Car t-cells for 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 therapeutic approaches for the treatment of 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 capable of targeting CD1a. [Background technology]

[0002] T-lineage acute lymphoblastic leukemia (T-ALL) is a malignant disorder resulting from leukemic transformation of thymic T-cell precursors. 1 T-ALL is phenotypically and genetically heterogeneous and is commonly associated with genetic alterations / mutations in transcription factors involved in hematopoietic stem / progenitor cell (HSPC) homeostasis and key 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 , median age at diagnosis is 9 years 5~7 Intensive chemotherapy regimens have led to improved survival of patients with T-ALL. However, event-free survival (EFS) and overall survival (OS) remain below 70%, with relapsed / refractory (R / R) T-ALL being associated with a particularly poor outcome. Currently, there are no more effective treatment options than hematopoietic cell transplantation and conventional chemotherapy, which are associated with a significant trade-off in toxicity. 4、8 There is an increasing need for novel targeted therapies. T-cell lymphoblastic lymphoma (TCL) is etiologically and pathogenetically distinct from T-ALL, but phenotypically they are very similar. The main difference is that TCL is found extramedullary, whereas T-ALL is a myeloid disease.

[0003] Immunotherapy has generated unprecedented promise in cancer treatment, relying on the immune system as a powerful weapon against cancer. In recent years, chimeric antigen receptor (CAR)-based adoptive cellular immunotherapy has shown great promise. In CAR therapy, engineered T cells are redirected to specifically recognize and eliminate tumor cells expressing a particular antigen, independent of major histocompatibility complexes.9、10 The success of redirecting CAR T cells (CART) against CD19 or CD22 is currently uncontroversial for B-cell malignancies (mainly B-ALL). 11~14 However, the strategy of targeting T-cell malignancies with CART remains challenging due to the common 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 hypoplasia leading to fatal immunodeficiency. 15~17 .

[0004] Recent and elegant studies have demonstrated that T cells transduced with either the most expressed pan-T cell antigens CD7, CD3, CD5 or TCR CARs can efficiently eliminate T-ALL blasts in vitro and control disease in vivo. 15~20 Nevertheless, approaches such as CRISPR / Cas9 genome editing or protein expression blockers remain far from the clinic and require the destruction of target antigens in T cells prior to CAR transduction to avoid fratricide induced by a wide range of self-antigens. 15~17、19 . Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there remains 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 problem]

[0006] The choice of antigen against which it is desired to redirect T cells depends on the antigen-specificity of normal and malignant T cells. “We represent a major advance towards resolving issues associated with the common expression of T cell markers between BRCA1 and BRCA2. We identify the lipid-presented molecule CD1a as a suitable target for the treatment of a large subset of T-ALL, namely cortical T-ALL.”

[0007] We developed and functionally characterized a CD1a-specific CART that exhibited robust 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 serially expanded 200-fold, similar to mock T cells, demonstrating that redirecting CART against the CD1a antigen does not induce T cell fratricide. Furthermore, the use of CD1a CART in cortical T-ALL obviates the need for sophisticated genome editing-based destruction of target antigens in T cells prior to CAR transduction as a strategy to avoid self-antigen-induced fratricide. 15~17、19 During steady-state hematopoiesis, CD1a is expressed only on a subset of cortical CD34+CD7+ thymic T precursors, whereas earlier CD34 high CD7 high We further demonstrated that T precursors lack CD1a. In addition, neither normal CD34+ HSPCs nor mature T cells derived from multiple tissues express CD1a during ontogeny, minimizing the risk of on-target / off-tumor toxicity. Indeed, when CD7+ thymocytes derived from human fetal thymus were exposed to CD1a CART, only CD1a+ cortical thymocytes were eliminated by CD1a CART, whereas developmentally earlier and later thymic T lineage populations (CD34+ and CD34-) were not targeted, restricting the on-target / off-tumor effects to a developmentally transient thymic population of cortical thymocytes, further confirming the fratricide resistance of CD1a CART.

[0008] The exclusive thymic localization of cortical thymocytes and the fact that a thymic subpopulation of CD34+CD7+CD1a- T cell precursors that physiologically / regularly mature into functional T cells exists upstream of CD1a+ cortical thymocytes provide an additional level of safety for the use of CD1a CART in R / R T-ALL patients. No irreversible toxicity or severe T cell aplasia due to CD1a CART is expected because: i) the CD1a+ thymocyte population is a transient thymic T cell fraction that is eventually regenerated by upstream CD1a- T cell precursors; ii) CD1a CART itself normally responds to viral antigens and thus may protect against pathogens; iii) the clinical use of specific antibodies against CD5 or CD7 42 did not show severe or irreversible toxicity; iv) there are several studies demonstrating extrathymic maturation of T cells and a balance between the innate and adaptive immune systems that may at least partially ensure immunological protection in patients who have undergone partial or total thymectomy. 45~47 .

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

[0010] The invention also provides a nucleic acid encoding a CAR of the invention. Additionally, the invention provides a cell comprising the nucleic acid and / or CAR of the invention. The invention also provides a pharmaceutical composition comprising a plurality of cells according to the invention and a pharma- ceutically acceptable carrier or diluent.

[0011] The cell of the invention or the pharmaceutical composition of the invention for use as a medicament is provided. In particular, the invention provides a method for treating CD1a-positive cancer, comprising administering the cell of the invention or the pharmaceutical composition of the invention to a patient in need thereof. [Brief description of the drawings]

[0012] [Figure 1]Figure 1: CD1a expression in T-ALL and normal hematopoietic and thymocyte proliferation. (A) Immunophenotype of de novo T-ALL samples (n=38) for the indicated markers. Upper and middle curly brackets identify CD1a+ / ++ and CD1alow / + coT-ALL patients, respectively. Bottom black circle represents 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 diagnosis-relapse coT-ALL vs.). Data shown as CD1a expression in relapse samples versus diagnosis-matched samples (diagnosis shown as 100% expression). (D) T cells and CD34+HSPCs do not express CD1a throughout ontogeny. (E) Scheme showing the phenotype of developing thymic T cell populations. (F) Representative FACS of pre-cortical (CD34highCD7++CD1a-) and cortical (CD34+CD7++CD1a+) thymocytes. DX: diagnosis. RX: relapse. [Diagram 2]Figure 1: CD1a CART specifically targets and eliminates CD1a+ T-ALL cell lines in vitro. (A) Scheme of the CD1aCAR construct used. (B) CAR detection in 293T 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) Robust increase in activated T cells transduced with either mock or CD1a CAR without signs of fratricide (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 the indicated 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 at an E:T ratio of 1:1. (J) Representative FACS analysis of cytotoxicity by eFluor670-labeled target cells. (K) ELISA showing high levels of production of the inflammatory cytokines IL-2, TNFα, and IFNγ by CD1a CART exposed to Jurkat and NALM6 (negative control) cells in a 16-hour assay at an E:T ratio of 1:1 (n=4). *p<0.05, **p<0.01, ***p<0.001. [Diagram 3]Figure 1: 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 patients / primografts. % of CD1a+ blasts are shown. (B) Cytotoxicity (in absolute number of eFluor+ cells) measured by FACS in a 48 hour cytotoxicity assay at an E:T ratio of 4:1 (n=3). (C) Representative FACS analysis of CD1a in eFluor-labeled target cells at the end of the cytotoxicity assay revealing the specificity of CD1a CART (n=3). (D) High levels of proinflammatory cytokine production by CD1a CART analyzed by ELISA in a 16 hour assay at an E:T ratio of 4:1 (n=3 independent supernatants). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4] Figure 1: CD1a CART fully controls coT-ALL cell progression in a mouse xenograft setting. (A) Scheme of the xenograft model. NSG mice (n=6 / group) were injected iv with 3x106 Luc-GFP expressing Jurkat cells followed by a single iv injection of 5x106 mock or CD1a CART 3 days later. Tumor burden was monitored by bioluminescence (BLI) using IVIS imaging once every 4-6 days. When mock-treated animals were fully leukemic, half of the CD1a CART-treated animals were sacrificed and analyzed by FACS (BM, PB and spleen) for leukemic burden and CART persistence. The remaining animals were rechallenged 6 weeks later with 1.5x106 Luc-Jurkat and followed as before. (B) IVIS imaging of tumor burden monitored by BLI at the indicated time points. (C) Total radiance quantification (p / sec / cm2 / sr) at the indicated time points. †: sacrifice. (D) Circulating Jurkat cells in the PB 17 days after CART infusion. (E) T cell persistence in the PB on day 17, and in the spleen and BM at sacrifice. Data are shown as mean ± SD (n=6 mice / group). *p<0.05, **p<0.01, ***p<0.001. [Diagram 5]Figure 2: CD1a CART completely abolishes the progression of primary CD1a+ coT-ALL blasts in a PDX setting. (A) Scheme of the PDX model. NSG mice (n=5 or 6 / group) were injected iv with 1x106 primary coT-ALL cells followed 3 days later by a single iv injection of 1x106 mock or CD1a CART. Tumor burden was monitored biweekly by FACS and by blood sampling and BM aspiration after 6 and 9 weeks. (B, C) Frequency of leukemic mice and level of leukemia in BM (B) and PB (C) 6 and 9 weeks after injection of CART. Left panel shows representative FACS plots. Primary CD1a+ T-ALL blasts are shown in the box (grey). Effector T cells are shown outside the box in grey. Mouse cells are shown in black. (D) 9-week OS of coT-ALL primografts given either CD1a CART or mock T cells. (E) Effector T cell persistence over time in the PB (weeks 2-9) and BM (weeks 6 and 9). Each dot represents an independent mouse. **p<0.01, Malcolm-Cox test. [Figure 6]Figure 2: CD1a CART retains the ability to control progression of CD1a+ cell lines and primary coT-ALL samples in the rechallenge PDX setting. (A) IVIS imaging of Jurkat cell burden in rechallenged mice. (B) Total radiance quantification (p / sec / cm2 / sr) over time in mice rechallenged with Jurkat cells. (C) Circulating Jurkat cells in PB 16 days after rechallenge. (D) Robust effector T cell persistence in PB, BM and spleen upon sacrifice of rechallenged animals. (E) Scheme of rechallenge PDX experiment with primary coT-ALL samples. CART-bearing PDX mice were rechallenged with 1x106 primary CD1a+ T-ALL 7 weeks after initial CART infusion. (F) Secondary coT-ALL burden in engrafted PB (left panel) and BM (right panel) 6 weeks after leukemia rechallenge. (G) Effector T cell persistence over time in PB (weeks 2, 4 and 6) derived from PDXs re-challenged with coT-ALL primary samples. Each dot represents an independent mouse. *p<0.05, **p<0.01, ***p<0.001, ****<0.0001. [Figure 7] Figure 1: CD1a CART derived from coT-ALL patients at disease onset specifically lyses autologous CD1a+ T-ALL blasts. (A) Scheme showing experimental design of autologous cytotoxicity assay. Mature (normal) CD3+CD1a- T cells were FACS purified from PB of coT-ALL patients, infected with CD1a CAR, expanded and exposed to autologous total PBMCs. (B) FACS analysis of 48 h autologous cytotoxicity assay with E:T of 1:1 and 4:1. eFluor670-labeled total PBMC target population contains CD1a+ T-ALL blasts (upper box) and mature CD3+CD1a- T cells (lower box). (C) Quantification of CD1a CART-mediated specific lysis for coT-ALL blasts (upper panel) and CD3+CD1a- mature T cells (lower panel). (D) ELISpot showing number of IFNγ SFC from mock vs. CD1a CART stimulated with pools of peptides derived from CMV, EBV and influenza (CEF). Staphylococcal enterotoxin B (SEB) was used as a positive control. [Figure 8] Figure 1 shows the immunophenotype for each of the individual CD1a++ coT-ALL patients presented in this study. (A) Gating strategy to distinguish mature normal T cells (CD3++CD1a-, either CD4+ or CD8+) and coT-ALL blasts (CD7+CD1a+). Note that coT-ALL blasts generally have aberrant 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] Figure 1 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. CD22 CART lyses the B-ALL line NALM6, but CD1a CART does not lyse NALM6. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 10] Figure 2: Dose-dependent in vivo cytotoxicity of CD1a CART. (A) Tumor burden monitored by BLI at the beginning of the experiment (scale: 3x104 p / sec / cm2 / sr to 1x105 p / sec / cm2 / sr) confirming early and efficient T-ALL engraftment. (B) IVIS imaging of tumor burden monitored by BLI at the indicated time points for CART doses of 2x106 p / sec / cm2 / sr and 5x106 p / sec / cm2 / sr. (C) Total radiance quantification (p / sec / cm2 / sr) at the indicated time points for CART doses of 2x106 and 5x106. N=3 or 4 mice / group. †: sacrifice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 11]Figure 1 shows that CD1a CART does not target CD7+CD1a- thymocytes. Cytotoxicity assays against fetal thymocytes were performed with CD1a CART and mock T cells at 16 and 72 hours with E:T of 4:1 (n=2). [Figure 12] FIG. 1 shows that the absolute numbers of CD1a- primary coT-ALL cells remain the same after either mock or CD1a CART exposure, confirming that CD1a expression was not lost / downregulated by immune pressure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] definition "Administering" a medication to a patient or "administration" of a medication to a patient (and grammatical equivalents of this phrase) refers to direct administration, which can be administration to the patient by a medical professional or self-administration, and / or indirect administration, which can be the act of prescribing a medication. For example, a physician who instructs a patient to self-administer a medication or prescribes a medication to a patient administers the medication to the patient.

[0014] The term "affibody" refers to proteins derived from the Z domain of Protein A and engineered to bind to specific targets (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 or is immunologically reactive with a specific target. The term includes whole antibodies and any antigen-binding portion thereof or single chains, and combinations thereof. For example, the term "antibody" specifically includes bivalent antibodies and bivalent, bispecific antibodies.

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

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

[0018] Each "light chain" comprises a "light chain variable domain" (abbreviated herein as "VL") and a "light chain constant domain" ("CL"). The light chain constant domain (CL) can be of the kappa or lambda type. The VH and VL domains can be further subdivided into regions of hypervariability, called complementarity determining regions ("CDRs"), interspersed with regions that are more conserved, called "framework regions" ("FW").

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

[0020] The precise amino acid sequence boundaries of a given CDR are set forth 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); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme). The determination can be made using any of a number of known schemes, including those described herein.

[0021] Therefore, it is understood by those skilled in the art that the sequences FW1, FW2, FW3 and FW4 are equally disclosed. For a particular 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 particular VL, FW1 is a subsequence between the N-terminus of VL and the N-terminus of L-CDR1, and 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 a binding target, also referred to herein as "antigen-binding sites" or "antigen binding sites." The constant domains of the antibody can mediate binding of the antibody to host tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Exemplary antibodies of the present disclosure include classical antibodies as well as bivalent fragments and variants thereof, 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 molecule that contains an antigen-binding site.

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

[0025] The term "anticalin" is derived from lipocalin and is a molecule that binds to a specific target. This refers to a protein that has been modified to bind to a ribosome (see Skerra, 2008. FEBS J. 275(11):2677-83). Please refer to.

[0026] The term "antigen-binding fragment" or "Fab" refers to an antibody fragment that contains one constant domain and one variable domain of each of the heavy and light chains. Fab fragments can be obtained by digestion of an intact monoclonal antibody with papain.

[0027] The term "cancer" refers to a cancer caused by uncontrolled, usually rapid, cell proliferation without physiological function. It refers to a group of diseases that can be defined as any abnormal benign or malignant neoplasm of tissue that arises from a tumor and has the potential to invade or metastasize to other parts of the body.

[0028] The term "CD1a" refers to a non-polymorphic MHC class 1-related cell surface glycoprotein expressed in association with β2-microglobulin. CD1a is expressed by cortical thymocytes, Langerhans cells, and interdigitating cells. CD1a is also expressed in some malignancies of the T-cell lineage 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 number P06126.

[0029] "CD1a positive" cancers, including "CD1a positive" cancerous diseases, are cancers comprising cells in which CD1a is present on the cell surface. The term "CD1a positive" also refers to cancers in which cells comprising the CAR of the present invention produce sufficient levels of CD1a on the cell surface to have a therapeutic effect mediated by binding of the CAR to CD1a. In some embodiments, the CD1a positive cancer is 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 the context of a CAR, the CD1a targeting moiety targets T cells to CD1a-positive cells, preferably cancer cells. It should be understood that in the context of a CAR, the CD1a targeting moiety can be 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 T cell function, metabolism, and persistence (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124). Similarly, "CART" The term refers to a T cell that comprises a CAR.

[0032] "Combination therapy," "in combination with," or "in conjunction with," as used herein, refers to any form of combined, parallel, simultaneous, sequential, or intermittent treatment with at least two different therapeutic modalities (i.e., compounds, components, targeted agents, or therapeutic agents). Thus, these terms refer to the administration of one therapeutic modality to a subject before, during, or after the administration of the other therapeutic modality. Modalities in a combination can be administered in any order. The therapeutic modalities are administered together (e.g., simultaneously in the same or separate compositions, formulations, or unit dosage forms) or separately (e.g., on the same or different days, in any order according to administration protocols appropriate for the separate compositions, formulations, or unit dosage forms), in a manner and dosing schedule prescribed by a medical professional or in accordance with a regulatory agency. In general, each therapeutic modality is administered at a dose and / or schedule determined for that therapeutic modality. Optionally, more than two modalities may be used in a combination therapy. In addition, the combination therapy provided herein can be used in combination with other types of treatment. For example, the other anti-cancer treatment may be selected from the group consisting of chemotherapy, surgery, radiotherapy (radiation), and / or hormonal therapy, among other treatments associated with the current standard of care for the subject.

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

[0034] The term "costimulatory signaling domain" refers to, for example, the CD3 It refers to a signaling portion that provides a signal to a T cell that mediates a T cell response, including, but not limited to, activation, proliferation, differentiation, cytokine secretion, etc., in addition to the primary signal provided by the ζ chain. A costimulatory domain can include, but is not limited to, all or a portion of CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds CD83. In some embodiments, a costimulatory signaling domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate a cellular response, including activation, proliferation, differentiation, and cytokine secretion.

[0035] The term "designed ankyrin repeat proteins" or "DARPins" refers to proteins that are derived from ankyrin repeats and engineered 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 that a patient remains free of disease.

[0037] As used herein, the term "effective amount" of an agent, e.g., a therapeutic agent such as CART, is an amount sufficient to produce a beneficial or desired result, e.g., a clinical result, and thus, an "effective amount" 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 amount can reduce the number of cancer cells, reduce tumor size or burden, inhibit (i.e., slow to a certain extent, and in certain embodiments, stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to a certain extent, and in certain embodiments stop) tumor metastasis, inhibit tumor growth to a certain extent, alleviate to a certain extent one or more symptoms associated with cancer, and / or produce a favorable response, such as an increase in progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), a complete response (CR), a partial response (PR), or in some cases stable disease (SD), a decrease in progressive disease (PD), a shortened time to progression (TTP), or any combination thereof. The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0038] The term "fynomer" is derived from the SH3 domain of human Fyn kinase. The term refers to proteins that have been engineered to bind to specific targets (Bertschinger et al., 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 in any way. An "individual", "patient" or "subject" may be of any age, sex and physical condition. The term "patient in need thereof" refers to a patient who is usually suffering from a CD1a positive cancer.

[0040] "Infusion" or "infusing" refers to the introduction of a solution containing a therapeutic agent into the body via a vein for therapeutic purposes. Typically, this is accomplished by an intravenous bag.

[0041] "Intracellular signaling domain," as used herein, refers to all or a portion 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 signals by interacting with cellular mediators, resulting in proliferation, differentiation, activation, and other effector functions. Examples of intracellular signaling domains for use in the CARs of the present invention include intracellular sequences of the CD3 zeta chain and / or co-receptors that act in concert to initiate signaling after CAR ligation, as well as any derivatives or variants of these sequences, as well as any synthetic sequences with the same function. T cell activation can be said to be mediated by two distinct 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 costimulatory signals (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain signaling motifs known as receptor tyrosine-based activation motifs or ITAMs. Examples of primary cytoplasmic signaling sequences that contain ITAMs include those derived from CD3zeta, FcRgamma, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0042] The term "monobody" refers to a protein derived from the fibronectin type III domain and engineered 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 that comprises 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 time from patient enrollment until death or censoring at last known alive date. OS includes an increase in life expectancy compared to untreated or untreated individuals or patients. Overall survival refers to the situation in which a patient remains alive for a defined period of time, e.g., 1 year, 5 years, etc., from the time of diagnosis or treatment.

[0045] "Partial response" or "PR" refers to a reduction in the sum of diameters of target lesions of at least 30% referenced to the baseline sum of diameters in response to treatment as defined in the RECIST v1.1 guideline.

[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 loop regions of stable protein scaffolds (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12):1082-101).

[0047] As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, additional buffering agents, preservatives, co-solvents, 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, 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 (e.g., cyclosporine, cyclopentasiloxane, cyclohex ... For example, organic sugars or sugar alcohols such as inositol, 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 pharma- ceutical acceptable polymers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), may be used. Acceptable carriers, excipients, or stabilizers may be included in the pharmaceutical compositions described herein so long as 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 overt 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 from the start of treatment due to an increase in either tumor mass or spread.

[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 Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 standard. Generally, progression-free survival refers to the situation in which a patient continues to live without the cancer getting worse.

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

[0051] The term "repebody" refers to proteins derived from leucine-rich repeat modules and engineered to bind to specific targets (see Lee et al., 2012. PNAS. 109(9): 3299-3304).

[0052] The term "respond favorably" generally refers to producing a beneficial state in a subject. In the context of cancer treatment, the term refers to producing a therapeutic effect on a subject. A favorable therapeutic effect in cancer can be measured in a number of 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 treatments. For tumor growth inhibition, according to NCI standards, T / C≦42% is the lowest level of anti-tumor activity. T / C<10% is considered a high level of anti-tumor activity, where T / C(%)=median tumor volume with treatment / median tumor volume of control×100. A favorable response may be assessed, for example, by an increase in progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), a complete response (CR), a partial response (PR), or in some cases stable disease (SD), a reduction in progressive disease (PD), a shortened time to progression (TTP), or any combination thereof.

[0053] The term "sequence identity" refers to the percentage value obtained when two sequences are compared using a pairwise sequence alignment tool. In the present case, sequence identity is obtained using the global alignment tool "EMBOSS Needle" using 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 term "single-chain antigen-binding fragment" or "scFab" refers to a fusion protein containing one variable and one constant domain of an antibody light chain attached to one variable and one constant domain of an antibody heavy chain, the heavy and light chains being linked by a short peptide.

[0055] The term "single-chain variable fragment" or "scFv" refers to a fusion protein containing the 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 by disulfide bonds are disclosed in Reiter et al., 1996. Nat Biotechnol. 14(10):1239-45. It has been done.

[0056] "Stable disease" refers to disease without progression or recurrence as defined by the RECIST v1.1 guidelines. Stable disease is neither sufficient tumor shrinkage to be considered a partial response nor sufficient tumor growth to be considered progressive disease.

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

[0058] The terms "treatment" and "therapy" as used in this application refer to a set of hygienic, pharmacological, surgical and / or physical measures used with the goal of improving a health problem with the objective of curing and / or alleviating a disease and / or symptom. The terms "treatment" and "therapy" include preventative and curative methods, since both are directed to maintaining and / or restoring the health of an individual or animal. The administration of suitable medicines to alleviate and / or cure a health problem, regardless of the cause of the symptoms, disease and disability, should be interpreted as a form of treatment or therapy within the context of this application.

[0059] Chimeric Antigen Receptor In one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular domain comprising 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, anticalin, lipibody, monobody, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, or peptide aptamer that specifically binds to CD1a.

[0061] Binding molecules that specifically bind to CD1a may be very useful in the diagnosis and treatment of the above-mentioned disorders. Several mouse monoclonal antibodies against CD1a are known in the art (Kelly (1994), Amiot et al. (1986), Furue et al. (1992)). However, mouse antibodies are limited for in vivo use due to problems associated with the administration of mouse antibodies to humans, such as short serum half-life, inability to elicit certain human effector functions, and the generation of undesirable immune responses against mouse antibodies (Van Kroonenburgh and Pauwels (1988)). New human antibodies have been developed in recent years that overcome these aforementioned drawbacks. In addition to NA1 / 34.HLK, other Hybridomas, such as SIGMA ALDRICH's OKT6 (IgG1 isotype), are commercially available. 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 (e.g., U.S. Pat. No. 5,223,409 to Ladner et al., WO 92 / 18619 to Kang et al., WO 91 / 17271 to Dower et al., WO 92 / 20791 to Winter et al., WO 92 / 15679 to Markland et al., WO 93 / 01288 to Breitling et al., WO 94 / 01366 to McCafferty et al., and WO 95 / 01101 to McCafferty et al.). No. 92 / 01047, Garrard et al., WO 92 / 09690, Ladner et al. 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 et al. (1991) Nuc Acid Res 19:4133-4137 and Barbas et al. (1991) PNAS 88:7978-7982, the contents of both of which are incorporated herein by reference.

[0064] Additionally, methods are known in the art for generating and selecting non-immunoglobulin scaffolds that bind to specific targets (see, e.g., 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, wherein LCDR1 consists solely of [QDINKY] (SEQ ID NO: 1), LCDR2 consists solely of [YTS], LCDR3 consists solely of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists solely of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists solely of [INPNSAST] (SEQ ID NO: 5) and HCDR3 consists solely 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, wherein LCDR1 consists solely of [QDINKY] (SEQ ID NO: 1), LCDR2 consists solely of [YTS], LCDR3 consists solely of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists solely of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists solely of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists solely of [ARGFYTMDY] (SEQ ID NO: 6).

[0067] 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 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 moiety is an scFv comprising 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.

[0069] VL domain (SEQ ID NO: 7) [RDIQMTQSPSSLSASLGGKVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYSFSISNLEPEDIATYYCLHYDNLPWTFGGGTKLEIKRA]

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

[0071] In some embodiments, the CD1a targeting moiety is an scFv comprising or consisting of SEQ ID NO:9.

[0072] scFv from clone NA1 / 34.HLK (SEQ ID NO: 9) [QVQLQQSGAELARPGASVKMSCKASGYAFSTYTMHWVKQRPRQGLEWIGYINPNSASTSYNENFKDKATLTADKSSNTAYMHLSSLTSEDSAVYYCARGFYTMDYWGQGTSVTVSSGGGGGSGG GGSGGGGSGGGGSRDIQMTQSPSSLSASLGGKVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYSFSISNLEPEDIATYYCLHYDNLPWTFGGGTKLEIKRA]

[0073] Transmembrane domain The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region may include at least the transmembrane region(s) 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 mutant of a naturally occurring transmembrane domain, hi some embodiments, the synthetic or mutant transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine.

[0075] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a variant thereof, wherein the variant has 95% sequence identity.

[0076] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a variant thereof, wherein the variant has 98% sequence identity.

[0077] In some embodiments, the transmembrane domain comprises the transmembrane domain 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 transmembrane domain of CD8 or a variant thereof, wherein the variant has 95% sequence identity.

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

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

[0081] In some embodiments, the transmembrane domain comprises SEQ ID NO:10 or a sequence having 95% sequence identity to SEQ ID NO:10.

[0082] In some embodiments, the transmembrane domain comprises SEQ ID NO:10 or a sequence having 98% sequence identity to SEQ ID NO:10.

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

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

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

[0086] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3zeta, FcRgamma, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD66b, or a variant thereof, wherein the variant has 95% sequence identity.

[0087] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3zeta, FcRgamma, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD66b, or a variant thereof, wherein the variant has 98% sequence identity.

[0088] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3zeta, FcRgamma, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, or CD66b.

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

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

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

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

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

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

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

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

[0097] Costimulatory Signaling Domains In some embodiments, the CAR may further comprise a costimulatory signaling domain, which in some embodiments comprises the intracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, or a variant thereof, which variant has 95% sequence identity.

[0098] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, or a variant thereof, which variant has 98% sequence identity.

[0099] In some embodiments, the costimulatory signaling domain comprises 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 costimulatory signaling domain comprises the intracellular domain of CD137 or a variant thereof, wherein the variant has 95% sequence identity.

[0101] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD137 or a variant thereof, wherein the variant has 98% sequence identity.

[0102] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD137.

[0103] In some embodiments, the costimulatory signaling domain comprises SEQ ID NO:12 or a sequence having 95% sequence identity to SEQ ID NO:12.

[0104] In some embodiments, the costimulatory signaling domain comprises SEQ ID NO:12 or a sequence having 98% sequence identity to SEQ ID NO:12.

[0105] In some embodiments, the costimulatory signaling domain comprises SEQ ID NO:12 or a sequence having 99% sequence identity to SEQ ID NO:12.

[0106] In some embodiments, the costimulatory signaling domain comprises SEQ ID NO: 12. In some embodiments, the costimulatory signaling domain consists solely of SEQ ID NO: 12.

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

[0108] Full sequence CAR according to the present invention In some embodiments, the 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, HC an scFv comprising LCDR1, LCDR2 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 SEQ ID NO: 10 or a sequence having 95% sequence identity to SEQ ID NO: 10; (iii) an intracellular signaling domain comprising SEQ ID NO:11 or a sequence having 95% sequence identity to SEQ ID NO:11; and (iv) a costimulatory signaling domain comprising SEQ ID NO:12, or a sequence having 95% sequence identity to SEQ ID NO:12; and Includes.

[0109] In some embodiments, the 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, in which 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 SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10; (iii) an intracellular signaling domain comprising SEQ ID NO:11 or a sequence having 98% sequence identity to SEQ ID NO:11; and (iv) a costimulatory signaling domain comprising SEQ ID NO:12, or a sequence having 98% sequence identity to SEQ ID NO:12; and Includes.

[0110] In some embodiments, the 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, in which LCDR1 consists solely of [QDINKY] (SEQ ID NO: 1), LCDR2 consists solely of [YTS], LCDR3 consists solely of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists solely of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists solely of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists solely of [ARGFYTMDY] (SEQ ID NO: 6); (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10; (iii) an intracellular signaling domain comprising SEQ ID NO:11 or a sequence having 99% sequence identity to SEQ ID NO:11; (iv) a costimulatory signaling domain comprising SEQ ID NO:12, or a sequence having 99% sequence identity to SEQ ID NO:12; and Includes.

[0111] In some embodiments, the 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, in which LCDR1 consists only of [QDINKY] (SEQ ID NO: 1), LCDR2 consists only of [YTS] and LCDR3 consists only of [LHYDNLP an scFv consisting only of [WT] (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) a transmembrane domain comprising SEQ ID NO: 10; and (iii) an intracellular signaling domain comprising SEQ ID NO:11; and (iv) a costimulatory signaling domain comprising SEQ ID NO:12; and Includes.

[0112] In some embodiments, the 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, in which LCDR1 consists solely of [QDINKY] (SEQ ID NO: 1), LCDR2 consists solely of [YTS], LCDR3 consists solely of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists solely of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists solely of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists solely of [ARGFYTMDY] (SEQ ID NO: 6); (ii) a transmembrane domain consisting of SEQ ID NO: 10; (iii) an intracellular signaling domain consisting of SEQ ID NO: 11; (iv) a costimulatory signaling domain consisting of SEQ ID NO: 12; and Includes.

[0113] In some embodiments, the CAR is (i) an scFv comprising 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; (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 95% sequence identity to SEQ ID NO: 10; (iii) an intracellular signaling domain comprising SEQ ID NO:11 or a sequence having 95% sequence identity to SEQ ID NO:11; and (iv) a costimulatory signaling domain comprising SEQ ID NO:12, or a sequence having 95% sequence identity to SEQ ID NO:12; and Includes.

[0114] In some embodiments, the CAR is (i) an scFv comprising 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; (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10; (iii) an intracellular signaling domain comprising SEQ ID NO:11 or a sequence having 98% sequence identity to SEQ ID NO:11; and (iv) a costimulatory signaling domain comprising SEQ ID NO:12, or a sequence having 98% sequence identity to SEQ ID NO:12; and Includes.

[0115] In some embodiments, the CAR is (i) an scFv comprising 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; (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10; (iii) an intracellular signaling domain comprising SEQ ID NO:11 or a sequence having 99% sequence identity to SEQ ID NO:11; (iv) a costimulatory signaling domain comprising SEQ ID NO:12, or a sequence having 99% sequence identity to SEQ ID NO:12; and Includes.

[0116] In some embodiments, the CAR is (i) an scFv comprising 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; (ii) a transmembrane domain comprising SEQ ID NO: 10; and (iii) an intracellular signaling domain comprising SEQ ID NO:11; and (iv) a costimulatory signaling domain comprising SEQ ID NO:12; and Includes.

[0117] In some embodiments, the CAR is (i) an scFv comprising 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; (ii) a transmembrane domain consisting solely of SEQ ID NO: 10; (iii) an intracellular signaling domain consisting only of SEQ ID NO: 11; (iv) a costimulatory signaling domain consisting solely of SEQ ID NO: 12; and Includes.

[0118] In some embodiments, the CAR comprises or consists of SEQ ID NO:2 or a sequence having 95% sequence identity to SEQ ID NO:2. In some embodiments, the CAR comprises or consists of SEQ ID NO:2 or a sequence having 98% sequence identity to SEQ ID NO:2. In some embodiments, the CAR comprises or consists of SEQ ID NO:2 or a sequence having 99% sequence identity to SEQ ID NO:2. In some embodiments, the CAR comprises or consists of SEQ ID NO:2.

[0119] Complete sequence of CAR (SEQ ID NO:2) [MALPVTGLLLSLGLLLHAARPTGQVQLQQSGAELARPGASVKMSCKASGYAFSTYTMHWVKQRPRQGLEWIGYINPNSASTSYNENFKDKATLTADKSSNTAYMHLSSLTSEDSAVYYCARG FYTMDYWGQGTSVTVSSGGGGSGGGGSGGGGGSGGGGSRDIQMTQSPSSLSASLGGKVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGGSGREYSFSISNLEPEDIATY YCLHYDNLPWTFGGGTKLEIKRATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR]

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

[0121] In some embodiments, the nucleic acid is suitable for transducing or transforming a cell, hi some embodiments, the nucleic acid is suitable for transducing or transforming a T cell for use in adoptive immunotherapy.

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

[0123] The nucleic acids of the invention can be included in gamma-retroviral or lentiviral vectors that can be used to transduce or transform T cells (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124). Nucleic acids can also be inserted into cells using fection or genome editing techniques such as TALEN, ZFN and CRISPR / Cas9 (see Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124).

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

[0125] In some embodiments, the cells are naive T cells, memory stem T cells, or central memory T cells. These cells are currently believed to be better 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 that is treated with any one of the methods of the present invention. It is 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 that contain the nucleic acid and / or CAR of the present invention.

[0127] In some embodiments, the cells are allo-tolerant T cells. The term "tolerant cells" refers to cells that have been modified to reduce the risk of graft-versus-host disease responses. In some embodiments, this is accomplished by deletion of the TCR and / or β2-microglobulin via genome editing. 15、19 Allogeneic tolerant cells are known in the art (Riviere & Sadelain, 2017. Mol Ther. 25(5):1117-1124, allogeneic T cells, (see chapter ).

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

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

[0130] Pharmaceutical Compositions In one aspect, the invention provides a pharmaceutical composition comprising a plurality of the cells of the invention and a pharma- ceutically 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 agents that provide stability to CART against freezing-induced stress. Non-limiting 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 commonly used.

[0132] In some embodiments, the cells are formulated by first harvesting them from their culture medium, then washing the cells and concentrating them in a therapeutically effective amount in a medium and container system suitable for administration (a "pharmaceutical acceptable" carrier). A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or lactated Ringer's solution can also be used. The infusion medium may be supplemented with human serum albumin, fetal bovine serum or other human serum components.

[0133] In one aspect, the present invention provides a cell according to the present invention or a pharmaceutical composition according to the present invention for use as a medicament.

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

[0135] In some embodiments, the patient is administered a therapeutically effective amount of cells. In some embodiments, the patient is administered 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 The number of cells will depend on the intended end use of the composition, as will the type of cells contained in the composition. For example, if cells specific for a particular antigen are desired, the population will contain more than 70%, typically more than 80%, 85% and 90%-95% of such cells. For the uses provided herein, the cells will generally be in a volume of 1 liter or less, and may be 500 ml or less, or even 250 ml or less, or 100 ml or less. A clinically relevant number of cells will be greater than 10, cumulatively. 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 The amount of the injected cell can be distributed over multiple injections equal to or exceeding 100 cells.

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

[0137] In some embodiments, the method comprises a combination therapy. In some embodiments, the method comprises further administering an immune checkpoint inhibitor (see Lim & June, 2017. Cell. 168(4):724-740). In further embodiments, the method comprises further administering an immune checkpoint inhibitor and / or an IAP inhibitor (see WO 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 first treated 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 avoided altogether.

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

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

[0141] In particular, in first-line trials of ALL in adults, there is broad consensus that relapse is defined as "detection of more than 5% blasts in the bone marrow or clear evidence of extramedullary leukemia involvement after achievement of a previous complete remission (CR)" (see Goekbuget (2017)). The European Working Group on Adult ALL (EWALL) states: , who recorded this statement in a consensus recommendation with the additional explanation that "in the case of 5%-20% blasts during the intensive treatment phase and / or at some stage during regeneration, the bone marrow evaluation should be repeated after 1 week to distinguish between bone marrow relapse and regeneration phenomena" (see Dohner (2010) The definitions cited are based on international recommendations for outcome parameters in acute myeloid leukemia that have been estimated for several subtypes of ALL as well as for T-ALL (see Cheson (2003) and Chantepie (213)).

[0142] In recent years, some studies have not even defined the concept of relapse. Thus, studies with chimeric antigen receptor (CAR) T cells have included patients with "measurable disease" and even hematological relapse (without further specification) or minimal residual disease (MRE) (see Lee (2015) and Maude (2014) and Goekbuget (2017)). See: 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 methods of the invention are in complete or near complete remission after treatment with another therapy. In the case of patients with highly aggressive relapsed / refractory cortical T-cell acute lymphoblastic leukemia, it is preferable to reduce the tumor burden before using the methods of the invention, since some alternative effector T cells exist. In some embodiments, the patient being treated with the methods of the invention has previously been treated with another therapy that resulted in a partial response, a complete response, stable disease, a decrease in progressive disease, a decrease in time to tumor progression, or any combination thereof. EXAMPLES

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

[0145] Generation of CAR-expressing lentivirus, transduction, activation and expansion of T cells VSV-G pseudotyped CAR-expressing viral particles were produced in 293T cells using a standard polyethylenimine transfection protocol and concentrated by ultracentrifugation as described elsewhere. 27 Viral titers were consistently 10 8 Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation from buffy coats derived from healthy volunteers. Buffy coats were obtained from the Barcelona Blood and Tissue Bank (BST) after IRB approval (HCB / 2018 / 0030). T cells were activated with plate-bound anti-CD3 (OKT3) and anti-CD28 antibodies (BD Biosciences) for 2 days 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 Cell surface expression of CD1aCAR was tracked by fluorescence-activated cell sorting (FACS) co-expression of GFP 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 2 days of expansion.

[0146] Immunophenotyping of healthy CD34+ precursors, T cells and primary T-ALL samples Expression of CD1a antigen on CD34+ stem / progenitor cells (HSPCs), CD34+CD7+ thymic T cell precursors and CD3+ T cells was prospectively 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 terminated at 18-22 weeks gestation from the MRC / Wellcome Trust Human Developmental Biology Resource after informed consent and approval by the local Ethics and Biohazard Board Committee (CMRBCEIC-26 / 2013). were collected as previously described 28、29 Neonatal and adult tissues were obtained from BST after IRB approval (HCB / 2018 / 0030). Primary T-ALL samples and diagnostic Predictive immunophenotyping data were obtained from the local hospitals Sant Joan de Den, Germans Trias i Pujol and Santa Creu i San Pau (Barcelona, ​​Spain). For immunophenotyping of primary samples, the following fluorochrome-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 fluorochrome-conjugated MoAbs b was always used as a fluorescence reference. Briefly, PB mononuclear cells (PBMCs, approximately 5 × 10 5 ) were incubated with red blood cell lysis solution (BDBiosciencies) for 10 min and then stained with MoAb (20 min at 4 °C in the dark). Stained cells were washed in phosphate-buffered saline (PBS) and analyzed by FACS on a FACSCanto-II flow cytometer (BDBiosciencies) equipped with FACSDiva software.30~32 .

[0147] In vitro cytotoxicity assays and determination of cytokine release The cell lines Jurkat, MOLT4, and NALM6 were purchased from DSMZ (Braunschweig, Germany). Luciferase (Luc) / GFP expressing cells were stably generated by retroviral transduction and FACS purification of GFP+ cells. 33 Target cells (cell lines and primary T-ALL blasts) were labeled with 3 μM eFluor670 (eBioscience) and co-expressed with different effector:targets along with CD1a, CD22 or mock CART. CART-mediated cytotoxicity was determined by analyzing remaining viable (7-AAD-) eFluor670+ target cells at each time point and E:T ratio. Absolute cell numbers were determined using Trucount absolute count beads (BD Biosciences). Additionally, cortical T-AL cells at onset were stained with 0.0001% CI, 0.01 to 0.0001. FACS-selected CD3+CD1a- mature T cells derived from the PB of patient L were activated, transduced with CD1a CAR and tested against their eFluor670-labeled autologous CD1a+ T-ALL blasts. Production of the proinflammatory cytokines IL-2, TNFα and IFNγ was measured by ELISA (Human ELISA SET, BD Biosciences) in supernatants harvested 16 hours later.

[0148] In vivo Jurkat and T-ALL patient-derived xenograft (PDX) models Six- to 12-week-old non-obese diabetic (NOD)-Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice (The Jackson Laboratory) were bred and housed under pathogen-free conditions in the animal facility of the Barcelona Biomedical Research Park (PRBB). Mice were irradiated (2 Gy) and administered 3 × 10 61 x 10 Luc-GFP expressing Jurkat cells or 1 x 10 6 Primary cortical CD1a+ T-ALL blasts (primary and expanded in primografts) were transplanted intravenously (iv) 34 1.5×10 6 pieces~5×10 6 Mice were injected iv with either CD1a or mock CART 3 days later. When using Luc-Jurkat cells, tumor burden was followed by bioluminescence (BLI) using a Xenogen IVIS 50 Imaging System (Perkin Elmer). To measure luminescence, mice were given 150 mg / kg D-luciferin intraperitoneally and tumor burden was monitored at the indicated time points. Total luminescence was visualized and calculated using Living Image software (Perkin Elmer). Tumor burden of primary T-ALL samples was followed by biweekly blood sampling and FACS analysis. When mock CART-treated animals became leukemic, mice were sacrificed and tumor burden (hHLA-ABC+hCD45+hCD1a+graft) and CART persistence (hHLA-ABC+hCD45+hCD3+hCD1a-GFP+) were analyzed by FACS in BM, PB and spleen. In the rechallenge experiment, 1.5 × 10 6 Luc-Jurkat cells or 1 × 10 6 One of the CD1a+ T-ALL primografts was re-infused and disease recurrence was followed by BLI and FACS as described above. All procedures were performed in accordance with the Animal Care and Use Committee of the PRBB (DAAM7393).

[0149] Enzyme-linked immunospot assay (ELISpot) ELISpot plates (Millipore) were coated with anti-human IFNγ antibody (1-D1K, Mabtech) and kept overnight at 4° C. The plates were then washed six times with PBS containing 1% fetal bovine serum, after which 5×10 cells from three independent donors were added. 5 ~1×10 6Cells / well were plated and cultured in triplicate for 20 h at 37°C and 5% CO2. IFNγ-secreting cells were measured in response to 1 μg / mL CEF, a peptide pool of T cell epitopes of cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza, and 1 μg / mL Staphylococcal enterotoxin B (SEB) as a positive control. Plates were then revealed 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 and was determined by 10 5 The number of IFNγ spot-forming units (SFU) per cell was calculated.

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

[0151] Example 1: CD1a specifically marks cortical T-ALL blasts Due to the shared expression of target antigens between CART and T-lineage blasts, immunotherapeutic approaches in T-ALL are limited due to CART-associated fratricide and potentially lethal T-cell aplasia. However, the CD1a antigen is expressed in cortical T-ALL, a major subset of T-ALL (Figure 1A, B), and is completely absent on functional T cells in all extrathymic tissues. 25 Steady-state CD34+ HSPCs lack CD1a expression at multiple hematopoietic sites across ontogeny (Figure 1C). T cell development is initially dominated by CD34 highIt begins in the thymus by colonizing CD7-CD1a- primitive HSPCs, which subsequently respond to the thymic microenvironment by adopting CD34 high Differentiate into CD7+CD1a- early T cell precursors 37 As T cell precursors progress through thymic differentiation, they maintain CD7 expression and gradually lose CD34 expression, whereas CD1a expression appears, which is transiently restricted to cortical thymocytes. 38 (Figure 1E, F). Approximately 50% of the CD34+ thymic population is made up of precortical T cell precursors (CD34 high CD7+CD1a-, Fig. 1E, Fig. 1F (gray cells), CD1a, has a fatal outcome 3、39~41 , it can be hypothesized that it may be a feasible and safe target for immunotherapy in R / R cortical T-ALL.

[0152] Example 2: CD1a redirected T cells (CD1a CART) expand without T cell fratricide We designed a second generation CD1a CAR consisting of an anti-CD1a scFv, a CD8TM spacer, and the intracellular signaling domains of 4-1BB (CD137) and CD3ζ linked in-frame to GFP via a T2A sequence (Figure 2A). Expression of the CD1a CAR was readily detected by co-expression of both scFv and GFP in 293T cells (Figure 2B) and primary CD4+ and CD8+ T cell subsets (Figure 2C). Importantly, activated (CD69+CD25+) CD1a CART (Figure 2D), as well as mock T cells (Figure 2E), continuously increased 200-fold over a 12-day period, demonstrating that redirection of CART against the CD1a antigen does not induce T cell fratricide.

[0153] Example 3: CD1a CART specifically eradicates T-ALL cell lines and primary blasts in vitro CD1a CART was then tested in vitro using the 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 CARTs (either mock T cells or CD22 CART), CD1a CART specifically eliminated CD1a+ T-ALL cells depending on the E:T ratio. Relatively low E:T ratios of 2:1 or 4:1 induced 50%-80% specific cell lysis in a 16-h assay (Figure 2H, Figure 2I, Figure 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-h assay (Figure 2I). CD1a CART produced high levels of the proinflammatory cytokines IL-2, TNFα, and IFNγ when co-cultured with CD1a+ T-ALL cells, confirming their effects (Figure 2K).

[0154] To further address their ability to eliminate primary tumors, CD1a CARTs were co-cultured with primary cortical T-ALL samples (freshly harvested or from PDX) with percentages of CD1a+ blasts ranging from 80% to 98% (Figure 3A). Compared with mock T cells, CD1a CARTs specifically eliminated primary CD1a+ cortical T-ALL cells at an E:T ratio of 4:1 in a 72-h cytotoxicity assay (Figure 3B, Figure 3C). Normal hematopoietic cells (CD1a-) coexisting with CD1a+ T-ALL blasts in the BM were not lysed by CD1a CART (Figure 3C). High levels of IFNγ and TNFα were also secreted upon co-culture with CD1a+ primary T-ALL cells (Figure 3D). Collectively, these results indicate that CD1a CARTs have potent and specific antileukemic activity against T-ALL cell lines and primary blasts in vitro.

[0155] Example 4: CD1a CART exhibits potent anti-leukemic activity in vivo We next examined the activity of CD1a CART in Luc-expressing Jurkat T-ALL cells (Figure 4, Figure 10) and in a primary cortical T-ALL xenograft model. 34In vivo evaluation was performed using both 2 × 10 6 pcs or 5 x 10 6 iv injection of either CD1a (or mock) CART 3 × 10 6 Luc-expressing Jurkat cells were transplanted and leukemia establishment was followed weekly by BLI (Figure 4A, Figure 10). In contrast to mice given mock T cells, which showed extensive tumor burden by BLI, mice given CD1a CART were virtually leukemia-free by day 25 (Figure 4B, Figure 4C, Figure 10). Control of leukemia progression was dose-dependent with CD1a CART cells (Figure 10B, Figure 10C). Flow cytometric analysis of tumor burden in PB at sacrifice confirmed the BLI data (Figure 4D). Importantly, FACS analysis revealed T cell persistence in all hematopoietic tissues analyzed (Figure 4E). However, there was a markedly increased biodistribution of CD1a CART in the BM and spleen compared to T cell biodistribution in mice given mock T cells (Figure 4E), indicating active control of disseminated leukemia by CD1a CART.

[0156] In a more clinically relevant PDX model of cortical T-ALL, NSG mice were initially treated with 1 × 10 6 of primary CD1a+ T-ALL blasts, followed 3 days later by 1 × 10 6 After infusion of 10 CD1a (or mock) CART, leukemic engraftment was tracked by biweekly blood sampling and end-point BM analysis (Figure 5A). Engraftment of CD1a+ cortical T-ALL cells gradually increased over time in mock T cell-treated PDXs in both the 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) and was associated with significantly lower week 9 OS (42% vs. 100%, p = 0.01; Figure 5D). In contrast, CD1a CART completely abolished T-ALL growth / engraftment (0.36% and 0% T-ALL blasts in BM and PB, respectively), remaining in BM and PB after 9 weeks (Figure 5B, Figure 5C, Figure 5E).

[0157] Example 5: In vivo persistent CD1a CART is functional in a rechallenge assay Since persistence of CART in hematopoietic tissues is a key biological parameter for their clinical success, we next assessed whether remaining CD1a CART after 40-50 days remained functional and efficient in controlling T-ALL progression. To this end, T-ALL-engrafted mice, in which leukemia was eradicated by treatment with CD1a CART, were rechallenged with either Luc-Jurkat cells (Figures 6A-6D) or primary T-ALL derived from primografts (Figures 6E-6G). In contrast to controls, in which secondary leukemia engrafted rapidly (as early as 2 weeks) and extensively, T-ALL engraftment was barely detectable by either BLI or FACS in Jurkat (Figure 6C) or primograft models after 6 weeks (Figure 6F).

[0158] Example 6: Patient-derived CD1a CART specifically targets autologous CD1a+ blasts and retains antiviral activity Appropriate selection of target antigens and avoidance of T cell fratricide are essential for the success of CART in the treatment of T-ALL. Therefore, we tested whether PB-derived CD3+CD1a- T cells from patients with cortical T-ALL could be isolated and genetically engineered to express a CD1a CAR (Figure 7). To this end, CD3+CD1a- T cells were isolated from patients (>95% pure, data not shown), activated with CD3 / CD28, and expressed a CD1a CAR. We then transduced the cells with lentiviruses containing CAR or mock (31%-70% transduction). We then transduced CD1a cells derived from primary T-ALL against PBMCs matched to active T-ALL patients. The cytolytic potential of CART was investigated (Figure 7A). Total PBMCs were used as targets because this allows for the assessment of both the degree of fratricide and the degree of cytotoxicity. In eFluor670-labeled target PBMCs, the majority were CD1a+ blasts, and approximately 15% were CD3+CD1a- normal T cells (Figure 7B). Compared with mock T cells, CD1a CART showed extensive and specific cytolytic ability against autologous CD1a+ blasts, but not against CD1a- normal T cells (Figure 6B), further demonstrating that CD1a CART is fratricide-resistant.

[0159] To further assess the potential thymic toxicity of CD1a CART, we next used CD7+ thymocytes from human normal fetal thymus as target cells. Only CD1a+ cortical thymocytes (second and third grey boxes) were eliminated by CD1a CART, whereas developmentally earlier and later CD1a- (first box) thymic T lineage populations (CD7+CD34+ and CD7+CD34-) were not targeted (Figure 1E, Figure 1F), suggesting that the on-target / off-tumor effect was restricted to a 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 immunosuppressed patients. To this end, we tested the reactivity of CD1a CART against CMV, EBV and influenza antigens (CEF) and quantified SCF by IFNγ ELISpot. Mock T cells and CD1a Both CARTs displayed highly similar responses to stimulation with viral peptides, suggesting that CD1a CART retains antiviral activity ( Fig. 7D ).

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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 of only [QDKY] (SEQ ID NO: 1), LCDR2 consisting of only [YTS], LCDR3 consisting of only [LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consisting of only [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consisting of only [INPNSAST] (SEQ ID NO: 5), and HCDR3 consisting of only [ARGFYTMDY] (SEQ ID NO: 6), an extracellular domain, (ii) A transmembrane domain, (iii) An intracellular signaling domain, A CD1a-negative T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising the above.

2. The T cell according to claim 1, wherein the transmembrane domain comprises the transmembrane domain 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 comprises the transmembrane domain of CD8.

4. The T cell according to any one of claims 1 to 3, wherein the intracellular signaling domain comprises the intracellular domain 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 comprises 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 co-stimulatory signaling domain comprises 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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