CAR-CD123 vector and its use

Abstract

The present invention relates to a chimeric antigen receptor (CAR) molecule, comprising from N-terminus to C-terminus: a) an extracellular domain and a transmembrane domain of human CD19, b) an antigen binding domain, c) a spacer domain, d) a transmembrane domain, and e) a cytoplasmic domain, preferably, wherein the CAR is a CD123-specific CAR (CD123CAR) and the antigen binding domain comprises VL and / or VH from a monoclonal anti-CD123 antibody, more preferably, the antigen binding domain comprises a single-chain variable fragment (scFv) that specifically binds to CD123.

Description

Technical Field

[0001] The present invention relates to the generation of novel chimeric antigen receptor (CAR)-CD123 vectors for the treatment of CD123+ tumors, such as acute myeloid leukemia (AML) [1], B-lymphocytic leukemia, blastic plasmacytoid dendritic neoplasm (BPDCN) [2], myelodysplastic syndrome [3] and hairy cell leukemia [4]. Background Art

[0002] Acute myeloid leukemia (AML) is a heterogeneous clonal disease characterized by the accumulation of abnormal immature blasts. Despite advances in the understanding of leukemia biology, standard upfront chemotherapy treatment of AML remains unchanged. Current induction therapy—also known as remission induction therapy—aims to clear immature blasts from the blood and bone marrow and achieve complete remission. This treatment is typically given over 1 week and can produce an initial complete remission in almost 70% of young adult patients. However, 43% of patients will eventually relapse, and 18% of patients never achieve complete remission on first-line induction therapy [5]. Immunotherapy using T cells genetically modified to express CARs represents an innovative approach to AML.

[0003] In the past few years, great interest has focused on identifying surface molecules preferentially expressed by AML cells and leukemic stem cells (LSCs) in order to selectively target the tumor population while avoiding affecting their normal counterparts, hematopoietic stem / progenitor cells (HSPCs) [6].

[0004] CD123 is a 360 amino acid glycoprotein also known as the transmembrane α subunit of the interleukin-3 receptor (IL-3Rα). CD123 and CD131 together form the high-affinity IL-3R. After binding to IL-3, IL-3R promotes cell proliferation and survival[7].

[0005] The basal expression of CD123 is low to negligible in HSPCs, monocytes, a subset of dendritic cells (DCs) called plasmacytoid DCs-pDCs[8], and endothelial cells[7][9]. In contrast to the low expression on HSPCs, AML progenitor cells extensively overexpress CD123

[10]

[11] . In addition, CD123 overexpression on AML cells is associated with apoptosis resistance, higher proliferation potential, and poor prognosis

[11]

[12]

[13] .

[0006] Notably, CD123 is selectively overexpressed not only by AML progenitors but also by the quiescent AML LSC population, which has been shown to be one of the major players in chemoresistance.

[14] The differential expression of CD123 on normal HSCs and LSCs makes CD123 an attractive target for AML therapy.

[0007] In addition, CD123 can be used to mark MDS stem cells in patients at high risk of MDS, and the CD123-positive population is biologically different from normal hematopoietic stem cells[3].

[0008] Several antibody-based therapies targeting CD123 have been developed

[15]

[16]

[14] . To date, CD123-targeted therapies have been shown to be safe, with no reported major adverse effects on hematopoiesis. Their anti-human leukemia activity is still under investigation.

[0009] Alternative AML treatment approaches utilize T cells expressing CARs that redirect T cell specificity to CD123 in an MHC-independent manner

[16] . CAR T cells have the potential to proliferate and persist in the human body for many years, thereby establishing long-term tumor immunity. The first generation of CARs consists of a single-chain variable fragment (scFv) from a monoclonal antibody (e.g., anti-CD123) fused to the CD3ζ signaling domain in the canonical structure scFv-spacer-CD3z. In addition to the structure just described, second generation CARs also have a co-stimulatory endodomain (e.g., CD28 or 41BB) that helps a) achieve T activation and b) avoid apoptosis by promoting IL2 secretion.

[0010] Preclinical studies using second-generation CAR-T cells with anti-CD123-CD28ζCAR and anti-CD123-41BBζCAR T cells have demonstrated effective leukemia-killing ability in vitro and in vivo, but have produced inconsistent results regarding their bone marrow depletion of healthy CD123+ cells

[17]

[18] . Multiple phase I trials (ClinicalTrials.gov numbers NCT03796390, NCT02937103, NCT03114670, NCT03672851) of CD123-directed CAR T cell therapy are currently underway to verify efficacy and safety.

[0011] Clearly, anti-CD123 CAR T technology has great potential for the treatment of AML and other CD123+ tumors. While the benefits of incorporating at least one co-stimulatory domain into the CAR structure are well established, the scFv and extracellular spacer domains, the selection of T cell subsets, and the manufacturing process remain an area of ​​focused research.

[0012] There is still a need to identify CAR T vectors for the treatment of CD123+ tumors.

[0013] Furthermore, CD123-redirected T-cell therapy in mice transplanted with normal human hematopoietic cells resulted in severe myeloablation, raising concerns about hematologic toxicity in AML patients who might receive such therapy.

[0014] Therefore, there remains a need for an effective CAR therapy that does not cause toxic effects to patients. DETAILED DESCRIPTION OF THE INVENTION

[0016] The inventors envision incorporating a new suicide gene into the CAR.CD123 method, which can minimize this bystander toxicity without compromising leukemia control, thereby increasing the therapeutic window of anti-leukemia CAR T cells or CAR innate cell immunotherapy. In summary, the present invention provides a novel second-generation CAR-CD123.

[0017] More specifically, the inventors constructed a CAR-CD123 structure ( Figure 1 ):ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41BB.CD3ζ (Table 1: OPBG-242 vector) retroviral vector.

[0018] The inventor has designed a bicistronic vector that allows simultaneous expression of two transgenes, i.e., ΔCD19 and CARCD123 (ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41BB.CD3ζ). The clonal retrovirus producer cell line produces a retrovirus supernatant characterized by high titer (using PCR to analyze the presence of vectors in the supernatant).

[0019] ΔCD19 represents the extracellular domain of human CD19 attached to the transmembrane portion. It has a triple function to help:

[0020] 1) Selection of genetically modified cells using clinical-grade microbeads;

[0021] 2) Phenotypic characterization of genetically modified cells;

[0022] 3) As an inducible suicide gene, it can be targeted by FDA / EMA-approved anti-CD19 bite antibodies (such as blinatumomab).

[0023] The CAR molecule is a single chain based on the human antigen CD123-specific monoclonal antibody 7G3 fused with the VK-VL region, in frame with the CD8 transmembrane domain and its internal domain, the 4.1BB co-stimulatory domain and the CD3ζ cytoplasmic domain for transduction of activator signals after antigen engagement. By using the 2A sequence, the CAR construct is cloned behind the gene cassette containing the ΔCD19 sequence in the retroviral vector.

[0024] Therefore, the functional and structural components of ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41BB.CD3ζ (after ΔCD19-2A-CAR.CD123-4.1BB-ζ) expression and activity are summarized in Table 1 and listed below:

[0025] 5'LTR - the retroviral long terminal repeat at the 5' end of the vector (functions as a promoter sequence)

[0026] ψ-retroviral encapsidation signal (psi; required for packaging RNA into virion particles)

[0027] SA-splicing acceptor site

[0028] ΔCD19 consists of the optimized human (extracellular and transmembrane) domains of the CD19 marker

[0029] 2A - encodes a synthetic 20 amino acid peptide from the lycopodiella virus that acts as a cleavable linker between the ΔCD19 protein and the CAR protein

[0030] • Signal peptide - a short amino acid sequence to allow the correct transport of secretory proteins from the endoplasmic reticulum to the cell membrane.

[0031] ΔCD34 consists of a short peptide derived from human CD34, which facilitates detection of CAR+ cells after transduction

[0032] The CAR-CAR molecule is based on the following elements indicated in the order of their position: a single chain of the fused VL(k)-VH regions of the monoclonal antibody 7G3, specific for the human antigen CD123, in frame with the ΔCD34 domain, the CD8 domain (spacer, TM, CD8Cyt), the 4.1BB co-stimulatory domain, and the CD3ζ cytoplasmic domain.

[0033] 3'LTR - Retroviral long terminal repeat at the 3' end of the vector (functions as terminator / polyadenylation sequence)

[0034] Table 1. Functional elements of OPBG-242 plasmid

[0035]

[0036]

[0037] Identity of vector ΔCD19-2A-CAR.CD123-4.1BB-ζ:

[0038] The reference electronic vector sequence was assembled by combining the DNA sequence files for each component of the vector construct. Since the retroviral genome is RNA-based, the plasmid DNA used for transfection (the initial step in the preparation of retroviral products) into the 293VEC cell line was sequenced. The inventors performed bidirectional sequencing on the entire OPBG-242 vector. The sequencing run was assembled using SnapGene software. No mismatched bases were identified compared to the theoretical reference electronic sequence.

[0039] According to the present invention, a second-generation novel CD123-specific chimeric antigen receptor (ΔCD19-2A-CAR.CD123-4.1BB-ζ) is now provided. In particular, the following clinical-grade second-generation CAR CD123SFG retroviral vector is provided:

[0040] ΔCD19-2A-CAR.CD123-4.1BB-ζ

[0041] (ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41BB.CD3ζ) composition:

[0042] ΔCD19 consists of an optimized human (extracellular and transmembrane) domain of the CD19 marker;

[0043] 2A peptide - encodes a synthetic 20 amino acid peptide from the Lymantria virus that functions as a cleavable linker between the ΔCD19 protein and the CAR protein;

[0044] Single chain variable fragment (scFv) from 7G3 hybridoma,

[0045] A traceable marker CD34-derived epitope (ΔCD34) of only 16 amino acids (aa) (as a traceable marker) for rapid identification by FACS (Fluorescence Activated Cell Sorting) system and / or selection by Cell Sorter System of genetically modified cells.

[0046] • The spacer is represented by the CD8 region to avoid the immunogenic IgG4 Fc region.

[0047] A transmembrane domain from the CD8 transmembrane domain to improve the stability of the molecule

[0048] A small portion of the CD8 cytoplasmic portion to facilitate signaling from the single chain to the 4.1BB-CD3ζ chain (4.1BB-ζ)

[0049] A co-stimulatory domain was added to the ΔCD19-2A-CAR.CD123-4.1BB-ζ vector: 4-1BB was fused to the CD3-ζ chain.

[0050] In ΔCD19-2A-CAR.CD123-4.1BB-ζ, the native nucleotide sequences of the traceable marker, co-stimulatory domain, and CD3-ζ chain were modified to obtain codon optimization to improve efficient protein expression.

[0051] Table 2: Comparison between CD123 CARs. This table shows the differences between known CD123 CARs and an object of the present invention (retroviral OPBG).

[0052]

[0053] Herein the inventors report strong evidence for a retroviral functional anti-CD123 CAR to transduce different subsets: T cells ( Figure 2 ), NK cells( Figure 4 ) and γδ T cells ( Figure 6 ).

[0054] They clearly showed that genetically modified CAR.CD123 T-cells ( Figure 8 )、CAR.CD123 NK cells( Fig. 9 ), CAR.CD123 γδ T cells ( Fig.11 ) in both in vitro and in vivo models (described in detail in Fig.14 ) has potent killing activity against leukemia cell line (THP-1).

[0055] In addition, the inventors herein demonstrated that ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ(CD123.CAR) T-cells ( Fig.15 ) or CAR.CD123 NK cells ( Fig.16 ) treatment, ΔCD19 acts as an inducible suicide gene. It should be noted that CD123.CAR T ( Fig.15 CD) and CD123.CAR NK cells ( Fig.16 B and 16E) were insensitive to the negative control, i.e., bite anti-CD19B-Gal+ antibody.

[0056] Therefore, the specificity of the suicide gene ΔCD19 in CD123 CAR NK cells is demonstrated by the fact that anti-hCD19-CD3+bite is active only in the presence of CD3 (T cells) in the in vitro model ( Fig.16 C and 16F).

[0057] Two co-expression markers (ΔCD34 and ΔCD19) are introduced into the CAR of the present invention ( Figure 1 ) allows the inventors to study genetically modified cells by considering dual channels (i.e., based on cytofluorimetric analysis using monoclonal antibodies to CD34 and / or CD19). This is particularly relevant to the suicide gene approach. In fact, if anti-CD19 antibodies are used to detect CAR.CD123 cells after anti-hCD19-CD3+bite exposure, masking effects associated with CD19 on bite-engaged CAR.CD123 modified cells may be encountered. However, the inclusion of dual markers represented by ΔCD34 has shown great advantages for analyzing residual genetically modified cells after treatment with anti-hCD19-CD3+bite.

[0058] There are also in vivo data confirming suicide gene activity in NSG mice receiving ΔCD19-2A-CAR.CD123-4.1BB-ζT cells ( Fig.17 ).like Fig.17 As shown in A, mice received a large number of ΔCD19-2A-CAR.CD123-4.1BB-ζT cells (10x10 6 / topo three times infusion) to simulate the worst case of highly expanded genetically modified cells. After 30 days, when the mice began to show painful clinical symptoms, the inventors performed bleeding to evaluate the expansion level of ΔCD19-2A-CAR.CD123-4.1BB-ζT cells ( Fig.17 B). Then, mice were treated with bite anti-hCD19-CD3 for five consecutive days (1 ug / day). On day 40, the inventors performed bleeding to assess the level of residual ΔCD19-2A-CAR.CD123-4.1BB-ζ T cells after treatment. Fig.17 As shown in C, after bite anti-hCD19-CD3 treatment, the amount of residual ΔCD19-2A-CAR.CD123-4.1BB-ζT was negligible.

[0059] Therefore, an object of the present invention is a CD123 chimeric antigen receptor molecule, from N-terminus to C-terminus, comprising or consisting of:

[0060] a) ΔCD19 extracellular and transmembrane domains, such as:

[0061] (Homo sapiens CD19 molecule (CD19), transcript variant 1, mRNA is disclosed in NCBI with the following accession number: nucleotide NM_001178098.2 and protein number: NP_001171569.1);

[0062] b) 2A peptide - a synthetic 20 amino acid peptide derived from the Lymantria virus, such as RRGRGGSLTCGDVEENPGP (SEQ ID NO: 2);

[0063] c) a signal peptide, such as MEFGLSWLFLVAILKGVQCSR (SEQ ID NO: 3) (Homo sapiens mRNA of IgG H chain is disclosed in NCBI with the following accession number, nucleotide number: AB776838.1 and protein number: BAN63131.1), which is in frame with:

[0064] d) an anti-CD123 single-chain antibody domain from a 7G3 hybridoma comprising or consisting of a 7G3 VL(K) sequence:

[0065] and connected to the 7G3 VH sequence via a second linker (GGGSGGGG (SEQ ID NO: 27)):

[0066] The 7G3 VL (K) and VH sequences were linked to:

[0067] e) a traceable marker consisting of ΔCD34:ELPTQGTFSNVSTNVS (SEQ ID NO: 6) (the Homo sapiens CD34 gene for the CD34 antigen is disclosed in NCBI with the following accession numbers: Nucleotide number: AB238231.1 and protein number: BAE46748.1);

[0068] f) Spacer, consisting of the extracellular part (hinge) of CD8α:

[0069] PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 7) (Homo sapiens T-cell surface glycoprotein CD8 α chain is disclosed in NCBI with the following accession numbers: nucleotide number: NM_001145873.1, and NM_001768.6, and protein number: NP_001139345, and NP_001759.3)

[0070] g) Transmembrane domain composed of CD8TM: IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 8) (Homo sapiens CD8a molecule (CD8A) transcript variant 1, mRNA disclosed in NCBI with the following accession numbers: nucleotides NM_001145873.1, and at NM_001768.6, and protein numbers: NP_001139345, and at NP_001759.3)

[0071] h) a cytoplasmic domain consisting of CD8a cyt: LYCNHRNRRRVCKCPR (SEQ ID NO: 9) (Homo sapiens CD8a molecule (CD8A) transcript variant 1, mRNA disclosed in NCBI with the following accession number: nucleotide number NM_001768.6 and protein number: NP_001759.3), connected via a short linker VD to:

[0072] i) Co-stimulatory signal transduction domain CD137 (4-1BB) sequence:

[0073] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 10) (human receptor protein 4-1BB mRNA: nucleotide number: U03397.1 and protein number: AAA53133.1);

[0074] j) and CD3-ζ chain:

[0075] (Human T cell receptor ζ-chain mRNA, complete cds is publicly available at NCBI with the following accession numbers: Nucleotide number: J04132.1 and Protein number: AAA60394.1)

[0076] According to a preferred embodiment of the present invention, the ΔCD19-2A-CAR.CD123-4.1BB-ζ chimeric antigen receptor molecule is:

[0077]

[0078]

[0079] That is, this sequence is also referred to herein as ΔCD19-2A-CAR.CD123-4.1BB-ζ.

[0080] The present invention also provides a nucleotide sequence encoding the CD123 chimeric antigen receptor.

[0081] According to an embodiment of the present invention, the nucleotide sequence is:

[0082]

[0083]

[0084]

[0085] That is, the nucleotide sequence, which encodes a sequence also referred to as ΔCD19-2A-CAR.CD123-4.1BB-ζ, comprises the following sequence:

[0086] ΔCD19

[0087]

[0088]

[0089] 2A peptide

[0090]

[0091] Signal peptide

[0092]

[0093] VL(7G3)

[0094]

[0095] Flex

[0096]

[0097] VH(7G3)

[0098]

[0099]

[0100] Link (BamH1 restriction site and connection sequence)

[0101]

[0102] ΔCD34

[0103]

[0104] Spacer (improved recognition by using anti-CD34 Ab)

[0105]

[0106] Spacer (CD8a)

[0107]

[0108] CD8a(TM) transmembrane

[0109]

[0110] CD8a cytoplasmic (CD8a cyt)

[0111]

[0112] connection

[0113]

[0114] 4-1BB sequence (co-stimulatory signal transduction domain)

[0115]

[0116] CD3ζ chain

[0117] (Nucleotide number: J04132.1)

[0118] Therefore, one object of the present invention is a chimeric antigen receptor (CAR) molecule comprising, from N-terminus to C-terminus:

[0119] a) the extracellular domain and transmembrane domain of human CD19 (ΔCD19),

[0120] b) an antigen binding domain,

[0121] c) a spacer domain,

[0122] d) transmembrane domain,

[0123] e) cytoplasmic domain,

[0124] The present invention also includes a CAR molecule comprising at least one of the above elements a)-e). The present invention also includes a CAR molecule wherein the elements are present in any order.

[0125] Preferably, the CAR molecule is a CD123-specific CAR (CD123 CAR) molecule, and the antigen binding domain comprises VL (or VK) and / or VH from a monoclonal anti-CD123 antibody, more preferably, the antigen binding domain comprises a single-chain variable fragment (scFv) that specifically binds to CD123.

[0126] Preferably, the antigen binding domain comprises or consists of the following sequence: and the 7G3 VK sequence: A sequence having at least 80% identity with the 7G3VH sequence:

[0127] A sequence having at least 80% identity, the VL and / or VK and / or VH are optionally humanized, wherein the VL (or VK) and VH are preferably separated by a first linker, more preferably the scFv comprises or consists of the following sequences: VK comprising or consisting of SEQ ID NO: 4 and VH comprising or consisting of SEQ ID NO: 5, more preferably the scFv comprises or consists of SEQ ID NO: 30.

[0128] Preferably, the above-mentioned antigen-binding domain may comprise an antigen-binding fragment of the sequence disclosed above, for example, at least one of the CDRs contained in the VH and VL sequences.

[0129] The first linker preferably comprises or consists of the sequence GGGSGGGG (SEQ ID NO: 27).

[0130] Preferably, the CAR molecule of the present invention further comprises a traceable marker.

[0131] The traceable marker is preferably comprised between the antigen binding domain and the spacer domain, preferably the antigen binding domain and the traceable marker are separated by a second linker.

[0132] Preferably, the traceable marker comprises or consists of the following sequence: a CD34-derived epitope (ΔCD34), or a peptide derived from the mouse Ig CH2CH3 region spacer (UNIPROTKB: P01861), which is:

[0133] or a peptide derived from the nerve growth factor receptor (NGFR) which is:

[0134]

[0135] Preferably, the traceable marker comprises or consists of SEQ ID NO: 6

[0136] (ELPTQGTFSNVSTNVS) has a sequence with at least 80% identity, even more preferably it comprises or consists of SEQ ID NO:6.

[0137] Preferably, the extracellular and transmembrane domains of human CD19 comprise or consist of SEQ ID NO: 1

[0138]

[0139] The invention relates to a sequence having at least 80% identity or a functional fragment, derivative or variant thereof.

[0140] Preferably, the extracellular domain and transmembrane domain of human CD19 comprise or consist of SEQ ID NO:1.

[0141] Preferably, the spacer domain comprises or consists of the extracellular region or hinge CH2-CH3 or hinge CH3 of the CD8 α chain or CD28, preferably it comprises or consists of the following sequence: a sequence having at least 80% identity to SEQ ID NO: 7, or a sequence having at least 80% identity to amino acids 12-42 of SEQ ID NO: 7 (corresponding to SEQ ID NO: 31 (IASQPLSLRPEACRPAAGGAVHTRGLDFACD)), and / or a sequence having at least 80% identity to amino acids 1-11 of SEQ ID NO: 7 (corresponding to SEQ ID NO: 32 (PAPRPPTPAPT)), or a sequence having at least 80% identity to SEQ ID NO: 7, or to IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 35) or to or with A sequence having at least 80% identity. More preferably, the spacer domain comprises or consists of SEQ ID NO:7.

[0142] Preferably, the transmembrane domain comprises or consists of a transmembrane domain of a protein selected from the group consisting of CD8, α, β or ζ chain of a T-cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD15, preferably comprising a transmembrane domain of CD8 or CD28, more preferably the transmembrane domain comprises or consists of a sequence having at least 80% identity to SEQ ID NO: 8 (IYIWAPLAGTCGVLLLSLVIT) or to FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 37). More preferably, the transmembrane domain comprises or consists of SEQ ID NO: 8.

[0143] Preferably, the cytoplasmic domain comprises or consists of:

[0144] a) The region and

[0145] b) 4-1BB co-stimulatory domain (co-stimulatory signal transduction domain CD137) or its CD28 cytoplasmic sequence or OX40 sequence and

[0146] c) CD3-ζ chain.

[0147] Preferably, the cytoplasmic domain comprises the region of the CD8a cytoplasmic portion and / or 4-1BB costimulatory domain and / or CD3-ζ chain. More preferably, the cytoplasmic domain comprises the region of the CD8a cytoplasmic portion and 4-1BB costimulatory domain and CD3-ζ chain.

[0148] Preferably, the cytoplasmic domain comprises a linker between the CD8a cytoplasmic and 4-1BB co-stimulatory domains.

[0149] Preferably, the CD8a cytoplasmic portion comprises or consists of SEQ ID NO: 9

[0150] (LYCNHRNRRRVCKCPR) has a sequence composition of at least 80% identity, even more preferably it comprises SEQ ID NO:9.

[0151] Preferably, the co-stimulatory signaling domain CD137(4-1BB) comprises or consists of a sequence having at least 80% identity to SEQ ID NO:10 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), even more preferably it comprises SEQ ID NO:10.

[0152] Preferably, the CD28 cytoplasmic sequence comprises or consists of

[0153] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 38) has a sequence composition that is at least 80% identical.

[0154] Preferably, the OX40 sequence comprises or consists of

[0155] RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 39) has a sequence composition of at least 80% identity.

[0156] Preferably, the CD3-ζ chain comprises or consists of SEQ ID NO: 11

[0157] A sequence having at least 80% identity, even more preferably it comprises SEQ ID NO:11.

[0158] Preferably, the sequence encoding the linker between the CD8a cytoplasm and the co-stimulatory signal transduction domain CD137 (4-1BB) is composed of 6 nucleotides. More preferably, it comprises or is composed of a linker of two amino acids, preferably VD.

[0159] The CAR molecules included in the present invention further include

[0160] - a cleavable linker, preferably a 2A peptide or an IRES, and / or

[0161] -Signal peptide and / or

[0162] - a second connector, and / or

[0163] - traceable markers,

[0164] In any order.

[0165] Preferably, the CAR molecule according to any one of the preceding claims further comprises:

[0166] - a cleavable linker, preferably a 2A peptide or an IRES, and / or

[0167] -Signal peptide.

[0168] Preferably, the cleavable linker is a 2A peptide comprising or consisting of a sequence having at least 80% identity to SEQ ID NO: 2 (RGRGRGSLLTCGDVEENPGP), more preferably, it comprises or consists of SEQ ID NO: 2; or a 2A self-cleaving peptide comprising the core sequence motif of DxExNPGP (SEQ ID NO: 40), preferably, comprising or consisting of AEGRGSLLTCGDVEENPGP (SEQID NO: 41); ATNFSLLKQAGDVEENPGP (SEQ ID NO: 42);

[0169] QCTNYALLKLAGDVESNPGP (SEQ ID NO: 43) or a peptide consisting of a sequence at least 80% identical to VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 44).

[0170] Preferably, the signal peptide comprises or consists of SEQ ID NO: 3

[0171] (MEFGLSWLFLVAILKGVQCSR), more preferably it comprises or consists of SEQ ID NO: 3; or it comprises or consists of a sequence at least 80% identical to MALPVTALLLPLALLLHAARP (SEQ ID NO: 45).

[0172] Preferably, between the antigen binding domain and the spacer domain there is:

[0173] - a second connector and / or

[0174] -Traceable markers.

[0175] Preferably, the CAR molecule has the above-mentioned cleavable linker, signal peptide and first linker from N-terminus to C-terminus, as well as the second linker and traceable marker.

[0176] Preferably, the second linker between the antigen binding domain and the traceable marker comprises or consists of SAGS (SEQ ID NO: 28).

[0177] Preferably, the sequence encoding at least one of the following is codon-optimized: the extracellular domain and the transmembrane domain of human CD19, a traceable marker, a spacer domain, a transmembrane domain, and a co-stimulatory domain.

[0178] Preferably, the CAR molecule according to the present invention comprises or consists of the following from N-terminus to C-terminus:

[0179] a) the extracellular domain and transmembrane domain of human CD19,

[0180] b) a cleavable linker,

[0181] c) a signal peptide,

[0182] d) an antigen binding domain,

[0183] e) traceable markers,

[0184] f) a spacer domain,

[0185] g) transmembrane domain,

[0186] h) Cytoplasmic domain.

[0187] In a preferred embodiment, the CAR molecule as defined above comprises or consists of from N-terminus to C-terminus:

[0188] a) the extracellular domain and transmembrane domain of human CD19, as defined above,

[0189] b) a cleavable linker, as defined above

[0190] c) a signal peptide, as defined above

[0191] d) an antigen binding domain, as defined above

[0192] e) Traceable marker, as defined above

[0193] f) a spacer domain, as defined above

[0194] g) a transmembrane domain, as defined above

[0195] h) a cytoplasmic domain, as defined above

[0196] Preferably, the CAR molecule comprises or consists of SEQ ID NO: 12

[0197]

[0198] It consists of a sequence having at least 80% identity, even more preferably it comprises or consists of SEQ ID NO:12.

[0199] Another object of the present invention is an isolated nucleic acid molecule encoding a chimeric antigen receptor molecule according to any of the preceding claims, wherein said isolated nucleic acid molecule is preferably operably linked to an expression control sequence. Preferably said molecule comprises at least one sequence having at least 80% identity to one of the following sequences: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,

[0200] Even more preferably said molecule comprises or consists of a sequence having at least 80% identity to SEQ ID NO:29, more preferably said molecule comprises or consists of SEQ ID NO:29.

[0201] Another object of the present invention is a vector comprising an isolated nucleic acid molecule as defined above, preferably an expression vector, more preferably a vector comprising an exogenous promoter controlling the expression of the CAR, preferably the vector is a DNA, RNA vector, plasmid, lentiviral vector, adenoviral vector, retroviral vector or non-viral vector.

[0202] Another object of the present invention is an engineered immune cell, preferably a T cell, more preferably an α / β or γ / δ T cell, or a NK cell, or a NK-T cell or a combination thereof, even more preferably of human origin, comprising the vector as defined above, or an isolated nucleic acid molecule as defined above, or expressing (preferably on the cell surface) at least one CAR molecule as defined above. Another object of the present invention is a pharmaceutical composition comprising an isolated nucleic acid molecule as defined above, or a vector as defined above, or a cell as defined above, and at least one pharmaceutically acceptable excipient and / or adjuvant.

[0203] Another object of the present invention is a CAR molecule as defined above, an isolated nucleic acid molecule as defined above, a vector as defined above, a cell as defined above, or a pharmaceutical composition for medical use as defined above, preferably for the treatment of CD123+ cancer, more preferably for acute myeloid leukemia or B-lymphoid leukemia, blastic plasmacytoid dendritic neoplasm (BPDCN), myelodysplastic syndrome or hairy cell leukemia, or for treatment before, after or during hematopoietic stem cell transplantation.

[0204] Another object of the present invention is the extracellular domain and the transmembrane domain of human CD19, preferably as defined above, for medical use, preferably as a death inducing agent in cells genetically modified with said extracellular domain and the transmembrane domain of human CD19, preferably after said cells have been exposed to at least one agent that binds to the extracellular and transmembrane domains of human CD19 on transduced cells.

[0205] Preferably, the reagent that binds to the extracellular and transmembrane domains of human CD19 is an antibody, a small molecule, a polypeptide, a nucleic acid or a combination thereof, more preferably a monoclonal antibody. More preferably, the reagent that binds to the extracellular and transmembrane domains of human CD19 is an anti-CD19 antibody, including (bispecific T-cell engagers) bite antibodies, such as blinatumomab.

[0206] Preferably, the activity of the antibody is independent of the mechanism of action of ADCC. In the context of the present invention, the elimination of genetically modified cells is preferably performed by a mechanism that is not based on ADCC.

[0207] Preferably the antibody is a bispecific antibody that engages ΔCD19 and CD3 to activate cytotoxicity in ΔCD19+ cells.

[0208] Preferably, the genetically modified cell is a transduced cell, such as an immune cell or a stem cell, which comprises a nucleic acid encoding the extracellular domain and transmembrane domain of human CD19, and a nucleic acid encoding one or more therapeutic gene products.

[0209] Preferably, the therapeutic gene product is an engineered receptor, more preferably a T cell receptor, a chimeric antigen receptor (CAR), a cytokine receptor, a homing receptor or a chemokine receptor, preferably the engineered receptor targets a cancer antigen.

[0210] Preferably, the nucleic acid encoding the extracellular domain and transmembrane domain of human CD19 and the nucleic acid encoding the therapeutic gene product are the same nucleic acid molecule, preferably the nucleic acid molecule is a vector, more preferably it is a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0211] Preferably, the nucleic acid encoding the extracellular domain and transmembrane domain of human CD19 is located at the N-terminus of the nucleic acid molecule, preferably at the N-terminus of the CAR molecule.

[0212] Preferably the CAR is a CAR according to the present invention.

[0213] Another object of the present invention is a composition comprising transduced cells, such as immune cells or stem cells, which comprises a nucleic acid encoding the extracellular domain and transmembrane domain of human CD19 and a nucleic acid encoding one or more therapeutic gene products, preferably the therapeutic gene product is an engineered receptor, more preferably a T cell receptor, a chimeric antigen receptor (CAR), a cytokine receptor, a homing receptor or a chemokine receptor, preferably the engineered receptor targets a cancer antigen.

[0214] Preferably, the nucleic acid encoding the extracellular domain and transmembrane domain of human CD19 and the nucleic acid encoding the therapeutic gene product are the same nucleic acid molecule, preferably the nucleic acid molecule is a vector, more preferably it is a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0215] Another object of the present invention is a method for inducing death of transduced cells expressing the extracellular domain and transmembrane domain of human CD19, which comprises providing an effective amount of at least one agent that binds to the extracellular domain and transmembrane domain of human CD19 on the transduced cells, wherein the agent is preferably an antibody, a small molecule, a polypeptide, a nucleic acid or a combination thereof, more preferably a monoclonal antibody, more preferably an anti-CD19 antibody, including a bite antibody, such as blinatumomab.

[0216] Preferably, the cell further expresses an engineered receptor, preferably a T cell receptor or a CAR, more preferably said engineered receptor targets a cancer antigen.

[0217] Preferably the CAR is a CAR according to the present invention.

[0218] Another object of the present invention is a vector comprising sequences encoding the extracellular domain and transmembrane domain of human CD19 and sequences encoding an engineered receptor.

[0219] Preferably, the sequences of the vector encoding the extracellular domain and transmembrane domain of human CD19 and the sequences of the vector encoding the engineered receptor are separated on the vector by a cleavable linker (preferably as defined above). Preferably, the sequences of the vector encoding the extracellular domain and transmembrane domain of human CD19 are upstream of the sequences of the vector encoding the engineered receptor.

[0220] Preferably, the engineered receptor is a CAR, more preferably a CAR according to the present invention.

[0221] In the context of the present invention, the extracellular domain and transmembrane domain of human CD19 comprise or consist of a sequence having the same structure as SEQ ID NO: 1

[0222] The polypeptide comprises a sequence with at least 80% identity or a functional fragment, derivative or variant thereof, more preferably, the extracellular and transmembrane domains of human CD19 comprise or consist of SEQ ID NO: 1.

[0223] Preferably, the method of the invention occurs in an individual suffering from a medical condition and the individual has been provided with a treatment comprising a plurality of transduced cells for the medical condition, preferably cancer. Preferably, the agent is provided to the individual once one or more adverse events from the treatment occur.

[0224] Another object of the present invention is a method for reducing the risk of cell therapy toxicity in an individual, comprising the step of modifying cells of cell therapy to express the extracellular and transmembrane domains of human CD19.

[0225] Preferably, the treatment is of cancer.

[0226] Preferably, the cell therapy comprises an engineered receptor targeting an antigen.

[0227] Another object of the invention is the extracellular domain and transmembrane domain of human CD19, in particular as defined above, for medical use, preferably as a death inducer in cells genetically modified with said extracellular domain and transmembrane domain of human CD19, preferably after said cells have been exposed to anti-CD19 antibodies (including bite antibodies, such as blinatumomab).

[0228] In the context of the present invention, it is preferred that the activity of the antibody is independent of the ADCC mechanism of action and / or that the elimination of the genetically modified cells is preferably via a mechanism not based on ADCC.

[0229] Preferably the antibody is a bispecific antibody that engages ΔCD19 and CD3 to activate cytotoxicity in ΔCD19+ cells.

[0230] The cell genetically modified by the extracellular domain and transmembrane domain of human CD19 can be a cell comprising and / or and / or transformed and / or transduced and / or expressing the CAR molecule of the present invention, for example, a CAR-T cell or a CAR-NK cell or a cell as defined above,

[0231] The choice of linker depends on the cloning strategy (insertion of restriction enzymes).

[0232] Preferably, the above nucleic acid is integrated into the genome of the cell.

[0233] The CAR-cells can be allogeneic or autologous and / or HLA matched to the subject.

[0234] In the context of the present invention, a cancer or tumor preferably expresses CD123.

[0235] Preferably, the polynucleotide is under the control of an endogenous promoter.

[0236] Preferably, the extracellular and transmembrane domains of human CD19 are encoded by a sequence comprising or consisting of a sequence having at least 80% identity to SEQ ID NO:13, preferably by a sequence comprising or consisting of SEQ ID NO:13.

[0237] Another object of the invention is a viral particle comprising an isolated nucleic acid molecule as defined above or a vector as defined above.

[0238] The antigen binding domain is preferably a chimeric antigen binding domain.

[0239] The chimeric antigen receptor (CAR) of the present invention may include an antibody or antibody fragment engineered to specifically bind to a CD123 protein or its fragment. On the one hand, the antigen binding domain of CAR includes a human or humanized CD123 antibody or its antibody fragment. Preferably, the antigen binding domain of CAR includes a human CD123 antibody fragment containing scFv (preferably human CD123 scFv).

[0240] Preferably, the antigen binding domain of the CAR comprises an anti-CD123 single-chain antibody domain from a 7G3 hybridoma, preferably comprising or consisting of a 7G3 VL (or VK) sequence and a 7G3 VH sequence, preferably connected by a linker.

[0241] In one aspect, the CAR123 binding domain comprises a scFv portion provided in SEQ ID NO: 30

[0242]

[0243] In the context of the present invention, the elements "extracellular domain and transmembrane domain" of human CD19 may be as defined above, or they may be ΔCD19 extracellular and transmembrane domains from other transcriptional variants of the Homo sapiens CD19 molecule (CD19), for example, those from the variants disclosed in NCBI with the following accession numbers NM_001770 and NP_001761.3 (isoform 2), XM_017023893.1 and XP_016879382.1 (isoform X2), XM_011545981.2 and XP_011544283.1 (isoform X3).

[0244] In the context of the present invention, the cleavable linker, which may be a 2A peptide as defined above or it may be a 2A self-cleaving peptide, has a core sequence motif of DxExNPGP (SEQ ID NO: 40), preferably a peptide selected from the group consisting of: T2A (derived from Lyme disease virus 2): AEGRGSLLTCGDVEENPGP (SEQ ID NO: 41) (nucleotide number: AF062037.1 and protein number: YP_009665206.1); P2A (derived from porcine teschovirus-1 2A): ATNFSLLKQAGDVEENPGP (SEQ ID NO: 42) (nucleotide number: AB038528.1 and protein number: BAB32828.1); E2A (derived from equine rhinitis A virus): QCTNYALLKLAGDVESNPGP (SEQ ID NO: 43) (nucleotide number: AF062037.1 and protein number: YP_009665206.1); NO: 43) (nucleotide number: NC_039209.1 and protein number: YP_009513027.1) and F2A (derived from foot-and-mouth disease virus): VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 44) (nucleotide number: AY593825.1 and protein number: AAT01768.1). Preferably, the modified polynucleotide T2ARGRGRGSLLTCGDVEENPGP (SEQ ID NO: 2) (nucleotide number: AF062037.1 and protein number: YP_009665206.1) is used.

[0245] Other standard signal peptides for CAR constructs can be used in place of the above signal peptides, including different signal peptides derived from proteins characterized as being highly expressed, such as the CD8a signal peptide

[0246] MALPVTALLLPLALLLHAARP (SEQ ID NO: 45) (nucleotide number: NM_001768; and protein number: NP_001759.3).

[0247] Other traceable markers may be used in place of the traceable markers disclosed above, such as those derived from the murine Ig CH2CH3 region spacer (UNIPROT KB: P01861):

[0248] Peptides or derived from

[0249] NGFR:

[0250] (Nucleotide number: AK313654.1 and protein number: BAG36408.1).

[0251] Other spacer domains (or sequences) can be used in place of the spacer sequences disclosed above, such as spacer domains selected from the group consisting of stalk sequences from membrane-bound proteins, including the following examples: CD28 stalk: CD28:

[0252] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 35) (nucleotide number: AJ517504.1 and protein number: CAD57003.1); hinge CH2-CH3

[0253] (UNIPROTKB: P01861):

[0254] Hinge CH3

[0255] (UNIPROTKB: P01861):

[0256] Preferred CD8 stems:

[0257] (Nucleotide number: M12828.1 and protein number: AAB04637.1).

[0258] Other transmembrane domains may be used in place of those disclosed above, such as CD28TM: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 37) (nucleotide number: J02988.1 and protein number: AAA60581.1).

[0259] The above co-stimulatory signal transduction domain can be a peptide comprising or consisting of the CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 38) (nucleotide number: AF222341.1 and protein number: AAF33792.1) or the CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQID NO: 10) (nucleotide number: U03397.1 and protein number: AAA53133.1) or the OX40 sequence RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 39) (nucleotide number: NM_003327.3 and protein number: NP_003318.1).

[0260] The domains of the CARs disclosed herein are linked and in the same reading frame to form a single fusion protein.

[0261] In the context of the present invention, the immune cell may be a T cell, a NK cell, a NKT cell, an iNKT cell, a B cell, a regulatory T cell, a monocyte, a macrophage, a dendritic cell or a mesenchymal stromal cell.

[0262] The cells of the present invention (e.g., immune effector cells, e.g., T cells or NK cells) can be engineered to express CAR, wherein cells expressing CAR (e.g., "CAR-T" or NK cells expressing CAR) exhibit anti-tumor properties. On the one hand, cells are transformed with CAR, and at least part of CAR is expressed on the cell surface. In some embodiments, cells (e.g., immune cells, immune effector cells, such as T cells or NK cells, engineered immune cells, such as T cells, more preferably α / β or γ / δT cells, or NK cells or NK-T cells or a combination thereof) are transduced with a viral vector encoding CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, cells can stably express CAR. In another embodiment, cells (e.g., immune effector cells, e.g., T cells or NK cells) are transfected with nucleic acids encoding CAR, such as mRNA, cDNA, DNA. In some such embodiments, cells can transiently express CAR. Preferably, the CD123 binding domain of CAR, such as the human or humanized CD123 binding domain of CAR is a scFv antibody fragment. On the one hand, the function of this antibody fragment is that they retain equivalent binding affinity, for example, they bind the same antigen with the same efficacy as IgG antibodies with the same heavy chain and light chain variable regions. On the one hand, this antibody fragment functions in providing a biological response, including but not limited to the activation of immune response, inhibition of signal transduction from its target antigen, inhibition of kinase activity, etc., as understood by those skilled in the art. The antibody of the present invention can be incorporated into a chimeric antigen receptor (CAR). On the one hand, the part of the CAR of the present invention, the CD123 binding domain (such as humanized or human CD123 binding domain) is encoded by a transgene, and its sequence has been codon optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons encoding the same amino acid) in coding DNA is biased in different species. This codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Various codon optimization methods are known in the art and include, for example, the methods disclosed in at least US Pat. Nos. 5,786,464 and 6,114,148.

[0263] "CD123" refers to an antigenic determinant known to be detected on leukemia precursor cells. The amino acid and nucleic acid sequences of humans and mice can be found in public databases, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD123 can be found in accession number NP_002174.1, and the encoding nucleotide sequence of human CD123 can be found in accession number NM_002183.4. On the one hand, the antigen binding moiety of CAR recognizes and binds to the antigen in the extracellular domain of CD 123 protein. On the one hand, CD123 protein is expressed on cancer cells. As used herein, the term "chimeric antigen receptor" or alternative "CAR" refers to a recombinant polypeptide construct comprising at least an antigen binding domain, a transmembrane domain and a cytoplasmic domain (also referred to herein as "intracellular signal transduction domain"), which includes a functional signal transduction domain derived from a stimulatory molecule defined herein. On the one hand, the stimulatory molecule is a ζ chain associated with a T cell receptor complex. On the one hand, the intracellular signal transduction domain also includes one or more functional signal transduction domains derived from at least one costimulatory molecule defined as follows.On the one hand, costimulatory molecules are selected from 4-1BB (i.e., CD137), CD27 and / or CD28.On the one hand, CAR includes a chimeric fusion protein containing an antigen recognition domain, a transmembrane domain, and a cytoplasmic signal transduction domain, and the cytoplasmic signal transduction domain includes a functional signal transduction domain derived from a stimulatory molecule.On the one hand, CAR includes a chimeric fusion protein containing an antigen recognition domain, a transmembrane domain, and a cytoplasmic signal transduction domain, and the cytoplasmic signal transduction domain includes a functional signal transduction domain derived from a costimulatory molecule and a functional signal transduction domain derived from a stimulatory molecule.On the one hand, CAR includes a chimeric fusion protein containing an antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, and the intracellular signal transduction domain includes two functional signal transduction domains derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR comprises a chimeric fusion protein containing an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain comprises at least two functional signal transduction domains derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. On the one hand, CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein, during the cell processing and localization to the cell membrane of CAR, the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv). As used herein, the terms intracellular (), cytoplasmic () and cytoplasmic () are used interchangeably.The term "signal transduction domain" refers to a functional portion of a protein that regulates cellular activity by transmitting information within the cell to act as an effector by generating second messengers or by responding to such messengers through a defined signal transduction pathway. As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen, e.g., non-covalently, reversibly, and in a specific manner. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or complete immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. For example, naturally occurring IgG antibodies are tetramers that contain at least two heavy (H) chains and two light (L) chains (wherein the L chains can be composed of κ or λ chains)

[20] interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of a domain CL. In the context of the present invention, the term VL also includes VK. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), with more conservative regions interspersed therebetween, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant domains of antibodies can mediate the binding of immunoglobulins 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. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camel antibodies, and chimeric antibodies. The antibody can be of any isotype / type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to the antigen binding domain (e.g., antigen-determining variable region) of an intact antibody that enables the antibody fragment to be sufficient to recognize and specifically bind to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain or "scFv" antibody fragments, linear antibodies, single-domain antibodies such as sdAb (VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain and heavy chain variable regions are continuously connected by a short flexible polypeptide linker and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated, as used herein, scFv can have VK or VL and VH variable regions in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, scFv can comprise VL (or VK)-linker-VH or can comprise VH-linker-VL (or VK). The CAR composition portion of the present invention comprising an antibody or an antibody fragment thereof can exist in a variety of forms, wherein the antigen binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York) [21, 22]. On the one hand, the antigen binding domain of the CAR composition of the present invention comprises an antibody fragment. On the other hand, the CAR comprises an antibody fragment containing scFv. As used herein, the term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, for example, an antibody expressed by a phage or yeast expression system. The term should also be understood to mean an antibody produced by synthesizing a DNA molecule encoding the antibody, and the DNA molecule expresses the antibody protein or specifies the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has a DNA or amino acid sequence obtained using recombinant or synthetic DNA or amino acid sequence technology available in the art and well-known. The term "antigen" or "Ag" as used herein is defined as a molecule that causes an immune response. This immune response may involve the production of antibodies, or the activation of specific immunocompetent cells, or both. It will be appreciated by those skilled in the art that any macromolecule, including nearly all proteins or peptides, can be used as an antigen.

[0264] As used herein, the phrase "disease associated with the expression of CD123" includes, but is not limited to, diseases associated with the expression of CD123 or disorders associated with cells expressing CD123, including, for example, proliferative diseases, such as cancer or malignant tumors or precancerous conditions, such as myelodysplasia, myelodysplastic syndrome or preleukemia; or non-cancer related indications associated with cells expressing CD123. On the one hand, the cancer associated with CD123 expression is a blood cancer. On the one hand, blood cancers include, but are not limited to, AML, myelodysplastic syndrome, ALL, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, blastic plasmacytoid dendritic cell tumors, etc. Other diseases associated with CD123 expression include, but are not limited to, for example, atypical and / or non-classical cancers, malignant tumors, precancerous conditions, or proliferative diseases associated with CD123 expression. Non-cancer related indications associated with CD123 expression may also be included.

[0265] In the present invention, one or more amino acid residues in the CAR of the present invention can be replaced by other amino acid residues from the same side chain family, and the ability of the altered CAR to bind to CD123 can be tested using functional assays known to those skilled in the art.

[0266] As used herein, the term "stimulatory molecule" refers to a molecule expressed by a T cell, which provides one or more first cytoplasmic signal transduction sequences (primary cytoplasmic signaling sequence), and regulates the first activation (primary activation) of the TCR complex in a stimulating manner in at least some aspects of the T cell signal transduction pathway. On the one hand, the first signal is caused by, for example, the combination of the TCR / CD3 complex and the MHC molecule loaded with a peptide, and leads to the mediation of T cell responses, including but not limited to proliferation, activation, differentiation, etc. The first cytoplasmic signal transduction sequence (also referred to as "the first signal transduction domain") that works in a stimulating manner can include a signal transduction motif called an activation motif based on immunoreceptor tyrosine or IT AM. Examples of IT AMs containing the first cytoplasmic signal transduction sequence used in the present invention include but are not limited to those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d. In the specific CAR molecules of the present invention, the intracellular signal transduction domain in any one or more CAR molecules of the present invention includes an intracellular signal transduction sequence, such as a first signal transduction sequence of CD3-ζ. In the specific CAR of the present invention, the first signal transduction sequence of CD3-ζ is a human sequence (SEQ ID NO: 11), or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, the intracellular signal transduction domain may include a first intracellular signal transduction domain. An exemplary first intracellular signal transduction domain includes those derived from molecules responsible for the first stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signal transduction domain may include a costimulatory intracellular domain. Exemplary costimulatory intracellular signal transduction domains include those derived from molecules responsible for costimulatory signals or antigen-dependent stimulation. For example, in the case of CART, the first intracellular signal transduction domain may include a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signal transduction domain may include a cytoplasmic sequence from a co-receptor or a costimulatory molecule. The first intracellular signal transduction domain can include a signal transduction motif, which is referred to as an immunoreceptor tyrosine-based activation motif or IT AM. Examples of IT AMs containing a first cytoplasmic signal transduction sequence include, but are not limited to, those derived from CD3ζ, FcRγ, FcRα, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, DAP10, and DAP12.

[0267] As used herein, "ζ" or alternatively "ζ chain", "CD3-ζ" or "TCR-ζ" is, for example, defined as a protein provided by NCBI Accession No. J04132.1, or equivalent residues from non-human species (such as mice, rodents, monkeys, apes, etc.), and "ζ stimulatory domain" or alternatively "CD3-ζ stimulatory domain" or "TCR-ζ stimulatory domain" is defined as amino acid residues from the cytoplasmic domain of the ζ chain that are sufficient to functionally transmit the initial signal required for T cell activation. "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which are required for an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la / CD18) and 4-1BB (CD137). The costimulatory intracellular signal transduction domain can be an intracellular part derived from a costimulatory molecule. Costimulatory molecules can be represented in the following protein families: TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein) and activation NK cell receptor. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand specifically binding CD83, etc. The intracellular signal transduction domain may include the entire intracellular portion of the molecule derived therefrom, or the entire native intracellular signal transduction domain, or a functional fragment thereof. "4-1BB" as used herein refers to a member of the TNFR superfamily, having, for example, an amino acid sequence provided by NCBI No. AAA53133.1, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, etc. On the one hand, the "4-1BB co-stimulatory domain" is a human sequence or an equivalent residue from a non-human species (e.g., mice, rodents, monkeys, apes, etc.). On the one hand, the "4-1BB co-stimulatory domain" is a sequence provided by SEQ ID NO: 10 or an equivalent residue from a non-human species (e.g., mice, rodents, monkeys, apes, etc.). "Encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA), as a template for synthesizing other polymers and macromolecules in a biological process having a determined nucleotide (e.g., rRNA, tRNA, and mRNA) sequence or a determined amino acid sequence, and the resulting biological properties.Therefore, if the mRNA corresponding to the gene, cDNA or RNA is transcribed and translated in a cell or other biological system to produce a protein, the gene, cDNA or RNA encodes a protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence) and the non-coding strand (used as a transcription template for a gene or cDNA) can be referred to as encoding a protein or other product of the gene or cDNA. Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Insofar as the nucleotide sequence encoding a protein may include one or more introns in some versions, the nucleotide sequence encoding a protein or RNA may also include introns. The term "vector" or "transfer vector" refers to a polynucleotide that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides bound to ions or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be understood to further include non-plasmid and non-viral compounds that promote nucleic acid transfer into cells, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like. "Expression vector" refers to a vector comprising a recombinant polynucleotide, which comprises an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression, and other elements for expression can be provided by a host cell or in an in vitro expression system. Expression vectors include all known vectors in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides. The term "homologous" or "identity" as used herein refers to the identity of the subunit sequence between two polymeric molecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit positions in the two molecules are occupied by the same monomer subunit; for example, if the positions of each of the two DNA molecules are occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer of 10 subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10), are matching or homologous, then the two sequences are 90% homologous.

[0268] As used herein, "lentivirus" refers to the genus of the Retroviridae family. Slow viruses are unique among retroviruses in that they are able to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, so they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of slow viruses.

[0269] A "lentiviral vector" is a vector derived from at least part of a lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided by Milone et al.

[23] . Other examples of lentiviral vectors that can be used clinically include, but are not limited to, those from Oxford BioMedica. Gene delivery technology, Lentigen's LENTIMAX TM Vector Systems, etc. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

[0270] The term "operably linked" or alternatively "transcriptional control" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, when a first nucleic acid sequence is in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to a coding sequence. Operably linked DNA sequences can be continuous with each other, for example, in the case where two protein coding regions need to be added, they are in the same reading frame. The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be obtained by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues

[24]

[25]

[26] . As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that make up a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to two short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and also refers to long chains, which are commonly referred to in the art as proteins, including many types. "Polypeptides" (such as domains, markers, peptides, linkers above) include, for example, biologically active fragments or functional fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides or combinations thereof. As used herein, the term "promoter" is defined as a DNA sequence that is recognized by the synthetic machinery of the cell or introduced into the synthetic machinery, which is required to initiate the specific transcription of the polynucleotide sequence. As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence that is operably connected to a promoter / regulatory sequence required for the expression of a gene product. In some cases, the sequence can be a core promoter sequence, and in other cases, the sequence can also include enhancer sequences and other regulatory elements required for the expression of gene products. For example, a promoter / regulatory sequence can be a sequence that expresses a gene product in a tissue-specific manner.In the case of scFv, "linker" refers to a peptide linker composed of amino acids (e.g., glycine and / or serine residues) used alone or in combination to connect the variable heavy chain region and the variable light chain region together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises an amino acid sequence (Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly)n (SEQ ID NO: 27), wherein n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10.

[0271] The linkers described in WO2012 / 138475 are also included within the scope of the present invention, which is incorporated herein by reference in its entirety.

[0272] The linker connecting the VL (or VK) and VH sequences is preferably selected from the following group: a rigid linker rich in proline, such as the upper hinge region of mouse IgG3 (mlgG3UFI): PKPSTPPGSS (SEQ ID NO: 46), (mlgG3UH)2: PKPSTPPGSSPKPSTPPGSS (SEQ ID NO: 47), or a flexible linker rich in glycine, such as the (G4S)2 linker: GGGGSGGGG (SEQ ID NO: 48), (G4S)4 linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 49), G4SG2 linker GGGGSGG (SEQ ID NO: 50) or G3SG4 linker: GGGSGGGG (SEQ ID NO: 27), preferably GGGSGGGG (SEQ ID NO: 27). As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected" or "transformed" or "transduced" cells are cells that have been transfected, transformed or transduced with exogenous nucleic acids. The cells include primary subject cells and their progeny. The cells include engineered immune cells, preferably T cells, more preferably α / β or γ / δ T cells, or NK cells or NK-T cells or a combination thereof. As used herein, "transient" refers to non-integrated transgene expression for hours, days or weeks, wherein the expression time is shorter than the expression time of the gene if it is integrated into the genome or contained in a stable plasmid replicon in the host cell. As used herein, the phrase "under transcriptional control" or "operably connected" refers to that the promoter is in the correct position and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0273] As used herein, the term "specific binding" refers to an antibody or its antigen binding fragment, or a ligand, which recognizes and binds to a cognate binding partner (e.g., a stimulatory and / or co-stimulatory molecule present on a T cell) protein present in a sample, but the antibody, its antigen binding fragment or ligand does not substantially recognize or bind to other molecules in the sample. It should be understood that the description in the form of a range is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the invention. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and each numerical value within the range. For example, it should be considered that a range description such as 1 to 6 has specifically disclosed a sub-range, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and each number within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6. As another example, a range such as 95-99% homogeneity includes some with 95%, 96%, 97%, 98% or 99% homogeneity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% homogeneity. This is generally applicable regardless of the breadth of the range. The term at least 80% homogeneity includes those with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% homogeneity.

[0274] The present invention also includes nucleic acid and amino acid sequences derived from the above sequences, such as functional fragments, mutants, derivatives, analogs and sequences having at least 70% identity with the above sequences.

[0275] The domains, peptides, molecules, markers, regions, parts, sequences and chains disclosed herein include their functional derivatives, fragments, variants, corresponding proteins encoded by corresponding orthologous or homologous genes, functional mutants, functional fragments or analogs, isoforms.

[0276] The terms polynucleotide and polypeptide also include derivatives and functional fragments thereof.Polynucleotides may be synthesized using oligonucleotide analogs or derivatives (eg, inosine or phosphorothioate nucleotides).

[0277] In the context of the present invention, genes / proteins as defined above are preferably characterized by sequences defined by their NCBI gene IDs and Gen Bank accession numbers. The term gene herein also includes its corresponding straight homology or homologous genes, isoforms, variants, allelic variants, functional derivatives, functional fragments. The expression "protein" is intended to also include corresponding proteins, functional mutants, functional derivatives, functional fragments or analogs, isoforms encoded by its corresponding straight homology or homologous genes.

[0278] In the context of the present invention, the term "polypeptide" or "protein" includes:

[0279] i. The whole protein, its allelic variants and orthologs;

[0280] ii. any synthetic, recombinant or proteolytic functional fragment;

[0281] iii. Any functional equivalent, such as a synthetic or recombinant functional analogue.

[0282] For "functionality", it is intended to maintain its activity. For example, a functional fragment, derivative or variant of the extracellular domain and transmembrane domain of human CD19 should maintain its activity as a suicide gene or the activity of inducing death in transduced cells expressing the extracellular domain and transmembrane domain of human CD19.

[0283] As used herein, the name "functional derivative" or "functional variant" in the context of a functional derivative of a sequence (whether a nucleic acid sequence or an amino acid sequence) means a molecule that retains biological activity (functional or structural) that is substantially similar to its original sequence. The functional derivative or equivalent can be a natural derivative or can be prepared synthetically. Such derivatives include amino acid sequences with substitutions, deletions or additions of one or more amino acids, provided that the biological activity of the protein is conservative. The same applies to derivatives of nucleic acid sequences that can have substitutions, deletions or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained. When it comes to protein sequences, the substituted amino acids generally have chemical and physical properties similar to the substituted amino acids. Similar chemical and physical properties include charge similarity, volume, hydrophobicity, hydrophilicity, etc. The term "functional derivative" is intended to include "fragments", "segments", "variants", "analogs" or "chemical derivatives" of the subject matter of the present invention. In one embodiment, the above-mentioned derivatives, variants or fragments are "antigenic derivatives, variants or fragments" (e.g., they have the ability to induce / trigger an immune response to a parent antigen).

[0284] Therefore, the term "variant" herein refers to a protein or nucleic acid molecule that is substantially similar in structure and biological activity to a protein or nucleic acid of the present invention, such as but not limited to a variant that retains all biological activities of the parent protein.

[0285] The functional derivatives of the present invention can be chemically synthesized or produced by recombinant DNA technology. All these methods are well known in the art.

[0286] As used herein, "chemical derivatives" are intended to encompass other chemical moieties that are not generally part of the subject matter of the present invention. Such moieties may affect the physicochemical characteristics of the derivative (e.g., solubility, absorption, half-life, reduced toxicity, etc.). Such moieties are illustrated in Remington's Pharmaceutical Sciences (1980). Methods for coupling these chemical-physical moieties to polypeptides or nucleic acid sequences are well known in the art. "Fragments" as used herein represent polypeptides having preferably at least 10 amino acids, more preferably at least 15, at least 17 amino acids, or at least 20 amino acids, even more preferably at least 25 amino acids, or at least 37 or 40 amino acids, and more preferably at least 50, or 100, or 150, or 200, or 250, or 300, or 350, or 400, or 450, or 500 amino acids in length.

[0287] The invention is illustrated by way of non-limiting example with reference to the following figures.

[0288] Figure 1 :Design of ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ (CAR.CD123-T) retroviral vector

[0289] The expression cassette of CAR.CD123 is shown in cartoon. The bicistronic vector is composed of two transgenics, i.e. ΔCD19 and CARCD123. ΔCD19 is composed of the (extracellular and transmembrane) domains of CD19 markers. It is connected to CARCD123 via a 2A peptide.

[0290] The scFv of CD123 and the CD8a spacer-transmembrane domain and the costimulatory domain represented by 4-1BB and the signal transduction domain CD3-ζ chain (ζ) were cloned in the frame. ΔCD34 was added as a traceable marker.

[0291] Figure 2.ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ is stably expressed on T cells. (AB) After long-term in vitro culture (up to 25 days) expansion, ΔCD34 (A) or ΔCD19 expression (B) on T cells genetically modified by CAR.CD123 was analyzed by flow cytometry, and stable expression of CAR.CD123 on the surface of T cells was observed. The histogram (AB) represents the mean + standard deviation (SD) of 8 HDs, monitoring its CAR expression over time (on the +3rd day and the +25th day). (C) CAR.CD123 is efficiently expressed in T cells after retroviral transduction. Representative donor non-transduced (NT) T cells (C, upper figure) and transduced (C, lower figure) are shown. CAR expression was evaluated by using anti-CD34 (PE) and CD19 (FITC) mAbs in combination with anti-CD3 (BV421).

[0292] Figure 3 : T cells expand multiple times over time. Untransduced T cells (NT-T) or T cells genetically modified by ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ (CAR.CD123-T) were counted weekly to monitor cell expansion. Each point is the average number of T cells from three donors ± SD.

[0293] Figure 4 : ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ is stably expressed on NK cells. (AB) After long-term in vitro culture (up to 25 days) amplification, flow cytometry analysis of the expression of ΔCD34 (A) or ΔCD19 (B) on NK cells genetically modified by CAR.CD123. The histogram represents the average of 8 HD+SDs for monitoring CAR expression. (C) CAR.CD123 is efficiently expressed in NK cells after retroviral transduction. Representative donor non-transduced (NT) NK cells (C, upper figure) and transduced (C, lower figure) are shown. CAR expression is assessed by using anti-CD34 (PE) and CD19 (FITC) mAbs in combination with anti-CD56 (BUV395).

[0294] Figure 5 : Innate cells expand multiple times over time. Untransduced innate cells (NT-innate cells) or innate cells genetically modified by ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ (CAR.CD123-innate cells) were counted weekly to monitor cell expansion. Each point is the average number of innate cells from three donors ± SD.

[0295] Figure 6: ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ is stably expressed on γδ T cells. CAR.CD123 was used to analyze the expression of ΔCD34 or ΔCD19 on genetically modified γδ T cells by flow cytometry. The first figure shows that representative samples are represented by pure γδ T cell populations without αβ T cells. Anti-TCR-γδ PE.CAR.CD123 is efficiently expressed in γδ T cells after retroviral transduction by using anti-TCRαβ (FITC) for immunophenotyping. Representative donor non-transduced (NT) γδ T cells (upper figure) and transduced (lower figure) are shown. CAR expression is evaluated by using anti-CD34 (PE) and CD19 (FITC) mAbs in combination with anti-CD3 (APC).

[0296] Figure 7 : Long-term anti-tumor activity of CAR.CD123-T cells. Three CD123+ cell lines (THP1, MOLM13, OCIAML3) and CD123- cells (KARPAS 299) have been co-cultured with untransduced T cells (NT-T) or T cells (CAR.CD123-T) genetically modified by ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ. The histogram shows the percentage of residual tumor cells after 5 days of co-culture with NT-T (light gray) or CAR.CD123-T cells (black columns) at a ratio of 1:1. The experiment was repeated in three times, and T cells came from three donors. The results are expressed as the mean ± SD of biological replicates. **p-value ≤ 0.01; ***p-value ≤ 0.001; ns = not significant.

[0297] Figure 8: Long-term anti-tumor activity of ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζT cells. Three CD123+ cell lines (THP1, MOLM13, OCI AML3) and CD123- cells (KARPAS 299) have been co-cultured with untransduced T cells (NT-T) or T cells (CAR.CD123-T) genetically modified by ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ from the same donor (n=8). Anti-tumor activity was assessed using a 6-day long-term co-culture assay. NT-T (light gray column) and CAR.CD123-T (black column) were co-cultured with effector: target (E: T) at a ratio of 1: 1. The histogram shows the percentage of residual tumor cells after 6 days of co-culture with NT-T or CAR.CD123-T cells at a ratio of 1: 1. Data from 8 donors are presented as mean ± SD. **p-value ≤ 0.01; ***p-value ≤ 0.001; ns = not significant.

[0298] Fig. 9 :Long-term anti-tumor activity of ACD19-2A-CAR.CD123-ACD34-CD8.4.1BB-ζNK cells. Three CD123+ cell lines (THP1, MOLM13, OCI AML3) and CD123- cells (KARPAS 299) have been co-cultured with untransduced NK cells (NT-NK) or NK genetically modified (CAR.CD123-NK). The histogram shows the percentage of residual tumor cells after 6 days of co-culture with NT-NK (light gray) or CAR.CD123-NK cells (black columns) at a ratio of 1:1. Data from 8 donors are expressed as mean ± SD. **p-value ≤ 0.01; ***p-value ≤ 0.001; ns = not significant.

[0299] Fig.10 : Long-term anti-tumor activity of ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζγδ T cells.Three CD123+ cell lines (THP1, MOLM3, OCI AML3) and CD123- cells (KARPAS 299) have been co-cultured with unmodified (NT-γδ) or γδ T cells (CAR.CD123-γδ) without ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ genetic modification with E: T ratio 1: 1 for 6 days. The histogram of a single donor shows the percentage of residual tumor cells (CD123+ or CD123-) after 6 days of co-culture with NT-γδ (light gray) or CAR.CD123-γδ (black column).

[0300] Fig.11 .Flow cytometric analysis of CD123 expression on bone marrow cells from patients with acute myeloid leukemia. The figure shows the surface expression of CD123 in primary human bone marrow (BM) acute myeloid leukemia (AML) patients at diagnosis (AML#1, AML#2). Flow cytometric analysis of CD123 expression was performed using anti-CD123 (BV421), CD34 (BB770), and CD38 (PeCy7).

[0301] Fig.12 Flow cytometry analysis of CD123 expression on bone marrow cells of healthy donors. The figure shows the low / negligible expression of CD123 on primary human bone marrow (BM) healthy donor (HD) samples. Flow cytometry analysis of CD123 expression was performed using anti-CD123 (BV421), CD34 (BB770) and CD38 (PeCy7).

[0302] Fig.13 .The off-tumour on-target effect of ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ cells was negligible. The histogram shows the comparison of the number of colony-forming units (CFU) in different experimental groups. CD34+ cells purified from BM of healthy donors (HD) were co-cultured with unmodified T and NK (NT-T, NT-NK) cells and modified T and NK cells (CAR.CD123-T, CAR.CD123-NK) at an effector:target ratio of 1:1. The co-culture experiment was plated in biological triplicates and hematopoietic colony scoring was performed after 14 days. (A) Absolute count score of BFU-E;

[0303] (B) Absolute count scores of GM-CFU. Data from 2 donors are expressed as mean ± SD.

[0304] Fig.14 The in vivo model of ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ cells showed activity in both T cells and NK cells. (A) Cartoon shows systemic infusion of THP-1-FF-Luc.GFP cells (0.01x10 6(A) A xenograft immunodeficient mouse model with 4 cells / mouse (100 μg / mL). At tumor establishment (day 3), mice were intravenously infused (day +3 and day +7). (B) Time course of in vivo bioluminescence imaging of treated NSG mice from day 3 to day 61. (C) 80-day overall survival (OS) probability of leukemic mice treated with two consecutive adoptive transfers: NT-T (red line; 4 mice), CAR.CD1213-T (black line; 4 mice), NT-NK (green line; 4 mice), and CAR.CD123-NK (blue line; 4 mice). (D) Statistical analysis of 80-day overall survival of leukemic mice treated with two consecutive adoptive transfers: NT-T, CAR.CD1213-T, NT-NK, and CAR.CD1123-NK cells.

[0305] Fig.15 .In vitro model of CD19 suicide gene activity in ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζT cells. Flow cytometric analysis of ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζT cells after CD19 selectively engaged the anti-hCD19-CD3 bite antibody. T cells from an exemplary donor were genetically modified by ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ. (AB) ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζT cells were characterized for CAR expression by Ab CD34-PE (A) and Ab CD19-FITC (B).

[0306] ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζT cells were exposed to a dose of 10 ng / ml of negative control (i.e., Ab CD19-β Gal bite) or a dose of 10 ng / ml anti-hCD19-CD3 bite for 24 hours. (CD) ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζT cells were not affected by the negative control (i.e., Ab CD19-β Gal bite engagement), as shown by flow cytometric analysis of CD34 (C) and CD19 (D). (EF) ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζT cells were selectively eliminated by anti-hCD19-CD3 bite engagement at the first evaluated time point (24 hours), as shown by flow cytometric analysis of CD34 (E) and CD19 (F).

[0307] Fig.16In vitro model of CD19 suicide gene activity in ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ NK cells. Flow cytometric analysis of ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ NK cells after CD19 selectively engages anti-hCD19-CD3 bite antibody in the presence of CD3+ T cells. (AC) NK cells from exemplary donors were genetically modified with ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ.

[0308] ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζNK cells are characterized by Ab CD56 PE-Cy7 and AbCD19-FITC (A) CAR expression. In the absence of CD3+T cells (control negative experimental conditions), ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζNK cells are exposed to a dose of 10ng / ml negative control (i.e. Ab CD19-β Galbite (B)) or a dose of 10ng / ml anti-hCD19-CD3bite (C) for 24 hours. (DF) NK cells from exemplary donors are genetically modified by ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ. ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζNK cells were characterized by Ab CD56 PE-Cy7 and Ab CD19-FITC (D) for CAR expression. In the presence of CD3+T cells, ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζNK cells were exposed to a dose of 10ng / ml negative control (ie, Ab CD19-β Gal bite (E)) or a dose of 10ng / ml anti-hCD19-CD3 bite (F) for 24 hours. In the presence of CD3+T cells, ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζNK cells were selectively eliminated by anti-hCD19-CD3 bite engagement.

[0309] Fig.17 In vivo model of CD19 suicide gene activity in ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ T cells. (A) Cartoon shows systemic infusion of ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ T cells (CAR.CD123-T cells; dose: 10x10 6 / mouse) xenograft immunodeficient mouse model. On days 0 (I dose), 4 (II dose) and 8 (III dose), effector cells were infused intravenously and IL2 was administered subcutaneously (twice a week; 1000U / mouse). 30 days after the first dose, when T cells showed significant expansion in vivo, NSG mice were treated with anti-hCD19-CD3 bite (5μg / mouse) for 5 consecutive days. (BC) FACS analysis of ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζT cells before (B) and after (C) administration of anti-hCD19-CD3bite in an exemplary bleed of NSG mice. CD19 expression analysis shows CAR+ cells (CD3+CD19+) before (B) and after (C) administration of anti-hCD19-CD3 bite.

[0310] Materials and methods

[0311] Design of CAR-CD123 plasmid (construct)

[0312] A clinical-grade “second” generation retroviral bicistronic vector, SFG, was designed to allow for the simultaneous expression of two transgenes, namely ΔCD19 and a cassette anti-CD123 single-chain variable fragment (scFv), a murine antibody derived from the IgG class (7G3), linked via a codon-optimized human CD8 spacer transmembrane domain to the codon-optimized signaling costimulatory domains 4-1BB (CD137) and CD3-ζ ( Figure 1 ).

[0313] In particular, ΔCD19 represents the extracellular domain of human CD19 connected to the transmembrane portion. The single-chain variable fragment (scFv) specific for CD123- is a fusion protein of 114 amino acids (aa) of the variable region of the immunoglobulin light chain (VL) connected to 118 amino acids of the immunoglobulin heavy chain (VH) through a bend (nex) of 8 amino acids (short linker peptide).

[0314] In particular, scFv 7G3 is cloned in frame with a codon-optimized CD34-derived 16 amino acid epitope (as a traceable marker), connected to a codon-optimized 21 amino acid human CD8-transmembrane domain (CD8aTM) by a 42 amino acid spacer (11 amino acids as a spacer plus 31 amino acids of the codon-optimized CD8 extracellular domain). The signal for the SFG:ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ (CAR.CD123-T) retroviral vector is transported from the extracellular part of the CD123scFv 7G3 to the intracellular part of the CD3-ζ chain (113 amino acids) through the costimulatory molecule: 4-1BB intracellular domain (42 amino acids).

[0315] Generation of eGFP-firefly-luciferase cell lines.

[0316] Retroviral vectors encoding eGFP-firefly-luciferase (eGFP-FFLuc) were used in selection experiments to label CD123-positive (CD123+) or CD123-negative (CD123-) tumor cells:

[0317] CD123+:

[0318] -Acute monocytic leukemia cell line THP-1

[0319] -Acute myeloid leukemia cell line MOLM-13

[0320] -Acute myeloid leukemia cell line OCI-AML-3

[0321] CD123-:

[0322] - Non-Hodgkin's Lymphoma (NHL) Karpas 299

[0323] Cell lines.

[0324] Acute monocytic leukemia cell line THP-1 cells were obtained from LGC Standards-ATCC. Acute myeloid leukemia cell lines MOLM-13 and OCI-AML-3 were obtained from DSMZ. Non-Hodgkin's lymphoma (NHL) Karpas 299 was obtained from Sigma-Aldrich.

[0325] THP-1, MOLM-13 and Karpas 299 cell lines are maintained with RPMI 1640 culture medium. OCI-AML-3 is maintained with IMDM culture. Cell line is supplemented with 10% fetal bovine serum (FBS, Hyclone, Thermo Scientific, Pittsburgh, Pennsylvania) and 2mM GlutaMax (Invitrogen, California, USA). Cells are maintained at 37°C and contain 5%CO2 in a humidified atmosphere. The surface expression of mycoplasma and target antigen CD123 of all cell lines is regularly tested. All cell lines have been verified by STR analysis in the certified laboratory "BMRGenomics srl".

[0326] Retroviral supernatant

[0327] Transient retroviral supernatants were produced by co-transfecting 293T with the MoMLV gag / pol expression plasmid PeqPam3(-env), the RD114env expression plasmid RDF, and the SFG vector at a ratio of 2:3:3, respectively, for a total of 10 μg of DNA. Transfection was facilitated using GeneJuice reagent (Calbiochem). Supernatants were collected 2 and 3 days after transfection, filtered (using a 0.45-mm filter), snap-frozen, and stored in 5-ml aliquots at -80°C

[27] .

[0328] Isolation, generation and transduction of effector cells.

[0329] Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood (PB) or buffy coat obtained from healthy donors (OPBG Hospital, Rome, Italy) using lymphocyte separation medium (Eurobio; France) according to the rules established by the Institutional Review Board (IRB) of OPBG (approval of the ethics committee N°969 / 2015prot. N°669LB) after obtaining signed informed consent. T lymphocytes were activated with immobilized OKT3 (1 μg / ml, e-Bioscience Inc.; San Diego, CA, USA) and anti-CD28 (1 μg / ml, BD Biosciences, Europe) antibodies in the presence of a combination of recombinant human interleukin-7 (IL7, 10 ng / ml; R&D; USA)

[28] and recombinant human interleukin-15 (IL15, 5 ng / ml; R&D)

[29]

[30] . Activated T cells were transduced on day 3 using specific retroviral supernatants and specific cytokines in 24-well plates pre-coated with recombinant human RetroNectin (Takara-Bio. Inc; Japan). On day 5 after transduction, T cells were expanded in “CTL complete medium” containing 45% RPMI 1640 and 45% Crick’s medium (Sigma-Aldrich; USA), supplemented with 10% FBS and 2 mM Glutamax, and supplemented with specific cytokines twice a week

[31] .

[0330] Isolation, generation and transduction of NK or γδ-T cells.

[0331] Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats or leukapheresis from healthy donors by density gradient technique (Ficool-Histopaque (Eurobio; France); the healthy donors had signed written informed consent according to the rules set by the Institutional Review Board of OPBG (ethics committee approval N_969 / 2015prot.N_669LB). CD56+CD3- NK cells were isolated using the NK Isolation Kit (Miltenyi Biotec, Inc., San Diego, CA, USA) and expanded using the NK Cell Activation / Expansion Kit (Miltenyi Biotec, Inc., San Diego, CA, USA) and recombinant human interleukin 2 (IL2, 500 U / ml; R&D; USA) or recombinant interleukin 15 (IL15 10 U / ml; R&D; USA). Retroviral supernatants were used to transduce activated NK cells in 24-well plates pre-coated with recombinant human RetroNectin (Takara Biotech, Ltd.; Japan). Enriched NK cells were cultured in GMP-compliant medium (NK MACS Miltenyi Biotech, San Diego, CA, USA).

[0332] exist γδ-T cells were obtained from αβ- and CD19-depleted donors after cryopreservation in cell cryopreservation medium (Sigma-Aldrich) and expanded in NK MACS medium after activation using the Activation / Expansion Kit (Miltenyi Biotec, San Diego, CA, USA) as described above.

[0333] Patient details

[0334] For in vitro studies, primary BM samples were collected from 2 pediatric patients with acute myeloid leukemia (AML) at the time of diagnosis. In addition, BM samples were collected from one HD donor (OPBG Hospital, Rome, Italy). Signed informed consent was obtained according to the rules established by the Institutional Review Board (IRB) of OPBG (Ethics Committee Approval N°969 / 2015prot. N°669LB)

[0335] Phenotypic analysis

[0336] The following mAbs were used: CD3, CD123, CD45, CD56, CD34, CD19 (all from BD Pharmigen, USA). Anti-CD34 Ab (R&D, USA) or Pierce recombinant biotinylated protein L

[32] (Thermo Fisher Scientific, USA) were used to evaluate the expression of CAR.CD123 on T cells. Samples were analyzed using BD LSRFortessa X-20 and Diva software (BD Biosciences). For each sample, a minimum of 20,000 events were analyzed.

[0337] Multiple amplification

[0338] Cells were counted weekly using the trypan blue method with a counting chamber.

[0339] In vitro anti-leukemic activity

[0340] For co-culture experiments, effector T lymphocytes and leukemia cell lines were seeded in 24-well plates. After 5 days of incubation at 37°C, tumor cells and T cells were collected and analyzed by FACS.

[0341] Positive selection of bone marrow CD34+ cells

[0342] CD34+ cells were isolated from bone marrow samples of healthy donors. First, CD34+ cells were magnetically labeled with CD34 microbeads (Miltenyi Biotec, San Diego, CA, USA). Flow cytometry analysis was performed using anti-CD34 (PE) to assess the purity of the CD34 positive fraction (CD34+).

[0343] Colony forming unit (CFU) assay

[0344] CD34+ and unmodified T and NK cells and

[0345] ΔCD19-2A-CAR.CD123-ΔCD34-CD8.4.1BB-ζ genetically modified T or NK cells were co-cultured at an E:T ratio of 1:1. After 4 hours of incubation, CD34+ cells were seeded into MethoCult GF H4434 medium. Following the manufacturer's instructions for CFU assay, cells were seeded at a density of 500 cells / dish. Each cell co-culture group was seeded in triplicate wells and the cultures were maintained at 37°C, 5% CO 2 and 20% O 2 After 14 days of culture, the number and type of CFU were determined.

[0346] Induction of apoptosis in CAR-123 cells

[0347] Bispecific antibodies for human CD19 and human CD3 (anti-hCD19-CD3 [Aurogene, catalog number: BIMAB-HCD19CD3) were added to NT or CAR.CD123T cells at specified concentrations and co-cultured with unmodified T cells and CAR.CD123-NK cells. The elimination of genetically modified CAR+ cells was assessed at 72 hours by FACS analysis of living cells.

[0348] In vivo xenogeneic leukemia model

[0349] NOD / SCID IL-2Rγ null (NSG) xenograft mice were infused with THP-1 cells to evaluate the antitumor effects of CAR-transduced NK or T cells in vivo. Mouse experiments were performed in accordance with ethical international, EU and national requirements and approved by the Italian Ministry of Health (N°88 / 2016-PR). THP-1 cells labeled with firefly luciferase (THP-1FF-Luc) (0.01x10 6 ) inoculated NGS mice (5 weeks old; Jackson Laboratory, USA). On days -3 and 7, mice were intravenously injected with two doses of 10x10 6 NT-NK or CAR.CD123-NK and 5x10 6 NT-T or CAR.CD123 T cells were cultured and subjected to weekly bioluminescence imaging (IVIS System, Perkin Elmer, USA). Signal quantification in photons / second was performed as described previously.

[0350] Bispecific antibodies against human CD19 and human CD3 in CAR.CD123-T cells induce apoptosis in vivo.

[0351] The activity of a bispecific antibody against human CD19 and human CD3 (anti-hCD19-CD3) was evaluated in an in vivo model of CAR.CD123 T cells. NGS mice were systemically (intravenously) infused with 3 doses of 20 × 10 6 The mice were treated with NT or CAR.CD123 T cells in combination with IL-2 (1000U). After T cell expansion, mice were treated with anti-hCD19-CD3 infusion every day for 5 consecutive days. Blood was collected from mice regularly before and after treatment for FACS analysis, and the expansion of CAR.CD123 T cells was monitored by anti-human-CD19 (FITC).

[0352] Statistical analysis

[0353] Statistical evaluation was performed using GraphPad Prism (GraphPad software). Differences between groups that produced p-values ​​<0.05 were considered significant. Mouse survival data were analyzed using Kaplan-Meier survival curves, and statistically significant differences were measured using the log-rank (Mantel-Cox) test. Samples without value were excluded from the analysis. Animals that died only after tumor implantation but before T or NK cell infusion were excluded. During the in vivo study, neither randomization nor blinding was performed. However, before control or genetically modified T (NK) cell infusion, mice were matched based on tumor signals in the control and treatment groups. In order to compare tumor growth over time, bioluminescent signal intensity was collected in a blinded manner. The bioluminescent signal intensity was logarithmically transformed and then compared using a t-test for two samples.

[0354] result

[0355] Compared with the known CAR-CD123 (Table 2), the above-mentioned CAR.CD123-4.1BB-ζ sequence according to the present invention provides unexpected advantages, such as:

[0356] Introducing ΔCD19 as a selectable marker and inducible suicide gene ( Figure 1 );

[0357] In addition, the inventors demonstrated high levels of stable CAR-CD123 expression in both T cells and innate cells (NK cells or γ / δ T cells) ( Figure 2 , 4 and 6).

[0358] Compared with control non-transduced (NT) T-cells ( Figure 3 ) or NK cells ( Figure 5 ), the ΔCD19-2A-CAR.CD123-4.1BB-ζ retroviral vector did not induce any significant proliferation changes in the genetically modified cells, demonstrating that the construct had no toxic or fratricide activity in the cellular platforms of abT, NK and gdT cells.

[0359] The in vitro results described herein show that polyclonal ΔCD19-2A-CAR.CD123-4.1BB-ζT cells / NK cells or γ / δT cells modified according to the present invention can effectively inhibit the proliferation of CAR-CD123 T cells ( Figure 8 ), NK cells( Fig. 9 ) and γ / δ T cells ( Fig.10 ) can very efficiently eliminate CD123+ tumors in long-term co-cultures ( Figure 7 ).

[0360] More specifically, the supernatant obtained by SFG retroviral vector can efficiently transduce activated T cells ( Figure 2 ), NK cells( Figure 4 ) or γ / δ T cells ( Figure 6 ) and have very high transduction levels. In addition, expression of retroviral vectors in T cells ( Figure 2 AB) or NK cells ( Figure 4 AB) is stable over time and does not interfere with cell proliferation ( Figure 3 and 5 ). A CD34-derived epitope was introduced into the construct as a traceable marker in γ / δ T cells (γ / δCD3+CD34+) in an in vitro model ( Figure 2 C. Figure 4 C and Figure 6 ), genetically modified T cells (α / β CD3+CD34+), NK cells (CD56+CD34+) can be easily tracked. The in vivo model of AML xenograft mice confirmed the in vitro results of CAR-CD123 efficacy ( Fig.14 ). FACS analysis confirmed that compared with progenitor cells derived from the bone marrow of healthy donors ( Fig.12 ), only leukemic stem cells from AML patients expressed high levels of CD123 ( Fig.11 In addition, absolute counts of colony-forming units ( Fig.13 ), we confirmed that the present invention is characterized in that CAR-CD123 has a high specificity for targeting leukemia cells and has negligible tumor off-target effects on hematopoietic stem cells derived from the bone marrow of healthy donors.

[0361] In addition, we demonstrated in vitro that CAR-CD123 T cells ( Fig.15 ) and CAR-CD123 NK cells ( Fig.16 ) to induce CD19 suicide gene activity. It should be noted that CAR-CD123 NK cells can only be expressed in the presence of T cells ( Fig.16 DF) were eliminated by anti-bCD19-CD3 bite treatment, demonstrating the high specificity and selectivity of this approach. As shown by FACS analysis, circulating CAR.CD123 T cells (CD3+CD19) ( Fig.17 BC), suicide gene activity has also been confirmed in in vivo models ( Fig.17 A).

[0362] The present invention also discloses the following items:

[0363] 1. A chimeric antigen receptor (CAR) molecule comprising, from N-terminus to C-terminus:

[0364] a) the extracellular domain and transmembrane domain of human CD19,

[0365] b) an antigen binding domain,

[0366] c) a spacer domain,

[0367] d) transmembrane domain,

[0368] e) cytoplasmic domain,

[0369] Preferably, wherein the CAR is a CD123-specific CAR (CD123 CAR) and the antigen binding domain comprises VH and / or VL from a monoclonal anti-CD123 antibody, more preferably, the antigen binding domain comprises a single-chain variable fragment (scFv) that specifically binds to CD123.

[0370] 2. The CAR molecule of item 1, wherein the extracellular and transmembrane domains of human CD19 comprise or consist of the amino acid sequence of SEQ ID NO: 1

[0371] A sequence having at least 80% identity, preferably it comprises or consists of SEQ ID NO:1.

[0372] 3. A CAR molecule as described in claim 1 or 2, wherein the antigen binding domain comprises a 7G3 VL sequence:

[0373] A sequence having at least 80% identity, the VH and / or VL are optionally humanized, wherein the VH and VL are preferably separated by a first linker, more preferably the scFv comprises a VL comprising SEQ ID NO: 4 and a VH comprising SEQ ID NO: 5.

[0374] 4. A CAR molecule as described in any of the preceding items, wherein the spacer comprises the extracellular region of the CD8α chain, preferably comprising or consisting of a sequence having at least 80% identity with amino acids 12-42 (IASQPLSLRPEACRPAAGGAVHTRGLDFACD) of SEQ ID NO: 7, and / or the spacer comprises or consists of a sequence having at least 80% identity with amino acids 1-11 (PAPRPPTPAPT) of SEQ ID NO: 7, more preferably the spacer comprises SEQ ID NO: 7.

[0375] 5. A CAR molecule as described in any of the preceding items, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD8, α, β or ζ chain of a T-cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD15, preferably it comprises the transmembrane domain of CD8, more preferably it comprises or consists of a sequence having at least 80% identity with SEQ ID NO: 8 (IYIWAPLAGTCGVLLLSLVIT), even more preferably it comprises SEQ ID NO: 8.

[0376] 6. A CAR molecule as described in any of the preceding items, wherein the cytoplasmic domain comprises a region of a CD8a cytoplasmic portion, a 4-1BB co-stimulatory domain, and a CD3-ζ chain, preferably, the cytoplasmic domain comprises a linker between the CD8a cytoplasm and the 4-1BB co-stimulatory domain, more preferably:

[0377] - said CD8a cytoplasm comprises or consists of a sequence having at least 80% identity to SEQ ID NO: 9 (LYCNHRNRRRVCKCPR), even more preferably it comprises SEQ ID NO: 9, and / or

[0378] - the co-stimulatory signal transduction domain CD137 (4-1BB) comprises or consists of a sequence having at least 80% identity to SEQ ID NO: 10 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), even more preferably it comprises SEQ ID NO: 10, and / or

[0379] - the CD3-ζ chain comprises or consists of SEQ ID NO: 11

[0380] having a sequence composition of at least 80% identity, even more preferably comprising SEQ ID NO: 11, and / or

[0381] -The sequence encoding the linker between the cytoplasmic CD8a and the co-stimulatory signal transduction domain CD137 (4-1BB) consists of 6 nucleotides.

[0382] 7. The CAR molecule according to any one of the preceding items further comprises:

[0383] i. Between the extracellular domain and transmembrane domain of human CD19 and the antigen binding domain:

[0384] - a cleavable linker, preferably a 2A peptide or an IRES, and / or

[0385] -Signal peptide and / or

[0386] - connectors, and / or

[0387] ii. Between the antigen binding domain and the spacer domain.

[0388] - connectors and / or

[0389] - traceable markers,

[0390] Preferably:

[0391] - said 2A peptide comprises or consists of a sequence having at least 80% identity to SEQ ID NO: 2 (RGRGRGSLLTCGDVEENPGP), even more preferably it comprises SEQ ID NO: 2, and / or

[0392] - said signal peptide comprises or consists of a sequence having at least 80% identity to SEQ ID NO: 51 (MEFGLSWLFLVAILKGVQC), even more preferably it comprises SEQ ID NO: 3, and / or

[0393] - said traceable marker comprises or consists of a sequence having at least 80% identity to SEQ ID NO:6 (ELPTQGTFSNVSTNVS), even more preferably it comprises SEQ ID NO:6.

[0394] 8. A CAR molecule as described in any of the preceding items, wherein the sequence encoding at least one of the following is codon-optimized: the extracellular domain and transmembrane domain of human CD19, a traceable marker, a spacer domain, a transmembrane domain, and a co-stimulatory domain.

[0395] 9. The CAR molecule of any of the preceding items, wherein the molecule comprises:

[0396] From N-terminus to C-terminus:

[0397] a) the extracellular domain and the transmembrane domain of human CD19, as defined in any one of items 1-2 and 8,

[0398] b) a cleavable linker as defined in item 7,

[0399] c) a signal peptide as defined in item 7,

[0400] d) an antigen binding domain as defined in any one of items 1 and 3,

[0401] e) A traceable marker as defined in items 7-8.

[0402] f) a spacer domain as defined in any one of items 1 and 4 and 8

[0403] g) a transmembrane domain as defined in any one of items 1 and 5 and 8

[0404] h) a cytoplasmic domain as defined in any one of items 1 and 6 and 8,

[0405] Preferably, the CAR molecule comprises or consists of a sequence having at least 80% identity to SEQ ID NO: 12, more preferably it comprises the sequence of SEQ ID NO: 12.

[0406] 10. An isolated nucleic acid molecule encoding a chimeric antigen receptor molecule as described in any of the preceding items, wherein the isolated nucleic acid molecule is preferably operably linked to an expression control sequence, and more preferably the molecule comprises at least one sequence having at least 80% identity to one of the following sequences: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,

[0407] Even more preferably, the molecule comprises or consists of a sequence that is at least 80% identical to SEQ ID NO:29.

[0408] 11. A vector comprising the isolated nucleic acid molecule as described in item 10, preferably an expression vector, more preferably the vector comprises an exogenous promoter that controls the expression of the CAR, preferably the vector is a DNA, RNA vector, plasmid, lentiviral vector, adenoviral vector, retroviral vector or non-viral vector.

[0409] 12. An engineered immune cell, preferably a T cell, more preferably an α / β and γ / δ T cell, or a NK cell, or a NK-T cell or a combination thereof, even more preferably of human origin, comprising the vector as described in item 11, or the isolated nucleic acid molecule as described in item 10, or the CAR expressed on the cell surface as described in any one of items 1-9.

[0410] 13. A pharmaceutical composition comprising the isolated nucleic acid molecule as described in item 10, or the vector as described in item 11, or the cell as described in item 12, and at least one pharmaceutically acceptable excipient and / or adjuvant.

[0411] 14. The CAR molecule as described in any one of items 1-9, the isolated nucleic acid molecule as described in item 10, the vector as described in item 11, the cell as described in item 12 or the pharmaceutical composition as described in item 13 is used for medical purposes, preferably for the treatment of CD123+ cancer, more preferably for the treatment of acute myeloid leukemia or B-lymphocytic leukemia, blastic plasmacytoid dendritic neoplasm (BPDCN), myelodysplastic syndrome or hairy cell leukemia, or before, after or during hematopoietic stem cell transplantation.

[0412] 15. The extracellular domain and transmembrane domain of human CD19, as defined in any one of items 1-2 and 8, are used as death-inducing agents in cells genetically modified with the extracellular domain and transmembrane domain of human CD19, after the cells are exposed to anti-CD19 antibodies (including bite antibodies, such as blinatumomab).

[0413] References

[0414] 1. Shi, M., et al., CD123 is a novel biomarker for diagnosis and treatment of leukemia. Cardiovasc Hematol Disord Drug Targets, 2019.

[0415] 2. Sapienza, MR, et al., Blastic Plasmacytoid Dendritic Cell Neoplasm: State of the Art and Prospects. Cancers (Basel), 2019. 11(5).

[0416] 3. Stevens, BM, et al., CD123 CART cells for the treatment of myelodysplastic syndrome. Exp Hematol, 2019. 74: p. 52-63e3.

[0417] 4. Salem, DA, et al., Differential Expression of CD43, CD81, and CD200 in Classic Versus Variant Hairy Cell Leukemia. Cytometry B Clin Cytom, 2019.

[0418] 5. Forman, SJ and JM Rove. The myth of the second remission of acute leukemia in the adult. Blood, 2013. 121(7): p. 1077-82.

[0419] 6. Majeti, R., Monoclonal antibody therapy directed against human acute myeloid leukemia stem cells. Oncogene, 2011. 30(9): p. 1009-19.

[0420] 7. Korpelainen, EI, et al. Interferon-gamma upregulates interleukin-3 (IL-3) receptor expression in human endothelial cells and synergizes with IL-3 in stimulating major histocompatibility complex class II expression and cytokine production. Blood, 1995. 86(1): p. 176-82.

[0421] 8. Li, K., et al., Expression of complement components, receptors and regulators by human dendritic cells. Mol Immunol, 2011. 48(9-10): p.1121-7.

[0422] 9. Taussig, DC, et al., Hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia. Blood, 2005. 106(13): p. 4086-92.

[0423] 10. Munoz, L., et al., Interleukin-3 receptor alpha chain (CD123) is widely expressed in hematologic malignancies. Haematologica, 2001. 86(12): p.1261-9.

[0424] 11. Testa, U., et al., Elevated expression of IL-3Ralpha in acute myelogenous leukemia is associated with enhanced blast proliferation, increased cellularity, and poor prognosis. Blood, 2002. 100(8): p. 2980-8.

[0425] 12. Graf, M., et al., Expression and prognostic value of hemopoietic cytokinereceptors in acute myeloid leukemia (AML): implications for future therapeutic strategies. (Eur J Haematol, 2004. 72(2): p.89-106.

[0426] 13. Vergez, F., et al., High levels of CD34+CD38low / -CD123+blasts are predictive of an adverse outcome in acute myeloidleukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study. Haematologica, 2011. 96(12): p.1792-8.

[0427] 14. Tettamanti, S., et al. Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor. Br J Haematol, 2013. 161(3): p. 389-401.

[0428] 15. Smith, BD, et al., First-in Man, Phase 1 Study of CSL362 (Anti-IL3Ra / Anti-CD123 Monoclonal Antibody) in Patients with CD123+ Acute Myeloid Leukemia (AML) in CR at High Risk for Early Relapse. Blood, 2014. 124(21): p. 120-120.

[0429] 16. Testa, U., E. Pelosi, and A. Frankel. CD 123 is a membrane biomarker and a therapeutic target in hematologic malignancies. Biomark Res, 2014. 2(1): p. 4.

[0430] 17. Mardiros, A., et al., T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia. Blood, 2013. 122(18): p. 3138-48.

[0431] 18. Gill, S., et al., Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells. Blood, 2014. 123(15): p. 2343-54.

[0432] 19. Thokala, R., et al., Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+Tumors. PLoS One, 2016. 11(8): p. e0159477.

[0433] 20. Schroeder, HW, Jr. and L. Cavacini. Structure and function of immunoglobulins. J Allergy Clin Immunol, 2010. 125(2 Suppl 2): ​​p. S41-52.

[0434] 21. Huston, JS, et al., Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA, 1988. 85(16): p.5879-83.

[0435] 22. Bird, RE, et al., Single-chain antigen-binding proteins. Science, 1988. 242(4877): p. 423-6.

[0436] 23. Milone, MC, et al., Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther, 2009. 17(8): p. 1453-64.

[0437] 24. Batzer, MA, JE Carlton and PL Deininger. Enhanced evolutionary PCR using oligonucleotides with inosine at the 3′-terminus. Nucleic Acids Res, 1991. 19(18): p. 5081.

[0438] 25. Ohtsuka, E., et al., An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions. J Biol Chem, 1985. 260(5): p. 2605-8.

[0439] 26. Rossolini, GM, et al. Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information. Mol Cell Probes, 1994. 8(2): p. 91-8.

[0440] 27. Pule, MA, et al. A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells. Mol Ther, 2005. 12(5): p. 933-41.

[0441] 28. Perna, SK, et al., Interleukin-7 mediates selective expansion of tumor-redirected cytotoxic T lymphocytes (CTLs) without enhancement of regulatory T-cell inhibition. Clin Cancer Res, 2014. 20(1): p. 131-9.

[0442] 29. Perna, SK, et al. Interleukin 15 provides relief to CTLs from regulatory T cell-mediated inhibition: implications for adoptive T cell-based therapies for lymphoma. Clin Cancer Res, 2013. 19(1): p. 106-17.

[0443] 30. Cieri, N., et al., IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors. Blood, 2013. 121(4): p. 573-84.

[0444] 31. Zheng, Z., N. Chinnasamy and R. A. Organ. Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry. J Transl Med, 2012. 10: p. 29.

[0445] 32. Klement, M., et al., Effect of linker flexibility and length on the functionality of a cytotoxic engineered antibody fragment. J Biotechnol, 2015. 199: p. 90-7. Sequence Listing <110> Bambino Gesu' Children's Hospital <120> CAR-CD123 vector and its use <130> PCT 143735 <150> EP 19188360.2 <151> 2019-07-25 <160> 51 <170> PatentIn version 3.5 <210> 1 <211> 333 <212> PRT <213> Homo sapiens <400> 1 Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met 1 5 10 15 Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp 20 25 30 Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln 35 40 45 Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu 50 55 60 Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile 65 70 75 80 Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu 85 90 95 Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110 Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125 Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140 Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala 145 150 155 160 Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro 165 170 175 Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro 180 185 190 Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser 195 200 205 Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser 210 215 220 Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp 225 230 235 240 Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255 Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265 270 Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly 275 280 285 Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu 290 295 300 Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg 305 310 315 320 Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe 325 330 <210> 2 <211> 20 <212> PRT <213> artificial sequence <220> <223> 2A peptide <400> 2 Arg Gly Arg Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu 1 5 10 15 Asn Pro Gly Pro 20 <210> 3 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Signal <400> 3 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Ser Arg 20 <210> 4 <211> 114 <212> PRT <213> Artificial sequence <220> <223> 7G3 VL <400> 4 Asp Phe Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 7G3 VH <400> 5 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val 115 <210> 6 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Traceable markers <400> 6 Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser 1 5 10 15 <210> 7 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Spacer <400> 7 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 1 5 10 15 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 20 25 30 His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 <210> 8 <211> twenty one <212> PRT <213> Homo sapiens <400> 8 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> 9 <211> 16 <212> PRT <213> Homo sapiens <400> 9 Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg 1 5 10 15 <210> 10 <211> 42 <212> PRT <213> Homo sapiens <400> 10 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 11 <211> 112 <212> PRT <213> Homo sapiens <400> 11 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 12 <211> 869 <212> PRT <213> Artificial Sequence <220> <223> ΔCD19-2A-CAR.CD123-4.1BB-ζ CAR <400> 12 Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met 1 5 10 15 Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp 20 25 30 Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln 35 40 45 Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu 50 55 60 Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile 65 70 75 80 Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu 85 90 95 Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110 Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125 Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140 Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala 145 150 155 160 Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro 165 170 175 Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro 180 185 190 Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser 195 200 205 Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser 210 215 220 Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp 225 230 235 240 Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255 Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265 270 Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly 275 280 285 Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu 290 295 300 Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg 305 310 315 320 Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Arg Gly Arg 325 330 335 Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly 340 345 350 Pro Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys 355 360 365 Gly Val Gln Cys Ser Arg Asp Phe Val Met Thr Gln Ser Pro Ser Ser 370 375 380 Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser 385 390 395 400 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 405 410 415 Leu Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser 420 425 430 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly 435 440 445 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala 450 455 460 Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly 465 470 475 480 Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Ser Gly Gly Gly Gly 485 490 495 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 500 505 510 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 515 520 525 Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 530 535 540 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 545 550 555 560 Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 565 570 575 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 580 585 590 Thr Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 595 600 605 Gly Thr Leu Val Thr Val Ser Ala Gly Ser Glu Leu Pro Thr Gln Gly 610 615 620 Thr Phe Ser Asn Val Ser Thr Asn Val Ser Pro Ala Pro Arg Pro Pro 625 630 635 640 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 645 650 655 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 660 665 670 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 675 680 685 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn 690 695 700 Arg Arg Arg Val Cys Lys Cys Pro Arg Val Asp Lys Arg Gly Arg Lys 705 710 715 720 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 725 730 735 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 740 745 750 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 755 760 765 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 770 775 780 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 785 790 795 800 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 805 810 815 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 820 825 830 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 835 840 845 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 850 855 860 Ala Leu Pro Pro Arg 865 <210> 13 <211> 999 <212> DNA <213> Homo sapiens <400> 13 atgccaccac ctcgcctgct gttctttctg ctgttcctga cacctatgga ggtgcgacct 60 gaggaaccac tggtcgtgaa ggtcgaggaa ggcgacaatg ccgtgctgca gtgcctgaaa 120 ggcacttctg atgggccaac tcagcagctg acctggtcca gggagtctcc cctgaagcct 180 tttctgaaac tgagcctggg actgccagga ctgggaatcc acatgcgccc tctggctatc 240 tggctgttca tcttcaacgt gagccagcag atgggaggat tctacctgtg ccagccagga 300 ccaccatccg agaaggcctg gcagcctgga tggaccgtca acgtggaggg gtctggagaa 360 ctgtttaggt ggaatgtgag tgacctggga ggactgggat gtgggctgaa gaaccgctcc 420 tctgaaggcc caagttcacc ctcagggaag ctgatgagcc caaaactgta cgtgtgggcc 480 aaagatcggc ccgagatctg ggagggagaa cctccatgcc tgccacctag agacagcctg 540 aatcagagtc tgtcacagga tctgacaatg gcccccgggt ccactctgtg gctgtcttgt 600 ggagtcccac ccgacagcgt gtccagaggc cctctgtcct ggacccacgt gcatcctaag 660 gggccaaaaa gtctgctgtc actggaactg aaggacgatc ggcctgccag agacatgtgg 720 gtcatggaga ctggactgct gctgccacga gcaaccgcac aggatgctgg aaaatactat 780 tgccaccggg gcaatctgac aatgtccttc catctggaga tcactgcaag gcccgtgctg 840 tggcactggc tgctgcgaac cggaggatgg aaggtcagtg ctgtgacact ggcatatctg 900 atcttttgcc tgtgctccct ggtgggcatt ctgcatctgc agagagccct ggtgctgcgg 960 agaaagagaa agagaatgac tgacccaaca agaaggttt 999 <210> 14 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> 2A Peptide <400> 14 cgcggccgcg gccgagggag cctgctgaca tgtggcgatg tggaggaaaa cccaggacca 60 <210> 15 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Signal Peptide <400> 15 atggagtttg gactttcttg gttgtttttg gtggcaattc tgaagggtgt ccagtgtagc 60 agg 63 <210> 16 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> 7G3 VL <400> 16 gactttgtaa tgacccaatc tccaagctct cttacggtaa cggcaggaga gaaagtcacc 60 atgtcatgta aatccagtca atccctcttg aactcaggca accagaaaaa ttatcttacg 120 tggtatcttc aaaagccggg gcaaccccca aaactcctga tctactgggc atcaaccagg 180 gagtccggcg tccccgaccg ctttacgggt agtggaagtg gaaccgattt tacccttact 240 atcagcagcg tacaagcgga agacttggct gtgtattatt gtcaaaatga ttattcatat 300 ccctatactt tcggtggagg gactaaactt gaaattaaac ga 342 <210> 17 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Bend <400> 17 ggcggaggaa gcggaggtgg gggc 24 <210> 18 <211> 354 <212> DNA <213> Artificial sequence <220> <223> VH 7G3 <400> 18 gaagtccagt tgcaacaatc tggccctgag ttggtaaagc ccggagcctc tgtgaagatg 60 agttgtaagg cttcagggta tacatttaca gactattata tgaaatgggt caaacaatct 120 cacggtaaat ccttggagtg gattggcgat attatcccga gtaacggtgc cacgttctac 180 aaccagaagt tcaagggcaa ggcaacactg acggtagacc gcagcagcag cacggcgtat 240 atgcacctga actcattgac ttcagaggat agtgcagttt actactgtac tcggagtcat 300 ttgctgagag cgagttggtt cgcctattgg ggtcagggca cactcgttac cgta 354 <210> 19 <211> 12 <212> DNA <213> Artificial sequence <220> <223> Ligation <400> 19 tctgcaggat cc 12 <210> 20 <211> 48 <212> DNA <213> Homo sapiens <400> 20 gaacttccta ctcaggggac tttctcaaac gttagcacaa acgtaagt 48 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Spacer <400> 21 cccgccccaa gaccccccac a 21 <210> 22 <211> 126 <212> DNA <213> Homo sapiens <400> 22 cccgccccaa gaccccccac acctgcgccg accattgctt ctcaacccct gagtttgaga 60 cccgaggcct gccggccagc tgccggcggg gccgtgcata caagaggact cgatttcgct 120 tgcgac 126 <210> 23 <211> 63 <212> DNA <213> Homo sapiens <400> 23 atctatatct gggcacctct cgctggcacc tgtggagtcc ttctgctcag cctggttatt 60 act 63 <210> 24 <211> 48 <212> DNA <213> Homo sapiens <400> 24 ctgtactgta atcaccggaa tcgccgccgc gttgtaagt gtcccagg 48 <210> 25 <211> 126 <212> DNA <213> Homo sapiens <400> 25 aaacggggca gaagaaact cctgtatata ttcaacaac catttatgag accagtacaa 60 actactcaag aggagatgg ctgtagctgc cgatttccag agagaga aggagatgt 120 failure 126 <210> 26 <211> 339 <212> DNA <213> Homo sapiens <400> 26 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgagagag gagtacgatg tttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagatagat ggcggaggcc tacagtgaga ttgggatga aggcgagcgc 240 cggaggggca aggggcacga tggctttac cagggtctca gtacagccac caggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctag 339 <210> 27 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Connectors <400> 27 Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 28 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Connectors <400> 28 Ser Ala Gly Ser 1 <210> 29 <211> 2610 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding CD123 chimeric antigen receptor <400> 29 atgccaccac ctcgcctgct gttctttctg ctgttcctga cacctatgga ggtgcgacct 60 gaggaaccac tggtcgtgaa ggtcgaggaa ggcgacaatg ccgtgctgca gtgcctgaaa 120 ggcacttctg atgggccaac tcagcagctg acctggtcca gggagtctcc cctgaagcct 180 tttctgaaac tgagcctggg actgccagga ctgggaatcc acatgcgccc tctggctatc 240 tggctgttca tcttcaacgt gagccagcag atgggaggat tctacctgtg ccagccagga 300 ccaccatccg agaaggcctg gcagcctgga tggaccgtca acgtggaggg gtctggagaa 360 ctgtttaggt ggaatgtgag tgacctggga ggactgggat gtgggctgaa gaaccgctcc 420 480. tctgaaggcc caagttcacc ctcagggaag ctgatgagcc caaaactgta cgtgtgggcc aaagatcggc ccgagatctg ggaggggaga cctccatgcc tgccacctag agacagcctg 540 aatcagagtc tgtcacagga tctgacaatg gcccccgggt ccactctgtg gctgtcttgt 600. ggagtcccac ccgacagcgt gtccagaggc cctctgtcct ggacccacgt gcatcctaag 660 gggccaaaaa gtctgctgtc actggaactg aaggacgatc ggcctgccag agacatgtgg 720 gtcatggaga ctggactgct gctgccacga gcaaccgcac aggatgctgg aaaatactat 780 tgccaccggg gcaatctgac aatgtccttc catctggaga tcactgcaag gcccgtgctg 840 tggcactggc tgctgcgac cggaggatgg aaggtcagtg ctgtgacact ggcatatctg 900 atcttttgcc tgtgctccct ggtgggcatt ctgcatctgc agagagccct ggtgctgcgg 960 1020. 1020. 1020. 1020. 1020. 1020. 1020. 1020. 1020. 1020 ctgctgacat gtggcgatgt ggaggaaaac ccaggaccaa tggagtttgg actttcttgg 1080 ttgtttttgg tggcaattct gaagggtgtc cagtgtagca gggactttgt aatgacccaa 1140 tctccaagct ctcttacggt aacggcagga gagaaagtca ccatgtcatg taaatccagt 1200 caatccctct tgaactcagg caaccagaaa aattatctta cgtggtatct tcaaaagccg 1260 gggcaacccc caaaactcct gatctactgg gcatcaacca gggagtccgg cgtccccgac 1320 cgctttacgg gtagtggaag tggaaccgat tttaccctta ctatcagcag cgtacaagcg 1380 gaagacttgg ctgtgtatta ttgtcaaaat gattattcat atccctatac tttcggtgga 1440 gggactaaac ttgaaattaa acgaggcgga ggaagcggag gtgggggcga agtccagttg 1500 caacaatctg gccctgagtt ggtaaagccc ggagcctctg tgaagatgag ttgtaaggct 1560 tcagggtata catttacaga ctattatatg aaatgggtca aacaatctca cggtaaatcc 1620 ttggagtgga ttggcgatat tatcccgagt aacggtgcca cgttctacaa ccagaagttc 1680 aagggcaagg caacactgac ggtagaccgc agcagcagca cggcgtatat gcacctgaac 1740 tcattgactt cagagtag tgcagtttac tactgtactc ggagtcattt gctgagagcg 1800 agttggttcg cctattgggg tcagggcaca ctcgttaccg tatctgcagg atccgaactt 1860 cctactcagg ggactttctc aaacgttagc aaaacgtaa gtcccgcccc aagacccccc 1920 acacctgcgc cgaccattgc ttctcaaccc ctgagtttga gacccgaggc ctgccggcca 1980 gctgccggcg gggccgtgca tacaagga ctcgatttcg cttgcgacat ctatatctgg 2040 gcacctctcg ctggcacctg tggagtcctt ctgctcagcc tggttattac tctgtactgt 2100 aatcaccgga atcgccgccg cgtttgtaag tgtcccaggg tcgaaacg gggcagaaag 2160 2220 gatggctgta gctgccgatt tccagaagaa gaaggag gatgtgaact gagagtgaag 2280 2340 ctcaatctag your hand ggagtacgat gttttggaca agagacgtgg ccgggaccct 2400 gagatggggg gaaagccgag aaagaac cctcaggaag gcctgtacaa tgaactgcag 2460 aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 2520 aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 2580 cttcacatgc aggccctgcc ccctcgctag 2610 <210> 30 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> scFv <400> 30 Asp Phe Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Gln Gln 115 120 125 Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Lys 145 150 155 160 Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser 165 170 175 Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu 180 185 190 Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr Met His Leu Asn Ser Leu 195 200 205 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser His Leu Leu 210 215 220 Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 <210> 31 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Amino acids 12-42 of SEQ ID NO: 7 <400> 31 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 1 5 10 15 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 20 25 30 <210> 32 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Amino acids 1-11 of SEQ ID NO:7 <400> 32 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 1 5 10 <210> 33 <211> 229 <212> PRT <213> House mouse (Mus musculus) <400> 33 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 34 <211> 222 <212> PRT <213> Homo sapiens <400> 34 Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys 1 5 10 15 Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn 20 25 30 Gln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val 35 40 45 Val Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu 50 55 60 Gln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg 65 70 75 80 Cys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala 85 90 95 Cys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp 100 105 110 Lys Gln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp 115 120 125 Glu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp 130 135 140 Thr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys 145 150 155 160 Glu Glu Ile Pro Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly 165 170 175 Ser Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu 180 185 190 Gln Asp Leu Ile Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met 195 200 205 Gly Ser Ser Gln Pro Val Val Thr Arg Gly Thr Thr Asp Asn 210 215 220 <210> 35 <211> 39 <212> PRT <213> Homo sapiens <400> 35 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro 35 <210> 36 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Hinge CH3 <400> 36 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Gly Gln Pro Arg 1 5 10 15 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 20 25 30 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 35 40 45 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 50 55 60 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 65 70 75 80 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 85 90 95 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 100 105 110 Leu Ser Leu Ser Leu Gly Lys 115 <210> 37 <211> 27 <212> PRT <213> Homo sapiens <400> 37 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 38 <211> 41 <212> PRT <213> Homo sapiens <400> 38 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 39 <211> 36 <212> PRT <213> Homo sapiens <400> 39 Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly 1 5 10 15 Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 20 25 30 Leu Ala Lys Ile 35 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Core sequence motif <220> <221> misc_features <222> (2)..(2) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_features <222> (4) <223> Xaa can be any naturally occurring amino acid <400> 40 Asp Xaa Glu Xaa Asn Pro Gly Pro 1 5 <210> 41 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Peptides <400> 41 Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 42 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Peptides <400> 42 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 43 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Peptides <400> 43 Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 44 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Peptides <400> 44 Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <210> 45 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Signal peptide <400> 45 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 46 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Connectors <400> 46 Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser 1 5 10 <210> 47 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Connectors <400> 47 Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Pro Lys Pro Ser Thr Pro 1 5 10 15 Pro Gly Ser Ser 20 <210> 48 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Connectors <400> 48 Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 49 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Connectors <400> 49 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 50 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Connectors <400> 50 Gly Gly Gly Gly Ser Gly Gly 1 5 <210> 51 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Signal peptide <400> 51 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys

Claims

1. A chimeric antigen receptor (CAR) molecule comprising, from N-terminus to C-terminus: a) The extracellular domain and transmembrane domain of human CD19 (ΔCD19), which consists of the following sequence: SEQ ID NO:1(MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQ LTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAP GSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRF), b) an antigen binding domain consisting of a 7G3 VK sequence, wherein the 7G3 VK sequence is: DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYLQKPGQP PKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIKR (SEQ ID NO:4), and a 7G3 VH sequence, wherein the 7G3 VH sequence is: EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWI GDIIPSNGATFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTR SHLLRASWFAYWGQGTLVTV (SEQ ID NO:5), c) a traceable marker consisting of a CD34-derived epitope (ΔCD34) consisting of the following sequence: SEQ ID NO: 6 (ELPTQGTFSNVSTNVS), or a peptide derived from the CH2CH3 region spacer of a murine Ig (UNIPROTKB: P01861). The CH2CH3 regional spacer (UNIPROTKB:P01861) is: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:33), or a peptide derived from a nerve growth factor receptor (NGFR), wherein the nerve growth factor receptor (NGFR) is: KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN (SEQ ID NO:34), d) a spacer domain consisting of the extracellular region of the CD8 alpha chain or CD28 or hinge CH2-CH3 or hinge CH3, e) a transmembrane domain consisting of a transmembrane domain of a protein selected from the group consisting of CD8, the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD15, as well as f) a cytoplasmic domain consisting of: i.) a region of the cytoplasmic portion of CD8a consisting of SEQ ID NO:9 (LYCNHRNRRRVCKCPR), and ii.) a 4-1BB co-stimulatory domain consisting of SEQ ID NO: 10 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), or an OX40 sequence consisting of RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 39), or a CD28 cytoplasmic sequence consisting of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 38), and iii.) CD3-zeta chain, which consists of SEQ ID NO: 11 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR*), Wherein the CAR molecule is a CD123-specific CAR (CD123 CAR) molecule and the antigen binding domain comprises VL or VK and VH from a monoclonal anti-CD123 antibody.

2. The CAR molecule of claim 1, wherein the spacer domain consists of the following sequence: SEQ ID NO:7, or amino acids 12-42 of SEQ ID NO:7 (IASQPLSLRPEACRPAAGGAVHTRGLDFACD), and / or amino acids 1-11 of SEQ ID NO:7 corresponding to SEQ ID NO:32 (PAPRPPTPAPT), or SEQ ID NO:7, or IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:35), or ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK,(SEQ ID NO: 33); or ESKYGPPCPSCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK (SEQ ID NO: 36).

3. The CAR molecule of claim 1, wherein the transmembrane domain consists of the following transmembrane domains: CD8 or CD28 or consists of SEQ ID NO: 8 (IYIWAPLAGTCGVLLLSLVIT), or FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 37).

4. The CAR molecule of claim 1, wherein the traceable marker consists of SEQ ID NO: 6, the spacer domain consists of SEQ ID NO: 7, and / or the transmembrane domain consists of SEQ ID NO:

8.

5. The CAR molecule of claim 1, wherein the VL or VK and VH are separated by a first linker.

6. The CAR molecule of claim 1, wherein the VL and / or VK and / or VH are humanized.

7. The CAR molecule of any one of claims 1-6, wherein the antigen binding domain and the traceable marker are separated by a second linker, And / or wherein the cytoplasmic domain comprises a linker between the CD8a cytoplasmic domain and the 4-1BB co-stimulatory domain.

8. A CAR molecule as described in claim 7, wherein the sequence encoding the linker located between the CD8a cytoplasmic domain and the 4-1BB co-stimulatory domain consists of 6 nucleotides, or the linker is VD.

9. The CAR molecule of any one of claims 1-6, further comprising: - a cleavable linker, and / or -Signal peptide.

10. The CAR molecule of claim 9, wherein the signal peptide consists of SEQ ID NO: 3 (MEFGLSWLFLVAILKGVQCSR), or it consists of SEQ ID NO: 3; or it consists of MALPVTALLLPLALLLHAARP (SEQ ID NO: 45).

11. The CAR molecule of claim 9, wherein the cleavable linker is a 2A peptide or an internal ribosome entry site.

12. The CAR molecule of claim 9 or 11, wherein the cleavable linker is a 2A peptide consisting of SEQ ID NO: 2 (RGRGRGSLLTCGDVEENPGP); or a 2A self-cleaving peptide consisting of the core sequence motif of DxExNPGP (SEQ ID NO: 40), or a peptide consisting of AEGRGSLLTCGDVEENPGP (SEQ ID NO: 41); ATNFSLLKQAGDVEENPGP (SEQ ID NO: 42); QCTNYALLKLAGDVESNPGP (SEQ ID NO: 43) or VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 44).

13. The CAR molecule of any one of claims 1-6, wherein the sequence encoding at least one of the following is codon-optimized: the extracellular domain and transmembrane domain of the human CD19, the traceable marker, the spacer domain, the transmembrane domain, and the co-stimulatory domain.

14. The CAR molecule of any one of claims 1-6, comprising or consisting of from N-terminus to C-terminus: a) the extracellular domain and transmembrane domain of human CD19, b) a cleavable linker, c) a signal peptide, d) an antigen binding domain, e) traceable markers, f) a spacer domain, g) transmembrane domain, h) Cytoplasmic domain.

15. The CAR molecule of any one of claims 1-6, consisting of SEQ ID NO:

12.

16. An isolated nucleic acid molecule encoding the chimeric antigen receptor molecule of any one of claims 1-15.

17. The isolated nucleic acid molecule of claim 16, operably linked to an expression control sequence.

18. The isolated nucleic acid molecule of claim 16 or 17, wherein the molecule comprises at least one of the following sequences: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or The molecule comprises or consists of SEQ ID NO:

29.

19. The isolated nucleic acid molecule of claim 18, wherein the molecule comprises or consists of SEQ ID NO:

29.

20. A vector comprising the isolated nucleic acid molecule of any one of claims 16-19. The vector according to claim 20 , which is an expression vector.

22. The vector of claim 20 or 21, wherein the vector comprises an exogenous promoter that controls the expression of the CAR.

23. The vector of any one of claims 20-22, wherein the vector is a DNA, RNA vector, plasmid, lentiviral vector, adenoviral vector, retroviral vector or non-viral vector.

24. An engineered immune cell comprising a vector as described in any one of claims 20-23, or an isolated nucleic acid molecule as described in any one of claims 16-19, or expressing a CAR as described in any one of claims 1-15 on the cell surface.

25. The cell of claim 24, which is a T cell, an α / β or γ / δ T cell, a NK cell or a NK-T cell or a combination thereof.

26. The cell of claim 25, which is of human origin.

27. A pharmaceutical composition comprising the isolated nucleic acid molecule of any one of claims 16-19, or the vector of any one of claims 20-23, or the cell of any one of claims 24-26, and at least one pharmaceutically acceptable excipient and / or adjuvant.

28. A CAR molecule as described in any one of claims 1-15, an isolated nucleic acid molecule as described in any one of claims 16-19, a vector as described in any one of claims 20-23, a cell as described in any one of claims 24-26, or a pharmaceutical composition as described in claim 27 for the preparation of a medicament for treating CD123+ cancer.

29. A CAR molecule as described in any one of claims 1-15, an isolated nucleic acid molecule as described in any one of claims 16-19, a vector as described in any one of claims 20-23, a cell as described in any one of claims 24-26, or a pharmaceutical composition as described in claim 27 for the preparation of a medicament for the treatment of acute myeloid leukemia or B-lymphocytic leukemia, blastic plasmacytoid dendritic neoplasm (BPDCN), myelodysplastic syndrome or hairy cell leukemia, or for use before, after or during hematopoietic stem cell transplantation.

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