WO2025032323A1

WO2025032323A1 - Methods and cell compositions

Info

Publication number: WO2025032323A1
Application number: PCT/GB2024/052062
Authority: WO
Inventors: Martin PULÉ
Original assignee: UCL Business Ltd; Autolus Ltd
Current assignee: UCL Business Ltd; Autolus Ltd
Priority date: 2023-08-04
Filing date: 2024-08-02
Publication date: 2025-02-13
Anticipated expiration: 2026-02-04
Also published as: GB202312009D0

Abstract

There is provided an anti-GD2 CAR T cell composition, a method for making such a composition and its use in the treatment of diseases such as Neuroblastoma.

Description

METHODS AND CELL COMPOSITIONS

FIELD OF THE INVENTION

The present invention relates to an anti-GD2 CAR T cell composition, a method for making such a composition and its use in the treatment of diseases such as Neuroblastoma.

BACKGROUND

Neuroblastoma is the most common extracranial solid tumour in childhood and disproportionality contributes to death due to childhood cancer. More than 1 ,200 children/year are diagnosed with neuroblastoma in USA and Europe. Half of those cases are considered high-risk disease either due to presence of metastasis or MYCN gene amplification.

In the UK/Europe, up to 28 countries follow the front-line treatment for high-risk neuroblastoma according to the SIOPEN HR-NBL1 study which incorporates induction chemotherapy, local treatment with surgery and radiotherapy, myeloablative chemotherapy with stem cell rescue, and maintenance treatment. Through randomised arms this study has shown (i) the benefit of the addition of granulocyte colony-stimulating factor (G-CSF) to avoid infections during induction chemotherapy; (ii) equivalent outcome but less acute toxicity using rapid COJEC as compared to modified N7 as induction chemotherapy regimen and (iii) higher event free survival (EFS) and less toxicity of busulfan-melphalan conditioning over carboplatin-etoposide-melphalan for myeloablative chemotherapy with stem cell support.

Neuroblastoma selectively accumulates metaiodobenzylguanidine (MIBG). MIBG based single-photon emission computed tomography (SPECT) imaging is widely used to measure response to treatment applying Curie or SIOPEN semi-quantitative scoring. Both in Children’s Oncology Group (COG) and SIOPEN studies, a post induction Curie score > 2 or SIOPEN score >3 has been shown to be associated with an inferior EFS independent of other prognostic factors including age and MYCN status. In the HR-NBL1 study clearance of metastatic disease is a requirement to proceed to myeloablative chemotherapy with stem cell rescue.

More recently, the introduction of immunotherapy into the multimodal treatment of neuroblastoma has shown promising results. This was first demonstrated in the COG phase II study showing an increase in 2-year EFS of up to 20% with the addition of the anti-GD2 monoclonal antibody ch14.18 combined with interleukin-2 and GM-CSF to cis retinoic acid standard treatment. This improved EFS is in part maintained at 5 years when incorporating anti-GD2 antibodies in maintenance treatment (56.6% vs 46.1 % for COG study and 57% vs 42% in the SIOPEN study), but late relapses do occur [Yu, A. L. et al. Clin Cancer Res 27, 2179-2189 (2021); Ladenstein, R. et al. Cancers (Basel) 12, E309 (2020)].

Overall, with the use of these intensive multi-modal treatment regimens, long-term survival for children with high-risk neuroblastoma has moderately improved over the past 30 years, but in long-term reports, overall survival is still below 50%.

A subgroup of patients with high-risk disease has a suboptimal response at end of induction chemotherapy based on MIBG assessment. A second line chemotherapy regimen consisting of topotecan, vincristine and doxorubicin (TVD) has been shown to achieve (near) complete clearance of metastatic disease in one third of these patients. Nevertheless, in the SIOPEN HR-NBL trial, 25% of the patients had primary refractory disease defined as suboptimal response to induction chemotherapy and second-line regimen TVD.

Of those patients who had good response to induction chemotherapy, and hence proceed with myeloablative chemotherapy still a significant number will suffer disease recurrence: 3- year EFS of 50% for those receiving busulfan-melphalan high dose chemotherapy and stem cells rescue, shows that still at least 50% of children with high-risk neuroblastoma experience relapse.

Relapsed or refractory disease neuroblastoma remains incurable for the vast majority of patients. In metastatic neuroblastoma, 10-year OS was 2% after relapse and 1.5% after progression according to Italian Registry data³. The International Neuroblastoma Risk Group (INRG), a database with outcomes from 8,800 children with neuroblastoma treated worldwide, shows 5-year overall survival after relapse of 8% for non-infants with relapsed metastatic neuroblastoma and 4% for those with MYCN amplification.

A number of second-line strategies have been tested for refractory or relapsed (r/r) neuroblastoma over the past thirty years including chemotherapy, immunotherapy or targeted radionuclide therapy. Most published clinical trials have concentrated on response as an endpoint and there is a lack of data on median survival. The interpretation of those studies remains very difficult given that the populations studied were heterogeneous, with different proportions of refractory/relapsed patients, numbers of prior treatments, different response criteria being used, prognostic factors were not considered, and most studies were singlearm non-randomised studies. The majority of these studies have only reported best responses during treatment, without data on median time-to-progression, PFS or OS; endpoints that might be more reflective of patient benefit. Overall, objective response rates (OR + VGPR + PR) range from 19 to 41 %, and 13 to 58% experience disease stabilisation (SD). Overall, patients with refractory disease seem to benefit more than those with relapsed disease.

Some of these regimens tested have been taken forward. Chemo-immunotherapy with irinotecan and temozolomide in combination with anti-GD2 antibody dinutuximab is now standard of care for r/r neuroblastoma in the USA following a phase II COG trial (ANBL1221). This regimen was shown to have anti-tumour activity for patients at first relapse irrespective of prior anti-GD2 antibody treatment; 41.5% of the patients showed objective responses and a further 41.5% had stable disease [Mody, R. et al. J Clin Oncol 38, 2160-2169 (2020)].

In the UK, treatment regimens for patients with r/r neuroblastoma have been tested in Innovative Therapies for Children with Cancer (ITCC)/SIOPEN studies. The ITCC phase II trial testing the combination of topotecan and temozolomide (TOTEM) showed a best objective response rate (ORR) of 24% with a median response duration of 8.5 months³⁶. This regimen has been taken forward to the BEACON-lmmuno study. The BEACON study aimed to assess the activity of backbone chemotherapy regimens with and without inhibiting angiogenesis with bevacizumab. The optimal combination was not determined but bevacizumab combined with irinotecan and temozolomide was identified as a regimen that achieved anti-tumour activity and warranted further randomized evaluation.

Experimental treatment approaches incorporating anti-GD2 antibodies or, for a subgroup of patients, targeted therapies are in development. In addition, CAR-engineered T and natural killer (NK) cell therapies are being evaluated in early phase clinical studies.

The expression of GD2 on neuroblastoma has led to exploration of GD2 CAR T cell targeting in the setting of r/r disease. Most CAR T cell studies to date have used the murine high affinity antibody 14g2a as the binding domain, but application of CARs based on humanized intermediate affinity huK666 and high affinity 3F8 has also been reported. Key clinical data from 6 phase I clinical studies is summarised in Table and then detailed below.

Table 1 . Clinical trials of CAR T cell therapy in systemic GD2+ tumours.

The first study of CAR T cells (14g2a- CAR T cells) targeting GD2 in r/r neuroblastoma used a first-generation receptor based on 14g2a. This was a double marking study which aimed to determine whether CAR T cells derived from peripheral blood T cells or from Epstein Barr virus (EBV) specific cytotoxic T cells (CTL) would result in improved persistence. Three dose levels were explored ranging from 2x10⁷/m² to 2x10⁸/m², all without prior lymphodepletion. GD2-CAR EBV-CTLs reached higher peak expansion in vivo compared to GD2-CAR non selected T cells. Long term follow up of an extended cohort of patients treated on this study, showed low-level persistence for up to 96 weeks for GD2-CAR EBV-CTLs and for up to 192 weeks for GD2-CAR unselected T cells [Louis et al., supra]. Treatment was well tolerated. Neurotoxicity was not observed. No CRS was reported. Three patients with active disease at the time of the GD2-CAR T cell infusion achieved a CR and two of these CRs were durable out to >21 and >60 months respectively. These three patients had persistent CAR T cells at 6 weeks.

The next study involved 14g2a-CD28-OX40-^/14G2a-CD28-z CAR T cells and tested a third generation receptor again based on 14g2a expressed in unselected autologous T cells [Heczey et al. (2017), supra]. Cohort 1 received GD2-CAR T cells (1x10⁷/m² - 1x10⁸/m²) without prior lymphodepletion, cohort 2 received GD2-CAR (1x10⁸/m²-1.5x10⁸/m²) cells using fludarabine and cyclophoshamide (Flu/Cy) lymphodepletion, and cohort 3 was treated with GD2-CAR T cells (1 ,5x10⁸/m²) following Flu/Cy lymphodepletion combined with programmed death-1 (PD-1) inhibitor. In total, eleven patients were treated with GD2-CAR T cells. The infusions were safe, and no neurotoxicity occurred. Peak expansion was increased in patients receiving GD2-CAR T cells following Flu/Cy lymphodepletion as compared to when no lymphodepletion was used. Addition of PD-1 inhibition did not increase in vivo expansion or persistence. Of the 11 evaluable patients, 6 had progressive disease and 5 had stable disease at 6-week follow-up.

A second generation version of this same receptor using CD28 and CD3z has subsequently been used in phase I clinical study using NKT cells as effector cells [Heczey et al. (2020), supra]. Here, IL-15 was co-expressed with the GD2-CAR to support persistence of CAR NKT cells. Interim report of the first 3 patients treated on this study showed that GD2-CAR NKT cells (3x10⁶/m²) were tolerated with no dose limiting toxicity (DLT), no neurotoxicity and no CRS. Presence of GD2-CAR NKT cells in a post infusion tumour biopsy in one patient and a post infusion bone marrow biopsy in another patient was demonstrated. Two patients had stable disease and one patient a partial response.

A third-generation receptor based on 14G2a incorporating CD28, 4-1 BB and CD3z endodomains, iCasp9/14g2a-CD28-41 BB- , has been tested in a phase l/ll study conducted by the Ospidale Bambino De Gesu [Del Bufalo et al., supra]. The suicide gene iCasp9 was co-expressed with the GD2-CAR. A total of 27 patients (26 with r/r neuroblastoma, 1 in CR after first line treatment) received 3-10x10⁶/kg GD2-CAR T cells with prior Flu/Cy lymphodepletion. Eleven patients received multiple (up to 4) doses of GD2-CAR T cells. Neuropathy was reported in 6 patients which was transient only. No neurotoxicity was observed and no DLT occurred. One of 27 patients had CRS grade 3. One patient received two infusions of the dimerizing agent intended to induce caspase 9 and destroy the infused GD2-CAR cells after an altered state of consciousness developed. Subsequent diagnostic workup revealed a brain haemorrhage as the causative event deemed unrelated to GD2 CAR T cells. Persistence of CAR T cells at 3 months was reported for 75% of patients. Best response for the 27 patients enrolled on this study included 8 CR, 1 maintained CR (patient with no evidence of disease at enrolment), 8 PR, 5 SD and 5 PD. In 4 patients induced CR was maintained > 12 months at the time of report.

In a Chinese study, a iCasp9/hu3F8-CD28-41 BB-z CAR based on humanized 3F8 was used [Yu et al., supra]. This CAR incorporates CD28, 41 BB and CDz endodomains. The suicide gene iCasp9 was co-expressed with the CAR. Ten children with r/r neuroblastoma received 0.13-34x10⁶/kg GD2-CAR T cells with prior Flu/Cy lymphodepletion. Eight patients received a single dose, one patient 2 doses and one patient 3 doses of CAR T cells. Neuropathy (grade 1) was reported in 3 patients which was transient only. No neurotoxicity was observed and no DLT occurred. One of 10 patients had grade 3 skin toxicity. Biopsy showed signs of inflammation. Symptoms resolved fully without treatment. Six patients had SD 6 months post CAR T cells. Of these, SD was sustained in 4 patients at one year post CAR T cells.

At UCL, a RQR8/huK666-CD28-z CAR based on a different anti-GD2 antibody, humanized KM666 which has intermediate affinity for GD2, has been developed. This is a second generation CAR incorporating CD28 and CDz endodomains. The sort-suicide gene RQR8 was co-expressed with the CAR. In brief, in a clinical study [Straathof et al., supra], 12 children with r/r neuroblastoma received escalating doses of GD2-CAR T cells (1x10⁷/m² - 1x10⁹/m²) and increasing intensity of preparative lymphodepletion. Of the 6 patients receiving >10⁸/m² CAR T cells after flu/cy conditioning, two experienced grade 2-3 CRS and 3 demonstrated regression of soft tissue and bone marrow disease. This clinical activity, while transient, was achieved without DLT and without neurotoxicity.

CAR T cell therapy of solid cancers has proven to be less effective than against lymphoid malignancies. For instance, complete remissions are achieved in patients with r/r B-ALL, Diffuse large B cell lymphoma, Mantle Cell Lymphoma and Multiple Myeloma. In contrast, much fewer studies have been reported to date in solid cancers with objective responses observed only in a minority of patients. Here, clinical activity most commonly is reduction in tumour burden and disease control and rarely complete and sustained responses.

Adoptive T cell therapy with tumour infiltrating lymphocytes (TILs) has demonstrated the potential of T cells to elicit complete responses in solid cancer [Kochenderfer, J. N. et al. Blood 116, 3875-3886 (2010)]. While only a proportion of patients exhibit long term, durable responses, these results suggest that T cells have the potential to eliminate solid tumours under adequate conditions. Barriers limiting the efficacy of CAR T cells for solid tumours include: (i) the lack of truly tumour-specific target antigens and tumour heterogeneity that can lead to tumour escape due to loss of antigen expression; (ii) delayed exposure to cognate antigen due to requirement of CAR T cells to traffic to tumour sites which impairs their in vivo expansion and persistence; (iii) impaired access of CAR T cells to the tumour sites due to disrupted tumour vasculature and tumour stromal and extracellular matrix barriers to T cell infiltration; (iv) immunosuppressive cells and soluble factors within the tumour microenvironment which inhibit CAR T cell function and persistence [Rodriguez-Garcia, A et al. Front Immunol 11 , 1109 (2020)].

In neuroblastoma, GD2 is homogenously expressed on all tumour cells and to date no modulation of GD2 expression in patients who received CAR T cells have been described. While in vivo expansion of GD2 CAR T cells is achieved, peak expansion is lower than that seen with CD19-CAR T cells where immediately encounter with cognate antigen occurs upon intravenous administration. While GD2-CAR T cells have been shown to home to bone, bone marrow and soft tissue sites of disease, only small amounts of CAR T cells are detectable at tumour sites in limited number of patients. Long term in vivo persistence of the CAR T cells was not achieved in the majority of patients in clinical studies of GD2 CAR T cells in r/r neuroblastoma. In the 1 RG-CART study, analysis of biopsies or recurring tumours showed the presence of myeloid cells such as macrophages with an immune suppressive ability i.e. expression of PD-L1 [Straathof et al., supra]. In addition, frequency of circulating myeloid derived suppressor cells (MDSC) was shown to inversely correlates with persistence of GD2- CAR T cells and clinical response in the Italian GD2-CAR T study [Tumino, N. et al. Journal of Hematology & Oncology 14, 191 (2021)].

GD2 is expressed on a range of solid tumours in addition to neuroblastoma, diffuse midline glioma (DMG), medulloblastoma, retinoblastoma, sarcomas (osteosarcoma, Ewing’s sarcoma, rhabdomyosarcoma), melanoma and small cell lung cancer.

GD2-CAR T cells are being tested in phase I clinical studies in patients with r/r GD2+ osteosarcoma (NCT02107963, NCT04539366, NCT03356782, NCT03373097). Results of these clinical studies have not been published to date.

For Diffuse Midline Glioma, recently the first experience with GD2 CAR T cells was reported [Majzner, R. G. et al. Nature 603, 934-941 (2022)] with subsequent additional data in a conference abstract [Majzner, R. et al. Abstract CT001 : Major tumor regressions in H3K27M- mutated diffuse midline glioma (DMG) following sequential intravenous (IV) and intracerebroventricular (ICV) delivery of GD2-CAR T cells, in (2022)] (total 11 patients). DMG has a dismal outcome with standard of care being palliative radiotherapy. On this study (NCT04196413), patients with brainstem or spinal DMG receive autologous T cells expressing a second-generation 41 BB- CAR based on 14g2a after completion of standard of care radiotherapy. CAR T cells (Dose level (DL)1 : 1 x10⁶/kg, DL2: 3 x10⁶/kg) are administered intravenously (IV) with prior Flu/Cy lymphodepletion. Patients with clinical benefit after IV CAR T cells are eligible for further GD2 CAR T cells administration intraventricularly via an Ommaya reservoir (flat dose of 10-30x10⁶).

Three patients on DL2 experienced Grade 4 CRS, successfully managed with tocilizumab, anakinra, and corticosteroids. On both dose levels, all subjects exhibited transient symptoms related to on-tumour inflammation, termed Tumour Inflammation-Associated Neurotoxicity (TIAN). This neurotoxicity was successfully managed with anakinra and, in some cases, CSF drainage and dexamethasone. No DLT due to TIAN has occurred. No on-target off-tumour neurotoxicity occurred. Nine of 10 patients evaluable for response at time of report experienced radiographic and/or clinical benefit after IV infusion, and they received subsequent intracerebroventricularly (ICV) GD2-CART infusions. ICV infusions were not associated with high-grade CRS, although some subjects developed transient fever, headache, meningism, nausea, and/or vomiting, and several subjects developed TIAN. Four patients who received ICV CAR T cells experienced continued clinical and radiographic benefit.

The neurotoxicity (TIAN) observed in this study is due to the CNS midline location of DMG [Mahdi, J. et al. Tumor-inflammation-associated neurotoxicity: a toxicity syndrome in patients treated with immunotherapy for CNS tumors. Nature Medicine in press, (2023)]. This toxicity was transient and fully reversable. Critically no on-target off-tumour neurotoxicity was observed which support safe targeting of GD2.

TGFb controls multiple cellular functions. During homeostasis, TGFb controls inflammatory responses triggered by exposure to the outside milieu in barrier tissues. Most cancers leverage TGFb to control several microenvironmental cell types including the adaptive immune system. TGFb has multiple effects on the adaptive cellular immune system. This includes prevention of differentiation of TH1 cells, promoting differentiation to TH 17 and Treg cells. Additionally, TGFb inhibits proximal T cell signalling events and more generally inhibits cytotoxic lymphocytes. TGFb has inhibitory effects on CAR T cells, and these inhibitory effects can be blocked by co-expression of a dominant-negative TGFb receptor.

Myeloid cell infiltration, including MDSC, is a hallmark of the neuroblastoma immune microenvironment. Myeloid cells from neuroblastoma patients have been shown to inhibit CAR T cell function. TGFb is secreted by MDSC. Additionally, TGFb was strongly expressed by primary neuroblastoma tumours and cell lines¹⁸, suggesting that the tumour cells are a major source of TGFb in addition to the microenvironment. This has been confirmed by more studies: for instance, a study of neuroblastomas of all clinical stages using reverse transcription polymerase chain reaction (RT-PCR) showed TGFb expression in 45 out of 51 cases. In a more recent study, microarray gene expression profiling of 249 untreated primary neuroblastomas was performed. This showed TGFBR1, TGFBR2, TGFB1, and TGFB2 expression both in high-risk tumours that have either amplified or nonamplified MYCN and in low-risk neuroblastomas.

Functional studies have shown that TGFb influences the neuroblastoma immune microenvironment. For instance, conditioning with neuroblastoma supernatant affects the chemokine receptor repertoire of human resting NK cells. In particular neuroblastoma cells upregulated the expression of CXCR4 and CXCR3 in all NK cells and downregulated CX(3)CR1 ; with this phenomenon being dependent on neuroblastoma secretion of TGF-pi . In addition, phospho-SMAD2, which accumulates in cell nuclei downstream of TGFPR1 signalling, can be detected in untreated neuroblastomas growing in NSG mice. Further, bone marrow and blood plasmas from neuroblastoma patients induce SMAD signalling in a reporter cell lines, and that galunisertib, a small-molecule inhibitor of the TGFPR1 signalling, blocks this activity.

PD-L1 (Programmed Death-Ligand 1) is a protein that plays a significant role in suppressing the immune response by binding to its receptor, PD-1 , which is expressed on activated T cells. The upregulation of PD-L1 in cancer cells or the tumour microenvironment can help cancer cells evade immune system detection and attack.

PD-L1 has been detected in neuroblastoma cell lines and tumours. For example, PD-L1 expression was present in 14% (17/118) primary neuroblastoma samples, with inferior survival of patients whose samples stained positive for PD-L1 as compared with those whose samples were PD-L1-negative. Immunohistochemistry on 31 neuroblastomas showed that 35% were positive for PD-L1 and similarly showed that expression was associated with worse prognosis. Immunohistochemistry on 500 paediatric tumours showed PD-L1 was found in 48/254 (18.9%) of neuroblastoma cases, again with high PD-L1 associated with increased risk of relapse. Finally, in a study testing Pembroluzimab, 80 neuroblastoma cases were secreened by immunohistochemistry, and 16 (20%) were PD-L1 positive.

These reports may underestimate the true role of PD-L1 in neuroblastoma since PD-L1 is upregulated in the face of immune activation, particularly in response to IFN-g. In addition, MYCN amplification, a hallmark of high-risk neuroblastoma, has been shown to upregulate PD-L1 IFNy upregulates or induces PD-L1 both in neuroblastoma cell lines as well as in metastatic neuroblasts isolated from bone marrow aspirates of patients with high-risk neuroblastoma.

Further, PD-L1 can also be expressed on tumour-infiltrating immune cells within the tumour microenvironment. In tumour samples of 104 patients with neuroblastoma, PD-L1 expression was observed in tumour-infiltrating myeloid cells. This study also found that PD-L1 expression on myeloid cells was associated with poorer overall survival.

Heterogeneity occurs between patients, between tumours (inter-tumour heterogeneity) and within tumours (intra-tumour heterogeneity). Multiple types of heterogeneity have been observed between tumour cells, stemming from both genetic and non-genetic variability. Heterogeneity between tumour cells can be further increased due to heterogeneity in the tumour microenvironment. Regional differences in the tumour (e.g. availability of oxygen) impose different selective pressures on tumour cells, leading to a wider spectrum of dominant subclones in different spatial regions of the tumour. The influence of microenvironment on clonal dominance is also a likely reason for the heterogeneity between primary and metastatic tumours seen in many patients, as well as the inter-tumour heterogeneity observed between patients with the same tumour type.

The heterogeneity of cancer cells introduces significant challenges in designing effective treatment strategies.

For example, heterogeneic tumours may exhibit different sensitivities to cytotoxic drugs among different clonal populations. This is attributed to clonal interactions that may inhibit or alter therapeutic efficacy.

Drug administration in heterogeneic tumours will seldom kill all tumour cells. The initial heterogeneic tumour population may bottleneck, such that few drug resistant cells (if any) will survive. This allows resistant tumour populations to replicate and grow a new tumour through the branching evolution mechanism (see above). The resulting repopulated tumour is heterogeneic and resistant to the initial drug therapy used. The repopulated tumour may also return in a more aggressive manner.

Additional “armouring”, which enhances CAR T cell function has been proposed as a means to enhance efficiency of CAR T activity in face of challenges posed by solid cancers. This additional engineering is usually distinct from the CAR and includes several possible strategies.

Some strategies involve rendering CAR T cells to be resistant to inhibitory signals. One of the most understood and ubiquitous inhibitory signals is that of TGFb. Several strategies have been developed to engineer resistance to TGFb. These include genomic editing of the TGFb receptor locus¹³⁵, chimeric TGFb receptors and dominant-negative TGFb receptors [Bollard, C. M. et al. Blood 99, 3179-3187 (2002)].

Another key inhibitory signal is that of PD1 receptor in response to interactions with PD-L1.

Several strategies have been developed to engineer resistance to PD1. These include genomic editing of the PD1 gene, dominant negative or PD1 switch receptors or more recently modulating second messengers of inhibitory signals such as SHP1/2.

Additional strategies act to increase the survival and expansion of CAR T cells. These typically involve transmission of cytokine or related intracellular signals. The simplest strategy is to co-express a mitogenic cytokine such as IL7 or IL15. More recently, strategies to constitutively transmit cytokine signals have been described [Krenciute, G. et al. Cancer Immunol Res 5, 571-581 (2017)]. There are several formats to accomplish this. For instance, a fusion of cytokine and its cognate receptor can be expression [Hurton, L. V. et al. PNAS 201610544 (2016) doi:10.1073/pnas.1610544113], Alternatively, mutations in signalling domains of cytokine receptors can be introduced which render the receptors active independent of ligation [Shum, T. et al. Cancer Discov 7, 1238-1247 (2017)]. More recently, heterodimerization domain can be used to link cytokine receptor chains to stimulate receptor signalling in the absence of ligand. Finally, JAK/STAT and AP1 can be directly activated [Lynn, R. C. et al. Nature 576, 293-300 (2019)]. These strategies act to enhance CAR T cell survival while trafficking to sites of disease and in response to hostile microenvironments.

Many other strategies have been proposed (reviewed by Kershaw et a/¹⁴⁰). These include secretion of immune activatory cytokines such as IL12 or immune modulating antibodies; genome editing to enhance CAR T survival by preventing exhaustion or differentiation and many other approaches. One of the current challenges of developing engineered T cell therapy is limited insight which pre-clinical data provides given that current in vivo models do not recapitulate tumour microenvironment. Given the complexity of solid cancers, it is likely that multiple strategies will need to be employed and their utility determined clinically.

There is thus a need for alternative neuroblastoma treatment approaches and cellular therapeutics.

DESCRIPTION OF THE FIGURES

Figure 1 - Schematic diagram illustrating the molecules expressed by the vectors used in the dual vector composition described in Example 1 . Vector 1 expresses a CAR with an antigenbinding domain which binds GD2 (GD2 CAR), a constitutively active cytokine receptor (CCR) and a sort/suicide gene (RQR8). Vector 2 expresses the same GD2 CAR, a dominant negative SHP-2 (ASHP2); a dominant negative transforming growth factor (TGF)pl I receptor (ATGFbRII) and the same sort/suicide gene (RQR8).

Figure 2 - Investigating the capacity of single and dual transduced T cell populations to kill GD2-expressing (SupT1 GD2) and non-expressing (SupT1 NT) target cells.

Figure 3 - Investigating the proliferation of single and dual transduced T cell populations following culture in cytokine-free complete cell culture media for 7 days without further antigen stimulus.

Figure 4 - Investigating the capacity of single and dual transduced T cell populations to kill GD2-expressing (SupT1 GD2) and non-expressing (SupT1 NT) target cells in the presence or absence of TGFp.

Figure 5 - Investigating cytokine production (IFNy) from single and dual transduced T cell populations following co-culture with GD2-expressing (SupT1 GD2) and non-expressing (SupT 1 NT) target cells in the presence or absence of TGFp.

Figure 6 - Results of an in vivo assay investigating the anti-tumour activity of T cells transduced with the dual vector composition by intravenous administration in an established neuroblastoma xenograft model in NSG mice. 1x10⁶ CHLA-255 Flue cells were injected i.v. into female NSG mice. Xenografts were left to establish for 15 days until stable engraftment was detectable by BLI . CAR-T cells were made either by transducing cells with a single vector expressing a GD2 CAR (GD2 CAR) or by transducing cells with the dual vector composition described in Example 1 and Illustrated in Figure 1 (GD2 CAR + IL7 CCR/GD2 CAR+dSHP2+dTGFbRII). CAR T-cells were administered i.v. at a dose of 3x10⁶ CAR T- cells/mouse. Quantitated bioluminescent signal of CHLA-255 Flue was plotted over time as total flux (photons/s) A. Graph showing fluorescent signal over time for mice receiving CAR- T cells expressing GD2 CAR alone (GD2 CAR); untransduced T cells (NT) or buffer alone (PBS). B Ventral images of mice obtained on days -1 , 2, 7, 10 and 14 following administration of CAR-T cells expressing GD2 CAR alone (GD2 CAR); untransduced T cells (NT) or buffer alone (PBS) C Graph showing fluorescent signal over time for mice receiving cells transduced with the dual vector composition described in Example 1 and Illustrated in Figure 1 (GD2 CAR + IL7 CCR/GD2 CAR+dSHP2+dTGFbRII); untransduced T cells (NT) or buffer alone (PBS). D Ventral images of mice obtained on days -1 , 2, 7, 10 and 14 following administration of cells transduced with the dual vector composition described in Example 1 and Illustrated in Figure 1 (GD2 CAR + IL7 CCR/GD2 CAR+dSHP2+dTGFbRII); untransduced T cells (NT) or buffer alone (PBS).

Figure 7- (a) Initial designs of CAR based on the anti-GD2 scFv huK666 used the human IgG 1 Fc domain as a spacer between the scFv and transmembrane domain. Use of the Fc domain risks binding to Fc Receptors potentially activating expressing cells, and potentially reducing persistence of CAR T cell. Mutations (-) in the Fc domain were introduced to reduce FcR binding. This original receptor (huKFc*28Z) possessed a CD28-Z endodomain, (b) A subsequent receptor, huK828Z, used in the present study was similar, but the spacer domain was replaced with the CD8a stalk

Figure 8- Structure of the y-retroviral vector transfer cassettes; 5’LTR: 5’ Long terminal repeat; SD: splice donor; Y: packaging signal; SA: Splice acceptor; each vector has a single open reading frame denoted by ®; self-cleaving foot-and-mouth disease virus 2A-like sequences are denoted by #, these allow multiple proteins to be encoded by a single frame; SAR: Scaffold attachment region; 3’LTR: 3’ Long terminal repeat, (a) Plasmid AU54280 encodes the sort-suicide gene RQR8 and the GD2 CAR huK828Z along with both chains of the constitutively active IL7 cytokine receptor (FabCCR-IL7); (b) Plasmid AU54281 also encodes RQR8 and huK828Z but additionally encodes truncated SHP2 (dSHP2) and truncated TGFb receptor II (dTBRII).

Figure 9- Proteins applied in the present study. dSHP2 is a truncated form of SHP2 which consists of the SH2 domains buck lacks the phosphatases; dTRBRII is a truncated form of the TGFb receptor which lacks most of its endodomain; RQR8 is a sort-suicide gene consisting of two copies of a disulfide bond constrained peptide mimetope of the CD20 major extracellular loop flanking the extreme amino-terminus of CD34 connected to the CD8a stalk and transmembrane domain; huK828Z is a second-generation GD-specific CAR based on the scFv from the humanized GD2-specific antibody huK666 with the endodomain of CD3 FabCCR-IL7 is a constitutively active IL7 receptor which consists of the two proteins: the first is a fusion between the human Ig k light chain constant region and the endodomain of the cytokine receptor common g chain. The second is a fusion between human IgG CH1 and the IL7 receptor a endodomain.

Figure 10- Key starting materials are AU54280 and AU54281 y-retroviral vectors (A1) and patient derived leukapheresis (B1). Vectors are manufactured in large batches and can be used for multiple CAR T cell productions. CAR T cell production is performed individually for each patient. Patient T cells are isolated from patient pheresate and activated (S1). These activated cells are then transduced with both vectors simultaneously (S2). Transduced T cells are expanded in culture for 6-8 days (S3). Expanded and transduced T cells constitute the drug substance. These are then cryopreserved in DMSO containing cryoprotectant (S4). Cryopreserved expanded and transduced T cells constitute the drug product.

Figure 11- Cytotoxicity, (a) SupT1 cells, (b) SupT1 cells engineered to express GD2 (SupT1.GD2), (c) Raji cells or (d) Raji cells engineered to express GD2 (Raji.GD2) were cultured alone, or 1 :1 with non-transduced T cells, or with RQR8/huKFc*28Z CAR T cells or with RQR8/huK828Z CAR T cells for 72 hours. Remaining target cells were quantified using flow- cytometry. Percentage surviving target cells normalized to NT T cell controls are shown. Experiment shows results from 8 healthy donor T cells.

Figure 12- Proliferation of T cells in response to Raji target cells. Non-transduced (NT) T cells, or huKFc*28Z CAR T cells, or huK828Z CAR T cells were loaded with CFSE dye. After resting overnight and washing, T cells were cultured alone, or co-incubated 1 :1 with either Raji cells or Raji.GD2 cells. After 5 days, T cells were analyzed by flow-cytometry. Cell division results in dilution of the CFSE and subsequent reduction in fluorescence. The percentage of T cells dividing was hence determined.

Figure 13- Cytokine release. Target cells were culture alone or in co-culture with either nontransduced (NT) T cells, or huKFc*28Z CAR T cells or huK828Z CAR T cells, (a) IFN-g secretion in response to SupT1 ; (b) IFN-g secretion in response to SupT1.GD2; (c) IL-2 release in response to SupT1 ; (d) IL-2 release in response to SupT1.GD2; (e) IFN-g release in response to Raji cells; (f) IFN-g release in response to Raji.GD2; (g) IL-2 release in response to Raji cells; (h) IL-2 release in response to Raji.GD2.

Figure 14- Effect of dSHP2 on cytolytic function in the presence of PDL1/PD1. (a) 1 :4 effector to target ratio and (b) is 1 :8 effector to target ratio. Conditions marked with a bar and PD1 are conditions where PD1 is artificially over-expressed in the CAR T cells via additional retroviral transduction with a vector encoding PD1. Lines represent the median value of 12 separate donors. Statistical significance was defined using One-way ANOVA and Tukey post hoc test. ****, p<0.0001 ; ***, p<0.001 ; ns, not statistically significant.

Figure 1- RQR8/huK828Z/CST CAR T cells overcome PD1/PDL1-driven inhibition - cytokine release restoration. CAR T cells or CAR + PD1 T cells were co-cultured with SupT1 NT, SupT 1 GD2 and SupT 1 GD2 PDL1 targets for 72h at ratio (a) 1 :4 and (b) 1 :8 (E:T) quantified by ELISA. Lines represent the median value of 12 separate donors. Statistical significance was defined using One-way ANOVA and Tukey post hoc test. ****, p<0.0001 ;***, p<0.001 ; ns, not statistically significant

Figure 16- Effect of TGFp on cytotoxicity. Non-transduced (NT) T cells, RQR8/huK828Z and RQR8/huK828Z/CST CAR T cells were co-cultured with SupT1 NT and SupT1 GD2 targets for 7 days at ratios of 1 :2 and 1 :8 (E:T) either in the presence or absence of 10 ng/ml TGF- p. Target cell killing was quantified by flow cytometry and normalised to targets alone. Remaining viable target cells were defined by their exclusion of Sytox Blue and the absence of CD2 and CD3 expression. Lines represent the median value of 10 separate donors. Statistical significance was defined using One-way ANOVA and Tukey post hoc test. ****, p<0.0001 ;***, p<0.001 ; ns, not statistically significant

Figure 17- Effect of TGFp on cytokine release. Non-transduced (NT), RQR8/huK828Z or RQR8/huK828Z/CST CAR T cells were co-cultured with SupT1 NT and SupT1 GD2 targets for 7 days at a ratio of 1 :2 and 1 :8 (E:T) either in the presence or absence of 10ng/ml TGF- p. IFN-y secretion was quantified by ELISA. Lines represent the median value of 10 separate donors. Statistical significance was defined using One-way ANOVA and Tukey post hoc test. ****, p<0.0001 ;***, p<0.001 ; ns, not statistically significant

Figure 18- RQR8/huK828Z/CST early proliferation in the absence of exogenous cytokines or Ag-stimulation. CAR T cells were Cell Trace Violet (CTV) dye labelled and left for 7 days in culture in the absence of exogenous cytokines or Ag-stimulation. (a) Percent proliferating CAR T cells was determined as percentage of cells having diluted CTV die. (b) shows CAR T cell level of CTV dye dilution on day 7 which indicates the extent of cell cycling. Lines represent the median value of 11 separate donors. Statistical significance was defined using the paired T test. ****, p<0.0001 ;***, p<0.001 ; **, p<0.01 ; *, p<0.05; ns, not statistically significant.

Figure 19- Exploration of RQR8/huK828Z/CST CAR T cell autonomous proliferation, (a) Nontransduced (NT) T cells, (b) RQR8/huK828Z or (c) RQR/huK828Z/CST CAR T cells were left in culture in the absence of exogenous cytokines or Ag-stimulation. Every 7 days CAR T cells were counted, re-suspended in fresh media and plated at 1x10⁶ cells/ml. T cell counts were plotted against time. Lines represent 7 individual donors. While RQR8/huK828Z/CST CAR T cells from some donors displayed initial independent proliferation, expansion contracted in all donors by day 42, with no long-term proliferation.

Figure 20- RQR8/huK828Z/CST CAR T cells maintain cytotoxic potential after repeated encounters with antigen. CAR T cells were co-cultured with either (a) SupT 1 cells or (b) SupT1 GD2 cells at 1 :1 ratio (E:T). Every 3- or 4-days, CAR T cells were re-stimulated with (a) 5x10⁴ SupT1 or (b) SupT1 GD2 cells/well. Target cell killing was quantified by FACS before each new re-stimulation. Remaining viable target cells were defined by their exclusion of Sytox Blue and the absence of CD2 and CD3 expression, while T cells were defined by the expression of CD2 and CD3. Lines represent the median value of 4 separate donors.

Figure 21- RQR8/huK828Z/CST CAR T cells do not expand autonomously in the absence of cytokines or Ag-stimulation CAR T cells were left in culture in the absence of exogenous cytokines or Ag-stimulation. Every 7 days CAR T cells were counted, re-suspended in fresh media and plated at 1x10⁶ cells/ml. T cell counts were plotted against time. Lines represent 7 individual donors.

Figure 22- Rituximab-mediated depletion of RQR8/huK828Z/CST CAR T cells. Either nontransduced or RQR8/huK828Z or RQR8/huK828Z/CST CAR T cells were incubated for 2h with 25% baby-rabbit complement and rituximab or isotype control at 100 pg/mL. Samples were stained with Sytox Blue and viable cells were assessed via flow cytometry, (a) Representative histograms from 1 donor showing depletion; (b) Percentage CAR T cells from total T cells with lines representing the median value of 15 separate donors. Statistical significance was defined using paired t-test. ****, p<0.0001

Figure 23- Cytotoxic potential of RQR8/huK828Z/CST CAR T cells post Rituximab-mediated depletion of transduced T cells. Following 2h CDC-mediated depletion of CAR T cells by rituximab or isotype control, T cells were co-cultured with SupT1 NT or SupT1 GD2 cells at 1 :1 effectortarget ratio for 24h. Target cell killing was quantified by flow cytometry and normalised to co-cultures with NT T cells. Remaining viable target cells were defined by their exclusion of viability dye (Sytox Blue) and the absence of CD2 and CD3 expression. Lines represent the mean value of 10 separate donors. Statistical significance was defined using paired t-test. ****, p<0.000

Figure 24- Schema of experimental methodology used to test RQR8/huK828Z/CST CAR T cells in vivo. NSG mice were burdened with 1x106 CHLA-255 cells by tail vein injection. Engraftment was confirmed by BLI at days 11 and 15. Mice received PBS, 1 x 10⁶ CAR T cells or an equivalent number of total NT T cells on day 16. BLI was repeated every 3-4 days until day 50. Mice were sacrificed at day 50 or sooner in case of disease progression, >10% weight loss or other signs of illness or distress.

Figure 25- 1x10⁶ CAR T cells or equivalent numbers of NT T cells were administered via tail vein at day 15 following administration of CHLA-255 Neuroblastoma cells. Following this, tumour burden was estimated by bioluminescence imaging every 3-4 days. Mice were sacrificed when humane endpoints were reached in terms of tumour burden or overall health and wellbeing or at the scientific endpoint of the experiment at day 50.

SUMMARY

The present inventors have developed a combinatorial approach to address the issues of tumour cell and microenvironment heterogeneity to CAR therapies, delayed antigen encounter reliant on CAR T cell trafficking to tumour sites and immunosuppressive mechanisms within the tumour environment.

When cells are transduced with multiple vectors simultaneously, the resulting product will be a mixture of cells which are singly and combinatorially transduced. For example, if cells are transduced with two vectors, one comprising transgene A and one comprising transgene B, the transduced cells will be a mixture of cells expressing A alone; B alone; and cell expressing both A and B. For cells transduced with three vectors each comprising a transgene, the resulting transduced cells will be a mixture of: A alone; B alone; C alone; A and B; A and C; B and C; and cells expressing A, B and C.

The present invention involves using such a mixture as a therapeutic CAR-T-cell product. The use of a combinatorial product gives in-built flexibility which enhances the product's capacity to adapt to differences in target cells and in tumour microenvironment.

The present disclosure provides CAR T cells such as RQR8/huK828Z/CST CAR T cells. The present disclosure provides methods for producing the CAR T cells by transduction of autologous T cells with two y-retroviral vectors. The first vector may be pSF.SV40.KanR.RQR8-2A-aGD2_Huk666-CD8STK-TyrpTM-CD28z-2A-Fab_IL7, which was designated AU54280 and encodes RQR8, huK828Z and FabCCR-IL7. The second vector may be pSF.SV40.KanR.dual_SH2-SHP2-2Aw-RQR8-2A-aGD2_huK666_CD8STK- TyrpTM-CD28z-2A-dnTGFbetaRII, which was designated AU54281 and encodes RQR8, huK828Z, dSHP2 and dTBRII.

Thus, in a first aspect, the present invention provides methods for treating a neuroblastoma in a patient comprising administering to the patient autologous anti-GD2 CAR T cells, wherein the CAR T cells comprise an anti-GD2 chimeric antigen receptor (CAR) comprising:

(i) a GD2 antigen-binding domain which comprises: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

CDR1 - SYNIH (SEQ ID No. 1),

CDR2 - VIWAGGSTNYNSALMS (SEQ ID No. 2), and

CDR3 - RSDDYSWFAY (SEQ ID No. 3) and b) a light chain variable region (VL) having CDRs with the following sequences:

CDR1 - RASSSVSSSYLH (SEQ ID No. 4), CDR2 - STSNLAS (SEQ ID No. 5), and CDR3 - QQYSGYPIT (SEQ ID No. 6);

(ii) a CD8a stalk; and

(iii) an a CD28-CD3 endodomain; and wherein the CAR T cells comprise at least one vector comprising a nucleic acid encoding an activity modulator which modulates the activity of the CAR, of a cell expressing the CAR, or of a target cell. The activity modulator may be selected from a dominant negative SHP-1 or SHP-2, a dominant negative transforming growth factor (TGF) p receptor and/or a constitutively active chimeric cytokine receptor. The GD2 binding domain may comprise a VH domain having the sequence shown as SEQ ID No. 7; and/or a VL domain having the sequence shown as SEQ ID No 8.

In the methods, the activity modulator may be a dominant negative SHP-2.

In the methods, the at least one vector may comprise a nucleic acid encoding a dominant negative SHP-2 and a dominant negative TGF receptor.

In the methods, the constitutively active chimeric cytokine receptor may comprise an IL-7 receptor a-chain endodomain. In the methods, the anti-GD2 CAR T cells may be RQR8/huK828Z/CST CAR T cells.

The patient may have relapsed or refractory neuroblastoma following at least one line of therapy.

In the methods, the patient may be administered a single dose of about 30 x 10⁶, 100 x 10⁶, or 300 x 10⁶ anti-GD2 RQR8/huK828Z/CST CAR T cells/m².

In the methods, the administration may be an intravenous injection through a Hickman line or peripherally inserted central catheter.

In a second aspect, the invention provides methods for producing a CAR T cell composition which comprises step of transducing a population of cells with a mixture of at least two y- retroviral vectors, wherein each vector comprises a nucleic acid sequence which encodes an anti-GD2 chimeric antigen receptor (CAR) comprising:

CDR1 - SYNIH (SEQ ID No. 1),

CDR2 - VIWAGGSTNYNSALMS (SEQ ID No. 2), and

(ii) a CD8a stalk; and

(iii) an a CD28-CD3 endodomain; and wherein at least one vector comprises a nucleic acid encoding an activity modulator which modulates the activity of the CAR, of a cell expressing the CAR, or of a target cell, wherein the activity modulator may be selected from a dominant negative SHP-1 or SHP-2, a dominant negative transforming growth factor (TGF) receptor and/or a constitutively active chimeric cytokine receptor. The GD2 binding domain may comprise a VH domain having the sequence shown as SEQ ID No. 7; and/or a VL domain having the sequence shown as SEQ ID No 8. In the production methods, the activity modulator may be a dominant negative SHP-2.

In the production methods, at least one vector may comprise a nucleic acid which encodes a dominant negative SHP-2 and a dominant negative TGF p receptor.

In the production methods, the activity modulator may be a constitutively active chimeric cytokine receptor.

In the production methods, in the mixture of viral vectors at least one vector may comprise a nucleic acid sequence which encodes a dominant negative SHP-2 and a dominant negative TGF receptor; and at least one vector may comprise a nucleic acid sequence which encodes a constitutively active chimeric cytokine receptor.

In the production methods, the constitutively active chimeric cytokine receptor may comprise an IL-7 receptor a-chain endodomain.

The production methods may further comprise selecting CAR-expressing cells from the transduced cell population.

In a third aspect, the invention provides a cell composition made by the production methods.

In a fourth aspect, the invention provides a cell composition according made by the production methods for use in treating and/or preventing Neurobastoma.

In a fifth aspect, the cell compositions may be used in the manufacture of a medicament for treating and/or preventing Neuroblastoma.

FURTHER ASPECTS

The present invention also provides additional aspects which are summarised in the following numbered paragraphs.

1. A method for treating a neuroblastoma in a patient comprising administering to the patient autologous anti-GD2 CAR T cells, wherein the CAR T cells comprise an anti-GD2 chimeric antigen receptor (CAR) comprising: (i) a GD2 antigen-binding domain which comprises: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

CDR1 - SYNIH (SEQ ID No. 1),

CDR2 - VIWAGGSTNYNSALMS (SEQ ID No. 2), and

CDR1 - RASSSVSSSYLH (SEQ ID No. 4),

CDR2 - STSNLAS (SEQ ID No. 5), and

CDR3 - QQYSGYPIT (SEQ ID No. 6);

(ii) a CD8a stalk; and

(iii) an a CD28-CD3 endodomain; and wherein the CAR T cells comprise at least one vector comprising a nucleic acid encoding an activity modulator which modulates the activity of the CAR, of a cell expressing the CAR, or of a target cell, wherein the activity modulator is selected from a dominant negative SHP-1 or SHP-2, a dominant negative transforming growth factor (TGF) p receptor and/or a constitutively active chimeric cytokine receptor.

2. The method of claim 1 , wherein the GD2 binding domain comprises a VH domain having the sequence shown as SEQ ID No. 7; and/or a VL domain having the sequence shown as SEQ ID No 8.

3. The method according to any preceding claim, wherein the activity modulator is a dominant negative SHP-2.

4. The method according to any preceding claim, wherein the at least one vector comprises a nucleic acid encoding a dominant negative SHP-2 and a dominant negative TGF receptor.

5. The method according to any preceding claim, wherein the constitutively active chimeric cytokine receptor comprises an IL-7 receptor a-chain endodomain.

6. The method of claim 1 wherein the anti-GD2 CAR T cells are RQR8/huK828Z/CST CAR T cells. 7. The method of any preceding claim wherein the patient has relapsed or refractory neuroblastoma following at least one line of therapy.

8. The method of any preceding claim wherein the patient is administered a single dose of about 30 x 10⁶, 100 x 10⁶, or 300 x 10⁶ anti-GD2 RQR8/huK828Z/CST CAR T cells/m².

9. The method of any preceding claim wherein the administration is an intravenous injection through a Hickman line or peripherally inserted central catheter.

10. A method for making a CAR T cell composition which comprises step of transducing a population of cells with a mixture of at least two y-retroviral vectors, wherein each vector comprises a nucleic acid sequence which encodes an anti-GD2 chimeric antigen receptor (CAR) comprising:

CDR1 - SYNIH (SEQ ID No. 1),

CDR2 - VIWAGGSTNYNSALMS (SEQ ID No. 2), and

CDR1 - RASSSVSSSYLH (SEQ ID No. 4),

CDR2 - STSNLAS (SEQ ID No. 5), and

CDR3 - QQYSGYPIT (SEQ ID No. 6);

(ii) a CD8a stalk; and

(iii) an a CD28-CD3 endodomain; and wherein at least one vector comprises a nucleic acid encoding an activity modulator which modulates the activity of the CAR, of a cell expressing the CAR, or of a target cell, wherein the activity modulator is selected from a dominant negative SHP-1 or SHP-2, a dominant negative transforming growth factor (TGF) receptor and/or a constitutively active chimeric cytokine receptor.

11 . The method of claim 10, wherein the GD2 binding domain comprises a VH domain having the sequence shown as SEQ ID No. 7; and/or a VL domain having the sequence shown as SEQ ID No 8. 12. A method according to claim 10 or 11 , wherein the activity modulator is a dominant negative SHP-2.

13. A method according to claim 10, 11 or 12, wherein at least one vector comprises a nucleic acid which encodes a dominant negative SHP-2 and a dominant negative TGF p receptor.

14. A method according to claim 10, 11 , 12 or 13, wherein the activity modulator is a constitutively active chimeric cytokine receptor.

15. A method according to claim 10, 11 , 12, 13 or 14, wherein in the mixture of viral vectors at least one vector comprises a nucleic acid sequence which encodes a dominant negative SHP-2 and a dominant negative TGF receptor; and at least one vector comprises a nucleic acid sequence which encodes a constitutively active chimeric cytokine receptor.

16. A method according to claim 10, 11 , 12, 13, 14 or 15, wherein the constitutively active chimeric cytokine receptor comprises an IL-7 receptor a-chain endodomain.

17. A method according to any claims 10 to 16 further comprising selecting CAR-expressing cells from the transduced cell population.

18. A cell composition made by a method according to any of claims 10 to 17.

19. A cell composition according to claim 18 for use in treating and/or preventing Neuroblastoma.

20. The use of a cell composition according to claim 18 in the manufacture of a medicament for treating and/or preventing Neuroblastoma.

The following detailed description, as it relates to nucleic acid and polypeptide sequences, polypeptide components, vectors, cells methods etc applies equally to the aspects laid out in the above paragraphs as to the aspects of the invention in the claims. DETAILED DESCRIPTION

The present invention relates to a method for making a cell composition which comprises step of transducing a population of cells with a mixture of at least two viral vectors.

The viral vectors may, for example, be retroviral vectors or lentiviral vectors.

Retroviruses are double stranded RNA enveloped viruses mainly characterized by the ability to “reverse-transcribe” their genome from RNA to DNA. Virions measure 100-120 nm in diameter and contain a dimeric genome of identical positive RNA strands complexed with the nucleocapsid proteins. The genome is enclosed in a proteic capsid that also contains enzymatic proteins, namely the reverse transcriptase, the integrase and proteases, required for viral infection. The matrix proteins form a layer outside the capsid core that interacts with the envelope, a lipid bilayer derived from the host cellular membrane, which surrounds the viral core particle. Anchored on this bilayer, are the viral envelope glycoproteins responsible for recognizing specific receptors on the host cell and initiating the infection process. Envelope proteins are formed by two subunits, the transmembrane (TM) that anchors the protein into the lipid membrane and the surface (SU) which binds to the cellular receptors.

Based on the genome structure, retroviruses are classified into simple retroviruses, such as MLV and murine leukemia virus; or complex retroviruses, such as HIV and EIAV. Retroviruses encode four genes: gag (group specific antigen), pro (protease), pol (polymerase) and env (envelope). The gag sequence encodes the three main structural proteins: the matrix protein, nucleocapsid proteins, and capsid protein. The pro sequence encodes proteases responsible for cleaving Gag and Gag-Pol during particle assembly, budding and maturation. The pol sequence encodes the enzymes reverse transcriptase and integrase, the former catalyzing the reverse transcription of the viral genome from RNA to DNA during the infection process and the latter responsible for integrating the proviral DNA into the host cell genome. The env sequence encodes for both SU and TM subunits of the envelope glycoprotein. Additionally, retroviral genome presents non-coding cis-acting sequences such as: two LTRs (long terminal repeats), which contain elements required to drive gene expression, reverse transcription and integration into the host cell chromosome; a sequence named packaging signal (i ) required for specific packaging of the viral RNA into newly forming virions; and a polypurine tract (PPT) that functions as the site for initiating the positive strand DNA synthesis during reverse transcription. In addition to gag, pro, pol and env, complex retroviruses, such as lentiviruses, have accessory genes including vif, vpr, vpu, nef, tat and rev that regulate viral gene expression, assembly of infectious particles and modulate viral replication in infected cells.

During the process of infection, a retrovirus initially attaches to a specific cell surface receptor. On entry into the susceptible host cell, the retroviral RNA genome is then copied to DNA by the viral ly encoded reverse transcriptase which is carried inside the parent virus. This DNA is transported to the host cell nucleus where it subsequently integrates into the host genome. At this stage, it is typically referred to as the provirus. The provirus is stable in the host chromosome during cell division and is transcribed like other cellular proteins. The provirus encodes the proteins and packaging machinery required to make more virus, which can leave the cell by a process known as “budding”.

When enveloped viruses, such as retrovirus and lentivirus, bud out of the host cells, they take part of the host cell lipidic membrane. In this way, host-cell derived membrane proteins become part of the retroviral particle. The present invention utilises this process in order to introduce proteins of interest into the envelope of the viral particle.

VIRAL VECTORS

Retroviruses and lentiviruses may be used as a vector or delivery system for the transfer of a nucleic acid sequence, or a plurality of nucleic acid sequences, to a target cell. The transfer can occur in vitro, ex vivo or in vivo. When used in this fashion, the viruses are typically called viral vectors.

Gamma-retroviral vectors, commonly designated retroviral vectors, were the first viral vector employed in gene therapy clinical trials in 1990 and are still one of the most used. More recently, the interest in lentiviral vectors, derived from complex retroviruses such as the human immunodeficiency virus (HIV), has grown due to their ability to transduce non-dividing cells. The most attractive features of retroviral and lentiviral vectors as gene transfer tools include the capacity for large genetic payload (up to 9 kb), minimal patient immune response, high transducing efficiency in vivo and in vitro, and the ability to permanently modify the genetic content of the target cell, sustaining a long-term expression of the delivered gene.

The retroviral vector can be based on any suitable retrovirus which is able to deliver genetic information to eukaryotic cells. For example, the retroviral vector may be an alpharetroviral vector, a gammaretroviral vector, a lentiviral vector or a spumaretroviral vector. Such vectors have been used extensively in gene therapy treatments and other gene delivery applications. The viral vector of the present invention may be a retroviral vector, such as a gamma- retroviral vector. The viral vector may be based on human immunodeficiency virus.

The viral vector of the present invention may be a lentiviral vector. The vector may be based on a non-primate lentivirus such as equine infectious anemia virus (EIAV).

NUCLEIC ACID SEQUENCES AND CONSTRUCTS

In the mixture of viral vectors used in the method of the present invention, each vector may comprise one or more nucleic acid sequences. For example, one or more of the vectors in the mixture may comprise a nucleic acid construct comprising a plurality of nucleic acid sequences which are co-expressed. The nucleic acid construct may, for example, be bicistronic or tri-cistronic. The nucleic acid construct may comprise 2, 3, 4 or 5 transgenes.

The nucleic acid sequences in the nucleic acid construct may be separated by a “coexpression” sequence which enables the two or more polypeptides, once translated, to be expressed separately in or on the cell.

The coexpression sequence may encode a cleavage site, such that the nucleic acid construct produces comprises two or more polypeptides joined by a cleavage site(s). The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual polypeptides without the need for any external cleavage activity.

The cleavage site may be any sequence which enables the two or more polypeptides to become separated.

The term “cleavage” is used herein for convenience, but the cleavage site may cause the polypeptides to separate into individual entities by a mechanism other than classical cleavage. For example, for the Foot-and-Mouth disease virus (FMDV) 2A self-cleaving peptide (see below), various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect [Donnelly et al (2001) J. Gen. Virol. 82:1027-1041], The exact mechanism of such “cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities. The cleavage site may be a furin cleavage site.

Furin is an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor. Furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X- (Arg/Lys)-Arg' (SEQ ID No. 9)) and is enriched in the Golgi apparatus.

The cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.

TEV protease is a highly sequence-specific cysteine protease which is chymotrypsin-like proteases. It is very specific for its target cleavage site and is therefore frequently used for the controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TEV cleavage site is ENLYFQ\S (where ‘V denotes the cleaved peptide bond) (SEQ ID No. 10). Mammalian cells, such as human cells, do not express TEV protease. Thus in embodiments in which the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell - exogenous TEV protease must also expressed in the mammalian cell.

The cleavage site may encode a self-cleaving peptide.

A ‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the proteins and the self-cleaving peptide is produced, it is immediately “cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.

The self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus. The primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus. In apthoviruses, such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating “cleavage” at its own C-terminus (Donnelly et al, supra). “2A-like” sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al, supra).

The cleavage site may comprise the 2A-like sequence shown as SEQ ID No. 11.

SEQ ID No. 11

RAEGRGSLLTCGDVEENPGP

The present invention provides a nucleic acid construct which comprises a nucleic acid sequence encoding a dominant negative SHP-1 or SHP-2 (dnSHP-1 or dnSHP-2, respectively) and a nucleic acid sequence encoding a dominant negative TGFp receptor.

Dominant negative SHP-1 or SHP-2 and TGFp receptors are described in more detail below.

The nucleic acid construct may have the structure: dnSHP-coexpr-dnTGFpR, or dnTGFpR-coexpr-dnSHP in which: dnSHP is a nucleic acid sequence encoding dominant negative SHP-1 or SHP-2

"coexpr" is a nucleic acid sequences enabling coexpression of the two polypeptides as separate entities dnTGFpR is a dominant negative TGFp receptor.

The nucleic acid construct may also comprise a nucleic acid sequence encoding a CAR. In which case the nucleic acid construct may have the structure:

CAR-coexpr1-dnSHP-coexpr2-dnTGFpR

CAR-coexpr1-dnTGFpR-coexpr2-dnSHP dnTGFpR -coexpr1-CAR-coexpr2-dnSHP dnTGFpR -coexprl- dnSHP -coexpr2-CAR dnSHP-coexprl-dnTGFpR -coexpr2-CAR or dnSHP -coexpr1-CAR-coexpr2-dnTGFpR in which: dnSHP dominant negative SHP-2

"coexprl" and "coexpr2" which may be the same or different, are nucleic acid sequences enabling coexpression of the three polypeptides as separate entities dnTGFpR is a is a nucleic acid sequence encoding dominant negative TGFp receptor; and CAR is a nucleic acid sequence encoding a chimeric antigen receptor.

The nucleic acid construct may have the structure: dnSHP2 -coexprl -CAR-coexpr2-dnTGFpR

The present invention also provides a nucleic acid construct which comprises a nucleic acid sequence encoding a the constitutively active chimeric cytokine receptor.

Constitutively active chimeric cytokine receptors are described in more detail below.

CAR-coexpr-CCR

CCR -coexpr-CAR in which:

CCR is a nucleic acid encoding a constitutively active chimeric cytokine receptor coexpr is a nucleic acid sequence enabling coexpression of the two polypeptides as separate entities

CAR is a nucleic acid sequence encoding a chimeric antigen receptor.

The nucleic acid construct may have the structure:

CAR-coexpr-CCR

SUICIDE GENE

A nucleic acid construct may also comprise a nucleic acid encoding a suicide gene.

Since T-cells engraft and are autonomous, a means of selectively deleting CAR T-cells in patients is desirable. Suicide genes are genetically encodable mechanisms which result in selective destruction of infused T-cells in the face of unacceptable toxicity. The earliest clinical experience with suicide genes is with the Herpes Virus Thymidine Kinase (HSV-TK) which renders T-cells susceptible to Ganciclovir. HSV-TK is a highly effective suicide gene. However, pre-formed immune responses may restrict its use to clinical settings of considerable immunosuppression such as haploidentical stem cell transplantation. Inducible Caspase 9 (iCasp9) is a suicide gene constructed by replacing the activating domain of Caspase 9 with a modified FKBP12. iCasp9 is activated by an otherwise inert small molecular chemical inducer of dimerization (CID). iCasp9 has been recently tested in the setting of haploidentical HSCT and can abort GvHD. Both iCasp9 and HSV-TK are intracellular proteins, so when used as the sole transgene, they have been co-expressed with a marker gene to allow selection of transduced cells.

WO2016/135470 describes a suicide gene which also comprises Caspase 9 but can be induced to dimerise using rapamycin or a rapamycin analog.

This suicide gene, sometimes termed Rapcasp9 or Rapacasp9, has or comprises the amino acid sequence shown as SEQ ID No. 12.

SEQ ID No. 12 (Rapcasp9) ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSLEGVQVETISPGDGRTF PKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK LTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGGSGGGGSGGGGSGVDGFGDVGA LESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEV KGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVYGTDGCPVSVEKI VNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGL RTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLL LRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSA

WO2013/153391 describes a marker/suicide gene known as RQR8 which can be detected with the antibody QBEndlO and expressing cells lysed with the therapeutic antibody Rituximab.

The sort/suicide gene RQR8 has or comprises the amino acid sequence shown as SEQ ID No. 13. SEQ ID No. 13 (RQR8)

CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVV

Including a suicide gene in one or more of the vectors in the viral vector composition of the invention enables the selective ablation of a proportion of transduced cells within the subject.

For example, for two vectors A and B, transduced cells will be a mixture of cells transduced with vector A alone, cells transduced with vector B alone, and cells transduced with both vectors A and B. If vector A expresses or co-expresses a suicide gene, activating the suicide gene will cause the deletion of cells transduced with vector A alone, or with vectors A and B, but cells transduced with vector B alone will be spared.

This is particularly useful where one vector in the mixture encodes a potentially dangerous or toxic gene. If a suicide gene is included on the cassette for that vector, then in the event of an unacceptable immunological or toxic event in the patient, cells expressing the gene in question can be selectively deleted by triggering the suicide gene. Cells expressing other vector combinations which do not include the potentially dangerous gene/suicide gene combination are spared and can continue their therapeutic effect.

VIRAL VECTOR COMPOSITION

The present invention provides a viral vector composition which comprises a mixture viral vectors. The composition may be made by simply mixing two of more viral vectors. The composition may comprise between 2 and 10 viral vectors, for example, 2, 3, 4, 5 or 6 viral vectors.

The viral vectors in the mixture may each comprise one or more transgenes. Two or more viral vectors in the composition may overlap in one or more transgenes. For example, two viral vectors in the composition may comprise a nucleic acid sequence encoding the same CAR, but may differ in the presence or type of activity modulator(s) encoded by other nucleic acid sequences.

One or more of the viral vector(s) in the composition may comprises a nucleic acid sequence encoding a dominant negative SHP-1 or SHP-2. One or more viral vector(s) in the composition may comprise a nucleic acid sequence encoding a dominant negative TGFp receptor. One or more viral vectors in the composition may comprise a nucleic acid sequence encoding a chimeric antigen receptor. One or more viral vectors in the composition may comprise a nucleic acid sequence encoding a constitutively active chimeric cytokine receptor.

The viral vector composition may comprise a vector which comprises a nucleic acid sequence encoding a dominant negative SHP-1 or SHP-2 and a nucleic acid sequence encoding a dominant negative TGFp receptor.

The viral vector composition may comprise a vector which comprises a nucleic acid sequence encoding a dominant negative SHP-2 and a nucleic acid sequence encoding a dominant negative TGFp receptor.

The constitutively active chimeric cytokine receptor may comprise an IL-2 receptor p-chain endodomain, an IL-7 receptor a-chain endodomain, an IL-15 receptor a-chain endodomain; or a common y-chain receptor endodomain. The constitutively active chimeric cytokine receptor may comprise an IL-7 receptor a-chain endodomain.

The viral vector composition may comprise a vector which comprises a nucleic acid sequence encoding a dominant negative SHP-2 and a nucleic acid sequence encoding a dominant negative TGFp receptor, and a vectorwhich comprises constitutively active chimeric cytokine receptor may comprise an IL-7 receptor a-chain endodomain.

The viral vector composition may comprise plurality of vectors, each of which encode different activity modulator(s) or activity modulator combinations.

CHIMERIC ANTIGEN RECEPTOR

In the method of the present invention at least one vector in the mixture of viral vectors may comprise a nucleic acid sequence which encodes a chimeric antigen receptor (CAR).

CHIMERIC ANTIGEN RECEPTORS (CARS)

CARs are chimeric type I trans-membrane proteins which connect an extracellular antigenrecognizing domain (binder) to an intracellular signalling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site. A spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of lgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the I gG 1 hinge alone, depending on the antigen. A trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FCER1 or CD3 (also termed CD3z, CDz or CD^). Consequently, these first generation receptors transmitted immunological signal 1 , which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3 results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal - namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related 0X40 and 41 BB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.

CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral or lentiviral vectors to generate cancer-specific T cells for adoptive cell transfer. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus, the CAR directs the specificity and cytotoxicity of the T cell towards tumour cells expressing the targeted antigen.

ANTIGEN-BINDING DOMAIN

The antigen-binding domain is the portion of CAR which recognizes antigen. Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain antibody; an artificial single binder such as a DARPin (designed ankyrin repeat protein); or a single-chain derived from a T-cell receptor.

In a classical CAR, the antigen-binding domain comprises: a single-chain variable fragment (scFv) derived from a monoclonal antibody. CARs have also been produced with domain antibody (dAb) or VHH antigen binding domains or which comprise a Fab fragment of, for example, a monoclonal antibody. A FabCAR comprises two chains: one having an antibodylike light chain variable region (VL) and constant region (CL); and one having a heavy chain variable region (VH) and constant region (CH). One chain also comprises a transmembrane domain and an intracellular signalling domain. Association between the CL and CH causes assembly of the receptor.

The two chains of a Fab CAR may have the general structure:

VH - CH - spacer - transmembrane domain - intracellular signalling domain; and

VL - CL or

VL - CL - spacer- transmembrane domain - intracellular signalling domain; and

VH - CH

For Fab-type chimeric receptors, the antigen binding domain is made up of a VH from one polypeptide chain and a VL from another polypeptide chain.

The polypeptide chains may comprise a linker between the VHA/L domain and the CH/CL domains. The linker may be flexible and serve to spatially separate the VHA/L domain from the CH/CL domain.

GD2

CARs have been developed which bind disialoganglioside (GD2) a sialic acid-containing glycosphinolipid. Such CARs may, for example, be based on the GD2 binder 14g2a, or huK666 as described in WO2015/132604.

A CAR which binds GD2 may have an antigen-binding domain which comprises: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

CDR1 - SYNIH (SEQ ID No. 1);

CDR2 - VIWAGGSTNYNSALMS (SEQ ID No. 2)

CDR3 - RSDDYSWFAY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences:

CDR1 - RASSSVSSSYLH (SEQ ID No. 4); CDR2 - STSNLAS (SEQ ID No. 5)

CDR3 - QQYSGYPIT (SEQ ID No. 6).

The GD2 binding domain may comprise a VH domain having the sequence shown as SEQ ID No. 7; and/or a VL domain having the sequence shown as SEQ ID No 8.

SEQ ID No. 7 (Humanised KM666 VH sequence)

QVQLQESGPGLVKPSQTLSITCTVSGFSLASYNIHWVRQPPGKGLEWLGVIWAGGSTNYN SALMSRLTISKDNSKNQVFLKMSSLTAADTAVYYCAKRSDDYSWFAYWGQGTLVTVSS

SEQ ID No. 8 (Humanised KM666 VH sequence) ENQMTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKSGKAPKVWIYSTSNLASGV PSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSGYPITFGQGTKVEIK

INTRACELLULAR T CELL SIGNALING DOMAIN (ENDODOMAIN)

The CAR may comprise or associate with an activating endodomain: the signal-transmission portion of the CAR. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling may be needed. For example, chimeric CD28 and 0X40 can be used with CD3- Zeta to transmit a proliferative/survival signal, or all three can be used together.

The endodomain of the CAR may comprise the CD28 endodomain and 0X40 and CD3-Zeta endodomain.

The endodomain may comprise:

(i) an ITAM-containing endodomain, such as the endodomain from CD3 zeta; and/or

(ii) a co-stimulatory domain, such as the endodomain from CD28; and/or

(iii) a domain which transmits a survival signal, for example a TNF receptor family endodomain such as QX-40 or 4-1 BB.

An endodomain which contains an ITAM motif can act as an activation endodomain in this invention. Several proteins are known to contain endodomains with one or more ITAM motifs. Examples of such proteins include the CD3 epsilon chain, the CD3 gamma chain and the CD3 delta chain to name a few. The ITAM motif can be easily recognized as a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/l (SEQ ID NO. 14). Typically, but not always, two of these motifs are separated by between 6 and 8 amino acids in the tail of the molecule (YxxL/lx(6-8)YxxL/l). Hence, one skilled in the art can readily find existing proteins which contain one or more ITAM to transmit an activation signal. Further, given the motif is simple and a complex secondary structure is not required, one skilled in the art can design polypeptides containing artificial ITAMs to transmit an activation signal (see WO 2000/063372, which relates to synthetic signalling molecules).

A number of systems have been described in which the antigen recognition portion of the CAR is on a separate molecule from the signal transmission portion, such as those described in W0015/150771 ; WO2016/124930 and WO2016/030691. One or more of the viral vectors used in the method of the invention may encode such a "split CAR". Alternatively, one vector may comprise a nucleic acid sequence encoding the antigen recognition portion and one vector may comprise a nucleic acid sequence encoding the intracellular signalling domain.

Where the composition of viral vectors includes more than one vector comprising a nucleic acid sequence encoding a CAR, the CARs may have different endodomains or different endodomain combinations. For example, one CAR may be a second generation CAR and one CAR may be a third generation CAR. Alternatively, both CARs may be a second generation CAR but may have different co-stimulatory domains. For example, different second generation CAR signalling domains include: 41 BB-CD3 OX40-CD3 and CD28- CD3 .

SIGNAL PEPTIDE

One or more nucleic acid sequences in the vector composition may encode a signal peptide so that when the CAR or activity modulator is expressed inside a cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed (or secreted).

The core of the signal peptide may contain a long stretch of hydrophobic amino acids that tends to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases.

The signal peptide may be at the amino terminus of the molecule.

A CAR may have the general formula:

Signal peptide - antigen binding domain - spacer domain - transmembrane domain - intracellular T cell signaling domain (endodomain).

SPACER

The CAR may comprise a spacer sequence to connect the antigen binding domain with the transmembrane domain and spatially separate the antigen binding domain from the endodomain. A flexible spacer allows to the antigen binding domain to orient in different directions to enable antigen binding.

The spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a CD8 stalk, or a combination thereof. The spacer may alternatively comprise an alternative sequence which has similar length and/or domain spacing properties as an IgG 1 Fc region, an IgG 1 hinge or a CD8 stalk.

Where the composition of viral vectors includes more than one vector comprising a nucleic acid sequence encoding a CAR, the CARs may have different spacers.

ACTIVITY MODULATOR

In the method of the present invention at least one vector in the mixture of viral vectors may comprise a nucleic acid sequence which encodes an activity modulator. When this is the case, at least a proportion of the transduced cells in the CAR-expressing cell composition of the invention will express one or more activity modulator(s). An activity modulator is a molecule made by the CAR-expressing cell which modulates the activity of the CAR, of a cell expressing the CAR, or of a target cell.

An activity modulator may be an intracellular molecule, expressed at the cell surface, or secreted by the CAR-expressing cell. MODULATING THE ACTIVITY OF THE CAR t _ Enhancing ITAM phosphorylation

During T cell activation in vivo, antigen recognition by the T-cell receptor (TCR) results in phosphorylation of Immunoreceptor tyrosine-based activation motifs (ITAMs) on CD3 . Phosphorylated ITAMs are recognized by the ZAP70 SH2 domains, leading to T cell activation.

T-cell activation uses kinetic segregation to convert antigen recognition by a TCR into downstream activation signals. Briefly: at the ground state, the signalling components on the T-cell membrane are in dynamic homeostasis whereby dephosphorylated ITAMs are favoured over phosphorylated ITAMs. This is due to greater activity of the transmembrane CD45/CD148 phosphatases over membrane-tethered kinases such as lek. When a T-cell engages a target cell through a T-cell receptor (or CAR) recognition of cognate antigen, tight immunological synapses form. This close juxtapositioning of the T-cell and target membranes excludes CD45/CD148 due to their large ectodomains which cannot fit into the synapse. Segregation of a high concentration of T-cell receptor associated ITAMs and kinases in the synapse, in the absence of phosphatases, leads to a state whereby phosphorylated ITAMs are favoured. ZAP70 recognizes a threshold of phosphorylated ITAMs and propagates a T- cell activation signal.

The process is essentially the same during CAR-mediated T-cell activation. An activating CAR comprises one or more ITAM(s) in its intracellular signalling domain, usually because the signalling domain comprises the endodomain of CD3 . Antigen recognition by the CAR results in phosphorylation of the ITAM(s) in the CAR signalling domain, causing T-cell activation.

Inhibitory immune-receptors such as PD1 cause the dephosphorylation of phosphorylated ITAMs. PD1 has ITIMs in its endodomain which are recognized by the SH2 domains of molecules such as PTPN6 (SHP-1) and SHP-2. Upon recognition, PTPN6 is recruited to the juxta-membrane region and its phosphatase domain subsequently de-phosphorylates ITAM domains inhibiting immune activation.

An activity modulator capable of modulating the activity of the CAR may be capable of directly or indirectly phosphorylating the ITAM(s) in the CAR signalling domain. MODULATING THE ACTIVITY OF THE CAR-T CELL

1. Dominant negative SHP

An activity modulator which blocks or reduces the inhibition mediated by inhibitory immunoreceptors such as CTLA4, PD-1 , LAG-3, 2B4 or BTLA 1 may tip the balance of phosphorylatiomdephosporylation at the T-cell:target cell synapse in favour of phosphorylation of ITAMs, leading to T-cell activation. For example, the activity modulator may block or reduce the phosphorylation of ITIMs in the endodmain of inhibitory receptor(s) or may block or reduce the dephosphorylation of ITAMs in the CAR signalling domain by proteins such as SHP-1 and SHP-2.

WO2016/193696 describes various different types of protein capable of modulating the balance of phosphorylatiomdephosporylation at the T-cell:target cell synapse. For example, the activity modulator may comprise a truncated form of SHP-1 or SHP-2 which comprises one or both SH2 domains, but lacks the phosphatase domain. When expressed in a CAR-T cell, these molecules act as dominant negative versions of wild-type SHP-1 and SHP-2 and compete with the endogenous molecule for binding to phosphorylated ITIMs.

The activity modulator may be a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain. The truncated protein may comprise one or both SHP-1 SH2 domain(s) but lack the SHP-1 phosphatase domain. Alternatively, the truncated protein may comprise one or both SHP-2 SH2 domain(s) but lack the SHP-2 phosphatase domain.

SHP-1

Src homology region 2 domain-containing phosphatase-1 (SHP-1) is a member of the protein tyrosine phosphatase family. It is also known as PTPN6.

The N-terminal region of SHP-1 contains two tandem SH2 domains which mediate the interaction of SHP-1 and its substrates. The C-terminal region contains a tyrosine-protein phosphatase domain.

SHP-1 is capable of binding to, and propagating signals from, a number of inhibitory immune receptors or ITIM containing receptors. Examples of such receptors include, but are not limited to, PD1, PDCD1, BTLA4, LILRB1, LAIR1 , CTLA4, KIR2DL1, KIR2DL4, KIR2DL5,

KIR3DL1 and KIR3DL3.

Human SHP-1 protein has the UniProtKB accession number P29350.

An activity modulator may comprise or consist of the SHP-1 tandem SH2 domain which is shown below as SEQ ID NO: 15.

SHP-1 SH2 complete domain (SEQ ID NO: 15)

MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGD FYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQ AETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVG GLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYY

SHP-1 has two SH2 domains at the N-terminal end of the sequence, at residues 4-100 and 110-213. An activity modulator may comprise one or both of the sequences shown as SEQ ID No. 16 and 17.

SHP-1 SH2 1 (SEQ ID NO: 16)

WFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDL YGGEKFATLTELVEYYTQQQGVLQDRDGTI I H LKYPL

SHP-2 SH2 2 (SEQ ID No. 17)

WYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIK VMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPY

The activity modulator may comprise a variant of SEQ ID NO: 15, 16 or 17 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence is a SH2 domain sequence has the required properties. In other words, the variant sequence should be capable of binding to the phosphorylated tyrosine residues in the cytoplasmic tail of at least one of PD1 , PDCD1, BTLA4, LILRB1 , LAIR1, CTLA4, KIR2DL1 , KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3 which allow the recruitment of SHP-1.

SHP-2

SHP-2, also known as PTPN11 , PTP-1 D and PTP-2C is is a member of the protein tyrosine phosphatase (PTP) family. Like PTPN6, SHP-2 has a domain structure that consists of two tandem SH2 domains in its N-terminus followed by a protein tyrosine phosphatase (PTP) domain. In the inactive state, the N-terminal SH2 domain binds the PTP domain and blocks access of potential substrates to the active site. Thus, SHP-2 is auto-inhibited. Upon binding to target phospho-tyrosyl residues, the N-terminal SH2 domain is released from the PTP domain, catalytically activating the enzyme by relieving the auto-inhibition.

Human SHP-2 has the UniProtKB accession number P35235-1.

An activity modulator may comprise or consist of the SHP-2 tandem SH2 domain which is shown below as SEQ ID NO: 20. SHP-2 has two SH2 domains at the N-terminal end of the sequence, at residues 6-102 and 112-216. An activity modulator may comprise one or both of the sequences shown as SEQ ID No. 18 and 19.

SHP-2 first SH2 domain (SEQ ID NO: 18)

WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLY GGEKFATLAELVQYYM EH HGQLKEKNGDVI ELKYPL

SHP-2 second SH2 domain (SEQ ID No. 19)

WFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGDDKGESNDGKSKVTHV MIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVLQLKQPL

SHP-2 both SH2 domains (SEQ ID No. 20)

WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLY GGEKFATLAELVQYYMEHHGQLKEKNGDVIELKYPLNCADPTSERWFHGHLSGKEAEKLL TEKGKHGSFLVRESQSHPGDFVLSVRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGE RFDSLTDLVEHYKKNPMVETLGTVLQLKQPL

The activity modulator may comprise a variant of SEQ ID NO: 18, 19 or 20 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence is a SH2 domain sequence has the required properties. In other words, the variant sequence should be capable of binding to the phosphorylated tyrosine residues in the cytoplasmic tail of at least one of PD1 , PDCD1, BTLA4, LILRB1 , LAIR1, CTLA4, KIR2DL1 , KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3 which allow the recruitment of SHP-2.

Alternatively, the activity modulator may comprise a non-cytokine receptor exodomain. WO2017/029512 describes chimeric cytokine receptors (CCR) comprising: an exodomain which binds to a ligand selected from a tumour secreted factor, a chemokine and a cellsurface antigen; and a cytokine receptor endodomain.

The chimeric cytokine receptor may comprise two polypeptides:

(i) a first polypeptide which comprises:

(a) a first antigen-binding domain which binds a first epitope of the ligand

(b) a first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide which comprises:

(a) a second antigen-binding domain which binds a second epitope of the ligand

(b) a second chain of the cytokine-receptor endodomain.

Alternatively, the chimeric cytokine receptor which comprises two polypeptides:

(i) a first polypeptide which comprises:

(a) a heavy chain variable domain (VH)

(b) a first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide which comprises:

(a) a light chain variable domain (VL)

(b) a second chain of the cytokine-receptor endodomain.

For example, the cytokine receptor endodomain may comprise:

(i) IL-2 receptor p-chain endodomain

(ii) IL-7 receptor a-chain endodomain;

(iii) IL-15 receptor a-chain endodomain; or

(iv) common y-chain receptor endodomain.

The cytokine receptor endodomain may comprise (i), (ii) or (iii); and (iv).

The cytokine receptor endodomain may comprise the a-chain endodomain and the p-chain endodomain from granulocyte-macrophage colony-stimulating factor receptor (GMCSF-R)

The ligand may be a tumour secreted factor, for example a tumour secreted factor selected from: prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF) and CA125. The ligand may be a chemokine, for example a chemokine selected from chemokine selected from: CXCL12, CCL2, CCL4, CCL5 and CCL22.

The ligand may be a cell-surface molecule, such as a transmembrane protein. The ligand may be, for example, CD22.

2. 1. Constitutively active chimeric cytokine receptors

The activity modulator may be a constitutively active chimeric cytokine receptor. The activity modulator may comprise two chains which dimerise, either spontaneously or in the presence of an agent (a chemical inducer of dimerization or CID) bringing together two cytokine receptor endodomains.

The activity modulator may therefore comprise a dimerization domain; and a cytokine receptor endodomain.

Dimerisation may occur spontaneously, in which case the chimeric transmembrane protein will be constitutively active. Alternatively, dimerization may occur only in the presence of a chemical inducer of dimerization (CID) in which case the transmembrane protein only causes cytokine-type signalling in the presence of the CID.

Suitable dimerization domains and CIDs are described in WO2015/150771 , the contents of which are hereby incorporated by reference.

For example, one dimerization domain may comprise the rapamycin binding domain of FK- binding protein 12 (FKBP12), the other may comprise the FKBP12-Rapamycin Binding (FRB) domain of mTOR; and the CID may be rapamycin or a derivative thereof.

One dimerization domain may comprise the FK506 (Tacrolimus) binding domain of FK- binding protein 12 (FKBP12) and the other dimerization domain may comprise the cyclosporin binding domain of cylcophilin A; and the CID may be an FK506/cyclosporin fusion or a derivative thereof.

One dimerization domain may comprise an oestrogen-binding domain (EBD) and the other dimerization domain may comprise a streptavidin binding domain; and the CID may be an estrone/biotin fusion protein or a derivative thereof. One dimerization domain may comprise a glucocorticoid-binding domain (GBD) and the other dimerization domain may comprise a dihydrofolate reductase (DHFR) binding domain; and the CID may be a dexamethasone/methotrexate fusion protein or a derivative thereof.

One dimerization domain may comprise an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain and the other dimerization domain may comprise a dihydrofolate reductase (DHFR) binding domain; and the CID may be an O6-benzylguanine derivative/methotrexate fusion protein or a derivative thereof.

One dimerization domain may comprise a retinoic acid receptor domain and the other dimerization domain may comprise an ecodysone receptor domain; and the CID may be RSL1 or a derivative thereof.

Where the dimerization domain spontaneously heterodimerizes, it may be based on the dimerization domain of an antibody. In particular it may comprise the dimerization portion of a heavy chain constant domain (CH) and a light chain constant domain (CL). The “dimerization portion” of a constant domain is the part of the sequence which forms the interchain disulphide bond.

The chimeric cytokine receptor may comprise the Fab portion of an antibody as exodomain. In this respect, the chimeric antigen may comprise two polypeptides:

(i) a first polypeptide which comprises:

(a) a heavy chain constant domain (CH)

(b) a first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide which comprises:

(a) a light chain constant domain (CL)

(b) a second chain of the cytokine-receptor endodomain.

The cytokine receptor endodomain may comprise:

(i) IL-2 receptor p-chain endodomain

(ii) IL-7 receptor a-chain endodomain; or

(iii) IL-15 receptor a-chain endodomain; and/or

(iv) common y-chain receptor endodomain. The cytokine receptor endodomain may comprise the a-chain endodomain and the p-chain endodomain from granulocyte-macrophage colony-stimulating factor receptor (GMCSF-R)

A constitutively active CCR having an IL-2, IL-7, IL-15 or GM-CSF receptor endodomain may have one of the following structures:

Fab_CCR_IL2: HuLightKappa-IL2RgTM-IL2RgEndo-2A-HuCH1-IL2bTM-IL2RbENDO

Fab_CCR_IL7: HuLightKappa-IL2RgTM-IL2RgEndo-2A-HuCH1-IL7RaTM-IL7RaENDO

Fab_CCR_IL15: HuLightKappa-IL2RgTM-IL2RgEndo-2A-HuCH1-IL15RaTM-IL15RaENDO Fab_CCR_GMCSF:

HuLightKappa-GMCSFRbTM-GMCSFRbEndo-2A-HuCH1-GMCSFRaTM-GMCSFRaENDO

In which:

HuLightKappa is a human light kappa chain

IL2RgTM is a transmembrane domain from human IL2R common gamma chain

IL2RgEndo is an endodomain derived from human IL2R common gamma chain

2A is a sequence enabling the co-expesion of the two polypeptides, which may be a selfcleaving peptide such as a 2A peptide HuCH1 is a human CH1

IL2bTM is a transmembrane domain from human IL-2R beta

IL2RbENDO is an endodomain from human IL2R beta

IL7RaTM is a transmembrane domain from human IL-7R alpha

IL7RaENDO is an endodomain from human IL-7R alpha

IL15RaTM is a transmembrane domain from human IL-15R alpha

IL15RaENDO is an endodomain from human IL-15R alpha

GMCSFRbTM is a transmembrane domain from Human GM-CSFR common beta chain

GMCSFRbEndo is an endodomain from GM-CSFR common beta chain

GMCSFRaTM is a transmembrane domain from Human GF-CSFR alpha GMCSFRaENDO is an endodomain Derived from Human GM-CSFR alpha

A constitutively active CCR having an IL-7 receptor endodomain may have the following structure:

Fab_CCR_IL7: HuLightKappa-IL2RgTM-IL2RgEndo-2A-HuCH1-IL7RaTM-IL7RaENDO

The sequences for the components for making a constitutively active cytokine receptor as shown below as SEQ ID NO. 21 to 33. SEQ ID No. 21 (Human Light Kappa Chain)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID No. 22 (Human Hinge)

EPKSCDKTHTCPPCPKDPK

SEQ ID No. 23 (Human CH1)

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

SEQ ID No. 24 (Transmembrane domain from human IL2R common gamma chain):

VVISVGSMGLIISLLCVYFWL

SEQ ID No. 25 (Transmembrane domain from human IL-2R beta)

IPWLGHLLVGLSGAFGFIILVYLLI

SEQ ID No. 26 (Transmembrane domain from human IL-7R alpha)

PI LLTISI LSFFSVALLVI LACVLW

SEQ ID No. 27 (Transmembrane domain from Human GF-CSFR alpha)

NLGSVYIYVLLIVGTLVCGIVLGFLF

SEQ ID No. 28 (Transmembrane domain from Human GM-CSFR common beta chain)

VLALIVIFLTIAVLLAL

SEQ ID No. 29 (Endodomain from human IL2R common gamma chain)

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGE

GPGASPCNQHSPYWAPPCYTLKPET

SEQ ID No. 30 (Endodomain from human IL-2R beta)

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPL

EVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDP

YSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPG

GSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAG

PREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID No. 31 (Endodomain from human IL-7R alpha)

KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQD TFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRS LDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEA YVTMSSFYQNEQ

SEQ ID No. 32 (Endodomain Derived from Human GM-CSFR alpha)

KRFLRIQRLFPPVPQIKDKLNDNHEVEDEIIWEEFTPEEGKGYREEVLTVKEIT

SEQ ID No. 33 (Endodomain from GM-CSFR common beta chain)

RFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFP ELEGVFPVGFGDSEVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSPQPGPPAASHT PEKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQL VPLAQAMGPGQAVEVERRPSQGAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPM SSGDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPG PVKSGFEGYVELPPIEGRSPRSPRNNPVPPEAKSPVLNPGERPADVSPTSPQPEGLLVLQ QVGDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKAL KQQDYLSLPPWEVNKPGEVC

The activity modulator may comprise a variant of one or more of SEQ ID NO: 21 to 33 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence has the required properties. For example, a variant CH or CL sequence should retain the capacity to dimerise with a CL/CH containing-chain. A variant chain from a cytokine receptor endodomain should retain the capacity to trigger cytokine-mediated signalling when coupled with the reciprocal chain for that cytokine receptor.

3. Modulating TGBB signalling

Engineered cells face hostile microenvironments which limit adoptive immunotherapy. One of the main inhibitory mechanisms within the tumour microenvironment is transforming growth factor beta (TGFP). The TGFp signalling pathway has a pivotal role in the regulatory signalling that controls a variety of cellular processes. TGFp play also a central role in T cell homeostasis and control of cellular function. Particularly, TGFp signalling is linked to an immuno-depressed state of the T-cells, with reduced proliferation and activation. TGFp expression is associated with the immunosuppressive microenvironment of tumour. A variety of cancerous tumour cells are known to produce TGFp directly. In addition to the TGFp production by cancerous cells, TGFp can be produced by the wide variety of non- cancerous cells present at the tumour site such as tumour-associated T cells, natural killer (NK) cells, macrophages, epithelial cells and stromal cells.

The transforming growth factor beta receptors are a superfamily of serine/threonine kinase receptors. These receptors bind members of the TGFp superfamily of growth factor and cytokine signalling proteins. There are five type II receptors (which are activatory receptors) and seven type I receptors (which are signalling propagating receptors).

Auxiliary co-receptors (also known as type III receptors) also exist. Each subfamily of the TGFp superfamily of ligands binds to type I and type II receptors.

The three transforming growth factors have many activities. TGFpi and 2 are implicated in cancer, where they may stimulate the cancer stem cell, increase fibrosis /desmoplastic reactions and suppress immune recognition of the tumour.

TGFpi , 2 and 3 signal via binding to receptors TpRI I and then association to TpRI and in the case of TGFP2 also to TpRI 11. This leads to subsequent signalling through SMADs via TpRI.

TGFps are typically secreted in the pre-pro-form. The “pre” is the N-terminal signal peptide which is cleaved off upon entry into the endoplasmic reticulum (ER). The “pro” is cleaved in the ER but remains covalently linked and forms a cage around the TGFp called the Latency Associated Peptide (LAP). The cage opens in response to various proteases including thrombin and metalloproteases amongst others. The C-terminal region becomes the mature TGFp molecule following its release from the pro-region by proteolytic cleavage. The mature TGFp protein dimerizes to produce an active homodimer.

The TGFp homodimer interacts with a LAP derived from the N-terminal region of the TGFp gene product, forming a complex called Small Latent Complex (SLC). This complex remains in the cell until it is bound by another protein, an extracellular matrix (ECM) protein called Latent TGFp binding protein (LTBP) which together forms a complex called the large latent complex (LLC). LLC is secreted to the ECM. TGFp is released from this complex to a biologically active form by several classes of proteases including metalloproteases and thrombin. The activity modulator of the present invention may modulate TGFp signalling.

3. 1. Dominant negative TGF3 receptor

The active TGFp receptor (TpR) is a hetero-tetramer, composed by two TGFp receptor I (TpRI) and two TGFp receptor II (TpRII). TGFpi is secreted in a latent form and is activated by multiple mechanisms. Once activated it forms a complex with the TpRII TpRI that phosphorylates and activates TpRI.

The activity modulator may be a dominant negative TGFp receptor. A dominant negative TGFp receptor may lack the kinase domain.

For example, the activity modulator may comprise or consist of the sequence shown as SEQ ID No. 34, which is a monomeric version of TGF receptor II

SEQ ID No. 34 (dn TGFp RII)

TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECN DN 11 FSEEYNTSN PDLLLVI FQVTGISLLPPLGVAISVI 11 FYCYRVN RQQKLSS

A dominant-negative TGF-pRII (dnTGF-pRII) has been reported to enhance PSMA targeted CAR-T cell proliferation, cytokine secretion, resistance to exhaustion, long-term in vivo persistence, and the induction of tumour eradication in aggressive human prostate cancer mouse models (Kloss et al (2018) Mol. Ther.26: 1855-1866).

CELL COMPOSITION

The present invention also provides a cell composition made by the method of the present invention.

The invention provides cell composition made by transduction of cells with a plurality of viral vectors such that the composition comprises a mixture of untransduced cells, singly transduced cells and combinatorially transduced cells.

At least one vector in the mixture of viral vectors used in the method of the present invention comprises a nucleic acid sequence encoding a CAR. The cell composition may therefore comprise a mixture of singly and combinatorially transduced CAR-expressing cells. "Combinatorially transduced" means that the cell is transduced with at least two viral vectors. For example, if cells are transduced with two vectors, one comprising transgene A and one comprising transgene B, the transduced cells will be a mixture of cells expressing A alone; B alone; and cell expressing both A and B. In this situation, cells expressing A and B are combinatorially transduced.

For cells transduced with three vectors each comprising a transgene, the resulting transduced cells will be a mixture of: A alone; B alone; C alone; A and B; A and C; B and C; and cells expressing A, B and C. In this situation, the three sub-populations, expressing A and B; A and C; B and C; and cells expressing A, B and C are combinatorially transduced.

The cell composition comprises a plurality of sub-populations derived by transduction with different vector combinations in the mixture of viral vectors.

The cell composition may comprise cytolytic immune cells such as a T cells and/or or NK cells.

T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarised below.

Helper T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1 , TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.

Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL- 10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis. Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.

Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive Treg cells.

Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.

Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.

Gamma delta T cells (yb T cells) are T cells that have a TCR that comprised of one y (gamma) chain and one 5 (delta) chain. Gamma delta T cells are typically less common than op T cells. In humans, in 95% of T cells the TCR consists of an alpha (a) chain and a beta (P) chain (encoded by TRA and TRB, respectively). However, in about 5% of T cells the TCR consists of gamma and delta (y/b) chains (encoded by TRG and TRD, respectively). Gamma delta T cells are abundant in the gut mucosa. Examples of gamma delta cells include Vy9Vb2 T cells. yb TCRs are MHC independent and may detect markers of cellular stress expressed by tumours. The yb TCR may be capable of binding to a phosphoantigen/butyrophilin 3A1 complex; major histocompatibility complex class I chain-related A (MICA); major histocompatibility complex class I chain-related B (MICB); NKG2D ligand 1-6 (LILBP 1-6); CD1c; CD1d; endothelial protein C receptor (EPCR); lipohexapeptides; phycoreythrin or histidyl-tRNA-synthase.

Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids.

Invariant natural killer T (iNKT) cells, also known as type I or classical NKT cells, are a distinct population of T cells that express an invariant ap T-cell receptor (TCR) and a number of cell surface molecules in common with natural killer (NK) cells. NKT cells express a restricted TCR repertoire that, in humans, is composed of a Va24-Ja18 TCRa chain preferentially coupled with a V i 1 TCR chain. Unlike conventional T cells, which mostly recognise peptide antigens presented by MHC molecules, iNKT cells recognise glycolipid antigens presented by the non-polymorphic MHC class l-like molecule, CD1d.

Natural Killer cells (or NK cells) form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner

NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.

The cells of the invention may be any of the cell types mentioned above.

The cells to be transduced with a method of the invention may be derived from a blood sample, for example from a leukapheresate. The cells may be or comprise peripheral blood mononuclear cells (PBMCs).

Cells may either be created ex vivo either from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party). Alternatively, cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to, for example, T or NK cells. Alternatively, an immortalized T- cell line which retains its lytic function and could act as a therapeutic may be used.

The cells may be activated and/or expanded prior to being transduced with nucleic acid encoding the molecules providing the chimeric polypeptide according to the first aspect of the invention, for example by treatment with an anti-CD3 monoclonal antibody.

After transduction, the cells may then by purified, for example, selected on the basis of expression of the CAR. It may be desirable to select cells on the basis of CAR expression when there may be a sub-population of cells post-transduction which express an activity modulator in the absence of a CAR. Where each of the vectors in the mixture of viral vectors comprises a nucleic acid sequence encoding a CAR, it may not be necessary to purify or sort cells on the basis of CAR-expression, as it should not be possible for any cells to express an activity modulator in the absence of a CAR.

The present invention provides a CAR T cell composition which comprises cells comprising at least one of two y-retroviral vectors, wherein one y-retroviral vector comprises a nucleic acid construct having the structure:

RQR8-coexpr1 -CAR-coexpr2- Fab_CCR_l L7 in which:

RQR8 is a nucleic acid sequence encoding a sort/suicide gene RQR8 as described above;

CAR is a nucleic acid sequence encoding an anti-GD2 CAR as described above;

Fab_CCR_IL7 is a nucleic acid sequence encoding a constitutively active IL-7 chimeric cytokine receptor as described above;

"coexprl" and "coexpr2" may be the same or different and are nucleic acid sequences enabling coexpression of the two polypeptides as separate entities; and wherein the other y-retroviral vector comprises a nucleic acid construct having the structure:

The nucleic acid construct may have the structure: d nS H P2 -coexpr3- RQ R8-coexpr4-CA R-coexpr3-d nT G F R in which: dnSHP2 is a nucleic acid sequence encoding a dominant negative SHP-2 as described above;

"coexpr3" and "coexpr4" may be the same or different and are nucleic acid sequences enabling coexpression of the two polypeptides as separate entities;

"coexprl", "coexpr2", "coexpr3" and "coexpr4" may be the same or different;

CAR is a nucleic acid sequence encoding an anti-GD2 CAR as described above; dnTGFpR is a nucleic acid sequence encoding a dominant negative TGFp receptor as described above.

The present invention provides a CAR T cell composition which comprises at least one of the polypeptide combinations: i) RQR8, CAR and Fab_CCR_IL7, and ii) dnSHP2, RQR8, CAR and dnTGFpR, in which:

RQR8 is a sort/suicide gene RQR8 as described above;

CAR is an anti-GD2 CAR having the structure Huk666-CD8STK-TyrpTM-CD28z as described above,

Fab_CCR_IL7 is a constitutively active IL-7 chimeric cytokine receptor as described above; dnSHP2 is a dominant negative SHP-2 as described above; and dnTGFpR is a dominant negative TGFp receptor as described above.

The present invention provides a CAR T cell composition which comprises RQR8, CAR and Fab_CCR_IL7, dnSHP2, and dnTGFpR, in which:

RQR8 is a sort/suicide gene RQR8 as described above;

Fab_CCR_IL7 is a constitutively active IL-7 chimeric cytokine receptor as described above; dnSHP2 is a dominant negative SHP-2 as described above; and dnTGFpR is a dominant negative TGFp receptor as described above. PHARMACEUTICAL COMPOSITION

The cell composition of the present invention, comprising a mixture of singly and combinatorially transduced CAR-expressing cells, may be administered to a patient as a pharmaceutical composition.

The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

METHOD OF TREATMENT

The present invention provides a method for treating a disease which comprises the step of administering a cell composition of the present invention (for example in a pharmaceutical composition as described above) to a subject.

A method for treating a disease relates to the therapeutic use of the cell composition of the present invention. The cell composition may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.

The method for preventing a disease relates to the prophylactic use of the cell composition of the present invention. The cell composition may be administered to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease. The subject may have a predisposition for, or be thought to be at risk of developing, the disease.

The method may involve the steps of:

(i) isolating a cell-containing sample;

(ii) transducing the cells with a mixture of at least two viral vectors;

(iii) administering the cells from (ii) to a subject.

The present invention also provides a cell composition of the present invention for use in treating and/or preventing a disease. The invention also relates to the use of a cell composition of the present invention in the manufacture of a medicament for the treatment of a disease.

The disease to be treated by the methods of the present invention may be a cancerous disease, in particular a cancerous disease associated with GD2 expression. The cancerous disease associated with GD2 expression may be a solid tumour.

The GD2-expressing solid tumour may be relapsed or refractory.

The cancer may be an ectodermal tumour.

Examples of cancers which correlate with elevated GD2 expression levels are: neuroblastoma, melanoma, medulloblastoma, soft-tissue sarcomas, osteosarcoma and small-cell lung cancers such as NSCLC.

The disease may be Neuroblastoma. The disease may be relapsed or refractory (r/r) Neuroblastoma.

The cells of the composition of the present invention may be capable of killing target cells, such as cancer cells. The target cell may be characterised by the presence of a tumour secreted ligand or chemokine ligand in the vicinity of the target cell. The target cell may be characterised by the presence of a soluble ligand together with the expression of a tumour- associated antigen (TAA) at the target cell surface.

Different subpopulations of cells within the composition of the invention may have different levels of ability to kill target cells, both between different patients having the same disease and within a patient at different disease sites (e.g. tumour sites).

The patient may be administered a single dose of about 10 x 10⁶, 30 x 10⁶, 50 x 10⁶, 100 x 10⁶, 150 x 10⁶, 200 x 10⁶, 300 x 10⁶, 400 x 10⁶, 500 x 10⁶, or 600 x 10⁶ anti-GD2 CAR T cells/m² as described above. The anti-GD2 CAR T cells may be RQR8/huK828Z/CST CAR T cells.

The patient may be 18 years of age or younger. The patient may be between equal or older than 1 year of age and equal or older of 18 years of age. The patient may be between equal or older than 1 year of age and equal or older of 16 years of age. If the patient is aged 10 or older, the patient’s performance status may be a Karnofsky score equal to or greater than 50%.

If the patient is younger than 10 years old, the patient’s performance status may be a Lansky score equal to or greater than 50%.

Patients who are unable to walk because of paralysis, but who are able to sit upright unassisted in a wheelchair, will be considered ambulatory for the purpose of assessing performance score.

The patient’s blood or serum level of creatinine may be equal to or lower than 1.5 ULN for age. It this level is higher, then the estimated (or calculated) creatinine clearance may be equal to or higher than 60 ml/min/1 .73 m².

The patient’s absolute lymphocyte count may be equal to or higher than 0.25 x 10⁹/L.

The disease may be relapsed or refractory disease after one or more lines of previous therapy.

The cell composition of the present invention may be administered at least 3 weeks or 5 halflives following treatment with another agent.

The patient may be administered conditioning (pre-conditioning) chemotherapy or lymphodepletion prior to receiving the CAR T-cells. The conditioning chemotherapy or lymphodepletion may include cyclophosphamide and fludarabine, such as 500mg/m² cyclophosphamide for 2 doses on Day -4 and Day -3 and 30mg/m² fludarabine for 4 doses over Day -5 to Day -3 prior to RQR8/huK828Z/CST CAR T cells infusion on Day 0.

At least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85 %, or at least 90%, or more of patients treated achieve are identified as responders. Response for the primary and metastatic soft tissue sites may be determined for patients with evaluable disease on ¹²³l-mlBG radionuclide scan (for patients with MIBG-avid disease only) using a semi-quantitative score (SIOPEN scoring). ¹²³l-mlBG scoring according to the SIOPEN-method as described by Matthay et al. [Matthay et al. Br. J. Cancer 102:1319-26 (2010)] is based on the presence of ¹²³l-mlBG uptake in 12 anatomical body segments as follows: the skull, the thoracic cage, the proximal right upper limb, the distal right upper limb, the proximal left upper limb, the distal left upper limb, the spine, the pelvis, the proximal right lower limb, the distal right lower limb, the proximal left lower limb and the distal left lower limb. The extent and pattern of skeletal mIBG involvement is scored using a 0-6 scale to discriminate between focal discrete lesions and patterns of more diffuse infiltration (Table 14):

Table 14

Score mIBG uptake 0 no sites per segment 1 one discrete site per segment 2 two discrete lesions three discrete lesions

>3 discrete foci or a single diffuse lesion involving <50% of a bone diffuse involvement of >50-95% whole bone diffuse involvement of the entire bone.

A combined response assessment based on cross-sectional imaging, ¹²³I-MIBG and bone marrow assessment may be performed based on the revised International Neuroblastoma Response Criteria [INRC, Park et al. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 35:2580-7 (2017); Table 13],

Table 13. Primary (soft tissue) tumour response¹

RESPONSE ANATOMICAL IMAGING AND MIBG² IMAGING

Complete Response • < 10 mm residual soft tissue at primary site AND

(CR) • Complete resolution of MIBG uptake at primary site

Partial Response • > 30% decrease in longest diameter of primary site AND

(PR) • MIBG uptake at primary site stable, improved or resolved Progressive disease • > 20% increase in longest diameter taking as reference the

(PD) smallest sum on study (this includes the baseline sum if that is the smallest on study) AND

• Minimum absolute increase of 5 mm in longest dimension2

Stable Disease (SD) Neither sufficient shrinkage for PR nor sufficient increase for PD at primary site

¹ Not for use in assessment of metastatic sites.

² A mass that has not met PD measurement criteria but has fluctuating 1231-MIBG avidity will not be considered progressive disease.

The response may be complete response, partial response, progressive disease or stable disease.

The patient may show progression-free survival of at least one month, three months, six months, 12 months, 18 months, 24 months, 26 months, 48 months, 60 months or longer after CAR T cells administration.

OTHER TERMINOLOGY AND DISCLOSURE

As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

When a range of values is provided herein, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials for the purpose for which the publications are cited.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. This disclosure is intended to provide support for all such combinations.

As used herein, “may,” “may comprise,” “may be,” “can,” “can comprise” and “can be” all indicate something envisaged by the inventors that is functional and available as part of the subject matter provided.

EXAMPLES

The invention will now be further described by way of examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLE 1 - Generation of an anti-GD2-CAR T-cell product with enhancement modules by dual transduction with two separate retroviral vectors

Neuroblastoma is the most common extracranial solid cancer in children with poor long-term survival in those with high-risk disease.

A currently ongoing phase I clinical study of GD2-targeted CART for refractory/relapsed neuroblastoma (NCT02761915) shows activity against disseminated disease without inducing on target/off tumor toxicity. However, CART persistence was limited and clinical activity transient and incomplete.

Building on the GD2 CAR used in that study, a next generation T-cell product was developed. The product provided herein consists of three distinct populations of GD2-targeted CAR T- cells, produced by dual transduction of T-cells with two separate retroviral vectors. The first vector directs the expression of a GD2-targeting CAR, co-expressed with RQR8 and either a constitutively signalling IL7 cytokine receptor (IL7R_CCR) or a constitutively signalling IL2 cytokine receptor (IL2R_CCR) (product A), while the second vector is a tri-cistronic retroviral vector encoding the same GD2 CAR and RQR8, co-expressed with dominant negative TGFbRII (dnTGFbRII) and truncated SHP2 (dSHP2) (product B). dSHP2 confers resistance to inhibitory signals such as those from PD1.

The vector design is illustrated schematically in Figure 1 .

The GD2 CAR is as described in WO2015/132604, with an antigen-binding domain with a VH domain having the sequence shown as SEQ ID No. 7 and a VL domain having the sequence shown as SEQ ID No. 8.

The constitutively signalling IL2 and IL7 cytokine receptors are as described in WO2017/029512, The IL2 CCR comprises a comprising a first polypeptide having an IL-2 receptor p-chain endodomain (SEQ ID No. 30) and a second polypeptide comprising a common y-chain receptor endodomain (SEQ ID No. 29); whereas the IL7 CCR comprises a first polypeptide having an IL-7 receptor a-chain endodomain (SEQ ID No. 31) and a second polypeptide comprising a common y-chain receptor endodomain (SEQ ID No. 29).

The sort/suicide gene RQR8 is as described in WQ2013/153391 and has the sequence shown as SEQ ID No. 13.

The dominant negative TGFbRII (dnTGFbRII) has the sequence shown above as SEQ ID No. 34.

The truncated SHP2 (dSHP2) has the sequence shown above as SEQ ID No. 20.

Human T-cells were either dual transduced with both vectors yielding a mix of product A/B/A+B or single transduced with each vector individually giving raise to product A or B. Controls included non-transduced cells (NT) and cells expressing GD2 CAR alone.

EXAMPLE 2 - Investigating the cytotoxic capacity of single and dual transduced cells and the function of various vector-expressed elements in vitro 1 Cytotoxicity assay

The various effector cell types were co-cultured with GD2-expressing SupT1 target cells (SupT1 GD2) or control, non-transduced target cells (SupT1 NT) for 72 hours and the percentage of target cell lysis was analysed by flow cytometry. The results are shown in Figure 2. All CAR-expressing effector cells were capable of killing GD2-expressing target cells. T-cells transduced with the dual vector composition (product A/B/A+B) were highly potent in cytotoxicity assays against GD2 positive tumour cell lines with no differences observed compared with single transduced CAR T-cells (product A or B).

2 _ Validation of the CCR

The various transduced CAR T-cells described above and control NT T-cells were labelled with Cell Trace Violet (CTV) and cultured in cytokine-free complete cell culture media for 7 days without further antigen stimulus. In vitro persistence was quantified as percent proliferating cells which have diluted the CTV dye and absolute CAR T-cell count after 7 days. The results are shown in Figure 3. T cells transduced with product A, expressing either the constitutively signalling IL7 cytokine receptor (IL7R_CCR) or the constitutively signalling IL2 cytokine receptor (IL2R_CCR); or transduced with product A+B showed increased proliferation compared with untransduced cells (NT), cells transduced with a vector expressing GD2 CAR alone, or cells transduced with the vector expressing product B alone (GD2 CAR + dSHP2 + dTGFbRII). Expression of either the IL2 or IL7R_CCR conferred exogenous-cytokine-independent viability and homeostatic proliferation of modified T-cells, without causing autonomous T-cell growth.

3 Validation of the dTGFbRII

The various transduced CAR T-cells described above and control NT T-cells were co-cultured with GD2-expressing SupT 1 target cells (SupT 1 GD2) or control, non-transduced target cells (SupT 1 NT) at a 1 :2 or 1 :8 E:T ratio, for 7 days, in the presence or absence of 10ng/ml TGFp. Killing of target cells was analysed by flow cytometry and secretion of IFNy was analysed by ELISA. The results are shown in Figures 4 and 5 respectively. CAR T cells transduced with product B, expressing dnTGFbRII; or transduced with product A+B, showed resistance to TGFp-mediated inhibition of target cell killing compared with CAR-T cells expressing GD2 CAR alone, or cells transduced with the vector expressing either product A alone (GD2 CAR + IL2 CCR or GD2 CAR + IL7 CCR). CAR T cells transduced with product B or product A+B, showed restoration of IFNy secretion in the presence of TGFp to a level comparable to that observed in the absence of TGFp. By contrast, IFNy secretion by CAR-T cells expressing GD2 CAR alone, or cells transduced with the vector expressing either product A alone, was significantly inhibited by the presence of TGFp. T-cells expressing dnTGFbRII therefore proved resistant to TGFp-mediated immunosuppression in vitro.

EXAMPLE 3 - Investigating anti-tumour activity of dual-transduced CAR-T cell product in vivo in a xenograft model of neuroblastoma

An in vivo assay was used to investigate the anti-tumour activity of T cells transduced with a dual vector composition by intravenous administration in an established neuroblastoma xenograft model in NSG mice. Ten- to 14-week-old female NSG mice were intravenously injected with 1 million Firefly luciferase expressing CHLA-255 cells (CHLA-255 FFIuc). Xenografts were left to establish for 15 days until stable engraftment was detectable by BLI. CAR-T cells were made either by transducing cells with a single vector expressing a GD2 CAR (GD2 CAR) or by transducing cells with the dual vector composition described in Example 1 and illustrated in Figure 1 (GD2 CAR + IL7 CCR/GD2 CAR+dSHP2+dTGFbRII). Mice were injected intravenously with 10x10⁶ CAR T-cells (50% transduction efficiency), 3x10⁶ CAR T-cells (50% transduction efficiency), 20x10⁶ NT T-cells (total T-cells equivalent to 10x10⁶ CAR T-cell dose) or PBS. Fourteen days later, tumour growth was assessed by biweekly bioluminescent imaging.

The results are shown in Figure 6. Intravenous delivery of CAR T cells expressing a simple GD2 CAR alone had no significant effect on tumour growth (Figure 6 A and B). By contrast, intravenous delivery of CAR T cells transduced with the dual vector composition at both the 3x10⁶ and 10x10⁶ doses exhibited potent anti-tumour activity and extended survival in NSG mice with established tumour burden (Figure 6 C and D).

EXAMPLE 4 - RQR8/huK828Z/CST CAR T cells for clinical study

The GD2 CAR T cells described above in Examples 1-3, which are referred to herein as “RQR8/huK828Z/CST CAR T cells”, are a mixed population of CAR T cells.

As explained above, the RQR8/huK828Z/CST CAR T cells were generated by cotransduction of autologous T cells with two multi-cistronic y-retroviral vectors. The first vector was pSF.SV40.KanR.RQR8-2A-aGD2_Huk666-CD8STK-TyrpTM-CD28z-2A-Fab_IL7, designated AU54280 and encodes RQR8, huK828Z and FabCCR-IL7. The second vector was pSF.SV40.KanR.dual_SH2-SHP2-2Aw-RQR8-2A-aGD2_huK666_CD8STK-TyrpTM- CD28z-2A-dnTGFbetaRII, designated AU54281 and encodes RQR8, huK828Z, dSHP2 and dTBRII.

1 _ y-retroviral vector transfer plasmids

The y-retroviral vectors are produced by transient transfection of 293T cells with transfer vector plasmid and two helper plasmids which supply retroviral env and gagpol. Two y- retroviral vectors were made. The transfer vector plasmids (AU54280 and AU54281) for each vector are detailed in Figure 8.

AU54280 and AU54281 are transfer vector plasmids for a gamma-retroviral vector derived from SFG (a vector cassette widely used in engineering T cells)¹⁴². These comprises 5’ and 3’ wild-type Moloney Mouse Leukemia Virus long-terminal repeats, the MoMLV packaging signal, the MoMLV splice acceptor (SA) and the MoMLV polypurine tract. The MoMLV packaging signal includes the MoMLV splice donor denoted by (y and SD); this codes for a portion of gag, but the stop codon is mutated from an ATG to a GTG. The transgene openreading frame (orf) is inserted just 3’ to the splice acceptor replacing the retroviral envelope orf. The scaffold attachment region (SAR) [Murray, L. et al. Human Gene Therapy 11 , 2039- 2050 (2000)] from p-interferon has been inserted immediately 3’ to the transgene orf and acts to enhance expression. The retention of MoMLV splicing signals, the SAR and codonoptimization ensure high homogenous expression of the transgene.

The orfs are codon-optimized to result in a stable transcript with little secondary structure and optimal codon-usage for efficient translation. The orf both codes for a single protein which is cleaved soon after translation to give multiple separate proteins by interposed FMD-2A-like self-cleaving peptides

AU54280 orf encodes a sort-suicide protein termed RQR8 and an anti-GD2 CAR termed huK828Z. In addition, a constitutively active IL7 receptor is expressed as two additional chains

AU54281 orf also encodes RQR8 and huK828Z. In addition, a truncated form of SHP2 is also expressed. This truncated SHP2 contains only the SH2 domains and lacks phosphatase, thereby inhibiting endogenous SHP2. In addition, a truncated from of TGFb receptor is also expressed. This acts in a dominant-negative manner hence inhibiting TGFb signalling. 2 Transcjenes expressed by RQR8/huK828Z/CST CAR T cells

The transgenes encoded by AU54280 and AU54281 are illustrated in Figure and detailed in the text below. huK828Z is a second-generation GD2 chimeric antigen receptor (CAR). A scFv derived from a humanized GD2 antibody is at the amino terminus. This leads on to the CD8a stalk (residues 137 to 183 of isoform 1 from uniprot P01732). This is further connected to the transmembrane domain of Tyrpl (residues 477 to 501 from uniprot P17643) which leads onto the endodomain of CD28 (residues 180 to 220 from uniprot P10747). Finally, this is connected to the endodomain of CD3z (residues 52 to 163 of isoform 3 - uniprot P20963-3).

RQR8 is a sort-suicide gene [Philip, B. et al. Blood (2014) doi:10.1182/blood-2014-01- 545020], It is a fusion of two copies of a rituximab binding mimetope (CPYSNPSLC)¹⁴⁵ separated by a 16 amino acid portion of human CD34 (residues 42 to 57 of the canonical sequence uniprot: P28906) which binds the monoclonal antibody QBEndlO. This structure is fused to the stalk, transmembrane domain and portion of the endodomain of human CD8a (residues 141 to 222 of the canonical sequence uniprot: P01732). RQR8 allows selective depletion of transgenic T cells with the therapeutic monoclonal antibody rituximab in the event of unmanageable toxicity. In addition, RQR8 allows convenient tracking of cells in vivo by QBEndlO staining.

FabCCR-IL7R is a chimeric cytokine receptor designed to provide constitutive IL7 signalling in the absence of any extracellular cytokines, thereby augmenting modified CAR T cell function while avoiding stimulating bystander lymphocytes. The IL7R CCR consists of two covalently paired polypeptide chains. The signal peptide of the first polypeptide chain is at the extreme amino-terminus and is derived from a mouse IgG Kappa Chain V (residues 1 to 19 of uniprot P01750). This leads on to the antibody K light chain (residues 1 to 107 of uniprot P01834) fused to a human lgG1 hinge (residues 218 to 232 of uniprot PODOX5) which is further connected to the endodomain of the common gamma chain (residues 263 to 369 of uniprot P31785). At the extreme amino-terminus the second polypeptide chain comprises of a signal peptide derived from a mouse IgG Kappa Chain V-lll (residues 1 to 20 of uniprot P01658) which is fused to the antibody heavy chain CH1 (residues 118 to 217 of uniprot PODOX5). This is further connected to a human lgG1 hinge (residues 218 to 232 of uniprot PODOX5) which is in turn fused to the IL7Ra endodomain (residues 240 to 459 of uniprot P16871). Truncated SHP2 protein (dSHP2) is designed to block the inhibitory signals from a range of ITIM-containing immunoinhibitory receptors, among which is PD1. It consists of two N- terminal SH2 domains (residues 5 to 216 from uniprot Q06124) and lacks the PTP (protein tyrosine phosphatase) domain from the conventional molecule (residues 217 to 593 from uniprot Q06124 have been deleted).

Truncated TGFbRII protein (dTBRII) is designed to block inhibitor signals from TGFb. The signal peptide of dTGFbRII is at the extreme amino-terminus and is derived from a mouse IgG Kappa Chain V (residues 1 to 19 of uniprot P01750). This leads on to the conventional TGFbRII ectodomain and transmembrane domain which leads onto the endodomain truncated following residue 199. When formed into a TGFb receptor with dTGFbRI, the complex does not signal in response to TGFb binding and acts to preventing TGFb associated functions.

3 Manufacture of the RQR8/huK828Z/CST CAR T cells y-retroviral vectorwas generated by transient transfection of 293T cells with plasmid encoding transfer, RD114 envelope and MoMLV gagpol. Viral vector was concentrated and purified by high-speed centrifugation, aliquoted and stored at -80°C. The methodology used has been described by Mekkaoui et al. [Mekkaoui, L. et al. Mol Ther Methods Clin Dev 28, 116-128 (2023)].

T cell production is generated on a per-patient basis. Fresh patient-derived (autologous) pheresis is the starting material. T cells in the pheresis are isolated using Miltenyi CliniMACS CD4/CD8 magnetic beads. T cells are then activated using Miltenyi MACS® GMP T cell Transact, a T cell mitogenic biodegradable polymeric nanomatrix. Activated T cells are then transduced simultaneously by exposure to aliquots of both vectors in the presence of Vectofusin®-1. T cells are expanded in media containing IL7/IL15 and the AKT inhibitor AKT VIII. The RQR8/huK828Z/CST CAR T cells are cryopreserved in DMSO and stored in vapour phase of Liquid Nitrogen. They are thawed at the patient’s bedside and administered intravenously.

Production of the RQR8/huK828Z/CST CAR T cells is summarized in Figure .

4 _ y-retroviral vector

Vectors were generated by transient transfection of a previously established MCB of 293T cells. The 293T cells were first expanded into multi-layered flasks. When confluent, 293T cells were transfected with respective transfer vector plasmid (either AU54280 or AU54281) and helper plasmids supplying MoMLV gagpol and RD114 envelope.

MP27000 pSF_CMV.optGAGPOL_KanR is a helper plasmid which expresses MoMLV gagpol

MP27001 pSF_Ferritin_optRD114_KanR is a helper plasmid which expresses the envelope glycoprotein from RD114

Supernatant was harvested, filtered and concentrated by high-speed centrifugation. Retroviral pellets were re-suspended, aliquoted, and frozen at -80°C. Concentrated supernatant and end-of-production cells were subjected to testing.

5 _ CAR T cell production

This is summarised in Figure 10. The starting material for generation of the RQR8/huK828Z/CST CAR T cells is a patient derived pheresis. This may be fresh or cryopreserved.

Fresh or thawed patient derived pheresis is transferred to a T cell tubing set (TS520) on the CliniMACS Prodigy®. T cells are then enriched using CD4 and CD8 immunomagnetic selection beads prior to activation with MACS GMP TransAct, IL-7 and IL-15.

Following up to 48 hours of activation, cells undergo transduction simultaneously with both AU54280 and AU54281 y-retroviral vectors. Transduction is facilitated by the polycationic polymer vectofusin. To remove residual activation reagent, vector and Vectofusin®-1 , cells are washed post activation using the automatic culture wash on the CliniMACS Prodigy.

The transduced cell population comprises a mixture of cells, which all express RQR8 and huK828Z, and which variously express FabCCR-IL7, dSHP2/dTBRII or all transgenes.

Cells are then expanded on the CliniMACS Prodigy in fresh media supplemented with IL-7 and IL-15, human AB serum and the AKT inhibitor AKT VIII. The cell culture is automatically shaken in order to allow for optimal gas exchange.

The final product is then frozen in cryopreservation bags using CliniMACS buffer, 1% Human Albumin Solution (HAS) and DMSO cryoprotectant. Aliquots taken at this time are then subjected to quality control assays to ensure the transduced T cell product meets the release criteria as listed in the ATMPD. y-retroviral vector was chosen due to experience with transducing T cells along with the excellent safety record. RD114 was selected as the pseudotype due to high tropism of T cells to RD114 and excellent stability of RD114 pseudotyped vector particles. Isolation of T cells improves consistence of manufacture. Culture in IL7/IL15 and AKT VIII results in a T cell product which is less differentiated and hence has a higher propensity for long-term engraftment.

The ClinicMACS Prodigy semi-automated system was selected for manufacturing of drug substance since it reduces workload and fewer operator dependent steps results in less in- process variability.

6 Physical Properties

The medicinal product is supplied as a frozen liquid suspension following cryopreservation in CliniMACS PBS/EDTA Buffer, 20% human albumin solution (HAS, 4% final concentration), and DMSO (7.5% final concentration). When thawed, the product will appear as an off-white, pink or yellow cloudy cell suspension. It will be supplied in cryovials or, or more usually, freezing bags within an overwrap bag.

7 Pharmaceutical Properties

Sufficient RQR8/huK828Z/CST CAR T cells will be cryopreserved for the proposed cell doses to be infused. Viability will be sufficient to allow survival of the cells post-infusion. It is anticipated that RQR8/huK828Z/CST CAR T cells will function in vivo to confer anti-tumour efficacy. Pre-clinical studies and prior clinical experience have demonstrated the cytotoxic potential of RQR8/huK828Z/CST CAR T cells against GD2-expressing tumours.

In addition, the following assays may be performed, though they do not constitute release criteria: flow cytometry to determine the immunophenotype and the percentage of non-T cell immune subsets in the RQR8/huK828Z/CST CAR T cells, viral copy number assessment by qPCR, and functional assays to assess cytotoxicity, proliferation and cytokine secretion.

8 Formulation

The final product is a cryopreserved liquid suspension of RQR8/huK828Z/CST CAR T cells in CliniMACS PBS buffer supplemented with EDTA and with human albumin solution and DMSO. The cells are cryopreserved at 10⁷ CD3⁺/mL. The concentrations of EDTA, DMSO and Human Albumin Solution are listed in the Table below. (Note, 1 ml DMSO = 1.101 g).

The composition of CliniMACS PBS buffer is listed in Table below.

Table 5. ATIMP excipients.

Table 6. Composition of cliniMACS.

CAR T cell production is an individualised process. Pheresis may vary in composition from donor to donor and hence differences may exist between RQR8/huK828Z/CST CAR T cells derived from different donors. These differences include the transduction efficiency, the proportion of cells which are CD4+ and CD8+ cells, the proportion of cells which are NK cells as well as differences in markers of T cell differentiation.

9 Storage and handling

The RQR8/huK828Z/CST CAR T cells are cryopreserved in DMSO and stored in clinical vapour phase nitrogen tanks or in a mechanical freezer (-130°C to -196°C). They are thawed at the patient’s bedside and administered intravenously.

10 Differences in CAR cassette and T cell manufacture between 1RG-CART and the present disclosure

Table 7 below summarizes the differences in CAR cassette(s) cand CAR T cell manufacture for 1 RG-CART and the present study. The CAR used in the present study is based on the same humanized K666 GD2 binder. In the present study, CD8a is used as linker as opposed to lgG1-derived CH2CH3 to connect the ectodomain to the same CD28 and CD3z endodomains. The present study incorporates the same RQR8 sort-suicide switch in the CAR cassettes as in 1 RG-CART. However, in the present study, a double transduction approach is used to incorporate three additional components (i.e., FabCCR-IL7 and, sHP2 and dTBRII). In both 1 RG-CART and the present study CAR cassettes 2A sequences are used to achieve coexpression of components.

CAR T cell manufacture for 1 RG-CART used an older process based on the WAVE bioreactor while in the present study a Miltenyi Prodigy based manufacture processes is used. A comparison of the two processes is included in Table 7 below. Use of the Miltenyi Prodigy was motivated by reduced cost and convenience of a semi-automated closed system. Removal of monocytes and other non T cells from the pheresate by CD4/CD8 selection may improve the robustness of manufacture [Highfill, S. L. et al. Cytotherapy 19, S14 (2017)]. Use of I L7/IL15 may result in CAR T cells with a differentiation state which favours long-term engraftment [Kaneko, S. et al. Blood 113, 1006-1015 (2009)].

The huK666/CD28-Z CARs used are near identical and differences in manufacture are unlikely to influence on-target off-tumour toxicity and hence clinical data from 1 RG-CART is considered informative for the present study.

Table 7. Comparison between 1 RG-CART and present manufacturing processes.

EXAMPLE 3 - Functional activity of GD2 receptor huK828Z and RQR8/huK828Z CAR T cells As described in Example 2, the GD2 receptor huK828Z was tested in vitro. Two target cells were used: SupT1 (a T cell leukemia cell line), and Raji (a B cell lymphoma cell line) which are both widely used to test CAR T cell function. They do not express GD2 but can easily be engineered to express GD2 by engineering expression of two biosynthetic enzymes (GM3 synthase and GD2 synthase) to result in SupT1.GD2 and Raji.GD2.

Specific cytotoxic function of huK828Z CAR T cells was tested by co-culture of huK828Z CAR T cells with target cells: SupT 1 , SupT 1.GD2, Raji or Raji.GD2 cells for 72 hours. Remaining target cells were enumerated by flow-cytometry. A comparison was also made with cytolytic function of huKFc*28Z CAR T cells using the same targets (Figure ). HuK828Z CAR T cells selectively lysed GD2 positive target cells. Cytolytic function was not different to that from huKFc*28Z CAR T cells.

Next, proliferation of huK828Z CAR T cells in response to target antigen expressing cells was determined. In this assessment, proliferation was tested only in response to Raji and Raji.GD2 as proliferation to SupT1 cells is typically poor. HuK828Z CAR T cells were first loaded with CFSE, a fluorescent dye which chelates intracellular proteins. Labelled T cells were co-cultured with Raji or Raji.GD2. After 5 days, dilution of CFSE dye, which indicates cell proliferation, was determined by flow cytometry (Figure ).

Next, the ability of huK828Z to trigger cytokine release was determined. huK828Z CAR T cells were co-cultured with target cells: SupT 1 , SupT 1 .GD2, Raji or Raji.GD2. After 72 hours, IFN-g cytokine in co-culture supernatants was measured by ELISA (Figure ).

Susceptibility of RQR8/huK828Z/CST CAR T cells to PD1 inhibition was determined. Nontransduced T cells, RQR8/huK828Z CAR T cells and RQR8/huK828Z/CST CAR T cells were co-cultured with SupT1 cells or SupT1.GD2 cells. In addition, T cells were co-cultured with SupT1.GD2 which were engineered to also express PDL1 (SupT1.PDL1.GD2).

Cytotoxicity was determined by flow-cytometry. To simulate scenarios where T cells would upregulate PD1 , low E:T ratios were used which would require serial killing by CAR T cells. To further explore dSHP2 function in the context of PD1 upregulation, in some conditions, PD1 was transgenically expressed in the CAR T cells using an additional retroviral vector. These cytotoxicity experiments showed that RQR8/huK828Z/CST CAR T cell cytotoxicity of target cells expressing PDL1 was superior to that of RQR8/huK828Z CAR T cells (Figure 14). Improved cytotoxicity was more apparent in lower E:T ratio and when PD1 was additionally co-expressed (Figure 5). RQR/huK828Z/CST CAR T cells display resistance to inhibition from PD1 in comparison with RQR8/huK828Z CAR T cells.

To determine resistance to TGFp, functional experiments were conducted in the presence of exogenous TGFp. Non-transduced T cells, RQR8/huK828Z and RQR8/huK828Z/CST CAR T cells were co-cultured with either SupT1 or SupT1.GD2 in the absence or presence of 10 ng/ml TGFp. Target cell killing was determined by flow cytometry and is shown in Figure . Cytokine release in response to target cells is shown in Figure . Exogenous TGFp inhibits target cell killing and CAR T cell cytokine release. RQR8/huK828Z/CST cytotoxicity and cytokine release function is resistant to exogenous TGFp.

CAR T cells were labelled with Cell Trace Violet (CTV) and were plated in a 96-well plate in the absence of exogenous cytokines or antigen stimulation for 7 days. RQR8/huK828Z/CST CAR T cells proliferated in the absence of exogenous stimulus due to the presence of the constitutively active IL7R CCR in those genetically engineered CAR T cells (Figure 18).

To determine the relative long-term effect of FabCCR-IL7, a long-term persistence assay was set up. 5x10⁶ CAR T cells were plated in a 6-well plate in the absence of exogenous cytokines or antigen stimulation. Every 7 days CAR T cells were counted and resuspended at 1x10⁶ cells/mL in fresh medium. Although dual transduced RQR8/huK828Z/CST CAR T cells persisted significantly longer in vitro in these antigen- and cytokine-depleted conditions than control RQR8/huK828Z CAR T cells, RQR8/huK828Z/CST CAR T cells started to contract by day 21 , with all cells dying off by day 84 after initiation of the persistence assay (Figure 19). These data demonstrate that FabCCR-IL7 does not sustain indefinite antigen-independent autonomous T cell expansion of RQR8/huK828Z/CST CAR T cells.

To evaluate whether RQR8/huK828Z/CST CAR T cells retain their cytotoxic potential and capacity to secrete IFN-y in response to Ag recognition, transduced CAR T cells were subjected to sequential rounds of re-stimulation. Transduced CAR T cells were co-cultured with either SupT1 (20. A) or SupT1 GD2 (Figure .B) targets at 1 :1 ratio (E:T). Every 3- or 4- days, CAR T cells were re-stimulated with fresh 5x10⁴ targets/well.

RQR8/huK828Z/CST CAR T cells maintained cytotoxicity after repeated encounters with Ag-positive tumour in comparison with controls while RQR8/huK828Z CAR T cells started failing to clear the targets by the 5^th re-challenge (Figure ). Additionally, RQR8/huK828Z/CST CAR T cells retained their capacity to secrete IFN-y upon GD2- recognition until the last re-stimulation cycle (Figure 1). In conclusion, RQR8/huK828Z/CST CAR T cells display early but not late cytokine/antigen independent expansion. RQR8/huK828Z/CST CAR T cells retain in vitro tumour killing and IFN-g in response to repeated antigen challenge compared with RQR8/huK828Z CAR T cells.

The ability of RQR8 to direct depletion of RQR8/huK828Z/CST CAR T cells was tested. Transduced T cells were exposed to 25% baby-rabbit complement with either isotype control or rituximab (100 pg/mL) to examine complement-dependent cytotoxicity (CDC)- mediated sensitivity. Following 2h incubation, CAR T cells were stained with the viability dye Sytox Blue and the presence of remaining transduced cells was assessed by flow cytometry. CDC by rituximab led to the deletion of >95% of the transduced populations in RQR8/huK828Z and RQR8/huK828Z/CST CAR T cells (Figure ).

Following this, an equal number of isotype-treated CAR T cells or rituximab-depleted CAR T cells were co-cultured with either SupT 1 or SupT 1 .GD2 cells to achieve an effectortarget ratio of 1 :1 for 24 hours. CAR-mediated cytotoxicity was assessed by flow cytometry.

Rituximab depletion resulted in greatly reduced specificity for GD2 positive target cells (Figure 23).

In conclusion, RQR8/huK828Z/CST CAR T cells were highly susceptible to in vitro rituximab-mediated depletion. Furthermore, this depleted population had reduced recognition of GD2-expressing targets.

EXAMPLE 5 - RQR8/huK828Z/CST CAR T cells are effective in vivo

As described in Example 3 above, the in vivo efficacy of RQR8/huK828Z/CST CAR T cells was tested in a neuroblastoma xenograft model using the CHLA-255 cell line. The CHLA-255 cell line was derived from a metastatic lesion in the brain of a patient with recurrent neuroblastoma by Sohara et al¹⁶⁵. Like most neuroblastoma, CHLA-255 have amplification of MYCN and are GD2 positive¹⁶⁵. CHLA-255 is widely used in to test adoptive immunotherapy in xenograft models of neuroblastoma^166-168.

CHLA-255 cells engineered to express firefly luciferase were injected intravenously (i.v.) in female NSG (NOD.Cg-PrkdcSCIDII2rgtm1Wjl/SzJ) immunodeficient mice (n=6/group). Stable engraftment of CHLA-255 cell lines was confirmed by bioluminescent imaging (BLI) 15 days later (day 16). Mice were randomized into groups and treated i.v. with either PBS, non-transduced T cells, RQR8/huK828Z CAR T cells or RQR8/huK828Z/CST CAR T cells. A low dose of 1x10⁶ CAR T cells was selected since this dose was known to be insufficient for RQR8/huK828Z CAR T cells to be effective in this model. BLI was conducted as shown in Figure below and the planned termination of experiment was day 50.

BLI showed control of CHLA-255 in mice receiving RQR8/huK828Z/CST, but not in mice receiving RQR8/huK828Z CAR T cells or controls. One mouse who received RQR8/huK828Z/CST developed an eye infection considered unrelated to therapy and had to be sacrificed, but remaining mice in this cohort were maintained until day 50.

RQR8/huK828Z/CST CAR T cell treatment was well tolerated and no adverse effects relating to the treatment were observed. RQR8/huK828Z/CST mice did not lose weight. Macroscopic examination of internal organs at sacrifice revealed no abnormality. Splenic weight of RQR8/huK828/CST mice was similar to that of controls. Control mice were sacrificed at day 37 due to excessive disease burden on BLI.

In conclusion, RQR8/huK828Z/CST CAR T cells can control established neuroblastoma xenograft for 50 days in NSG mice in a dose-stress model. This was superior to RQR8/huK828Z CAR T cells. No signs of severe toxicity were observed in terms of animal behaviour, weight and macroscopic organ examination.

A toxicity study for RQR8/huK828Z/CST was performed. Twelve- to 14-week-old naive female NSG (NOD.Cg-Prkdc^SCIDll2rg^tm1Wjl/SzJ) immunodeficient mice (n=5/group) received intravenous infusions of 3x10⁶ of RQR8/huK828Z/CST CAR T cells. Control groups received non-transduced T cells (NT), CD19 CAR T cells or PBS. Mice were monitored for signs of neurotoxicity (head tilt, gait disturbance or seizures) and were weighted weekly. Mice were sacrificed on day 36 and organs were harvested by Charles River staff and sent for processing and pathology (Error! Reference source not found.).

No signs of toxicity were observed during the experiment, specifically no neurotoxicity or weight loss. On necropsy, no organ weight changes, or microscopic findings related to RQR8/huK828Z/CST CAR T cells were found.

EXAMPLE 6 Clinical Treatment of Neuroblastoma Patients

1 Inclusion criteria Age > 1 and < 16 years. Tissue diagnosis of neuroblastoma. If sufficient biopsy material is available, GD2 expression on the tumour will be confirmed. As GD2 is consistently expressed in neuroblastoma demonstration of GD2 is not mandated. Relapsed or refractory disease after one or multiple lines of previous treatment. Measurable disease by cross sectional imaging or evaluable disease by uptake on 1²³I-MIBG scan. Patients with only bone marrow detectable disease (bone marrow aspirate or trephine) are NOT eligible for the study. At least 3 weeks or 5 half-lives, whichever is shorter, after treatment with agents on other early phase clinical trial. Performance status: Karnofsky (age > 10 years) or Lansky (age < 10) score > 50%. Patients who are unable to walk because of paralysis, but who are able to sit upright unassisted in a wheelchair, will be considered ambulatory for the purpose of assessing performance score. Creatinine <1.5 ULN for age, if higher, an estimated (calculated) creatinine clearance must be > 60 ml/min/1.73 m². Absolute lymphocyte count > 0.25 x 10⁹/L. For post-pubertal subjects agreement to have a pregnancy test, use adequate contraception (if applicable). Written informed consent. Exclusion criteria for study overall Patients with only bone marrow detectable disease in the absence of measurable disease by cross sectional imaging or evaluable disease by uptake on ¹²³I-MIBG scan. Patients with active, inoperative CNS disease including leptomeningeal disease. Active hepatitis B, C or HIV infection. Inability to tolerate leukapheresis. Clinically significant systemic illness or medical condition (e.g., significant cardiac, pulmonary, hepatic or other organ dysfunction), that in the judgement of the investigator is likely to interfere with assessment of safety or efficacy of the investigational regimen and its requirements. Any contraindication to lymphodepletion or to the use of Cyclophosphamide or Fludarabine as per the local SmPC. Any contraindication to the use of Anticoagulant Citrate Dextrose Solution. Known allergy to albumin, EDTA or DMSO. 9. Primary immunodeficiency or history of autoimmune disease (e.g., Crohn’s, rheumatoid arthritis, systemic lupus) requiring systemic immunosuppression /systemic disease modifying agents within the last 2 years.

10. Prior treatment with investigational or approved gene therapy or cell therapy products.

11. Life expectancy <3 months.

12. Use of rituximab (or rituximab biosimilar) within the last 3 months prior to of RQR8/huK828Z/CST CAR T cells infusion.

13. Systemic corticosteroid therapy > 0.05 mg/kg dexamethasone daily (or equivalent) at time of RQR8/huK828Z/CST CAR T cells infusion.

14. Post-pubertal subjects who are pregnant or breastfeeding. Exclusion criteria for RQR8/huK828Z/CST CAR T cells infusion

1. Uncontrolled fungal, bacterial, viral, or other infection.

Previously diagnosed infection for which the patient continues to receive antimicrobial therapy is permitted if responding to treatment and clinically stable at the time of scheduled RQR8/huK828Z/CST CAR T cells infusion.

2. Systemic corticosteroid therapy > 0.05 mg/kg dexamethasone daily (or equivalent) at time of RQR8/huK828Z/CST CAR T cells infusion.

3. Use of rituximab (or rituximab biosimilar) within the last 3 months prior to RQR8/huK28Z CAR T cell infusion _ Treatment Summary

1. Leukapheresis: unstimulated leukapheresis is performed according to local standard practice.

2. RQR8/huK828Z/CST CAR T cells: following transport of the leukapheresis to the manufacturer, the manufacturing period will take 5-8 days. During this period, patients may receive holding therapy as per institutional practice to maintain disease control if needed. If manufacture is successful, i.e. , RQR8/huK828Z/CST CAR T cells meet the release criteria and sufficient dose is generated, the patient can proceed on the study to be assessed for lymphodepletion and RQR8/huK828Z/CST CAR T cell administration.

3. Lymphodepletion (LD): Fludarabine and Cyclophosphamide will be administered on day -6 to -3

4. Pre-CAR T cells infusion assessment. If patients meet the exclusion criteria, they cannot proceed to CAR T cells infusion.

5. CAR T cells infusion (at a dose assigned by UCL CTC prior to eligibility assessment for CAR T cells infusion) is administered. All patients are monitored similarly post CAR T cells infusion.

Table 8. Treatment schedule on the present study.

★Delays of up to 7 days for infection are allowed.

It is expected that most patients will receive a single infusion of RQR8/huK828Z/CST CAR T cells. However, patients may receive a second dose if they tolerated the first dose of RQR8/huK828Z/CST CAR T cells (no DLT) and showed evidence of anti-tumour activity (CR, PR or MR at day+28 or subsequent assessment) but who subsequently showed progressive disease. These patients may receive a second dose. Patient monitoring will be as after the first RQR8/huK828Z/CST CAR T cells infusion.

5 Treatment Details

5.1 Leukapheresis

Leukapheresis should be performed once the patient has been registered into the trial.

The starting material for generation of RQR8/huK828Z/CST CAR T cells is an unstimulated leukapheresis from the patient which will be performed at the study site. This may require insertion of central venous access and is a day case procedure. For patients with a circulating absolute lymphocyte count of > 0.5 x 10⁹/L a double blood volume leukapheresis will be performed, according to local institutional practice. For patients with absolute lymphocyte count between 0.25 x 10⁹/L - 0.5 x 10⁹/L, a 2.5 volume leukapheresis will be carried out.

The leukapheresis is transferred to the CCGTT-RFH under the HTA licence of the referring site, in accordance with a validated local policy and with an approved courier or the manufacture scientists. Further details for leukapheresis shipment to manufacturer can be found in the Summary of Drug Arrangements.

Following transport of the leukapheresis to the manufacturer, the manufacturing period will take 5 - 8 days, and a QP release a further ~15 days. During this period, patients may receive holding therapy as per institutional practice to maintain disease control if needed. 5.2 Lymphodepletion (days -6 to -3)

Lymphodepletion is administered prior to CAR T cells infusion to enhance the expansion of adoptively transferred T cells.

Patients will be admitted onto the in-patient ward at Great Ormond Street Hospital from the initiation of lymphodepletion chemotherapy.

All trial patients will be assessed by the site physician prior to commencing lymphodepletion to ensure they meet the eligibility criteria for RQR8/huK828Z/CST CAR T cells infusion. A repeat pregnancy test will also be performed (if applicable).

5.3 Lymphodepletion will consist of the following.

1. Fludarabine 30 mg/m² iv once daily on day -6 to -3 (total dose 120 mg/m²). Fludarabine is given as an intravenous infusion over 30 minutes. For patients with renal impairment or those weighing < 9 kg, the dose of fludarabine can be reduced according to institutional practice.

2. Cyclophosphamide 500 mg/m² iv once daily on day -4 to -3 (total dose 1 ,000 mg/m²). Cyclophosphamide is given as an intravenous infusion over 1 hour. Hydration and mesna are recommended from 4 hours prior to starting the cyclophosphamide until 24 hours after completing the infusion as per institutional guidelines. Anti-emetic prophylaxis and transfusion support should be administered as per standard local policy.

Antimicrobial prophylaxis. It is recommended that all patients receive prophylactic aciclovir (from Day -6; to continue) and prophylactic co-trimoxazole (from Day -6 until Day -1 when this stops and is recommenced at the point of blood count recovery, estimated to be at Day 8-10). All patients should additionally be flagged to Blood Bank with a new requirement for Irradiated CMV seromatched blood products. All patients should be expectantly managed with prophylactic allopurinol or other locally agreed measures to prevent tumour lysis syndrome.

Eligibility assessment prior to RQR8/huK828Z/CST CAR T cell infusion (Day 0)

On day 0, patients will be evaluated to determine eligibility prior to infusion of RQR8/huK828Z/CST CAR T cells.

Patients will not be eligible for infusion of the RQR8/huK828Z/CST CAR T cells if they have: 1. Uncontrolled fungal, bacterial, viral, or other infection. Previously diagnosed infection for which the patient continues to receive antimicrobial therapy is permitted if responding to treatment and clinically stable at the time of scheduled RQR8/huK828Z/CST CAR T cell infusion.

2. Systemic corticosteroid therapy > 0.05 mg/kg dexamethasone daily (or equivalent) at time of RQR8/huK828Z/CST CAR T cell infusion.

3. Use of rituximab (or rituximab biosimilar) within the last 3 months prior to RQR8/huK28Z CAR T cell infusion.

In this situation, administration of the CAR T cell therapy can be delayed by up to 7 days of the planned date, during which time supportive medication should be administered as per local policy to resolve the issue and the RQR8/huK828Z/CST CAR T cells administered if the patient improves clinically. For patients who recover after 7 days, a further course of lymphodepletion may be given prior to administration of RQR8/huK828Z/CST CAR T cells, provided any chemotherapy induced cytopenias have resolved and the patient remains eligible.

5.4 Administration of RQR8/huK828Z/CST CAR T cells

Premedication with chlorpheniramine and paracetamol may be given prior to infusion of the RQR8/huK828Z/CST CAR T cells as per standard local institutional protocols but steroids should NOT be given as part of the premedication.

Immediately prior to infusion and up to and including 4 hours after infusion (hourly observations), the patient should be monitored for the following. temperature pulse blood pressure respiratory rate oxygen saturation level

The RQR8/huK828Z/CST CAR T cells is administered intravenously at a dose of:

Dose Level 1 : 30 x 10⁶ CAR T cells/m²

Dose Level 2: 100 x 10⁶ CAR T cells/m² or

Dose Level 3: 300 x 10⁶ CAR T cells/m²

On occasion, RQR8/huK828Z/CST CAR T cells manufacture may fail to generate the required dose. In these circumstances, a minimum dose of not less than 10 x 10⁶ RQR8/huK828Z/CST CAR T cells/m² can be administered. This minimum dose represents the proposed cell dose for dose level -1 if required. This dose is within the dose range of GD2 CAR T used in r/r neuroblastoma⁵ [Chang, H. R. et al. Cancer 70, 633-638 (1992)] and patients may benefit even with this small dose. If manufacture generates <10 x 10⁶ RQR8/huK828Z/CST CAR T cells/m², the patient will not be able to receive CAR T cells on study.

5.5 Patient monitoring post RQR8/huK828Z/CST CAR T cell infusion

Patients will be monitored post infusion at the participating trial site. Patients will be observed as an inpatient for a minimum of 14 days post-infusion of the RQR8/huK828Z/CST CAR T cells with regular observations (as described above) at frequency as clinically indicated, and monitoring for blood counts, biochemistry, clotting, CRP and samples for serum cytokines as outlined in Table 10.

EXAMPLE 7 Assessments

1 Pre-treatment Assessments

Full medical history and physical examination

Weight

Body Surface Area (BSA)

Vital signs (temperature, pulse, blood pressure and respiratory rate) Oxygen saturation

Karnofsky/Lansky score (see Appendix 1) Assessment of fitness to undergo leukapheresis Serum pregnancy test in WOCBP Full blood count

Clotting screen (prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen)

Biochemical assessment of renal and liver function (LDH, urea, creatinine (and estimated creatinine clearance if creatinine >1.5 ULN for age), ALT/AST, Alkaline Phosphatase, bilirubin) Virology

Electrocardiogram (ECG)

Tumour biopsy specimen (if available) to confirm histological diagnosis of neuroblastoma Cross-sectional imaging (MRI) of disease sites and/or ¹²³I-MIBG scan and MRI brain (this does not need to be repeated if performed at the referring hospital as part of standard of care)

Patients will be tested for infectious diseases as outlined below prior to (within 30 days of) leukapheresis and repeated at the time of donation (or, if not possible, within seven days post donation).

Table 9. Biological screening for infectious diseases.

* A validated testing algorithm must be applied to exclude the presence of active infection with Treponema pallidum. A non-reactive test, specific or non-specific, can allow tissues and cells to be released. When a nonspecific test is performed, a reactive result will not prevent procurement or release if a specific Treponema confirmatory test is non-reactive. A donor whose specimen tests reactive on a Treponema-specific test will require a thorough risk assessment to determine eligibility for clinical use. A positive test result will not necessarily prevent the tissues or cells or any product derived from them being stored, processed and reimplanted, if appropriate isolated storage facilities are available to ensure no risk of cross-contamination with other grafts and/or no risk of contamination with adventitious agents and/or mix-ups.

Positive serological tests will be confirmed by PCR. A positive serological test in the absence of viraemia detected by PCR will not necessarily prevent the cells or RQR8/huK828Z/CST CAR T cells derived from them being stored, processed and infused.

Where required, patients will be tested for SARS-CoV-2.

Patients will be assessed clinically, and the following tests should be performed prior to starting lymphodepleting chemotherapy (within 48 hours prior to D-6, unless otherwise specified below); tests are also summarised in Table 10. Physical examination

Weight

Body surface area (BSA)

Vital signs (temperature, pulse, blood pressure and respiratory rate)

Oxygen saturation level

Full blood count

Clotting (PT, aPTT, fibrinogen)

Biochemistry (LDH, urea, creatinine (or estimated creatinine clearance), bilirubin, ALT/AST, ALP) CRP

Ferritin

Serum pregnancy test for WOCBP

Cross-sectional imaging (MRI) of disease sites and ¹²³I-MIBG scan within 7 days prior to staring lymphodepletion

Bilateral bone marrow aspirates and trephines within 7 days prior to starting lymphodepletion

Urine HVA/VMA within 7 days prior to starting lymphodepletion

4 Pre-treatment Assessments before CAR T cell infusion (Day 0)

Patients will be assessed clinically, and the following tests should be performed at DO prior to starting RQR8/huK828Z/CST CAR T cell infusion (tests also summarised in Table 10):

Karnofsky I Lansky score

Oxygen saturation level

Full blood count,

Biochemistry (LDH, urea, creatinine, bilirubin, ALT/AST, ALP),

Clotting (PT, aPTT, fibrinogen),

CRP

Ferritin

For 1 year after the RQR8/huK828Z/CST CAR T cells infusion, patients will be evaluated as outlined in Table 10 and Table further below. The time-points are in relation to RQR8/huK828Z/CST CAR T cells infusion. After patients complete 1 year, longer term follow up will be carried out annually for a further 14 years (i.e., until 15 years post RQR8/huK828Z/CST CAR T cells infusion). 5 Assessments from infusion until 28 days follow! no RQR8/huK828Z/CST CAR T cells

Immediately prior to the RQR8/huK828Z/CST CAR T cell infusion and for 4 hours after infusion (hourly observations) the nurse/doctor administering the RQR8/huK828Z/CST CAR T cells should monitor the patient for temperature, pulse, blood pressure, respiratory rate and oxygen saturation level.

Following this, patients will remain under paediatric oncology inpatient care (from the day of RQR8/huK828Z/CST CAR T cell infusion to approximately 2-4 weeks after the dose of RQR8/huK828Z/CST CAR T cells) and monitored as outlined below and in Table 10.

Karnofsky I Lansky score on D28

Twice daily clinical observations (temperature, pulse, blood pressure, respiratory rate and oxygen saturations) and toxicity assessment (in particular for cytokine release syndrome and neurological disturbance).

Blood tests at time points as summarized in Table 10.

Cross-sectional imaging (MRI) of disease sites and ¹²³I-MIBG scan - performed on D28 +/- 3 business days for response assessment

Bilateral bone marrow aspirates and trephines - performed on D28 +/- 3 business day for response assessment

Urine HVA/VMA - performed on D28 +/- 3 business days

able 10. Assessments on study until D28 post RQR8/huK828Z/CST CAR T cells infusion ay 0 = day of RQR8/huK828Z/CST CAR T cell administration. Assessments beyond Day 21 can be performed +/- 3 business days of the visit.

Assessments at lymphodepletion (LD) start (D-6) can be performed within 48 hours prior to LD start. Day 0 = day of RQR8/huK828Z/CST CAR T cells administration. Patients will be monitored immediately prior to infusion and for 4 hours post RQR8/huK828Z/CST CAR T cell infusion (hourly observations) for temperature, pulse, blood pressure, respiratory rate, oxygen saturation level. Following this observation at least twice per day until D14. Bloods for coagulation: PT, aPTT and fibrinogen Bloods for biochemistry: LDH, urea, creatinine, bilirubin, ALT/AST, ALP Estimated creatinine clearance if creatinine >1 .5 ULN for age Virology screen Cross-sectional imaging (MRI) and/or 1231-MIBG will be performed at Registration to confirm if the disease is measurable and/or evaluable. The imaging assessment do not need to be repeated if performed at the referring hospital as part of standard of care. Cross-sectional imaging (MRI), ¹²³I-MIBG and bilateral bone marrow aspirates within 7 days prior to starting LD and +/- 3 business day after reaching D28 post CAR T cells infusion. Surplus bone marrow samples will be used to assess exploratory endpoints. . Urine HVA/VMA will be performed within 7 days prior to starting lymphodepletion and +/- 3 business days for the visit on Day 28. . Serum collected for exploratory endpoints (including cell-free DNA as biomarker of disease burden/response). . Blood collected for exploratory endpoints (to characterise CAR T cells and other immune cells present by flow cytometry and/or scRNAseq). . Blood for cryopreservation of PBMC and serum in case replication-competent retrovirus (RCL) testing or integration site analysis is required and for other assays as developed and sent to Immunology Laboratory, Great Ormond Street Hospital

6 Assessments during one year follow up (interventional phase of the study)

Following discharge, patients will be followed up at intervals as outlined in Table . At each of these time points (+/- 7days), patients will be reviewed clinically and with the investigations outlined below:

Table 11. Subsequent assessments during interventional phase.

Assessments from Day +42 onwards can be performed +/- 7 days of the visit.

1 . Bloods for biochemistry: LDH, urea, creatinine, bilirubin, ALT/AST, ALP

2. Surplus bone marrow samples will be used to assess exploratory endpoints.

In the event of disease progression following RQR8/huK828Z/CST CAR T cell infusion, a second RQR8/huK828Z/CST CAR T cell dose may be administered, and the participant will be monitored as after the first RQR8/huK828Z/CST CAR T cells infusion.

After completing the interventional phase of the study patients will enter long term follow up until the end of trial is declared. Long term follow up will be carried out annually with data reporting to UCL CTC for the following: Table 12. Subsequent assessments during long term follow-up.

Assessments can be performed +/-1 month of the visit

*After 15 years data on patient’s status will be obtained annually from patient’s medical records until end of trial is declared but no further hospital visits are required.

In the event of disease progression during the long-term follow-up, the patient will cease follow up and no further trial visits will be required.

Limited safety, disease and survival status data for all patients (regardless of whether they have progressed or not) will continue to be collected annually from routine standard of care visits.

7 _ Assessment of Toxicity

Toxicity will be graded using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v5.0 criteria. The incidence of NCI CTCAE grade 3-5 toxicity occurring within 28 days of RQR8/huK828Z/CST CAR T cells infusion will be determined.

In particular, the incidence of Grade 3-4 Cytokine Release Syndrome (using the revised ASTCT grading system) and Grade 3-4 neurotoxicity (ICANS) occurring within 28 days of RQR8/huK828Z/CST CAR T cells infusion (using the revised ASTCT ICANS score) will be determined.

8 _ Dose Limiting Toxicity (DLT)

Dose limiting toxicity will be defined as any of the following RQR8/huK828Z/CST CAR T cells related adverse events which occur within the DLT period (between DO and D28 of RQR8/huK828Z/CST CAR T cells infusion):

CRS toxicity Grade 4 in severity for greater than 96 hours CRS toxicity Grade 3 not resolved < Grade 1 at D28 assessment

ICANS Grade 4 in severity

ICANS Grade 3 in severity not resolved to < Grade 1 at D28 assessment Grade >2 Infusion Reaction with RQR8/huK828Z/CST CAR T cells infusion Any other fatal event (Grade 5), or life-threatening event (Grade 4) that cannot be managed with conventional supportive measures, or which in the opinion of the TMG necessitates modification to trial treatment to avoid a similar occurrence in future patients

Any event that in the opinion of the TMG put patient at undue risk may also be considered a DLT

9 Assessments of Efficacy

Responses will be evaluated at the time points listed in Table 8 and Table 9.

Response for the primary and metastatic soft tissue sites will be determined for patients with evaluable disease on ¹²³I-MIBG radionuclide scan (for patients with MIBG-avid disease only) using a semi-quantitative score (see Appendix 2 - SIOPEN scoring).

A combined response assessment based on cross-sectional imaging, ¹²³I-MIBG and bone marrow assessment will be performed based on the revised International Neuroblastoma Response Criteria⁶⁷ (INRC, Appendix 3).

EXAMPLE 8 - Further biological studies

1 Expansion and persistence of RQR8/huK828Z/CST CAR T cells in peripheral blood

The expansion and persistence of RQR8/huK828Z/CST CAR T cells is determined. This will be examined by flow cytometry and quantitative PCR, using established assays. For PCR assays, T cell population transduced with AU54280, AU54281 or both will be identified separately to study the contribution of FabCCR-IL7, dSHP2/dTRBII to engraftment and persistence.

2 Serum cytokines

Serum cytokines will be examined by an established assay measuring concentration of multiple cytokines including IL-6, IL-10 and TNF-a. Maximum and kinetics of cytokine concentrations will contribute to assessment of CAR T induced immune activation and will be correlated with clinical symptoms. 3 Cell free DNA in serum as marker for tumour response

Cell free DNA will be assessed at indicated time points post CAR T administration as indicated in Error! Reference source not found, to assess whether changes in the level of cfDNA in the serum can provide prognostic information. Findings will be correlated with the response assessment.

4 Characterization of RQR8/huK828Z/CST CAR T cells

Proteomic and transcriptomic characterization of RQR8/huK828Z/CST CAR T cells will be performed at time points post CAR T administration as indicated in Error! Reference source not found, to assess whether parameters can be identified which are associated with peak expansion and persistence of administered CAR T cells. For these studies, T cell populations transduced with AU54280, AU54281 or both will be identified separately to study the contribution of FabCCR-IL7, dSHP2/dTRBII to engraftment and persistence.

EXAMPLE 9 - Clinical results

One patient with relapsed neuroblastoma was treated with RQR8/huK828Z/CST CAR T cell. This patient received a dose of 30x10⁶ RQR8/huK828Z/CST CAR T cells/m². This patient later developed Grade 3 CRS and there were no symptoms or signs of neurotoxicity.

ABBREVIATIONS

Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. A method for treating a neuroblastoma in a patient comprising administering to the patient autologous anti-GD2 CAR T cells, wherein the CAR T cells comprise an anti-GD2 chimeric antigen receptor (CAR) comprising:

CDR1 - SYNIH (SEQ ID No. 1),

CDR2 - VIWAGGSTNYNSALMS (SEQ ID No. 2), and

CDR1 - RASSSVSSSYLH (SEQ ID No. 4),

CDR2 - STSNLAS (SEQ ID No. 5), and

CDR3 - QQYSGYPIT (SEQ ID No. 6);

(ii) a CD8a stalk; and

6. The method of claim 1 wherein the anti-GD2 CAR T cells are RQR8/huK828Z/CST CAR T cells.

7 The method of any preceding claim wherein the patient has relapsed or refractory neuroblastoma following at least one line of therapy.

CDR1 - SYNIH (SEQ ID No. 1),

CDR2 - VIWAGGSTNYNSALMS (SEQ ID No. 2), and

(ii) a CD8a stalk; and

(iii) an a CD28-CD3 endodomain; and wherein at least one vector comprises a nucleic acid encoding an activity modulator which modulates the activity of the CAR, of a cell expressing the CAR, or of a target cell, wherein the activity modulator is selected from a dominant negative SHP-1 or SHP-2, a dominant negative transforming growth factor (TGF) p receptor and/or a constitutively active chimeric cytokine receptor.

11 . The method of claim 10, wherein the GD2 binding domain comprises a VH domain having the sequence shown as SEQ ID No. 7; and/or a VL domain having the sequence shown as SEQ ID No 8.

12. A method according to claim 10 or 11 , wherein the activity modulator is a dominant negative SHP-2.

13. A method according to claim 10, 11 or 12, wherein at least one vector comprises a nucleic acid which encodes a dominant negative SHP-2 and a dominant negative TGF receptor.

15. A method according to claim 10, 11 , 12, 13 or 14, wherein in the mixture of viral vectors at least one vector comprises a nucleic acid sequence which encodes a dominant negative SHP-2 and a dominant negative TGF p receptor; and at least one vector comprises a nucleic acid sequence which encodes a constitutively active chimeric cytokine receptor.

17. A method according to any claims 10 to 16 further comprising selecting CAR- expressing cells from the transduced cell population.

18. A cell composition made by a method according to any of claims 10 to 17.

19. A cell composition according to claim 18 for use in treating and/or preventing Neurobastoma.