% sequence identity with sequence number 12, Includes.

[0115] In some embodiments, CAR is (i) an scFv containing a VL domain and a VH domain, wherein the VL domain consists only of sequence number 7 and the VH domain consists only of sequence number 8, (ii) A transmembrane domain comprising a sequence having 98% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 10, (iii) an intracellular signaling domain comprising sequence number 11 or a sequence having 99% sequence identity with sequence number 11, (iv) A co-stimulatory signaling domain comprising sequence number 12 or a sequence having 99% sequence identity with sequence number 12, Includes.

[0116] In some embodiments, CAR is (i) an scFv containing a VL domain and a VH domain, wherein the VL domain consists only of sequence number 7 and the VH domain consists only of sequence number 8, (ii) A transmembrane domain containing Sequence ID No. 10, (iii) an intracellular signaling domain including sequence number 11, (iv) Co-stimulatory signaling domain including Sequence ID No. 12, Includes.

[0117] In some embodiments, CAR is (i) an scFv containing a VL domain and a VH domain, wherein the VL domain consists only of sequence number 7 and the VH domain consists only of sequence number 8, (ii) A transmembrane domain consisting only of Sequence ID No. 10, (iii) an intracellular signaling domain consisting only of Sequence ID No. 11, (iv) A co-stimulatory signaling domain consisting only of Sequence ID No. 12, Includes.

[0118] In some embodiments, CAR includes or consists solely of sequence number 2 or a sequence having 95% sequence identity with sequence number 2. In some embodiments, CAR includes or consists solely of sequence number 2 or a sequence having 98% sequence identity with sequence number 2. In some embodiments, CAR includes or consists solely of sequence number 2 or a sequence having 99% sequence identity with sequence number 2. In some embodiments, CAR includes or consists solely of sequence number 2.

[0119] Complete sequence of CARs (Sequence ID 2) [MALPVTGLLLSLGLLLHAARPTGQVQLQQSGAELARPGASVKMSCKASGYAFSTYTMHWVKQRPRQGLEWIGYINPNSASTSYNENFKDKATLTADKSSNTAYMHLSSLTSEDSAVYYCARG FYTMDYWGQGTSVTVSSGGGGSGGGGSGGGGGSGGGGSRDIQMTQSPSSLSASLGGKVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGGSGREYSFSISNLEPEDIATY YCLHYDNLPWTFGGGTKLEIKRATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR]

[0120] nucleic acid In one embodiment, the present invention provides a nucleic acid encoding any one of the CARs of the present invention, which includes any one of the CARs disclosed above. The nucleic acid sequence encoding the chimeric receptor connects a number of modular components that can be excised and replaced with other components to customize the chimeric receptor for efficient T cell activation and CD1a recognition.

[0121] In some embodiments, nucleic acids are suitable for the transduction or transformation of cells. In some embodiments, nucleic acids are suitable for the transduction or transformation of T cells used in adoptive immunotherapy.

[0122] In some embodiments, nucleic acids are codon-optimized for expression in mammalian cells. Codon optimization methods are known in the art (see, for example, Parret et al., 2016. Curr Opin Struct Biol. 39: 155-162).

[0123] The nucleic acids of the present invention may be included in γ-retroviral vectors or lentiviral vectors that can be used for transduction or transformation of T cells (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124). DNA transposons, RNA transposons. Nucleic acids can also be inserted into cells using infection or genome editing technologies such as TALEN, ZFN, and CRISPR / Cas9 (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124).

[0124] cell In one embodiment, the present invention provides cells comprising the nucleic acid and / or CAR of the present invention. In some embodiments, the cells are T cells (referred to as CART).

[0125] In some embodiments, the cells are naive T cells, memory stem T cells, or central memory T cells. These cells are currently considered well-suited for adaptive immunotherapy (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124). stomach).

[0126] In some embodiments, the cells are autologous T cells. The term “autologous cells” refers to cells obtained from the same patient being treated with any one of the methods of the present invention. It should be noted that flow cytometry analysis of peripheral blood obtained from 40 patients with active T-cell acute lymphoblastic leukemia revealed the presence of normal CD3+CD1a-T cells in all patients. Therefore, it is entirely possible to treat patients with autologous T cells containing the nucleic acids and / or CARs of the present invention.

[0127] In some embodiments, the cells are allo-tolerant T cells. The term "tolerant cells" refers to cells modified to reduce the risk of graft-versus-host disease response. In some embodiments, this is achieved by genome-edited deletion of TCR and / or β2-microglobulin. 15、19 Allogeneic tolerance cells are known in the field of this technology (Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124 allogeneic T cells). (See the relevant chapter.)

[0128] In some embodiments, the T cells are CD3-positive and CD1a-negative T cells.

[0129] In some embodiments, the cells are lymphocyte precursors, embryonic stem cells, or induced pluripotent stem cells that have the ability to differentiate into mature T cells (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124).

[0130] Pharmaceutical composition In one embodiment, the present invention provides a pharmaceutical composition comprising a plurality of cells of the present invention and a pharmaceutically acceptable carrier or diluent.

[0131] The pharmaceutical compositions described herein may also contain other substances. These substances include, but are not limited to, cryoprotectants, surfactants, antioxidants, and stabilizers. As used herein, the term “cryoprotectant” includes substances that provide stability to CART against freeze-induced stress. Non-exclusive examples of cryoprotectants include sugars such as sucrose, glucose, trehalose, mannitol, mannose, and lactose; polymers such as dextran, hydroxyethyl starch, and polyethylene glycol; surfactants such as polysorbates (e.g., PS-20 or PS-80); and amino acids such as glycine, arginine, leucine, and serine. Cryoprotectants with low toxicity in biological systems are generally used.

[0132] In some embodiments, cells are first collected from their culture medium, then washed, and formulated in a therapeutically effective dose in a medium and container system suitable for administration ("pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), and 5% dextrose in water or Ringer's lactate solution may also be used. Human serum albumin, fetal bovine serum, or other human serum components may be added to the infusion medium.

[0133] In one embodiment, the present invention provides cells or pharmaceutical compositions according to the present invention that can be used as pharmaceuticals.

[0134] Treatment method In one embodiment, the present invention provides a method for treating CD1a-positive cancer, comprising administering the cells of the present invention or the pharmaceutical composition of the present invention to a patient in need thereof.

[0135] In some embodiments, a therapeutically effective dose of cells is administered to the patient. In some embodiments, at least 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces or 10 10 A certain number of cells are administered. The number of cells depends on the final intended use of the composition, as well as the type of cells contained in the composition. For example, if cells specific to a particular antigen are desired, the population may contain more than 70%, generally more than 80%, 85%, and 90% to 95% of such cells. For the uses provided herein, the volume of cells is generally less than 1 liter, and may be less than 500 ml, even less than 250 ml, or less than 100 ml. The number of clinically relevant cells is cumulative to 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces or 10 10 It can be distributed for multiple injections equal to or exceeding the number of individual cells.

[0136] In some embodiments, cells or pharmaceutical compositions are administered intravenously, intraperitoneally, into the bone marrow, into lymph nodes and / or into the cerebrospinal fluid.

[0137] In some embodiments, the method includes combination therapy. In some embodiments, the method includes further administration of an immune checkpoint inhibitor (see Lim & June, 2017. Cell. 168(4):724-740). In further embodiments, the method includes further administration of an immune checkpoint inhibitor and / or an IAP inhibitor (see International Publication No. 2016 / 054555).

[0138] In some embodiments, the cells or pharmaceutical compositions described herein are administered in combination with chemotherapeutic agents and / or immunosuppressants. In one embodiment, the patient is treated first with a chemotherapeutic agent that inhibits or destroys other immune cells, followed by the cells or pharmaceutical compositions described herein. In some cases, chemotherapy can be completely avoided.

[0139] In some embodiments, CD1a-positive cancer is cortical T-cell acute lymphoblastic leukemia or Langerhans cell histiocytosis. In some embodiments, CD1a-positive cancer is cortical T-cell acute lymphoblastic leukemia. In some embodiments, CD1a-positive cancer is relapsed / refractory cortical T-cell acute lymphoblastic leukemia.

[0140] Generally, leukemia relapses can occur months or years after the initial remission, but most relapses occur within two years of initial treatment. Refractory is a term that means a patient has become unresponsive to at least one treatment strategy after a relapse.

[0141] In particular, in first-line trials for ALL in adults, there is a broad consensus that relapse is defined as "the detection of more than 5% blast cells in the bone marrow after the achievement of previous complete remission (CR) or clear evidence of extramedullary leukemia involvement" (see Goekbuget (2017)). The European Working Group on Adult ALL (EWALL) This statement was included in the consensus recommendation, along with the additional explanation that "if 5% to 20% of blast cells are present at any stage of intensive care and / or regeneration, bone marrow evaluation should be repeated after one week to distinguish between bone marrow relapse and regeneration" (see Dohner (2010)). (i) The definitions cited are based on international recommendations regarding outcome parameters in acute myeloid leukemia, as well as for several subtypes of ALL, as is the case with T-ALL (see Cheson (2003) and Chantepie (213)).

[0142] In recent years, some studies have not even defined the concept of relapse. Therefore, studies using chimeric antigen receptor (CAR) T cells have included patients with “measurable disease,” and further, patients with hematological relapse (without additional designation) or minimal residual disease (MRE) (see Lee (2015), Maude (2014), and Goekbuget (2017)). See below: Dohner H, Estey EH, Amadori S, et al, Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European Leukemia Net. Blood 2010;115:453-74. Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol 2003;21:4642-9. Chantepie SP, Cornet E, Salaun V, Reman O. Hematogones: an overview. Leuk Res 2013;37:1404-11. 13. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014;371:1507-17. Goekbuget N, Dombret H, Bassan R, Wadleigh M, Doubek M, Ribera J. Inclusion and response criteria for clinical trials in relapsed / refractory acute lymphoblastic leukemia and usefulness of historical control trials. Haematologica. 2017;102(3):e118-e119.

[0143] In some embodiments, patients treated with the method of the present invention are in a state of complete or near-complete remission after treatment with another therapy. In patients with highly active relapsed / refractory cortical T-cell acute lymphoblastic leukemia, several alternative effector T cells are present, so it is preferable to reduce the tumor burden before using the method of the present invention. In some embodiments, patients treated with the method of the present invention have previously been treated with other therapies that have resulted in a partial response, a complete response, stabilization of disease, a reduction in progressive disease, a reduction in time to tumor progression, or any combination thereof. [Examples]

[0144] Materials and methods CD1a-specific scFv generation and CAR design CD1a-specific single-strand variable fragments (scFv) derived from the NA1 / 34.HLK clone of a CD1a-specific antibody were obtained using commercial synthesis (Sigma-Aldrich) with the mouse IgG Library Primer Set (Progen) and human CD8 transmembrane (T) The M) domain, human CD137 and CD3ζ endodomains, and a T2A-GFP cassette were cloned into a second-generation pCCL lentivirus-based CAR skeleton. A control was used with either GFP alone (mock vector) or the same lentiviral vector expressing either the CD22 CAR skeleton (Figure 1D and Figure 8A).

[0145] Production of CAR-expressing lentiviruses, transduction, activation, and increase of T cells. VSV-G pseudotyped CAR-expressing virus particles were generated in 293T cells using a standard polyethyleneimine transfection protocol and concentrated by ultracentrifugation as described elsewhere. 27 The viral titer remained consistently 10. 8 The values ​​were within the TU / mL range. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats derived from healthy volunteers by Ficol-Hypak gradient centrifugation. Buffy coats were obtained from the Barcelona Blood and Tissue Bank (BST) after IRB approval (HCB / 2018 / 0030). T cells were activated for 2 days with plate-bound anti-CD3 (OKT3) and anti-CD28 antibodies (BD Biosciences), and then transduced with CAR-expressing lentivirus (MOI=10) in the presence of interleukin-7 (IL-7) and IL-15 (10 ng / mL, Mitenyi Biotec). 16、18 The cell surface expression of CD1aCAR was tracked by co-expression of GFP-fluorescence-activated cell sorting (FACS) and confirmed using AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG(H+L) (Jackson ImmunoResearch). Appropriate activation of CAR-transduced T cells was demonstrated by staining for CD25 and CD69 after a 2-day increase.

[0146] Immunophenotyping of healthy CD34+ precursors, T cells, and primary T-ALL samples CD1a antigen expression in CD34+ stem / progenitor cells (HSPCs), CD34+CD7+ thymic T cell precursors, and CD3+ T cells was predicted and analyzed in fresh human thymus, fetal liver and bone marrow (BM), umbilical cord blood, and adult BM and peripheral blood (PB) (n=3). Fetal tissues were obtained from developing embryos aborted between 18 and 22 weeks of gestation, obtained from the MRC / Wellcome Trust Human Developmental Biology Resource after informed consent and approval by the local Ethics and Biohazard Board of Directors (CMRBCEIC-26 / 2013). As previously described, it was collected. 28、29 Neonatal and adult tissues were obtained from BST after IRB approval (HCB / 2018 / 0030). Primary T-ALL samples and clinical samples were obtained from BST. Discrete immunophenotyping data was obtained from local hospitals Sant Joan de Den, Germans Trias i Pujol, and Santa Creu i San Pau (Barcelona, ​​Spain). T-ALL For immunophenotyping of primary samples, the following fluorescent dye-conjugated monoclonal antibodies (MoAbs) were used: anti-CD2-PE, CD7-FITC / PE, CD13-PerCP-Cy5.5, CD34-APC, CD3-PE, CD5-FITC, CD4-BV-421, CD8-APC-Cy7, CD45-AmCyan, CD1a-BV-421 / APC / PE, CD33-APC, and CD123-APC (BDBiosciencies or Miltenyi Biotec). Isotype-matched non-reactive fluorescent dye-conjugated MoAbs. b was always used as a fluorescence reference. In short, PB mononuclear cells (PBMCs, approximately 5 × 10⁻⁶) 5 Cells were incubated with erythrocyte lysate (BDBiosciencies) for 10 minutes and then stained with MoAb (in the dark at 4°C for 20 minutes). The stained cells were washed in phosphate-buffered saline (PBS) and analyzed by FACS using a FACSCanto-II flow cytometer (BDBiosciencies) equipped with FACSDiva software.30~32 .

[0147] In vitro cytotoxicity assay and cytokine release determination Cell lines Jurkat, MOLT4, and NALM6 are used in DSMZ (Braunschweig, Germany) We purchased the cells and increased their numbers according to DSMZ's recommendations. Luciferase (Luc) / GFP-expressing cells were stably generated by retroviral introduction and FACS purification of GFP+ cells. 33 Target cells (cell lines and primary T-ALL blasts) were labeled with 3 μM eFluor670 (eBioscience) and then labeled with CD1a, CD22, or mock CART with different effectors: Target cells were incubated at the specified (E:T) ratio for a specified period. CART-mediated cytotoxicity was determined by analyzing residual surviving (7-AAD-)eFluor670+ target cells at each time point and at the E:T ratio. Absolute cell counts were determined using Trucount absolute count beads (BD Biosciences). Furthermore, cortical T-AL at the time of disease onset was also analyzed. FACS-selected CD3+CD1a-mature T cells derived from patient PB (Patient B1) were activated, transduced with CD1a CAR, and tested against eFluor670-labeled auto-CD1a+T-ALL blast cells. Production of pro-inflammatory cytokines IL-2, TNFα, and IFNγ was measured by ELISA (Human ELISA SET, BD Biosciences) in the supernatant collected 16 hours later.

[0148] In vivo xenograft (PDX) models derived from Jurkat and T-ALL patients Non-obese diabetic (NOD)-Cg-Prkdcscid Il2rgtm1Wjl / SzJ(NSG) mice (The Jackson Laboratory) aged 6 to 12 weeks were bred and reared under pathogen-free conditions at the Barcelona Biomedical Research Park (PRBB) animal facility. The mice were irradiated (2 Gy) and 3 × 10⁶ mice were irradiated. 61 x 10⁶ Luc-GFP expressing Jurkat cells or 1 x 10⁶ 6 (iv) Intravenous transplantation was performed on (iv) primary cortical CD1a+ T-ALL blasts (increased in primary and primografts). 34 1.5 × 10 6 pieces~5×10 6 Individual CD1a or mock CART cells were intravenously injected 3 days later. When Luc-Jurkat cells were used, tumor burden was tracked by bioluminescence (BLI) using the Xenogen IVIS 50 Imaging System (Perkin Elmer). To measure luminescence, mice were intraperitoneally administered 150 mg / kg of D-luciferin, and tumor burden was monitored at specified time points. Total luminescence was visualized and calculated using Living Image software (Perkin Elmer). Tumor burden of primary T-ALL samples was tracked every two weeks by blood sampling and FACS analysis. Mice treated with mock CART cells were sacrificed when they developed leukemia, and tumor burden (hHLA-ABC+hCD45+hCD3+hCD1a-GFP+) and CART persistence (hHLA-ABC+hCD45+hCD3+hCD1a-GFP+) were analyzed by FACS in the BM, PB, and spleen. In the re-challenge experiment, animals that were not leukemia and had received CD1a CART injections 5-6 weeks prior were given 1.5 × 10⁶ 6 individual Luc-Jurkat cells or 1 × 10⁶ 6 One of the CD1a+T-ALL primografts was reinfused, and disease recurrence was tracked by BLI and FACS as described above. All procedures were performed in accordance with the PRBB Animal Experimentation Committee (DAAM7393).

[0149] Enzyme-linked immunoassay spot assay (ELISpot) ELISpot plates (Millipore) were coated with anti-human IFNγ antibody (1-D1K, Mabtech) and maintained at 4°C overnight. The plates were then washed six times with PBS containing 1% fetal bovine serum, and cells from three independent donors were placed in 5 × 10⁶ cells. 5 ~1 × 10 6Cells were plated in a cell / well configuration and cultured in triplicates at 37°C and 5% CO2 for 20 hours. IFNγ-secreting cells were measured in response to 1 μg / mL CEF, a peptide pool of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza T cell epitopes, as well as 1 μg / mL Staphylococcus enterotoxin B (SEB) as a positive control. The plates were then decontaminated with biotinylated anti-human IFNγ, streptavidin-alkaline phosphatase (Mabtech), as previously described. 3 5、36 The frequency of IFNγ-secreting cells was quantified using ImmunoCapture and ImmunoSpot software. 5 The number of IFNγ spot-forming units (SFUs) per individual was calculated.

[0150] statistical analysis Data from at least three individual donors were shown in all figures, and experimental replication was always performed. At least five animals were used in each in vivo condition. All p-values ​​were calculated using unpaired two-tailed Student's t-tests with Prism software (GraphPad). Event-free survival (EFS) of mice was determined using the Mantel-Cox test. P-values ​​less than 0.05 were considered statistically significant.

[0151] Example 1: CD1a specifically marks cortical T-ALL blasts. The common expression of target antigens between CART and T-series blasts limits immunotherapeutic approaches in T-ALL due to CART-associated fracturisides and potential lethal T-cell malformation. However, the CD1a antigen is expressed in cortical T-ALL, the major subset of T-ALL (Figure 1A, Figure 1B), and is not found at all in functional T cells in all extrathymic tissues. 25 Steady-state CD34+ HSPCs lack CD1a expression in multiple hematopoietic sites throughout individual development (Figure 1C). T cell development begins with CD34, which possesses lymphoid-myelin activity. highCD7-CD1a-primitive HSPCs initiate in the thymus upon establishment, which then responds to the thymic microenvironment with CD34 high CD7+CD1a differentiates into early T cell precursors. 37 As T cell precursors progress through thymic differentiation, they maintain CD7 expression and gradually lose CD34 expression, but CD1a expression appears, which is temporarily limited to corticothymocytes. 38 (Figure 1E, Figure 1F). In the CD34+ thymic population, approximately 50% are procortical T cell precursors (CD34 high CD7+CD1a- (Figure 1E, Figure 1F (gray cells)), CD1a has a fatal outcome. 3、39~41 Therefore, it can be hypothesized that this could be a feasible and safe target for immunotherapy in R / R cortical T-ALL.

[0152] Example 2: CD1a redirected T cells (CD1a CART) increase without T cell fracturing. We designed a second-generation CD1a CAR consisting of an anti-CD1a scFv, a CD8™ spacer, and intracellular signaling domains of 4-1BB (CD137) and CD3ζ ligated in-frame with GFP via a T2A sequence (Figure 2A). CD1a CAR expression was readily detected by co-expression of both scFv and GFP in 293 T cells (Figure 2B) and primary CD4+ and CD8+ T cell subsets (Figure 2C). Importantly, activated (CD69+CD25+) CD1a CAR (Figure 2D) increased 200-fold over 12 days, similar to mock T cells (Figure 2E), demonstrating that redirection of the CAR to the CD1a antigen does not induce T cell fracturing.

[0153] Example 3: CD1a CART specifically eradicates T-ALL cell lines and primary blasts in vitro. Next, CD1a CART was tested in vitro using CD1a+T-ALL cell lines Jurkat and MOLT4, as well as the B-ALL cell line NALM6 as a negative control (Figure 2F). Compared to control CART (either mock T cells or CD22 CART), CD1a CART specifically eliminated CD1a+T-ALL cells in an E:T ratio-dependent manner. Relatively low E:T ratios of 2:1 or 4:1 induced 50%–80% specific cell lysis in a 16-hour assay (Figures 2H, 2I, and 9). Importantly, most CD1a+T-ALL cells did not survive exposure to CD1a CART at an E:T ratio of 1:1 in a 72-hour assay (Figure 2I). CD1a CART produced high levels of pro-inflammatory cytokines IL-2, TNFα, and IFNγ when co-cultured with CD1a+T-ALL cells, and their effects were confirmed (Figure 2K).

[0154] To further explore their ability to eliminate primary tumors, CD1a CART cells were co-cultured with primary cortical T-ALL samples (freshly collected or PDX-derived) to achieve a CD1a+ blast cell ratio ranging from 80% to 98% (Figure 3A). Compared to mock T cells, CD1a CART cells specifically eliminated primary CD1a+ cortical T-ALL cells with a 4:1 E:T ratio in a 72-hour cytotoxicity assay (Figures 3B and 3C). Normal hematopoietic cells (CD1a-) coexisting with CD1a+ T-ALL blast cells in BM cells were not lysed by CD1a CART (Figure 3C). High levels of IFNγ and TNFα were also secreted during co-culture with CD1a+ primary T-ALL cells (Figure 3D). In summary, these results demonstrate that CD1a CART cells possess potent and specific antileukemic activity against T-ALL cell lines and primary blast cells in vitro.

[0155] Example 4: CD1a CART exhibits potent antileukemia activity in vivo. Next, the activity of CD1a CART was measured in Luc-expressing Jurkat T-ALL cells (Figures 4 and 10) and in a primary cortical T-ALL xenograft model. 34Both methods (Figure 5) were used for in vivo evaluation. NSG mice were given 2 × 10⁶ mice. 6 individual or 5 x 10 6 Three days before the IV infusion of any CD1a (or mock) CART, 3 × 10 6 Luc-expressing Jurkat cells were transplanted, and the establishment of leukemia was tracked weekly by BLI (Figure 4A, Figure 10). In contrast to mice given mock T cells, which showed a significant tumor burden by BLI, mice given CD1a CART were virtually leukemia-free until day 25 (Figure 4B, Figure 4C, Figure 10). The control of leukemia progression was dose-dependent with CD1a CART cells (Figure 10B, Figure 10C). BLI data were confirmed by flow cytometry analysis of tumor burden in PB at sacrifice (Figure 4D). Importantly, FACS analysis revealed T cell persistence in all hematopoietic tissues analyzed (Figure 4E). However, compared to the T cell distribution in mice given mock T cells, a significantly increased in vivo distribution of CD1a CART was observed in BM and spleen (Figure 4E), indicating active control of disseminated leukemia by CD1a CART.

[0156] In the clinically relevant PDX model of cortical T-ALL, NSG mice were initially given 1 × 10⁶ mice. 6 Individual primary CD1a+ T-ALL blast cells were transplanted, followed by 1 × 10⁶ cells three days later. 6 After infusion of individual CD1a (or mock) CART cells, leukemia engraftment was tracked every two weeks by blood sampling and endpoint BM analysis (Figure 5A). Engraftment of CD1a+ cortical T-ALL cells gradually increased over time in both BM (Figure 5B, 50%±13% and 55%±11% at weeks 6 and 9, respectively) and PB (Figure 5C, 4.4%±2% and 18%±6% at weeks 6 and 9, respectively) in mock T cell-treated PDX and was associated with a significantly lower OS at week 9 (42% vs. 100%, p=0.01; Figure 5D). In contrast, CD1a CART completely eliminated the growth / engraftment of T-ALL (0.36% and 0% of T-ALL blasts in BM and PB, respectively), which remained present in BM and PB after 9 weeks (Figures 5B, 5C, and 5E).

[0157] Example 5: In vivo sustained CD1a CART is functional in rechallenge assays. Since the persistence of CART in hematopoietic tissue is a key biological parameter for their clinical success, we next assessed whether residual CD1a CART remained functional and efficient in controlling T-ALL progression after 40–50 days. For this reason, mice transplanted with T-ALL, whose leukemia had been eliminated by CD1a CART treatment, were re-challenged with either Luc-Jurkat cells (Figures 6A–6D) or primary T-ALL derived from primografts (Figures 6E–6G). In contrast to controls, where secondary leukemia engrafted rapidly (as early as 2 weeks) and significantly, T-ALL engraftment was barely detectable by either BLI or FACS in the Jurkat (Figure 6C) or primograft models after 6 weeks (Figure 6F).

[0158] Example 6: Patient-derived CD1a CART specifically targets autologous CD1a+ blast cells and retains antiviral activity. Appropriate selection of target antigens and avoidance of T cell fracturing are essential for the success of CART in the treatment of T-ALL. Therefore, we investigated whether CD3+CD1a-T cells derived from PB from patients with cortical T-ALL could be isolated and genetically engineered to express CD1a CAR (Figure 7). For this purpose, CD3+CD1a-T cells were isolated from patients (purity over 95%, data not shown), activated with CD3 / CD28, and then CD1a Lentiviral transduction was performed using CAR or mock cells (31%–70% transduction). Next, CD1a derived from primary T-ALL was applied to PBMCs suitable for active T-ALL patients. The cell-lysing ability of CART was investigated (Figure 7A). The autocytotoxic properties and the degree of fractorides were examined. Since it was possible to assess both degrees, all PBMCs were used as targets. In the eFluor670-labeled target PBMCs, the majority were CD1a+ blast cells, and about 15% were CD3+CD1a-normal T cells (Figure 7B). Compared to mock T cells, CD1a CART showed great and specific cytolytic activity against self-CD1a+ blast cells, but showed no cytolytic activity against CD1a-normal T cells (Figure 6B), further demonstrating that CD1a CART is fructicide-resistant.

[0159] To further assess the potential thymic toxicity of CD1a CART, CD7+ thymocytes derived from normal human fetal thymus were then used as target cells. Only CD1a+ cortical thymocytes (second and third gray boxes) were eliminated by CD1a CART, while developmentally earlier and later CD1a- (first box) thymic T-series populations (CD7+CD34+ and CD7+CD34-) were not targeted (Figure 1E, Figure 1F), indicating that the on-target / off-tumor effect was limited to the developmentally transient thymic population of cortical thymocytes. Finally, we sought to determine whether CD1a CART could protect the host alone by targeting the most common pathogens causing viremia in immunocompromised patients. For this purpose, the responsiveness of CD1a CART to CMV, EBV, and influenza antigen (CEF) was tested, and SCF was quantified by IFNγ ELISpot. Mock T cells and CD1a Both CARTs showed very similar responses to stimulation with viral peptides, suggesting that CD1a CART retains antiviral activity (Figure 7D).

[0160] literature 1. Karrman K, Johansson B. Pediatric T-cell acute lymphoblastic leukemia. Genes Chromosomes Cancer. 2017;56(2):89-116. 2. Weng AP, Ferrando AA, Lee W, et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science. 2004;306(5694):269-271. 3. Hunger SP, Mullighan CG. Acute Lymphoblastic Leukemia in Children. N Engl J Med. 2015;373(16): 1541-1552. 4. Litzow MR, Ferrando AA. How I treat T-cell acute lymphoblastic leukemia in adults. Blood. 2015; 126(7):833-841. 5. Schneider NR, Carroll AJ, Shuster JJ, et al. New recurring cytogenetic abnormalities and association of blast cell karyotypes with prognosis in childhood T-cell acute lymphoblastic leukemia: a pediatric oncology group report of 343 cases. Blood. 2000;96(7):2543-2549. 6. Ballerini P, Landman-Parker J, Cayuela JM, et al. Impact of genotype on survival of children with T-cell acute lymphoblastic leukemia treated according to the French protocol FRALLE-93: the effect of TLX3 / HOX11L2 gene expression on outcome. Haematologica. 2008;93(11): 1658-1665. 7. Karrman K, Forestier E, Heyman M, et al. Clinical and cytogenetic features of a population-based consecutive series of 285 pediatric T-cell acute lymphoblastic leukemias: rare T-cell receptor gene rearrangements are associated with poor outcome. Genes Chromosomes Cancer. 2009;48(9):795-805. 8. Sutton R, Shaw PJ, Venn NC, et al. Persistent MRD before and after allogeneic BMT predicts relapse in children with acute lymphoblastic leukaemia. Br J Haematol. 2015;168(3):395-404. 9. Qasim W, Thrasher AJ. Progress and prospects for engineered T cell therapies. Br J Haematol. 2014;166(6):818-829. 10. Humphries C. Adoptive cell therapy: Honing that killer instinct. Nature. 2013;504(7480):S13-15. 11. Brentjens RJ, Davila ML, Riviere I, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5(177): 177ra138. 12. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. NEngl J Med. 2014;371(16): 1507-1517. 13. Gardner RA, Finney O, Annesley C, et al. Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults. Blood. 2017;129(25):3322-3331. 14. Fry TJ, Shah NN, Orentas RJ, et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat Med. 2018;24(1):20-28. 15. Cooper ML, Choi J, Staser K, et al. An "off-the-shelf1 fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies. Leukemia. 2018. 16. Gomes-Silva D, Srinivasan M, Sharma S, et al. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood. 2017;130(3):285-296. 17. Png YT, Vinanica N, Kamiya T, Shimasaki N, Coustan-Smith E, Campana D. Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies. Blood Adv. 2017;1(25):2348-2360. 18. Mamonkin M, Rouce RH, Tashiro H, Brenner MK. A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies. Blood. 2015;126(8):983-992. 19. Rasaiyaah J, Georgiadis C, Preece R, Mock U, Qasim W. TCRalphabeta / CD3 disruption enables CD3-specific antileukemic T cell immunotherapy. JCIInsight. 2018;3(13). 20. Maciocia PM, Wawrzyniecka PA, Philip B, et al. Targeting the T cell receptor beta-chain constant region for immunotherapy of T cell malignancies. Nat Med. 2017;23(12): 1416-1423. 21. Bene MC, Castoldi G, Knapp W, et al. Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia. 1995;9(10): 1783-1786. 22. Burger R, Hansen-Hagge TE, Drexler HG, Gramatzki M. Heterogeneity of T-acute lymphoblastic leukemia (T-ALL) cell lines: suggestion for classification by immunophenotype and T-cell receptor studies. Leuk Res. 1999;23(1): 19-27. 23. Niehues T, Kapaun P, Harms DO, et al. A classification based on T cell selection-related phenotypes identifies a subgroup of childhood T-ALL with favorable outcome in the COALL studies. Leukemia. 1999;13(4):614-617. 24. van Grotel M, Meijerink JP, van Wering ER, et al. Prognostic significance of molecularcytogenetic abnormalities in pediatric T-ALL is not explained by immunophenotypic differences. Leukemia. 2008;22(1): 124-131. 25. Bechan GI, Lee DW, Zajonc DM, et al. Phage display generation of a novel human anti-CD1A monoclonal antibody with potent cytolytic activity. Br J Haematol. 2012;159(3):299-310. 26. Carrera Silva EA, Nowak W, Tessone L, et al. CD207(+)CD1a(+) cells circulate in pediatric patients with active Langerhans cell histiocytosis. Blood. 2017; 130(17): 1898-1902. 27. Prieto C, Stam RW, Agraz-Doblas A, et al. Activated KRAS Cooperates with MLL-AF4 to Promote Extramedullary Engraftment and Migration of Cord Blood CD34+ HSPC But Is Insufficient to Initiate Leukemia. Cancer Res. 2016;76(8):2478-2489. 28. Munoz-Lopez A, van Roon EH, Romero-Moya D, et al. Cellular Ontogeny and Hierarchy Influence the Reprogramming Efficiency of Human B Cells into Induced Pluripotent Stem Cells. Stem Cells. 2016;34(3):581-587. 29. Munoz-Lopez A, Romero-Moya D, Prieto C, et al. Development Refractoriness of MLLRearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency. Stem Cell Reports. 2016;7(4):602-618. 30. Bueno C, Roldan M, Anguita E, et al. Bone marrow mesenchymal stem cells from patients with aplastic anemia maintain functional and immune properties and do not contribute to the pathogenesis of the disease. Haematologica. 2014;99(7): 1168-1175. 31. Menendez P, Catalina P, Rodriguez R, et al. Bone marrow mesenchymal stem cells from infants with MLL-AF4+ acute leukemia harbor and express the MLL-AF4 fusion gene. J Exp Med. 2009;206(13):3131-3141. 32. Rodriguez R, Tornin J, Suarez C, et al. Expression of FUS-CHOP fusion protein in immortalized / transformed human mesenchymal stem cells drives mixoid liposarcoma formation. Stem Cells. 2013;31(10):2061-2072. 33. Recasens-Zorzo C, Cardesa-Salzmann T, Petazzi P, et al. Pharmacological modulation of CXCR4 cooperates with BET bromodomain inhibition in diffuse large B-cell lymphoma. Haematologica. 2018. 34. Garcia-Peydro M, Fuentes P, Mosquera M, et al. The NOTCH1 / CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model. J Clin Invest. 2018.35. Dalmau J, Rotger M, Erkizia I, et al. Highly pathogenic adapted HIV-1 strains limit host immunity and dictate rapid disease progression. AIDS. 2014;28(9): 1261-1272. 36. Addo MM, Yu XG, Rathod A, et al. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J Virol. 2003;77(3):2081-2092. 37. Martin-Gayo E, Gonzalez-Garcia S, Garcia-Leon MJ, et al. Spatially restricted JAG1-Notch signaling in human thymus provides suitable DC developmental niches. J Exp Med. 2017;214(11):3361-3379. 38. Galy A, Verma S, Barcena A, Spits H. Precursors of CD3+CD4+CD8+ cells in the human thymus are defined by expression of CD34. Delineation of early events in human thymic development. J Exp Med. 1993; 178(2): 391-401. 39. Lepretre S, Graux C, Touzart A, Macintyre E, Boissel N. Adult T-type lymphoblastic lymphoma: Treatment advances and prognostic indicators. Exp Hematol. 2017;51:7-16. 40. Marks DI, Paietta EM, Moorman AV, et al. T-cell acute lymphoblastic leukemia in adults: clinical features, immunophenotype, cytogenetics, and outcome from the large randomized prospective trial (UKALL XII / ECOG 2993). Blood. 2009;114(25):5136-5145. 41. Mendes RD, Cante-Barrett K, Pieters R, Meijerink JP. The relevance of PTEN-AKT in relation to NOTCH1-directed treatment strategies in T-cell acute lymphoblastic leukemia. Haematologica. 2016; 101(9): 1010-1017. 42. LeMaistre CF, Rosen S, Frankel A, et al. Phase I trial of H65-RTA immunoconjugate in patients with cutaneous T-cell lymphoma. Blood. 1991;78(5): 1173-1182. 43. Frankel AE, Laver JH, Willingham MC, Burns LJ, Kersey JH, Vallera DA. Therapy of patients with T-cell lymphomas and leukemias using an anti-CD7 monoclonal antibody-ricin A chain immunotoxin. Leuk Lymphoma. 1997;26(3-4):287-298. 44. Junca J, Botin T, Vila J, Navarro JT, Milla F. Adult T-cell leukemia / lymphoma with an unusual CD1a positive phenotype. Cytometry B Clin Cytom. 2014;86(4):292-296. 45. van den Broek T, Madi A, Delemarre EM, et al. Human neonatal thymectomy induces altered Bcell responses and autoreactivity. Eur J Immunol. 2017;47(11): 1970 -1981. 46. Kurobe H, Tominaga T, Sugano M, et al. Complete but not partial thymectomy in early infancy reduces T-cell-mediated immune response: three-year tracing study after pediatric cardiac surgery. J Thorac Cardiovasc Surg. 2013;145(3):656-662, 662 e651-652; discussion 662. 47. van den Broek T, Delemarre EM, Janssen WJ, et al. Neonatal thymectomy reveals differentiation and plasticity within human naive T cells. J Clin Invest. 2016; 126(3): 1126-1136.

Claims

1. (i) an extracellular domain comprising a CD1a targeting moiety, wherein the CD1a targeting moiety is an antibody, scFV, Fab, or scFab comprising a VL domain and a VH domain, the VL domain comprising LCDR1, LCDR2, and LCDR3 polypeptides, the VH domain comprising HCDR1, HCDR2, and HCDR3 polypeptides, LCDR1 consisting only of [QDINKY] (SEQ ID NO: 1), LCDR2 consisting only of [YTS], LCDR3 consisting only of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consisting only of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consisting only of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consisting only of [ARGFYTMDY] (SEQ ID NO: 6), (ii) Transmembrane domain and (iii) Intracellular signaling domain and CD3-positive and CD1a-negative T cells containing nucleic acids encoding a chimeric antigen receptor (CAR).

2. The T cell according to claim 1, wherein the transmembrane domain includes the transmembrane domains of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

3. The T cell according to claim 2, wherein the transmembrane domain includes the transmembrane domain of CD8.

4. The T cell according to any one of claims 1 to 3, wherein the intracellular signaling domain includes the intracellular domains of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66b.

5. The T cell according to claim 4, wherein the intracellular signaling domain includes the intracellular domain of CD3ζ.

6. The T cell according to any one of claims 1 to 3, wherein the CAR further comprises a co-stimulatory signaling domain.

7. The T cell according to claim 6, wherein the aforementioned co-stimulatory signaling domain includes the intracellular domain of CD137.

8. A pharmaceutical composition comprising a plurality of T cells according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or diluent.

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