The present invention concerns CAR-CD30 T cells for treatment of CD30+ Tumors. In particular, the present invention concerns a third generation of CAR-CD30 T cells for treatment of CD30+ Tumors such as lymphoid malignancies, leukemia, solid tumors.

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

The PD-1 blockade for relapsed lymphoma post alio HSCT appears to be highly efficacious but frequently complicated by rapid onset of severe and treatment-refractory GVHD(3). CAR-T cells are emerging as a novel treatment modality for these patients.

CD30 (Ki-1 ) is a cell membrane protein derived from the tumor necrosis factor receptor superfamily 8 (TNFRSF8), and its normal expression is restricted to activated T and B cells. In tumor cells, CD30 expression is most commonly associated with lymphoid malignancies (Hodgkin and non-Hodgkin lymphomas, CD30+ acute lymphoblastic leukemia (ALL), of either T-cell(4) or B-cell lineage(5)). CD30 expression has been reported also in mostly adult non-lymphoid malignancies. Based on the published data, 24.5% of all solid tumors are also CD30+, most notably among germ cell tumors (myofibroblasticsarcoma (93%), embryonal carcinoma (77%), mesothelioma (77%), mixed Germ Cell Tumor (GCT) (65%), head and neck carcinoma (24%), yolk sac tumor (18%), angiosarcoma (14%), pituitary adenoma (1 1 %) and seminomas (6%)), raising the possibility of CD30-targeted therapy for additional tumors(6). While 90% of early-stage HL patients can be cured with conventional treatment, only 70% of advanced-stage patients are cured with standard therapeutic approaches. For HL patients with relapsed disease, only half are cured with standard salvage therapies (7).

Targeting CD30 with monoclonal antibodies in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) has had profound clinical success. However, adverse events, mainly mediated by the toxin component of the conjugated antibodies, cause treatment discontinuation in many patients. Targeting CD30 with T cells expressing a CD30-specific chimeric antigen receptor (CAR) may reduce the side effects and augment antitumor activity.

Immunotherapeutic approaches targeting CD30 by CAR has been demonstrated of value in preclinical models(8, 9)and confirmed in two different independent clinical trials(10, 1 1 ), although clinical benefit was not optimal.

First-generation 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 an anti-CD30 CAR have been shown to have activity against CD30+ cancer cell lines in vitro, as well as in vivo, in a mouse xenograft model, improving the persistence of T cells in vivo(8).

Notably, the presence of soluble CD30 did not attenuate cytolysis while eliminating CD30+ lymphoma cells, suggesting that CD30 shed from HL cells into the blood would not inhibit the efficacy of anti CD30 CAR T cells in vivo(14).

In a first trial, an inconsistent response of lymphoma was observed, with the majority of patients presenting stable disease after CAR T cell multiple infusion, or no response at all. Overall, lymph nodes presented a better response than extranodal lesions, the response of lung lesions seemed to be relatively poor, and infused CAR T did not persisted more than 60 days after infusion. Notably, several clinical data(15, 16) clearly showed that the in vivo persistence of CAR-T cells is associate to better outcome of the treated patients. As summarized in table 1 , in the first clinical trial described the authors considered a lentiviral platform carrying a second generation CAR characterized by the single-chain fragment variable (scFv) sequence specific for the CD30 antigen derived from AJ878606.1 hybridoma, the costimulatory domain derived from human CD137 in frame with Oϋ3z signaling domains(10).

The first one open-label phase I clinical trial of anti-CD30 CAR T cells that were gene-modified with a lentiviral vector to express CD137 co- stimulatory domain involved eighteen patients suffering from relapsed or refractory Hodgkin lymphoma. The 18 patients included one with primary cutaneous anaplastic large cell lymphoma (ALCL) and 17 with Hodgkin lymphoma of 3 different subtypes, most of which were nodular sclerosis. Thirteen patients received 1 cycle of CAR T-cell infusion and five received 2 cycles.

Preliminary results of this study demonstrated seven achieved partial remission and six achieved stable disease. The objective response was 39%(10).

In a second trial, the majority of patients were treated with multiple infusions of CD30.CAR T cells achieving a transient response, and CD30. CAR-T cells were not more detectable after 6 weeks from infusion. As summarized in table 1 , in this clinical trial the authors considered a retroviral platform carrying a second generation CAR characterized by the single-chain fragment variable (scFv) sequence specific for the CD30 antigen derived from HRS3 hybridoma, the costimulatory domain derived from human CD28 in frame with ΰϋ3z signaling domains.

Table 1

Particularly, in the second clinical trial, 9 patients with relapsed/refractory HL or ALCL were infused with autologous T cells that were gene-modified with a retroviral vector to express the CD30-specific CAR (CD30.CAR-T) encoding the CD28 costimulatory endodomain. Of note, seven of these patients had brentuximab-refractory disease. Preliminary results of this study demonstrated complete response in 3 of 9 patients, and 3 had transient stable disease. CAR-T-cell persistence was <8 weeks in this study, but tumour biopsies showed efficient trafficking of T cells to lymphoma sites(1 1 ).

Both clinical trials teach that multiple CD30.CAR-T cells infusion was well tolerated. Host lymphodepletion before CAR-T infusion would be beneficial in further improving of CAR T cells expansion and their antitumor activity. More important the CAR-T-cell persistence correlate with clinical response.

All these data show that CD30. CAR-T cells are safe and can lead to clinical responses in patients with HL, although further optimization of this therapy is warranted to achieve longer in vivo persistence, and higher anti tumor control especially at lymphoma recurrence.

In particular, the optimization of the approach should consider that the classical Hodgkin lymphoma (cHL) and the anaplastic large T-cell lymphoma are characterized by only a few malignant Reed-Sternberg and Hodgkin cells (HRS) and by an abundance of inflammatory cells. These non-malignant cells produce soluble or membrane-bound molecules involved in tumor immune-evasion. Moreover, HL tumor generates a chemokine milieu that significantly influences which T-cell subtypes traffic to and accumulate in the tumor(17). Indeed, HRS cells produce the chemokines TARC and MDC that attract T helper (Th2) cells and regulatory T cells (Tregs), which express CCR4, the receptor for these chemokines. The abundance of Tregs (and Th2 cells) in tumors including HL create a hostile immune microenvironment by impairing the antitumor activity of the few cytotoxic-effector T lymphocytes able to reach the tumor site. Forced expression of CCR4 on CD30-specific chimeric antigen receptor (CAR-CD30) improve the migration of CAR-CD30 T -redirected, effector T lymphocytes toward an HL-generated TARC gradient (9). HRS cells often express high level of PDL1 and produce the immunosuppressive IL-10, TGF-beta, Galectinl and Prostaglandin E2, which inhibit T cell effector functions and induce apoptosis of activated Th1 and CD8+ T cells, through induction of CD95 ligand. It has been also recently showed that IL-15 selectively favors the survival, proliferation, and effector function of Epstein-Barr virus (EBV)-CTLs in the presence of T- regs(18). Moreover recently it has been shown that CAR-CD30 T cells grow in IL-7/IL-15 expressing higher levels of CXCR4 and CXCR3, which are chemokine receptors known to promote T cell migration to peripheral tissues(1 1 ).

Moreover, preclinical study showed that third generation of CAR-T cells combining CD28 and 4-1 BB co-receptors may have superior in vitro activation and proliferation capacity compared with second generation CAR-T cells carrying CD28 signal domains, and both kinds of cells displayed in vivo comparable efficacy in eliminating CD19+ B cells (19), although it was never demonstrated for CAR.CD30. Other CAR-CD30 T cells are known, such as those which are described in WO2017066122, WO2016134284 and CN107759699. For example, WO2017066122 compares 5F1 1 -28Z, AC10-28Z and XmAb-28Z cells produced in IL2 condition.

High level of transduction efficiency of all CAR used is reported, however higher transduction efficiency is obtained with 5F1 1 in comparison to AC10 and XmAb (table A), namely higher percentage of transduction of CD8+CAR (80,7%) is obtained respect to AC10 (61.90%) or XmAb (64.70%) when the cells are growth in IL2 for 7 days. In addition, functional experiments are described concerning IFNy production by co culturing CAR-T cells with CD30+tumors: SUDHL-1 , HH and BV173. 5F1 1 -28Z (growth in IL2) showed higher IFNy production in comparison to AC10-28Z, when co-cultured with BV173, namely, TABLE D-1 shows that 5F1 1 -28Z produced 3781 pg/ml of IFNy, whereas AC10-28Z produced 538 pg/ml of IFNy. Moreover 5F1 1 -28Z produced 3534 pg/ml of IFNy when co-cultured with FIDML-2 cell line.

In the light of the above, it is therefore apparent the need to provide for further CAR CD30 T cells, which are able to overcome the disadvantages of the known CAR CD30 T cells.

According to the present invention, two novel CD30-specific chimeric antigen receptors (CAR-CD30) of third generation are now provided. Particularly, the following two clinical grade third generation of CAR CD30 SFG retroviral vectors are provided:

SFG.CAR.CD30(AC10)ACD34.CD8aTM.CD28cyto.4-1 BB.z (28.4-

1 BB.z)

SFG.CAR.CD30(AC10)ACD34.CD8aTM.CD28cyto.OX40^

(28.0X40. z)

which comprise:

a single chain variable fragment (scFv) from AC10 hybridoma, which was never applied for CAR therapy before;

a trackable marker CD34 derived epitope (ACD34) of only 16 amino acid (aa) (as trackable marker) for a rapid identification by FACS

(Fluorescence-activated cell sorting) System and/or selection by Cell Sorter System of gene modified T cells;

an hinge represented by CD8 regions to avoid the immunogenic CFI2-CFI3 murine sequence applied in the vast majority of the similar CAR(20);

a transmembrane domain from the transmembrane domain of CD8 to improve molecule stabilization;

- two costimulatory domains were added to the CAR-CD30 vector: CD28(21 , 22) and 0X40(23, 24) or CD28 and 4-1 BB(25), both fused respectively to Oϋ3-z chain. Therefore, the two SFG vectors can be distinguished by a single costimulatory domain (4-1 BB for the first one and 0X40 for the second vector).

In both CAR-CD30 the region, the trackable marker, the costimulatory domains and the Oϋ3-z chain were codon optimized to improve the efficient protein expression.

Table 2 shows the differences of the CAR-CD30 according to the present invention in comparison with known CAR-CD30.

Table 2

The above mentioned sequence of the CAR-CD30 according to the present invention as a whole provides unexpected advantages in comparison with the known CARs-CD30 such as a more efficient stable CAR-CD30 expression in T cells which is obtained by the use of a retroviral platform and CD8 TM domain, a longer in vivo persistence in comparison with that of the known CAR-CD30 T cells which depends on the costimulatory domain, high anti-tumor activities even in the presence of immunomodulation and one single CAR-CD30 T cell administration thanks to the affinity of the scFv with the antigen and the choice of the production methods, such as the use of IL7/IL15 instead of IL2.

The in vitro and in vivo results herewith described show that modified polyclonal CD30CAR T cells according to the present invention were able to eliminate very efficiently, in long-term co-culture, CD30+ tumours. The biological products according to the present invention in xenograft in vivo model show to eliminate the Hodgkin and Non Hodgkin lymphomas and to establish a long immunological memory.

More in detail, the supernatants obtained by both SFG retroviral vector were able to transduce efficiently activated T cells, with very high level of transduction. The introduction in both construct of CD34 derived epitope as trackable marker let easily to track the genetically modified T cells (CD3+CD34+) in vitro and in vivo xenograft mouse model. The switching from IL2 to combination of IL7/IL15 improve the stability of expression of CAR-CD30 T cells, as showed by long-term in vitro culture, in particular for 28.0X40. z CAR T cells. In the setting of experiment the combination of IL7/IL15 improve the kinetics of proliferating T cells, in particular significantly evident after day +20 of in vitro expansion.

The in vitro culture for 15 days of CAR-CD30 T cells in IL7/IL15 induce a preferentially expansion of Effector Memory (EfM) T cells compartment respect T cell growth in IL2. Evaluating a day+15, CAR- CD30 T cells (IL2) for them exhaustion profile, a significative basal expression of PD1 and TIM3 was found, in particular in 28.0X40. z T cells. In vivo xenograft experiment model a long-term immunological memory which is able to eradicate for the second time the re-challenged tumour has been demonstrated for the first time.

The switching from IL2 to IL7/IL15 reduces significantly the PD1 expression, but increases only moderately TIM3 in both CAR T cells.

As reported by different authors, the presence of 4.1 BB, by itself, reduces the exhaustion profile in CAR T cells (26). The culture condition (IL7/IL15) improves further the reduction of PD1 expression, in particular in 28.0X40. z T cells. To assess the role of basal PD1 expression on potency of CAR-modified T cells against PDL1 + tumor, CAR modified T cells were co-cultured with L428-PDL1 lymphoma cell line permanently transduced with PDL1 , showing that no significant difference is found respect to Wild Type (WT) L428 cell line, even a lower effector/target ratios. Notable, in stressed long-term co-culture, unexpectedly the 28.0X40. z T cells show a significative superior lytic activity respect to 28.4-1 BB.z T cells, against Karpas 299, a lower effector/target ratios (at ratio E:T 1 :8 and 1 :16) and HDML-2.

The results according to the present invention clearly show that (AC10) 28.4-1 BB.z T cells or 28.0X40. z, growth in IL2, when cultured with HDML-2 (ratio effectontarget 1 :1 ), produce about 10303±3321.63 pg/ml and 29872.1718572.18 pg/ml of IFNy rispectively (Figure 9B), i.e. more than three time the IFNy produced by 5F1 1 -28Z described in WO2017066122, which produced 3534 pg/ml of IFNy when co-cultured with HDML-2 cell line.

Moreover, 28.4-1 BB.z T cells growth in IL7/IL15 show even a higher IFNy production: 21270.1711 1621.21 pg/ml (FIGURE 9E). In addition, 28.0X40. z T cells according to the present invention, when cultured with HDML-2, show even higher IFNy production: 29872.1718572.18 pg/ml (when they growth with IL2 (Figure 9B)) and 34444.67118872.62 pg/ml (when they growth with IL7/IL15)(Figure 9E).

It was also observed that, when CAR-T cells according to the present invention are prepared in conditions comprising IL7 and IL15, higher long-term stability of expression of the CAR on T cells is obtained (figure 1 D) in comparison to the CAR T cells prepared in conditions comprising IL2, especially with 28.0X40. z. The stability of the detectable CAR of the present invention provides a stable expression of the CAR into the membrane of T cells (Figure 1 D). In particular, the ratio of transduction of CD8/CD4 at day+5, +15 and +30 was evaluated. Although the level of CD8 is lower respect to CD4 at DAY+5, the level of CD8 CAR+ increased over times from day+5 to day+15 in favor to CD8 (Figure 1 E-F). In addition, the presence of IL7/IL15 in the culture conditions improved significantly the fold expansion of CAR-T cells in comparison to conditions comprising IL2 as shown in figure 1 L and 1 M.

Figure 2 shows also that the presence of IL7/IL15 in the culture conditions are important in connection to the reduction of exhaustion profile of CAR-T Cells.

A long term persistence of 28.0X40. z T cells infused up to 240 days (Figure 1 1 ) was also observed.

In addition, according to the present invention it was found that CAR.CD30 T cells with CD28.OX40 costimulatory domain were able to control Karpass 299 more efficiently with respect to CAR.CD30 T cells with 4.1 BB costimulatory domain, during the sequential additions of CD30+ lymphoma up to 4 time (“stressed” co-culture) (Figure 13A-B).

Interestingly, the percentage of CAR+ positive cells increased after the first tumor challenging, raising from 61.7%±18.4% and 74.0%±1 1.3% (day 0) to 93.4%±3.4% and 93.5%±3.1 % (day +5) in 28.4-1 BB.z T and 28.0X40. z T cells, respectively (p=0.049 and p=0.026)(Figure 13C). Furthermore, with the subsequent tumor re-challenging the percentage and the Median Fluorescence Intensity (MFI) of genetically modified T cells remained stable over-time only for 28.0X40. z T cells (figure 13C-D).

Moreover, CAR.CD30 T cells with CD28.OX40 costimulatory domain produced significantly higher amount of IFN-gamma (Figure 13 E), IL-2 (Figure 13 F) and TNF-alpha (Figure 13G) respect to 28.4-1 BB.z when co-cultured with Karpa299 tumor cell line.

Moreover, both CAR-CD30 T cells show a cytotoxic effect also against solid CD30+ tumours, as the Desmoplastic cerebellar medulloblastoma DAOY (Figure 5D and Figure 7G), the Rhabdomyosarcoma RD tumour cell line (Figure 7I-J) and the Embryonal Carcinoma. Overall, all this results make it highly plausible that the constructs according to the present invention can be used to treat efficiently CD30+ tumour patients.

Therefore, it is an object of the present invention a CD30 chimeric antigen receptor molecule comprising or consisting of, from the N-terminus to the C- terminus: a) a signal peptide, such as 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 by a first linker to;

b) an anti CD30 single chain antibody domain from AC10 hybridoma comprising or consisting of the AC10 VL sequence:DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQ KPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC QQSNEDPWTFGGGTKLEIK (SEQ ID NO:2) and AC10 VH sequence :QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPG QGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDT AVYFCANYGNYWFAYWGQGTQVTVSA (SEQ ID NO: 3), said AC10 VL and VFI sequences being linked by a second linker;

c) a trackable marker chosen from the group consisting of

ACD34:ELPTQGTFSNVSTNVS (SEQ ID NO:4) (nucleotide ID NO AB238231.1 and Protein ID NO:

BAE46748.1 ;ACD19:PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWS RESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPS EKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSG KLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLW LSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMET GLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK(S EQ ID.NO:5)(nucleotide ID NO: M21097.1 and Protein ID NO: AAA35533.1 );

NGFR:KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSV TFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETT GRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLP CTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEP EAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN (SEQ ID NO:6) (nucleotide ID NO: AK313654.1 and Protein ID NO: BAG36408.1 ); preferably ACD34:ELPTQGTFSNVSTNVS (SEQ ID NO:4) (nucleotide ID NO: AB238231.1 and Protein ID NO: BAE46748.1 );

d) an hinge chosen from the group consisting of hingeCD8cc PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (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 ):

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:9); hinge CH3

(UNIPROTKB:P01861 ):ESKYGPPCPSCPGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:10), preferably hinge CD8cc

PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO:7) (nucleotide ID NO: M12828.1 and Protein ID NO: AAB04637.1 ); e) a trans membrane domain chosen from the group consisting of

CD28TM: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID

NO:13)(nucleotide ID NO: BC1 12085.1 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

f) a co-stimulatory signalling domain chosen from the group consisting of the sequence obtained by linking CD28 cytoplasmic sequence:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:21 ) (nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1), CD137 (4-1 BB) sequence:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:22) (nucleotide ID NO: U03397.1 and Protein NO: AAA53133.1), and CD3-Zeta chain:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR ^* (SEQ ID NO: 23) (nucleotide ID NO: J04132.1 And Protein ID: AAA60394.1 ) or the sequence obtained by linking CD28 cytoplasmic sequence

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:21 ) (nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1), 0X40 sequence RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:24) (nucleotide ID NO: NM_003327.3 and Protein NO: NP_003318.1) and CD3Zeta chain:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR ^* (SEQ ID NO:23) (nucleotide ID NO: J04132.1 and Protein ID NO:AAA60394.1).

Hinge CD8a which is mentioned above comprises the sequence PAPRPPTPAPT (SEQ ID NO: 1 1 ) (spacer) and lASQPLSLRPEACRPAAGGAVHTRGLDFAiSEQ ID NO:12) (nucleotide ID NO NM 001768.6 and Protein ID NO: NP_001759.3).

The first linker can be a linker of two or three amino acids, such as SR.

The second linker which links AC10 VL and VH sequences can be chosen from the group consisting of a rigid linker prolines-rich, such as mouse igG3 upper hinge (mlgG3UH): PKPSTPPGSS (SEQ ID NO:15), (mlgG3UH) ₂ : PKPSTPPGSSPKPSTPPGSS (SEQ ID NO:16), or a flexible linker glycines-rich, 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).

In addition, a third linker can be used between AC10 VH sequence and the trackable marker, such as the short sequence GS.

One or more linkers (forth linker) can be present between the trans membrane domain and the co-stimulatory signalling domain such as CD8a cytoplasmic (cyto): LYCNHRN(SEQ ID NO:25) (nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3)and EF. According to an embodiment of the present invention, CD30 chimeric antigen receptor molecule comprises or consists of:

a) the signal peptide which comprises or consists of MEFGLSWLFLVAILKGVQC (SEQ ID NO:1 ), which is linked by a first linker to;

b) an anti CD30 single chain antibody domain from AC10 hybridoma comprising or consisting of the AC10 VL sequence:DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQ KPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC QQSNEDPWTFGGGTKLEIK (SEQ ID NO:2) and AC10 VH sequence :QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPG QGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDT AVYFCANYGNYWFAYWGQGTQVTVSA (SEQ ID NO: 3), said AC10 VL and VFI sequences being linked by the second linker (G4S)2 linker: GGGGSGGGG (SEQ ID NO:17);

c) a trackable marker comprising or consisting of ACD34:ELPTQGTFSNVSTNVS (SEQ ID NO:4); d) the hinge CD8a

PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO:7);

e) the trans membrane domain CD8aTM CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:14), which is linked by one or more linkers, which comprise or consist of the linker CD8a cytoplasmic (cyto): LYCNHRN (SEQ ID NO:25), to

f) the co-stimulatory signalling domain consisting of the sequence obtained by linking CD28 cytoplasmic sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:21 ), 0X40 sequence

RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:24) and CD3Zeta chain:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR ^* (SEQ ID NO:23).

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

a) the signal peptide which comprises or consists of MEFGLSWLFLVAILKGVQC (SEQ ID NO:1 ), which is linked by a first linker to;

b) an anti CD30 single chain antibody domain from AC10 hybridoma comprising or consisting of the AC10 VL sequence:DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQ KPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC QQSNEDPWTFGGGTKLEIK (SEQ ID NO:2) and AC10 VH sequence :QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPG QGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDT AVYFCANYGNYWFAYWGQGTQVTVSA (SEQ ID NO: 3), said AC10 VL and VFI sequences being linked by the second linker (G4S)2 linker: GGGGSGGGG (SEQ ID NO:17); c) a trackable marker comprising or consisting of ACD34:ELPTQGTFSNVSTNVS (SEQ ID NO:4);

d) the hinge CD8a

PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO:7);

e) the trans membrane domain CD8aTM CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:14), which is linked by one or more linkers, which comprise or consist of CD8a cytoplasmic (cyto): LYCNHRN(SEQ ID NO:25), to

f) the co-stimulatory signalling domain consisting of the sequence obtained by linking CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:21 ), CD137 (4-1 BB) sequence:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:22), and CD3-Zeta chain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR ^* (SEQ ID NO: 23).

According to a preferred embodiment of the present invention CD30 chimeric antigen receptor molecule is:

MEFGLSWLFLVAILKGVQCSRDIVLTQSPASLAVSLGQRATISCKASQSV DFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTL NIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKGGGSGGGGQIQLQQ SGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGS GNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWF AYWGQGTQVTVSAGSELPTQGTFSNVSTNVSPAPRPPTPAPTIASQPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH RNEFRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR ^* (SEQ ID NO:26).

Namely, this sequence, herewith named also as

SFG.CAR.CD30(AC10)ACD34.CD8aTM.CD28cyto.4-1 BB.z,

comprises the following sequences:

Signal peptide

MEFGLSWLFLVAILKGVQC (SEQ ID NO:1 ) (nucleotide ID NO: AB776838.1 and Protein ID NO: BAN63131.1 )

Link

SR (connection sequence)

VL (AC10)

DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPP KVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNED PWTFGGGTKLEIK (SEQ ID NO:2)

Flex

GGGSGGGG (G3SG4 Linker) (SEQ ID NO:20) VH (AC10)

QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIG WIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCAN YGNYWFAYWGQGTQVTVSA (SEQ ID NO:3)

Link

GS (connection sequence)

ACD34

ELPTQGTFSNVSTNVS (SEQ ID NO:4) (nucleotide ID NO: AB238231.1 and Protein ID NO:BAE46748.1 )

Hinge (spacer) extracellular

PAPRPPTPAPT (spacer) (SEQ ID NO:11 )

Hinge (CD8a) extracellular

IASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO:12) (nucleotide ID NO:NM_001768.6 and Protein ID NO: NP_001759.3)

CD8a (TM) transmembrane

CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:14) (nucleotide ID NO:

NM_001768.6 and Protein ID NO: NP_001759.3)

CD8a cytoplasmic (cyto) link of connection

LYCNHRN(SEQ ID NO:25) (nucleotide ID NO: NM_001768.6 and Protein

ID NO: NP_001759.3)

Link of connection

EF(connection sequence)

CD28 cyto

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:21 ) (nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1) 4.1 BB

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:22) (nucleotide ID NO: U03397.1 and Protein NO: AAA53133.1 )

CD3 Zeta chain

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG

KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR ^* (SEQ ID NO: 23) (nucleotide ID NO: J04132.1 and Protein ID NO:AAA60394.1)

According to a further preferred embodiment of the present invention, CD30 chimeric antigen receptor is

MEFGLSWLFLVAILKGVQCSRDIVLTQSPASLAVSLGQRATISCKASQSV

DFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTL

NIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKGGGSGGGGQIQLQQ

SGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGS

GNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWF

AYWGQGTQVTVSAGSELPTQGTFSNVSTNVSPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH

RNEFRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRD

QRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQ

GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL

QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP

PR ^* (SEQ ID NO:27).

Namely, this sequence, herewith named also as

SFG.CAR.CD30(AC10)ACD34.CD8aTM.CD28cyto.OX40.

comprises the following sequences:

Signal peptide

MEFGLSWLFLVAILKGVQC (SEQ ID NO:1 ) (nucleotide ID NO:AB776838.1 and Protein ID NO: BAN63131.1 )

Link

SR (connection sequence)

VL (AC10)

DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPP KVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNED PWTFGGGTKLEIK(SEQ ID NO:2)

Flex

GGGSGGGG(G3SG4 Linker)(SEQ ID NO:20)

VH (AC10) QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIG WIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCAN YGNYWFAYWGQGTQVTVSA(SEQ ID NO:3)

Link

GS (connection sequence)

ACD34

ELPTQGTFSNVSTNVS(SEQ ID NO:4) (nucleotide ID NO:AB238231.1 and Protein ID NO:BAE46748.1 )

Hinge (spacer) extracellular

PAPRPPTPAPT (spacer) (SEQ ID NO:11 )

Hinge (CD8a) extracellular

IASQPLSLRPEACRPAAGGAVHTRGLDFA(SEQ ID NO:12) (nucleotide ID NO:NM_001768.6 and Protein ID NO: NP_001759.3)

CD8a (TM) transmembrane

CDIYIWAPLAGTCGVLLLSLVIT(SEQ ID NO:14) (nucleotide ID NO:

NM_001768.6 and Protein ID NO: NP_001759.3)

CD8a cytoplasmic (cyto) link of connection

LYCNHRN(SEQ ID NO:25) (nucleotide ID NO: NM_001768.6 and Protein

ID NO: NP_001759.3)

Link of connection

EF(connection sequence)

CD28 cyto

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:21 ) (nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1) 0X40

RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI(SEQ ID NO:24) (nucleotide ID No:NM_003327.3 and Protein NO: NP_003318.1 )

CD3 Zeta chain

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR ^* (SEQ ID NO:23) (nucleotide ID NO:J04132.1 and Protein ID NO:AAA60394.1 )

The present invention concerns also a nucleotide sequence which encodes CD30 chimeric antigen receptor described above.

According to an embodiment of the present invention, the nucleotide sequence is

AT GG AGTTTGGGCT CT CCTGGCT CTT CCTGGT CGCG ATT CT G AAGGG GGT CCAGT GTT CACG AG AT AT CGT CCT GACT CAG AGT CCTGCCAGCC TGGCAGT CT CCCTGGG ACAG AG AGCT ACCAT AAGTT GT AAAGCAT CA CAGT CT GTT G ATTT CG ATGGCG ACAGCT AT AT G AATTGGT ACCAGCAA AAACCCGGCCAGCCCCCG AAAGTTTT GAT CT ATGCAGCCT CT AACTT GG AAAGCGGCATT CCTGCGCG ATT CAGTGGCAGCGGG AGT GGT ACA GATTT CACCCT G AACAT ACACCCAGT CGAAG AGG AGGACGCAGCCAC AT ATT ACTGCCAACAAT CT AACGAGG AT CCATGGACTTTTGGGGGCG GCACT AAACT CG AAAT CAAGGGCGGAGGTT CAGGCGG AGG AGGGCA GATT CAACTGCAGCAAT CAGG ACCCG AGGT GGT CAAACCAGGTGCC AGT GT CAAG AT AT CTTGCAAGGCAT CCGG AT AT ACATTT ACCG ACT AT T ACATT ACCTGGGT CAAGCAG AAACCCGGGCAAGGACTT G AATGG AT TGG ATGG AT CT ACCCTGGT AGCGGCAACACCAAAT ACAACG AAAAGT TT AAAGGGAAGGCAACCCT GACT GT AGACACCT CCAGCT CCACAGCA TT CATGCAGCT CT CCT CACT G ACCT CCGAGG ACACAGCAGT GT ATTT CT GTGCT AATT ACGGT AATT ACTGGTT CGCCT ATTGGGGCCAGGG AA CCCAAGT G ACCGTTT CAGCTGG AT CCG AACTT CCT ACT CAGGGG ACT TT CT CAAACGTT AGCACAAACGT AAGT CCCGCCCCAAG ACCCCCCAC ACCTGCGCCG ACCATTGCTT CT CAACCCCT G AGTTT GAG ACCCG AGG CCTGCCGGCCAGCTGCCGGCGGGGCCGTGCAT ACAAG AGG ACT CG ATTT CGCTTGCG ACAT CT ACAT CTGGGCT CCCCT CGCTGGCACCT GT GGGGTGCTGCTGCT GT CACT CGT GAT CACCCTTT ATTGCAACCAT CG AAACG AATT CAG AAGT AAACGGT CAAGGCTT CTGCACAGCG ATT AT AT GAAT AT G ACACCAAG AAG ACCTGGT CCAACCCGG AAACACT AT CAGC CCT ACGCGCCCCCT AG AG ACTT CGCAGCAT ACCGCT CT AAGAG AGG GAG AAAAAAATTGCT CT AT ATTTTT AAACAACCATTT AT G AGGCCCGT A CAG ACAACT CAGG AAG AGG ATGGCT GT AGTTGCCGCTT CCCAG AGG AGG AGG AAGGAGGCTGCG AGTT GAG AGTT AAATT CAGT AGAAGTGC GG ATGCGCCTGCTT ACCAGCAGGGCCAGAACCAACT GT ACAAT G AAC T GAAT CT CGGGCGCCG AG AAG AGT AT G ACGT CCT CGAT AAGCGG AG GGGT AGGG AT CCT GAAATGGGTGGG AAGCCAAG AAG AAAAAACCCC CAGG AAGG ACT GT AT AACG AACTT CAG AAGG ACAAG ATGGCAG AGG CCTACTCTGAGATTGGCATGAAAGGCGAACGACGGCGCGGTAAAGG T CAT G ACGGGCT GT ACCAGGGCCT GT CCACAGCG ACG AAGG ACACT T ACG ACGCCCTGCACATGCAGGCACT CCCCCCCAGGT G A (SEQ ID NO:28).

Namely, this nucleotide sequence, which encodes the sequence named also as SFG.CAR.CD30(AC10)ACD34.CD8aTM.CD28cyto.4- 1 BB.z, comprises the following sequences:

Signal peptide

AT GG AGTTTGGGCT CT CCTGGCT CTT CCTGGT CGCG ATT CT G AAGGG GGT CCAGT GTT CACG A(SEQ ID NO: 29) (nucleotide ID NO:

AB776838.1 )

VL (AC10)

GAT AT CGT CCT G ACT CAG AGT CCTGCCAGCCTGGCAGT CT CCCTGGG ACAG AG AGCT ACCAT AAGTT GT AAAGCAT CACAGT CT GTT G ATTT CG A TGGCG ACAGCT AT AT G AATTGGT ACCAGCAAAAACCCGGCCAGCCCC CG AAAGTTTT GAT CT ATGCAGCCT CT AACTTGGAAAGCGGCATT CCT G CGCGATT CAGTGGCAGCGGG AGTGGT ACAG ATTT CACCCT G AACAT A CACCCAGT CG AAG AGG AGG ACGCAGCCACAT ATT ACTGCCAACAAT C T AACG AGG AT CCATGG ACTTTT GGGGGCGGCACT AAACT CG AAAT CA AG(SEQ ID NO:30)

Flex

GGCGGAGGTTCAGGCGGAGGAGGG(G3SG4 Linker)(SEQ ID NO:31 )

VH (AC10)

GAT AT CGT CCT G ACT CAG AGT CCTGCCAGCCTGGCAGT CT CCCTGGG ACAG AG AGCT ACCAT AAGTT GT AAAGCAT CACAGT CT GTT G ATTT CG A T GGCG ACAGCT AT AT G AATTGGT ACCAGCAAAAACCCGGCCAGCCCC CGAAAGTTTT GAT CT ATGCAGCCT CT AACTTGGAAAGCGGCATT CCT G CGCG ATT CAGTGGCAGCGGG AGTGGT ACAG ATTT CACCCT G AACAT A CACCCAGT CG AAG AGG AGG ACGCAGCCACAT ATT ACTGCCAACAAT C T AACG AGG AT CCATGG ACTTTT GGGGGCGGCACT AAACT CG AAAT CA AG(SEQ ID NO:32)

Link (BamH1 restriction site)

GGATCC (BamH1 restriction site and connection sequence)(SEQ ID NO:33)

ACD34

GAACTT CCT ACT CAGGGG ACTTT CT CAAACGTT AGCACAAACGT AAGT

(SEQ ID NO: 34) (nucleotide ID NO:AB238231.1 )

Hinge (CD8a) extracellular

CCCGCCCCAAG ACCCCCCACACCTGCGCCG ACCATTGCTT CT CAAC CCCT GAGTTT G AGACCCG AGGCCTGCCGGCCAGCTGCCGGCGGGG CCGT GCAT ACAAG AGG ACT CGATTT CGCT (SEQ ID NO:35)

(NM_001768.6)

CD8a (TM) transmembrane

TGCGACAT CT ACAT CTGGGCT CCCCT CGCTGGCACCT GTGGGGTGC TGCTGCTGT CACT CGT GAT CACC(SEQ ID NO:36) (NM_001768.6)

CD8a cytoplasmic (cyto) link of connection

CTTT ATTGCAACCAT CG AAAC(SEQ ID NO:37) (NM_001768.6)

Link (EcoR1 restriction site and connection sequence)

GAATTC (SEQ ID NO:38)

CD28 cyto

AG AAGT AAACGGT CAAGGCTT CTGCACAGCG ATT AT AT G AAT AT G ACA CCAAG AAG ACCTGGT CCAACCCGG AAACACT AT CAGCCCT ACGCGC CCCCT AG AG ACTT CGCAGCAT ACCGCT CT (SEQ ID NO:39)

(AF222341.1 )

4.1 BB

AAG AGAGGGAG AAAAAAATTGCT CT AT ATTTTT AAACAACCATTT AT G A GGCCCGT ACAG ACAACT CAGG AAGAGG ATGGCT GT AGTTGCCGCTT CCCAG AGG AGGAGG AAGG AGGCTGCG AGTT G (SEQ ID NO:40)

(U03397.1 )

CD3 Zeta chain

AG AGTT AAATT CAGT AG AAGTGCGG ATGCGCCTGCTT ACCAGCAGGG CCAG AACCAACT GT ACAAT G AACT GAAT CT CGGGCGCCG AG AAG AGT AT GACGT CCT CG AT AAGCGG AGGGGT AGGG AT CCT GAAATGGGTGG GAAGCCAAG AAG AAAAAACCCCCAGG AAGG ACT GT AT AACG AACTT C AG AAGG ACAAGATGGCAG AGGCCT ACT CT G AG ATTGGCAT GAAAGG CGAACG ACGGCGCGGT AAAGGT CAT G ACGGGCT GT ACCAGGGCCT G T CCACAGCG ACGAAGG ACACTT ACG ACGCCCTGCACATGCAGGCAC T CCCCCCCAGGT G A(SEQ ID N0:41 ) (J04132.1 )

According to a further embodiment of the present invention, the nucleotide sequence is

AT GG AGTTTGGGCT CT CCTGGCT CTT CCTGGT CGCG ATT CT G AAGGG GGT CCAGT GTT CACG AG AT AT CGT CCT GACT CAG AGT CCTGCCAGCC TGGCAGT CT CCCTGGG ACAG AG AGCT ACCAT AAGTT GT AAAGCAT CA CAGT CT GTT G ATTT CG ATGGCG ACAGCT AT AT G AATTGGT ACCAGCAA AAACCCGGCCAGCCCCCG AAAGTTTT GAT CT ATGCAGCCT CT AACTT GG AAAGCGGCATT CCTGCGCG ATT CAGTGGCAGCGGG AGT GGT ACA G ATTT CACCCT G AACAT ACACCCAGT CGAAG AGG AGGACGCAGCCAC AT ATT ACTGCCAACAAT CT AACGAGG AT CCATGGACTTTTGGGGGCG GCACT AAACT CG AAAT CAAGGGCGGAGGTT CAGGCGG AGG AGGGCA GATT CAACTGCAGCAAT CAGG ACCCG AGGT GGT CAAACCAGGTGCC AGT GT CAAG AT AT CTTGCAAGGCAT CCGG AT AT ACATTT ACCG ACT AT T ACATT ACCTGGGT CAAGCAG AAACCCGGGCAAGGACTT G AATGG AT TGG ATGG AT CT ACCCTGGT AGCGGCAACACCAAAT ACAACG AAAAGT TT AAAGGGAAGGCAACCCT GACT GT AGACACCT CCAGCT CCACAGCA TT CATGCAGCT CT CCT CACT G ACCT CCGAGG ACACAGCAGT GT ATTT CT GTGCT AATT ACGGT AATT ACTGGTT CGCCT ATTGGGGCCAGGG AA CCCAAGT G ACCGTTT CAGCTGG AT CCG AACTT CCT ACT CAGGGG ACT TT CT CAAACGTT AGCACAAACGT AAGT CCCGCCCCAAG ACCCCCCAC ACCTGCGCCG ACCATTGCTT CT CAACCCCT G AGTTT G AG ACCCG AGG CCTGCCGGCCAGCTGCCGGCGGGGCCGTGCATACAAGAGGACTCG ATTT CGCTTGCG ACAT CT ACAT CTGGGCT CCCCT CGCTGGCACCT GT GGGGTGCTGCTGCT GT CACT CGT GAT CACCCTTT ATTGCAACCAT CG AAACG AATT CAG AAGT AAACGGT CAAGGCTT CTGCACAGCG ATT AT AT GAAT AT G ACACCAAG AAG ACCTGGT CCAACCCGG AAACACT AT CAGC CCT ACGCGCCCCCT AG AG ACTT CGCAGCAT ACCGCT CT CGCGAT CAA AG ACT CCCGCCCG ATGCCCACAAACCCCCTGGCGGGGGCAGCTTT A GG ACACCCATT CAAG AAG AGCAGGCAG ACGCCCACAGCACCTTGGC CAAAATT AGAGTT AAATT CAGT AG AAGTGCGG ATGCGCCTGCTT ACCA GCAGGGCCAG AACCAACT GT ACAAT G AACT GAAT CT CGGGCGCCG A GAAG AGT AT G ACGT CCT CG AT AAGCGG AGGGGT AGGG AT CCT G AAA T GGGTGGG AAGCCAAG AAG AAAAAACCCCCAGG AAGGACT GT AT AA CGAACTT CAG AAGGACAAG AT GGCAG AGGCCT ACT CT G AG ATTGGCA T GAAAGGCG AACG ACGGCGCGGT AAAGGT CAT G ACGGGCT GT ACCA GGGCCT GT CCACAGCG ACG AAGGACACTT ACG ACGCCCTGCACAT G CAGGCACTCCCCCCCAGGTGA (SEQ ID NO: 42)

Namely, this nucleotide sequence, which encodes the sequence named also as

SFG.CAR.CD30(AC10)ACD34.CD8aTM.CD28cyto.OX40^, comprises the following sequences:

Signal peptide

AT GG AGTTTGGGCT CT CCTGGCT CTT CCTGGT CGCG ATT CT G AAGGG GGT CCAGT GTT CACG A(SEQ ID NO:29) (AB776838.1 )

VL (AC10)

GAT AT CGT CCT G ACT CAG AGT CCTGCCAGCCTGGCAGT CT CCCTGGG ACAG AG AGCT ACCAT AAGTT GT AAAGCAT CACAGT CT GTT G ATTT CG A T GGCG ACAGCT AT AT G AATTGGT ACCAGCAAAAACCCGGCCAGCCCC CGAAAGTTTT GAT CT ATGCAGCCT CT AACTTGGAAAGCGGCATT CCT G CGCG ATT CAGTGGCAGCGGG AGTGGT ACAG ATTT CACCCT G AACAT A CACCCAGT CG AAG AGG AGG ACGCAGCCACAT ATT ACTGCCAACAAT C T AACG AGG AT CCATGG ACTTTT GGGGGCGGCACT AAACT CG AAAT CA AG(SEQ ID NO: 30)

Flex

GGCGGAGGTTCAGGCGGAGGAGGG(G3SG4 Linker) (SEQ ID NO:31 )

VH (AC10)

GAT AT CGT CCT G ACT CAG AGT CCTGCCAGCCTGGCAGT CT CCCTGGG ACAG AG AGCT ACCAT AAGTT GT AAAGCAT CACAGT CT GTT G ATTT CG A TGGCG ACAGCT AT AT G AATTGGT ACCAGCAAAAACCCGGCCAGCCCC CG AAAGTTTT GAT CT ATGCAGCCT CT AACTTGGAAAGCGGCATT CCT G CGCG ATT CAGTGGCAGCGGG AGTGGT ACAG ATTT CACCCT G AACAT A CACCCAGT CG AAG AGG AGG ACGCAGCCACAT ATT ACTGCCAACAAT C T AACG AGG AT CCATGG ACTTTT GGGGGCGGCACT AAACT CG AAAT CA AG(SEQ ID NO:32)

Link (BamH1 restriction site and connection sequence)

GGATCC (SEQ ID NO:33)

ACD34

GAACTT CCT ACT CAGGGG ACTTT CT CAAACGTT AGCACAAACGT AAGT

(SEQ ID NO: SEQ ID NO:34)(AB238231.1 )

Hinge (CD8a) extracellular

CCCGCCCCAAG ACCCCCCACACCTGCGCCG ACCATTGCTT CT CAAC CCCT G AGTTT G AGACCCG AGGCCTGCCGGCCAGCTGCCGGCGGGG CCGTGCAT ACAAG AGG ACT CGATTT CGCT (SEQ ID NO:35)

(NM_001768.6)

CD8a (TM) transmembrane

TGCG ACAT CT ACAT CTGGGCT CCCCT CGCTGGCACCT GTGGGGTGC TGCTGCTGT CACT CGT GAT CACC(SEQ ID NO:36) (NM_001768.6)

CD8a cytoplasmic (cyto) link of connection

CTTT ATTGCAACCAT CG AAAC(SEQ ID NO:37) (NM_001768.6)

Link (EcoR1 restriction site and connection sequence)

GAATTC (SEQ ID NO:38) CD28 cyto

AG AAGT AAACGGT CAAGGCTT CTGCACAGCG ATT AT AT G AAT AT G ACA CCAAG AAG ACCTGGT CCAACCCGG AAACACT AT CAGCCCT ACGCGC CCCCT AG AG ACTT CGCAGCAT ACCGCT CT (SEQ ID NO:39)

(AF222341.1 )

0X40

CGCG AT CAAAG ACT CCCGCCCG ATGCCCACAAACCCCCTGGCGGGG GCAGCTTT AGG ACACCCATT CAAG AAG AGCAGGCAG ACGCCCACAG C ACCTT G G CC A AAATT (SEQ ID NO:43) (NM_003327.3 )

CD3 Zeta chain

AG AGTT AAATT CAGT AG AAGTGCGG ATGCGCCTGCTT ACCAGCAGGG CCAG AACCAACT GT ACAAT G AACT GAAT CT CGGGCGCCG AG AAG AGT AT GACGT CCT CG AT AAGCGG AGGGGT AGGG AT CCT GAAATGGGTGG GAAGCCAAG AAG AAAAAACCCCCAGG AAGG ACT GT AT AACG AACTT C AG AAGG ACAAGATGGCAG AGGCCT ACT CT G AG ATTGGCAT GAAAGG CGAACG ACGGCGCGGT AAAGGT CAT G ACGGGCT GT ACCAGGGCCT G T CCACAGCG ACGAAGG ACACTT ACG ACGCCCTGCACATGCAGGCAC T CCCCCCCAGGT G A(SEQ ID NO:41 ) (J04132.1 )

The present invention concerns also a vector comprising the nucleotide sequence as described above, wherein said vector is a DNA vector, a RNA vector, a plasmid, a lentivirus vector, adenoviral vector, retrovirus vector or non viral vector.

In addition, the present invention concerns a cell, such as T cell, such as alfa/beta and gamma/delta T cell, NK cells, NK-T cells, comprising the vector or plasmid mentioned above.

According to an embodiment of the present invention, the above mentioned cell is obtained in the presence of recombinant human IL-2, or with a combination of recombinant IL-7 and IL15. For example, said interleukins can be present in at least one or all of the steps of the process of preparation of the cell such as activation, transduction and expansion.

The present invention concerns also a pharmaceutical composition comprising the nucleotide sequence, or the vector, or the cell, all of them mentioned above, together with one or more pharmaceutically acceptable eccipients and/or adjuvants.

It is a further object of the present invention, the CD30 chimeric antigen receptor molecule, the nucleotide sequence, the vector, the cell, the pharmaceutical composition, all of them mentioned above, for medical use.

It is a further object of the present invention the CD30 chimeric antigen receptor molecule, the nucleotide sequence, the vector, the cell, the pharmaceutical composition, all of them mentioned above, for use in the treatment of CD30+ cancers, for example at diagnosis or refractory/relapsed disease, such as lymphoma, such as Hodgkin and non-Hodgkin lymphomas, solid tumors such as myofibroblastic sarcoma, rhabdoid, histiocytic sarcoma, embryonal carcinoma, adenocarcinoma, mesothelioma, mixed germ cell tumors (GCT), non-seminomas GCT, head and neck carcinoma, yolk sac tumor, angiosarcoma, pituitary adenoma, dysgerminoma, teratoma or seminomas. Moreover the present invention can be used also to treat CD30+PDL1 + tumor (L428-PDL1 ) as shown by potency assay in Figure 7D .

In addition, the present invention concerns also a process for the preparation of a cell as defined above, wherein at least one or all the steps of activation (such as with immobilized OKT3 and anti-CD28 antibodies), transduction and expansion of said cell, such as T lymphocyte, are carried out in the presence of recombinant human IL-2, or with a combination of recombinant IL-7 and IL15.

The present invention is described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the enclosed drawings, wherein:

Figure 1 CAR-CD30 T cells with CD28.OX40 or CD28.4-1 BB costimulation exhibit similar transduction level, CD4+/CD8+ distribution, and in vitro proliferation upon initial antigen stimulation. (A) The expression cassette of two CAR-CD30 shown in cartoon. The scFv of CD30 was cloned in frame with CD8aTM, CD28 cytoplasmic moiety, and a second costimulatory domain represented by either 4-1 BB (upper figure) or 0X40 (lower figure), as well as the signaling domain CD3-zeta chain (z). As a trackable marker, ACD34 was added. (B) Flow- cytometry analyses shows the level of transduction of T cells by CD34 expression (upper panel) in an exemplificative donor, growth in IL2, of un transduced (NT) T cells, as negative control (left panels), or genetically modified T cells with CAR. CD30.ACD34.28.4.1 BB.z (28.4.1 BB.z) (middle panel) and genetically modified T cells with

CAR. CD30.ACD34.CD28.0X40. z (28.OC40.z) (right panel). (C) The level of transduction of T cells were confirmed also by Biotinylated Protein L; able to binds efficiently the scFv.(D-F) The 3 panels shows the average of the percentage of positive CAR+ T cells, profiled by FACS at three time of in vitro culture. First panel show CAR+CD3+ expression (D); the second panel (E)show the sub-population CAR+CD4+; and the last panel (F) the CAR+CD8+ T cells. For T cells growth in IL2 NT (white bar), 28.4.1 BB.z (white bar with horizontal lines)and 28.0X40. z (black bar); or in IL7/IL15: NT (white bar with vertical lines), 28.4.1 BB.z (grid bar)and 28.0X40. z (chessboard bar). Data are expressed as average ± standard deviation (SD) from six healthy donors (FIDs) at day 5, 15 and 30 of in vitro culture. (G-H) graph show the fold expansion in IL2, continuous lines (G) or IL7/IL15, dotted lines (H) of NT T cells and CAR-CD30 T cells, evaluated by trypan-blue count assay. Data represent results from 6 HDs. (I-M) Effect of cytokine usage of in vitro long term fold expansion of NT T cells (I), 28.4.1 BB.z T cells (L) and 28.0X40. z T cells (M). Significance were reported with an asterisk, while significance of variance of transduction level during the in vitro culture were reported with an asterisk encircled. ^* p-value=<0.05, ^** p-value=<0.01.

Figure 2. Exhaustion profiles of gene modified

CAR-CD30 T Cells. Basal exhaustion profile of CD3 T cells representative of 4 HDs, either NT (white bar), 28.4-1 BB.z (white bar with horizontal lines) or 28.OC40z (black bar) expanded for 15 days in the presence of either IL2 (left side); or in IL7/IL15, (white bar with vertical lines for NT; squared white bar for CARGD2.28-41 BBz T cells and chequered bar for OABOϋ30.28-OC40z T cells respectively). The circle around the asterisk(s) indicates the p-value for comparison between the same population of T cells cultured in presence of either IL7/IL15 or IL2. Data from four HDs are expressed as average ± SD. ^* p-value=<0.05; ^** p-value=<0.01 .

Figure 3. Basal and/or induced proliferation of NT

or CAR-CD30 T cells.

To evaluate the influence of retroviral modification or culture condition on safety profile of modified T cells, for NT (A) or CAR-CD30 T cells (B-C) the basal proliferation or cytokine or/and antigen specific proliferation were evaluated. T cells were labeled at day zero with the fluorescent cell staining CFSE and plated for five days with/out cytokines, or co-cultured in the presence of tumor cell line CD30 positive (Karpas299) or tumor cell line CD30 negative (BV173). The basal or induced proliferation (measured by CFSE dye dilution) of CD3+ (left side), CD8+(right side) and CD4+ T cells (left side) has been evaluated by FACS analysis.

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

Figure 5. CAR-CD30 T cells growth in complete CTL media with IL2, and transduced with 28.4-1 BB or 28.0X40 costimulatory domains, show comparable short-term cytotoxic effect in vitro experiment. In vitro ⁵¹ Cr release assay evaluating cytolytic activity of NT T cells (line with white circle), 28.4-1 BB.z T cells (line with white square) or 28.0X40. z T cells (line with black circle), on CD30+ lymphoma (Karpas 299 cell line (A), HDML-2 cell line (B) and L428 cell line (C)) on CD30+ medulloblastoma DAOY (D), and in CD30 negatives as the medulloblastoma D283 (E) and the lymphoma BV173 (F). Assays were performed 15 days after initial activation and expansion in the presence of IL2. Data from six healthy donors (HDs) are expressed as average ± SD. ^* p-value<0.05; ^** p-value<0.01 ; ^*** p-value<0.001 and ^**** <0.0001.

Figure 6. CAR-CD30 T cells growth in complete CTL media with IL7/IL15, and transduced with 28.4-1 BB or 28.0X40 costimulatory domains, show comparable short-term cytotoxic effect in vitro experiment. In vitro ⁵¹ Cr release assay evaluating cytolytic activity of NT T cells (dotted line with white circle), 28.4-1 BB.z T cells (dotted line with white square) and 28.0X40. z T cells (dotted line with black circle), on CD30+ lymphoma (Karpas 299 cell line (A), HDML-2 cell line (B) and L428 cell line (C)) on CD30+ medulloblastoma DAOY (D), and in CD30 negatives as the medulloblastoma D283 (E) and the lymphoma BV173 (F). Assays were performed 15 days after initial activation and expansion in the presence of IL7/IL15. Data from six healthy donors (HDs) are expressed as average ± SD. ^* p-value<0.05; ^** p-value<0.01 ; ^*** p- value<0.001 and ^**** <0.0001.

Figure 7. Long-term co-culture of both CAR-CD30 T cells against CD30+ tumor cell lines confirm their equal specific cytotoxic potency, independently of cytokines used.(A-J) Representative FACS analysis of residual tumor cells (identified as GFP+ cells) (or CD45-CD3- for D283 cells) after 7 days-coculture at the ratio E/T 1 :1 with effector NT cells (top panels), CD30-CAR T cells: 28.4-1 BBz T cells (middle panel) and 28-OC40z T cells (lower panels).

(K-L) Average representation of remaining tumor cells, after 7 days- coculture at the ratio E/T 1 :1 with NT (white bar), CARGD2.28-41 BBz T cells (white bar with horizontal lines), and OABOϋ30.28-OC40z T cells (black bar) growth in IL2 (K) or in IL7/IL15, (white bar with vertical lines for NT; squared white bar for CARGD2.28-41 BBz T cells and chequered bar for OABOϋ30.28-OC40z T cells respectively) (L). Data from six healthy donors (HDs) are expressed as average ± SD. ^* p-value<0.05; ^** p- value<0.01 ; ^*** p-value<0.001 and ^**** <0.0001 .

Figure 8. Stressed long-term co-culture to evaluate and to quantify the functional activities of CAR-CD30 T cells. (A-F) Evaluation of efficiency tumor control of lymphoma tumor cell, after 7 days-coculture at low E/T ratio with CARGD2.28-41 BBz T cells (bar graph with horizontal lines), or OABOϋ30.28-OC40z T cells (black bar) growth in IL2 (A-C); or in IL7/IL15, (squared bar for CARGD2.28-41 BBz T cells or chequered-bar for OABOϋ30.28-OC40z T cells respectively) (D-F). Tumor alone is indicated by white bar Data from six healthy donors (HDs) are expressed as average ± SD. ^* p-value<0.05; ^** p-value<0.01 ; ^*** p-value<0.001 and ^**** <0.0001 .

Figure 9. IFN-gamma profile of CAR-CD30 T cells co-cultured with CD30+ tumors cells. (A-F) Specific IFN-gamma production after 24h of Effector:Target co-culture. The diagram shows IFN-gamma production of CAR.CD30 T cells growth in IL2 (A-C) or in IL7/IL15, (D-F), after stimulation by tumor Lymphoma cell lines. OABOϋ30.28-OC40z T cells co cultured 24h with Karpas 299 (A and D) or HDML-2 (B and E) produce a significative higher level of IFN-gamma respect to CARGD2.28-41 BBz T cells, in particular a lower ratio Effector: Target. When the CAR-CD30 T cells were co-cultured with the tumor cell line L428, no difference was observed between two CARs (C and F). CARCD30.28-OX4C^ T cells growth also in IL7/IL15 produce higher level the IFN-gamma, when co- cultured with the CD30+ tumours cells (D-E), except when co-cultured with L428 tumor cell line (F).

Figure 10. In vivo activity of CAR.CD30 T cells generated and expanded in the presence of IL2 against the NHL Karpas 299. (A-C) In vivo bioluminescence imaging of NSG mice bearing sistemic Karpas 299- FF-Luc.GFP cells and treated with NT, CARCD30.28.4-1 BBz or OABOϋ30.28.OC40z T cells generated and expanded in the presence of IL2. (A) Schematic model of in vivo experiments. Mice receive i.v. Karpas 299-FF-Luc.GFP cells. After three days when the bioluminescence of the tumor became stable they are divided in three cohort and treated with NT or one of two CAR-CD30 T Cells. The tumors growth was evaluated by I VIS evaluation every weeks for 140 days. (B) Bioluminiscence imaging of tumor growth measured weekly in three cohort of mice; (C) representation of bioluminescence of each single mouse treated with NT (IL2) (lines with white circle; 8 mice), 28.4-1 BBz (IL2) (lines with white square; 10 mice) and 28.OC40z (IL2) T cells (lines with black square; 10 mice). (D) Kaplan- Meier overall survival (OS) analysis of tumor-bearing mice treated with NT (IL2) (lines with white circle; 8 mice), 28.4-1 BBz (IL2) (lines with white square; 10 mice) and 28.OC40z (IL2) T cells (lines with black square; 10 mice). ^* P-value=<0.05; ^** P-value=<0.001 ; ^*** P-value=<0.0001. Log-rank (Mantel-Cox).

Figure 11. Re-Challenging model: The establishment of longterm immunological memory in NHL mice model. (A-C) In vivo bioluminescence imaging of cured NSG mice re-challenged at day +140 i.v. with 0.2x10 ⁶ Karpas 299-FF-Luc.GFP cells, and followed for other 100 days. (A) Schematic model of in vivo experiments. Mice received i.v. Karpas 299-FF-Luc.GFP cells two times: at day 0 and at day 140. At days 3 when the bioluminescence became stable, they are divided in three cohort and treated with NT or one of two CAR-CD30 T Cells. The tumor growth was evaluated by IVIS evaluation every weeks for 240 days. At day 140 cured mice and a new cohort of mice (added to the experiment as positive control (CTR Mice) of the engraftment of the tumor) were re challenged i.v. with Karpas 299-FF-Luc.GFP cells.

(B) Bioluminiscence imaging of tumor growth measured weekly from day140 until day 240. (C) Representation of bioluminescence of each single mouse treated with NT (lines with white circle; 8 mice), CARCD30.28.4-1 BBz (lines with white square; 10 mice) and CARCD30.28.OX4C^ T cells (lines with black square; 10 mice) and CTR mice added to the experiment at day 140 as positive control of the engraftment of second tumor (dotted lines; 6 mice). (D) Kaplan-Meier overall survival (OS) analysis of tumor-bearing mice treated only one time at day +3 with NT (lines with white circle; 8 mice), CARCD30.28.4-1 BBz (lines with white square; 10 mice) and CARCD30.28-OX4C^ T cells (lines with black square; 10 mice) and re-challenged with the second tumor at day +140. Days of survival of CTR mice (dotted lines with white triangle; 6 mice) were added considering day140 as zero. ^* P-value=<0.05; ^** P- value=<0.01 ; ^*** P-value=<0.001 ; ^**** P-value=<0.0001. Log-rank (Mantel- Cox). (E) Representative picture of circulating human T cells bleed at day indicated, after challenging with the first tumor (day+6) and before and after re-challenging of the second tumor (day132 and 180 respectively). NT (first line), CARCD30.28-41 BBz T cells (second line) and CARCD30.28-OX4(^ (third line).

Figure 12. Evaluation of in vivo activity of CAR.CD30 T cells generated and expanded in the presence of IL2 or IL7/IL15 against the HL L428. (A-C) In vivo bioluminescence imaging of NSG mice bearing systemic L428-FF-Luc.GFP cells treated at day 6 with NT, 28.OC40z or 28.4-1 BBz T cells generated and expanded in vitro in the presence of IL2 or IL7/IL15. The tumor growth was evaluated by I VIS evaluation every weeks for 165 days. (A) Schematic model of in vivo experiments. Mice received i.v. 2x10 ⁶ L428-FF-Luc.GFP cells and after 6 days, when the bioluminescence became stable, they were divided in six cohort and treated with NT or CAR-CD30 T Cells. The tumor growth was evaluated by I VIS evaluation every weeks for 165 days. (B) Bioluminescence imaging of tumor growth measured weekly from day 6 until day 165. (C) Bioluminescence of each single xenograft mouse treated with NT(IL2) T cells (lines with white circle; 5 mice); 28.4-1 BBz(II_2)T cells (lines with white square; 5 mice); 28.OC40z(II_2)T cells (lines with black square; 5 mice); NT (IL7/IL15)T cells (dotted lines with white circle; 5 mice); 28.4- 1 BBz(II_7/II_15)T cells (dotted lines with white square; 5 mice) and 28.OC40z(II_7/II_15)T cells (dotted lines with black square; 5 mice). (D) Kaplan-Meier overall survival (OS) analysis of tumor-bearing mice treated with NT(IL2) (lines with white circle), 28.4-1 BBz(II_2) (lines with white square) and OABOϋ30.28-OC40z(II_2) (lines with black square); NT (IL7/IL15) (dotted lines with white circle), CARCD30.28.4-1 BBz (IL7/IL15) (dotted lines with white square) and OABOϋ30.28.OC40z (IL7/II15) (dotted lines with black square); ^* P-value=<0.05; ^** P-value=<0.001 ; ^*** P-value=<0.0001. Log-rank (Mantel-Cox). (E) The Table report the significance of OS of L428 xenograft mice treated with NT or CAR-CD30 T Cells growth in IL2 or IL7/IL15.

^* P-value=<0.05 and ^** P-value=<0.01. (F-G) Average of human circulating T cells, in NSG mice bearing systemic L428-FF-Luc.GFP tumor cells and treated at day+6 with human NT or CAR.CD30 T cells, evaluated either as percentage CD45+CD3+ cells (F) and either as CAR-CD30 T cells (CD3+CD34+) at days 15, 30, 56, 80, 100, 130 and 160. (G).

Figure 13. Stressed long-term co-culture. (A) The experimental design of “stressed co-culture” shown in a cartoon. T cells, at day +15 after transduction, were co-cultured in contact with Karpas 299 tumor cell line at E/T ratio of 1 :1 (0.5E+06 of T cells vs 0.5E+06 of Karpas 299 in 24 well plate). Tumor cells were administrated every five days until day 20 of co-culture (I, II, III and IV administration). At each time point, supernatant was collected at 24 hours and analysed for the presence of citokines IFNy, TNFa, IL-2 and IL-10. After five days of each administration cells were collected and analyzed by FACS. (B) A bar graph showing the percentage of residual tumor in the culture after 5 days of each tumor administration. Both CAR.CD30 T cells controlled tumor growth efficiently. Nevertheless, 28-OC40z T cells shown an increased tumor control at day +20. (C) A bar graph showing the percentage of CAR positive T cells on the total of CD3 positive T cells present in the co-culture at each time point. The percentage in both CAR.CD30 molecule significantly increased after the first co-culture, i.e. day+5. The re-challenging of the tumor negatively influenced the level of transduction only for 28.4-1 BB.z T cells whilst the percentage remained stable in 28.0X40. z T cells. (D) The graph underlines significant higher values of MFI in 28.0X40. z T cells (black bars) respect to 28.4-1 BB.z T cells (white bars). (E-H) Cytokine profile obtained from ELLA assay, performed on the supernatants collected after 24 hours by the tumor stimulation. Data from 7 healthy donors (HDs) are expressed as average ± SD. ^* p-value<0.05; ^** p-value<0.01 ; ^*** p- value<0.001 and ^**** <0.0001. (I): Tumor modulation of Memory and Exhaustion profiles in CAR.CD30 T Cells. Flow cytometry analysis of proportion of Na ^' ive, CM, EM and EMRA subsets at day+15 of in vitro culture of CD3+ T cells either NT (white bar), 28.4.1 BB.z (horizontal lines bar) or 28.0X40. z (black bar), expanded in the presence of IL2 or IL7/IL15 cytokines. (J) Long-term“stressed” co-culture induced a selection of EM and CM compartments in both 28.4-1 BB.z and 28.0X40. z T cells, but not in NT T cells. (K) Exhaustion profile of CD3+ T cells, either NT (white bar), 28.4.1 BB.z (horizontal lines bar) or 28.0X40. z (black bar) expanded for 15 days in IL2 or IL7/15 cytokines. Significance between NT T or CAR-CD30 T cells growths in the same culture condition were reported in black, while the encircled asterisks indicates the p-value for comparison between the same populations of T cells cultured in presence of IL2 or IL7/IL15. (L) Long-term“stressed” co-culture induced an upregulation of the exhaustion markers, especially of PD1 and TIM3, in both types of CAR.CD30 T cells, although the upregulation of these molecules did not interfere with their lytic activity. Data from 4 HDs are expressed as average ± SD. ^* p- value=<0.05; ^** p-value=<0.001.

EXAMPLE 1 : Design and study in vitro and in vivo of CAR-CD30 according to the present invention Material and Methods

Design of CAR-CD30 plasmid (Constructs)

Two clinical grade“third” generation of retrovirus Vector SFG have been designed which carry the cassette anti-CD30 single-chain variable fragment (scFv), derived from a murine antibody of IgG (AC10) class, linked via a codon optimized human CD8 hinge-transmembrane domain, to the codon optimized signaling domains of the two costimulatory domains CD28, 4-1 BB (CD137) or 0X40 and Oϋ3-z (Figure 1A). The single chain variable fragment (scFv) specific for CD30 is a fusion protein of 1 1 1 amino acid (aa) of the variable regions of the light chains (VL) of immunoglobulins connected by flex (27)(a short linker peptide) of 8 amino acids to 117 aa of heavy chains (VFI) of immunoglobulins. In particular the scFv AC10 is cloned in frame with codon optimized CD34 derived epitope of 16 aa (as trackable marker), linked by hinge of 40 aa (11 aa as spacer plus 29 aa of codon optimized CD8 extracellular domain) to bind the codon optimized human CD8-transmembrane domain (CD8aTM) of 30aa. The signal run from extracellular portion of CD30 scFv AC10 to intracellular portion of Oϋ3-z chain (1 13aa) through two costimulatory molecules: CD28 endodomain (41 aa) and 4-1 BB endodomain (42aa) for the

SFG.CAR.CD30(AC10)ACD34.CD8aTM.CD28cyto.4.1 BB.z retroviral vector (28.4-1 BB.z).

The switch from the costimulatory molecules 4.1 BB to 0X40 (36aa) allow to obtain the

SFG.CAR.CD30(AC10)ACD34.CD8aTM.CD28cyto.OX40^ retroviral vector (28.0X40. z).

Generation of eGFP-Firefly-Luciferase cell lines.

The retroviral vector encoding eGFP-Firefly-Luciferase (eGFP- FFLuc) was used in selected experiments to label CD30+tumor cells:

- Non-Flodgkin’s Lymphoma (NHL) Karpas 299,

- Hodgkin's Lymphomas (HL) HDML-2 and L428; - Rhabdomyosarcoma RD,

- Desmoplastic cerebellar medulloblastoma DAOY.

The retroviral vector encoding eGFP-Firefly-Luciferase (eGFP- FFLuc) was used in selected experiments to label CD30 negative control:

- B cell precursor leukemia BV173,

- Chronic Myelogenous Leukemia K562,

These cells lines were used for in vitro and in vivo study as previously described(9).

Cell lines.

Non-Flodgkin’s Lymphoma (NHL) Karpas 299 was obtained from Sigma-Aldrich. Hodgkin's Lymphomas (HL) HDML-2 and L428 and the B cell precursor leukemia Ph+ BV173 were obtained from DSMZ. The rhabdomyosarcoma RD, the desmoplastic cerebellar medulloblastoma DAOY, the chronic myelogenous leukemia K562, the medulloblastoma D283and the embryonic kidney 293T cell line were obtained from LGC Standards-ATCC.

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

Retroviral supernatant

Transient retroviral supernatant was produced by cotransfection of 293T with the MoMLV gag/pol expression plasmid PeqPam3(-env), the RD114 env expression plasmid RDF, and SFG vectors at a ratio of 2:3:3, respectively, with a total of 10 pg DNA. The transfection was facilitated with GeneJuice reagent (Calbiochem). The supernatant was harvested 2 and 3 days after transfection, filtered (using a 0.45-mm filter), snap-frozen, and stored at -80 °C in 5-ml aliquots(28).

Isolation, generation and transduction of effector cells.

Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood (PB) or buffy coat obtained from healthy donors (OPBG Hospital, Rome, Italy) after that signed informed consent was obtained, in accordance with rules set by Institutional Review Board (IRB) of OPBG (Approval of Ethical Committee N °969/2015 prot. N ° 669LB), using Lymphocytes separation medium (Eurobio; France). T lymphocytes were activated with immobilized OKT3 (1 pg/ml, e-Bioscience lnc.;San Diego, CA, USA) and anti-CD28 (1 pg/ml, BD Biosciences, Europe) antibodies in the presence of recombinant human interleukin-2 (IL-2, 100 U/ml; R&D; USA)(28), or with 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). Activated T cells were transduced on day 3 in 24-well plates pre-coated with recombinant human RetroNectin (Takara-Bio. Inc; Japan) using a specific retroviral supernatant and the specific above-described cytokines. At day 5 from transduction the T cells are expanded in “CTL complete medium” containing 45% RPMI1640 and 45% Click’s medium (Sigma-Aldrich,Co.; Usa) supplemented with 10% FBS and 2 mM Glutamax, and fed twice a week with the specific above described cytokines.

Phenotypic analysis. Expression of cell surface molecules was determined by flow cytometry using standard methodology. 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. The expression of CAR-CD30 on T cells was detected using a specific anti-CD34+ (QBENdl OV Clone) or the Pierce Recombinant Biotinylated Protein L, able to binds efficiently the scFv. T- cell receptor (TCR)-Vp repertoire on NT T cells and CAR-T cells, was evaluated at day+15 and day+30, using a panel of 24 different TCR nb- specific mAbs (IO TEST Beta Mark TCR-nb repertoire kit, BC) used in association with CD3 specific mAb (BD Biosciences) and isotype control mAb (BD Biosciences)(31 ). Samples were analyzed with a BD LSRFortessa X-20. Flow cytometry profiles were analyzed using the FACSDiva software (BD Biosciences). For each sample, a minimum of 20,000 events have been analyzed.

TCR V beta (b) repertoire

To evaluate the relative TCR nb repertoire distribution between NT and CAR modified T cells at day+15 the lOTest® Beta Mark Kit (Beckman Coulter) was used. This method use a multi-parametric analysis tool designed for quantitative determination of the TCR nb repertoire of human T lymphocytes by flow cytometry.

CFSE dilution method assay

To evaluate whether the CAR-CD30 T cells proliferate only in the presence of specific antigen or cytokine usage, T cell was labeled with the fluorescent cell staining dye carboxyfluorescein succinimidyl ester (CFSE), using the CellTrace™ CFSE Cell Proliferation Kit, for flow cytometry (Invitrogen).

Chromium release assay. The cytotoxic activity of transduced effector cells was evaluated using a 6-hour chromium release assay as previously described(9). Target cells (Karpas 299, FIDML-2, L428 and BV173) were labeled with radioactive chromium ( ⁵¹ Cr, PerkinElmer, cat no NEZ030S) and subsequently washed prior to co-culture with CAR T cells at different ratio for 4 hours. Co-culture supernatants were analyzed on the Microbeta ² 2450 Microplate Counter ( Pekin Elmer). The mean percentage of specific lysis of triplicate wells was calculated as follows: [(Experimental release-spontaneous release)/(maximal release-spontaneous release)]x100.

Co-culture assay. For co-culture experiments, control non transduced (NT) and CAR-CD30 T lymphocytes were plated at 1 x10 ⁶ cells/well in 24-well plates at the indicated Effector:Target (E:T) ratios. Following 7 days of incubation at 37 °C, tumor cells and T cells were collected and residual tumor cells and T cells assessed by fluorescence- activated cell-sorting (FACS) analysis based on CD3 expression (Effector T cells) and GFP or CD30+ (tumor cell line).

Enzyme-linked immunosorbent assay and cytometric bead array

The production of the IFNgamma was quantified by specific ELISA using commercially available kits (R&D Systems, Pepro-Tech, Rocky Hill, NJ). Supernatants tested with ELISA were collected from the co-cultures assay.

In vivo experiments

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

In vivo NHL mouse model (Karpas 299)

NSG (strain NOD.Cg-Prkdcscid N2rgtm1 Wjl/SzJ; from Charles River) mice 6 weeks of age were engrafted with 0.2x10 ⁶ CD30+Karpas299-F-Luc.GFP by intravenous (i.v.) injection. Three days later, when the light emission of the tumor was consistently measurable, the mice received at i.v. injection of 10 x 106 control (non-transduced, NT) lymphocytes or T cells genetically modified with either the CAR.CD30.ACD34.28.4.1 BB.z (28.4.1 BB.z) or

CAR. CD30.ACD34.CD28.0X40. z (28.0X40. z) grown for 12-15 days in IL2 or in a cocktail of IL7/IL15. Tumor growth was evaluated using I VIS imaging system (Xenogen). The intensity of the signal of the tumor was measured as total photon/sec/cm2/sr (p/s/cm2/sr). The signal of bioluminescence below of 5x10 ⁵ p/sec/cm2/sr (measured of mice without tumor) was considered negative. The in vivo experiments was followed for 140 days. The circulating T cells on mice peripheral blood were evaluated periodically.

Re-Challenging model: The establishment of long-term immunological memory in NHL mice model.

Mice engrafted with the NHL CD30+Karpas299-F-Luc.GFP tumor cell lines and treated with one single doses of CAR.CD30 T cells were monitored for 140 days and they were considered cured when a complete eradication of the tumor was observed (with a bioluminescence signal below to 5x10 ⁵ p/sec/cm2/sr.) for an least seventy days. To evaluate the establishment of long-term immunological memory, cured mice were re challenged at day +140 i.v. with 0.2x10 ⁶ CD30+ Karpas299-F-Luc.GFP tumor cell line. The mice were followed for at least other 1 10 days. A new cohort of control mice (CTR mice) were added to the experiment as positive control of the engraftment of the tumor. The circulating T cells on mice peripheral blood were evaluated before and after re-challenged the CD30+ Tumor. The mice were euthanized on day 250.

In vivo HL mouse model (L428)

NSG mice 6 weeks of age were engrafted with 2x10 ⁶ CD30+ L428- FF-Luc.GFP by intravenous (i.v.) injection. Six days later, when the light emission of the tumour was consistently measurable, the mice received intravenous (iv) injection of 10 x 10 ⁶ control (non-transduced, NT) lymphocytes or T cells genetically modified with either the CAR.CD30.ACD34.28.4.1 BB.z (28.4.1 BB.z) or

CAR. CD30.ACD34.CD28.0X40. z (28.0X40. z) grown for 12-15 days in IL2 or in a cocktail of IL7/IL15. Tumor growth was evaluated using I VIS imaging system (Xenogen).

Statistical analysis

Statistical Evaluation were performed using Graph Pad Prism (GraphPad Software), Differences between groups generating P-values <0.05 were considered significantly. When multiple comparison analyses were required, statistical significance was evaluated by a repeated measures ANOVA followed by a Log-rank (Mantel-Cox) test for multiple comparisons. The mouse survival data were analyzed using the Kaplan-Meier survival curve and Fisher’s exact test was used to measure statistically significant differences. No valuable samples were excluded from the analyses. Animals were excluded only in the event of their death after tumor implant but before T- cell infusion. Neither randomization nor blinding was done during the in vivo study. However, mice were matched based on the tumor signal for control and treatment groups before infusion of control or gene modified T cells. To compare the growth of tumors over time, bioluminescence signal intensity was collected in a blind fashion. Bioluminescence signal intensity was log transformed and then compared using a two-sample t-test. The analysis of the pathologist, aimed at quantifying tumor volume, was performed in a blind fashion.

Results

Generation, characterization of CAR-CD30 T Cells

Two potent third generation of CAR-CD30 (CAR-CD30) T cells have been generated, containing the single chain variable fragment (scFv) derived from a murine antibody of IgG (AC10), in frame with CD28, and a second costimulatory domain represented by either 4-1 BB or 0X40, as well as the signaling domain CD3-zeta chain (z). As a selectable marker a small molecule derived from the phosphoglycoprotein CD34 (ACD34), figure 1 A has been added. Activated T-cells (ATCs), growth in CTL complete medium with IL2 or a cocktail of IL7/IL15, were established from six healthy donors and transduced with retroviral supernatant encoding the CAR.CD30.ACD34.28.4.1 BB.z(28.4.1 BB.z)ot

CAR. CD30.ACD34.CD28.0X40. z (28.OC40.z) SFG vectors respectively. As a negative control, non-transduced (NT) ATCs were cultured in parallel. Transduced T cells were detected by flow cytometry using efficiently or CD34 antibody (clone QBEndI O), as shown in representative figure 1 B, or in alternative the protein L reagent(32), figure 1 C. As shown in figure 1 D, T-cells transduced with either 28.0X40. z or the 28.4.1 BB.z construct expressed high levels of CAR-CD30. However at day +5 the level of transduction was significant higher in T cells transduced in IL2 with the vector encoding 28.0X40. z (IL2) respect to 28.4.1 BB.z (IL2) (87.3%±5.1 % vs. 76.1 %±9.8%, respectively, p<0.05; (average! standard deviation (SD) is reported here and throughout the manuscript unless otherwise specified). Similar results were obtained also for T cells transduced in IL7/IL15. At day+5 the level of transduction was similarly higher in 28.0X40. z (IL7/IL15) T cells (84.1 %±2.2%) than in 28.4.1 BB.z(II_7/II_15) T cells (78.6%±3.9%), p<0.05. Notable in both CD3+ CAR.CD30 T cells growth in IL2, the level of transduction significantly decreased at day +15 (65.1 %±10.9% for 28.0X40. z(II_2) and 49.2%±13.3%, for 28.4.1 BB.z(II_2) respectively, p<0.05; blue asterisk), remaining for the next two weeks more stable at least until day +30 (73.0%±6.4% vs. 55.9%±18.1 %, 28.OC40.z(II_2) and 28.4.1 BB.z(II_2) respectively). The switching in IL7/IL15, independently of constructs used, significantly improve the stability and the level of transduction in CD4+ and/or CD8+ T cells.

In CAR T cells the CD4+/CD8+ ratio decrease weekly, figure 1 E (CAR+CD4+), coming out in favors of CAR+CD8+, figure 1 F . A day+15 a predominance of CD8+ in both CAR.CD30 T cells were obtained. The same trend for NT T cells was observed. The expansion rate of modified T cells did not change significantly from NT T cell when cultured in IL2 (figure 1 G) or in IL7/IL15 (figure 1 H). However, the cocktail IL7/IL15, in long-term in vitro culture, improve significantly the fold expansion of NT (figure 1 I) and transduced T cells (Figure 1 L and 1 M).

Memory and exhaustion profiles of gene modified CAR-CD30 T

Cells

To evaluate the influence of specific costimulatory domains and cytokines on CAR.CD30 T-cell compartment, CD3+ CAR-T cells were characterized for the expression of memory markers. 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 substantial difference between NT and the two kinds of CAR.CD30 T cells. However, the switching in IL7/IL15 reduced significantly the Central Memory (CM) compartment a favor of EfM and Effector Terminal (EfT) in CAR.CD30 T cells. After 30 days of in vitro culture, only a significative (only for T cells cultured in IL2) reduction of Naive CAR.CD30 T cells was noticed.

The pattern of inhibitory-receptors (PD-1 , LAG3 and TIM3) simultaneously expressed by CAR+ T cells was also evaluated in order to define their exhaustion status (figure 2). It was observed that, when T cells were transduced with (28.0X40. z), in IL2 , at day+15 of in vitro culture, a significative upregulation of PD1 and TIM3 has been observed respect to NT or CARCD30.28.4-1 BB.z T cells (Figure 2). Notable the switching from IL2 to IL7/IL15 reduced significantly the PD1 expression in CARCD30.28.0X40. z T cells (15.33%±4.75% in IL2 and 7.95%±4.93% in IL7/IL15, respectively, p=0.006), but upregulate TIM3 expression (2.45%±0.41 % in IL2 and 7.28%±1.26% in IL7/IL15, respectively, p=0.009). A day+30 of in vitro culture the exhaustion profile of NT and CARCD30 modified T cells were typically determined by PD1 and TIM3 expression.

Safety profile of CAR-CD30 T cells

To evaluate the influence of retroviral modification or culture condition on safety profile of modified T cells, for NT or CAR-CD30 T cells the basal proliferation or cytokine or/and antigen specific proliferation were evaluated. T cells were labeled at day zero with the fluorescent cell staining CFSE and plated for five days with/out cytokines, or co-cultured in the presence of tumor cell line CD30 positive (Karpas299) or tumor cell line CD30 negative (BV173). The basal proliferation of CD3+ cells has been evaluated, but also of CD8+ cells and CD4+ cells.

NT T cells proliferate only when cultured with IL2 (50U/ml) (figure 3A-II) or combination of IL7 (10ng/ml)/ IL15 (5ng/ml)(figure 3A-III), as shown by CFSE dye dilution. The proliferation was preferentially due to CD8+ cells (A middle panel). In contrast for CAR-CD30 T cells a specific CFSE dye dilution was observed also when they were co-cultured in presence of Karpas299 cell lines (figure 3B-IV and figure 3C-IV) but not in presence of BV173 tumor cell line (figure 3B-V and figure 3C-V)or when plated without cytokines (B-l and C-l). Moreover, to evaluate the polyclonal expansion of cultured T cells, at day+15 and day+30 of in vitro culture, whether there was the concordance of TCR nb repertoire distribution between NT and both CAR-CD30 T cells growth in IL2 or IL7/IL15 was determined. No significant preferential expansion of specific clone’s cytokine or CAR dependent was observed, even when the cells were cultured up to 30 days (data not shown).

CAR-CD30 T cells efficiently lyse in vitro CD30+ lymphoma, but also solid tumor as medulloblastoma and sarcoma cell lines

The capacity of CAR-CD30 T cells to kill CD30+ human tumor cell lines was then evaluated.

As well known, the cell membrane protein CD30 was expressed on 2 out of 2 Flodgkin’s lymphoma cell lines, figure 4A (FIDML-2, L428) and 1 out of 1 NFIL cell line (Karpas 299). Interesting CD30+ was also 1 out of 5 sarcoma cell lines (CD30 positive: RD and CD30 negative: SK-ES-1 , A673, CW9019 and CT-10) figure 4B; 1 out of 2 medulloblastoma cell lines tested (CD30 positive: DAOY and CD30 negative: D283) figure 4C; and one T lymphoblastic cell line T2 (CEM.T2, but not in one B cell leukemia cell line BV173 (figure 4D).

Moreover, the follow CD30+ tumor cell lines expressed also high level of PDL1 (KARPAS 299 and FIDML-2). To evaluate the relative influence of PDL1 on intrinsic resistance of CD30+ tumor cell line to be killed by CARCD30 T cells, the HL L428 (PDL1 negative) was transduced to stably express PDL1 , figure 4E.

In ⁵¹ Cr release assays both CARCD30 T cells were able to lyse, specifically and with high efficiency, the CD30+ lymphomas, as Karpas 299 (figure 5A), the HDML-2 (figure 5B), L428 (figure 5C), but not CD30- leukemia cell line BV173 (figure 5F). Notable both CAR-CD30 T cells showed to kill also the desmoplastic cerebellar medulloblastoma DAOY (figure 5D). As CD30 negative control for solid tumor, the medulloblastoma D283 (figure 5E) and the leukemia cell line BV173 (figure 5F) were tested. The switching from IL2 to the cocktail IL7/IL15 did not improve the potency of CAR-CD30 T cells (figure 6A-F).

As shows by representative donor, in 7 days long-term co-culture, using the ratio effector target one to one (R1 :1 ), a specific and comparable potency of both CAR-CD30 T cells on GFP+ lymphoma cell lines (figure 7A-D) was observed. Notable the expression of PDL1 on L428 tumor cell line (figure 7D) did not influence apparently the sensitivity of L428 tumor cell line to both CAR-CD30 T cells, respect to L428 wild type (figure 7C). Moreover a significative tumor control was observed also in other CD30+ tumor cell lines, as CD30+GFP+ leukemia cell lines (figure 7E) but not in CD30 negative BV173 (figure 7F); in CD30+GFP tumor DAOY medulloblastoma cell line (figure 7G) and in CD30+ RD sarcoma cell lines (figure 7I) but not in CD30 negative CD45 negative D283 medulloblastoma cell line (Figure 7H). Notable for solid tumors the 28.0X40. z kills significantly better than 28.4.1 BB.z (figure 7G and 7I), but not in CD30 negative SK-ES-1 (figure 7J). These results were confirmed for six different donors, expanded in IL2 (figure 7K) or in IL7/IL15 (figure 7L).

In the present study, the cytokine culture conditions did not influence the in vitro specific cytolytic activity of CAR-CD30 T cells against CD30+cells, when tested in a standard Chromium cytotoxic assay (Figure 5-6), or in a long-term co-culture (Figure 7). To evaluate the real power of the lytic potency between two CAR-CD30 T cells, the in vitro long-term co culture potency assay was stressed increasing the target tumor cells from R 1 :1 to R 1 :32. Noteworthy, the activity of CAR.CD30 T cells at low effector/target ratios showed a significant improvement of the in vitro tumor control of 28.0X40. z(II_2) T cells for the Karpas 299 (in R1 :8 and R1 :16) and HDML-2 cell lines (in R 1 :8, R1 :16 and R1 :32) (Figure 8A-B, respectively), however for L428 no significative difference of cytolytic activity between 28.OC40.z(II_2) and 28.4.1 BB.z (IL2) was observed.

For CAR-CD30 T cells (IL7/IL15) superior lytic activity of 28.0X40. z was confirmed, in particular at lower effector/target ratios for Karpas 299 and FIDML-2 cell lines, although it cannot reach the significance respect to 28.4-1 BB.z (Figure 8D and 8F, respectively),

Overall these data confirmed of superior CD30+ specific activation of 28.0X40. z (IL7/IL15), when co-cultured with Karpas 299 and FIDML-2, in term of specific IFN-gamma production, evaluated on supernatant collected 24 hours from co-culture potency assay (Figure 9A-B and 9D-E). Both CAR-CD30 T cells produce specific and equal level of IFN-gamma when co-cultured with the CD30+Tumor cell line L428 (Figure 9C and 9F).

The establishment of long-term immunological memory in NHL mice model.

The in vivo efficacy and persistence of CAR-CD30 T cells were compared against the NHL Karpas299-FF-Luc.GFP tumor cell line (Figure 10A) in a xenograft model, using immunodeficient NSG mice.

While in the group treated with NT (IL2) T cells, the bioluminescence of the tumor progressively increased (Figure 10B-C), in mice treated with 10x10 ⁶ CAR-CD30 T cells (IL2) a significative tumor control was observed, as measured by reduction or control of bioluminescence signal. The median survival of the mice treated with NT cells (IL2) reach only 45,5 days, 30% of mice treated with 28.4.1 BB.z(Iί2) and 60% of mice treated with 28.OC40.z(Iί2) respectively experienced long-term tumor control (Figure 10D). Specifically the median survival of mice treated with 28.4.1 BB.z (IL2) was 58 days (p=0.05), and undefined for mice treated with 28.0X40. z (IL2) (p=0,0002) (Figure 10D).

After 140 days of treatments, cured mice (3 mice treated at day 0 with 28.4.1 BB.z (IL2) and 6 mice with 28.0X40. z (IL2) were re-challenged i.v. with the same tumor (0.2x10 ⁶ ), and the mice were followed for other 100 days. In this setting of experiments, 6 new mice were added as positive control mice (CTR mice), that received CD30+Karpas299-F- Luc.GFP by intravenous injection (i.v.) (Figure 1 1 A). By evaluation of bioluminescence of the Karpas299-F-Luc.GFP reinfused at day +140, it was taken note of rapid progression of the tumour in CTR mice (lines with white circle) and 28.4-1 BB.z (IL2) treated mice (lines with white square) (Figure 1 1 B-C). In contrast in 28.0X40. z (IL2) treated mice (lines with black square), after an initial expansion of the tumor for the first 40 days, the 66,67% (4 out 6) of the mice eradicate for the second time the re challenged tumor, with a significative survival benefit (Figure 10D). To confirm the establishment a long-term immunological memory, the blood was sampled and analysed in treated mice, after the first tumour infusion (day+6, +56, +103 and +132) and the second tumour infusion (day+180, +221 and +254). In particular, for mice treated with 28.0X40. z (IL2) a significative expansion of circulating T cells (Figure 1 1 E) was observed in conjunction with the infusion of the Lymphoma (2.49%±1.03%, p<0.005) respect of mice treated with 28.4-1 BB.z (IL2) (0.275%±0.109%) or NT (IL2) (0.347%±0.071 %). Interesting after the eradication of the first tumor, when at day +132, the circulating T cells in cohort of mice treated with CAR-CD30 T cells was evaluated, only 0.022%±0.027% and 0.090%±0.1355% of T cells were quantified, for 28.0X40. z (IL2) and 28.4.1 BB.z (IL2) respectively. Forty days after the tumor re-challenging (day+180) a slow, but impressive expansion of circulating 28.0X40. z (IL2) T cells (7.216%±1 1 ,259%), respect to 28.4.1 BB.z (IL2) (0.093%±0.129%) was appreciated. The complete eradication of the second tumour followed the simultaneous reduction of circulating 28.0X40. z (IL2) T cells to undetectable percentage, as measured at day+254 (0.001 %±0.0018%).

Evaluation of efficacy and persistence of CAR-CD30 T cells in NHL mice model.

Successively the influence of cytokine usage of in vivo efficacy of CAR-CD30 T cells grow for 15 days in IL2 or IL7/IL15, against the more aggressive HL L428 was evaluated. Mice received at day-6 i.v. 2x10 ⁶ L428-FF-Luc.GFP cell line and, when the light emission of the tumor was consistently measurable, the mice were treated i.v. with NT or genetically modified T cells. To evaluate the persistence of human circulating T cells, the treated mice were blood sampled at day +15, +30, +56, +80, +100, +130, +160 (Figure 12A). The bioluminescence of the L428 cell line in HL- tumor-bearing mice, treated with NT T cells, rapidly increase up to five log in less of 50 days (figure 12B and figure 12C), and the mice died or were sacrificed for morbidity. The macroscopic analysis of organs in sacrificed mice shown a large tumor mass preferentially present on the liver. HL- tumor-bearing mice treated with 28.4-1 BB.z (IL2) survived on median slightly significantly longer (79±10 days) respect FIL-tumor-bearing mice treated with NT (IL2), NT (IL7/IL15) (52±9 and 58±1 days respectively) (Figure 12B-E). The switching in IL7/IL15 did not improve the cytotoxic in activity of 28.4-1 BB.z (IL7/IL15) (Figure 12B-D). The median of survival of FIL-tumor-bearing mice treated with 28.0X40. z (IL2) improve significantly up to 133±4 days respect to mice treated with NT(IL2) or 28.4-1 BB.z (IL2). When the FIL-tumor-bearing mice were treated with 28.0X40. z (IL7/IL15) the median of survival became undefined, without however any significant difference of overall survival between mice treated with two 28.0X40. z CAR T cells (p=0.0876 and figure 12E). To evaluate the persistence of infused T cell, the blood circulating T cells in NSG mice bearing L428 tumors were monitored periodically and treated with NT or genetically modified T cells for all the period of the experiment (Figure 12F). Although, also mice treated with NT T Cells, showed a significative increase of human circulating CD45+CD3+ cells with a peak evaluated at day+56 (26.69%±7.02% and 5.97%±9.63%, for NT (IL2) and NT(IL7/IL15) respectively, no tumor control was observed.

At day 80 in only one survived mouse treated with 28.4-1 BB.z (IL2), a very high number of circulating human T cells (CD45 ⁺ CD3 ⁺ = 20.56%) was measured, but with low level of transduction (CD3 ⁺ CD34 ⁺ = 4,57%). In contrast in all four mice treated with 28.0X40. z (IL2) the circulating T cells (CD45 ⁺ CD3 ⁺ ) at day +80 was in average 9.79%± 5.24% (range 3.08%- 15.37%), with a stable level of transduction equal to 34.23%±5.87%. Interesting in mice treated with 28.0X40. z (IL7/IL15) a significative reduced level of circulating T cells (0.92%±0.56%, p=0.0065) with higher percentage of transduced T cells (41 89%±2.25%, p=0.0300) was measured. The complete eradication of the tumor infused in the mice treated with 28.0X40. z CAR T cells was followed by the reduction of circulating T cells. The percentage of circulating CAR-CD30 T cells remain stable during the first 100 days. A day+165 residual circulating T cells were found in only in two mouse treated on four (0.06%±0.02%). All four mice resulted cured at this time. In this two mice the CAR-CD30 T cells show to be equally distribute between CD4+ and CD8+, as central memory (CM), defined as CD45RA-CD62L+ and Effector memory (EM), defined as CD45RA-CD62L-; Figure 12G.

EXAMPLE 2: Tumor modulation of Memory and Exhaustion profiles in CAR.CD30 T Cells according to the present invention (28.0X40. z T cells and 28.4-1 BB.z T cells)

Materials and methods

Stressed Co-culture assay

For stressed co-culture experiments, non-transduced (NT) control and CAR.CD30 T lymphocytes (28.0X40. z T cells and 28.4-1 BB.z T cells) were plated at 1 x10 ⁶ cells/well in 24-well plates at the indicated Effector: Target (E:T) ratios 1 :1. To evaluate how long CAR.T cells were able to eliminate the tumor when added more than one time, tumor cells were added to the well at day zero, 5, 10 and 15. The residual tumor cells and persistence of T cells by FACS analysis based on CD3 expression (Effector T cells) and GFP or CD30+ (tumor cell line) up to 20 days of co- colture were then evaluated.

Enzyme-linked lectin assay Supernatant from E:T co-culture was collected at 24 hours to evaluate the level of lnterferon-g (IFNy), lnterleukin-2 (IL-2), Interleukin-10 (IL-10) and Tumor Necrosis Factor-a (TNF-a) using ELLA protocol (R and D System).

Results

In order to evaluate the lytic effectiveness of CAR.CD30 T cells in a more complex contest, the co-culture conditions were “stressed” by re challenging the tumor (Karpas 299) every five days (at day 0, +5, +10 and +15 of co-culture) (Figure 13A). At each time point, the percentage of residual tumor and the behavior of CAR.CD30 T cells were evaluated, by evaluating the single chain expression and the relative mean fluorescence intensity (MFI), the memory and exhaustion profile and the relative specific cytokines production as IFNy, TNFa, IL-2 and IL-10 (at day 1 , +6, +1 1 and +16 of co-culture). Both CAR.CD30 T cells exhibited high tumor control even after multiple exposures to Karpas 299. Although significant differences in lytic activity at day +5 and day+10 between them were not observed, 28.0X40. z T cells shown a major tumor control at day+20 (8.6%±5.3% for 28.OC40.z and 27.9%±29.5% for 28.4-1 BB z T cells) (Figure 13B). Interesting the percentage in both CAR.CD30 molecules significantly increased after the first co-culture, from 66.9%±15.23% (day+5) to 93.8%±1 1 .3% (day 10) for 28.4-1 BB.z T cells, p= 0.022 and from 74.9%±1 1 .3% (day+5) to 93.2%±1 1 .3% (day+10) for 28.OC40.z T cells, p=0.007). Indeed, the next tumor re-challenging negatively influenced the level of transduction only for 28.4-1 BB.z T cells from 93.8%±3,06% (day +10) to 67.9%±32.33% (day+20) while in 28.OC40.z T cells the percentage remained stable from 93.22%±2,75% (day+10), to 92.53%±3,45% (day+20) (Figure 13C).

Moreover, the MFI of 28.0X40. z T cells was significantly higher than 28.4-1 BB.z T cells at each time point (1 1294.83±580453 vs 2400411 1365.6 at day 0, p=0.006; 1688316703.47 vs 40671 .21 12162.2 at day 5, p=0.004; 7382.7514042.01 vs 25329.75114746.76 at day 10, p=0.037; 1 1729.8311 1 158.66 vs 29552.831234643.32 at day 15, p=0.035; 8344.5 vs 23834.8311 1272.55 at +20, p=0.001 ) (Figure 13D).

Furthermore, the cytokine profile confirmed a prompter activation of 28.0X40. z T cells, resulting in a significant IFNy production, also at day+20 of stressed co-culture (4768.86 pg/ml±3708.34 pg/ml) compared to 28.4-1 BB.z T cells (2699.54 pg/ml ±2517.10 pg/ml, p= 0.012) (Figure 13E) and TNFa production (8439.32 pg/ml ±6187.27 pg/ml) compared to 28.4-1 BB.z T cells (2983.40 pg/ml ±2497.48 ng/ml, p= 0.013) (Figure 13F).

Furthermore, the difference between CAR.CD30 T cells, in the production of IL-2, was significantly different until day +10, that correspond to tumor challenging number II) (8480.82 Pg/ml ±5065.76 Pg/ml for 28.0X40. z T cells) compared to 28.4-1 BB.z T cells (2778.64 pg/ml ±3852.82 pg/ml, p= 0.006) (Figure 13G). The alternative manner to evaluate the tumor control is the detection of the cytokine IL-10 (produced by Karpas299 cell line). As shown in Figure 13H, from day +10 the level of IL-10 detected in NT T cells culture media (white bars) was similar to the quantity detected plating tumor alone (horizontal lines bar) while high level of IL-10 was detected only after 24 hour of the first co-culture (challenging number I) with CAR.CD30 T cells (Figure 13H).

The results show that CAR.CD30 T cells with CD28.OX40 costimulatory domain were able to control the tumor more efficiently with respect to 4.1 BB costimulatory domain during the sequential additions of CD30+ lymphoma up to 4 time (“stressed” co-culture), producing significantly higher amount of IFN-gamma, TNF-alpha and IL-2 when co cultured with Karpa299 tumor cell line. Bibliography

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