CAR-CD30 T cells for the treatment of CD30+ tumors

Abstract

The present invention relates to third-generation CAR-CD30 T cells for the treatment of CD30+ tumors such as lymphoid malignancies, leukemias, and solid tumors.

Description

Technical Field

[0001] The present invention relates to CAR-CD30 T cells for treating CD30+ tumors. In particular, the present invention relates to third-generation CAR-CD30 T cells for treating CD30+ tumors (such as lymphoid malignancies, leukemias, solid tumors). Background Art

[0002] It is well known that the prognosis for most patients with chemotherapy-refractory or multiply relapsed non-Hodgkin lymphoma (NHL) or Hodgkin lymphoma (HL) remains poor (1). Although allogeneic HSCT (allo-HSCT) offers the potential to cure patients of various lymphoma subtypes, transplant-related mortality remains high, and long-term sequelae, including chronic graft-versus-host disease (GVHD), can have a substantial negative impact on quality of life (2).

[0003] PD-1 blockade appears to be highly effective in lymphoma relapse after allo-HSCT but is often accompanied by rapid onset of severe and refractory GVHD (3). CAR-T cells are emerging as a novel treatment option for these patients.

[0004] CD30 (Ki-1) is a cell membrane protein derived from the tumor necrosis factor receptor superfamily 8 (TNFRSF8), whose normal expression is restricted to activated T cells and B cells. Among tumor cells, CD30 expression is most commonly found in lymphoid malignancies (Hodgkin lymphoma and non-Hodgkin lymphoma, CD30+ acute lymphoblastic leukemia (ALL) of T cell (4) or B cell lineage (5)). CD30 is also reported to be expressed in most adult non-lymphoid malignancies. According to published data, 24.5% of solid tumors are also CD30+, most notably germ cell tumors (myofibroblastic sarcoma (93%), embryonal carcinoma (77%), mesothelioma (77%), mixed germ cell tumor (GCT) (65%), head and neck cancer (24%), yolk sac tumor (18%), angiosarcoma (14%), pituitary adenoma (11%), and seminoma (6%)), raising the possibility of CD30-targeted therapy for other tumors (6).

[0005] While 90% of patients with early-stage HL can be cured with conventional treatment, only 70% of patients with advanced stage HL can be cured with standard treatments. For patients with relapsed HL, only half of patients are cured with standard salvage therapy (7).

[0006] Targeting CD30 with monoclonal antibodies has achieved great success in the treatment of Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). However, adverse events, mainly mediated by the toxic component of the chimeric antibodies, have led many patients to discontinue treatment. Targeting CD30 with T cells expressing CD30-specific chimeric antigen receptors (CARs) can reduce side effects and enhance anti-tumor activity.

[0007] Immunotherapy approaches targeting CD30 via CAR have demonstrated value in preclinical models (8,9) and have been confirmed in two different independent clinical trials (10,11), although the clinical benefit was suboptimal.

[0008] The first generation of anti-CD30 CAR T cells were developed in the 1990s, and preclinical studies demonstrated the ability of these cells to lyse CD30-expressing HL cell lines in vitro (12,13). Indeed, Epstein–Barr virus-specific cytotoxic T cells transduced with anti-CD30 CARs showed activity against CD30+ cancer cell lines in vitro and in vivo in mouse xenograft models and improved T cell persistence in vivo (8).

[0009] Notably, the presence of soluble CD30 did not attenuate cell lysis while clearing CD30+ lymphoma cells, suggesting that CD30 shedding from HL cells into the blood does not inhibit the efficacy of anti-CD30 CAR T cells in vivo (14).

[0010] In the first trial, inconsistent responses of lymphoma were observed, with most patients showing stable disease after multiple infusions of CAR T cells or no response at all. In general, lymph nodes showed better responses than extranodal lesions, lung lesions seemed to respond relatively poorly, and no infused CAR T cells persisted for more than 60 days after infusion. Of note, several clinical data (15,16) clearly showed that CAR-T cells that persist in vivo are associated with better outcomes for patients treated. As summarized in Table 1, in the first clinical trial described, the authors considered a lentiviral platform carrying a second-generation CAR, characterized by a single-chain variable region fragment (scFv) sequence derived from the AJ878606.1 hybridoma specific for the CD30 antigen, a co-stimulatory domain derived from human CD137 and the CD3ζ signaling domain (10) in the same reading frame.

[0011] The first open-label phase I clinical trial of anti-CD30 CAR T cells involved 18 patients with relapsed or refractory Hodgkin lymphoma that were genetically modified with a lentiviral vector to express the CD137 co-stimulatory domain. The 18 patients included 1 patient with primary cutaneous anaplastic large cell lymphoma (ALCL) and 17 patients with 3 different subtypes of Hodgkin lymphoma, most of which were tuberous sclerosis. 13 patients received 1 cycle of CAR T cell infusion, and 5 patients received 2 cycles.

[0012] Preliminary results of this study showed that 7 patients achieved partial remission and 6 patients had stable disease, with an objective response rate of 39% (10).

[0013] In the second trial, most patients received multiple infusions of CD30.CAR T cells, achieved temporary remission, and 6 weeks after infusion, CD30.CAR T cells could no longer be detected. As summarized in Table 1, in this clinical trial, the authors considered a retroviral platform carrying a second-generation CAR, characterized by a single-chain variable region fragment (scFv) sequence derived from the HRS3 hybridoma specific for the CD30 antigen, a costimulatory domain derived from human CD28, and a CD3ζ signaling domain in the same reading frame.

[0014] Table 1

[0015]

[0016] Specifically, in a second clinical trial, nine patients with relapsed / refractory HL or ALCL were infused with autologous T cells that were genetically modified with a retroviral vector to express a CD30-specific CAR (CD30.CAR-T) encoding the CD28 co-stimulatory intracellular domain. Notably, seven of these patients had brentuximab-refractory disease. Preliminary results from this study showed that three of the nine patients had a complete response and three had transient stable disease. In this study, the CAR-T cells persisted for <8 weeks, but tumor biopsies showed that they were able to effectively traffic T cells to the lymphoma site (11).

[0017] Both clinical trials showed that multiple CD30.CAR-T cell infusions were well tolerated. Host lymph node clearance before CAR-T infusion will help further improve the expansion of CAR T cells and their anti-tumor activity. More importantly, the retention of the CAR-T cells is associated with clinical response.

[0018] All these data suggest that CD30.CAR-T cells are safe and can lead to clinical responses in patients with HL, although further optimization of this therapy is necessary to achieve longer in vivo retention and higher antitumor control, especially at lymphoma relapse.

[0019] In particular, optimization of this approach should take into account that classical Hodgkin lymphoma (cHL) and anaplastic large T-cell lymphoma are characterized by only a few malignant Reed-Sternberg and Hodgkin cells (HRS) and a large number of inflammatory cells. These non-malignant cells produce soluble or membrane-bound molecules that are involved in tumor immune evasion. In addition, the chemokine environment produced by HL tumors can significantly affect which T cell subtypes migrate and accumulate in the tumor (17). In fact, HRS cells produce the chemokines TARC and MDC, which attract helper T cells (Th2) and regulatory T cells (Tregs), the latter expressing the receptor for these chemokines CCR4. The high presence of Tregs (and Th2 cells) in tumors, including HL, creates an unfavorable immune microenvironment by impairing the antitumor activity of the few cytotoxic effector T lymphocytes that are able to reach the tumor site. Forced expression of CCR4 on the CD30-specific chimeric antigen receptor (CAR-CD30) improves the migration of CAR-CD30 T-redirected effector T lymphocytes toward the TARC gradient produced by HL (9). HRS cells typically express high levels of PDL1 and produce immunosuppressive IL-10, TGF-β, galectin 1, and prostaglandin E2, which inhibit T cell effector cell function and induce apoptosis of activated Th1 and CD8+ T cells by inducing CD95 ligands. It has also been recently shown that IL-15 selectively supports the survival, proliferation, and effector cell function of Epstein–Barr virus (EBV)-CTLs in the presence of T-regs (18). In addition, it has recently been shown that CAR-CD30 T cells grown in IL-7 / IL-15 express higher levels of CXCR4 and CXCR3, which are chemokine receptors known to promote T cell migration to peripheral tissues (11).

[0020] In addition, preclinical studies have shown that third-generation CAR-T cells that bind CD28 and 4-1BB co-receptors may have better in vitro activation and proliferation capabilities than second-generation CAR-T cells carrying the CD28 signaling domain, and both cells show comparable efficacy in clearing CD19+ B cells in vivo (19), although this has never been demonstrated for CAR.CD30. Other CAR-CD30 T cells are known, such as those described in WO2017066122, WO2016134284, and CN107759699. For example, WO2017066122 compared the production of 5F11-28Z, AC10-28Z, and XmAb-28Z cells under IL2 conditions.

[0021] All CARs used were reported to have high levels of transduction efficiency, however, higher transduction efficiency was obtained using 5F11 compared to AC10 and XmAb (Table A), i.e., when cells were grown in IL2 for 7 days, 5F11 obtained a higher transduction percentage of CD8+CAR (80.7%) relative to AC10 (61.90%) or XmAb (64.70%). In addition, functional experiments were described regarding the production of IFNγ by co-culturing CAR-T cells with CD30+ tumors: SUDHL-1, HH, and BV173. When co-cultured with BV173, 5F11-28Z (grown in IL2) showed higher IFNγ production than AC10-28Z, i.e., Table D-1 shows that 5F11-28Z produced 3781pg / ml of IFNγ, while AC10-28Z produced 538pg / ml of IFNγ. In addition, 5F11-28Z produced 3534 pg / ml of IFNγ when co-cultured with the HDML-2 cell line.

[0022] In view of the above, therefore, there is a clear need to provide more CAR CD30 T cells that can overcome the shortcomings of the known CAR CD30 T cells. Summary of the invention

[0023] According to the present invention, two new third-generation CD30-specific chimeric antigen receptors (CAR-CD30) are now provided. In particular, the following two clinical-grade third-generation CAR CD30 SFG retroviral vectors are provided:

[0024] -SFG.CAR.CD30(AC10)ΔCD34.CD8aTM.CD28cyto.4-1BB.ζ(28.4-1BB.ζ)

[0025] -SFG.CAR.CD30(AC10)ΔCD34.CD8aTM.CD28cyto.OX40.ζ(28.OX40.ζ) contains:

[0026] - Single-chain variable region fragment (scFv) from AC10 hybridoma, which has never been used in CAR therapy before;

[0027] - 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 of genetically modified T cells by Cell Sorter system;

[0028] - The hinge is represented by the CD8 region to avoid the immunogenic CH2-CH3 murine sequence used in the vast majority of similar CARs (20);

[0029] -The transmembrane region from the CD8 transmembrane region is used to improve the molecular stability;

[0030] - Two co-stimulatory domains were added to the CAR-CD30 vectors: CD28 (21, 22) and OX40 (23, 24), or CD28 and 4-1BB (25), each fused to the CD3-ζ chain. Thus, two SFG vectors can be distinguished by a single co-stimulatory domain (4-1BB in the first vector and OX40 in the second).

[0031] In all CAR-CD30, the region, the traceable tag, the co-stimulatory domain and the CD3-ζ chain were codon-optimized to improve efficient protein expression.

[0032] Table 2 shows the differences between the CAR-CD30 according to the present invention and the known CAR-CD30.

[0033] Table 2

[0034]

[0035] Compared with known CAR-CD30, the above sequence of CAR-CD30 according to the present invention provides unexpected advantages as a whole, such as more effective and stable CAR-CD30 expression in T cells obtained by using a retroviral platform and a CD8 TM domain, longer in vivo retention compared with known CAR-CD30 T cells that rely on costimulatory domains, high anti-tumor activity even in the presence of immunomodulatory and single CAR-CD30 T cell administration due to the affinity of the single chain antibody to the antigen and the choice of production method (e.g., using IL7 / IL15 instead of IL2).

[0036] The in vitro and in vivo results described herein show that the modified polyclonal CD30CAR T cells according to the present invention are very effective in clearing CD30+ tumors in long-term co-culture. The biological products according to the present invention have been shown to clear Hodgkin's lymphoma and non-Hodgkin's lymphoma in xenograft in vivo models and establish long-term immune memory.

[0037] In more detail, the supernatants obtained from the two SFG retroviral vectors were able to effectively transduce activated T cells with very high transduction levels. The introduction of CD34-derived epitopes as traceable markers in both constructs made it easy to track genetically modified T cells (CD3+CD34+) in vitro and in vivo xenograft mouse models. As shown in long-term in vitro culture, the replacement of IL2 with an IL7 / IL15 combination improved the stability of CAR-CD30 T cell expression, especially for 28.OX40.ζCAR T cells. In an experimental setting, the combination of IL7 / IL15 improved the kinetics of proliferating T cells, especially after +20 days of in vitro expansion.

[0038] CAR-CD30 T cells cultured in vitro for 15 days in IL7 / IL15 induced preferential expansion of effector memory (EfM) T cells relative to T cells grown in IL2. Evaluation of exhaustion of CAR-CD30 T cells (IL2) at day +15 revealed significant basal expression of PD1 and TIM3, particularly in 28.OX40.ζ T cells. In an in vivo xenograft experimental model, long-term immune memory capable of second-time clearance of re-challenged tumors was demonstrated for the first time.

[0039] Switching from IL2 to IL7 / IL15 significantly reduced PD1 expression but only modestly increased TIM3 in both CAR T cells.

[0040] As reported by different authors, the presence of 4.1BB itself reduces exhaustion in CAR T cells (26). The culture conditions (IL7 / IL15) further enhanced the reduction of PD1 expression, especially in 28.OX40.ζ T cells. To evaluate the role of basal PD1 expression on the potency of CAR-modified T cells against PDL1+ tumors, CAR-modified T cells were co-cultured with the L428-PDL1 lymphoma cell line permanently transduced with PDL1, showing no significant differences and even a lower effector / target ratio relative to the wild-type (WT) L428 cell line. Of note, in stressed long-term co-cultures, unexpectedly, 28.OX40.ζ T cells showed significantly superior lytic activity against Karpas 299 (and HDML-2) and a lower effector / target ratio (ratios E:T of 1:8 and 1:16) compared to 28.4-1BB.ζ T cells.

[0041] The results according to the present invention clearly show that (AC10) 28.4-1BB.ζ T cells or 28.OX40.ζ T cells grown in IL2, when cultured with HDML-2 (effector:target ratio of 1:1), produced about 10303±3321.63 pg / ml and 29872.17±8572.18 pg / ml of IFNγ, respectively. Fig. 9 B), i.e., more than three times the IFNγ produced by 5F11-28Z described in WO2017066122, in which 5F11-28Z produced 3534 pg / ml of IFNγ when co-cultured with HDML-2.

[0042] In addition, 28.4-1BB.ζT cells grown in IL7 / IL15 showed even higher IFNγ production: 21270.17±11621.21 pg / ml ( Fig. 9 E). Furthermore, when cultured with HDML-2, 28.OX40.ζT cells according to the present invention showed an even higher IFNγ production: 29872.17±8572.18 pg / ml (when they were grown with IL2, Fig. 9 B) and 34444.67±18872.62 pg / ml (when they were grown with IL7 / IL15, Fig. 9 E).

[0043] It was also observed that when the CAR-T cells according to the present invention were prepared under conditions containing IL7 and IL15, a higher long-term stability of CAR expression on T cells was obtained compared to CAR-T cells prepared under conditions containing IL2 ( Figure 1 D), in particular 28.OX40.ζ. The stability of the detectable CAR of the present invention provides stable expression of CAR in the T cell membrane ( Figure 1 D). In particular, the CD8 / CD4 transduction ratio was assessed at days 5, 15, and 30. Although the levels of CD8 were lower relative to CD4 on day +5, the levels of CD8 CAR+ increased over time from day +5 to day +15 in favor of CD8 ( Figure 1 EF). In addition, Figure 1 As shown in Figures 1L and 1M, the presence of IL7 / IL15 in the culture conditions significantly improved the fold expansion of CAR-T cells compared to the conditions containing IL2.

[0044] Figure 2 It was also shown that the presence of IL7 / IL15 under culture conditions is important for reducing the exhaustion of CAR-T cells.

[0045] Long-term persistence of infused 28.OX40.ζT cells up to 240 days was also observed ( Fig.11 ).

[0046] Furthermore, it was found according to the present invention that CAR.CD30 T cells with CD28.OX40 co-stimulatory domains were able to more effectively control Karpass 299 ( ) during the continuous addition of CD30+ lymphoma up to 4 times ("stressed" co-culture) compared to CAR.CD30 T cells with 4.1BB co-stimulatory domains. Fig.13 A-13B).

[0047] Interestingly, after the first tumor challenge, the percentage of CAR+ positive cells increased from 61.7% ± 18.4% and 74.0% ± 11.3% (day 0) to 93.4% ± 3.4% and 93.5% ± 3.1% (day 5) in 28.4-1BB.ζT cells and 28.OX40.ζT cells, respectively (p = 0.049 and p = 0.026) ( Fig.13 C). Furthermore, with subsequent tumor re-challenge, the percentage and median fluorescence intensity (MFI) of gene-modified T cells remained stable over time only in 28.OX40.ζ T cells ( Fig.13 CD).

[0048] Furthermore, when co-cultured with the Karpa299 tumor cell line, CAR.CD30 T cells harboring the CD28.OX40 co-stimulatory domain produced significantly more IFN-γ ( Fig.13 E), IL-2 ( Fig.13 F) and TNF-α( Fig.13 G) is significantly higher.

[0049] In addition, both CAR-CD30 T cells also showed cytotoxic effects against solid CD30+ tumors, such as cerebellopontine angle medulloblastoma DAOY ( Figure 5 D and Figure 7 G), rhabdomyosarcoma RD tumor cell line ( Figure 7 Overall, all these results make it very plausible that the constructs according to the invention can be used to effectively treat patients with CD30+ tumors.

[0050] Therefore, an object of the present invention is a CD30 chimeric antigen receptor molecule, which comprises or consists of the following from N-terminus to C-terminus:

[0051] a) a signal peptide, for example a signal peptide comprising or consisting of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 1) (nucleotide ID NO: AB776838.1 and protein ID NO: BAN63131.1), which is linked via a first linker to:

[0052] b) an anti-CD30 single-chain antibody domain from an AC10 hybridoma, comprising an AC10 VL sequence: DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 2) and an AC10 VH sequence: QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA (SEQ ID NO: 3), wherein the AC10 VL and VH sequences are connected by a second linker, or consist of the same;

[0053] c) a traceable marker selected from the group consisting of: ΔCD34: ELPTQGTFSNVSTNVS (SEQ ID NO: 4) (nucleotide ID NO AB238231.1 and protein ID NO: BAE46748.1); ΔCD19: PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK ​​(SEQ ID. NO: 5) (nucleotide ID NO:M21097.1 and protein ID NO:AAA35533.1);

[0054] NGFR: KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN (SEQ ID NO: 6) (nucleotide ID NO: AK313654.1 and protein ID NO: BAG36408.1); preferably ΔCD34: ELPTQGTFSNVSTNVS (SEQ ID NO: 4) (nucleotide ID NO: AB238231.1 and protein ID NO: BAE46748.1);

[0055] d) a hinge selected from the group consisting of: hinge CD8α: PAPRPPTPAPT IASQPLSLRPEACRPAAGGAVH TRGLDFA (SEQ ID NO: 7) (nucleotide ID NO: M12828.1 and protein ID NO: AAB04637.1); hinge CD28: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 8) (nucleotide ID NO: AJ517504.1 and protein ID NO: CAD57003.1); hinge CH2-CH3 (UNIPROTKB: P01861):

[0056] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:9);Hinge CH3(UNIPROTKB:P01861):ESKYGPPCPSCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 10), preferably, hinge CD8α: PAPRPPTPAPT IASQPLSLRPEACRPAAG GAVHTRGLDFA (SEQ ID NO:7) (nucleotide ID NO:M12828.1 and protein ID NO:AAB04637.1);

[0057] e) a transmembrane domain selected from the group consisting of: CD28TM: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 13) (nucleotide ID NO: BC112085.1 and protein ID NO: AAI12086.1); CD8aTM (SEQ ID NO: 14), preferably, CD8aTM CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 14) (nucleotide ID NO NM_001768.6 and protein ID NO: NP_001759.3); and

[0058] f) a co-stimulatory signaling domain selected from the group consisting of: a CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 21) (nucleotide ID NO: AF222341.1 and protein ID NO: AAF33792.1), a CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 22) (nucleotide ID NO: U03397.1 and protein NO: AAA53133.1), and a CD3-zeta chain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO: 23) (nucleotide ID The sequences were obtained by ligating the CD28 cytoplasmic sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 21) (nucleotide ID NO: AF222341.1 and protein ID NO: AAF33792.1), the OX40 sequence RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 24) (nucleotide ID NO: NM_003327.3 and protein NO: NP_003318.1) and the CD3 zeta chain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO: 23) (nucleotide ID NO: NO:J04132.1 and protein ID NO:AAA60394.1).

[0059] The hinge CD8α mentioned above includes the sequence PAPRPPTPAPT (SEQ ID NO: 11) (spacer) and IASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 12) (Nucleotide ID NO NM_001768.6 and Protein ID NO: NP_001759.3).

[0060] The first linker can be a two or three amino acid linker, such as SR.

[0061] The second linker connecting the AC10 VL and VH sequences can be selected from the group consisting of: a proline-rich rigid linker, such as mouse IgG3 upper hinge (mIgG3UH): PKPSTPPGSS (SEQ ID NO: 15), (mIgG3UH)2: PKPSTPPGSSPKPSTPPGSS (SEQ ID NO: 16), or a glycine-rich flexible linker, such as (G4S)2 linker: GGGGSGGGG (SEQ ID NO: 17), (G4S)4 linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18), G4SG2 linker GGGGSGG (SEQ ID NO: 19) or G3SG4 linker: GGGSGGGG (SEQ ID NO: 20), preferably GGGSGGGG (SEQ ID NO: 20).

[0062] In addition, a third linker, such as a short sequence of GS, may be used between the AC10 VH sequence and the traceable label.

[0063] One or more linkers (fourth linker) may be present between the transmembrane domain and the co-stimulatory signaling domain, such as CD8a cytoplasm (cell): LYCNHRN (SEQ ID NO: 25) (nucleotide ID NO: NM_001768.6 and protein ID NO: NP_001759.3) and EF.

[0064] According to one embodiment of the present invention, the CD30 chimeric antigen receptor molecule comprises or consists of:

[0065] a) a signal peptide comprising or consisting of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 1), which is linked via a first linker to:

[0066] b) an anti-CD30 single-chain antibody domain from an AC10 hybridoma, comprising an AC10 VL sequence: DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 2) and an AC10 VH sequence: QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA (SEQ ID NO: 3), wherein the AC10 VL and VH sequences are connected by, or consist of, a second linker: (G4S)2 linker: GGGGSGGGG (SEQ ID NO: 17);

[0067] c) a traceable marker comprising or consisting of ΔCD34:ELPTQGTFSNVSTNVS (SEQ ID NO: 4);

[0068] d) hinge CD8αPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 7);

[0069] e) transmembrane domain CD8aTM CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 14), which is connected by one or more linkers, which linkers comprise or consist of the linker CD8a cytoplasmic (cellular): LYCNHRN (SEQ ID NO: 25),

[0070] f) a co-stimulatory signaling domain comprising a sequence obtained by connecting the CD28 cytoplasmic sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 21), the OX40 sequence RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 24), and the CD3 zeta chain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO: 23).

[0071] Alternatively, the CD30 chimeric antigen receptor molecule according to the present invention comprises or consists of:

[0072] a) a signal peptide comprising or consisting of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 1), which is linked via a first linker to:

[0073] b) an anti-CD30 single-chain antibody domain from an AC10 hybridoma, comprising an AC10 VL sequence: DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 2) and an AC10 VH sequence: QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA (SEQ ID NO: 3), wherein the AC10 VL and VH sequences are connected by, or consist of, a second linker: (G4S)2 linker: GGGGSGGGG (SEQ ID NO: 17);

[0074] c) a traceable marker comprising or consisting of ΔCD34:ELPTQGTFSNVSTNVS (SEQ ID NO: 4);

[0075] d) hinge CD8αPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 7);

[0076] e) transmembrane domain CD8aTM CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 14), connected by one or more linkers comprising or consisting of CD8a cytoplasmic (cellular): LYCNHRN (SEQ ID NO: 25),

[0077] f) a co-stimulatory signaling domain comprising a sequence obtained by connecting the CD28 cytoplasmic sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 21), the CD137 (4-1BB) sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 22), and the CD3 zeta chain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO: 23).

[0078] According to a preferred embodiment of the present invention, the CD30 chimeric antigen receptor molecule is: MEFGLSWLFLVAILKGVQCSRDIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKGGGSGGGGQIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA GS ELPTQGTFSNVSTNVSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNEFRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*(SEQID NO:26).

[0079] That is, the sequence is also referred to herein as SFG.CAR.CD30(AC10)ΔCD34.CD8aTM.CD28cyto.4-1BB.ζ, which includes the following sequence:

[0080] Signal peptide

[0081] MEFGLSWLFLVAILKGVQC (SEQ ID NO: 1) (Nucleotide ID NO: AB776838.1 and Protein ID NO: BAN63131.1)

[0082] Connectors

[0083] SR (connection sequence)

[0084] VL(AC10)

[0085] DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK(SEQ ID NO:2)

[0086] Flexible Area

[0087] GGGSGGGG (G3SG4 linker) (SEQ ID NO: 20)

[0088] VH(AC10)

[0089] QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA(SEQ ID NO:3)

[0090] Connectors

[0091] GS (Connection sequence)

[0092] ΔCD34

[0093] ELPTQGTFSNVSTNVS (SEQ ID NO: 4) (Nucleotide ID NO: AB238231.1 and Protein ID NO: BAE46748.1)

[0094] Extracellular hinge (spacer)

[0095] PAPRPPTPAPT(spacer) (SEQ ID NO:11)

[0096] Extracellular hinge (CD8a)

[0097] IASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 12) (Nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3)

[0098] CD8a(TM) transmembrane region

[0099] CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 14) (Nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3)

[0100] CD8a cytoplasmic (cell) junction adapter

[0101] LYCNHRN (SEQ ID NO: 25) (Nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3)

[0102] Connection joints

[0103] EF (connection sequence)

[0104] CD28 cells

[0105] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 21) (Nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1)

[0106] 4.1BB

[0107] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 22) (Nucleotide ID NO: U03397.1 and Protein ID NO: AAA53133.1)

[0108] CD3 Zeta chain

[0109] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO: 23) (Nucleotide ID NO: J04132.1 and Protein ID NO: AAA60394.1)

[0110] According to a further preferred embodiment of the present invention, the CD30 chimeric antigen receptor is * (SEQ ID NO: 27).

[0111] That is, the sequence is also referred to herein as SFG.CAR.CD30(AC10)ΔCD34.CD8aTM.CD28cyto.OX40.ζ, which includes the following sequence:

[0112] Signal peptide

[0113] MEFGLSWLFLVAILKGVQC (SEQ ID NO: 1) (Nucleotide ID NO: AB776838.1 and Protein ID NO: BAN63131.1)

[0114] Connectors

[0115] SR (connection sequence)

[0116] VL(AC10)

[0117] DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK(SEQ ID NO:2)

[0118] Flexible Area

[0119] GGGSGGGG (G3SG4 linker) (SEQ ID NO: 20)

[0120] VH(AC10)

[0121] QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA(SEQ ID NO:3)

[0122] Connectors

[0123] GS (linking sequence)

[0124] ΔCD34

[0125] ELPTQGTFSNVSTNVS (SEQ ID NO: 4) (Nucleotide ID NO: AB238231.1 and Protein ID NO: BAE46748.1)

[0126] Extracellular hinge (spacer)

[0127] PAPRPPTPAPT(spacer) (SEQ ID NO:11)

[0128] Extracellular hinge (CD8a)

[0129] IASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 12) (Nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3)

[0130] CD8a(TM) transmembrane region

[0131] CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 14) (Nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3)

[0132] CD8a cytoplasmic (cell) junction

[0133] LYCNHRN (SEQ ID NO: 25) (Nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3)

[0134] Connection joints

[0135] EF (connection sequence)

[0136] CD28 cells

[0137] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 21) (Nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1)

[0138] OX40

[0139] RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 24) (Nucleotide ID No: NM_003327.3 and Protein NO: NP_003318.1)

[0140] CD3 Zeta chain

[0141] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO: 23) (Nucleotide ID NO: J04132.1 and Protein ID NO: AAA60394.1)

[0142] The present invention also relates to a nucleotide sequence encoding the above CD30 chimeric antigen receptor.

[0143]

[0144] That is, the nucleotide sequence encodes a sequence also referred to as SFG.CAR.CD30(AC10)ΔCD34.CD8aTM.CD28cyto.4-1BB.ζ, which comprises the following sequence:

[0145] Signal peptide

[0146] ATGGAGTTTGGGCTCTCCTGGCTCTTCCTGGTCGCGATTCTGAAGGGGGTCCAGTGTTCACGA (SEQ ID NO: 29) (Nucleotide ID NO: AB776838.1)

[0147] VL(AC10)

[0148] GATATCGTCCTGACTCAGAGTCCTGCCAGCCTGGCAGTCTCCCTGGGACAGAGAGCTACCATAAGTTGTAAAGCATCACAGTCTGTTGATTTCGATGGCGACAGCTATATGAATTGGTACCAGCAAAAACCCGGCCAGCCCCCGAAAGTTTTGATCTATGCAGCCTCT AACTTGGAAAGCGGCATTCCTGCGCGATTCAGTGGCAGCGGGAGTGGTACAGATTTCACCCTGAACATACACCCAGTCGAAGAGGAGGACGCAGCCACATATTACTGCCAACAATCTAACGAGGATCCATGGACTTTTGGGGGCGGCACTAAACTCGAAATCAAG(SEQ ID NO:30)

[0149] Flexible Area

[0150] GGCGGAGGTTCAGGCGGAGGAGGG (G3SG4 linker) (SEQ ID NO: 31)

[0151] VH(AC10)

[0152] GATATCGTCCTGACTCAGAGTCCTGCCAGCCTGGCAGTCTCCCTGGGACAGAGAGCTACCATAAGTTGTAAAGCATCACAGTCTGTTGATTTCGATGGCGACAGCTATATGAATTGGTACCAGCAAAAACCCGGCCAGCCCCCGAAAGTTTTGATCTATGCAGCCTCT AACTTGGAAAGCGGCATTCCTGCGCGATTCAGTGGCAGCGGGAGTGGTACAGATTTCACCCTGAACATACACCCAGTCGAAGAGGAGGACGCAGCCACATATTACTGCCAACAATCTAACGAGGATCCATGGACTTTTGGGGGCGGCACTAAACTCGAAATCAAG(SEQ ID NO:32)

[0153] Linker (BamH1 restriction site)

[0154] GGATCC (BamH1 restriction site and linker sequence) (SEQ ID NO: 33)

[0155] ΔCD34

[0156] GAACTTCCTACTCAGGGGACTTTCTCAAACGTTAGCACAAACGTAAGT (SEQ ID NO: 34) (Nucleotide ID NO: AB238231.1)

[0157] Extracellular hinge (CD8a)

[0158] CCCGCCCCAAGACCCCCCACAACCTGCGCCGACCATTGCTTCTCAACCCCTGAGTTTGAGACCCGAGGCCTGCCGGCCAGCTGCCGGCGGGGCCGTGCATACAAGAGGACTCGATTTCGCT(SEQ ID NO:35)(NM_001768.6)

[0159] CD8a(TM) transmembrane region

[0160] TGCGACATCTACATCTGGGCTCCCCTCGCTGGCACCTGTGGGGTGCTGCTGCTGTCACTCGTGATCACC(SEQ ID NO:36)(NM_001768.6)

[0161] CD8a cytoplasmic (cell) junction adapter

[0162] CTTTATTGCAACCATCGAAAC(SEQ ID NO:37)(NM_001768.6)

[0163] Adapter (EcoR1 restriction site and linker sequence)

[0164] GAATTC(SEQ ID NO:38)

[0165] CD28 cells

[0166] AGAAGTAAACGGTCAAGGCTTCTGCACAGCGATTATATGAATATGACACCAAGAAGACCTGGTCCAACCCGGAAACACTATCAGCCCTACGCGCCCCCTAGAGACTTCGCAGCATACCGCTCT(SEQ ID NO:39)(AF222341.1)

[0167] 4.1BB

[0168] AAGAGAGGGAGAAAAAAATTGCTCTATATTTTTAAACAACCATTTATGAGGCCCGTACAGACAACTCAGGAAGAGGATGGCTGTAGTTGCCGCTTCCCAGAGGAGGAGGAAGGAGGCTGCGAGTTG(SEQ ID NO:40)(U03397.1)

[0169] CD3 Zeta chain

[0170] AGAGTTAAATTCAGTAGAAGTGCGGATGCGCCTGCTTACCAGCAGGGCCAGAACCAACTGTACAATGAACTGAATCTCGGGCGCCGAGAAGAGTATGACGTCCTCGATAAGCGGAGGGGTAGGGATCCTGAAATGGGTGGGAAGCCAAGAAGAAAAAACCCCCAGGAAGGACTGTATAACGAACTTCAGAAGGACAAGATGGCAGAGGCCTACTCTGAGATTGGCATGAAAGGCGAACGACGGCGCGGTAAAGGTCATGACGGGCTGTACCAGGGCCTGTCCACAGCGACGAAGGACACTTACGACGCCCTGCACATGCAGGCACTCCCCCCCAGGTGA(SEQ ID NO:41)(J04132.1)

[0171]

[0172] That is, the nucleotide sequence encodes a sequence also referred to as SFG.CAR.CD30(AC10)ΔCD34.CD8aTM.CD28cyto.OX40.ζ, which comprises the following sequence:

[0173] Signal peptide

[0174] ATGGAGTTTGGGCTCTCCTGGCTCTTCCTGGTCGCGATTCTGAAGGGGGTCCAGTGTTCACGA(SEQID NO:29)(AB776838.1)

[0175] VL(AC10)

[0176] GATATCGTCCTGACTCAGAGTCCTGCCAGCCTGGCAGTCTCCCTGGGACAGAGAGCTACCATAAGTTGTAAAGCATCACAGTCTGTTGATTTCGATGGCGACAGCTATATGAATTGGTACCAGCAAAAACCCGGCCAGCCCCCGAAAGTTTTGATCTATGCAGCCTCT AACTTGGAAAGCGGCATTCCTGCGCGATTCAGTGGCAGCGGGAGTGGTACAGATTTCACCCTGAACATACACCCAGTCGAAGAGGAGGACGCAGCCACATATTACTGCCAACAATCTAACGAGGATCCATGGACTTTTGGGGGCGGCACTAAACTCGAAATCAAG(SEQ ID NO:30)

[0177] Flexible Area

[0178] GGCGGAGGTTCAGGCGGAGGAGGG (G3SG4 linker) (SEQ ID NO: 31)

[0179] VH(AC10)

[0180] GATATCGTCCTGACTCAGAGTCCTGCCAGCCTGGCAGTCTCCCTGGGACAGAGAGCTACCATAAGTTGTAAAGCATCACAGTCTGTTGATTTCGATGGCGACAGCTATATGAATTGGTACCAGCAAAAACCCGGCCAGCCCCCGAAAGTTTTGATCTATGCAGCCTCT AACTTGGAAAGCGGCATTCCTGCGCGATTCAGTGGCAGCGGGAGTGGTACAGATTTCACCCTGAACATACACCCAGTCGAAGAGGAGGACGCAGCCACATATTACTGCCAACAATCTAACGAGGATCCATGGACTTTTGGGGGCGGCACTAAACTCGAAATCAAG(SEQ ID NO:32)

[0181] Linker (BamH1 restriction site and connection sequence)

[0182] GGATCC (SEQ ID NO:33)

[0183] ΔCD34

[0184] GAACTTCCTACTCAGGGGACTTTCTCAAACGTTAGCACAAACGTAAGT(SEQ ID NO:SEQ ID NO:34)(AB238231.1)

[0185] Extracellular hinge (CD8a)

[0186] CCCGCCCCAAGACCCCCCACAACCTGCGCCGACCATTGCTTCTCAACCCCTGAGTTTGAGACCCGAGGCCTGCCGGCCAGCTGCCGGCGGGGCCGTGCATACAAGAGGACTCGATTTCGCT(SEQ ID NO:35)(NM_001768.6)

[0187] CD8a(TM) transmembrane region

[0188] TGCGACATCTACATCTGGGCTCCCCTCGCTGGCACCTGTGGGGTGCTGCTGCTGTCACTCGTGATCACC(SEQ ID NO:36)(NM_001768.6)

[0189] CD8a cytoplasmic (cell) junction adapter

[0190] CTTTATTGCAACCATCGAAAC(SEQ ID NO:37)(NM_001768.6)

[0191] Adapter (EcoR1 restriction site and linker sequence)

[0192] GAATTC(SEQ ID NO:38)

[0193] CD28 cells

[0194] AGAAGTAAACGGTCAAGGCTTCTGCACAGCGATTATATGAATATGACACCAAGAAGACCTGGTCCAACCCGGAAACACTATCAGCCCTACGCGCCCCCTAGAGACTTCGCAGCATACCGCTCT(SEQ ID NO:39)(AF222341.1)

[0195] OX40

[0196] CGCGATCAAAGACTCCCGCCCGATGCCCACAAACCCCCTGGCGGGGGCAGCTTTAGGACACCCATTCAAGAAGAGCAGGCAGACGCCCACAGCACCTTGGCCAAAATT(SEQ ID NO:43)(NM_003327.3)

[0197] CD3 Zeta chain

[0198] AGAGTTAAATTCAGTAGAAGTGCGGATGCGCCTGCTTACCAGCAGGGCCAGAACCAACTGTACAATGAACTGAATCTCGGGCGCCGAGAAGAGTATGACGTCCTCGATAAGCGGAGGGGTAGGGATCCTGAAATGGGTGGGAAGCCAAGAAGAAAAAACCCCCAGGAAGGACTGTATAACGAACTTCAGAAGGACAAGATGGCAGAGGCCTACTCTGAGATTGGCATGAAAGGCGAACGACGGCGCGGTAAAGGTCATGACGGGCTGTACCAGGGCCTGTCCACAGCGACGAAGGACACTTACGACGCCCTGCACATGCAGGCACTCCCCCCCAGGTGA(SEQ ID NO:41)(J04132.1)

[0199] The present invention also relates to a vector comprising the above nucleotide sequence, wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector or a non-viral vector.

[0200] In addition, the present invention relates to cells comprising the above-mentioned vector or plasmid, such as T cells, such as α / β and γ / δ T cells, NK cells, NK-T cells.

[0201] According to one embodiment of the present invention, the above-mentioned cells are obtained in the presence of recombinant human IL-2, or a combination of recombinant IL-7 and IL 15. For example, the interleukin may be present in at least one or all steps of the cell preparation process, such as activation, transduction and amplification.

[0202] The present invention also relates to a pharmaceutical composition comprising a nucleotide sequence, or a vector, or a cell, or all of the above substances, together with one or more pharmaceutically acceptable excipients and / or adjuvants.

[0203] Another object of the present invention is the CD30 chimeric antigen receptor molecule, nucleotide sequence, vector, cell, pharmaceutical composition, all of the above, for medical use.

[0204] Another object of the present invention is to use the CD30 chimeric antigen receptor molecule, the nucleotide sequence, the vector, the cell, the pharmaceutical composition, all of which are mentioned above, for the treatment of CD30+ cancers, such as in-diagnosis or refractory / recurrent diseases, such as lymphomas, such as Hodgkin's lymphoma and non-Hodgkin's lymphoma; solid tumors, such as myofibroblastic sarcoma, rhabdomyosarcoma, histiocytic sarcoma, embryonal carcinoma, adenocarcinoma, mesothelioma, mixed germ cell tumor (GCT), non-seminoma GCT, head and neck cancer, yolk sac tumor, angiosarcoma, pituitary adenoma, dysgerminoma, teratoma or seminoma. In addition, the present invention can also be used to treat CD30+PDL1+ tumors (L428-PDL1), such as Figure 7 The titer assay in D is shown.

[0205] Furthermore, the present invention also relates to a method for preparing cells as defined above, wherein at least one or all steps of activation (e.g. with fixed OKT3 and anti-CD28 antibodies), transduction and expansion of the cells (e.g. T lymphocytes) are carried out in the presence of recombinant human IL-2, or a combination of recombinant IL-7 and IL15. BRIEF DESCRIPTION OF THE DRAWINGS

[0206] The present invention is described in an illustrative and non-limiting manner according to preferred embodiments of the present invention, with particular reference to the accompanying drawings, in which:

[0207] Figure 1After initial antigen stimulation, CAR-CD30 T cells with CD28.OX40 or CD28.4-1BB co-stimulation showed similar transduction levels, CD4+ / CD8+ distribution, and in vitro proliferation.

[0208] (A) The expression cassettes of two CAR-CD30 are shown in the figure. The scFv of CD30 is cloned in the same reading frame with CD8aTM, CD28 cytoplasmic part and the second costimulatory domain represented by 4-1BB (upper figure) or OX40 (lower figure) and the signaling domain CD3-ζ chain (ζ). ΔCD34 is added as a traceable marker. (B) Flow cytometry analysis shows the transduction level of T cells (upper figure) analyzed by CD34 expression in exemplary donors (grown in IL2), untransduced (NT) T cells (as negative control) (left figure) or genetically modified T cells (middle figure) with CAR.CD30.ΔCD34.28.4.1BB.ζ (28.4.1BB.ζ) and genetically modified T cells (right figure) with CAR.CD30.ΔCD34.CD28.OX40.ζ (28.OX40.ζ). (C) The transduction level of T cells was also confirmed by biotinylated protein L; it was able to effectively bind scFv. (DF) These three figures show the average of the percentage of positive CAR+T cells detected by FACS in three in vitro cultures. The first group shows CAR+CD3+ expression (D); the second group (E) shows CAR+CD4+ subsets; and the last group (F) is CAR+CD8+T cells. For T cells grown in IL2:NT (white bars), 28.4.1BB.ζ (white bars with horizontal lines) and 28.OX40.ζ (black bars); or in IL7 / IL15:NT (white bars with vertical lines), 28.4.1BB.ζ (grid bars) and 28.OX40.ζ (checkerboard bars). Data are expressed as mean ± standard deviation (SD) on days 5, 15 and 30 of in vitro culture from six healthy donors. (GH) Figures show the fold expansion of NT T cells and CAR-CD30 T cells in IL2 (continuous line (G)) or IL7 / IL15 (dashed line (H)) assessed by trypan blue counting assay. Data represent the results of 6 HDs. (IM) Effect of cytokine use on in vitro long-term fold expansion of NT T cells (I), 28.4.1BB.ζT cells (L), and 28.OX40.ζT cells (M). Significance is reported with an asterisk, and the significance of changes in transduction levels during in vitro culture is indicated by enclosed asterisks. *p value = <0.05, **p value = <0.01.

[0209] Figure 2 .Exhaustion of genetically modified CAR-CD30 T cells.

[0210] Baseline exhaustion curves representing CD3 T cells from four HDs, expanded for 15 days in the presence of IL2 (left): NT (white bars), 28.4-1BB.ζ (white bars with horizontal lines), or 28.OX40ζ (black bars); or in IL7 / IL15, (white bars with vertical lines represent NT; white bars with squares represent CARGD2.28-41BBζ T cells and checkerboard bars represent CARGD30.28-OX40ζ T cells). Circles around asterisks represent p values ​​for comparisons between the same T cell populations cultured in the presence of IL7 / IL15 or IL2. Data from four HDs are expressed as mean ± SD. *p value = <0.05, **p value = <0.01.

[0211] Figure 3 .Basal and / or induced proliferation of NT or CAR-CD30 T cells.

[0212] To evaluate the impact of retroviral modification or culture conditions on the safety of modified T cells, basal proliferation or cytokine or / and antigen-specific proliferation was assessed for NT (A) or CAR-CD30 T cells (BC). T cells were labeled with the fluorescent cell dye CFSE at day 0 and inoculated for 5 days in the presence / absence of cytokines or co-cultured in the presence of tumor cell lines CD30 positive (Karpas299) or tumor cell lines CD30 negative (BV173). Basal or induced proliferation (determined by CFSE dye dilution) of CD3+ (left), CD8+ (right), and CD4+ T cells (left) was assessed by FACS analysis.

[0213] Figure 4 CD30 and / or PDL1 expression in solid and hematological tumor cell lines. (AD) Representative FACS analysis of constitutive expression of CD30+ and / or PDL1 in three lymphoma cell lines: L428, HDML2, and Karpas 299 (A), five sarcoma cell lines: RD, A673, SK-ES-1, CW9019, and CT-10 (B), two medulloblastoma cell lines: DAOY and D283 (C), and two leukemia cell lines: CEM-T2 and BV-173 (D). The last picture shows the FACS analysis of the lymphoma cell line L428 genetically modified with the retroviral vector SFG containing the cassette PDL1 to obtain the L428-PDL1 lymphoma cell line (E).

[0214] Figure 5 CAR-CD30 T cells grown in complete CTL medium containing IL2 and transduced with either 28.4-1BB or 28.OX40 co-stimulatory domains showed comparable short-term cytotoxic effects in vitro.51 Cr release assays evaluate lytic activity of NT T cells (line with white circles), 28.4-1BBζT cells (line with white squares), or 28.OX40.ζT cells (line with black squares) in CD30+ lymphomas (Karpas 299 cell line (A), HDML-2 cell line (B), and L428 cell line (C)), in CD30+ medulloblastoma DAOY (D), and in CD30-negative (such as medulloblastoma D283 (E) and lymphoma BV173 (F)). The assay was performed in the presence of IL2 15 days after initial activation and expansion. Data from 6 healthy donors (HD) are expressed as mean ± SD. *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.001, and **** ≤ 0.0001.

[0215] Figure 6 CAR-CD30 T cells grown in complete CTL medium containing IL7 / IL15 and transduced with 28.4-1BB or 28.OX40 co-stimulatory domains showed comparable short-term cytotoxic effects in vitro. 51 Cr release assays evaluate lytic activity of NT T cells (dashed line with white circles), 28.4-1BBζT cells (dashed line with white squares), or 28.OX40.ζT cells (dashed line with black squares) in CD30+ lymphomas (Karpas 299 cell line (A), HDML-2 cell line (B), and L428 cell line (C)), in CD30+ medulloblastoma DAOY (D), and in CD30-negative (such as medulloblastoma D283 (E) and lymphoma BV173 (F)). The assay was performed in the presence of IL7 / IL15 15 days after initial activation and expansion. Data from 6 healthy donors (HD) are expressed as mean ± SD. *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.001, and **** ≤ 0.0001.

[0216] Figure 7 Long-term co-culture of both CAR-CD30 T cells against CD30+ tumor cell lines confirmed their identical specific cytotoxic potency, independent of the cytokines used. (AJ) Representative FACS analysis of residual tumor cells (identified as GFP+ cells) (or CD45-CD3- for D283 cells) after 7 days of co-culture at an E / T 1:1 ratio with effector NT cells (upper panel), CD30-CAR T cells:28.4-1BBζT cells (middle panel), and 28-OX40ζT cells (lower panel).

[0217] (KL) Average representation of remaining tumor cells after 7 days - co-cultured at an E / T ratio of 1:1 in IL2 (K) with NT (white bars), CARGD2.28-41BBζ T cells (white bars with horizontal lines), and CARCD30.28-OX40ζ T cells (black bars), or in IL7 / IL15 (L) (white bars with vertical lines represent NT; square white bars represent CARGD2.28-41BBζ T cells and checkerboard bars represent CARCD30.28-OX40ζ T cells). Data from 6 healthy donors (HD) are expressed as mean ± SD. *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.001, and **** ≤ 0.0001.

[0218] Figure 8 . Stressful long-term coculture to assess and quantify the functional activity of CAR-CD30 T cells. (AF) Tumor control efficiency of lymphoma tumor cells was assessed after 7 days of coculture at low E / T ratios: in IL2 (AC) with CARGD2.28-41BBζ T cells (bars with horizontal lines), or CARCD30.28-OX40ζ T cells (black bars); or in IL7 / IL15 (DF), (square bars represent CARGD2.28-41BBζ T cells or checkerboard bars represent CARCD30.28-OX40ζ T cells). Individual tumors are represented by white bars, and data from six healthy donors (HD) are presented as mean ± SD. *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.001, and **** ≤ 0.0001.

[0219] Fig. 9 .IFN-γ profile of CAR-CD30 T cells co-cultured with CD30+ tumor cells. (AF) Specific IFN-γ produced after 24 hours of effector cell: target cell co-culture. The figure shows the IFN-γ production of CAR.CD30 T cells grown in IL2 (AC) or in IL7 / IL15 (DF) after stimulation with tumor lymphoma cell lines. Relative to CARCD 2.28-41bbζ T cells, CARCD30.28-OX40ζ T cells co-cultured with Karpas 299 (A and D) or HDML-2 (B and E) for 24 hours produced significantly higher levels of IFN-γ, specifically lower ratios of effector cells: target cells. When CAR-CD30 T cells were co-cultured with the tumor cell line L428, no differences were observed between the two CARs (C and F). In addition to co-culture with the L428 tumor cell line (F), CARCD30.28-OX40ζ T cells grown in IL7 / IL15 also produced higher levels of IFN-γ when co-cultured with CD30+ tumor cells (DE).

[0220] Fig.10 .In vivo activity of CAR.CD30 T cells generated and expanded against NHL Karpas 299 in the presence of IL2. (AC) In vivo bioluminescence imaging of NSG mice carrying systemic Karpas299-FF-Luc.GFP cells and treated with NT, CARCD30.28.4-1BBζ or CARCD30.28.OX40ζ T cells generated and expanded in the presence of IL2. (A) Schematic model of the in vivo experiment. Mice received intravenous injections of Karpas 299-FF-Luc.GFP cells. Three days after the bioluminescence of the tumors became stable, they were divided into three groups and treated with NT or one of the two CAR-CD30 T cells. Tumor growth was assessed weekly by IVIS assessment for 140 days. (B) Bioluminescent imaging of tumor growth measured weekly in three groups of mice; (C) Representation of bioluminescence of each mouse treated with NT(IL2) (line with white circles; 8 mice), 28.4-1BBζ(IL2) (line with white squares; 10 mice), and 28.OX40ζ(IL2) T cells (line with black squares; 10 mice). (4) Kaplan-Meier overall survival (OS) analysis of tumor-bearing mice treated with NT(IL2) (line with white circles; 8 mice), 28.4-1BBζ(IL2) (line with white squares; 10 mice), and 28.OX40ζ(IL2) (line with black squares; 10 mice) T cells. *P value = <0.05; **P value = <0.001; ***P value = <0.0001. Log rank (Mantel-Cox).

[0221] Fig.11 Rechallenge model: Establishment of long-term immune memory in NHL mouse model. (AC) 0.2x10 6 Karpas 299-FF-Luc.GFP cells re-stimulated in vivo bioluminescence imaging of cured NSG mice and then for another 100 days. (A) Schematic model of the in vivo experiment. Mice received intravenous injections of Karpas 299-FF-Luc.GFP cells twice: on day 0 and day 140. On the third day when the bioluminescence of the tumor became stable, they were divided into three groups and treated with NT or one of the two CAR-CD30 T cells. Tumor growth was assessed weekly by IVIS assessment for 240 days. On day 140, cured mice and a new group of mice (added to the experiment as a positive control for tumor transplantation (CTR mice)) were re-stimulated by intravenous injection of Karpas 299-FF-Luc.GFP cells.

[0222] (B) Bioluminescent imaging of tumor growth measured weekly from day 140 to day 240. (C) Bioluminescent performance of each mouse treated with NT (line with white circles; 8 mice), CARCD30.28.4-1BBζ (line with white squares; 10 mice), and CARCD30.28.OX40ζ T cells (line with black squares; 10 mice), and CTR mice (dashed line; 6 mice) added to the experiment as a positive control for secondary tumor transplantation on day 140. (D) Kaplan-Meier overall survival (OS) analysis of tumor-bearing mice treated only once on day 3 with NT (line with white circles; 8 mice), CARCD30.28.4-1BBζ (line with white squares; 10 mice), and CARCD30.28.OX40ζ T cells (line with black squares; 10 mice), and mice re-challenged with tumors for the second time on day 140. Survival days of CTR mice were added (dashed line with white triangles; 6 mice), and day 140 was considered zero. *P value = <0.05; **P value = <0.01; ***P value = <0.001; ****P value = <0.0001. Logarithmic scale (Mantel-Cox). (E) Representative pictures of circulating human T cells on the indicated days are shown after the first tumor challenge (day 6) and before and after the second tumor re-challenge (days 132 and 180, respectively). NT (first row), CARD 30.28-41BBζ T cells (second row), and CARD 30.28-OX40ζ (third row).

[0223] Fig.12 .Evaluation of in vivo activity of CAR.CD30 T cells generated and expanded against HL L428 in the presence of IL2 or IL7 / IL15. (AC) In vivo bioluminescence imaging of NSG mice carrying systemic L428-FF-Luc.GFP cells treated on day 6 with NT, 28.OX40ζ, or 28.4-1BBζ T cells generated and expanded in vitro in the presence of L2 or IL7 / IL15. Tumor growth was assessed weekly by IVIS assessment for 165 days. (A) Schematic model of the in vivo experiment. Mice were injected intravenously with 2x10 6L428-FF-Luc.GFP cells, and after 6 days, when bioluminescence becomes stable, they are divided into 6 groups and treated with NT or CAR-CD30 T cells. Tumor growth is assessed weekly by IVIS assessment for 165 days. (B) Bioluminescence imaging of tumor growth measured weekly from day 6 to day 165. (C) Bioluminescence of each xenograft mouse treated with NT (IL2) T cells (line with white circles; 5 mice), 28.4-1BBζ (IL2) T cells (line with white squares; 5 mice), 28.OX40ζ (IL2) T cells (line with black squares; 5 mice), NT (IL7 / IL15) T cells (dashed line with white circles; 5 mice), 28.4-1BBζ (IL7 / IL15) T cells (dashed line with white squares; 5 mice) and 28.OX40ζ (IL7 / IL15) T cells (dashed line with black squares; 5 mice). (D) Kaplan-Meier overall survival (OS) analysis of tumor-bearing mice treated with NT (IL2) (line with white circles), 28.4-1BBζ (IL2) (line with white squares), and CARCD30.28-OX40ζ (IL2) (line with black squares), NT (IL7 / IL15) (dashed line with white circles), CARCD30.28.4-1BBζ (IL7 / IL15) (dashed line with white squares), and CARCD30.28.OX40ζ (IL7 / Il15) (dashed line with black squares); *P value = <0.05; **P value = <0.001; ***P value = <0.0001. Log rank (Mantel-Cox). (E) The table reports the significance of OS in L428 xenograft mice treated with NT or CAR-CD30 T cells grown in IL2 or IL7 / IL15.

[0224] *P value = <0.05 and **P value = <0.01. (FG) Mean values ​​of human circulating T cells in NSG mice bearing systemic L428-FF-Luc.GFP tumor cells and treated with human NT or CAR.CD30 T cells on day 6, CD45+CD3+ cell percentage (F) and CAR-CD30 T cells (CD3+CD34+) (G) assessed on days 15, 30, 56, 80, 100, 130, and 160.

[0225] Fig.13.Stressed long-term co-culture. (A) The experimental design of "stressed co-culture" is shown in the figure. On day 15 after transduction, T cells were co-cultured with the Karpas 299 tumor cell line at an E / T ratio of 1:1 (0.5E+06 for T cells and 0.5E+06 for Karpas 299 in a 24-well plate). Tumor cells were applied every five days until the 20th day of co-culture (applications I, II, III and IV). At each time point, the supernatant was collected at 24 hours and analyzed for the presence of cytokines IFNγ, TNFα, IL-2 and IL-10. After 5 days of each application, cells were collected and analyzed by FACS. (B) A bar graph showing the percentage of residual tumor in the culture 5 days after each tumor application. Both CAR.CD30 T cells can effectively control tumor growth. However, 28-OX40ζT cells showed increased tumor control at day 20. (C) Bar graph showing the percentage of CAR-positive T cells in the total number of CD3-positive T cells in the co-culture at each time point. After the first co-culture, i.e., on day 5, the percentages of both CAR.CD30 molecules increased significantly. Re-stimulation of the tumor only had a negative impact on the transduction level of 28.4-1BB.ζT cells, while the percentage remained stable in 28.OX40.ζT cells. (D) The figure emphasizes that the MFI values ​​in 28.OX40.ζT cells (black bars) are significantly higher than those in 28.4-1BB.ζT cells (white bars). (EH) Cytokine profiles obtained from ELLA analysis, analysis of supernatants collected 24 hours after tumor stimulation. Data from 7 healthy donors (HD) are expressed as mean ± SD. *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.001, and **** ≤ 0.0001. (I): Tumor regulation of memory and exhaustion conditions in CAR.CD30 T cells. CD3+ T cells (NT (white bars), 28.4.1BB.ζ (bars with horizontal lines), or 28.OX40.ζ (black bars)) expanded in the presence of IL2 or IL7 / IL15 cytokines were cultured on day 15 in vitro. Flow cytometric analysis of the proportion of CM, EM, and EMRA subsets. (J) Long-term "stressed" co-culture induced selection for EM and CM distinction in 28.4-1BB.ζ and 28.OX40.ζ T cells, but not in NT T cells. (K) Exhaustion of CD3+ T cells, NT (white bars), 28.4.1BB.ζ (horizontal bars), or 28.OX40.ζ (black bars) expanded in IL2 or IL7 / 15 cytokines for 15 days. Significance between the growth of NT T cells or CAR-CD30 T cells under the same culture conditions is indicated in black, while the circled asterisks indicate the p value for comparison between the same T cell populations cultured in the presence of IL2 or IL7 / IL15. (L) Long-term "stressed" co-culture induced upregulation of exhaustion markers (especially PD1 and TIM3) in both CAR.CD30 T cell types, although upregulation of these molecules did not affect their lytic activity. Data from four HDs are expressed as mean ± SD. *p value = < 0.05; **p value = < 0.001. DETAILED DESCRIPTION

[0226] Example 1: In vitro and in vivo design and study of CAR-CD30 according to the present invention

[0227] Materials and methods

[0228] Design of CAR-CD30 plasmid (construct)

[0229] Two clinical-grade "third" generation retroviral vectors, SFG, have been designed that carry an anti-CD30 single-chain variable fragment (scFv) derived from a murine antibody of the IgG (AC10) class, an expression cassette of a codon-optimized human CD8 hinge-transmembrane domain connected to the two co-stimulatory domains CD28, 4-1BB (CD137) or OX40 and a codon-optimized signaling domain of CD3-ζ ( Figure 1 A). The CD30-specific single-chain variable fragment (scFv) is a fusion protein of 111 amino acids (aa) of the variable region of the immunoglobulin light chain (VL), connected to 117 aa of the immunoglobulin heavy chain (VH) by an 8 amino acid flexible region (27) (short linker peptide). In particular, the scFv AC10 is cloned in the same reading frame with a codon-optimized CD34-derived 16aa epitope (as a traceable marker), connected to a codon-optimized human CD8 transmembrane region (CD8aTM) binding to 30aa via a 40aa hinge (11aa as a spacer plus a 29aa codon-optimized CD8 extracellular domain). By

[0230] SFG.CAR.CD30(AC10)ΔCD34.CD8aTM.CD28cyto.4.1BB.ζ retroviral vector (28.4-1BB.ζ) contains two co-stimulatory molecules, the CD28 intracellular domain (41aa) and the 4-1BB intracellular domain (42aa). The signal is transmitted from the extracellular part of CD30 scFvAC10 to the intracellular part of CD3-ζ chain (113aa).

[0231] The co-stimulatory molecule 4.1BB can be obtained by replacing OX40 (36aa)

[0232] SFG.CAR.CD30(AC10)ΔCD34.CD8aTM.CD28cyto.OX40.ζ retroviral vector (28.OX40.ζ).

[0233] Generation of eGFP-firefly-luciferase cell lines.

[0234] In selected experiments, CD30+ tumor cells were labeled using a retroviral vector encoding eGFP-firefly-luciferase (eGFP-FFLuc):

[0235] - Non-Hodgkin's Lymphoma (NHL) Karpas 299,

[0236] -Hodgkin lymphoma (HL) HDML-2 and L428;

[0237] - Rhabdomyosarcoma RD,

[0238] - Hyperplastic cerebellar medulloblastoma DAOY.

[0239] In selected experiments, a retroviral vector encoding eGFP-firefly-luciferase (eGFP-FFLuc) was used to label the CD30 negative control:

[0240] - B-cell precursor leukemia BV173,

[0241] - Chronic myeloid leukemia K562,

[0242] These cell lines were used for in vitro and in vivo studies as described previously ( 9 ).

[0243] Cell lines

[0244] Non-Hodgkin's lymphoma (NHL) Karpas 299 was obtained from Sigma-Aldrich. Hodgkin's lymphoma (HL) HDML-2 and L428 and B-cell precursor leukemia Ph+ BV173 were obtained from DSMZ. Rhabdomyosarcoma RD, hyperplastic cerebellar medulloblastoma DAOY, chronic myeloid leukemia K562, medulloblastoma D283 and embryonic kidney 293T cell lines were obtained from LGC Standards-ATCC.

[0245] Karpas 299, HDML-2, L428, BV173 and le K562 cell lines were cultured in RPMI 1640 medium (Gibco; USA). The RD, DAOY tumor cell lines and 293T cells were cultured in DMEM medium (Gibco, Invitrogen TM , Carlsbad, CA), and the D283 was cultured in IMDM (Life Technologies Corporation, USA); the cell lines were supplemented with 10% fetal bovine serum (FBS, Hyclone, Thermo Scientific, Pittsburgh, PA) and 2mM GlutaMax (Invitrogen, California, USA). The cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. All cell lines were routinely tested for mycoplasma and surface expression of target antigens. All cell lines have been authenticated by STR analysis in the certified laboratory "BMRGenomics srl".

[0246] Retroviral supernatant

[0247] Transient transfection retroviral supernatants were generated by co-transfecting 293T cells with MoMLV gag / pol expression plasmid Peqp m3(-env), RD114 env expression plasmid RDF, and SFG vector at a 2:3:3 ratio (total DNA 10 μg). Transfection was assisted by 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 (28).

[0248] Isolation, generation and transduction of effector cells.

[0249] Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood (PB) or buffy coats obtained from healthy donors (OPBG Hospital, Rome, Italy) using lymphocyte separation medium (Eurobio; France) after obtaining informed consent from the signatory according to the rules established by the Institutional Review Board (IRB) of OPBG (Approval of Ethical Committee N°969 / 2015prot. N°669LB). T lymphocytes were activated with fixed 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 recombinant human interleukin-2 (IL-2, 100 U / ml; R&D; USA) (28), or a combination of recombinant human interleukin 7 (IL7, 10 ng / ml; R&D; USA) (29) and recombinant human interleukin 15 (IL15, 5 ng / ml; R&D) (18, 30). On day 3, activated T cells were transduced with specific retroviral supernatants and the above-mentioned cytokines in 24-well plates pre-coated with recombinant human RetroNectin (Takara-Bio. Inc; Japan). On day 5 after transduction, the T cells were expanded in "CTL complete medium" containing 45% RPMI1640 and 45% Click medium (Sigma-Aldrich, Co.; USA) supplemented with 10% FBS and 2 mM Glutamax, and the above-mentioned specific cytokines were added twice a week.

[0250] Phenotypic analysis. Expression of cell surface molecules was determined by flow cytometry using standard methods. The following monoclonal antibodies (mAbs) were used: CD3, CD4, CD8, CD25, CD27, CD28, CD45RA, CD45RO, CD56, CD57, CD62L, CD62E, CD62P, CD95, CD106, CD127, CD137, CD197, CD223 (Lag3), CD274 (PDL1), CD279 (PD1), and TIM3. CAR-CD30 expression on T cells was detected using specific anti-CD34+ (QBENd10V clone) or Pierce Recombinant Biotinylated Protein L, which can effectively bind to scFv. T cell receptor (TCR)-Vβ repertoires on NT T cells and CAR-T cells were assessed on days 15 and 30 using a panel of 24 different TCR Vβ-specific mAbs (IO TEST Beta Mark TCR-Vβ Repertoire Kit, BC) in combination with CD3-specific mAbs (BD Biosciences) and isotype control mAbs (BD Biosciences) (31). Samples were analyzed using a BD LSRFortessa X-20. Flow cytometric analysis was performed using FACSDiva software (BD Biosciences). For each sample, at least 20,000 events were analyzed.

[0251] TCR V beta (β) spectrum

[0252] To assess the relative TCR Vβ repertoire distribution between NT and CAR-modified T cells at day 15, we used Beta Mark Kit (Beckman Coulter). This method uses a multiparameter analysis tool designed for the quantitative determination of the TCR Vβ repertoire of human T lymphocytes by flow cytometry.

[0253] CFSE dilution assay

[0254] To assess whether CAR-CD30 T cells proliferate only in the presence of specific antigens or cytokines, T cells were labeled with the fluorescent cell staining dye carboxyfluorescein succinimidyl ester (CFSE) using CellTrace TM Flow cytometry was performed using CFSE Cell Proliferation Kit (Invitrogen).

[0255] Chromium release assay. The cytotoxic activity of transduced effector cells was assessed using a 6-hour chromium release assay as previously described (9). Target cells (Karpas 299, HDML-2, L428, and BV173) were treated with radioactive chromium ( 51 Cr, PerkinElmer, cat no NEZ030S) were labeled, washed, and then co-cultured with CAR T cells at different ratios for 4 hours. The co-culture supernatant was incubated in Microbeta 2 The cells were analyzed on a 2450 Microplate Counter (Pekin Elmer). The average percentage of specific lysis of the three wells was calculated as follows: [(experimental release-spontaneous release) / (maximum release-spontaneous release)] x100.

[0256] Co-culture assay. In the co-culture assay, control non-transduced (NT) and CAR-CD30 T lymphocytes were cultured at 1x10 6 Cells / well were plated in 24-well plates at the indicated effector cell:target cell (E:T) ratios. After 7 days of incubation at 37°C, tumor cells and T cells were collected and residual tumor cells and T cells were assessed by fluorescence activated cell sorting (FACS) analysis based on CD3 expression (effector T cells) and GFP or CD30+ (tumor cell lines).

[0257] ELISA and flow cytometry bead array

[0258] IFNγ production was quantified by specific ELISA using a commercially available kit (R&D Systems, Pepro-Tech, Rocky Hill, NJ). Supernatants for ELISA testing were collected from the co-culture assays.

[0259] In vivo experiments

[0260] All in vivo experiments complied with international, EU and national ethical requirements and were approved by the Italian Health Minster (N°88 / 2016-PR).

[0261] In vivo NHL mouse model (Karpas 299)

[0262] Six-week-old NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ strain; Charles River) were implanted with 0.2×10 6 CD30+Karpas299-F-Luc.GFP. Three days later, when the tumor light emission was continuously measurable, mice were injected intravenously with 10×10 6Control (non-transduced, NT) lymphocytes or T cells genetically modified with CAR.CD30.ΔCD34.28.4.1BB.ζ (28.4.1BB.ζ) or CAR.CD30.ΔCD34.CD28.OX40.ζ (28.OX40.ζ) were grown in IL2 or in a mixture of IL7 / IL15 for 12-15 days. Tumor growth was assessed using an IVIS imaging system (Xenogeneic). The signal intensity of the tumor was measured as total photons / sec / cm2 / sr (p / s / cm2 / sr). Less than 5x10 5 Bioluminescent signals of p / sec / cm2 / sr (measured in mice without tumors) were considered negative. In vivo experiments were performed for 140 days. Circulating T cells in the peripheral blood of mice were assessed regularly.

[0263] Rechallenge model: establishing long-term immune memory in a mouse model of NHL.

[0264] Mice transplanted with the NHL CD30+Karpas299-F-Luc.GFP tumor cell line and treated with one dose of CAR.CD30 T cells were monitored for 140 days, and complete tumor eradication (less than 5x10 5 p / sec / cm2 / sr.) for at least 70 days and they were considered cured. To evaluate the establishment of long-term immune memory, 0.2x10 6 The cured mice were re-challenged with CD30+Karpas299-F-Luc.GFP tumor cell line. These mice were followed for at least 110 more days. A new group of control mice (CTR mice) were added to the experiment as a positive control for tumor implantation. Circulating T cells in the peripheral blood of mice were evaluated before and after re-challenging CD30+ tumors. Mice were euthanized on day 250.

[0265] In vivo HL mouse model (L428)

[0266] 2x10 6 CD30+L428-FF-Luc.GFP was transplanted into 6-week-old NSG mice. After 6 days, when the light emission of the tumor was continuously measurable, the mice received an intravenous (iv) injection of 10×10 6 Control (non-transduced, NT) lymphocytes or T cells genetically modified with CAR.CD30.ΔCD34.28.4.1BB.ζ (28.4.1BB.ζ) or CAR.CD30.ΔCD34.CD28.OX40.ζ (28.OX40.ζ) (grown in IL2 or in a mixture of IL7 / IL15 for 12-15 days). Tumor growth was assessed using an IVIS imaging system (Xenogeneic).

[0267] Statistical analysis

[0268] Statistical evaluation was performed using GraphPad Prism (GraphPad Software), and differences between groups yielding P values ​​< 0.05 were considered significant.

[0269] When multiple comparison analysis was required, statistical significance was assessed by repeated measures ANOVA followed by a log-rank (Mantel-Cox) test for multiple comparisons. Mouse survival data were analyzed using Kaplan-Meier survival curves, and statistically significant differences were measured using Fisher's exact test. No valuable samples were excluded from the analysis. Animals were excluded only in cases of death after tumor implantation but before T cell infusion. Neither randomization nor blinding was performed in the in vivo study. However, mice were matched based on tumor signals in the control and treatment groups before infusion of control or genetically modified T cells. In order to compare tumor growth over time, bioluminescent signal intensity was collected in a blinded manner. The bioluminescent signal intensity was log-transformed and then compared using a two-sample t-test. Analysis by pathologists aimed at quantifying tumor volume was performed in a blinded manner.

[0270] result

[0271] Generation and identification of CAR-CD30 T cells

[0272] Two effective third-generation CAR-CD30 (CAR-CD30) T cells have been produced, which contain a murine antibody single-chain variable fragment (scFv) derived from IgG (AC10) in the same reading frame as CD28, a second co-stimulatory domain represented by 4-1BB or OX40, and a signaling domain CD3-ζ chain (ζ). As a selective marker, a small molecule derived from the phosphoglycoprotein CD34 (ΔCD34) was added, Figure 1 A. Activated T cells (ATCs) were established from 6 healthy donors, grown in CTL complete medium containing IL2 or IL7 / IL15 mixture, and transduced with retroviral supernatant encoding CAR.CD30.ΔCD34.28.4.1BB.ζ (28.4.1BB.ζ) or CAR.CD30.ΔCD34.CD28.OX40.ζ (28.Ox40.ζ) SFG vectors, respectively. Non-transduced (NT) ATCs were cultured in parallel as negative controls. ATCs were efficiently expressed by flow cytometry using CD34 antibody (clone QBEnd10) (as shown in representative Figure 1 B), or alternatively, protein L reagent (32) ( Figure 1 C) Detection of transduced T cells. Figure 1As shown in D, T cells transduced with 28.OX40.ζ or 28.4.1BB.ζ constructs expressed high levels of CAR-CD30. However, on day 5, in T cells transduced in IL.2, the transduction level of the vector encoding 28.OX40.ζ(IL2) was significantly higher than that of the vector encoding 28.4.1BB.ζ(IL2) (87.3%±5.1% vs. 76.1%±9.8%, respectively, p<0.05; (mean±standard deviation (SD) is reported here and throughout the article unless otherwise stated). Similar results were obtained for T cells transduced in IL7 / IL15. On day 5, the transduction level of 28.OX40.ζ(IL7 / IL15) T cells (84.1%±2.2%) was also higher compared with 28.4.1BB.ζ(IL7 / IL15) T cells (78.6%±3.9%), p<0.05. Both CD3+CAR.CD30 T cells, the transduction level dropped significantly at day 15 (65.1% ± 10.9% for 28.OX40.ζ(IL2) and 49.2% ± 13.3% for 28.4.1BB.ζ(IL2), respectively, p<0.05, blue asterisk), and then remained stable for at least two weeks to day 30 (73.0% ± 6.4% vs. 55.9% ± 18.1% for 28.OX40.ζ(IL2) and 28.4.1BB.ζ(IL2), respectively). Switching to IL7 / IL15 significantly improved the stability and transduction level of CD4+ and / or CD8+ T cells, regardless of which construct was used.

[0273] In CAR T cells, the CD4+ / CD8+ ratio decreased by one week ( Figure 1 E(CAR+CD4+)), the results favor CAR+CD8+( Figure 1 F). CD8+ was found to be dominant in both CAR.CD30 T cells on day 15. The same trend was observed for NT T cells. Figure 1 G) or IL7 / IL15( Figure 1 When cultured in NT (H), the expansion rate of the modified T cells did not change significantly compared with that of NT T cells. However, in long-term in vitro culture, the IL7 / IL15 mixture significantly increased the expansion rate of NT ( Figure 1 I) and transduced T cells ( Figure 1 L and 1M) fold amplification.

[0274] Memory and exhaustion of genetically modified CAR-CD30 T cells

[0275] To evaluate the impact of specific co-stimulatory domains and cytokines on CAR.CD30 T cell differentiation, the expression of memory markers on CD3+CAR-T cells was characterized. At day 15 of culture, the majority of expanded T cells generated after CD3 / CD28 stimulation and culture with IL2 had an effector memory (EfM) phenotype, with no significant differences between NT and both CAR.CD30 T cells. However, switching to IL7 / IL15 significantly reduced central memory (CM) differentiation in CAR.CD30 T cells in favor of EfM and effector terminal (EfT) differentiation. After 30 days of in vitro culture, only EfM and EfT differentiation was observed. There was a significant (only in T cells cultured in IL2) reduction in CAR.CD30 T cells.

[0276] The pattern of inhibitory receptors (PD-1, LAG3, and TIM3) co-expressed by CAR+ T cells was also evaluated to determine their exhaustion status ( Figure 2 ). It was observed that when T cells were transduced with (28.OX40.ζ) in IL2, significant upregulation of PD1 and TIM3 was observed on day 15 of in vitro culture relative to NT or CARCD30.28.4-1BB.ζ T cells ( Figure 2 ). Notably, the switch from IL2 to IL7 / IL15 significantly reduced the expression of PD1 in CARCD30.28.OX40.ζT cells (15.33% ± 4.75% for IL2 and 7.95% ± 4.93% for IL7 / IL15, respectively, p = 0.006), but upregulated the expression of TIM3 (2.45% ± 0.41% for IL2 and 7.28% ± 1.26% for IL7 / IL15, respectively, p = 0.009). After +30 days of in vitro culture, the exhaustion of NT and CARCD30-modified T cells was generally determined by PD1 and TIM3 expression.

[0277] Safety of CAR-CD30 T cells

[0278] To evaluate the impact of retroviral modification or culture conditions on the safety of modified T cells, basal proliferation or cytokine or / and antigen-specific proliferation was assessed for NT or CAR-CD30 T cells. T cells were labeled with the fluorescent cell dye CFSE on day 0 and inoculated for 5 days in the presence or absence of cytokines or co-cultured in the presence of tumor cell lines CD30 positive (Karpas299) or tumor cell lines CD30 negative (BV173). Basal proliferation of CD3+ cells was assessed, as was basal proliferation of CD8+ cells and CD4+ cells.

[0279] NT T cells only in the presence of IL2 (50 U / ml) ( Figure 3A-II) or IL7 (10 ng / ml) / IL15 (5 ng / ml) combination ( Figure 3 A-III) proliferation when cultured as shown by CFSE dye dilution. Proliferation is preferentially due to CD8+ cells (middle panel). When they are cultured in the Karpas299 cell line ( Figure 3 B-IV and Figure 3 C-IV) but not in the BV173 tumor cell line ( Figure 3 BV and Figure 3 Specific CFSE dye dilution was also observed when co-cultured in the presence of IL2 (CV) or when there was no cytokine (BI and CI), CAR-CD30 T cells were contrasted. In addition, to evaluate the polyclonal expansion of cultured T cells, it was determined whether there was consistency in TCR Vβ spectrum distribution between NT and CAR-CD30 T cells grown in IL2 or IL7 / IL15 on days 15 and 30 of in vitro culture. No significant preferential expansion of cytokine or CAR dependence of specific clones was observed, even when cells were cultured for 30 days (data not shown).

[0280] CAR-CD30 T cells can effectively lyse CD30+ lymphomas in vitro, but also solid tumors (such as medulloblastoma and sarcoma cell lines)

[0281] The ability of CAR-CD30 T cells to kill CD30+ human tumor cell lines was then evaluated.

[0282] It is well known that the cell membrane protein CD30 is expressed in two Hodgkin lymphoma cell lines ( Figure 4 A (HDML-2, L428) and 1 NHL cell line (Karpas 299). Interestingly, CD30+ was also expressed in 1 of 5 sarcoma cell lines (CD30 positive: RD, CD30 negative: SK-ES-1, A673, CW9019 and CT-10) Figure 4 B; In 1 of the 2 medulloblastoma cell lines tested (CD30 positive: DAOY, CD30 negative: D283) Figure 4 C; and one T lymphocyte cell line T2 (CEM.T2, but not one B cell leukemia cell line BV173) ( Figure 4 D).

[0283] In addition, the following CD30+ tumor cell lines also express high levels of PDL1 (KAPAS 299 and HDML-2). To evaluate the relative impact of PDL1 on the intrinsic resistance of CD30+ tumor cell lines to be killed by CARCD30 T cells, HLL428 (PDL1 negative) was transduced to stably express PDL1 ( Figure 4 E).

[0284] exist 51 In the Cr release assay, both CARCD30 T cells were able to specifically and efficiently lyse CD30+ lymphomas, such as Karpas 299 ( Figure 5 A) HDML-2 Figure 5 B) L428( Figure 5 C), but not the CD30- leukemia cell line BV173 ( Figure 5 F). Notably, both CAR-CD30 T cells were also shown to kill DAOY ( Figure 5 D). As a CD30 negative control for solid tumors, medulloblastoma D283 ( Figure 5 E) and leukemia cell line BV173 ( Figure 5 F). Switching from IL2 to a mixture of IL7 / IL15 did not improve the potency of CAR-CD30 T cells ( Figure 6 AF).

[0285] As shown in representative donors, specificity and comparable titers of both CAR-CD30 T cells were observed on GFP+ lymphoma cell lines using a 1:1 ratio of effector cells to target cells (R1:1) in 7-day long-term co-culture ( Figure 7 AD). It is worth noting that compared with the L428 wild type ( Figure 7 C), PDL1 expression in L428 tumor cell line ( Figure 7 D) did not significantly affect the sensitivity of the L428 tumor cell line to the two CAR-CD30 T cells. In addition, significant tumor control was also observed in other CD30+ tumor cell lines, such as in the CD30+GFP+ leukemia cell line ( Figure 7 E), but not observed in CD30-negative BV173 ( Figure 7 F); in CD30+GFP tumor DAOY medulloblastoma cell line ( Figure 7 G) and in CD30+RD sarcoma cell lines ( Figure 7 I), but not in the CD30-negative CD45-negative D283 medulloblastoma cell line ( Figure 7 H). It is worth noting that for solid tumors, the killing ability of 28.OX40.ζ is significantly better than that of 28.4.1BB.ζ( Figure 7 G and 7I), but not in CD30-negative SK-ES-1 ( Figure 7 J). These results were not found in IL2 ( Figure 7 K) or IL7 / IL15( Figure 7 L) was confirmed in 6 different donors.

[0286] In this study, when tested in a standard chromium cytotoxicity assay ( Figure 5-6 ) or long-term co-culture ( Figure 7 ) tested, cytokine culture conditions did not affect the in vitro specific cell lytic activity of CAR-CD30 T cells against CD30+ cells. To evaluate the actual efficacy of the lytic titer between the two CAR-CD30 T cells, the in vitro long-term co-culture capacity assay was strengthened by increasing the target tumor cells from R1:1 to R1:32. Notably, at low effector cell / target cell ratios, the activity of CAR.CD30 T cells showed significant improvement in in vitro tumor control over 28.OX40.ζ(IL2) T cells against Karpas299 (at R1:8 and R1:16) and HDML-2 cell lines (at R1:8, R1:16, and R1:32, respectively). Figure 8 AB), but for L428, no significant difference in cytolytic activity was observed between 28.OX40.ζ(IL2) and 28.4.1BB.ζ(IL2).

[0287] For CAR-CD30 T cells (IL7 / IL15), superior lytic activity of 28.OX40.ζ was demonstrated, especially at lower effector / target cell ratios in the Karpas 299 and HDML-2 cell lines, although it failed to reach significance relative to 28.4-1BB.ζ (see Table 2 , respectively). Figure 8 D and 8F),

[0288] Overall, these data demonstrate the superior CD30+ specific activation of 28.OX40.ζ(IL7 / IL15) when co-cultured with Karpas 299 and HDML-2, in terms of specific IFN-γ production, as assessed in supernatants collected 24 h after co-culture titer assays ( Fig. 9 AB and 9D-E). When co-cultured with the CD30+ tumor cell line L428, both CAR-CD30 T cells produced specific and equivalent levels of IFN-γ ( Fig. 9 C and 9F).

[0289] Establishment of long-term immune memory in a mouse model of NHL.

[0290] Using immunodeficient NSG mice, the in vivo efficacy and persistence of CAR-CD30 T cells were compared with the NHL Karpas299-FF-Luc.GFP tumor cell line in a xenograft model. Fig.10 A) Make a comparison.

[0291] In the group treated with NT(IL2)T cells, the bioluminescence of the tumors gradually increased ( Fig.10 BC), and using 10x10 6 Significant tumor control was observed in mice treated with CAR-CD30 T cells (IL2), as measured by reduction or control of bioluminescent signals. The median survival of mice treated with NT cells (IL2) reached only 45.5 days, while 30% of mice treated with 28.4.1BB.ζ(IL2) and 60% of mice treated with 28.OX40.ζ(IL2) experienced long-term tumor control, respectively. Fig.10 D). Specifically, the median survival of mice treated with 28.4.1BB.ζ(IL2) was 58 days (p=0.05), whereas the median survival of mice treated with 28.OX40.ζ(IL2) was not determined ( Fig.10 D).

[0292] After 140 days of treatment, cured mice (3 mice treated with 28.4.1BB.ζ(IL2) and 6 mice treated with 28.OX40.ζ(IL2) on day 0) were re-challenged with the same tumors (0.2x10 6 ) and the mice were followed for an additional 100 days. In this set of experiments, six new mice were added as positive control mice (CTR mice) that received CD30+Karpas299-F-Luc.GFP ( Fig.11 A). Rapid tumor progression was noted in CTR mice (line with white circles) and 28.4-1BB.ζ(IL2)-treated mice (line with white squares) by assessing the bioluminescence of Karpas299-F-Luc.GFP injected again on day +140 ( Fig.11 In contrast, in 28.OX40.ζ(IL2) treated mice (line with black squares), 66.67% (4 of 6) of mice eradicated the re-challenged tumors a second time 40 days after the initial tumor expansion, with a significant survival benefit ( Fig.10 D). To confirm the establishment of long-term immune memory, blood was sampled and analyzed in treated mice after the first tumor infusion (days 6, 56, 103, and 132) and the second tumor infusion (days 180, 221, and 254). In particular, a significant expansion of circulating T cells associated with lymphoma infusion (2.49% ± 1.03%, p < 0.005) was observed for mice treated with 28.OX40.ζ(IL2) compared to mice treated with 28.4-1BB.ζ(IL2) (0.275% ± 0.109%) or NT(IL2) (0.347% ± 0.071%). Fig.11E). Interestingly, after the first tumor eradication, when circulating T cells in the cohort of mice treated with CAR-CD30 T cells were assessed at day +132, only 0.022% ± 0.027% and 0.090% ± 0.1355% of T cells were quantified for 28.OX40.ζ(IL2) and 28.4.1BB.ζ(IL2), respectively. 40 days after tumor re-challenge (day 180), a slow but impressive expansion of circulating 28.OX40.ζ(IL2) T cells (7.216% ± 11,259%) was found relative to 28.4.1BB.ζ(IL2) (0.093% ± 0.129%). After the second complete tumor eradication, circulating 28.OX40.ζ(IL2) T cells simultaneously decreased to undetectable percentages measured at day 254 (0.001% ± 0.0018%).

[0293] Evaluation of the potency and persistence of CAR-CD30 T cells in a mouse model of NHL.

[0294] We next evaluated the effect of cytokine administration on the in vivo efficacy of CAR-CD30 T cells grown in IL2 or IL7 / IL15 for 15 days against the more stimulatory HL L428. Mice received 2x10 6 L428-FF-Luc.GFP cell line, and when the light emission of the tumor continued to be measurable, the mice were intravenously injected with NT or genetically modified T cells. To evaluate the persistence of human circulating T cells, blood samples were taken from treated mice on days +15, +30, +56, +80, +100, +130, +160 ( Fig.12 A). In HL tumor-bearing mice treated with NT T cells, the bioluminescence of the L428 cell line rapidly increased to 5 log( Fig.12 B and Fig.12 C), and the mice died or were sacrificed due to morbidity. Macroscopic analysis of organs from sacrificed mice showed that large tumor masses preferentially appeared in the liver. The median survival time of HL tumor-bearing mice treated with 28.4-1BBζ(IL2) was slightly longer (79±10 days) than that of tumor-bearing mice treated with NT(IL2) and NT(IL7 / IL15) (52±9 days and 58±1 days, respectively). Fig.12 BE). IL7 / IL15 switching did not enhance the cytotoxic activity of 28.4-1BBζ(IL7 / IL15) ( Fig.12BD). The median survival of HL tumor-bearing mice treated with 28.OX40.ζ(IL2) was significantly improved to 133±4 days relative to mice treated with NT(IL2) or 28.4-1BB.ζ(IL2). Although the median survival of HL tumor-bearing mice treated with 28.OX40.ζ(IL7 / IL15) was uncertain, the overall survival rate was not significantly different between mice treated with the two 28.OX40.ζCAR T cells (p=0.0876, Fig.12 E). To assess the persistence of the infused T cells, circulating T cells were regularly monitored throughout the experiment in NSG mice bearing L428 tumors and treated with NT or genetically modified T cells ( Fig.12 F). Although mice treated with NT T cells also showed a significant increase in human circulating CD45+CD3+ cells, with a peak at day 56 assessment (NT(IL2) and NT(IL7 / IL15) of 26.69%±7.02% and 5.97%±9.63%, respectively), no tumor control was observed.

[0295] On day 80, very high numbers of circulating human T cells (CD45 + CD3 + =20.56%), but the transduction level was low (CD3 + CD34 + =4,57%). In contrast, in all four mice treated with 28.OX40.ζ(IL2), circulating T cells (CD45+CD3+) averaged 9.79%±5.24% (range 3.08%-15.37%) on day 80, and the transduction stable level was 34.23%±5.87%. Interestingly, in mice treated with 28.OX40.ζ(IL7 / IL15), significantly reduced circulating T cell levels (0.92%±0.56%, p=0.0065) and higher transduced T cell percentages (41.89%±2.25%, p=0.0300) were detected. Injected tumors were completely eradicated in mice treated with 28.OX40.ζCART cells, followed by a decrease in circulating T cells. The percentage of circulating CAR-CD30 T cells remained stable within the first 100 days. At day 165, circulating T cells were found in only two of the four treated mice (0.06% ± 0.02%). All four mice recovered at this time. In both mice, CAR-CD30 T cells showed an even distribution between CD4+ and CD8+, as central memory (CM), defined as CD45RA-CD62L+, and effector memory (EM), defined as CD45RA-CD62L-( Fig.12 G).

[0296] Embodiment 2: Tumor modulation of memory and exhaustion conditions in CAR.CD30 T cells (28.OX40.ζT cells and 28.4-1BB.ζT cells) according to the present invention

[0297] Materials and methods

[0298] Stress co-culture assay

[0299] In the stress co-culture experiment, non-transduced (NT) control and CAR-CD30 T lymphocytes (28.OX40.ζT cells and 28.4-1BB.ζT cells) were cultured at 1x10 6 Cells / well were plated in 24-well plates at the indicated effector cell: target cell (E:T) ratios of 1:1. To assess how long CAR.T cells were able to eliminate tumors when added more than once, tumor cells were added to the wells on days 0, 5, 10, and 15. The persistence of residual tumor cells and T cells was then assessed by FACS analysis based on CD3 expression (effector T cells) and GFP or CD30+ (tumor cell lines) up to 20 days of co-culture.

[0300] ELISA

[0301] Supernatants of E:T co-cultures were collected at 24 h to assess levels of interferon gamma (IFNγ), interleukin 2 (IL-2), interleukin 10 (IL-10), and tumor necrosis factor-α (TNF-α) using the ELLA method (R and D Systems).

[0302] result

[0303] To evaluate the lytic potency of CAR.CD30 T cells in a more complex comparison, the co-culture conditions were “enhanced” by re-stimulating the tumors (Karpas 299) every five days (on days 0, 5, 10, and 15 of co-culture). Fig.13A). At each time point (on days 1, 6, 11 and 16 of co-culture), the percentage of residual tumor and the behavior of CAR.CD30 T cells were evaluated by assessing single chain expression and relative mean fluorescence intensity (MFI), memory and exhaustion status, and relative specific cytokine production (such as IFNγ, TNFα, IL-2 and IL-10). Even after multiple exposures to Karpas 299, both CAR.CD30 T cells showed high tumor control ability. Although no significant difference in lytic activity between them was observed on days +5 and +10, 28.OX40.ζT cells exhibited strong tumor control on day 20 (8.6% ± 5.3% for 28.OX40.ζT cells and 27.9% ± 29.5% for 28.4-1BBζT cells) ( Fig.13 B). Interestingly, after the first co-culture, the percentages of both CAR.CD30 molecules increased significantly, from 66.9% ± 15.23% (day 5) to 93.8% ± 11.3% (day 10) for 28.4-1BB.ζT cells, p = 0.022, and from 74.9% ± 11.3% (day 5) to 93.2% ± 11.3% (day 10), p = 0.007 for 28.OX40.ζT cells. In fact, the next tumor re-challenge had a negative impact only on the transduction levels of 28.4-1BB.ζT cells (from 93.8% ± 3,06% (day 10) to 67.9% ± 32.33% (day 20)), while in 28.OX40.ζT cells the percentages remained stable (from 93.22% ± 2,75% (day 10) to 92.53% ± 3,45% (day 20)) ( Fig.13 C).

[0304] In addition, the MFI of 28.OX40.ζT cells was significantly higher than that of 28.4-1BB.ζT cells at each time point (11294.83±580453 vs 24004±11365.6 on day 0, p=0.006; 16883±6703.47 vs 40671.2±12162.2 on day 5, p=0.004; 7382.75±4042.01 vs 25329.75±14746.76 on day 10, p=0.037; 11729.83±11158.66 vs 29552.83±234643.32 on day 15, p=0.035; 8344.5 vs 29552.83±234643.32 on day 20, p=0.0 23834.83±11272.55,p=0.001)( Fig.13 D).

[0305] In addition, cytokine profiles confirmed that 28.OX40.ζT cells were activated more rapidly, resulting in significant IFNγ production (4768.86 pg / ml±3708.34 pg / ml) compared to 28.4-1BB.ζT cells (2699.54 pg / ml±2517.10 pg / ml, p=0.012) on day 20 of stressed co-culture ( Fig.13 E), and TNFα production (8439.32 pg / ml±6187.27 pg / ml), compared to 28.4-1BB.ζ T cells (2983.40 pg / ml±2497.48 ng / ml, p=0.013) ( Fig.13 F).

[0306] In addition, in IL-2 production, the difference between CAR.CD30 T cells was significantly different before day +10, which corresponds to tumor stimulation number II) (28.OX40.ζ T cells were 8480.82 pg / ml ± 5065.76 pg / ml) compared to 28.4-1BB.ζ T cells (2778.64 pg / ml ± 3852.82 pg / ml, p = 0.006) ( Fig.13 G). Another method to evaluate tumor control is to measure the cytokine IL-10 (produced by the Karpas 299 cell line). Fig.13 As shown in H, starting from day 10, the levels of IL-10 detected in the culture medium of NT T cells (white bars) were similar to those detected in the tumor alone (horizontal lines), while high levels of IL-10 were only detected 24 hours after the first co-culture with CAR CD30 T cells (stimulation number I) ( Fig.13 H).

[0307] The results showed that during the process of CD30+ lymphoma being added continuously for up to 4 times ("stressed" co-culture), CAR.CD30 T cells with CD28.OX40 co-stimulatory domains were able to control tumors more effectively than 4.1BB co-stimulatory domains, and when co-cultured with the Karpa299 tumor cell line, they could produce significantly higher levels of IFN-γ, TNF-α and IL-2.

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Sequence Listing <110> Baby Jesus Children's Hospital <120> CAR-CD30 T cells for the treatment of CD30+ tumors <130> PCT41700 <150> IT102018000003464 <151> 2018-03-13 <160> 43 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Signal peptide <400> 1 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys <210> 2 <211> 111 <212> PRT <213> Artificial sequence <220> <223> AC10 VL sequence <400> 2 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Phe Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 3 <211> 117 <212> PRT <213> Artificial sequence <220> <223> AC10 VH sequence <400> 3 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Thr Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Asn Tyr Gly Asn Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ala 115 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> trackable marker DeltaCD34 <400> 4 Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser 1 5 10 15 <210> 5 <211> 272 <212> PRT <213> Artificial sequence <220> <223> Traceable label DeltaCD19 <400> 5 Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp Asn Ala Val 1 5 10 15 Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln Gln Leu Thr 20 25 30 Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu Ser Leu Gly 35 40 45 Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile Trp Leu Phe 50 55 60 Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu Cys Gln Pro 65 70 75 80 Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr Val Asn Val 85 90 95 Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp Leu Gly Gly 100 105 110 Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser Pro 115 120 125 Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp Arg 130 135 140 Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro Arg Asp Ser 145 150 155 160 Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser Thr 165 170 175 Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser Arg Gly Pro 180 185 190 Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser Leu Leu Ser 195 200 205 Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp Val Met Glu 210 215 220 Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala Gly Lys Tyr 225 230 235 240 Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu Glu Ile Thr 245 250 255 Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly Gly Trp Lys 260 265 270 <210> 6 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Traceable marker NGFR <400> 6 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> 7 <211> 40 <212> PRT <213> Artificial sequence <220> <223> HingeCD8alpha <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 35 40 <210> 8 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Hinge CD28 <400> 8 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> 9 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> Hinge CH2CH3 <400> 9 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> 10 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Hinge CH3 <400> 10 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> 11 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Spacer <400> 11 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 1 5 10 <210> 12 <211> 29 <212> PRT <213> Artificial sequence <220> <223> HingeCD8alpha <400> 12 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 20 25 <210> 13 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Transmembrane domain CD28TM <400> 13 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> 14 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Transmembrane domain CD8aTM <400> 14 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 1 5 10 15 Leu Leu Ser Leu Val Ile Thr 20 <210> 15 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Mouse IgG3 upper hinge (mIgG3UH) <400> 15 Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser 1 5 10 <210> 16 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Mouse IgG3 upper hinge (mIgG3UH)2 <400> 16 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> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> (G4S)2 connector <400> 17 Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 18 <211> 20 <212> PRT <213> Artificial sequence <220> <223> (G4S)4 connector <400> 18 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> 19 <211> 7 <212> PRT <213> Artificial sequence <220> <223> G4SG2 connector <400> 19 Gly Gly Gly Gly Ser Gly Gly 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial sequence <220> <223> G3SG4 connector <400> 20 Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> twenty one <211> 41 <212> PRT <213> Artificial sequence <220> <223> CD28 cytoplasmic sequence <400> twenty one 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> twenty two <211> 42 <212> PRT <213> artificial sequence <220> <223> CD137 (4-1BB) sequence <400> 22 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> 23 <211> 112 <212> PRT <213> artificial sequence <220> <223> CD3-Zeta chain <400> 23 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> twenty four <211> 36 <212> PRT <213> Artificial sequence <220> <223> OX40 sequence <400> twenty four 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> 25 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CD8a cytoplasmic <400> 25 Leu Tyr Cys Asn His Arg Asn 1 5 <210> 26 <211> 542 <212> PRT <213> Artificial sequence <220> <223> CD30 Chimeric Antigen Receptor Molecule <400> 26 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 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu 20 25 30 Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln 35 40 45 Ser Val Asp Phe Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys 50 55 60 Pro Gly Gln Pro Pro Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu 65 70 75 80 Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Ser Asn Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Gly Gly Gly Ser Gly Gly Gly Gly Gln Ile Gln Leu 130 135 140 Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala Ser Val Lys Ile 145 150 155 160 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile Thr Trp 165 170 175 Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Trp Ile Tyr 180 185 190 Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala 195 200 205 Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Phe Met Gln Leu Ser 210 215 220 Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala Asn Tyr Gly 225 230 235 240 Asn Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 245 250 255 Ala Gly Ser Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr 260 265 270 Asn Val Ser Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280 285 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 290 295 300 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 305 310 315 320 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 325 330 335 Ile Thr Leu Tyr Cys Asn His Arg Asn Glu Phe Arg Ser Lys Arg Ser 340 345 350 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 355 360 365 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 370 375 380 Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 385 390 395 400 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 405 410 415 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 420 425 430 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 435 440 445 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 450 455 460 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 465 470 475 480 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 485 490 495 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 500 505 510 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 515 520 525 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 530 535 540 <210> 27 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> CD30 Chimeric Antigen Receptor Molecule <400> 27 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 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu 20 25 30 Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln 35 40 45 Ser Val Asp Phe Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys 50 55 60 Pro Gly Gln Pro Pro Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu 65 70 75 80 Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Ser Asn Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Gly Gly Gly Ser Gly Gly Gly Gly Gln Ile Gln Leu 130 135 140 Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala Ser Val Lys Ile 145 150 155 160 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile Thr Trp 165 170 175 Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Trp Ile Tyr 180 185 190 Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala 195 200 205 Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Phe Met Gln Leu Ser 210 215 220 Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala Asn Tyr Gly 225 230 235 240 Asn Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 245 250 255 Ala Gly Ser Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr 260 265 270 Asn Val Ser Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280 285 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 290 295 300 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 305 310 315 320 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 325 330 335 Ile Thr Leu Tyr Cys Asn His Arg Asn Glu Phe Arg Ser Lys Arg Ser 340 345 350 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 355 360 365 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 370 375 380 Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro 385 390 395 400 Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp 405 410 415 Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala 420 425 430 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 435 440 445 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 450 455 460 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 465 470 475 480 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 485 490 495 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 500 505 510 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 515 520 525 His Met Gln Ala Leu Pro Pro Arg 530 535 <210> 28 <211> 1629 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO:26 <400> 28 atggagtttg ggctctcctg gctcttcctg gtcgcgattc tgaagggggt ccagtgttca 60 cgagatatcg tcctgactca gagtcctgcc agcctggcag tctccctggg acagagagct 120 accataagtt gtaaagcatc acagtctgtt gatttcgatg gcgacagcta tatgaattgg 180 taccagcaaa aacccggcca gcccccgaaa gttttgatct atgcagcctc taacttggaa 240 agcggcattc ctgcgcgatt cagtggcagc gggagtggta cagatttcac cctgaacata 300 cacccagtcg aagaggagga cgcagccaca tattactgcc aacaatctaa cgaggatcca 360 tggacttttg ggggcggcac taaactcgaa atcaagggcg gaggttcagg cggaggaggg 420 cagattcaac tgcagcaatc aggacccgag gtggtcaaac caggtgccag tgtcaagata 480 tcttgcaagg catccggata tacatttacc gactattaca ttacctgggt caagcagaaa 540 cccggggcaag gacttgaatg gattggatgg atctaccctg gtagcggcaa caccaataac aacgaaagt ttaaagggaa ggcaaccctg actgtagaca cctccagctc cacagcattc atgcagctct cctcactgac ctccgaggac acagcagtgt atttctgtgc taattacggt aattactggt tcgcctattg gggccaggga acccaagtga ccgtttcagc tggatccgaa 780 cttcctactc aggggacttt ctcaaacgtt agcacaaacg tagtcccgc cccaagaccc 840 cccacacctg cgccgaccat tgcttctcaa cccctgagtt tgagacccga ggcctgccgg 900 ccagctgccg gcggggccgt gcatacaaga ggactcgatt tcgcttgcga catctacatc tgggctcccc tcgctggcac ctgtggggtg ctgctgctgt cactcgtgat caccctttat 1020 tgcaaccatc gaaacgaatt cagaagtaaa cggtcaaggc ttctgcacag cgattatatg aatatgacac caagaagacc tggtccaacc cggaaacact atcagcccta cgcgccccct agagacttcg cagcataccg ctctaagaga gggagaaaaa aattgctcta tatttttaaa caccattta tgaggcccgt acagacaact caggagagg atggctgtag ttgccgcttc ccagaggagg aggaaggagg ctgcgagttg agagttaaat tcagtagaag tgcggatgcg 1320 cctgcttacc agcagggcca gaaccaactg tacaatgaac tgaatctcgg gcgccgagaa 1380 gagtatgacg tcctcgataa gcggaggggt agggatcctg aaatgggtgg gaagccaaga 1440 agaaaaaacccccaggaagg actgtataac gaacttcaga aggacaagat ggcagaggcc 1500 tactctgaga ttggcatgaa aggcgaacga cggcgcggta aaggtcatga cgggctgtac 1560 cagggcctgt ccacagcgac gaaggacact tacgacgccc tgcacatgca ggcactcccc 1620 cccaggtga 1629 <210> 29 <211> 63 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO: 1 <400> 29 atggagtttg ggctctcctg gctcttcctg gtcgcgattc tgaagggggt ccagtgttca 60 cga 63 <210> 30 <211> 333 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO: 2 <400> 30 gatatcgtcc tgactcagag tcctgccagc ctggcagtct ccctgggaca gagagctacc 60 ataagttgta aagcatcaca gtctgttgat ttcgatggcg acagctatat gaattggtac 120 cagcaaaaac ccggccagcc cccgaaagtt ttgatctatg cagcctctaa cttggaaagc 180 ggcattcctg cgcgattcag tggcagcggg agtggtacag atttcaccct gaacatacac 240 ccagtcgaag aggaggacgc agccacatat tactgccaac aatctaacga ggatccatgg 300 acttttgggg gcggcactaa actcgaaatc aag 333 <210> 31 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO: 20 <400> 31 ggcggaggtt caggcggagg aggg 24 <210> 32 <211> 333 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO:3 <400> 32 gatatcgtcc tgactcagag tcctgccagc ctggcagtct ccctgggaca gagagctacc 60 ataagttgta aagcatcaca gtctgttgat ttcgatggcg acagctatat gaattggtac 120 cagcaaaaac ccggccagcc cccgaaagtt ttgatctatg cagcctctaa cttggaaagc 180 ggcattcctg cgcgattcag tggcagcggg agtggtacag atttcaccct gaacatacac 240 ccagtcgaag aggaggacgc agccacatat tactgccaac aatctaacga ggatccatgg 300 acttttgggg gcggcactaa actcgaaatc aag 333 <210> 33 <211> 6 <212> DNA <213> Artificial sequence <220> <223> BamH1 restriction enzyme site <400> 33 ggatcc 6 <210> 34 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO:4 <400> 34 gaacttccta ctcaggggac tttctcaaac gttagcacaa acgtaagt 48 <210> 35 <211> 120 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO:7 <400> 35 cccgccccaa gaccccccac acctgcgccg accattgctt ctcaacccct gagtttgaga 60 cccgaggcct gccggccagc tgccggcggg gccgtgcata caagaggact cgatttcgct 120 <210> 36 <211> 69 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO: 14 <400> 36 tgcgacatct acatctgggc tcccctcgct ggcacctgtg gggtgctgct gctgtcactc 60 gtgatcacc 69 <210> 37 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO:25 <400> 37 ctttattgca accatcgaaa c 21 <210> 38 <211> 6 <212> DNA <213> Artificial sequence <220> <223> EcoR1 restriction enzyme cutting site and linker sequence <400> 38 gaattc 6 <210> 39 <211> 123 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO:21 <400> 39 agaagtaaac ggtcaaggct tctgcacagc gattatatga atatgacacc aagaagacct 60 120. ggtccaaccc ggaaacacta tcagccctac gcgcccccta gagacttcgc agcataccgc tct 123 <210> 40 <211> 126 <212> DNA <213> The snowstorm <220> <223> Sequence SEQ ID NO:22 is available <400> 40 aagagagga gaaaaaaatt gctctatatt tttaaacaac catttatgag gcccgtacag acaactcagg aagaggatgg ctgtagttgc cgcttcccag aggaggagga aggaggctgc gagttg 126 <210> 41 <211> 339 <212> DNA <213> The snowstorm <220> <223> Sequence SEQ ID NO:23 is available <400> 41 agcttaat tcagtagaag tgcggatgcg cctgcttacc agcagggcca gaaccaactg 120. tcaatgaac tgaatctcgg gcgccgaga gagtatgacg tcctcgataa gcggaggggt agggatcctg aaatgggtgg aagaaaaacc cccaggagg actgtataac gaacttcaga aggacaagat ggcagaggcc tactctgaga ttggcatgaa aggcgaacga cggcgcggta aaggtcatga cgggctgtac cagggcctgt ccacagcgac gaaggacact 300 tacgacgccc tgcacatgca ggcactcccc cccaggtga 339 <210> 42 <211> 1611 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding SEQ ID NO:27 <400> 42 atggagtttg ggctctcctg gctcttcctg gtcgcgattc tgaagggggt ccagtgttca 60 cgagatatcg tcctgactca gagtcctgcc agcctggcag tctccctggg acagagagct 120 accataagtt gtaaagcatc acagtctgtt gatttcgatg gcgacagcta tatgaattgg 180 taccagcaaa aacccggcca gcccccgaaa gttttgatct atgcagcctc taacttggaa 240 agcggcattc ctgcgcgatt cagtggcagc gggagtggta cagatttcac cctgaacata 300 cacccagtcg aagaggagga cgcagccaca tattactgcc aacaatctaa cgaggatcca 360 tggacttttg ggggcggcac taaactcgaa atcaagggcg gaggttcagg cggaggaggg 420 cagattcaac tgcagcaatc aggacccgag gtggtcaaac caggtgccag tgtcaagata 480 tcttgcaagg catccggata tacatttacc gactattaca ttacctgggt caagcagaaa cccggggcaag gacttgaatg gattggatgg atctaccctg gtagcggcaa caccaataac aacgaaagt ttaaagggaa ggcaaccctg actgtagaca cctccagctc cacagcattc atgcagctct cctcactgac ctccgaggac acagcagtgt atttctgtgc taattacggt aattactggt tcgcctattg gggccaggga acccaagtga ccgtttcagc tggatccgaa 780 cttcctactc aggggacttt ctcaaacgtt agcacaaacg tagtcccgc cccaagaccc 840 cccacacctg cgccgaccat tgcttctcaa cccctgagtt tgagacccga ggcctgccgg 900 ccagctgccg gcggggccgt gcatacaaga ggactcgatt tcgcttgcga catctacatc tgggctcccc tcgctggcac ctgtggggtg ctgctgctgt cactcgtgat caccctttat 1020 tgcaaccatc gaaacgaatt cagaagtaaa cggtcaaggc ttctgcacag cgattatatg aatatgacac caagaagacc tggtccaacc cggaaacact atcagcccta cgcgccccct agagacttcg cagcataccg ctctcgcgat caaagactcc cgcccgatgc ccacaaaccc cctggcgggg gcagctttag gacacccatt caagaagagc aggcagacgc ccacagcacc 1260 ttggccaaaa ttagagttaa attcagtaga agtgcggatg cgcctgctta ccagcagggc 1320 cagaaccaac tgtacaatga actgaatctc gggcgccgag aagagtatga cgtcctcgat 1380 aagcggaggg gtagggatcc tgaaatgggt gggaagccaa gaagaaaaaa cccccaggaa 1440 ggactgtata acgaacttca gaaggacaag atggcagagg cctactctga gattggcatg 1500 aaaggcgaac gacggcgcgg taaaggtcat gacgggctgt accagggcct gtccacagcg 1560 acgaaggaca cttacgacgc cctgcacatg caggcactcc cccccaggtg a 1611 <210> 43 <211> 108 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding SEQ ID NO:24 <400> 43 cgcgatcaaa gactcccgcc cgatgcccac aaaccccctg gcgggggcag ctttaggaca 60 cccattcaag aagagcaggc agacgcccac agcaccttgg ccaaaatt 108 agctttag gacacccatt caagaagagc aggcagacgc ccacagcacc 1260 ttggccaaaa ttagagttaa attcagtaga agtgcggatg cgcctgctta ccagcagggc 1320 cagaaccaac tgtacaatga actgaatctc gggcgccgag aagagtatga cgtcctcgat 1380 aagcggaggg gtagggatcc tgaaatgggt gggaagccaa gaagaaaaaa cccccaggaa 1440 ggactgtata acgaacttca gaaggacaag atggcagagg cctactctga gattggcatg 1500 aaaggcgaac gacggcgcgg taaaggtcat gacgggctgt accagggcct gtccacagcg 1560 acgaaggaca cttacgacgc cctgcacatg caggcactcc cccccaggtg a 1611 <210> 43 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding SEQ ID NO:24 <400> 43 cgcgatcaaa gactcccgcc cgatgcccac aaaccccctg gcgggggcag ctttaggaca 60 cccattcaag aagagcaggc agacgcccac agcaccttgg ccaaaatt 108

Claims

1. CD30 chimeric antigen receptor, which consists of the following substances from the N-terminus to the C-terminus: a) signal peptide consisting of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 1) and connected to by a first linker of two or three amino acids; : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : A second linker of the VL and VH sequences consists of which the second linker is a proline-rich rigid linker or a glycine-rich flexible linker connected directly to or through a third linker; c)可追踪标记,其选自由如下组成的组:ΔCD34:ELPTQGTFSNVSTNVS(SEQ ID NO:4)、ΔCD19:PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGL PGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK(SEQ ID NO:5)、NGFR:KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSD VVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN(SEQ ID NO:6); : : : : : : : : : : : : : : : Hinge CH3: ESKYGPPCPSCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10); e) The transmembrane domain selected from the group consisting of: CD28TM:FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 13) and CD8aTM:CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 14) which is directly connected to or connected to by a linker; : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : sequence composition.

2. The CD30 chimeric antigen receptor of claim 1, wherein as the second linker connecting the AC10 VL and VH sequences, the proline-rich rigid linker is selected from the mouse igG3 upper hinge (mIgG3UH): PKPSTPPGSS (SEQ ID NO:15) and (mIgG3UH) 2 : PKPSTPPGSSPKPSTPPGSS (SEQ ID NO: 16), the glycine-rich flexible linker is selected from the group consisting of (G4S)2 linker: GGGGSGGGGG (SEQ ID NO: 17), (G4S)4 linker: GGGGSGGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 18), G4SG2 Connectors GGGGSGG (SEQ ID NO:19) and G3SG4 connectors: GGGSGGGGG (SEQ ID NO:20).

3. The CD30 chimeric antigen receptor according to claim 1 or 2, wherein the second linker connecting the AC10 VL and VH sequences is GGGSGGGGG (SEQ ID NO: 20).

4. The CD30 chimeric antigen receptor of claim 1 or 2, wherein the third linker is GS.

5. The CD30 chimeric antigen receptor according to claim 1 or 2, wherein the linker connecting the transmembrane domain sequence and the costimulatory signaling domain sequence is selected from the group consisting of LYCNHRN (SEQ ID NO: 25) and EF.

6. The CD30 chimeric antigen receptor according to claim 1 or 2, wherein: a) signal peptide consisting of MEFGLSWLFLVAILKGVQC (SEQ ID NO: 1) connected to: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : The second linker (G4S)2 linker of the VL and VH sequences consists of GGGGSGGGG (SEQ ID NO: 17), which is connected to by the third linker GS; c) Traceable mark ΔCD34:ELPTQGTFSNVSTNVS (SEQ ID NO: 4); d) Hinge CD8α:PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 7); e) Transmembrane domain CD8aTM CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 14), which is connected to: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : sequence composition.

7. The CD30 chimeric antigen receptor of claim 1 or 2, wherein the CD30 chimeric antigen receptor is MEFGLSWLFLVAILKGVQCSRDIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKGGGSGGGGQIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSAGSELPTQGTFSNVSTNVSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNEFRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*(SEQ ID NO:27)。 8. A nucleotide sequence encoding a CD30 chimeric antigen receptor according to any one of claims 1-7. 9.根据权利要求8所述的核苷酸序列,其是ATGGAGTTTGGGCTCTCCTGGCTCTTCCTGGTCGCGATTCTGAAGGGGGTCCAGTGTTCACGAGATATCGTCCTGACTCAGAGTCCTGCCAGCCTGGCAGTCTCCCTGGGACAGAGAGCTACCATAAGTTGTAAAGCATCACAGTCTGTTGATTTCGATGGCGACAGCTATATGAATTGGTACCAGCAAAAACCCGGCCAGCCCCCGAAAGTTTTGATCTATGCAGCCTCTAACTTGGAAAGCGGCATTCCTGCGCGATTCAGTGGCAGCGGGAGTGGTACAGATTTCACCCTGAACATACACCCAGTCGAAGAGGAGGACGCAGCCACATATTACTGCCAACAATCTAACGAGGATCCATGGACTTTTGGGGGCGGCACTAAACTCGAAATCAAGGGCGGAGGTTCAGGCGGAGGAGGGCAGATTCAACTGCAGCAATCAGGACCCGAGGTGGTCAAACCAGGTGCCAGTGTCAAGATATCTTGCAAGGCATCCGGATATACATTTACCGACTATTACATTACCTGGGTCAAGCAGAAACCCGGGCAAGGACTTGAATGGATTGGATGGATCTACCCTGGTAGCGGCAACACCAAATACAACGAAAAGTTTAAAGGGAAGGCAACCCTGACTGTAGACACCTCCAGCTCCACAGCATTCATGCAGCTCTCCTCACTGACCTCCGAGGACACAGCAGTGTATTTCTGTGCTAATTACGGTAATTACTGGTTCGCCTATTGGGGCCAGGGAACCCAAGTGACCGTTTCAGCTGGATCCGAACTTCCTACTCAGGGGACTTTCTCAAACGTTAGCACAAACGTAAGTCCCGCCCCAAGACCCCCCACACCTGCGCCGACCATTGCTTCTCAACCCCTGAGTTTGAGACCCGAGGCCTGCCGGCCAGCTGCCGGCGGGGCCGTGCATACAAGAGGACTCGATTTCGCTTGCGACATCTACATCTGGGCTCCCCTCGCTGGCACCTGTGGGGTGCTGCTGCTGTCACTCGTGATCACCCTTTATTGCAACCATCGAAACGAATTCAGAAGTAAACGGTCAAGGCTTCTGCACAGCGATTATATGAATATGACACCAAGAAGACCTGGTCCAACCCGGAAACACTATCAGCCCTACGCGCCCCCTAGAGACTTCGCAGCATACCGCTCTCGCGATCAAAGACTCCCGCCCGATGCCCACAAACCCCCTGGCGGGGGCAGCTTTAGGACACCCATTCAAGAAGAGCAGGCAGACGCCCACAGCACCTTGGCCAAAATTAGAGTTAAATTCAGTAGAAGTGCGGATGCGCCTGCTTACCAGCAGGGCCAGAACCAACTGTACAATGAACTGAATCTCGGGCGCCGAGAAGAGTATGACGTCCTCGATAAGCGGAGGGGTAGGGATCCTGAAATGGGTGGGAAGCCAAGAAGAAAAAACCCCCAGGAAGGACTGTATAACGAACTTCAGAAGGACAAGATGGCAGAGGCCTACTCTGAGATTGGCATGAAAGGCGAACGACGGCGCGGTAAAGGTCATGACGGGCTGTACCAGGGCCTGTCCACAGCGACGAAGGACACTTACGACGCCCTGCACATGCAGGCACTCCCCCCCAGGTGA(SEQ ID NO:42)。 10. A vector comprising the nucleotide sequence of claim 8 or 9, wherein the vector is a DNA vector or an RNA vector.

11. A vector comprising the nucleotide sequence of claim 8 or 9, wherein the vector is a plasmid, a lentiviral vector, adenovirus vector, a retroviral vector or a nonviral vector.

12. A cell comprising a vector according to claim 10 or 11.

13. The cell of claim 12, wherein the cell is a T cell.

14. The cell of claim 13, wherein the T cells are α / β and γ / δ T cells or NK cells.

15. The cell of claim 13, wherein the T cells are α / β and γ / δNK-T cells.

16. The cell of any one of claims 12-15 obtained under culture conditions in which both IL-7 and IL-15 are present.

17. The cell of claim 16 obtained under the culture conditions of the activation step, the transduction step and / or the amplification step of the method of preparing the cell.

18. A pharmaceutical composition comprising a nucleotide sequence according to claim 8 or 9, or a vector according to claim 10 or 11, or a cell according to any one of claims 12-17, Together with one or more excipients and / or adjuvants.

19. The CD30 chimeric antigen receptor according to any one of claims 1-7, the nucleotide sequence according to claim 8 or 9, the vector according to claim 10 or 11, the The cell according to any one of 12-17, the pharmaceutical composition according to claim 18, for the use of a drug for the treatment of CD30+ cancer, which is lymphoma, sarcoma, leukemia or mastoma Celloma.

20. The use of claim 19, wherein the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma.

21. The use of claim 19, wherein the sarcoma is myofibroblastic sarcoma or rhabdomyosarcoma.

22. The use of claim 19, wherein the medulloblastoma is proliferative cerebellar medulloblastoma.

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