Humanized CD1a targeting moieties for treatment of CD1a positive cancers

Abstract

Recurrent / refractory T cell acute lymphoblastic leukemia (T-ALL) has the outcome of depression, and there is no effective targeted immunotherapy against T-ALL. The CD1a is only expressed in one main subclass of the T-ALL and in the cortex T-ALL. Expression of CD1a is limited to that neither cortical thymus cells nor CD34 + progenitor cells nor T cells express CD1a in the individual development process, and therefore the risk of targeted / non-tumor toxicity is limited. The present invention provides a CD1a targeting moiety comprising CD1a, which can be placed in a T cell. The CART thus generated is suitable for the treatment of cortex T-ALL.

Description

Technical Field

[0001] The present invention provides therapies for treating CD1a positive cancers such as T-cell acute lymphoblastic leukemia. In particular, the present invention provides humanized CD1a targeting moieties. Background Art

[0002] T-cell lineage acute lymphoblastic leukemia (T-ALL) is a malignant disease arising from leukemic transformation of thymic T-cell precursors[1]. T-ALL is phenotypically and genetically heterogeneous and is often associated with genetic alterations / mutations in transcription factors involved in master regulators of hematopoietic stem / progenitor cell (HSPC) homeostasis and T-cell development[2]. T-ALL accounts for 10% to 15% and 20% to 25% of all acute leukemias diagnosed in children and adults, respectively[3,4], with a median age of diagnosis of 9 years[5]. Intensive chemotherapy regimens have improved the survival of patients with T-ALL. However, event-free survival (EFS) and overall survival (OS) remain <70%, and the outcomes of relapsed / refractory (R / R) T-ALL are particularly poor. There are currently no potential therapeutic options other than hematopoietic cell transplantation and conventional chemotherapy, which are associated with significant trade-offs in toxicity [4,6], reinforcing the need for novel targeted therapies.

[0003] Immunotherapy has generated unprecedented expectations in cancer treatment and relies on the immune system as a powerful weapon against cancer. In recent years, adoptive cell immunotherapy based on chimeric antigen receptor (CAR) has shown great potential. CAR therapy redirects genetically modified T cells to specifically recognize and eliminate tumor cells expressing specific antigens in a manner independent of the major histocompatibility complex [7,8]. The success of CAR T cells (CAR T-cell, CART) redirected to CD19 or CD22 is now undisputed for B-cell malignancies (mainly B-ALL) [9,10]. However, due to the co-expression of target antigens between CART and T-lineage tumor cells, the strategy of using CART to target T-cell malignancies remains challenging. In this regard, CARTs targeting pan-T cell antigens have two major disadvantages: i) CART self-targeting / fratricide, and ii) T cell agenesis, resulting in life-threatening immunodeficiency [11-13].

[0004] Recent studies have shown that T cells transduced with the most expressed pan-T cell antigens CD7, CD3, CD5, or TCR CARs can effectively eliminate T-ALL blasts in vitro and control the disease in vivo [11-15]. However, before CAR transduction, methods such as CRISPR / Cas9 genome editing or protein expression blockers, which are far from clinical use, need to be used to destroy the target antigen in T cells to avoid extensive self-antigen-driven cannibalism [11-15].

[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. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 .Transformation of vectors from murine to humanized versions of CAR-CD1a (mCAR-CD1a and hCAR-CD1a, respectively).

[0007] Figure 2 .(A) FACS histogram analysis of CD1a expression using two different antibodies, where CD1a positive cells (Jurkat and Molt4) were compared to NALM6 (CD1a negative cells). Isotype, IgG control antibody. (B) Cytotoxicity of CD1a CART and UT T cells against coT-ALL and B-ALL cell lines. Absolute counts of live eFluor positive target cells measured by FACS in a 72 h cytotoxicity assay at an E:T ratio of 1:1. ***p<0.001; ****p<0.0001. T test. One-tailed. AMP-GFP: mCAR-CD1a-AMP-GFP; AMP: mCAR-CD1a-AMP; KAN: mCAR-CD1a; ns: not significant; UT: untransduced PBMC.

[0008] Figure 3 .In vivo activity of CD1a CART. (A) IVIS imaging of tumor burden monitored by BLI at the indicated time days (D: days). (B) Quantification of total radiation at the indicated time points shows that all mCAR-CD1a versions can effectively eliminate the progression of coT-ALL cells in mouse xenografts. (C) Circulating Jurkat in PB and BM after animal euthanasia. *p<0.05, **p<0.01, ***p<0.001. T test. One-tailed. AMP-GFP: mCAR-CD1a-AMP-GFP; AMP: mCAR-CD1a-AMP; KAN: mCAR-CD1a.

[0009] Figure 4.A) Structure of the chimeric antigen receptor (CAR) of OC_1. (B) Sequence alignment of the heavy chain (VH) (top) and light chain (VL) (bottom) of the scFv between murine and humanized (h2). The connecting sequence between the heavy and light chains is also shown (middle). The CDR and backbone sequences are marked. The altered amino acids and the individual CDR and backbone sequences (underlined) have been highlighted.

[0010] Figure 5 .FACS histogram analysis of PBMCs transduced with mCAR-CD1a and h2CAR-CD1a. A) Comparable detection using anti-(a)-mouse and anti-(a)-human scFv from the same transduced donor; B) 3 different donors were transduced with lentiviral particles encoding mCAR-CD1a and h2CAR-CD1a. Percentages show scFv membrane expression relative to untransduced cells (UT); C) Representative experiment determining similar expansion of different CAR-CD1a transduced PBMCs and UT cells over time. UT: untransduced PBMCs.

[0011] Figure 6 . FACS analysis of PBMCs transduced with mCAR-CD1a and h2CAR-CD1a. Percentages show membrane expression of scFv, CD4+ and CD8+ cells. UT: untransduced PBMCs. 7-ADD, 7-aminoactinomycin D, negative (live) cells are shown.

[0012] Figure 7 .Cytotoxicity assays performed at different effector-target ratios (E / T) using the indicated cell lines. Viable cell levels were quantified by FACS analysis of eFluor-positive cells (A, B, and C) or GFP-positive cells (D) after 24 h of co-culture. **p<0.01, ***p<0.001 relative to UT. T-test. One-tailed. UT: untransduced PBMCs. NE: non-effector cells. ns: not significant.

[0013] Figure 8.Cytotoxicity assay of eFluor-labeled Jurkat cells using the Annexin V method. Cell death was quantified by FACS analysis of Annexin V-positive and 7-ADD-positive cells at 2, 4, 6 and 24 h of co-culture with the indicated CD1a-CAR-T or UT cells. A) Representative FACS showing the gating strategy (e-Fluor+ / Annexin V+ / 7-ADD+). B) Quantification of cell death using CAR-T from three different donors. UT: untransduced PBMCs. NE: non-effector cells. C) Quantification of viable cell levels by FACS analysis of eFluor+ / Anexin V- / 7-ADD- cells after 6 h of co-culture as in A. ****p<0.0001; ***p<0.001; **p<0,01; ns: not significant. Two-way ANOVA test (Tukey correction for multiple comparisons).

[0014] Fig. 9 . Levels of proinflammatory cytokines measured by ELISA (A, B, and C) in CD1a-positive cells (Jurkat) compared to NALM6 (CD1a-negative cells) after co-culture with the indicated CD1a-CAR-T or UT cells for 24 h. Data are pooled from two independent experiments with 3 different donors in each experiment, **p<0.01, ***p<0.001 relative to UT cells. T-test. One-tailed. ELISA: enzyme-linked immunosorbent assay; UT: untransduced PBMC.

[0015] Fig.10 In vivo activity of mCAR-CD1a and h2CAR-CD1a against Jurkat cells. NSG mice were injected intravenously with 3x10 6 Luc / GFP-expressing Jurkat cells were injected intravenously 3 days later with a single injection of 5x10 6Mock (UT) or CD1a CAR-T cells. A) IVIS imaging of tumor burden monitored by BLI at the indicated days (D). Representative BLI images of one experiment are shown. Color bars indicate intensity levels. B) Mean radiance quantification (p / sec / cm2 / sr) at the indicated days shows that all CAR-CD1a versions can effectively eliminate the progression of coT-ALL cells in mouse xenografts (n=6 mice / group). Statistical analysis: unpaired two-sample t-test related to UT. C) Circulating Jurkat cells in PB and BM after animals were euthanized. Statistical analysis: ANOVA test related to UT (Tukey correction for multiple comparisons). BLI: bioluminescence; BM: bone marrow; PB: peripheral blood; UT: untransduced PBMC. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Fig.35 The complete data for h1 are also shown. Arbitrary cutoff values ​​are indicated.

[0016] Fig.11 In vivo activity of mCAR-CD1a and h2CAR-CD1a against coT-ALL blasts in a PDX setting. NSG mice (n=6 / group) were injected intravenously with 0.5x10 6 3 days later, 0.5x10 primary coT-ALL cells were injected intravenously once 6 or 1x10 6 Mock (UT) or CD1a-CAR-T. After 6 weeks, tumor burden was monitored by FACS via bleeding and BM aspiration. A) Representative FACS showing the gating strategy for quantifying blasts and CAR-T cells. T-ALL blasts CD45+ / HLA-ABC+ / CD34+ cell populations were confirmed using CD1a+ and CCR9 markers. B) and C) T-ALL blasts in PB and BM, respectively, after animals were euthanized after 6 weeks. Statistical analysis: One-way ANOVA was used to correct for multiple comparisons. BM: bone marrow; PB: peripheral blood; UT: untransduced PBMC. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.

[0017] Fig.12 FACS histogram analysis of CD1a expression in several adherent cell lines of different origins using the indicated antibodies (A and B). HEK-CD1a is HEK-WT infected with CD1a lentivirus.

[0018] Fig.13.At 72 hours, the viability of HEK293T cells WT and cells stably expressing CD1a was monitored by MTT incorporation. UT: untransduced PBMC; WT: wild type. ****p<0.001, *p<0.05. Two-way ANOVA was performed to correct for multiple comparisons.

[0019] Fig.14 .At 72 hours, cell viability of the indicated cell lines was monitored by MTT incorporation. UT: untransduced PBMCs.

[0020] Fig.15 Cell proliferation, which was quantified as Normalized Cell Index, was monitored by the XCELLigence system in a real-time dependent manner. UT: Untransduced PBMCs. HEK293T-CD1a was used as a positive control for cell death in the presence of mCAR-CD1a and h2CAR-CD1a (upper right panel).

[0021] Fig.16 h2CD1a-CAR-T derived from a coT-ALL patient specifically lyses autologous CD1a+ T-ALL naive cells. (A) Scheme describing the experimental design of the autologous cytotoxicity assay. Mature (normal) CD3+CD1a– T cells were purified by MACS from PB of a patient with coT-ALL, infected with h2CAR-CD1a, expanded, and exposed to autologous total PBMCs. (B) Representative FACS plots showing transduction efficiency and CD4+ and CD8+ percentages of transduced mature T cells (CD3+CD1a-). (C) Cytotoxicity assay at E:T ratios of 1:2 and 2:1 for 48 hours. Representative FACS analysis of eFluor-labeled target cells co-cultured with UT or h2CD1a-CAR-T cells (eFluor- / CD3+) is shown. The right panel shows quantification of naive T cells (CD3- / CD1a+) and normal T cells (CD3+ / CD1a). D) Graph shows quantification of absolute cell numbers of live mature CD3+CD1a- T cells and cortical CD3-CD1a+ T-ALL blasts.

[0022] Fig.17.Recombinant protein CD1a (rCD1a) was bound to h2CD1a and mCD1a-CAR-T cells. A) Coomassie blue stained gel of purified recombinant CD1a dimerized with hb2-microglobulin. MW: standard molecular weight marker. B) FACS analysis of h2CD1a-CAR-T and mCD1a-CAR-T incubated with 25nM rCD1a under the same conditions. The percentage of rCD1a positive binding cells is indicated. C) Quantification of rCD1a binding of CAR-T from three different PBMC donors infected with h2CD1a-CAR or mCD1a-CAR, expressed as the percentage of rCD1a bound to cells (1x10 5 cells / condition; 25 nM rCD1a). D) Binding quantification was calculated as median fluorescence intensity (geometric) MFI. ***p<0.001; ****p<0.0001. Two-tailed unpaired T-test.

[0023] Fig.18 .Binding of recombinant protein CD1a (rCD1a) to h2CD1a and mCD1a-CAR-T cells co-expressing GFP (for data normalization). A) Murine and humanized CAR-CD1a co-expressing CAR and GFP. B) Representative FACS histograms showing similar GFP expression by mCD1a and h2CD1a-CAR-T. C) FACS histogram overlay showing binding of rCD1a (25nM) to the indicated CAR-T (solid line) compared to control cells without rCD1a (dashed line). Median fluorescence intensity (geometric) MFI of rCD1a+ cells in the GFP+ cell population. D) Quantification of dose-dependent specific binding of GFP+CAR-T to rCD1a from three different PBMC donors infected with h2CD1a-CAR(GFP) or mCD1a-CAR(GFP), expressed as median fluorescence intensity (geometric) MFI (1x10 5 cells / condition). For h2CD1a-CAR-T cells, the curve was adjusted to single site binding, Kd = 3,41 nM, Bmax = 2304 nM. E) Binding quantification, calculated as the percentage of rCD1a binding to GFP+ cells (1x10 5 cells / condition).

[0024] Fig.19Comparative binding analysis of PBMC transduced with h2CAR-CD1a(GFP) versus PBMC transduced with GFP (as control), showing detection of GFP, anti-human scFv, and rCD1a binding. A similar analysis was performed on h2CAR-CD1a (without GFP), comparing CAR detection of anti-human scFv as well as rCD1a binding. Quantitative analysis showed better detection of rCD1a than anti-scFv. The table inset shows the quantitative results.

[0025] Fig. 20 . Summarized scheme of the humanization strategy followed to obtain the different humanized constructs h1CAR-CD1a (h1), h2CARCD1a (h2), h3CAR-CD1a (h3) and h4CAR-CD1a (h4).

[0026] Fig.21 Structure-based humanization. Computer prediction of the structural overlap between the anti-CD1a murine scFv 3D structure (left), and the structures of the murine and proposed human scFv 5wn9 (right). Calculated root mean square deviation (RMSD) (RMSD < 2 indicates high structural similarity.

[0027] Fig. 22 . Structure-based humanization. Sequence alignment between murine anti-CD1a scFv and human 5wn9. CDRs and stems are indicated for the heavy chain (H1 to H3) and light chain (L1 to L3).

[0028] Fig.23 . Structure-based humanization. Comparison between murine and human 5wn9 heavy chains and the proposed humanized sequence (excluding CDRs and backbone). Boxes and squares indicate introduced amino acid changes (see Examples section for detailed explanation).

[0029] Fig.24 . Structure-based humanization. Comparison between murine and human 5wn9 light chains and the proposed humanized sequence (excluding CDRs and backbone). Boxes and squares indicate introduced amino acid changes (see Examples section for detailed explanation).

[0030] Fig.25 . Sequence-based humanization. Alignment between the murine heavy chain and the germline IGHV1-46*02 sequence (note that the sequence of the CDRs in the heavy chain differs slightly from the original sequence. This was done in order not to submit the real sequence to a public server).

[0031] Fig.26 . Sequence-based humanization. Alignment of the murine light chain and germline IGKVD1D-33*01 sequences (note that the sequences of the CDRs in the heavy chain differ slightly from the original sequence. This was done in order not to submit the true sequence to a public server).

[0032] Fig. 27 .Sequence-based humanization. Conservation of heavy and light chains between different human sequences.

[0033] Fig.28 .Sequence-based humanization. The top panel is a sequence-based "strict" (minimal changes introduced) humanized heavy chain candidate. The bottom panel is a sequence-based "loose" (more changes allowed) humanized candidate.

[0034] Fig.29 .“Demurinization” method. The mouse scFv protein sequence was submitted to the NetMHC-4.0NetMHC service, which returned predictions of peptide binding to MHC class I molecules and the corresponding sequence motifs. In addition, the DiscoTope server predicted discontinuous B-cell epitopes (squares).

[0035] Fig.30 . "Demurinization" approach. Alignment of murine scFv protein sequences (top) and corresponding "demurinization" candidates (bottom) for both heavy and light chains. Unchanged amino acid sequences are underlined, amino acid changes are highlighted.

[0036] Fig.31 Viral titration of different humanized CAR-CD1a constructs in HEK293T cells, detection with anti-human scFv (anti-hscFv) antibodies showed that all constructs were detectable except h4CAR-CD1a, which was still only detectable with anti-mouse antibodies (anti-mscFv, right). Percentages show scFv membrane expression relative to uninfected cells.

[0037] Fig.32 . Two different donors were transduced with lentiviral particles encoding versions h1 to h3 of mCAR-CD1a and hCAR-CD1a. Percentages show scFv membrane expression relative to untransduced cells (UT).

[0038] Fig.33.A) Cytotoxicity assays were performed using the indicated cell lines (Jurkat CD1a+, Nalm6 CD1a- control) and different scFv versions of anti-CD1a for humanized and murine CAR-T at different effector-target ratios. The level of viable cells was quantified by FACS analysis of eFluor-positive cells after 24 h of co-culture. UT: untransduced PBMC. NE: non-effector cells. B) Levels of proinflammatory cytokines were determined by ELISA. Data are pooled from two independent experiments with 3 different donors, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns: not significant. Two-way ANOVA was used for multiple comparisons. ELISA: enzyme-linked immunosorbent assay; UT: untransduced PBMC.

[0039] Fig.34 .A) FACS analysis of PBMCs transduced with mCAR-CD1a, as well as h1 CAR-CD1a and h2 CAR-CD1a. Percentages show membrane expression of scFv, CD4+ and CD8+ cells. UT: untransduced PBMCs. B) Proliferation of CAR-T transduced with the indicated constructs.

[0040] Fig.35 .In vivo activity of mCAR-CD1a and hCAR-CD1a (h1 and h2). A) IVIS imaging of tumor burden monitored by BLI at the indicated time points (days, D). (A) Representative BLI images of one experiment are shown. The color bar indicates the intensity level. B) Mean radiation quantification (p / sec / cm2 / sr) at the indicated days shows that all CAR-CD1a versions can effectively eliminate the progression of coT-ALL cells in mouse xenografts (n=6 mice / group). C) Circulating Jurkat cells in PB and BM after animals were euthanized. BLI: bioluminescence; BM: bone marrow; PB: peripheral blood; UT: untransduced PBMC. Statistical analysis: ANOVA test related to UT (Tukey correction for multiple comparisons). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Arbitrary cutoff values ​​are indicated.

[0041] Fig.36 . Generation of recombinant proteins h2scFv-CD1a and mscFv-CD1a. Experiments comparing their affinity with rCD1a.

[0042] A) Affinity purification of His-Tag proteins mscFv-CD1a(1) and h(2)scFv-CD1a(2) on nickel resin. Equilibration and binding were performed with PBS pH7.5, and washing and elution were performed by imidazole shift. Final sample QC was qualitatively characterized by SDS-PAGE, quantitatively by Bradford method (2ug per row), and Coomassie Brilliant Blue staining was shown: 1) mouse_scFv-anti-CD1a, 0.35mg / ml; 2) humanized 2_scFv-anti-CD1a: 0.25mg / ml. Imidazole was removed by dialysis, and the final buffer was PBS, pH7.5. Purity: >95%. B) Surface plasmon resonance (SPR) binding studies of serially diluted soluble humanized (top) and mouse (bottom) anti-scFv on immobilized rCD1a and CM5 sensor chips (Series S, Cytiva). Single cycle kinetic data (n=2) were recorded using a Biacore T-200 (GE Healthcare Bio-Science). Proteins were dissolved in 0.05% PBS-Tween-20. Multi-cycle kinetics were recorded at specified times, with each sample injected in a separate cycle and surface regeneration between cycles. Concentration series are presented as overlays aligned at the start of injection (time 0). Kinetic fitting model considered 1:1 binding. Temperature: 298K. Summary of the invention

[0043] In one aspect, the present invention provides a humanized CD1a targeting moiety, wherein the CD1a targeting moiety is an antibody, F(ab')2, Fab, scFab or scFv, and the CD1a targeting moiety comprises a VL domain consisting of SEQ ID NO:1 and a VH domain consisting of SEQ ID NO:2.

[0044] Preferably, the CD1a targeting moiety is a scFv comprising a VL domain consisting of SEQ ID NO: 1 and a VH domain consisting of SEQ ID NO: 2. More preferably, the CD1a targeting moiety is a scFv consisting of SEQ ID NO:3.

[0045] In another aspect, the present invention provides a chimeric antigen receptor (CAR), comprising:

[0046] a) an extracellular domain comprising a CD1a targeting moiety, wherein the CD1a targeting moiety is a scFv comprising a VL domain consisting of SEQ ID NO: 1 and a VH domain consisting of SEQ ID NO: 2;

[0047] b) a transmembrane domain; and

[0048] c) Intracellular signaling domain.

[0049] Preferably, the transmembrane domain comprises a transmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. Preferably, the intracellular domain of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b or CD66b. Preferably, CAR also comprises a costimulatory signaling domain, preferably the costimulatory signaling domain comprises an intracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or CD276. Most preferably, CAR consists of SEQ ID NO: 13.

[0050] In another aspect, the present invention provides a nucleic acid encoding a CAR according to the preceding aspects.

[0051] In one aspect, the invention provides a cell comprising a nucleic acid according to the preceding aspects. Preferably, the cell is a T cell.

[0052] In another aspect, the present invention provides a pharmaceutical composition comprising a plurality of cells according to the aforementioned aspects and a pharmaceutically acceptable carrier or diluent.

[0053] In another aspect, the invention provides a cell according to the preceding aspects for use as a medicament. Preferably, the use is for a method of treating a CD1a positive cancer, wherein the method comprises administering the cell or composition to a patient in need thereof. Preferably, the CD1a positive cancer is cortical T-cell acute lymphoblastic leukemia, preferably relapsed / refractory cortical T-cell acute lymphoblastic leukemia.

[0054] definition

[0055] "Giving" or "administering" a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be by a medical professional to a patient or may be self-administered, and / or indirect administration, which may be the act of prescribing the drug. For example, a physician who instructs a patient to self-administer or provides a prescription for a drug to a patient is administering the drug to the patient.

[0056] The term "affibroid" refers to a protein derived from the Z domain of protein A and engineered to bind to a specific target (see Frejd & Kim, 2017. Exp Mol Med. 49(3): e306).

[0057] The term "antibody" refers to a molecule comprising at least one immunoglobulin domain that binds to or immunoreacts with a specific target. The term includes intact antibodies and any antigen-binding portion or single chain thereof and combinations thereof; for example, the term "antibody" specifically includes bivalent antibodies and bivalent bispecific antibodies.

[0058] A typical type of antibody comprises at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds.

[0059] Each "heavy chain" is comprised of a "heavy chain variable domain" (abbreviated herein as "VH") and a "heavy chain constant domain" (abbreviated herein as "CH"). The heavy chain constant domain generally comprises three constant domains: CH1, CH2 and CH3.

[0060] 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 κ or λ type. The VH domain and the VL domain can be further subdivided into highly variable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FWs).

[0061] Each VH and VL consists 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, FW4. The present disclosure provides, inter alia, VH sequences and VL sequences and subsequences corresponding to CDR1, CDR2 and CDR3.

[0062] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (numbering scheme "Kabat"), Al-Lazikani et al., (1997) JMB 273, 927-948 (numbering scheme "Chothia").

[0063] Therefore, it will be understood by those skilled in the art that the sequences of FW1, FW2, FW3 and FW4 are also 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, 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.

[0064] The variable domains of the heavy and light chains contain regions that interact with the binding target, and this region that interacts with the binding target is also referred to herein as an "antigen-binding site" or "antigen binding site". The constant domains of antibodies can mediate the binding of antibodies to host tissues or 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 typical antibodies, but also include bivalent fragments and variants thereof, such as F(ab')2.

[0065] As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, bivalent antibody fragments (e.g., F(ab')2), multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecules that contain an antigen binding site.

[0066] Antibodies can be of any of the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on their heavy chain constant domains, referred to as α, δ, ε, γ, and μ, respectively. Different classes of immunoglobulins have different and known subunit structures and three-dimensional configurations. Antibodies can be naked antibodies or conjugated to other molecules, such as therapeutic or diagnostic agents, to form immunoconjugates.

[0067] The term "anticalin" refers to a protein derived from a lipocalin and engineered to bind to a specific target (see Skerra, 2008. FEBS J. 275(11):2677-83).

[0068] 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 digesting intact monoclonal antibodies with papain.

[0069] The term "cancer" refers to a group of diseases that can be defined as any abnormal benign or malignant new growth of non-functional tissue that results from uncontrolled, usually rapid, cell proliferation and has the potential to invade or spread to other parts of the body.

[0070] The term "CD1a" refers to a non-polymorphic MHC class 1-associated cell surface glycoprotein expressed in association with beta-2-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 associated with major histocompatibility complex (MHC) proteins and forms heterodimers with beta-2-microglobulin. Exemplary sequences and data associated with human CD1a have been deposited in the UniProtKB database under ID number P06126.

[0071] "CD1a positive" cancer, including "CD1a positive" cancerous diseases, is a cancer comprising cells with CD1a on their cell surface. The term "CD1a positive" also refers to a cancer that produces sufficient levels of CD1a on its cell surface, which enables the CAR-containing cells of the present invention to have a therapeutic effect mediated by binding of CAR to CD1a. In some embodiments, the CD1a positive cancer is cortical T cell acute lymphoblastic leukemia or Langerhans cell histiocytosis (LCH).

[0072] The term "CD1a targeting moiety" refers to a substance that can bind to CD1a. In the context of CAR, the CD1a targeting moiety targets T cells to CD1a positive cells, preferably cancer cells. In the context of CAR, it should be understood that the CD1a targeting moiety is genetically encodable.

[0073] "Specific binding" or "specifically binds" means that the antibody or ligand recognizes and binds to a binding partner (e.g., a stimulatory tumor antigen) protein present in a sample, but the antibody or ligand does not substantially recognize or bind to other molecules in the sample. Those skilled in the art are well aware of the various experimental procedures that can be used to test binding and binding specificity. In many protein-protein interactions, some cross-reactivity or background binding may be unavoidable; this does not reduce the "specificity" of the binding between the antibody and the epitope. When considering the term "specificity" in understanding the interaction between the antibody and the epitope, the term "directed against" also applies.

[0074] The term "chimeric antigen receptor" or "CAR" refers to a synthetic receptor that targets T cells to selected antigens and reprograms T cell function, metabolism, and persistence (see Rivière & Sadelain, 2017. Mol Ther. 25(5): 1117-1124). Similarly, the term "CART" refers to a T cell containing a CAR.

[0075] As used herein, "combination therapy", "combined with" or "in combination with" means any form of concurrent, parallel, simultaneous, sequential or intermittent treatment using at least two different treatment modalities (i.e., compounds, components, targeting agents or therapeutic agents). Therefore, the term refers to the administration of another treatment modality before, during or after a treatment modality is administered to a subject. The combined modality can be administered in any order. The therapeutically active modality is administered together (e.g., simultaneously in the same or separate compositions, preparations or unit dosage forms) or alone (e.g., on the same day or on different days, and in any order according to the appropriate dosing regimen of a separate composition, preparation or unit dosage form) by a medical caregiver or according to a regulatory agency. In general, each treatment modality will be administered in a dose and / or schedule determined for the treatment modality. Alternatively, three or more modalities can be used in combination therapy. In addition, the combination therapy provided herein can be used in combination with other types of treatments. For example, other anticancer treatments can be selected from a group consisting of chemotherapy, surgery, radiotherapy (radiation) and / or hormone therapy, and other treatments associated with the current standard of care for the subject.

[0076] A "complete response" or "complete remission" or "CR" means that all target lesions have disappeared, as defined in the RECIST v1.1 guidelines. This does not always mean that the cancer is cured.

[0077] The term "co-stimulatory signaling domain" refers to a signaling portion that provides a signal to a T cell, which, in addition to the main signal provided by the CD3ζ chain of the TCR / CD3 complex, also mediates T cell responses, including but not limited to activation, proliferation, differentiation, cytokine secretion, etc. The co-stimulatory domain may include but is not limited to all or part of CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, 1COS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. In some embodiments, the co-stimulatory signaling domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate cellular responses (including activation, proliferation, differentiation, and cytokine secretion, etc.).

[0078] The term "Designed ankyrin repeat protein" or "DARPin" refers to a protein derived from ankyrin repeat sequences that has been engineered to bind to a specific target (see Plückthun, 2015. Annu Rev Pharmacol Toxicol. 55:489-511).

[0079] “Disease free survival (DFS)” refers to the length of time a patient remains disease-free during and after treatment.

[0080] As used herein, the term "effective amount" of a preparation, such as a therapeutic agent, such as CART, is sufficient to achieve a beneficial or desired result, such as a clinical result, and therefore "effective amount" depends on the context of its application. For example, in the context of administering a therapeutic agent for treating T-ALL, an effective amount can: reduce the number of cancer cells; reduce tumor size or load; inhibit (i.e., slow down to a certain extent, and stop in a certain embodiment) cancer cell infiltration into peripheral organs; inhibit (i.e., slow down to a certain extent, and stop in a certain embodiment) tumor metastasis; inhibit tumor growth to a certain extent; alleviate one or more symptoms associated with cancer to a certain extent; and / or lead to a favorable response, for example, progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR) increase, or in some cases, stable disease (SD), progressive disease (PD) reduction, time to progression (TTP) shortened or any combination thereof. The term "effective amount" is used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0081] The term "fynomer" refers to a protein derived from the SH3 domain of human Fyn kinase that has been engineered to bind to a specific target (see Bertschinger et al., 2007. Protein Eng Des Sel. 20(2):57-68).

[0082] The terms "individual", "patient" or "subject" are used interchangeably in this application, which designates humans and is not intended to be limiting in any way. An "individual", "patient" or "subject" can be of any age, gender and physical condition. The term "patient in need thereof" generally refers to a patient suffering from a CD1a-positive cancer.

[0083] "Infusion (or infusing) is the introduction of a solution containing a therapeutic agent into the body through a vein for therapeutic purposes. Typically, this is done through an IV bag.

[0084] 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 provides lymphocyte activation. The intracellular domain of such molecules mediates signals by interacting with cell mediators, resulting in proliferation, differentiation, activation, and other effector functions. Examples of intracellular signaling domains for CARs of the present invention include intracellular sequences of CD3ζ chains and / or synergistic co-receptors that initiate signal transduction after CAR engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same function. T cell activation can be said to be mediated by two different types of cytoplasmic signaling sequences: cytoplasmic signaling sequences (primary cytoplasmic signaling sequences) that initiate antigen-dependent primary activation and provide T cell receptor-like signals and cytoplasmic signaling sequences (secondary cytoplasmic signaling sequences) that act in an antigen-independent manner to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences that act in a stimulating manner may contain signaling motifs, which are referred to as receptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signaling sequences include those derived from CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0085] The term "monomer" refers to a protein derived from a type III fibronectin domain that has been engineered to bind to a specific target (see Koide et al., 2013. J Mol Biol. 415(2):393-405).

[0086] The term "nanobody" refers to a protein comprising 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).

[0087] "Overall survival" (OS) refers to the time from patient enrollment to death or censoring at the last known date of survival. OS includes an extension of life expectancy compared to a newly diagnosed or untreated individual or patient. Overall survival refers to the situation in which a patient remains alive for a determined period of time (e.g., one year, five years, etc.), such as from the time of diagnosis or treatment.

[0088] A “partial response” or “PR” is a response to treatment with a reduction in the sum of target lesion diameters of at least 30% relative to the baseline summed diameter, as defined in the RECIST v1.1 guidelines.

[0089] The term "peptide aptamer" refers to a short sequence of 5 to 20 amino acid residues that can bind to a specific target. Peptide aptamers are usually inserted into the loop region of a stable protein scaffold (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12): 1082-101).

[0090] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, without limiting the scope of the invention, 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, polyols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylindole, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1-nitropropene, 1-hydroxy-1- Alanine, glutamic acid and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbitol, xylose, ribose, ribitol, myoinisitose, inositol, galactose, galactitol, glycerol, cyclic alcohols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents, such as glutathione, lipoic 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 carriers, excipients or stabilizers, such as those described in the 16th edition of Remington's Pharmaceutical Sciences (Osol, A. Ed. (1980)), edited by Osol, A. in 1980, can also be included in the pharmaceutical compositions described herein, provided that they do not adversely affect the desired properties of the pharmaceutical compositions.

[0091] "Progressive disease" or "progressed disease" refers to the appearance of a new lesion or tumor and / or unequivocal progression of an existing non-target lesion, as defined in the RECIST v1.1 guidelines. Progressive disease or progressive disease may also refer to tumor growth of more than 20% since the start of treatment due to an increase in tumor mass or spread.

[0092] "Progression-free survival" (PFS) refers to the time from enrollment to disease progression or death. PFS is usually measured using the Kaplan-Meier method and the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. Generally speaking, progression-free survival refers to a situation where the patient is still alive and the cancer has not worsened.

[0093] The term "RECIST" refers to Response Evaluation Criteria in Solid Tumors. The RECIST guidelines, criteria, or criteria describe standard methods for solid tumor measurement and definitions for objective assessment of changes in tumor size for use in adult and pediatric cancer clinical trials. RECISTv1.1 refers to version 1.1 of the revised RECIST guidelines, which was published in European Journal of Cancers 45 (2009) 228-247.

[0094] The term "repebody" refers to a protein derived from a leucine-rich repeat module and engineered to bind to a specific target (see Lee et al., 2012. PNAS. 109(9): 3299-3304).

[0095] The term "positive response" generally refers to causing a beneficial state in a subject. With regard to cancer treatment, the term refers to providing a therapeutic effect to a subject. The positive therapeutic effect of cancer can be measured in a variety 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 efficacy of anticancer therapy. For example, progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR) increase, or in some cases, stable disease (SD), progressive disease (PD) reduction, progression time (TTP) shortening, or any combination thereof to assess positive response.

[0096] The term "sequence identity" refers to the percentage value obtained when using a pairwise sequence alignment tool to compare two sequences. In this example, the sequence identity was 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 obtained from: https: / / www.ebi.ac.uk / Tools / psa / .

[0097] The term "single-chain antigen-binding fragment" or "scFab" refers to a fusion protein comprising one variable domain and one constant domain of an antibody light chain linked to one variable domain and one constant domain of an antibody heavy chain, wherein the heavy and light chains are linked together by a short peptide.

[0098] The term "single-chain variable fragment" or "scFv" refers to a fusion protein comprising the variable domains of the heavy and light chains of an antibody, the variable domains being linked to each other by a peptide linker. The term also includes disulfide-stabilized Fv (dsFv). Methods for stabilizing scFv with disulfide bonds are disclosed in Reiter et al., 1996. Nat Biotechnol. 14(10): 1239-45.

[0099] "Stable disease" refers to disease that has not progressed or relapsed, as defined in the RECIST v1.1 guidelines. In stable disease, there is neither enough tumor shrinkage to qualify as a partial response nor enough tumor increase to qualify as progressive disease.

[0100] "Time to Tumor Progression" (TTP) is defined as the time from enrollment to disease progression. TTP is usually measured using the RECIST v1.1 criteria.

[0101] As used in this application, the terms "treatment" and "therapy" refer to a group of hygienic, pharmacological, surgical and / or physical means intended to cure and / or alleviate diseases and / or symptoms with the goal of repairing health problems. The terms "treatment" and "therapy" include preventive and curative methods, as both methods are directed to the maintenance and / or reconstruction of the health of an individual or animal. Regardless of the origin of symptoms, diseases and disabilities, in the context of this application, the administration of appropriate drugs to alleviate and / or treat health problems should be interpreted as a form of treatment or therapy.

[0102] When referring to a given amount or quantity, the term "about" means that the number may vary between ±20%, preferably ±10%, of its specified value. For example, if a therapeutically effective amount or effective dose is said to be about 0.5 x 10 6 cells / kg body weight, then in the context of the present invention, this amount should be understood as 0.4x10 6 -0.6x10 6 cells / kg body weight, preferably 0.45x10 6 -0.55x10 6 cells / kg body weight. DETAILED DESCRIPTION

[0103] In the present invention, three humanization methods (called h1, h2 and h3) and one demurinization method (h4) were tested to develop new, optimized CD1a targeting parts that have high cytotoxic potential and lower immunogenicity than their murine counterparts. After computer simulation and functional comparative analysis of murine and different humanized or demurinized scFvs (h1-h4), it was found that h1 and h2 did not show any liabilities and had higher stability than h3 (Example 3). The demurinized version h4 was discarded at the beginning because it was not recognized by anti-human antibodies (Example 4). In addition, comparative functional analysis showed that the killing potential of h3 was completely ineffective, so it was also discarded (Example 4). H2 showed higher killing efficacy than h1 or mouse (Example 4) and had the lowest immunogenicity compared to the remaining humanized versions (Example 3). For these reasons, h2 was finally selected for further experiments and was named h2CD1a-CAR or OC_1. (See Table 12 for a summary of properties).

[0104] Once optimal humanization of CAR-CD1a is achieved and the best candidate (h2CD1a-CART, h2) is selected, the first experiment is performed to analyze the CAR expression and amplification of h2CAR-CD1a (OC_1). As shown in Example 6, OC_1scFv CAR-CD1a can be expressed on the membrane of transduced peripheral blood mononuclear cells (PBMCs), and its expression and ratio of CD4+ / CD8+ cells are similar to mCAR-CD1a. Next, two studies were conducted to evaluate the activity of OC_1 in Example 7: i) the first study evaluated product cytotoxicity, and ii) the second assay determined the levels of proinflammatory cytokines induced by the product. In summary, the results show that: i) h2CAR-CD1a has a strong inhibitory effect on Jurkat cells ( Figure 7 A) and MOLT4 cells ( Figure 7 B) showed significant reduction in cell viability at all analyzed ratios, and h2CD1a-CART was more effective than the mouse version; in addition, OC_1 had a stronger killing efficiency than the mouse version and manifested itself in a shorter time than the mouse version (see Figure 8 B, 2 h), and was true at most of the E:T ratios analyzed ( Figure 8 B and 8C); ii) OC_1 induced the production of proinflammatory cytokines in Jurkat cells at the same level as mCAR-CD1a.

[0105] Next, the in vivo efficacy and activity of h2CAR-CD1a were compared with mCAR-CD1a. In NSG animals, tumors developed very rapidly without treatment. Fig.10The data showed that both groups of mice (mCD1a-CAR-T and h2CD1a-CAR-T) had almost no leukemia on day 17, while mice receiving UT T cells showed a large tumor burden via BLI ( Fig.10 A). BLI quantification by IVIS showed that h2CAR-CD1a had improved efficacy compared to the mouse version ( Fig.10 B). FACS of tumor burden in PB and BM confirmed that all CD1a CAR-T cells effectively eliminated Jurkat cells ( Fig.10 C), but the number of mice with less than 0.1% circulating blasts in PB (chosen as an arbitrary cutoff) was lower in mice receiving h2CD1a-CAR-T cells (10 of 12 mice) than in mice receiving mCD1a-CAR-T cells (5 of 12 mice). These results suggest that h2CD1a-CAR-T exhibits potent anti-leukemic activity, higher than that of mCD1a-CAR-T ( Fig.10 B). In addition, regarding the number of mice with greater than 1% CAR-T cells detected in the BM, mice receiving h2CD1a-CAR-T (6 out of 12 mice) were significantly higher than mice receiving mCD1a-CAR-T cells (2 out of 12 mice). Comparative data with h1 are also shown in Example 4. In addition, the use of the PDX in vivo model OC_1 was able to better eliminate tumor progression (measured by tumor implantation in the bone marrow and peripheral blood) at a lower dose than the murine version ( Fig.11 ). All these data suggest that humanization of CAR-CD1a improves the ability of CAR-T to reduce tumor burden and reduces the amount required to produce a significant biological effect.

[0106] In addition, the newly developed h2CD1a-CAR-T was tested for off-target and cannibalistic effects, and the results showed that h2CD1a-CAR-T cells could not affect the cell viability of CD1a-negative cell lines from different sources, and humanization did not affect the specificity of CAR-CD1a (Example 9, Figure 14-15 ). In addition, h2CD1a-CAR-T has anti-fratricidal properties (Example 10, Fig.16 ).

[0107] Finally, affinity assays were performed to understand the improved and unexpectedly increased cytotoxicity of the h2CAR-CD1a developed in this study. The results presented in Example 10 were completely unexpected as they showed that the binding of rCD1a to h2CD1a-CAR-T cells was greatly improved compared to the binding of the murine counterpart ( Figure 17-18). In fact, the method described here using recombinant proteins improves the detection of OC_1 used in clinical trials (see Fig.19 , 58% using anti-scFv and 71% using rCD1a).

[0108] Thus, the present invention provides a humanized CD1a targeting moiety that is not only superior to its murine counterpart in terms of low immunogenicity, but it also possesses surprisingly increased cytotoxicity and affinity for rCD1a, which results in tumor reduction in in vivo efficacy assays.

[0109] In view of the above results, in a first aspect, the present invention provides a humanized CD1a targeting moiety 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:

[0110] - LCDR1 comprises, consists of, or consists essentially of: [QASQDINKYIA] (SEQ ID NO:4), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:4;

[0111] -LCDR2 comprises, consists of, or consists essentially of: [IHYTSTL] (SEQ ID NO: 5), or in combination with SEQ ID NO:

[0112] 5 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity;

[0113] -LCDR3 comprises, consists of, or consists essentially of: [LHYDNLPWT] (SEQ ID NO: 6), or a

[0114] NO:6 A sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity;

[0115] - HCDR1 comprises, consists of or consists essentially of: [SGYAFSTYTMH] (SEQ ID NO: 7), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 7;

[0116] - HCDR2 comprises, consists of, or consists essentially of: [YINPNSASTS] (SEQ ID NO: 8), or a residue with SEQ ID NO: 1

[0117] NO: 8 has a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity;

[0118] as well as

[0119] - HCDR3 comprises, consists of, or consists essentially of: [ARGFYTMDY] (SEQ ID NO: 9), or a residue with SEQ ID NO: 1

[0120] NO:9 has a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity.

[0121] In some embodiments, the CD1a targeting moiety is an antibody, anticalin, repebody, monomer, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, or peptide aptamer that specifically binds to CD1a.

[0122] In some embodiments, the CD1a targeting portion is an antibody, F(ab')2, Fab, preferably scFv or scFab, 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, and LCDR1 consists of [QASQDINKYIA] (SEQ ID NO: 4), LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5), LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 6), HCDR1 consists of [SGYAFSTYTMH] (SEQ ID NO: 7), HCDR2 consists of [YINPNSASTS] (SEQ ID NO: 8), and HCDR3 consists of [ARGFYTMDY] (SEQ ID NO: 9).

[0123] Preferably, the humanized CD1a targeting moiety is an antibody, F(ab')2, Fab, preferably scFv or scFab, comprising a VL domain and a VH domain, wherein the VL domain comprises, consists of or consists essentially of SEQ ID NO: 1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 1; and wherein the VH domain comprises, consists of or consists essentially of SEQ ID NO: 2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 2.

[0124] VL domain SEQ ID NO: 1:

[0125] RDIQMTQSPSSSLSASVGDRVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLEPAIPSRFSGSGSGREYTFTISSLQPEDIATYYCLHYDNLPWTFGGGTKVEIKRA

[0126] VH domain SEQ ID NO: 2:

[0127] QVQLQQSGAEVKKPGASVKVSCKASGYAFSTYTMHWVRQAPGQGLEWMGYINPNSASTSYAQKF QGRVTMTADKSTNTAYMELSSLRSEDTAVYYCARGFYTMDYWGQGTLVTVSS

[0128] 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 of SEQ ID NO: 1 and the VH domain consists of SEQ ID NO: 2. In a preferred embodiment, the VL and VH domains contained in the CD1a targeting moiety are connected by a peptide linker. In some embodiments, the linker comprises at least 5 amino acids, preferably 5 to 25 amino acids, preferably 10 to 20 amino acids, and most preferably 20 amino acids. Preferably, the amino acid is G. Preferably, the peptide linker comprises, consists of or consists essentially of SEQ ID NO: 14, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 14.

[0129] Linker between heavy chain and light chain SEQ ID NO: 14: GGGGSGGGGSGGGGSGGGGS

[0130] In a preferred embodiment, the CD1a targeting moiety further comprises a signal peptide, which is preferably located at the N-terminal region of the CD1a targeting moiety. Preferably, the signal peptide comprises, consists of, or consists essentially of SEQ ID NO: 15, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 15.

[0131] Signal peptide (SEQ ID NO: 15): MALPVTGLLLSLGLLLHAARPTG

[0132] Preferably, the CD1a targeting moiety comprises, consists of, or consists essentially of SEQ ID NO: 3, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 3. Most preferably, the CD1a targeting moiety consists of SEQ ID NO: 3.

[0133] Humanized CD1a targeting portion SEQ ID NO: 3:

[0134] QVQLQQSGAEVKKPGASVKVSCKASGYAFSTYTMHWVRQAPGQGLEWMGYINPNSASTSYAQKFQGRVTMTADKSTNTAYMELSSLRSEDTAVYYCARGFYTMDYWGQGTLVTVSSGGGGSGG GGSGGGGSGGGGSRDIQMTQSPSSSLSASVGDRVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLEPAIPSRFSGSGSGREYTFTISSLQPEDIATYYCLHYDNLPWTFGGGTKVEIKRA

[0135] In some embodiments, the humanized CD1a targeting moiety is a 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:

[0136] - LCDR1 comprises, consists of, or consists essentially of: [QASQDINKYIA] (SEQ ID NO:4), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:4;

[0138] -LCDR2 comprises, consists of, or consists essentially of: [IHYTSTL] (SEQ ID NO: 5), or in combination with SEQ ID NO:

[0139] 5 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity;

[0140] - LCDR3 comprises, consists of, or consists essentially of: [LHYDNLPWT] (SEQ ID NO:6), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:6;

[0141] - HCDR1 comprises, consists of or consists essentially of: [SGYAFSTYTMH] (SEQ ID NO: 7), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 7;

[0142] - HCDR2 comprises, consists of, or consists essentially of: [YINPNSASTS] (SEQ ID NO: 8), or a residue with SEQ ID NO: 1

[0143] NO: 8 has a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity;

[0144] as well as

[0145] - HCDR3 comprises, consists of, or consists essentially of: [ARGFYTMDY] (SEQ ID NO: 9), or a residue with SEQ ID NO: 1

[0146] NO:9 has a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity.

[0147] In some embodiments, the CD1a targeting moiety is a 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, and LCDR1 consists of [QASQDINKYIA] (SEQ ID NO: 4), LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5), LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 6), HCDR1 consists of [SGYAFSTYTMH] (SEQ ID NO: 7), HCDR2 consists of [YINPNSASTS] (SEQ ID NO: 8), and HCDR3 consists of [ARGFYTMDY] (SEQ ID NO: 9).

[0148] Preferably, the humanized CD1a targeting moiety is a scFv comprising a VL domain and a VH domain, wherein the VL domain comprises, consists of, or consists essentially of SEQ ID NO: 1, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 1; and wherein the VH domain comprises, consists of, or consists essentially of SEQ ID NO: 2, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the CD1a targeting moiety is a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 1 and the VH domain consists of SEQ ID NO: 2.

[0149] Preferably, the CD1a targeting moiety is a scFv comprising, consisting of, or consisting essentially of SEQ ID NO: 3, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 3. Most preferably, the CD1a targeting moiety is a scFv consisting of SEQ ID NO: 3.

[0150] Binding molecules that specifically bind to CD1a may be very useful for the diagnosis and treatment of the above-mentioned conditions. Several mouse monoclonal antibodies against CD1a are known in the art. However, due to the problems associated with the administration of mouse antibodies to humans, such as short serum half-life, inability to trigger certain human effector functions, and the generation of undesirable immune responses to mouse antibodies, the use of mouse antibodies in vivo is limited. In recent years, new human antibodies have been developed that overcome these aforementioned shortcomings. In addition to NA1 / 34.HLK, other hybridomas are commercially available from SIGMA ALDRICH, such as OKT6 (IgG1 isotype).

[0151] Phage display and combinatorial methods for generating antibodies are known in the art. In addition, methods for generating and selecting non-immunoglobulin scaffolds that bind to a specific target are known in the art.

[0152] In an alternative first aspect, the present invention provides a humanized CD1a targeting portion comprising a VL domain and a VH domain, wherein the VL domain comprises, consists of, or consists essentially of SEQ ID NO: 17 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 17; and wherein the VH domain comprises, consists of, or consists essentially of SEQ ID NO: 16 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 16.

[0153] Heavy chain (SEQ ID NO: 16)

[0154] QVQLVQSGPEVAKPGASVRLSCKASGYAFSTYTMHWVRQAPGQGLEWMGYINPNSASTSYNQNFK ARVTLTADKSTNTAYMHLSSLTSDDTAVYYCGRGFYTMDYWGQGTLVTVSSA

[0155] Light chain (SEQ ID NO: 17)

[0156] DIQMTQSPSSVSASVGDRVTISCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYTLTISSLQPEDFATYYCLHYDNLPWTFGGGTKVEVRRA

[0157] In some embodiments, the CD1a targeting moiety of the alternative first aspect is an antibody, scFv, Fab or scFab comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 17 and the VH domain consists of SEQ ID NO: 16. Preferably, the humanized CD1a targeting moiety is a scFv.

[0158] In a preferred embodiment, the VL and VH domains contained in the CD1a targeting moiety are connected by a peptide linker. The peptide linker is the same as defined above. In a preferred embodiment, the CD1a targeting moiety further comprises a signal peptide, which is preferably located in the N-terminal region of the CD1a targeting moiety. The signal peptide is the same as defined above.

[0159] Preferably, the CD1a targeting moiety comprises, consists of, or consists essentially of SEQ ID NO: 22, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 22. Most preferably, the CD1a targeting moiety consists of SEQ ID NO: 22. Preferably, the CD1a targeting moiety is a scFV.

[0160] Humanized CD1a targeting portion SEQ ID NO: 22:

[0161] QVQLVQSGPEVAKPGASVRLSCKASGYAFSTYTMHWVRQAPGQGLEWMGYINPNSASTSYNQNFKARVTLTADKSTNTAYMHLSSLTSDDTAVYYCGRGFYTMDYWGQGTLVTVSSA

[0162] GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTISCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYTLTISSLQPEDFATYYCLHYDNLPWTFGGGTKVEVRRA

[0163] The humanized CD1a targeting moiety as defined above (in the first aspect and the alternative first aspect) may be part of a chimeric antigen receptor. Therefore, in a preferred embodiment of the first aspect, the invention provides a chimeric antigen receptor (CAR) comprising:

[0164] a) comprising an extracellular domain of a CD1a targeting moiety as defined in the first aspect or in any of the above embodiments,

[0165] b) a transmembrane domain; and

[0166] c) Intracellular signaling domain.

[0167] Each element of the CAR according to this embodiment of the first aspect is further developed as follows:

[0168] a) an extracellular domain comprising a CD1a targeting moiety as defined in the first aspect or any embodiment thereof.

[0169] In some embodiments, the CD1a targeting moiety is a 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

[0170] - LCDR1 comprises, consists of, or consists essentially of: [QASQDINKYIA] (SEQ ID NO:4), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:4;

[0171] -LCDR2 comprises, consists of, or consists essentially of: [IHYTSTL] (SEQ ID NO: 5), or in combination with SEQ ID NO:

[0172] 5 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity;

[0173] - LCDR3 comprises, consists of, or consists essentially of: [LHYDNLPWT] (SEQ ID NO:6), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:6;

[0174] - HCDR1 comprises, consists of or consists essentially of: [SGYAFSTYTMH] (SEQ ID NO: 7), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 7;

[0175] - HCDR2 comprises, consists of, or consists essentially of: [YINPNSASTS] (SEQ ID NO: 8), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 8; and

[0176] - HCDR3 comprises, consists of or consists essentially of: [ARGFYTMDY] (SEQ ID NO: 9), or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 9.

[0177] In some embodiments, the CD1a targeting moiety is a 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, and LCDR1 consists of [QASQDINKYIA] (SEQ ID NO: 4), LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5), LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 6), HCDR1 consists of [SGYAFSTYTMH] (SEQ ID NO: 7), HCDR2 consists of [YINPNSASTS] (SEQ ID NO: 8), and HCDR3 consists of [ARGFYTMDY] (SEQ ID NO: 9).

[0178] In some embodiments, the CD1a targeting moiety (preferably a scFv) comprises a VL domain and a VH domain, wherein the VL domain comprises or consists of a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 polypeptides, wherein:

[0179] - LCDR1 consists of [QASQDINKYIA] (SEQ ID NO: 4),

[0180] - LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5),

[0181] - LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 6), and

[0182] wherein the VH domain comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2, wherein the VH domain comprises a HCDR1, HCDR2 and HCDR3 polypeptide, wherein

[0183] - HCDR1 consists of [SGYAFSTYTMH] (SEQ ID NO: 7),

[0184] - HCDR2 consists of [YINPNSASTS] (SEQ ID NO: 8), and

[0185] - HCDR3 consists of [ARGFYTMDY] (SEQ ID NO: 9),

[0186] And wherein the CD1a targeting portion specifically binds to an epitope of human CD1a protein.

[0187] In one embodiment, the CD1a targeting portion, preferably a scFv, comprises 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 of [QASQDINKYIA] (SEQ ID NO: 4), LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5), LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 6), HCDR1 consists of [SGYAFSTYTMH] (SEQ ID NO: 7), HCDR2 consists of [YINPNSASTS] (SEQ ID NO: 8), and HCDR3 consists of [ARGFYTMDY] (SEQ ID NO: 9), and wherein the adjacent sequences of the CDR regions (i.e., the framework sequences of the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 regions that are not part of SEQ ID NOs: 4-9, respectively) are derived from mouse or human antibodies. Preferably, the framework sequence in the VL domain that is not part of LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO:6 has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:1. Preferably, the framework sequences in the VH domain that are not part of HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8 and HCDR3 of SEQ ID NO:9 have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:2.

[0188] In one embodiment, the CD1a targeting moiety (preferably a scFv) binds to an epitope contained in a human CD1a protein and comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, and wherein the VH domain comprises a CDR 1 region, a CDR 2 region, and a CDR 3 region consisting of SEQ ID NO: 7, 8 and 9, respectively, and wherein the VL domain comprises a CDR 1 region, a CDR 2 region, and a CDR 3 region consisting of SEQ ID NO: 7, 8 and 9, respectively. NO:1 has a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and wherein the VL domain comprises a CDR 1 region, a CDR 2 region and a CDR 3 region consisting of SEQ ID NOs:3, 4 and 5, respectively.

[0189] As shown in Example 11, a CD1a targeting portion comprising a VL domain and a VH domain binds to a CD1a protein with surprisingly high affinity. Therefore, in one embodiment, a CD1a targeting portion (preferably a scFv) comprises a VL domain and a VH domain, wherein the VL and VH domains are capable of binding with high affinity to an epitope contained in a CD1a protein (preferably a human CD1a protein, preferably a human CD1a protein that forms a dimer with its natural partner b2-microglobulin (hB2M)). In one embodiment, a CD1a targeting portion (preferably a scFv) comprises a VL domain and a VH domain, wherein the VL and VH domains are capable of binding with high affinity to an epitope contained in a CD1a protein (preferably a human CD1a protein), thereby "destroying" or "blocking" the binding of a natural ligand to the CD1a protein. In one embodiment, a CD1a targeting portion (preferably a scFv) binds to an epitope contained in a human CD1a protein, destroying or blocking the TCR-CD1a interaction.

[0190] In one embodiment, the CD1a targeting portion (preferably scFv) is capable of binding to an epitope contained in the human CD1a protein with a binding affinity of about ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% of the Kd, at which the amino acid sequence of SEQ ID NO: 3 (humanized CD1a targeting portion) or SEQ ID NO: 13 (the complete sequence of the CAR of the present invention) binds to an epitope contained in the human CD1a protein, and the Kd is measured by surface plasmon resonance.

[0191] In one embodiment, the CD1a targeting moiety (preferably a scFv) binds to an epitope contained in a human CD1a protein and comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, and wherein the VH domain comprises a CDR 1 region, a CDR 2 region, and a CDR 3 region consisting of SEQ ID NO: 7, 8 and 9, respectively; and wherein the VL domain comprises a CDR 3 region consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 NO:1 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and wherein the VL domain comprises a CDR 1 region, a CDR 2 region and a CDR 3 region consisting of SEQ ID NO:3, 4 and 5, respectively, thereby disrupting TCR-CD1a interaction.

[0192] In an embodiment of the first aspect, the extracellular domain comprising a CD1a targeting moiety is a scFv comprising a VL domain and a VH domain, wherein the VL domain comprises, consists of, or consists essentially of SEQ ID NO: 1, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1; and wherein the VH domain comprises, consists of, or consists essentially of SEQ ID NO: 2, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2.

[0193] Most preferably, the CD1a targeting moiety is a scFv comprising, consisting of, or consisting essentially of SEQ ID NO: 3, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 3. Most preferably, the CD1a targeting moiety is a scFV consisting of SEQ ID NO: 3.

[0194] In an alternative embodiment of the first aspect, the extracellular domain comprising a CD1a targeting moiety is a scFv comprising a VL domain and a VH domain, wherein the VL domain comprises, consists of, or consists essentially of SEQ ID NO: 17 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 17; and wherein the VH domain comprises, consists of, or consists essentially of SEQ ID NO: 16 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 16. Preferably, the humanized CD1a targeting moiety is a scFv. Preferably, the VL domain consists of SEQ ID NO: 17 and the VH domain consists of SEQ ID NO: 16.

[0195] In some embodiments, the CD1a targeting moiety is a scFv comprising, consisting of, or consisting essentially of SEQ ID NO: 22, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 22. Most preferably, the CD1a targeting moiety consists of SEQ ID NO: 22.

[0196] In a preferred embodiment, the VL and VH domains contained in the scFv are connected by a peptide linker. Preferably, the peptide linker comprises, consists of, or consists essentially of SEQ ID NO: 14, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity with SEQ ID NO: 14.

[0197] In a preferred embodiment, the CD1a targeting moiety is a scFv, and it further comprises a signal peptide located at the N-terminal region of the CD1a targeting moiety. Preferably, the signal peptide comprises, consists of, or consists essentially of SEQ ID NO: 15, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 15.

[0198] b) Transmembrane domain

[0199] The transmembrane domain can be derived from a natural or synthetic source. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. The transmembrane region can at least comprise the transmembrane region of the α chain, β chain or ζ chain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

[0200] The transmembrane domain can be a synthetic transmembrane domain, or a variant of a naturally occurring transmembrane domain. In some embodiments, the synthetic or variant transmembrane domain comprises mainly hydrophobic residues, such as leucine and valine.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8 or a variant thereof, wherein the variant has 95% sequence identity.

[0205] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8 or a variant thereof, wherein the variant thereof has 98% sequence identity.

[0206] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8.

[0207] In some embodiments, the transmembrane domain comprises SEQ ID NO:10 or a sequence having 95% sequence identity to SEQ ID NO:10.

[0208] In some embodiments, the transmembrane domain comprises SEQ ID NO:10 or a sequence having 98% sequence identity to SEQ ID NO:10.

[0209] In some embodiments, the transmembrane domain comprises SEQ ID NO: 10. In some embodiments, the transmembrane domain consists of SEQ ID NO:10.

[0210] Derived from the transmembrane domain of CD8 (SEQ ID NO: 10)

[0211] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0212] c) Intracellular signaling domain

[0213] The intracellular signaling domain activates at least one function of a cell expressing CAR after binding to a ligand expressed on a tumor cell. In some embodiments, the intracellular signaling domain comprises 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 provides activation of at least one function of a cell comprising CAR.

[0214] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66b, or a variant thereof, wherein the variant has 95% sequence identity.

[0215] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66b, or a variant thereof, wherein the variant has 98% sequence identity.

[0216] In some embodiments, the intracellular signaling domain comprises an intracellular domain of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66b.

[0217] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ or a variant thereof, wherein the variant thereof has 95% sequence identity.

[0218] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ or a variant thereof, wherein the variant thereof has 98% sequence identity.

[0219] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3ζ.

[0220] In some embodiments, the intracellular signaling domain comprises SEQ ID NO:11 or a sequence having 95% sequence identity to SEQ ID NO:11.

[0221] In some embodiments, the intracellular signaling domain comprises SEQ ID NO:11 or a sequence having 98% sequence identity to SEQ ID NO:11.

[0222] In some embodiments, the intracellular signaling domain comprises SEQ ID NO:11 or a sequence having 99% sequence identity to SEQ ID NO:11.

[0223] In some embodiments, the intracellular signaling domain comprises SEQ ID NO: 11. In some embodiments, the intracellular signaling domain consists of SEQ ID NO:11.

[0224] Intracellular signaling domain derived from CD3ζ (SEQ ID NO: 11)

[0225] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0226] Optionally, according to the first aspect or any embodiment thereof, at least one co-stimulatory signaling domain may also be present in the CAR:

[0227] co-stimulatory signaling domain

[0228] In some embodiments, CAR may further include a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain comprises an 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, wherein the variant has 95% sequence identity.

[0229] In some embodiments, the co-stimulatory 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, wherein the variant has 98% sequence identity.

[0230] 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.

[0231] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD137 or a variant thereof, wherein the variant thereof has 95% sequence identity.

[0232] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD137 or a variant thereof, wherein the variant thereof has 98% sequence identity.

[0233] In some embodiments, the costimulatory signaling domain comprises the intracellular domain of CD137.

[0234] In some embodiments, the costimulatory signaling domain comprises SEQ ID NO: 12 or a sequence with 95% sequence identity to SEQ ID NO: 12.

[0235] In some embodiments, the costimulatory signaling domain comprises SEQ ID NO: 12 or a sequence with 98% sequence identity to SEQ ID NO: 12.

[0236] In some embodiments, the costimulatory signaling domain comprises SEQ ID NO: 12 or a sequence with 99% sequence identity to SEQ ID NO: 12.

[0237] In some embodiments, the costimulatory signaling domain comprises SEQ ID NO: 12. In some embodiments, the costimulatory signaling domain consists of SEQ ID NO:12.

[0238] Co-stimulatory signaling domain derived from CD137 (SEQ ID NO: 12)

[0239] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0240] The full sequence CAR according to the first aspect of the present invention

[0241] In some embodiments, the CAR comprises:

[0242] (i) a 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, and LCDR1 is

[0243] [QASQDINKYIA] (SEQ ID NO: 4), LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5), LCDR3 consists of

[0244] The HCDR1 is composed of [SGYAFSTYTMH] (SEQ ID NO: 7), the HCDR2 is composed of [YINPNSASTS] (SEQ ID NO: 8), and the HCDR3 is composed of [ARGFYTMDY] (SEQ ID NO:

[0245] 9) Composition;

[0246] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 95% sequence identity to SEQ ID NO: 10;

[0247] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0248] 11 sequences with 95% sequence identity; and

[0249] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 95% sequence identity to SEQ ID NO: 12.

[0250] In some embodiments, the CAR comprises:

[0251] (i) a 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, and LCDR1 consists of [QASQDINKYIA] (SEQ ID NO: 4), LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5), LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 6), HCDR1 consists of [SGYAFSTYTMH] (SEQ ID NO: 7), HCDR2 consists of [YINPNSASTS] (SEQ ID NO: 8), and HCDR3 consists of [ARGFYTMDY] (SEQ ID NO:

[0252] 9) Composition;

[0253] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0254] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0255] 11 sequences with 98% sequence identity; and

[0256] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 98% sequence identity to SEQ ID NO: 12.

[0257] In some embodiments, the CAR comprises:

[0258] (i) a 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, and LCDR1 consists of [QASQDINKYIA] (SEQ ID NO: 4), LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5), LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 6), HCDR1 consists of [SGYAFSTYTMH] (SEQ ID NO: 7), HCDR2 consists of [YINPNSASTS] (SEQ ID NO: 8), and HCDR3 consists of [ARGFYTMDY] (SEQ ID NO:

[0259] 9) Composition;

[0260] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0261] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0262] 11 sequences with 99% sequence identity; and

[0263] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 99% sequence identity to SEQ ID NO: 12.

[0264] In some embodiments, the CAR comprises:

[0265] (i) a 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, and LCDR1 consists of [QASQDINKYIA] (SEQ ID NO: 4), LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5), LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 6), HCDR1 consists of [SGYAFSTYTMH] (SEQ ID NO: 7), HCDR2 consists of [YINPNSASTS] (SEQ ID NO: 8), and HCDR3 consists of [ARGFYTMDY] (SEQ ID NO:

[0266] 9) Composition;

[0267] (ii) a transmembrane domain comprising SEQ ID NO: 10;

[0268] (iii) an intracellular signaling domain comprising SEQ ID NO: 11; and

[0269] (iv) a costimulatory signaling domain, the costimulatory signaling domain comprising SEQ ID NO:12.

[0270] In some embodiments, the CAR comprises:

[0271] (i) a 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, and LCDR1 consists of [QASQDINKYIA] (SEQ ID NO: 4), LCDR2 consists of [IHYTSTL] (SEQ ID NO: 5), LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 6), HCDR1 consists of [SGYAFSTYTMH] (SEQ ID NO: 7), HCDR2 consists of [YINPNSASTS] (SEQ ID NO: 8), and HCDR3 consists of [ARGFYTMDY] (SEQ ID NO:

[0272] 9) Composition;

[0273] (ii) a transmembrane domain consisting of SEQ ID NO: 10;

[0274] (iii) an intracellular signaling domain consisting of SEQ ID NO: 11; and

[0275] (iv) a costimulatory signaling domain consisting of SEQ ID NO: 12.

[0276] In some embodiments, the CAR comprises:

[0277] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 1 and the VH domain consists of SEQ ID NO: 2;

[0278] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 95% sequence identity to SEQ ID NO: 10;

[0279] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0280] 11 sequences with 95% sequence identity; and

[0281] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 95% sequence identity to SEQ ID NO: 12.

[0282] In some embodiments, the CAR comprises:

[0283] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 17 and the VH domain consists of SEQ ID NO: 16;

[0284] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 95% sequence identity to SEQ ID NO: 10;

[0285] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0286] 11 sequences with 95% sequence identity; and

[0287] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 95% sequence identity to SEQ ID NO: 12.

[0288] In some embodiments, the CAR comprises:

[0289] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 1 and the VH domain consists of SEQ ID NO: 2;

[0290] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0291] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0292] 11 sequences with 98% sequence identity; and

[0293] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 98% sequence identity to SEQ ID NO: 12.

[0294] In some embodiments, the CAR comprises:

[0295] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 17 and the VH domain consists of SEQ ID NO: 16;

[0296] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0297] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0298] 11 sequences with 98% sequence identity; and

[0299] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 98% sequence identity to SEQ ID NO: 12.

[0300] In some embodiments, the CAR comprises:

[0301] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 1 and the VH domain consists of SEQ ID NO: 2;

[0302] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0303] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0304] 11 sequences with 99% sequence identity; and

[0305] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 99% sequence identity to SEQ ID NO: 12.

[0306] In some embodiments, the CAR comprises:

[0307] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 17 and the VH domain consists of SEQ ID NO: 16;

[0308] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0309] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0310] 11 sequences with 99% sequence identity; and

[0311] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 99% sequence identity to SEQ ID NO: 12.

[0312] In some embodiments, the CAR comprises:

[0313] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 1 and the VH domain consists of SEQ ID NO: 2;

[0314] (ii) a transmembrane domain comprising SEQ ID NO: 10;

[0315] (iii) an intracellular signaling domain comprising SEQ ID NO: 11; and

[0316] (iv) a costimulatory signaling domain, the costimulatory signaling domain comprising SEQ ID NO:12.

[0317] In some embodiments, the CAR comprises:

[0318] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 17 and the VH domain consists of SEQ ID NO: 16;

[0319] (ii) a transmembrane domain comprising SEQ ID NO: 10;

[0320] (iii) an intracellular signaling domain comprising SEQ ID NO: 11; and

[0321] (iv) a costimulatory signaling domain, the costimulatory signaling domain comprising SEQ ID NO:12.

[0322] In some embodiments, the CAR comprises:

[0323] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 1 and the VH domain consists of SEQ ID NO: 2;

[0324] (ii) a transmembrane domain consisting of SEQ ID NO: 10;

[0325] (iii) an intracellular signaling domain consisting of SEQ ID NO: 11; and

[0326] (iv) a costimulatory signaling domain consisting of SEQ ID NO: 12.

[0327] In some embodiments, the CAR comprises:

[0328] (i) a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 17 and the VH domain consists of SEQ ID NO: 16;

[0329] (ii) a transmembrane domain consisting of SEQ ID NO: 10;

[0330] (iii) an intracellular signaling domain consisting of SEQ ID NO: 11; and

[0331] (iv) a costimulatory signaling domain consisting of SEQ ID NO: 12.

[0332] In some embodiments, the CAR comprises:

[0333] (i) scFv, scFv consisting of SEQ ID NO: 3;

[0334] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 95% sequence identity to SEQ ID NO: 10;

[0335] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0336] 11 sequences with 95% sequence identity; and

[0337] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 95% sequence identity to SEQ ID NO: 12.

[0338] In some embodiments, the CAR comprises:

[0339] (i) scFv, scFv consisting of SEQ ID NO: 3;

[0340] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0341] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0342] 11 sequences with 98% sequence identity; and

[0343] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 98% sequence identity to SEQ ID NO: 12.

[0344] In some embodiments, the CAR comprises:

[0345] (i) scFv, scFv consisting of SEQ ID NO: 3;

[0346] (ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0347] (iii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0348] 11 sequences with 99% sequence identity; and

[0349] (iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 99% sequence identity to SEQ ID NO: 12.

[0350] In some embodiments, the CAR comprises:

[0351] (i) scFv, scFv consisting of SEQ ID NO: 3;

[0352] (ii) a transmembrane domain comprising SEQ ID NO: 10;

[0353] (iii) an intracellular signaling domain comprising SEQ ID NO: 11; and

[0354] (iv) a costimulatory signaling domain comprising SEQ ID NO:12.

[0355] In some embodiments, the CAR comprises:

[0356] (i) scFv, scFv consisting of SEQ ID NO: 3;

[0357] (ii) a transmembrane domain consisting of SEQ ID NO: 10;

[0358] (iii) an intracellular signaling domain consisting of SEQ ID NO: 11; and

[0359] (iv) a costimulatory signaling domain consisting of SEQ ID NO: 12.

[0360] In some embodiments, the CAR comprises:

[0361] (v) scFv, scFv consisting of SEQ ID NO: 22;

[0362] (vi) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 95% sequence identity to SEQ ID NO: 10;

[0363] (vii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0364] 11 sequences with 95% sequence identity; and

[0365] (viii) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 95% sequence identity to SEQ ID NO: 12.

[0366] In some embodiments, the CAR comprises:

[0367] (v) scFv, scFv consisting of SEQ ID NO: 22;

[0368] (vi) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0369] (vii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0370] 11 sequences with 98% sequence identity; and

[0371] (viii) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 98% sequence identity to SEQ ID NO: 12.

[0372] In some embodiments, the CAR comprises:

[0373] (v) scFv, scFv consisting of SEQ ID NO: 22;

[0374] (vi) a transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity to SEQ ID NO: 10;

[0375] (vii) an intracellular signaling domain comprising SEQ ID NO: 11 or a sequence homologous to SEQ ID NO:

[0376] 11 sequences with 99% sequence identity; and

[0377] (viii) a costimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 99% sequence identity to SEQ ID NO: 12.

[0378] In some embodiments, the CAR comprises:

[0379] (v) scFv, scFv consisting of SEQ ID NO: 22;

[0380] (vi) a transmembrane domain comprising SEQ ID NO: 10;

[0381] (vii) an intracellular signaling domain comprising SEQ ID NO: 11; and

[0382] (viii) a costimulatory signaling domain, the costimulatory signaling domain comprising SEQ ID NO:12.

[0383] In some embodiments, the CAR comprises:

[0384] (v) scFv, scFv consisting of SEQ ID NO: 22;

[0385] (vi) a transmembrane domain consisting of SEQ ID NO: 10;

[0386] (vii) an intracellular signaling domain consisting of SEQ ID NO: 11; and

[0387] (viii) a costimulatory signaling domain consisting of SEQ ID NO: 12.

[0388] In some embodiments, the CAR comprises or consists of SEQ ID NO: 13 or a sequence with 95% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR comprises or consists of SEQ ID NO: 13 or a sequence with 98% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR comprises or consists of SEQ ID NO: 13 or a sequence with 99% sequence identity to SEQ ID NO: 13. In some embodiments, the CAR comprises or consists of SEQ ID NO: 13.

[0389] In some embodiments, the CAR comprises or consists of SEQ ID NO: 23 or a sequence with 95% sequence identity to SEQ ID NO: 23. In some embodiments, the CAR comprises or consists of SEQ ID NO: 23 or a sequence with 98% sequence identity to SEQ ID NO: 23. In some embodiments, the CAR comprises or consists of SEQ ID NO: 13 or a sequence with 99% sequence identity to SEQ ID NO: 23. In some embodiments, the CAR comprises or consists of SEQ ID NO: 23.

[0390] Full sequence of CAR (SEQ ID NO: 13)

[0391] MALPVTGLLLSLGLLLHAARPTGQVQLQQSGAEVKKPGASVKVSCKASGYAFSTYTMHWVRQAPGQGLEWMGYINPNSASTSYAQKFQGRVTMTADKSTNTAYMELSSLRSEDTAVYYCARGFYTMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSRDIQMTQSPSSLSASVGDRVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLEPAIPSRFSGSGSGREYTFTISSLQPEDIATYYCLHYDNLPWTFGGGTKVEIKRATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR

[0392] Full sequence of CAR (SEQ ID NO: 23)

[0393] MALPVTGLLLSLGLLLHAARPTGQVQLVQSGPEVAKPGASVRLSCKASGYAFSTYTMHWVRQAPGQGLEWMGYINPNSASTSYNQNFKARVTLTADKSTNTAYMHLSSLTSDDTAVYYCGRGF YTMDYWGQGTLVTVSSAGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTISCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYTLTISSLQPEDFATY YCLHYDNLPWTTFGGGTKVEVRRATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0394] Nucleic Acids

[0395] In a second aspect, the invention provides nucleic acids encoding any CD1a targeting moiety of the invention (including any CAR disclosed above). Nucleic acid sequences encoding chimeric receptors connect many modular components together, which can be removed and replaced with other components to customize chimeric receptors to effectively activate T cells and recognize CD1a.

[0396] In some embodiments, the nucleic acid is suitable for transducing or transforming a cell. In some embodiments, the nucleic acid is suitable for transducing or transforming a T cell for adoptive immunotherapy.

[0397] In some embodiments, the nucleic acid is codon optimized for expression in mammalian cells. Codon optimization methods are known in the art.

[0398] The nucleic acid of the present invention may be contained in a γ-retroviral or lentiviral vector that can be used to transduce or transform T cells. Preferably, the lentiviral vector is infectious but not replicable. Preferably, the lentiviral vector lacks the sequences required to form a lentivirus with replication ability.

[0399] Nucleic acids can also be inserted into cells by using DNA transposons, RNA transfection, or genome editing techniques (e.g., TALEN, ZFN, and CRISPR / Cas9).

[0400] cell

[0401] In a third aspect, the present invention provides cells comprising nucleic acid of the present invention and / or CAR of the present invention. In some embodiments, the cell is a T cell (referred to as CART).

[0402] In some embodiments, the cell is a naive T cell, a memory stem T cell, or a central memory T cell. These cells are currently considered more suitable for adoptive immunotherapy.

[0403] In some embodiments, the cell is an autologous T cell. The term "autologous cell" refers to a cell obtained from the same patient to be treated using any of the methods of the present invention. It is noteworthy that flow cytometry analysis of peripheral blood obtained from 40 patients with active T-cell acute lymphoblastic leukemia showed that normal CD3+CD1a-T cells were present in all patients. Therefore, it is entirely possible to treat patients with autologous T cells comprising the nucleic acid and / or CAR of the present invention.

[0404] In some embodiments, the cell is an allotolerant T cell. The term "allotolerant cell" refers to a cell engineered to reduce the risk of a graft-versus-host disease reaction. In some embodiments, this is achieved by genome editing-mediated TCR and / or β2-microglobulin deletion. Allotolerant cells are known in the art (see Rivière & Sadelain, 2017. Mol Ther. 25 (5): 1117-1124 in the allotolerant T cell section).

[0405] In some embodiments, the T cells are CD3 positive and CD1a negative T cells. In one embodiment, the T cells are human cells, preferably autologous human cells.

[0406] In some embodiments, the cell is a lymphoid precursor, embryonic stem cell, or induced pluripotent stem cell that has the ability to differentiate into a mature T cell.

[0407] Pharmaceutical composition

[0408] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a plurality of cells of the present invention and a pharmaceutically acceptable carrier or diluent.

[0409] The pharmaceutical compositions described herein may also contain other substances. These substances include, but are not limited to, cryoprotectants, surfactants, antioxidants, and stabilizers. The term "cryoprotectant" as used herein 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 that exhibit low toxicity in biological systems are generally used. In one embodiment, the pharmaceutical composition includes albumin, preferably human albumin, as a stabilizer.

[0410] In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells in a culture medium and container system suitable for administration in a therapeutically effective amount (a "pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin, fetal bovine serum or other human serum components.

[0411] In one aspect, the invention provides a cell according to the invention or a pharmaceutical composition according to the invention for use as a medicament.

[0412] Treatment

[0413] In a fifth aspect, the present invention provides a method for treating CD1a-positive cancer, the method comprising administering the cell of the present invention or the pharmaceutical composition of the present invention to a patient in need thereof.

[0414] In some embodiments, a therapeutically effective amount of cells is administered to a patient. In some embodiments, at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10The number of cells will depend on the ultimate use of the composition and the cell types contained therein. For example, if cells specific for a particular antigen are desired, the population will contain greater than 70%, typically greater than 80%, 85%, and 90% to 95% of such cells. For the uses provided herein, the volume of cells is typically one liter or less, and may be 500 ml or less, even 250 ml or less, or 100 ml or less. Clinically relevant cell counts may be allocated to cumulatively equal to or greater than 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 Multiple infusions of cells.

[0415] The optimal therapeutically effective amount or effective dose can be determined by a person skilled in the art based on the route of administration, treatment regimen and / or administration schedule, combined with existing toxicity and efficacy data and the patient's medical history. However, in a preferred embodiment, the therapeutically effective amount or effective dose of cells (for administration) is determined to be about 0.3 x 10 6 Up to 7x10 6 More preferably, the therapeutically effective amount or effective dose is about 0.4 x 10 6 Up to 6x10 6 cells / kg body weight, most preferably about 0.5 x 10 6 -5x10 6 Preferably, the therapeutically effective amount or effective dose of cells is about 0.5 x 10 6 cells / kg body weight, about 1x10 6 cells / kg body weight, about 2x10 6 cells / kg body weight, about 3x10 6 cells / kg body weight, about 4x10 6 cells / kg body weight, or about 5x10 6 Cells / kg body weight. Preferably, only living (viable) cells are considered to calculate the dose. In some embodiments, such a therapeutically effective amount or effective dose can be administered to a patient once or more than once within a determined or predetermined time period. In a specific embodiment, such a therapeutically effective amount or effective dose can be administered to a patient at least once, twice, three times, four times, five times, six times, seven times or more within a determined or predetermined time period.

[0416] In one embodiment, the therapeutically effective amount or effective dose may be administered 1, 2, 3, 4, 5, 6 or more times per month or year, or administered over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 months or years. In one embodiment, the therapeutically effective amount or effective dose may be administered once, twice, three times, four times, five times, six times, seven times or more than seven times over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more.

[0417] In one embodiment, regardless of the number of times such a therapeutically effective amount or effective dose is administered to a patient, the therapeutically effective amount or effective dose may be fractionated to reduce the likelihood of toxicity. The term "fractionated" refers to administration in several portions over several days to provide an effective dose, wherein each administration comprises a partial dose. This is also referred to herein as a fractionated administration regimen.

[0418] In one embodiment, the fractional administration regimen comprises at least 2, preferably 3 or 4, or more than 4 fractions of a therapeutically effective amount or effective dose. Preferably, such fractions of a therapeutically effective amount or effective dose include any one of the following: about 0.5x10 6 cells / kg body weight, about 1x10 6 cells / kg body weight, about 3x10 6 cells / kg body weight, about 5x10 6 cells / kg body weight, or any combination thereof.

[0419] In one embodiment, each portion of the therapeutically effective amount or effective dose can be used in a time period of about 10,11,12,13,14,15,16,17,18,19,20,22,25,30 or more than 30 days.In one embodiment, the time between each portion of the effective dose can be 1,2,3,4,5,6,7,8,9,10 days or more days, or any combination thereof.Preferably, the therapeutically effective amount or effective dose can be used in three times in a total of about 12 days.Preferably, the therapeutically effective amount or effective dose can be used in four times, wherein the first three times are used in a total of about 12 days, and the fourth time is used about 10 days from the third time.

[0420] In one embodiment, the non-fractionated dose of the therapeutically effective amount or effective dose can be administered before, during or after the fractionated dose regimen is administered. Preferably, the non-fractionated dose of the therapeutically effective amount or effective dose is administered at least one month after the last portion of the fractionated dose regimen is administered, preferably when the patient is at risk or has experienced early relapse or refractory disease.

[0421] In a preferred embodiment, according to the fourth aspect or any embodiment thereof, a given therapeutically effective amount or effective dose specified in any of the above paragraphs is provided in the composition, preferably a pharmaceutical composition, of the present invention.

[0422] In one embodiment, the cells are administered with lymphodepleting chemotherapy. In one embodiment, long-term immunosuppression is not applied to the patient.

[0423] In some embodiments, the cells or pharmaceutical compositions are administered intravenously, intraperitoneally, into the bone marrow, lymph nodes, and / or cerebrospinal fluid.

[0424] In some embodiments, the method comprises a combination therapy. In some embodiments, the method comprises further administering an immune checkpoint. In a further embodiment, the method comprises further administering an immune checkpoint inhibitor and / or an IAP inhibitor (see WO2016 / 054555).

[0425] In some embodiments, a cell or pharmaceutical composition as described herein is administered in combination with a chemotherapeutic agent and / or an immunosuppressant. In one embodiment, a patient is first treated with a chemotherapeutic agent that suppresses or destroys other immune cells and then treated with a cell or pharmaceutical composition as described herein. In some cases, chemotherapy can be avoided entirely.

[0426] In one embodiment, the patient is an adult or a child ≥ 2 years old. Preferably, the patient has advanced or severe R / R T-ALL / LL cancer.

[0427] 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.

[0428] Generally, relapse of leukemia can manifest months or years after initial remission; however, most relapses occur within two years of initial treatment. Refractory is a term that indicates that a patient no longer responds to at least one treatment strategy after relapse.

[0429] There is broad consensus in first-line trials for ALL, particularly in adults, that relapse is defined as “detection of more than 5% blasts in the bone marrow after previously achieving complete remission (CR) or clear demonstration of extramedullary leukemic involvement” (see (2017)). This statement was recorded in a consensus recommendation by the European Working Group on Adult ALL (EWALL) (see Dohner (2010)), with the additional explanation that “if there is 5% to 20% blasts at some stage during the intensive treatment phase and / or during regeneration, the bone marrow should be evaluated again one week later to distinguish between bone marrow relapse and regeneration phenomena”. The definitions cited are based on international recommendations for outcome parameters in acute myeloid leukemia (see Cheson (2003) and Chantepie (213)); this has been extrapolated to several subtypes of ALL, such as the case of T-ALL.

[0430] Recently, some trials have not even defined the concept of relapse. Therefore, studies of chimeric antigen receptor (CAR) T cells include patients with “measurable disease” as well as patients with hematologic relapse (unspecified) or minimal residual disease (MRE) (see Lee (2015) and Maude (2014) and (2017)).

[0431] In some embodiments, the patient to be treated with the methods of the invention is in complete remission or near complete remission following treatment with another therapy. Reducing tumor burden prior to using the methods of the invention may be preferred because there are several alternative effector T cells in the case of patients with highly active relapsed / refractory cortical T-cell acute lymphoblastic leukemia. In some embodiments, the patient 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 reduction in progressive disease, a shortened time to tumor progression, or any combination thereof.

[0432] Quantitative methods

[0433] As shown in Example 10, the detection and quantification of cells transduced with h2CAR-CD1a were greatly improved when recombinant rCD1a was used, while the detection results with scFv were reduced by about 15% (see Fig.19 : 58% of positive cells were detected with scFv, while 71% were detected with rCD1a). These results are the result of the increased affinity between the h2CAR-CD1a developed herein and its target protein CD1a. In addition, when co-expressed with the T2A-GFP modification of the h2CAR-CD1a construct, the detection of rCD1a was similar to that of GFP (see Fig.19: 65% GFP positive cells and 65% rCD1a positive cells), which is 15% higher than the positive cells detected by anti-scFv. Therefore, the method used herein represents an improved method for detecting and / or quantifying the binding between rCD1a and h2CAR-CD1a. Therefore, in the sixth aspect, the present invention provides a method for detecting the binding between CD1a protein (preferably rCD1a) and h2CAR-CD1a defined above. Preferably, the CD1a protein is a recombinant CD1a human protein. Preferably, the CD1a protein consists of the extracellular portion of the protein. Preferably, the CD1a protein comprises a tag sequence, which preferably includes a 6xHIS tag. Preferably, the tag sequence is located at the C-terminus. Preferably, CD1a is in the form of a dimeric protein bound to cell membrane human b2-microglobulin (hB2M).

[0434] In some embodiments, the method comprises the steps of: i) mixing and incubating a sample containing h2CAR-CD1a with a sample containing at least one dimer formed by CD1a and cell membrane b2-microglobulin (hB2M), ii) washing the mixture to wash away unbound proteins, and iii) detecting the presence of binding between CD1a and h2CAR-CD1a in the sample.

[0435] In some embodiments, the method comprises the steps of: i) incubating a sample comprising at least one dimer formed by CD1a and cell membrane b2-microglobulin (hB2M) with a sample comprising h2CAR-CD1a, ii) washing the mixture to wash away unbound proteins, and iii) detecting the presence of binding between CD1a and h2CAR-CD1a in the mixture.

[0436] In a seventh aspect, the present invention relates to a recombinant CD1a protein as defined in the sixth aspect.

[0437] Example

[0438] List of Abbreviations and Acronyms Used in the Examples

[0439]

[0440] Example 1: Experimental Introduction and Summary

[0441] OC_1 (also known as humanized h2CD1a-CAR-T) is a product classified as advanced therapy-gene therapy because the active substance is an autologous T lymphocyte that is transduced (genetically modified) by a lentiviral vector so that they express the chimeric antigen receptor CARCD1a with anti-CD1a specificity on their membrane. OC_1 is suitable for patients with refractory or relapsed T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma (T-ALL / LL). CART recognizes CD1a on the surface of lymphoproliferative cells, allowing T lymphocytes to exert cytotoxic functions and eliminate tumor cells. OC_1 is administered by intravenous infusion in divided doses.

[0442] CAR-T is based on modifying the patient's T lymphocytes by inducing the expression of a specific anti-tumor receptor in the form of a chimeric membrane molecule, the chimeric antigen receptor (CAR), on the surface. CAR binds to the recognition region of a construct that combines the specificity of a single-chain antibody (scFv) with the structural and signaling domains that activate the function of the T lymphocyte expressing the CAR. In this case, hCD1a-CAR T, the CAR molecule recognizes the CD1a molecule, a specific marker expressed only in cortical T-ALL and T-LL (T-cell acute lymphoblastic leukemia and lymphoblastic lymphoma) through the scFv

[16] .

[0443] CARs are usually expressed as dimers on the cell membrane, and native antigens can be recognized in a non-MHC-restricted manner, so they can be used in all individuals regardless of their HLA type [17,18]. However, under normal physiological conditions, the sustained and complete activation and proliferation of T cells by the T cell receptor (TCR) occurs through a complex process that involves the primary initiation signal (signal 1) as well as the activation of secondary co-stimulatory receptors (signal 2), and the involvement of third survival receptors (such as the TNF family or cytokine receptors) (signal 3). Since CAR-T cells do not function in an MHC-restricted manner, their lack of interaction with antigen presenting cells (APCs) is usually insufficient, so if CAR T is not added or modified, signals 2 and 3 will be severely impaired. After several generations of development, the modification and addition of CAR design has been gradually improved and has reached the third generation design. As a result, it has been shown that the addition of CD137 (4-1BB) with CD28 and CD3ζ to the CAR gene construct increased cytokine secretion and more effectively induced inhibition of tumor growth in mice

[18] . In fact, the first generation of CARs contained only CD3ζ as an intracellular signaling domain, while the second generation of CARs added a unique co-stimulatory domain derived from CD28 or 4-1BB; the third generation of CARs included two co-stimulatory domains, such as CD28, 4-1BB or other co-stimulatory molecules

[17] . Recently, it has been speculated that a "fourth generation CAR" approach has been developed that regulates antigen-independent proliferation and the resulting increase in cell-mediated toxicity and / or raises concerns about the immortalization of infused CAR-T cells, such as the inclusion of an inducible suicide gene in the construction of the CAR. OC_1 is a second generation CAR-T that contains 4-1BB as a co-stimulatory domain.

[0444] Therefore, the mechanism of action of OC_1 (h2CD1a-CAR-T) is the same as other CAR-T, which is to produce an immune response against the antigen for which they are designed (in this case, CD1a). This response leads to the expansion and activation of a population of T lymphocytes with cytotoxic capacity. When OC_1 is cultured against tumor cells with an antigen (CD1a), they produce an activation of the immune system against that antigen. When specific recognition occurs, the activation of a population of effector T lymphocytes is able to lyse and kill tumor cells, as is the case in vivo, resulting in a clinical response.

[0445] Summarize:

[0446] 1. OC_1 is a human T cell expressing h2CD1a-CAR, which is a humanized version of the previously described murine CAR. In addition, OC_1 specifically targets human CD1a.

[0447] 2. The active substance of OC_1 is autologous human cells, and the preferred route of administration of the cells is through the patient's intravenous (infusion).

[0448] 3. OC_1 only recognizes human CD1a molecules. In this sense, it is preferred that both the T cells (in which the CAR is expressed) and the target cells are from humans.

[0449] To develop OC_1, different experiments were performed to evaluate the efficacy of the product. As mentioned above, the initial development of OC_1 was carried out using mouse scFv and a lentiviral vector. The CD1a-specific mouse scFv was derived from the NA1 / 34.HLK clone of the CD1a-specific antibody and was commercially synthesized using a mouse IgG library primer set (Progen). The mouse scFv was cloned (upstream of the CAR backbone) into a second-generation CAR backbone based on the pCCL DNA lentivirus, which contains the human CD8 transmembrane domain, human 4-1BB and CD3ζ terminal domains, and a T2A-GFP (green fluorescent protein) box together with an ampicillin (AMP) resistance gene (lentiviral vector pCCLsinPPT-hEF1A-IntronGFP.WPRE). This original CAR-T cell product was named mCAR-CD1a-AMP-GFP and is the product used in the study published by Sánchez-Martínez et al.

[16] and is also the product disclosed in PCT / EP2020 / 053769.

[0450] Subsequently, in order to obtain a new vector suitable for clinical use, the GFP cassette was removed to generate the mCAR-CD1a-AMP vector, and the AMP resistance gene was subsequently replaced with the kanamycin (KAN) resistance gene to obtain a product containing KAN and murine scFv, termed murine CAR-CD1a (mCAR-CD1a) ( Figure 1 ).

[0451] Finally, since mCAR-CD1a is derived from mice, the mouse scFv was humanized to avoid human immunogenicity and make the CAR T cell product more suitable for clinical use. In general, the lentiviral vector containing KAN (without AMP / GFP) and the humanized sequence of the scFv in the CAR structure constitutes a humanized CAR-CD1a (hCAR-CD1a or h2CAR-CD1a) or OC_1 ( Figure 1 ).

[0452] Lentiviral particles were generated from the original mCAR-CD1a-AMP-GFP and the modified versions mCAR-CD1a-AMP, mCAR-CD1a, and hCAR-CD1a.

[0453] Table 1 below summarizes all studies performed using the original CAR T cell product (containing AMP / GFP and mouse scFv) included in the previous patent PCT / EP2020 / 053769. Table 1 also contains bridge experiments for mCAR-CD1a (containing KAN and mouse scFv) and the final OC_1 (hCAR-CD1a, containing KAN and humanized scFv). The experiments marked with circles are included in PCT / EP2020 / 053769 because they are part of the scientific paper published by Sánchez-Martínez et al.

[16] , while all black crosses are described in detail in different parts of the present invention.

[0454]

[0455]

[0456] Table 1. Nonclinical studies using the original CART cell product mCAR-CD1a and OC_1 product. #A GFP version of the construct hCAR-CD1a (KAN+humanized scFv) was also generated for binding experiments. AMP: ampicillin; CAR: chimeric antigen receptor; CD: cluster of differentiation; KAN: kanamycin; mCAR-CD1a: murine CAR-CD1a; scFv: single-chain variable fragment; T-ALL: T-cell acute lymphoblastic leukemia.

[0457] Sánchez-Martínez et al. have previously described the efficacy and safety of the original murine CAR-T cell product (AMP-GFP), demonstrating potent and specific anti-leukemic activity against coT-ALL cell lines and primary blasts in vitro, and potent anti-leukemic activity in vivo

[16] . All of these experiments are described in PCT / EP2020 / 053769 and can be summarized as follows:

[0458] - In vitro, murine CD1a CART specifically eliminated CD1a+ T-ALL cell lines Jurkat and MOLT4, but not B-ALL cell line NALM6 (as a negative control). In vitro, murine CD1a CART produced high levels of proinflammatory cytokines IL-2, TNFα, and IFN-γ when co-cultured with CD1a+ cell lines, confirming its cytotoxicity.

[0459] - In vitro, mouse CD1a CART specifically eliminated primary CD1a+coT-ALL cells, but not BM normal hematopoietic cells (CD1a-) and CD1a-T-ALL primitive cells, further confirming the specificity of mouse CD1a CAR. High levels of IFN-α and TNFγ were also secreted when co-cultured with CD1a+ primary T-ALL cells.

[0460] - In vivo, murine CD1a CARTs prevented leukemia formation in mice transplanted with Luc-expressing Jurkat T-ALL cells and in primary coT-ALL xenograft models.

[0461] - In vivo, murine CD1a CARTs are functional and durable in rechallenge assays; that is, after treatment with murine CD1a CARTs

[0462] Leukemia-cleared T-ALL-transplanted mice were reinjected with Luc / Jurkat cells or primary T-ALL from the primary transplant. The persistence of effector T cells in the PB, BM, and spleen of the rechallenged animals was able to control the disease.

[0463] Example 2: Humanization strategy

[0464] The humanization of mCAR-CD1a followed three strategies: Fig. 20 ):

[0465] 1) Structure-based methods

[0466] 2) Sequence-based "strict" mode

[0467] 3) Sequence-based “loose” mode

[0468] 4) Derafening method.

[0469] Notice : Although the fourth strategy "de-murinization" was also adopted, it is not a humanization method per se. This method relies on identifying immunologically compromised regions in the murine sequence and changing the sequence to eliminate this compromise. However, because the clone is still completely murine (it is only recognized by anti-mouse antibodies, not anti-human antibodies, as shown in Example 4 below), this strategy was not evaluated further.

[0470] The main features of each method are as follows:

[0471] 1. Structure-based methods

[0472] It includes:

[0473] - Modeling of scFv CD1a ( Fig.21 )

[0474] - Search the PDB database (www.rcsb.org) for known structures of similar human scFv

[0475] - Structural comparison of human scFv and mouse scFv ( Figure 21-22 )

[0476] - Identify the most similar human scFv in structure and sequence ( Figure 21-22 )

[0477] - Replace human CDR+stem with mouse ( Figure 23-24 )

[0478] - Identify other structurally compromised positions on the mouse scFv and transfer them to human-mouse (e.g. buried residues, sterically compromised residues, etc.).

[0479] The CAR-CD1a generated according to this method is designated as h1CAR-CD1a or h1 below.

[0480] After comparing the structural model of the murine scFv with the most similar human scFv (structure 5wn9), we can conclude that, apart from the CDRs, the sequence similarity (and identity) is also very high ( Fig. 22 ). The human scFv has a longer H3 (hence the vacancy in the mouse scFv). In summary, 5wn9 is a good candidate for a human scaffold to transfer the mouse CDRs. Once similarity is determined, the mouse CDR+stem on the human 5wn9 sequence can be replaced with the mouse CDR+stem (heavy chain see Fig.23 ).

[0481] Next, we decided on each amino acid modification to retain the human or murine version based on biochemical / structural criteria, heavy chain amino acids such as Fig.23 As shown:

[0482] - Position a: N-terminal, mouse Q, human V. This aa is exposed before the first β sheet. We keep the human V.

[0483] - Position b: mouse A, human P. This aa is exposed in the loop and away from the CDR region and the backbone region. In addition, from a physicochemical point of view, P is a very special aa and is therefore difficult to replace. We keep human P.

[0484] - Position c: mouse LAR, human VKK. This is the second important change between the two parallel b chains before H1. We decided to change it to VAK. Therefore, aa A was retained (changed to mouse, smaller in size).

[0485] - Position d: mouse R, human A. The distal loop (from the CDR) is changed and fully exposed. We keep human A.

[0486] - Position e: mouse NENFKDKATLTADK, human AQKLKARVTMTTDT. It is a long segment that includes the loop after H2 and follows the β strand. We keep some mouse amino acids close to H2 (NNF) and Vernier (LAK):

[0487] NENFKDKATLTADK (retained amino acids are in bold). Final humanized NQNFKARVTLTADK sequence (changed amino acids are in grey, retained amino acids are in bold).

[0488] - Position f: mouse HLSSLTS, human ELRSLRS. Structurally adjacent to region "e". We retained some mouse amino acids because they paired with the retained mouse amino acids in "e": HLSSLT (retained amino acids are in bold). The final humanized sequence is identical to mouse (HLSSLTS).

[0489] - Position g: mouse CA, human GR. Both amino acids are very different but are located in a region that does not appear to be important for antigen recognition. We kept human (GR).

[0490] - Position h: mouse S, human L. Not a conservative change but not structural (exposed in the last beta strand). We keep the human one.

[0491] For the light chain, we adopted a similar strategy, comparing Fig.24 As shown, the amino acid substitutions are also marked (from "a" to "h"). For the light chain, we have the following changes similar to the heavy chain:

[0492] - Position a: mouse IQ, human TP. N-terminal region, facing the L3 trunk. We decided to keep the mouse one.

[0493] - Position b: mouse L, human V. Packing the distal end of the b strand. We decided to keep the human one.

[0494] - Position c: mouse G, human D. Fully exposed mid-loop. We decided to keep the human.

[0495] - Position d: Change all amino acids to murine amino acids, as these are located at the interface with heavy chain CDR H3. In addition, the nature of the amino acids makes them critical for loop structure (P, G, and R)

[0496] - Position e: mouse REYSFS, human TDFTLT. We decided to keep some mouse and interact with CDR H2 of the heavy chain:

[0497] REYTLT (bold is retained). Final humanized REYTLT (grey is changed).

[0498] - Position f: mouse NLE, human SLQ. The loop is exposed at the distal position. We decided to keep the human SLQ.

[0499] - Position g: mouse I, human F. Buried in a distal position, it may be an important structure for scFv. We decided to keep human F.

[0500] - Position h: mouse G, human P. Located at the base of L3. Might be important for the entropy of L3. We decided to change to mouse G.

[0501] In summary, the humanized sequences proposed based on the structural approach are as follows:

[0502] Heavy chain (SEQ ID NO: 16)

[0503] QVQLVQSGPEVAKPGASVRLSCKASGYAFSTYTMHWVRQAPGQGLEWMGYINPNSASTSYNQNFK ARVTLTADKSTNTAYMHLSSLTSDDTAVYYCGRGFYTMDYWGQGT LVTVSSA

[0504] Light chain (SEQ ID NO: 17)

[0505] DIQMTQSPSSVSASVGDRVTISCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYTLTISSLQPEDFATYYCLHYDNLPWTFGGGTKVEVRRA

[0506] 2. Sequence-based "strict" mode

[0507] The method follows the following scheme:

[0508] - Use the scFv sequence to query the IgG database ( http: / / www.imgt.org / ).

[0509] -The search returns a list of human Ig genes, sorted by E value.

[0510] - Select the sequence with the highest sequence identity to the heavy and light chains ( Figure 25-26 ).

[0511] - Changes in human Ig gene sequences are limited to the CDR and stem regions and non-conserved positions (see below).

[0512] - Individual assessment of the remaining non-conserved positions in the sequence. For example, non-conserved positions are compared between different human sequences retrieved in the search (second point). Changes are made based on the degree of conservation, i.e. changes from human to mouse, or human will be retained.

[0513] - Two different strategies, "strict" and "relaxed", were designed to decide how many changes to make at non-conserved positions. The sequence-based "strict mode" attempts to make minimal changes to achieve humanization. In contrast, the "relaxed mode" allows more changes to the human sequence (see point 3).

[0514] The CAR-CD1a generated according to this method is hereinafter referred to as h2CAR-CD1a or h2.

[0515] For the heavy chain, a query to the IgG database gave the germline IGHV 1-46*02 (66%) with the highest sequence identity to the heavy chain ( Fig.25 ). In this case, without considering the CRD, the number of different residues is 26.

[0516] Regarding the light chain, we found that the germline IGKVD 1D-33*01 has the highest sequence identity with the heavy chain (72%). In this case, without considering the CRD, the number of different residues is 21 ( Fig.26 ). The conservation between heavy and light chains of different human sequences is as follows Fig. 27 As shown, and used to assess the degree of conservation in the human sequence at non-conserved positions (not CRD and stem regions) between the mouse and human sequences.

[0517] According to this methodology, the final selected sequence in "strict mode" is as follows:

[0518] Heavy chain (SEQ ID NO: 2)

[0519] QVQLQQSGAEVKKPGASVKVSCKASGYAFSTYTMHWVRQAPGQGLEWMGYINPNSASTSYAQKFQGRVTMTADKSTNTAYMELSSLRSEDTAVYYCARGFYTMDYWGQGTLVTVSS

[0520] Light chain (SEQ ID NO: 1)

[0521] RDIQMTQSPSSSLSASVGDRVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLEPAIPSRFSGSGSGREYTFTISSLQPEDIATYYCLHYDNLPWTFGGGTKVEIKRA

[0522] 3. Sequence-based "relaxed" mode

[0523] Different humanized sequences were chosen so that the human sequence had more amino acid changes relative to the murine sequence. Fig.28 The "strict" and "relaxed" sequences for mouse are shown. The "relaxed" sequence is shown below:

[0524] Heavy chain (SEQ ID NO: 18)

[0525] QVQLVQSGAELVKPGASVRVSCKASGYAFSTYTMHWVRQAPGKGLEWVGYINPNSASTSYNDSVKGRFTISRDTSKNTAYMEMNSLRSEDTAVYYCARGFYTMDYWGQGTLVTVSS

[0526] Light chain (SEQ ID NO: 19)

[0527] RDIVLTQSPSSLSASLGQRVTISCQASQIDNKYIAWYQQKPGQAPKLLIHYTSTLQPGIPDRFSGSGSGTDFTLTISGLQPEDFATYYCLHYDNLPWTFGGGTKLEIKRT

[0528] The CAR-CD1a generated according to this method is hereinafter referred to as h3CAR-CD1a or h3.

[0529] 4. Derafinization method.

[0530] In this approach, we identify immunogenic regions in the mouse sequence and make single amino acid changes to eliminate them. The identification of immunogenic regions is based on computational predictions. This is not a humanization process per se.

[0531] The protocol followed is as follows:

[0532] - Prediction of antigenic regions of murine scFv sequences using computational methods: MCH-I binders and B-cell epitopes ( Fig.29 )(see below).

[0533] - In the regions identified as potentially immunogenic, the contributing amino acids are identified and replaced with different amino acids from the multiple sequence alignment obtained previously ( Fig.30 )

[0534] - Three different computational approaches were used; two sequence-based and one structure-based:

[0535] Sequence-based methods: MCH-I (HLA supertype):

[0536] Artificial neural networks: Application to the MHC class I system

[19] .

[0537] T cell epitope prediction

[20] .

[0538] Both prediction methods are sequence-based artificial intelligence calculation methods.

[0539] • Structure-based approach: B cell epitopes (3D)

[21] . Here, a structural model of a murine scFv generated for structure-based humanization was used.

[0540] The CAR-CD1a generated according to this method is named h4CAR-CD1a or h4 below. The final "de-murinized" candidate is as follows:

[0541] Heavy chain (SEQ ID NO: 20)

[0542] EVQLQQSGAELARPGASVKMSCKASGYAFSTYTMHWVQQRPRQGLEWIGYINPNS

[0543] ASTSYNQNFKDKATLTADKASNTAYMHLSSVRVSDSAVFFCARGFYTMDYWGQGT

[0544] SVTVSS

[0545] Light chain (SEQ ID NO: 21)

[0546] RDIQMTQSASSASASPGGRVTITCQASQDINKYIAWYQFKGPKGPRLVIHYTSTL

[0547] QPAIPSRFSGGSGREYSFSISNLEPDDIATYYCIHYDNLPWTFGGGTKLEIKRP

[0548] Example 3: Computer analysis of murine and humanized scFv

[0549] Comparative analysis of the murine and three humanized versions: structure-based version (h1), sequence-based "strict" version (h2), and sequence-based "relaxed" version (h3) was performed according to three different types of analysis:

[0550] - Immunogenicity (sequence-based and structure-based)

[0551] -Sequence "liabilities" (sequence-based)

[0552] - Stability (structure-based):

[0553] o Protein: Rosetta and SPServer

[0554] oInterface: Rosetta and SPServer

[0555] 1) Immunogenicity

[0556] To evaluate the immunogenicity level of different scFvs, we used a bioinformatics approach to predict potential MHC-I / II binders. The following resources were used:

[0557] NetMHCcons

[0558] Artificial neural network (NN) for predicting MCH-I binders

[22]

[0559] NetMHCstabpan

[0560] Artificial NN for predicting the stability of MCH-I conjugates

[23]

[0561] NetMHCIIpan

[0562] Artificial NN for predicting MCH-II binders

[24]

[0563] The number of immunogenic peptides obtained by different bioinformatics analyses is summarized in Table 2. The humanized scFv presenting a greater reduction in immunogenic peptides was h2.

[0564]

[0565] Table 2. Number of immunogenic peptides for different scFvs considering several bioinformatics resources. ScFv h2 is the peptide with the lowest content of immunogenic peptides.

[0566] 2) Sequence "liabilities" (sequence-based)

[0567] The humanization process necessarily involves amino acid substitutions of the original murine scFv sequence. Sometimes, these modifications may introduce "pitfalls", potential changes in the protein sequence, most often post-translational modifications that can affect the function and stability of the scFv. Table 3 summarizes the different sequence pitfalls that have been analyzed, and Table 4 summarizes the sequence pitfalls found in different scFvs. As shown in Table 4, h3 presents a new N-linked glycosylation site NDS at amino acid positions 61-63 of the heavy chain, which is absent in the rest of the humanized forms and in the mouse. The remaining pitfalls observed already exist in the mouse scFv.

[0568]

[0569] Table 3. Summary of sequence hazards.

[0570]

[0571] Table 4. Summary of sequence pitfalls found in different scFvs. Grey indicates those found in CDRs; black highlights indicate the only new discovery in h3 (glycosylation site, not present in other humanized forms and in mice). Numbers indicate the position of amino acids in the heavy (H) and light (L) chains.

[0572] 3) Stability

[0573] The stability of different scFvs has been evaluated by using different structural predictions and calculations. SPServer analyzes protein folding and protein interfaces, allowing the quality of the structure to be evaluated based on statistical potential energy converted into energy. The analysis report shows that h1 and h2 have more powerful protein folding and better interface energy than mouse. The results are shown in Table 5 (folding analysis, h1's PAIR energy is better than the rest, h2's ES3DC energy is better than mouse) and Table 6 (interface analysis, h1 and h2's PAIR energy is better than mouse).

[0574]

[0575] In contrast, h3 was similar to mouse in terms of protein folding (Table 7) and interface analysis (Table 8).

[0576]

[0577] On the other hand, the Rosetta computing suite allows computational modeling and analysis of protein structures, estimating many metrics, including estimates of changes in free energy (dG). Rosetta analysis showed that h3 appears to have a less favorable stability (score and free energy (dG) and the worst interface score compared to other humanized constructs and mouse.

[0578]

[0579] Table 9. Protein scFv structures were analyzed using Rosetta software and the corresponding calculations were performed. The scores of h3 in terms of stability (scores / dG) and interface (int_score, packstat) seem to be less than ideal (indicated in gray)

[0580] 4) Summary of results

[0581] Summarizing the different analyses, we can conclude that all humanized scFv versions have low immunogenicity and that h2 is the best version, with a smaller number of putative immunogenic peptides. As for potential risks, only h3 has important concerns about new N-glycosylation sites that do not exist in mice. Importantly, no potential risks were observed for h1 or h2. Glycosylation of h3 may impair CAR function and weaken functional target recognition of scFv. As for stability, both h1 and h2 improved the stability of the protein according to the analysis of SPServer, and in any case, using the Rosetta method, it was learned that h3 had poor stability. With all these computer predictions, we can conclude that h3 is the worst of all humanized scFvs (Table 10).

[0582]

[0583] Table 10. Summary of properties of different scFvs. h3 appears to be the worst among all humanized versions.

[0584] Example 4: Comparative analysis of the functions of humanized scFv

[0585] In addition to the in silico predictions reported in the previous examples, we performed several functional analyses to select the final scFv humanization candidates. First, we analyzed whether the four humanized scFvs could produce CAR lentiviruses with similar efficiency in HEK293T and whether they could be detected with anti-human antibodies to check whether the humanization worked effectively ( Fig.31 ). Humanized scFv 1, scFv 2, and scFv 3 can be detected with anti-human antibodies in a dose-dependent manner, except for humanized 4 scFvCAR, which was discarded because it could not be detected with anti-human antibodies but could be detected with anti-mouse antibodies. This is because humanized 4 corresponds to the dehumanization strategy (see Example 2) and is not humanized per se, so it is not considered for further use.

[0586] Calculation of virus titers for different batches (n=6) yielded similar results for h1 and h2 (similar to the historical results obtained for mice shown in Table 11), whereas the titer for h3 was much lower (Table 11). Although the virus volumes were standardized accordingly, with a similar MOI of 10, the infection efficiency of PBMCs was still reduced for h3 ( Fig.32 ). Infection of PBMC from three different donors and toxicity assays with Jurkat (CD1a+) cells co-cultured within 24 hours showed that humanized 1 was as effective as murine in killing assays ( Fig.33 , A). Interestingly, at this observation time point, humanized 2 showed slightly better efficacy than murine or humanized 1 ( Fig.33, A). In contrast, humanized 3 showed little or no killing ability ( Fig.33 , A). No significant cytotoxic effect of any humanized construct on NALM6 (CD1a-) cells was observed ( Fig.33 , A). Subsequently, the release of proinflammatory cytokines IL-2 and IFNγ cytokines reached similarly high levels at 24 h in Jurkat cells using m, h1, and h2CD1a-CARs, but no significant increase was detected in Nalm6 cells (data pooled from two independent experiments performed with three different donors). Interestingly, h1CD1a-CAR-Ts had lower levels of TNFα compared to h2CD1a and mCD1a-CAR-Ts, and, as expected, h3CD1a-CAR-Ts had significant reductions in all quantified cytokines, with no significant differences associated with UT cells ( Fig.33 , B).

[0587]

[0588] Table 11. Summary of properties of CD1a-CAR-T with different scFv anti-CD1a (virus titer in HEK293T and cytotoxicity in Jurkat cells; see Fig.31 and Fig.33 ).

[0589] In summary, h3CAR-CD1a was discarded due to its low viral titer, low infectivity, and low cytotoxic activity (Table 11). These data are consistent with the pitfalls and lower stability found in h3CAR-CD1a. h1 and h2-CAR-CD1a appeared to be the best of all humanized versions and were therefore selected for further study.

[0590] h1 and h2CD1a-CAR-T can be expressed on the membrane of transduced peripheral blood mononuclear cells (PBMCs), and their expression and ratio of CD4+ / CD8+ cells are similar to those of mCAR-CD1a ( Fig.34 , A), and transduced PBMCs were expanded in a similar manner ( Fig.34 , B).

[0591] In vitro data showed that h2CD1a-CAR-T was slightly superior to h1CD1a-CAR-T ( Fig.33 ). In NSG animals, e.g. Fig.10 As shown, the in vivo activity of h2CAR-CD1a was slightly higher than that of mCAR-CD1a. Fig.35 Complete data for h1CAR-CD1a are also included. BLI quantification by IVIS showed that h2CAR-CD1a had improved efficacy relative to h1CAR-CD1a and the mouse version ( Fig.35 B). FACS of tumor burden in PB and BM confirmed that all CD1a CAR-T cells effectively eliminated Jurkat cells ( Fig.35 C), but the number of mice with less than 0.1% circulating blasts in PB (chosen as an arbitrary cutoff) was lower in mice receiving h2CD1a-CAR-T (10 of 12 mice) than in mice receiving mCD1a-CAR-T (5 of 12 mice) or h1CD1a-CAR T cells (6 of 12 mice). These results suggest that h2CD1a-CAR-Ts exhibit potent anti-leukemic activity, higher than h1 or mCD1a-CAR-T ( Fig.35 B). Finally, the number of mice with greater than 1% CAR-T cells detected in the BM was significantly higher in mice receiving h1 and h2CD1a-CAR-T (6 out of 12 mice in both groups) than in mice receiving mCD1a-CAR-T cells (2 out of 12 mice).

[0592] Example 5: Bridge experiments between murine and humanized constructs

[0593] As mentioned above, the initial development of OC_1 was carried out using a lentiviral vector containing a T2A-green fluorescent protein (GFP) cassette with an ampicillin (AMP) resistance gene, while for the CAR construct, the lentiviral vector contained a murine CD1a-specific single-chain variable fragment (scFv)

[16] ( Figure 1 A). Subsequently, in order to obtain a new vector suitable for clinical use, the GFP cassette was removed ( Figure 1 B), the AMP resistance gene was replaced with the kanamycin (KAN) resistance gene to obtain a product containing KAN and mouse scFv, called mouse CAR-CD1a (mCAR-CD1a) ( Figure 1 C).

[0594] To demonstrate that the vector modification does not affect the biological function of the original CAR-T cell product, the efficacy of different CD1a murine CARTs was evaluated. The CD1a-expressing T-ALL cell lines Jurkat and MOLT4 ( Figure 2 Two different antibodies were used to verify CD1a+ expression in A) and the B-ALL cell line Nalm6 ( Figure 2Different CD1a murine CARTs, mCAR-CD1a-AMP-GFP (original construct, abbreviated as AMP-GFP), mCAR-CD1a-AMP (abbreviated as AMP), and mCAR-CD1a (abbreviated as KAN to emphasize the replacement of AMP with KAN) were tested in vitro. In a 72-hour assay, all CD1a CARTs eliminated CD1a+ T-ALL cells (Jurkat, MOLT4) at a 1:1 E:T ratio compared to control (UT) ( Figure 2 B, absolute cell number), but not the CD1a- cell line (NALM6).

[0595] The in vivo activity of murine CD1a-CAR-T was also evaluated using Jurkat T-ALL cells expressing Luc / GFP ( Figure 3 ). Intravenous infusion of 4x10 6 Three days before the CD1a CART or UT PBMC, NSG mice were transplanted with 3x10 6 In addition, the mice given UT cells were given Luc-expressing Jurkat cells and the establishment of leukemia was followed weekly using BLI quantification. Compared with mice receiving UT cells, which showed a large tumor burden by BLI, mice given CD1a CART had almost no leukemia until day 17 ( Figure 3 A). BLI quantification by IVIS showed no differences between different murine CAR-CD1a versions ( Figure 3 B). Flow cytometric analysis (FACS) of tumor burden in PB and BM confirmed that all CD1a-CAR-T cells effectively eliminated Jurkat cells ( Figure 3 C).

[0596] Note: For in vivo studies, the Jurkat cell line, which is considered to be equivalent to a pre-T cell precursor (similar to what is defined for T-ALL), constitutes a classic example of T-ALL tumor cell implantation and growth. To track tumors in mice, cells are labeled with traceable molecules. In our case, the Jurkat cell line co-expresses green fluorescent protein (GFP) and luciferase. This enzyme, when oxidized by luciferin, catalyzes a reaction that emits photons that can be detected by photon detection equipment (IVIR or Hamamatsu). The reaction catalyzed by luciferase uses D-luciferin as a substrate, which is further converted into a luminescent product and can be detected outside the animal. In this animal model, effector cells can also be infused, like in our case mCD1a-CART cells. These transduced lymphocytes (or non-transduced equivalents, added as controls) can slow or eliminate tumor growth in mice. The most commonly used in vivo model is the mouse that has received human elements by modeling these components in vitro. However, there are several important limitations when human cells interact with the murine environment. In most immunocompromised mice, human T cell transplantation has limited success because residual elements of the mouse immune system continue to challenge the human T cell transplant

[25] . Therefore, highly immunocompromised models have been developed. The most widely used model is the so-called NSG, which is a lymphocyte-deficient mouse model that allows engraftment of human cells without rejection. The NSG or NOD-SCIDγ mouse (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) is an inbred laboratory mouse that is one of the most immunodeficient mice described to date. NSG mice lack mature T cells, B cells, and natural killer (NK) cells. NSGs also lack multiple cytokine signaling pathways and have various defects in innate immunity. The compound immunodeficiency of the NSG mouse allows engraftment of a variety of primary or derived human cells and allows for sophisticated modeling of many areas of human biology and disease. The NSG mouse was developed by Dr. Leonard Shultz's group at the Jackson Laboratory. At our center, we maintain a colony of these mice. Although the tumor cell engraftment (in distribution and number) is not equivalent to what would be expected from its natural development in humans, this xenogeneic model (NSG-modified mice) constitutes the best animal model for the development of tumor cells of human origin in a complete animal environment.

[0597] Overall, these results suggest that the modified version of mCAR-CD1a is suitable for clinical use and is indistinguishable from the original vector in terms of efficacy and lack of off-target effects. Therefore, the change in the vector does not affect the biological function of mCAR-CD1a.

[0598] Example 6: scFv humanization, hCAR-CD1a expression and OC_1 amplification

[0599] In murine CARs, the scFv sequence is often derived from a mouse mAb, raising legitimate concerns that it may be rejected by the host immune system, either through humoral or cellular responses. Furthermore, because CAR-Ts are synthetic biologics, the linker region between the different parts may result in the generation of new immunogenic peptides

[26] . In nonhuman primate models, cellular immune responses against the rodent CAR scFv components have been documented, suggesting that xenogeneic immune barriers may be problematic when using murine scFvs [26,27]. Furthermore, the murine scFv sequence may be recognized by the host immune system, rendering second and subsequent cell infusions ineffective. In some cases, this response has been associated with early loss of the CAR

[28] , a problem that has been addressed by infusing humanized or human CAR-Ts.

[0600] Since mCAR-CD1a is derived from mice, the mouse scFv was humanized to avoid human immunogenicity and make the CAR T cell product more suitable for clinical use. To this end, we implemented four types of strategies, as described in Example 2:

[0601] -Structure-based approach (h1CAR-CD1a, h1)

[0602] - Sequence-based approach with minimal changes to non-CDR regions: Sequence-stringent (h2CAR-CD1a, h2)

[0603] - Sequence-based methods, non-CDR regions have more variations: Sequence-relaxed (h3CAR-CD1a, h3)

[0604] - Demurinization method (h4CAR-CD1a, h4).

[0605] We first selected a structure-based CAR (h1) and a sequence-based "strict" CAR (h2) based on the computer prediction results explained in Example 3, which reported that they had no hidden dangers and were more stable than the sequence-based "relaxed" CAR h3. The demurinized version h4 was discarded at the beginning because it was not recognized by anti-human antibodies (Example 4). In addition, comparative functional analysis showed that the killing potential of h3 was completely ineffective, so it was also discarded (Example 4). H2 showed higher killing efficacy than h1 or mice, which behaved very similarly (Example 4). Finally, h2 showed the lowest immunogenicity compared to the remaining humanized versions (Example 3). For all these reasons, h2 was finally selected and named h2CD1a-CAR or OC_1 in the next section (see Table 12 for a summary of properties). At some point, when other comparisons were made between h1 and h2, h2 always showed better in vitro and in vivo properties (Example 4).

[0606]

[0607] Table 12. Summary of the different analyses explained in Examples 3-4.

[0608] Sequence-based humanization is explained in detail in Example 2. Briefly, by this method, the complementarity determining regions (CDRs) of murine origin are grafted onto the human scFv backbone. First, the CDR sequences and backbone sequences are identified, such as Figure 4 As shown in B (the backbone is the position before and after the CDR, also known as the vernier region / vernier area). In order to prevent loss of binding affinity, in addition to CDR transplantation, all mouse residues in the vernier region are considered to be retained. The mouse scFv sequence is used to search the human immunoglobulin gene list, sorted by E value similarity ( http: / / www.imgt.org / ). The genes with the highest sequence identity to the heavy and light chains were selected. In this regard, the germline IGHV 1-46*02 and the variable heavy chain V L The highest sequence identity (66%) is obtained with 26 different residues without considering the CRD. H , germline IGKVD 1D-33*01 has the highest sequence identity with VH (72%), and without considering the CRD, the number of different residues is 21. In addition to the CDR regions and the backbone regions, other changes (non-conservative changes) were analyzed one by one and evaluated for alteration taking into account conservation between different human sequences, always trying to introduce the fewest changes*.

[0609] *Note: deciding whether to change a specific amino acid is not as straightforward with sequence-based methods as it is with structure-based methods (the fact that we know the structure allows us to make changes that do not affect that structure), so to some extent sequence-based methods may follow other criteria that are more relevant to the researcher's experience.

[0610] Figure 4 B shows the proposed humanized sequence, where V L and V H The CDR sequences and modified amino acids of the heavy and light chains are highlighted. The connecting sequence of the heavy and light chains is also marked and is the same as in the mouse clone. Note that V H and V L 21 and 9 amino acids were altered in the chains, respectively, resulting in a humanized version of the scFv.

[0611] In general, the lentiviral vector containing KAN (without AMP / GFP) and the humanized sequence of scFv in the CAR structure constituted humanized CAR-CD1a (h2CAR-CD1a), and the final product was called OC_1 ( Figure 1 D).

[0612] Specifically, the OC_1 structure (second generation CART) contains ( Figure 4 A):

[0613] Single-chain variable fragment (scFv): recognizes CD1a on the membrane of T-ALL / LL malignant plasma cells. The scFv region is derived from a mouse monoclonal antibody that was subsequently humanized, while the signaling domain corresponds exactly to the corresponding region of the natural human protein. Mouse CD1a-specific single-chain variable fragment (scFv, with extracellular recognition site: V H (heavy chain) and V L The (light chain) domain) was derived from the NA1 / 34.HLK clone of a CD1a-specific antibody and was commercially synthesized (Millipore Sigma) using a mouse IgG library primer set (Progen)

[16] . The NA1 / 34.HLK monoclonal antibody has been shown to be specific for T-ALL / LL malignant cells (in vitro and in vivo). Later, this sequence was humanized and the efficacy and safety of OC_1 (h2CD1a-CART) were demonstrated in several comparative assays ( Figures 5 to 15 ). CD8a: transmembrane (TM) spacer.

[0614] CD137 (4-1BB); signal 2 and co-stimulatory domain / combined intracellular signaling domain: 4-1BB ligation induces a signaling cascade that leads to cytokine production, expression of anti-apoptotic molecules, and enhanced immune response

[29] . The combination of 4-1BB and CD3ζ in CAR enhances its ability to trigger T cell expansion and anti-tumor activity [26, 29].

[0615] CD3ζ; signal 1 and intracellular domain: The signaling domain mediated by the ζ chain of the TCR-CD3 complex. During interaction with tumor antigens (in this case, CD1a), aggregation of the CAR leads to recruitment of relevant molecules, phosphorylation of the signaling domain, and activation of downstream kinase cascades, resulting in gene transcription, cell activation, and cellular responses to diseased cells. CD3ζ may mediate responses to CD1a

[0616] The cytokine production in response is increased, thereby mediating tumor regression.

[0617] Immunogenicity

[0618] Clinical studies of CD19-CAR T cells using murine scFv reported that immunogenicity was a major problem in some patients, and that patients who underwent repeated infusions had worse responses

[30] . Computational comparative analysis of mCAR-CD1a and h2CAR-CD1a using different artificial neural networks (ANNs) was performed to assess the immunogenicity level of scFvs. To this end, the following resources were used to predict potential MHC-I / II binders:

[0619] NetMHCcons: ANN for predicting MCH-I binders

[22]

[0620] NetMHCstabpan: an ANN for predicting the stability of MCH-I binders

[23]

[0621] NetMHCIIpan: ANN for predicting MCH-II binders

[24]

[0622] As shown in Table 13, in the case of humanized scFv relative to the mouse counterpart, the number of immunogenic peptides was significantly reduced to half or less, depending on the algorithm used. When the CDRs (regions that cannot be modified) were not considered for analysis, the immunogenicity was indeed lower. For a complete comparison of all humanized versions (h1, h2 and h3) with mice, see Example 3.

[0623]

[0624] Table 13. Prediction of immunogenic peptides in the heavy and light chains of murine and humanized (h2) anti-CD1a scFvs. Numbers in bold indicate net reduction. Analyses were performed using the complete scFv sequence or without CDRs.

[0625] Once humanization of CAR-CD1a was achieved, the first experiment was performed to analyze CAR expression and amplification of h2CAR-CD1a (OC_1). Lentiviral particles of h2CAR-CD1a and mCAR-CD1a were generated to infect PBMCs. Lentiviral particles expressing CAR pseudotyped with vesicular stomatitis virus G glycoprotein were generated in HEK 293T cells using a standard polyethylenimine transfection protocol and concentrated by ultracentrifugation as previously described by Sánchez-Martínez et al.

[16] . Viral titers were consistently around 10 7 Up to 10 8TU / mL range (for a complete comparison of all humanized constructs, see Example 4). PBMCs were isolated from the buffy coat of healthy donors using Ficoll-Hypaque gradient centrifugation. T cells were activated for 2 days with plate-bound anti-CD3 (1 μg / ml) and anti-CD28 (1 μg / ml) antibodies. IL-17 and IL-15 (10 ng / mL) were added the next day, and this concentration was maintained throughout the transduction and amplification phases. On the third day, cells were collected, counted, and transduced. 500,000 or 1 million cells were transduced with a lentivirus expressing CAR (multiplicity of infection, MOI = 10). A two-step staining method was used to track cell surface expression of CAR-CD1a by fluorescence-activated cell sorting (FACS). First, the cells were incubated with biotin-SP-conjugated AffiniPure F(ab')2 fragment goat anti-mouse or anti-human IgG, and then incubated with streptavidin-PE antibody ( Figure 5 After 2 days of expansion, the effective activation of CAR-transduced T cells was also examined by FACS staining for CD25 and CD69 (not shown). The frequency of T cell subsets was analyzed by staining for CD3, CD4, and CD8 on day 5 after transduction ( Figure 6 The expression of CD1a on different cell lines was measured by FACS using anti-human CD1a antibodies (clone NA1 / 34-HLK, Invitrogen; clone HI149, Becton Dickinson). Figure 2 A and Fig.12 ).

[0626] First, it was demonstrated that scFv humanization occurred efficiently. Figure 5 As shown, scFv in h2CAR-CD1a-transduced PBMCs can be easily detected by anti-human and anti-mouse scFv antibodies, while mCAR-CD1a can only be detected by anti-mouse antibodies, proving that humanization has been successful ( Figure 5 A). Infection of 3 different donors resulted in PBMC transduction efficiencies of h2CAR-CD1a ranging from 30% to 60%, comparable to expression detected by anti-mouse scFv antibody for mCAR-CD1a ( Figure 5 B). The median membrane expression of mCAR-CD1a was 53% ± 15, and the median membrane expression of h2CAR-CD1a was 56 ± 18 (n = 3). In addition, the transduced PBMCs were expanded in a similar manner ( Figure 5 C). Example 4 also shows a comparative analysis with h1, h2, and h3-CAR-CD1a.

[0627] Finally, CAR-T generated under these conditions showed similar proportions of CD4+ and CD8+ cytotoxic T cells ( Figure 6). From these results, it can be concluded that humanized scFv h2CAR-CD1a can be expressed on the membrane of transduced peripheral blood mononuclear cells (PBMCs), and the expression and ratio of CD4+ / CD8+ cells are similar to those of mCAR-CD1a. Example 4 also shows a comparative analysis with h1-CAR-CD1a.

[0628] Example 7: Study on in vitro activity of OC_1

[0629] The activity of OC_1 was evaluated in different studies: i) the first study evaluated product cytotoxicity, ii) the second study determined the levels of pro-inflammatory cytokines induced by the product. Each study is further explained in the following subsections.

[0630] i) In vitro cytotoxicity of cell lines

[0631] The ability of h2CD1a-CAR T cells to eliminate target (T) CD1a+Jurkat and MOLT4 cells was analyzed. To this end, target cells (100,000 cells per condition) were labeled with 3 μM eFluor670 and incubated with UT PBMC and CART at different effector (E): target (T) ratios for 24 h. CD1a-NALM6 was used as a specificity control as previously described by Sánchez-Martínez et al.

[16] . Cell viability was monitored by eFluor labeling of target cells and FACS detection following the protocol described by these authors. CART-mediated cytotoxicity was determined by analyzing the residual live (7-aminoactinomycin D negative) eFluor 670-positive target cells and E:T ratio at each time point. The percentage of the cell population was calculated by FACSDiva software analysis.

[0632] like Figure 7 As shown, in Jurkat ( Figure 7 A) and MOLT4( Figure 7 h2CAR-CD1a significantly reduced cell viability at all ratios analyzed in B), with the humanized CAR being more effective than the murine version. Excluding nonspecific effects, no mortality was observed in UT cells. Similar analyses in NALM6 cells showed only a slight effect at the highest E:T ratio of 1:1 in both murine and humanized CAR-CD1a, although this was not statistically significant compared to UT cells ( Figure 7 C). Finally, the protocol was simplified by using Jurkat-GFP cells, which yielded similar results to eFluor staining ( Figure 7 D).

[0633] In summary, these experiments showed that human T lymphocytes transduced with h2CAR-CD1a (OC_1) significantly reduced the cell viability of CD1a+Jurkat and MOLT4 cells, more effectively than mCAR-CD1a. However, no statistically significant differences between murine and humanized CARs were observed within the observation time (24 hours). Nevertheless, the differences were highly reproducible and showed a clear trend. The lack of statistical significance may be due to an inappropriate observation time: that is, cell lysis was very strong at 24 hours, which may compensate for the small differences between the two CAR types.

[0634] In order to obtain statistically significant differences between humanized and murine CARs, different methods were used to assess early cell death. To this end, Jurkat cells labeled with eFluor were co-cultured with CAR-T cells for 2 to 24 hours, and the fraction of Jurkat apoptotic cells was quantified after incubation with Annexin V protein and 7-ADD. The fraction of eFluor+ / AnnexinV+ / 7ADD+ was considered as apoptotic / dead cells ( Figure 8 A), analysis of three different donors showed that: at 2 hours, statistically significant differences were observed between UT and h2CAR-CD1a, but not between UT and mCAR-CD1a, at E:T ratios of 1:2 and 1:4 (murine CAR showed differences from UT only at an E:T ratio of 1:1), and a trend towards higher cell death rate induced by h2CD1a-CAR T was observed; at 4 hours, higher cell death rates were observed in the presence of h2CD1a-CART compared with mCD1a-CAR T cells at E:T ratios of 1:1, 1:2, and 1:4 ( Fig. 9 B); At 1:8, a significant difference was observed between UT and h2CAR-CD1a, but not between UT and mCAR-CD1a. In the following 6 hours, the statistical difference shifted to lower ratios, especially at 24 hours, where a statistical difference was only observed at a ratio of 1:8.

[0635] right Figure 8 The 6-hour data shown in B is as follows Figure 8 A was repeated and showed viable cells (eFluor+ / AnnexinV- / 7ADD-) at 6 hours ( Figure 8C). As expected, the viable cell fraction at 6 hours was reduced more in the presence of h2CD1a-CART cells than in mCD1a-CAR T cells at ratios of 1:8, 1:4, and 1:2. Taken together, these results clearly demonstrate that h2CD1a-CART cells are more effective than mCD1a-CAR-T cells, reducing the time and dose required to promote cell death.

[0636] ii) Pro-inflammatory cytokine levels

[0637] Subsequently, the amount of proinflammatory cytokines released by CD1a+ and CD1a- cell lines after co-culture with mCD1a- and h2CD1a-CART cells was determined at 24 hours. The production of proinflammatory cytokines interleukin (IL)-2, tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ) in the supernatant harvested after 24 hours of co-culture was measured using an enzyme-linked immunosorbent assay (ELISA). Fig. 9 As shown, in Jurkat cells, both CD1a CARs produced the same high levels of IL-2, TNFα and IFNγ cytokines, but no significant increase was detected in NALM6 cells (data are summarized from two independent experiments performed by 3 different donors). Comparative data including h1 and h3-CD1a-CAR T are also shown in Example 4.

[0638] Taken together, these results suggest that OC_1 induces the production of proinflammatory cytokines in Jurkat cells at the same level as mCAR-CD1a.

[0639] Example 8: In vivo efficacy studies

[0640] To investigate the in vivo efficacy of OC_1 cells, several experiments were performed in a murine leukemia xenograft model using Jurkat T-ALL cells expressing Luc / GFP. Thus, as in the in vitro studies, the in vivo efficacy and activity of h2CAR-CD1a were compared to mCAR-CD1a.

[0641] In the experiment, 6- to 12-week-old NSG mice were transplanted intravenously (iv) via the lateral tail vein with 3x10 6 Luc / GFP-expressing Jurkat cells were intravenously infused 3 days later with 5x10 6mCD1a-CAR-T and h2CD1a-CAR-T, or untransduced (UT) PBMCs, as controls. For disease progression, quantification of bioluminescence emitted by tumor cells was used, as these cells had previously been modified to express GFP-luciferase. In addition, since the disease eventually spreads throughout the body, this measurement method allows for more precise quantification. Therefore, leukemia establishment or tumor burden was tracked weekly by bioluminescence (BLI). To measure bioluminescence, mice were intraperitoneally given 150 mg / kg of D-luciferin, which emits a bioluminescent signal proportional to the number of tumor cells when interacting with luciferase. The signal was measured on a Xenogen IVIS 50 imaging system (PerkinElmer). Tumor burden was monitored on days 0 (the day of CAR infusion), 3, 6, 10, 14, and 17. Total luminescence was visualized using Living Image software and calculated as mean radiation quantification (p / sec / cm 2 / sr).

[0642] In order to analyze the persistence of tumor circulating cells and transferred CART, mice were killed at the end of the experiment (day 17), and their circulating PB and hind limbs were taken. BM cells were isolated by rinsing the hind limbs with PBS containing 2% FBS. For FACS analysis, 100 μl Pb and 100 μl BM cell suspensions were stained with PBS / 2% FBS containing fluorescent dye-linked antibodies for the following markers: human leukocyte antigen (HLA)-ABC, CD45, CD3, CD1a, CD38, stained at 4 degrees for 30 minutes. 1 ml BD FACSTM fixed lysis buffer (BD Biosciences) was added to each sample to eliminate red blood cells. The sample was run in a FACSCantoTM-II flow cytometer.

[0643] To reduce the number of mice used in each group without affecting the statistical power, previous studies were considered

[31] . Therefore, the number of animals in each group was reduced to a minimum (six), sufficient to prove the hypothesis without losing statistical significance. P values ​​for IVIS quantification experiments were calculated using multiple unpaired two-sample t-tests, assuming a Gaussian distribution and corrected for multiple comparisons using the Holm-Sidak method. Post hoc P values ​​for leukemia and CAR-T cells quantified by FACS were calculated using ANOVA tests for multiple comparisons (Tukey's multiple comparison test). P < 0.05 (*) was considered statistically significant.

[0644] In NSG animals, tumors develop very rapidly if left untreated. Fig.10The data showed that both groups of mice (mCD1a-CAR-T and h2CD1a-CAR-T) had almost no leukemia on day 17, while mice receiving UT T cells showed a large tumor burden via BLI ( Fig.10 A). BLI quantification by IVIS showed that h2CAR-CD1a had improved efficacy compared to the mouse version ( Fig.10 B). FACS analysis of tumor burden in PB and BM confirmed that all CD1a CAR-T cells effectively eliminated Jurkat cells ( Fig.10 C, data are pooled from two independent experiments). Regarding the number of mice with less than 0.1% circulating primitive cells in PB (chosen as an arbitrary cutoff), mice receiving h2CD1a-CART cells (10 of 12 mice) were less than those receiving mCD1a-CAR T cells (5 of 12 mice). Regarding the number of mice with greater than 1% CAR-T cells detected in BM, mice receiving h2CD1a-CAR-T (6 of 12 mice) were significantly higher than those receiving mCD1a-CAR T cells (2 of 12 mice). These results indicate that h2CD1a-CAR-T exhibits potent anti-leukemic activity, slightly higher than that of mCD1a-CAR-T ( Fig.10 B). These data also show that humanization of CAR-CD1a improves the ability of CAR-T to reduce tumor burden. Example 4 also shows comparative data with h1CD1a-CAR T.

[0645] The Jurkat in vivo model develops leukemic engraftment within a week and should be stopped at day 17 to avoid animal suffering. We also used a PDX mouse model with slow progression of disease. For this purpose, animals were randomized and injected with 0.5x10 6 Three days later, the mice were divided into three groups and received 0.5x10 6 or 1x10 6 After 6 weeks, mice were sacrificed and circulating PB and hindlimbs were isolated. Fig.10 As described previously, FACS analysis was performed using the following markers: human leukocyte antigen (HLA)-ABC, CD45, CD3, CD1a, and CCR9, which are selectively and frequently expressed in T-ALL

[32] . Fig.11The results were performed as shown in A. The percentage of primitive cells was determined as HLA-ABC+CD45+CD34+CD3- and confirmed as CD1a+ (and CCR9+) and differentiated from CAR-T cells (HLA-ABC+CD45+CD3+). The results showed that the tumor burden in the PB and BM of mice receiving 1 million humanized or murine CAR-Ts was significantly reduced relative to UT. Interestingly, mice receiving a limited amount of h2CD1a-CAR-T (500,000) showed a significantly better reduction in tumor implantation in the PB relative to UT than mCD1a-CAR-T. It was observed that h2CD1a-CAR-T had a stronger and more significant inhibitory effect on tumor implantation in PB and BM compared with the 0.5M dose of mCD1a-CAR-T. At the highest dose of 1 million CAR-Ts, the difference between humanized CAR-T and murine CAR-T was minimized, but the reduction of h2CD1a-CAR-T was still higher compared to mCD1a-CAR-T ( Fig.11 B). All these results suggest that humanization of CAR-CD1a improves the ability of CAR-T to reduce tumor burden and reduces the amount required to produce a significant biological effect.

[0646] Example 9: Specificity studies: off-target analysis

[0647] To demonstrate that h2CD1a-CAR T cells do not bind to CD1a-negative cells or do not nonspecifically affect the viability of CD1a-negative cells, a series of off-target analyses were performed in a panel of 10 cell lines from different embryonic origins. Fig.12 As shown, two different anti-CD1a antibodies were used: a commercial antibody (Becton Dickinson) ( Fig.12 A) and NA1 / 34-HLK (same as the mouse anti-CD1a scFv used to obtain mCAR-CD1a ( Fig.12 B)) After examining its expression, cell lines from kidney (HEK293T), colon (HCT-116), microvascular endothelium (HMEC), lung (L55), breast (MCF7, MDA-MB-231), pancreas (PANC-1, RWP-1), skin (SKMEL), ovary (SKOV3), and brain (PG88) were all negative for CD1a. As a positive control, a HEK293T cell line ectopically expressing CD1a, which is recognized by both antibodies, was generated (see Fig.12 A and 12B).

[0648] Cell viability of adherent cell lines was assessed in the presence of UT and mCAR-CD1a and h2CAR-CD1a T cells and quantified by two different methods: an endpoint colorimetric assay using MTT reagent to examine cell viability at a total exposure of 72 h ( Fig.13 and Fig.14 ), and the other is to use the XCELLigence system to quantify proliferation in real time ( Fig.15 ).like Fig.13 As shown, HEK293T cells expressing only CD1a (used as a positive control for cell death) died in the presence of CD1a-CART mice or humanized Figure 7 In contrast, CD1a-negative cell lines were not affected by CD1a-CART ( Fig.13 and 14 ). Similar results were obtained using the XCELLigence system ( Fig.15 ). Both methods clearly showed that adherent CD1a cell lines were not affected by co-culture with mCAR-CD1a or h2CAR-CD1a T cells during 3 days. These results clearly show that CD1a-CAR T cells are unable to affect the cell viability of CD1a-negative cell lines of different origins. In addition, these results indicate that humanization does not affect CAR-CD1a specificity.

[0649] Example 10: Experiment to ensure that h2CD1a-CAR T cells do not cause cannibalism

[0650] As previously described, autologous mature T cells recovered from PB of T-ALL patients, modified to express mCAR-CD1a, retained cytolytic activity against tumor cells expressing the target antigen (CD1a+ T-ALL blasts) without affecting non-tumor cells (CD1a-T cells), suggesting that mCD1a-CAR-Ts have anti-cancer properties

[16] . To ensure that h2CD1a-CAR-Ts also have anti-cancer properties, CD3+CD1a-T cells were isolated from patients by magnetic activated cell sorting (MACS), activated with CD3 / CD28, and transduced with lentivirus, with a transduction rate of 19.7% using h2CAR-CD1a, showing a similar CD4+ / CD8+ ratio relative to UT cells (see Fig.16 AFor general solutions, see Fig.16B to understand the transduction efficiency). Subsequently, the cytolytic ability of h2CD1a-CAR-T targeting total PBMCs was studied to evaluate the potential for autologous cell toxicity and the degree of cannibalism. Among the target PBMCs labeled with eFluor 670, the vast majority (>60%) were CD1a+ primitive cells, and less than 20% were CD3+CD1a– mature T cells. Compared with UT cells, h2CD1a-CAR-T showed strong and specific cytolytic ability against autologous CD1a+ primitive cells ( Fig.16 C, lower quantitative graph), but not for CD1a– mature T cells ( Fig.16 C, upper quantitative graph), which further indicates that h2CD1a-CAR-T has anti-fratricidal properties. Similar results were obtained after quantification of absolute cell numbers using TrueCount (BD Biosciences) ( Fig.16 D; Left panel, the absolute number of CD1a-negative T cells was not affected after incubation with h2CAR-CD1a-T or UT cells; Right panel, CD1a-positive cells were eliminated by h2CAR-CD1aT cells but not by UT cells).

[0651] Example 11: Experiment comparing the affinity of h2CAR-CD1a and mCAR-CD1a for CD1a

[0652] The improved cytotoxicity of OC_1 (humanized version 2) can be explained by better recognition of the target. To verify this, we generated the human recombinant protein extracellular domain of CD1a (rCD1a, 34.9 KDa) and formed a dimer with its natural partner b2-microglobulin (hB2M, 13.7 KDa) on the cell membrane ( Fig.17 A). rCD1a was produced with a 6xHis tag, allowing its purification and detection by FACS using an anti-HIS antibody ( Fig.17 B). H2CD1a-CAR-T and mCD1a-CART cells were generated from three different donors and had similar membrane CAR expression, as determined by anti-scFv staining. Figure 5 As shown in B, when membrane CAR expression is different, its expression is compensated by UT cells. 5 Each CART cell was diluted in 100 μL PBS-2% FBS containing AB serum to block nonspecific binding. CAR T cells were incubated with 200 nM rCD1a at 4°C for 15 minutes, washed with PBS-2% FBS, and then re-incubated with anti-HIS-APC antibody. Fig.17 B shows a representative FACS analysis showing high and specific binding of rCD1a to h2CAR-CD1a T cells, greater than 70% ( Fig.17B). In contrast, quantitative results from three different PBMC donor samples showed that mCAR-CD1a T cell binding was approximately 20% ( Fig.17 C). Binding quantification was also performed based on mean fluorescence intensity (MFI, calculated as geometric mean using FACSDiva software), which is proportional to the amount of antibody that recognizes and binds to rCD1a, and similar results were obtained ( Fig.17 D).

[0653] h2CD1a-CAR-T and mCD1a-CAR T cells were generated by infection with a construct co-expressing T2A-GFP ( Fig.18 A). This method allows monitoring the infection efficiency of both constructs at the same level and with high precision ( Fig.18 B). Furthermore, it allows specific quantification of rCD1a binding in the GFP-positive cell population ( Fig.18 C). h2CD1a-CAR-T(GFP) and mCD1a-CAR-T(GFP) cells were incubated with 200 nM rCD1a-HIS at 4°C for 15 min, and a higher h2CD1a-CAR-T positive cell detection rate was obtained at the same GFP detection level ( Fig.18 C). In contrast, mCD1a-CAR-T cells showed poor binding ability and decreased labeling ( Fig.18 C). Quantification of median fluorescence intensity (MFI) from three different donors showed that rCD1a had stronger dose-dependent binding to CAR-T infected with h2CAR-CD1a compared with mCAR-CD1a ( Fig.18 D). Binding of rCD1a to h2CD1a CAR-T cells can be adjusted to a one-site equation curve by nonlinear regression after subtracting nonspecific binding (Bmax=2304, Kd=3.41). A similar analysis was performed on mCD1a CART cells, which showed much lower binding capacity (Bmax=91.63, Kd=1.26). Reliable data from three different PBMC donors clearly showed improved binding of rCD1a to h2CD1a-CAR T cells, as measured by the percentage of rCD1a-positive / GFP-positive cells ( Fig.18 E).

[0654] The results showing binding of rCD1a to h2CD1a-CAR-T cells were completely unexpected. In fact, the detection and quantification of humanized CAR-transduced cells has been greatly improved and will be the method used in the OC_1 clinical trial. In a comparative analysis with h2CD1a-CAR T cells co-expressing GFP, quantification with rCD1a gave the same results as GFP ( Fig.19 In the examples shown in A and 19B, the detection rate was 65%, while the detection with scFv was 15% lower. In fact, the method described here using recombinant protein improved the detection rate of h2CAR-CD1a (without GFP) used in clinical trials (see Fig.19 A and 19B, where scFv was 58% and rCD1a was 71%). For all these reasons, we can conclude that the unexpected binding of h2CD1a-CART cells to rCD1a is not only a significant difference from the murine original version, but is actually a novel quantitative method with clinical use.

[0655] Example 12: Experiment comparing the affinity of mscFv-CD1a and hscFv-CD1a for rCD1a.

[0656] To analyze the binding of humanized and murine scFv anti-CD1a to rCD1a, we produced recombinant proteins with a 6xHis tag by XtenCHO cells (ProteoGenix). Affinity purification of the His-Tag protein was performed on nickel resin and the protein was eluted by imidazole shift. Qualitative SDS-PAGE as Fig.36 A. The yield of mscFv-CD1a (lane 1) and h(2)scFv-CD1a (scFv with H2 humanized version) (lane 2) proteins was high with purity >95%. The affinity of the recombinant scFv to immobilized rCD1a was determined by surface plasmon resonance (SPR)-based binding studies. Fig.36 B. The results summarized in Table 14 below show that the affinity of the humanized recombinant scFv anti-CD1a for immobilized rCD1a is 2.4 times higher than that of the murine version, as shown by the calculated dissociation constant (KD).

[0657]

[0658] Table 14 summarizes the kinetic parameters calculated using Biacore T200 Evaluation Software Version 3.0. ka: association rate constant (M-1s-1); kd: dissociation rate constant (s-1). KD (nM): equilibrium dissociation constant.

[0659] References

[0660] 1.Karrman, K. and B. Johansson, Pediatric T-cell acute lymphoblasticleukemia. Genes Chromosomes Cancer, 2017.56(2):p.89-116.

[0661] 2.Weng,A.P.,et al.,Activating mutations of NOTCH1 in human T cellacute lymphoblastic leukemia.Science,2004.306(5694):p.269-71.

[0662] 3.Hunger,S.P.and C.G.Mullighan,Acute Lymphoblastic Leukemia inChildren.N Engl J Med,2015.373(16):p.1541-52.

[0663] 4.Litzow,M.R.and A.A.Ferrando,How I treat T-cell acute lymphoblasticleukemia in adults.Blood,2015.126(7):p.833-41.

[0664] 5.Karrman,K.,et al.,Clinical and cytogenetic features of apopulation-based consecutive series of 285pediatric T-cell acutelymphoblastic leukemias:rare T-cell receptor gene rearrangements areassociated with poor outcome.Genes Chromosomes Cancer,2009.48(9):p.795-805.

[0665] 6.Sutton,R.,et al.,Persistent MRD before and after allogeneic BMTpredicts relapse in children with acute lymphoblastic leukaemia.Br JHaematol,2015.168(3):p.395-404.

[0666] 7.Qasim,W.and A.J.Thrasher,Progress and prospects for engineered Tcell therapies.Br J Haematol,2014.166(6):p.818-29.

[0667] 8.Humphries,C.,Adoptive cell therapy:Honing that killerinstinct.Nature,2013.504(7480):p.S13-5.9.Gardner,R.A.,et al.,Intent-to-treatleukemia remission by CD19 CAR T cells of defined formulation and dose inchildren and young adults.Blood,2017.129(25):p.3322-3331.

[0668] 10.Fry,T.J.,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):p.20-28.

[0669] 11.Cooper,M.L.,et al.,An"off-the-shelf"fratricide-resistant CAR-T forthe treatment of T cell hematologic malignancies.Leukemia,2018.32(9):p.1970-1983.

[0670] 12.Gomes-Silva,D.,et al.,CD7-edited T cells expressing a CD7-specificCAR for the therapy of T-cell malignancies.Blood,2017.130(3):p.285-296.

[0671] 13.Png,Y.T.,et al.,Blockade of CD7 expression in T cells foreffective chimeric antigen receptor targeting of T-cell malignancies.BloodAdv,2017.1(25):p.2348-2360.

[0672] 14.Mamonkin,M.,et al.,A T-cell-directed chimeric antigen receptor forthe selective treatment of T-cell malignancies.Blood,2015.126(8):p.983-92.

[0673] 15.Rasaiyaah,J.,et al.,TCRalphabeta / CD3 disruption enables CD3-specific antileukemic T cell immunotherapy.JCI Insight,2018.3(13).

[0674] 16.Sanchez-Martinez,D.,et al.,Fratricide-resistant CD1a-specific CART cells for the treatment of cortical T-cell acute lymphoblasticleukemia.Blood,2019.133(21):p.2291-2304.

[0675] 17.Maus,M.V.,et al.,Antibody-modified T cells:CARs take the frontseat for hematologic malignancies.Blood,2014.123(17):p.2625-35.

[0676] 18.Kershaw,M.H.,J.A.Westwood,and P.K.Darcy,Gene-engineered T cellsfor cancer therapy.Nat Rev Cancer,2013.13(8):p.525-41.

[0677] 19.Andreatta,M.and M.Nielsen,Gapped sequence alignment usingartificial neural networks:application to the MHC class Isystem.Bioinformatics,2016.32(4):p.511-7.

[0678] 20.Nielsen,M.,et al.,Reliable prediction of T-cell epitopes usingneural networks with novel sequence representations.Protein Sci,2003.12(5):p.1007-17.

[0679] 21.Kringelum,J.V.,et al.,Reliable B cell epitope predictions:impactsof method development and improved benchmarking.PLoS Comput Biol,2012.8(12):p.e1002829.

[0680] 22.Karosiene,E.,et al.,NetMHCcons:a consensus method for the majorhistocompatibility complex class I predictions.Immunogenetics,2012.64(3):p.177-86.

[0681] 23.Rasmussen,M.,et al.,Pan-Specific Prediction of Peptide-MHC Class IComplex Stability,a Correlate of T Cell Immunogenicity.J Immunol,2016.197(4):p.1517-24.

[0682] 24.Reynisson,B.,et al.,Improved Prediction of MHC II AntigenPresentation through Integration and Motif Deconvolution of Mass SpectrometryMHC Eluted Ligand Data.J Proteome Res,2020.19(6):p.2304-2315.

[0683] 25.Kalaitsidou,M.,et al.,CAR T-cell therapy:toxicity and therelevance of preclinical models.Immunotherapy,2015.7(5):p.487-97.

[0684] 26.Norelli,M.,et al.,Clinical pharmacology of CAR-T cells:Linkingcellular pharmacodynamics to pharmacokinetics and antitumor effects.BiochimBiophys Acta,2016.1865(1):p.90-100.

[0685] 27.Berger,C.,et al.,Safety of targeting ROR1 in primates withchimeric antigen receptor-modified T cells.Cancer Immunol Res,2015.3(2):p.206-16.

[0686] 28.Wang,J.,Y.Hu,and H.Huang,Acute lymphoblastic leukemia relapseafter CD19-targeted chimeric antigen receptor T cell therapy.J Leukoc Biol,2017.102(6):p.1347-1356.

[0687] 29.Campana,D.,H.Schwarz,and C.Imai,4-1BB chimeric antigenreceptors.Cancer J,2014.20(2):p.134-40.

[0688] 30.Wagner,D.L.,et al.,Immunogenicity of CAR T cells in cancertherapy.Nat Rev Clin Oncol,2021.18(6):p.379-393.

[0689] 31.Sanjuan-Pla,A.,et al.,Platelet-biased stem cells reside at theapex of the haematopoietic stem-cell hierarchy.Nature,2013.502(7470):p.232-6.

[0690] 32.Qiuping,Z.,et al.,Selectively increased expression and functionsof chemokine receptor CCR9 on CD4+T cells from patients with T-cell lineageacute lymphocytic leukemia.Cancer Res,2003.63(19):p.6469-77.

Claims

1. A humanized CD1a targeting moiety, wherein the CD1a targeting moiety is an antibody, F(ab')2, Fab, scFab or scFv, and the CD1a targeting moiety comprises a VL domain consisting of SEQ ID NO: 1 and a VH domain consisting of SEQ ID NO:

2.

2. The humanized CD1a targeting moiety according to claim 1, wherein the CD1a targeting moiety is a scFv comprising a VL domain consisting of SEQ ID NO: 1 and a VH domain consisting of SEQ ID NO:

2.

3. A chimeric antigen receptor (CAR), comprising: a) an extracellular domain comprising a CD1a targeting moiety, wherein the CD1a targeting moiety is a scFv comprising a VL domain consisting of SEQ ID NO: 1 and a VH domain consisting of SEQ ID NO: 2; b) a transmembrane domain; and c) Intracellular signaling domain.

4. The CAR of claim 3, wherein the transmembrane domain comprises a transmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

5. The CAR of claim 4, wherein the transmembrane domain comprises the transmembrane domain of CD8.

6. The CAR according to any one of claims 1 to 5, wherein the intracellular signaling domain comprises the intracellular domain of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b or CD66b.

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

8. The CAR according to any one of claims 1 to 7, wherein the CAR consists of SEQ ID NO:

13.

9. A nucleic acid encoding the CAR according to any one of claims 3 to 8.

10. A cell comprising the nucleic acid according to claim 9 and / or the CAR according to any one of claims 3 to 8.

11. The cell of claim 10, wherein the cell is a T cell.

12. A pharmaceutical composition comprising a plurality of cells according to claim 11 and a pharmaceutically acceptable carrier or diluent.

13. The cell according to claim 11 or the pharmaceutical composition according to claim 12 for use as a medicament.

14. The cell according to claim 11 or the pharmaceutical composition according to claim 12, for use in a method of treating a CD1a-positive cancer, wherein the method comprises administering the cell or the composition to a patient in need thereof.

15. The cell or pharmaceutical composition for use according to claim 14, wherein the CD1a-positive cancer is cortical T-cell acute lymphoblastic leukemia, preferably relapsed / refractory cortical T-cell acute lymphoblastic leukemia.

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