Mixture of CD19 car t cells and CD22 car t cells for the treatment of high-risk / relapsed haematological malignancies

Abstract

The present disclosure relates to CD19 / 22 CAR T-cell products and methods for treating CD19+ or CD22+ haemotological malignancies.

Description

[0001] METHOD OF TREATMENT

[0002] FIELD OF THE INVENTION

[0003] The disclosure relates to cell populations comprising CD19 and CD22 CAR T-cells and methods for treating high risk or relapsed, CD19+ or CD22+ haemotological malignancies.

[0004] BACKGROUND OF THE INVENTION

[0005] B- cell acute lymphoblastic leukaemia (B-ALL) is a disease that occurs in both children and adults. As an acute leukaemia, it is a serious and life-threatening The disclosure relates to CD19 / 22 CAR T-cell products and methods for treating high risk or relapsed, CD19+ or CD22+ haemotological malignancies.

[0006] Pediatric B-cell acute lymphoblastic leukemia (ALL) accounts for about 30% of childhood cancer diagnoses. It is a serious and life-threatening disease and will progress rapidly if left untreated. B-ALL is characterized by the rapid proliferation of poorly differentiated lymphoid progenitor cells inside the bone marrow. Standard of care is combination chemotherapy. Typically, treatment procedures are divided into several phases: steroid pre-phase, induction, consolidation, intensification, and maintenance, and involve the administration of steroids, chemotherapy, targeted career drugs and / or bone marrow or stem cell transplant (SCT). The overall survival (OS) rate for pediatric B cell ALL patients is 90%, however 10-20% of pediatric B cell ALL patients relapse with chemoresistant disease. The clinical outcomes for relapsing pediatric patients have not changed over the past two decades. The long-term OS of children suffering a relapse of ALL remains 40-50% despite considerable effort to optimize standard approaches. Current treatment strategies for relapsed ALL include either further intensive chemotherapy and often consolidation with allogeneic SCT, but these patients have poor outcomes. Since most of these patients have frequently had the maximal tolerable dose of chemo / radiotherapy, novel therapies are urgently required for such patients.

[0007] A number of immunotherapeutic agents have been described for use in cancer treatment, including therapeutic monoclonal antibodies (mAbs), immunoconjugated mAbs, radioconjugated mAbs and bi-specific T-cell engagers. Typically these immunotherapeutic agents target a single antigen: for instance, Rituximab targets CD20; Myelotarg targets CD33; and Alemtuzumab targets CD52.Chimeric antigen receptors are proteins which graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus (binder), and a transmembrane domain connected to an endodomain which transmits T-cell activation signals. The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, which recognize a target antigen, fused via a transmembrane domain to a signalling endodomain. Such molecules result in activation of the T-cell in response to recognition by the scFv of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. CARs have been developed against various tumor-associated antigens and many are currently undergoing clinical trials.

[0008] The human CD19 antigen is a 95 kd transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is expressed very early in B-cell differentiation and is only lost at terminal B-cell differentiation into plasma cells. Consequently, CD19 is expressed on all B-cell malignancies apart from multiple myeloma. Since loss of the normal B-cell compartment is an acceptable toxicity, CD19 has been a CAR target and clinical studies targeting CD19 with CARs have been conducted.

[0009] CD19-directed CAR therapy has shown efficacy in treating ALL. The first studies in ALL were published in Spring 2013, by groups from Memorial Sloane Kettering [Brentjens et al., Leukemia. Sci. Transl. Med., 5’. 177ra38) (2013)] and the University of Pennsylvania. An updated report of the University of Pennsylvania study was made [Maude et al., N. Engl. J. Med., 371: 1507-1517 (2014)]. In that latter study, twenty-five patients under the age of 25 years and five over that age were treated. 90% achieved a complete response at one month, 22 of 28 evaluable cases achieved a minimal residual disease (MRD) negative status and the 6 month event free survival rate was 67%. Fifteen patients received no further therapy after the study.

[0010] The Memorial Sloane Kettering study was in the adult setting, and treated five ALL patients (two with refractory relapse, two with MRD positive disease and one who was MRD negative) with autologous T cells retrovirally transduced to express a CD19 CAR incorporating an scFv derived from the SJ25C1 hybridoma and a CD28 co-stimulatory domain. All of these patients achieved a deep molecular remission, enabling four of these patients to receive an allogeneic SCT. This precluded assessment of the durability of responses, but CAR T cells were only detectable in the blood or bone marrow for 3-8 weeks after infusion. The patient who was not transplanted relapsed at 90 days with CD19+ disease. Subsequently, Davila et al., Sci. Transl. Med. 6: 224:ra25 (2014) provided anupdate of this cohort 14 of 16 adult patients had detectable disease at the point of CAR T cell infusion, despite salvage chemotherapy and cyclophosphamide conditioning. 14 of 16 achieved a complete remission with or without count recovery including 7 of 9 patients with morphologic evidence of residual disease detectable after salvage chemotherapy. 12 of 16 patients achieved MRD negativity and this allowed seven to undergo allogeneic transplantation by the time of publication. Responses were durable in some patients with 4 of 8 non-transplanted patients continuing in morphological remission at up to 24 months follow-up although the survival curves for this cohort are not yet stable.

[0011] Another published study of a cohort of pediatric and young adult patients predominantly with ALL provides the first intention-to-treat analysis of its outcomes. This may help remove the bias inherent in excluding patients who do not receive the anticipated dose of CAR T cells [Lee et al., Lancet (2014) doi:10.1016 / S0140-6736(14)61403-3], Twenty-one patients were treated with a CD28 domain-containing second generation CAR. All but two patients received the anticipated T cell dose, highlighting the feasibility of delivering this treatment to those with refractory or multiply-relapsed ALL. This study showed 67% achieving a complete remission and 60% of those with ALL achieving MRD negative status.

[0012] The first trial showing clinical activity of CD22 CAR T cells in children and adults with B-ALL was reported in Fry et al., Nature Med., 24.20-28 (2018). 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy, received CD22 CAR T cells. Complete remission obtained in 73% (11 / 15) of patients receiving >1 x 106CD22-CAR T cells per kg body weight, including 5 of 5 patients who enrolled with CD19 dim / neg relapse. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22+ cell escape from killing by CD22-CAR T cells.

[0013] A particular problem in the field of oncology is provided by the Goldie-Coldman hypothesis: which describes that the sole targeting of a single antigen may result in tumor escape by modulation of said antigen due to the high mutation rate inherent in most cancers. This modulation of antigen expression may reduce the efficacy of known immunotherapeutics, including those which target CD19. Despite the excellent clinical responses to CD19-directed T cell therapies, a significant number of patients still relapse. The major cause of disease relapse is either the emergence of CD19 negative leukaemic clones or non-persistence of the CAR T cells [Sotillo etal., Cancer Di scov., 5:1282-1295 (2015); Gardner etal., Blood 727:2406-2410 (2016)].Thus a problem with immunotherapeutics targeted against CD19 is that a B-cell malignancy may mutate and become CD19-negative. This may result in relapse with CD19-negative cancers which are not responsive to CD19 targeted therapeutics. The emergence of CD19 negative escape clones has been reported in all the major studies in ALL and may relate to selection of leukaemic clones with either somatic mutations or expressing an alternatively spliced CD19 mRNA lacking exon 2 that prevent recognition by the CD19 CAR (Sotillo etal., supra). In the paediatric B cell ALL studies at the University of Pennsylvania, two-thirds of patients relapsed due to CD19 negative disease, while the remaining one-third relapsed due to poor CAR T cell engraftment [Grupp etal., Blood -128(2.2^.2.2.^ (2016)]. CD19 negative relapse has also been reported by NCI in a lymphoma patient treated with fully human anti-CD19 CAR (HuCAR-19) [Brudno etal., Blood, 128(22): 999(2016)]. Another study has recently reported that CD19-negative relapses were more frequently observed, post Tisagenlecleucel infusion, in patients with high tumor burden [Dourthe et al., Leukaemia, 35:3383-3393 (2021)].

[0014] There is thus a need for immunotherapeutic agents which are capable of targeting more than one cell surface structure to reflect the complex pattern of marker expression that is associated with many cancers, including CD19-positive cancers.

[0015] Although CAR-T cell-mediated treatment has shown success towards compact target antigens such as CD19 or GD2, chimeric antigen receptors have failed to signal in response to antigens with bulky extracellular domains.

[0016] An optimum synaptic distance is required for efficient triggering of downstream signaling after antigen encounter. Upon T cell encounter with an antigen presenting cell (via TCR interaction with peptide MHC), proteins at the interface segregate passively based on size. Phosphatases such as CD45 and CD148, which have large ectodomains, are excluded from regions of close contact between the T cell and APC. The synapse formed through interaction of peptide MHC and TCR is optimal for occlusion of CD45. In the case of CAR-T cells targeting smaller antigens such as CD19, there is no barrier to synapse formation and such antigens can be targeted efficiently at multiple epitopes. Large proteins such as CD22 pose a unique problem. Targeting a membrane distal epitope on such proteins is likely to provide a suboptimal synapse length allowing phosphatases to enter the synapse and inhibit tyrosine phosphorylation. Targeting membrane proximal regions may improve synapse formation, however steric occlusion of the epitope is likely to lead to suboptimal ligation of the target allowing the presence of phosphatases within the synapse, dampening tyrosine phosphorylation, kinase activity and thus CAR signaling.There is therefore a need for alternative CAR T-cell approaches, capable of killing target cells expressing a large or bulky target antigen.

[0017] T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections and cancer. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Recently, a clearer picture of the functional and phenotypic profile of exhausted T cells has emerged with expression of inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, being a key feature [Day etal., Nature, 443 350-354 (2006)].

[0018] Responses in CD19 CAR studies suggest that persistence of T-cells for a protracted period at high levels seems to be important in effecting durable responses [Mueller et al., Blood, 130, 2317-2325 (2017)].

[0019] In cohort 3 of the CARPALL study, T cells co-transduced with lentiviral vectors encoding CD19CAT CAR and a novel CD22CAR (9A8 CAR) derived from the rat 9A8 hybridoma, which recognises targets expressing CD22 at low antigen density, were tested. This CAR consists of scFv from 9A8 incorporated into a second-generation CAR with an identical design as CATCAR with the stalk and transmembrane region derived from CD8 connected to the 4-1 BB and CD3zeta endodomains. Dual targeting CD19 / 22CAR T cells were found to be safe (no grade 3-5 Cytokine Release Syndrome) with comparable efficacy to Tisagenlecleucel (10 / 12 patients 83% achieved molecular remission). Importantly no cases of relapse due to antigen-negative escape were seen suggesting dual targeting effectively prevented relapse due to antigen escape. However, 5 / 10 responding patients had antigen positive disease recurrence associated with loss of CAR T cell persistence. This has been a major limitation in many studies of dual antigen targeting in ALL.

[0020] There remains a need in the art for effective and persistent CAR therapies for CD19+ or CD22+ haematological malignancies which are not associated with the foregoing disadvantages.

[0021] SUMMARY OF THE INVENTION

[0022] The disclosure provides methods for treating high risk / relapsed CD19+ or CD22+ haematological malignancy in a patient comprising administering to the patient a cell population of autologous CD19 CAR-T cells and CD22 CAR T-cells (for example, theautologous CD19+CD22 CAR T-cell product comprising CAT19CAR and 9A8CAR CARs described in Example 1 herein).

[0023] A cell population comprising anti-CD19 CAR T-cells and anti-CD22 CAR-T cells for use in a method of treating a high risk / relapsed CD19+ or CD22+ haematological malignancy in a patient is provided.

[0024] Use of a cell population comprising anti-CD19 CAR T-cells and anti-CD22 CAR-T cells for the manufacture of a medicament to use in a method of treating a high risk / relapsed CD19+ or CD22+ haematological malignancy in a patient is also provided.

[0025] A method of treating a high risk / relapsed CD19+ or CD22+ haematological malignancy in a patient is also provided, comprising administering a cell population comprising anti-CD19 CAR T-cells and anti-CD22 CAR-T cells to the patient.

[0026] The age of the patient may be twenty-four years or younger.

[0027] The haematological malignancy may be acute lymphoblastic leukemia (ALL), or a CD19+ and / or CD22+ lymphoma.

[0028] The lymphoma may be Burkitt’s lymphoma.

[0029] The patient may have:

[0030] a. Resistant disease (>5% blasts) at end of ALLTogether-1 protocol (NCT03911128) or equivalent induction;

[0031] b. ALL with persisting high level MRD at second time point of frontline national protocol (currently MRD >10'4at week 9 ALLTogether-1 Protocol or equivalent); c. High risk infant ALL (age < 6 months at diagnosis with MLL gene rearrangement and either presenting white cell count > 300 x 109 / L or poor steroid early response (i.e. circulating blast count >1x109 / L following 7 day steroid pre-phase of induction as per national guidelines or equivalent);

[0032] d. Any patient with t(17, 19) TCF3-HLF rearrangement;

[0033] e. High risk first relapse (defined as very early (relapse within 18 months of diagnosis) and early relapses (any patient relapsing on therapy or within 6 months of completing treatment) and any relapse with high risk genetics, namely (KMT2A (MLL) rearrangements, low hypodiploidy / near haploidy, t(17;19)(q22;p13) / TCF3- HLF, iAMP21 and t(1;19)(q21;p13) / TCF3- PBX1, t(9;22)(34.1 q11.2) / BCR-ABL1); f. Any patient aged 16 to 24 on therapy relapse;

[0034] g. Any relapse of infant ALL;

[0035] h. ALL post > 2nd relapse;i. Any refractory relapse of ALL (defined as > 1 % blasts by flow cytometry after at least one cycle of standard chemotherapy)

[0036] j. ALL with MRD >10-4 prior to planned stem cell transplant

[0037] k. Any relapse of ALL eligible for stem cell transplant (SCT) but with no available HLA matched donor or other contraindication to transplant;

[0038] l. Any relapse of ALL after SCT as long as planned time of infusion of anti-CD19 CAR-T cells and anti-CD22 CAR T cells is longer than 4 months post-transplant; or

[0039] m. Early (defined as < 6 months post-infusion) loss of B cell aplasia or any CD19+CD22+ relapse following CD19CAR T cell therapy with Tisagenlecleucel. The patient may have an isolated CNS relapse meeting one or more of a) - m).

[0040] The cell population may comprise a 1:1 ratio of anti-CD19 CAR-T cells and anti-CD22 CAR-T cells.

[0041] The patient may be administered a total dose of 4 x105anti-CD19 and anti-CD22 CAR T-cells / kg, 5 x105anti-CD19 and anti-CD22 CAR T-cells / kg, 8 x105anti-CD19 and anti-CD22 CAR T-cells / kg, 1 x106anti-CD19 and anti-CD22 CAR T-cells / kg, 1.5 x 106anti-CD19 and anti-CD22 CAR T-cells / kg, or 2 x 106anti-CD19 and anti-CD22 CAR T-cells / kg.

[0042] The method of treating the high risk / relapsed CD19+ or CD22+ haematological malignancy in the patient may comprise administering a first dose and a second dose of the cell population comprising anti-CD19 CAR T-cells and anti-CD22 CAR-T cells, wherein the second dose is administered between about 12 days and about 21 days after the administration of the first dose.

[0043] The second dose may be administered about 14 days after the administration of the first dose.

[0044] Alternatively, the method of treating the high risk / relapsed CD19+ or CD22+ haematological malignancy in the patient may comprise administering a single dose of the cell population comprising CD19 CAR T-cells and CD22 CAR-T cells to the patient.

[0045] The method of treating the high risk / relapsed CD19+ or CD22+ haematological malignancy in the patient may comprise administering a single dose of the cell population comprising 4 x105CD19 and CD CAR T-cells / kg, which may comprise 2 x105CD19 CAR T-cells / kg and 2 x105CD22 CAR T-cells / kg, to the patient.

[0046] The anti-CD19 CAR-T cells may express a chimeric antigen receptor (CAR) comprising a CD19-binding domain which comprises:i) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

[0047] CDR1 - GYAFSSS (SEQ ID NO: 1);

[0048] CDR2 - YPGDED (SEQ ID NO: 2)

[0049] CDR3 - SLLYGDYLDY (SEQ ID NO: 3); and

[0050] ii) a light chain variable region (VL) having CDRs with the following sequences:

[0051] CDR1 - SASSSVSYMH (SEQ ID NO: 4);

[0052] CDR2 - DTSKLAS (SEQ ID NO: 5)

[0053] CDR3 - QQWNINPLT (SEQ ID NO: 6).

[0054] The CD19-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 or a variant thereof having at least 95% sequence identity.

[0055] The CD19-binding domain may comprise the sequence shown as SEQ ID NO: 9 or a variant thereof having at least 90% sequence identity.

[0056] The anti-CD22 CAR T-cells may express a chimeric antigen receptor (CAR) comprising a CD22-binding domain which comprises:

[0057] i) a heavy chain variable region (VH) having CDRs with the following sequences:

[0058] CDR1 - NFAMA (SEQ ID NO: 58);

[0059] CDR2 - SISTGGGNTYYRDSVKG (SEQ ID NO: 59)

[0060] CDR3 - QRNYYDGSYDYEGYTMDA (SEQ ID NO: 60); and

[0061] ii) a light chain variable region (VL) having CDRs with the following sequences:

[0062] CDR1 - RSSQDIGNYLT (SEQ ID NO: 61);

[0063] CDR2 - GAIKLED (SEQ ID NO: 62)

[0064] CDR3 - LQSIQYP (SEQ ID NO: 63).

[0065] The CD22-binding domain may comprise a VH domain having the sequence shown as SEQ ID NO: 64 and / or or a VL domain having the sequence shown as SEQ ID NO: 65 or a variant thereof having at least 95% sequence identity.

[0066] The CD22-binding scFv may comprise the sequence shown as SEQ ID NO: 66 or a variant thereof having at least 90% sequence identity.

[0067] The patient may receive lymphodepletion comprising fludarabine, cyclophosphamide and low dose total body irradiation prior to receiving the cell population comprising anti-CD19 CAR T-cells and anti-CD22 CAR-T cells.The lymphodepletion may comprise:

[0068] a) fludarabine 30 mg / m2once daily on day -6 to -3;

[0069] b) cyclophosphamide 0.5 g / m2once daily on day -6 to -5; and

[0070] c) low dose Total Body Irradiation 2Gy on day -2.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1. A) Cartoons of CD19 CATCAR (which is ALITO1) (left) and CD229A8CAR (right). These CARs are type I transmembrane proteins. Both CARs are identical except for the scFv. The scFv are at the amino-terminus are linked to a CD8 stalk and transmembrane domain which is linked to an endodomain comprised of a fusion between 4-1 BB and CD3 . B) Dual lentiviral vectors encoding CATCAR (top) and 9A8CAR (bottom).

[0073] Figure 2. Comparison of the transduction efficiencies for CD19+CD22CAR T cells manufactured with a 9-day or 5-day process. CD19+CD22 CART cells were generated from three patients (CPL-27, CPL-29 and CPL-31). Transduction efficiencies were determined by flow cytometry at the end of the 9-day process (Cohort 3) or 5-day process (Cohort 4) using anti-idiotype antibodies specific for the CD19 CAR or CD22 CAR and gating on viable CD45+CD3+singlet cells in the lymphocyte gate.

[0074] Figure 3. In vitro function of CD19+CD22 CAR T cells and comparison of functionality following manufacture with a 9-day or 5-day process. Functional assays were performed with PBMCs from leukapheresis or CD19 / 22 CAR T cells from three patients (CPL-27, CPL-29 and CPL-31) produced using either Cohort 3 (9-day process) or Cohort 4 (5-day process) methods. (A) Cytotoxicity assays were carried with co-cultures of CD19 / 22 CAR T cells with SupT1 (CD19-), Raji (CD19+) or Nalm6 cells (CD19+) for~18h (E:T ratio = 1:1). Flow cytometric analysis was used to determine the number of viable target cells. Percentage killing was determined relative to number of target cells in the co-cultures of leukapheresis + target cells. (B)3H-thymidine incorporation assays were carried out with CAR T cells cocultured with irradiated target cells at 1:1 E:T ratio for 72 hours. P / I=phorbol 12-myristate 13-acetate / ionomycin. Cells were pulsed with3H-thymidine for the last 18h of co-culture.

[0075] Cytometric bead assays were performed using supernatant taken at 48 h of co-culture (from the same wells as for panel B) to determine levels of (C) IFN-y or (D) IL-2. All data are mean ± s.d. for triplicate wells. Statistical analysis was carried out using Students t-test to compare Cohort 3 vs Cohort 4.Figure 4: In vivo functionality comparison of Cohort 3 (9-day process) and Cohort 4 (5-day process) CD19+CD22 CAR T cells. A. Xenograft experimental schematic. NSG mice were irradiated with 2.5Gy, followed by i.v. injection of 1x105GFP+ / Luc+NALM6 cells. PBS, 2x106Cohort 3 (CPL-27), or2x106Cohort 4 (CPL-27) CD19+CD22 CAR T were delivered i.v. on the following day. Leukaemia progression was monitored by weekly bioluminescence imaging. On day 28, the three mice with the highest I VIS signal from each CAR-treated group were harvested to examine early CAR-T cells persistence. B.

[0076] Bioluminescence images of GFP+ / Luc+NALM6 xenograft mice. C. Percentage of GFP+ / Luc+NALM6 cells of total mononuclear cells (MNCs) in blood, bone marrow, spleen, and liver in the sacrificed mice on day 28. One-way ANOVA test was performed. D. Percentage of CAR T cells (hCD45+hCD3+) cells of total MNCs in blood, bone marrow, spleen, and liver in the sacrificed mice on day 28. Mann-Whitney test was performed. E. Survival curves for xenograft mice up to 40 days post NALM6 injection. Logrank test was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001, ns, not significant.

[0077] Figure 5: CARPALL Cohort 4 manufacture flow diagram.

[0078] Figure 6: Memory phenotype of scale up products using cohort 4 manufacturing methodology compared to Cohort 3 ATIMPs manufactured using starting material from the same patient donors. Phenotyping of CAR T cells products by CCR7, CD45RA, and CD95 expression to determine central memory (TCM), Stem cell memory (TSCM), effector memory (TEM) and effector memory CD45RA+(TEMRA), T cell subsets. The populations shown were pre-gated on the viable, CD3+ T cells. All cells within the CCR7+ CD45RA+ population expressed CD95 and were designated TSCM. Each Cohort 4 scale up run was manufactured using cryopreserved starting material from the same donor for the corresponding Cohort 3 ATIMP. Cohort 3: Co-transduced CD19 / CD22 CAR-T cells; Cohort 4: CD19+CD22 CAR-T cell population.

[0079] Figure 7: Clinical trial schema (Cohort 4).

[0080] Figure 8. Annotated DNA sequence (SEQ ID NO: 88) encoding the CD19 CATCAR. Figure 9. Annotated amino acid sequence (SEQ ID NO: 89) of the CD19 CATCAR . Figure 10. Annotated DNA sequence (SEQ ID NO: 90) encoding the CD229A8CAR. Figure 11. Annotated amino acid sequence (SEQ ID NO: 91) of the CD229A8CAR. Figure 12. Clinical trial schema (Cohort 4). Single dose of CAR-T cells.DETAILED DESCRIPTION

[0081] In the CARPALL clinical study [Ghorashian et al., J. Haematol., 169: 463-478 (2015)] which tested the efficacy of CD19 CATCAR (sometimes also referred to as CAT19CAR or AUTO1 herein), an autologous fast off-rate CAR, in children and young adults with relapsed / refractory (r / r) B-ALL, AUTO1 was efficacious with molecular remission in twelve of fourteen patients. However, of the seven patients that relapsed, five relapses were due to CD19 antigen negative escape. To address antigen escape relapse in AUTO1, an additional ligand could be targeted.

[0082] CD22 is contemplated herein as another target for B-ALL malignancies, although generating an effective CAR to CD22 is challenging due to the size, density and rigidity of this ligand. Furthermore, in the B-ALL setting, CD22 expression levels are known to down regulate in response to selective CAR pressure. In a clinical trial of the CD22 CAR2, this resulted in patients relapsing with lower CD22 density post-treatment (2,839 epitopes / cell) presumably due to the target density falling below the sensitivity threshold for the CD22 CAR2 (Fry et al., supra)].

[0083] In cohort 3 of CARPALL (Ghorashian, S., et al. Blood, 2024. 143:118-23), patients were treated with autologous T cells co-transduced with lentiviral vectors encoding CD19CAT CAR (pCCL.PGK.aCD19CAT-41BB-zeta), and a CD229A8 CAR (pCCL.EF1a.aCD229A8-41BB-zeta). This resulted in products with a predominance of CD19 / 22 dual transduced CAR T cells with lower, balanced populations of CD19 and CD22 single positive CAR T cells. Twelve paediatric patients with advanced r / r ALL were treated. The toxicity profile remained favourable with no severe (grade > 3) CRS and only 1 case of late grade 4 neurotoxicity which was felt likely to be fludarabine-related although ICANS could not be excluded. In terms of efficacy, 10 / 12 patients achieved an MRD negative CR / CRi, including 2 of 3 patients with CD19 negative disease demonstrating the efficacy of the CD22CAR. Of the 10 responding patients, 3 relapsed and 2 others had emergence of MRD prompting further therapy. In all 5 patients with recurrent disease this was CD19+CD22+and associated with loss of CAR T cell persistence in 4 / 5 cases. At a median follow-up 8.7 months, 5 / 10 responders were alive and disease-free giving a 1 year EFS of 60%. Most importantly, no cases of relapse due to antigen-negative escape were seen, suggesting that dual targeting with co-transduction may have effectively prevented antigen negative relapse after CAR T-cell therapy. Indeed, this was the first dual targeting study where antigen-negative relapse had been eliminated. The major limitation of cohort 3 (and other CD19 / 22 dual targeting studies to date) was poor CAR T cell persistence in some patients leading to antigenpositive relapse.It has been hypothesized that higher CAR T expression and increased signalling in dual transduced CAR T cells may result in activation induced cell death (AICD) or exhaustion. The cell populations, uses and methods provided herein improve the treatment of B-ALL by combining T cells expressing a highly sensitive CD22 CAR capable of targeting cells that express less than one thousand CD22 molecules per cell (CD229A8 CAR) with ALITO1, T cells expressing a fast off-rate CD19 CAR (CAT19 CAR-T cells) for the treatment of pediatric B-ALL. The present inventors additionally combined the following approaches in order to enhance the persistence of the CAR T cells targeting CD19 and CD22 (‘CD19+CD22 CAR T cells’):

[0084] i) abbreviated manufacture to enrich for stem cell-like memory T cells (TSCM) and separate transduction of T cells with CD19CAR and CD22CAR lentiviral vectors to reduce activation induced cell death (AICD);

[0085] ii) optimised lymphodepletion with a reduced dose of fludarabine and cyclophosphamide to decrease toxicity, and low dose (2Gy) Total Body Irradiation to prevent CAR T cell rejection;

[0086] and, optionally,

[0087] iii) split dosing to mitigate activation-induced cell death / exhaustion of CD19+CD22 CAR T cells post-infusion. The present inventors hypothesised that by the time the second dose is infused, the majority of CD19+CD22+targets will have been deleted, so that CD19+CD22CAR T cells of the second dose, whilst receiving some antigenic stimulation, may not expand as much as CAR T cells infused in the first dose and therefore may be less likely to undergo AICD or become exhausted.

[0088] The CD19 CAR T cells and CD22 CAR T cells (CD19+CD22 CAR T cells) were obtained by separately transducing patient T cells with lentiviral vectors encoding the CD19CAT CAR and CD229A8 CAR and pooling them at a 1:1 ratio prior to cryopreservation. Results described in the Examples below were obtained as part of an extension cohort of the CARPALL clinical trial (NCT02443831).

[0089] 1. Chimeric

[0090]

[0091] A classical chimeric antigen receptor (CAR) is a chimeric type I trans-membrane protein which connects an extracellular antigen-binding domain to an intracellular signalling domain (endodomain). The antigen-binding domain 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 fragment or an antibody-like antigen-binding site. Otherexamples include, but are not limited to: a natural ligand of the target antigen, a peptide with sufficient affinity for the target, a F(ab) fragment, a F(ab’)2 fragment, a F(ab’) fragment, a single domain antibody (sdAb), a domain antibody (dAb), a VHH antigen-binding domain or nanobody, an artificial single binder such as a DARPin (designed ankyrin repeat protein), an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a VNAR, an iBody, an affimer, a fynomer, an abdurin / nanoantibody, a centyrin, an alphabody, a nanofitin, or a single-chain derived from a T-cell receptor which is capable of binding the target antigen. A spacer is usually necessary to isolate the antigen-binding domain from the membrane and to allow it a suitable orientation. A common spacer used is the Fc of I gG 1. More compact spacers can suffice, e.g., the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen. A transmembrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

[0092] Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FCER1 or CD3 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. One common co-stimulatory domain 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.

[0093] When the CAR binds the target antigen, an activating signal is transmitted to the T-cell on which the CAR is expressed thereby directing the specificity and cytotoxicity of the T cell towards cells expressing the target antigen.

[0094]

[0095] A ‘target antigen’ is an entity which is specifically recognized and bound by the antigenbinding domains of a chimeric receptor provided herein.

[0096] The target antigen may be an antigen present on a cancer cell, for example, a tumor- associated antigen. CD19 and CD22 are target antigens contemplated herein.3. Binding domains specific for CD19 target antigen

[0097] The human CD19 antigen is a 95 kd transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is classified as a type I transmembrane protein, with a single transmembrane domain, a cytoplasmic C-terminus, and extracellular N-terminus. CD19 is expressed very early in B-cell differentiation and is only lost at terminal B-cell differentiation into plasma cells. CD19 is a biomarker for normal B cells as well as follicular dendritic cells. CD19 primarily acts as a B cell co-receptor in conjunction with CD21 and CD81. Upon activation, the cytoplasmic tail of CD19 becomes phosphorylated, which leads to binding by Src-family kinases and recruitment of PI-3 kinase.

[0098] CD19 is also expressed on all B-cell malignancies but not multiple myeloma cells. It is not expressed on other haematopoietic populations or non-haematopoietic cells and therefore targeting this antigen should not lead to toxicity to the bone marrow or non-haematopoietic organs. Loss of the normal B-cell compartment is considered an acceptable toxicity when treating lymphoid malignancies, because although effective CD19 CAR T cell therapy will result in B cell aplasia, the consequent hypogammaglobulinaemia can be treated with pooled immunoglobulin.

[0099] Different designs of CARs have been tested against CD19 in various clinical trials, as outlined in the following Table 1.

[0100] Table 1

[0101]

[0102] As shown above, most of the studies conducted to date have used an scFv derived from the hybridoma fmc63 as part of the binding domain to recognize CD19.

[0103] The antigen-binding domain of a CAR which binds to CD19 (referred to as a CD19 CAR herein) may be any domain which is capable of binding CD19.For example, the antigen-binding domain may comprise a CD19 antigen-binding domain as described in Table 2.

[0104] Table 2

[0105]

[0106] The gene encoding CD19 comprises ten exons: exons 1 to 4 encode the extracellular domain; exon 5 encodes the transmembrane domain; and exons 6 to 10 encode the cytoplasmic domain. The antigen-binding domain of a CD19 CAR herein may bind an epitope of CD19 encoded by exon 1 of the CD19 gene. The antigen-binding domain of a CD19 CAR herein may bind an epitope of CD19 encoded by exon 2 of the CD19 gene. The antigen-binding domain of a CD19 CAR herein may bind an epitope of CD19 encoded by exon 3 of the CD19 gene. The antigen-binding domain of a CD19 CAR herein may bind an epitope of CD19 encoded by exon 4 of the CD19 gene.

[0107] A CD19-binding domain exemplified herein comprises variable regions with complementarity determining regions (CDRs) from an antibody referred to as CAT19,

[0108] a heavy chain variable region (VH) having CAT19 CDRs with the following sequences: CDR1 - GYAFSSS (SEQ ID NO: 1);

[0109] CDR2 - YPGDED (SEQ ID NO: 2)

[0110] CDR3 - SLLYGDYLDY (SEQ ID NO: 3); and

[0111] b) a light chain variable region (VL) having CAT 19 CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID NO: 4);CDR2 - DTSKLAS (SEQ ID NO: 5)

[0112] CDR3 - QQWNINPLT (SEQ ID NO: 6).

[0113] The CAT19 antibody is described in WO2016 / 139487.

[0114] It is contemplated that one or more mutations (substitutions, additions or deletions) can be introduced into one or more CDRs without negatively affecting CD19-binding activity. Each CDR may, for example, have one, two or three amino acid mutations.

[0115] The CDRs may be in the format of a single-chain variable fragment (scFv), which is a fusion protein of the heavy variable region (VH) and light chain variable region (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The scFv may be in the orientation VH-VL, i.e., the VH is at the amino-terminus of the CAR molecule and the VL domain is linked to the spacer and, in turn the transmembrane domain and endodomain. The CDRs may be grafted on to the framework of a human antibody or scFv. For example, the CAR may comprise a CD19-binding domain consisting or comprising one of the following sequences.

[0116] The CD19 CAR may comprise the following VH sequence.

[0117] SEQ ID NO: 7 - VH sequence from CAT19 murine monoclonal antibody QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTN YSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSS

[0118] The CD19 CAR may comprise the following VL sequence.

[0119] SEQ ID NO: 8 - VL sequence from CAT19 murine monoclonal antibody QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDR FSGSGSGTSYFLTI N N M EAEDAATYYCQQWN I N PLTFGAGTKLELKR

[0120] The CD19 CAR may comprise the following scFv sequence.

[0121] SEQ ID NO: 9 - VH-VL scFv sequence from murine monoclonal antibody QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTN YSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSG GGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPK RWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLEL KR

[0122] The CAR may consist of or comprise one of the following sequences.SEQ ID NO: 10 - CAT19 CAR using “Campana” architecture MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVK QRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARS LLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCS ASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAAT YYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR

[0123] “Campana” architecture refers to a CAR with a CD8a spacer and transmembrane domain, 4-1BB endodomain and TCR CD3z endodomain.

[0124] SEQ ID NO: 11 - CAT19 CAR with an OX40-Zeta endodomain MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVK QRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARS LLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCS ASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAAT YYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRDQRLPPDAHKPPGGGSFRTPIQEEQA DAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO: 12 - CAT19 CAR with a CD28-Zeta endodomain MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVK QRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARS LLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCS ASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAAT YYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR SEQ ID NO: 13 - Third generation CD19 CARMGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVK QRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARS LLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCS ASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAAT YYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 14 - CD19 CAR with lgG1 hinge spacer MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVK QRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARS LLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCS ASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAAT YYCQQWNINPLTFGAGTKLELKRSDPAEPKSPDKTHTCPPCPKDPKFWVLWVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 15 - CD19 CAR with hinge-CH2-CH3 of human lgG1 with FcR binding sites mutated out MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVK QRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARS LLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCS ASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAAT YYCQQWNINPLTFGAGTKLELKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDT LMIARTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMN MTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR

[0125] The CAR provided herein may comprise a variant of the polypeptide of SEQ ID NO: 1-15 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variantsequence retain the capacity to bind CD19 (when in conjunction with a complementary VL or VH domain, if appropriate).

[0126] The percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0127] The CD19 CAR exemplified herein ( / .e., the CAT19CAR using “Campana” architecture, SEQ ID NO: 10) has properties contemplated by the disclosure to result in lower toxicity and better efficacy in treated patients. When compared with an fmc63-Campana CAR, the CAT19CAR exemplified herein effected killing of target cells expressing CD19 and proliferated in response to CD19 expressing targets, but Interferon-gamma release was less. Further, a small animal model of an aggressive B-cell lymphoma showed equal efficacy and equal engraftment between the fmc63- and CAT19-based CAR-T cells, but surprisingly, less of the CAT19 CAR T-cells were exhausted than fmc63 CAR T-cells. See, Examples 2 and 3 of US Publication No.: 2018-0044417.

[0128] The CAT19CAR provided herein may cause 25, 50, 70 or 90% lower IFNy release in a comparative assay involving bringing CAR T cells into contact with target cells.

[0129] The CAT19CAR provided herein may result in a smaller proportion of CAR T cells becoming exhausted than fmc63 CAR T cells. T cell exhaustion may be assessed using methods known in the art, such as analysis of PD-1 expression. The CAR may cause 20, 30, 40, 50, 60 of 70% fewer CAR T cells to express PD-1 that fmc63 CAR T cells in a comparative assay involving bringing CAR T cells into contact with target cells.

[0130] Another exemplary CD19 antigen-binding domain contemplated by the disclosure is based on the CD19 antigen-binding domain CD19ALAb (described in WO2016 / 102965) and comprises:

[0131] a) a heavy chain variable region (VH) having CDRs with the following sequences:

[0132] CDR1 - SYWMN (SEQ ID NO: 16);

[0133] CDR2 - QIWPGDGDTNYNGKFK (SEQ ID NO: 17)

[0134] CDR3 - RETTTVGRYYYAMDY (SEQ ID NO: 18); and

[0135] b) a light chain variable region (VL) having CDRs with the following sequences:

[0136] CDR1 - KASQSVDYDGDSYLN (SEQ ID NO: 19);

[0137] CDR2 - DASNLVS (SEQ ID NO: 20)

[0138] CDR3 - QQSTEDPWT (SEQ ID NO: 21).It is contemplated that it is possible to introduce one or more mutations (substitutions, additions or deletions) into one or more CDRs without negatively affecting CD19-binding activity. Each CDR may, for example, have one, two or three amino acid mutations.

[0139] The CAR may comprise one of the following amino acid sequences.

[0140] SEQ ID NO: 22 - Murine CD19ALAb scFv sequence QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDT NYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQG TTVTVSSDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDA SNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK SEQ ID NO: 23 - Humanized CD19ALAb scFv sequence - Heavy 19, Kappa 16 QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDT NYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKG TLVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDA SNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPWTFGQGTKVEIKR SEQ ID NO: 24 (Humanized CD19ALAb scFv sequence - Heavy 19, Kappa 7) QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDT NYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKG TLVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYD ASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPWTFGQGTKVEIKR

[0141] The scFv may be in a VH-VL orientation (as shown in SEQ ID NO:s 9, 22, 23 and 24) or a VL-VH orientation.

[0142] The CAR may comprise one of the following VH sequences:

[0143] SEQ ID NO: 25 - Murine CD19ALAb VH sequence QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDT NYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQG TTVTVSS SEQ ID NO: 26 - Humanized CD19ALAb VH sequence QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDT NYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKG TLVTVSSThe CAR may comprise one of the following VL sequences:

[0144] SEQ ID NO: 27 - Murine CD19ALAb VL sequence DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGI PPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK SEQ ID NO: 28 (Humanized CD19ALAb VL sequence, Kappa 16) DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDASNLVSG VPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPWTFGQGTKVEIKR SEQ ID NO: 29 - Humanized CD19ALAb VL sequence, Kappa 7 DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYDASNLVSG VPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPWTFGQGTKVEIKR

[0145] The CAR provided herein may comprise a variant of the sequence shown as any of SEQ ID NO: 16-29 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD19 (when in conjunction with a complementary VL or VH domain, if appropriate).

[0146] The percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST which is freely available at blast.ncbi.nlm.nih.gov.

[0147] 4. Binding domains specific for CD22 target antigen

[0148] The human CD22 antigen is a molecule belonging to the SIGLEC family of lectins. It is found on the surface of mature B cells and on some immature B cells. Generally speaking, CD22 is a regulatory molecule that prevents the overactivation of the immune system and the development of autoimmune diseases.

[0149] CD22 is a sugar-binding transmembrane protein, which specifically binds sialic acid with an immunoglobulin (Ig) domain located at its N-terminus. The presence of Ig domains makes CD22 a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling.

[0150] CD22 is a molecule of the IgSF which may exist in two isoforms, one with seven domains and an intra-cytoplasmic tail comprising of three ITIMs (immune receptor tyrosine-based inhibitory motifs) and an ITAM; and a splicing variant which instead comprises of five extracellular domains and an intra-cytoplasmic tail carrying one ITIM. CD22 is thought to be an inhibitory receptor involved in the control of B-cell responses to antigen. Like CD19, CD22 is widely considered to be a pan-B antigen, although expression on some non-lymphoid tissue has been described. CD22-targeted therapeutic monoclonal antibodies and immunoconjugates have entered clinical testing.

[0151] The antigen-binding domain of the CAR which binds to CD22 may be any domain which is capable of binding CD22. For example, the antigen-binding domain may comprise a CD22 binder as described in Table 3.

[0152] Table 3

[0153]

[0154] Other anti-CD22 antibody-binding domains are known, such as the mouse anti-human CD22 antibodies 1D9-3, 3B4-13, 7G6-6, 6C4-6, 4D9-12, 5H4-9, 10C1-D9, 15G7-2, 2B12-8, 2C4-4 and 3E10-7; and the humanized anti-human CD22 antibodies LT22 and Inotuzumab (G5_44). Table 4 presents VH, VL and CDR sequences (in bold and underlined) and the position of the target epitope on CD22 for each antibody.Table 4

[0155]

[0156]

[0157]

[0158] Examples of CD22 CARs are described by Haso etal., Blood, 121(7): 1165-74 (2013). Specifically, CD22 CARs with antigen-binding domains derived from m971, HA22 and BL22 scFvs are described.

[0159] CD22 has seven extracellular IgG-like domains, which are commonly identified as Ig domain 7 to Ig domain 1, with Ig domain 1 being most proximal to the B cell membrane and Ig domain 7 being the most distal from the Ig cell membrane.The positions of the Ig domains in terms of the amino acid sequence of CD22 (UniProt Accession No. P20273, entry version 224, http: / / www.uniprot.org / uniprot / P20273) are summarized in the following Table 5:

[0160] Table 5

[0161]

[0162] The antigen-binding domain of the second CAR may bind to a membrane-distal epitope on CD22, for example, Ig domain 7. The antigen-binding domain of the second CAR may bind to an epitope on Ig domain 7, 6, 5 or 4 of CD22, for example, on Ig domain 5 of CD22. The antigen-binding domain of the second CAR may bind to an epitope located between amino acids 20-416 of CD22, for example, between amino acids 242-326 of CD22.

[0163] The antigen-binding domain of the second CAR may bind to a membrane-proximal epitope on CD22. The antigen-binding domain of the second CAR may bind to an epitope on Ig domain 3, 2 or 1 of CD22. The antigen-binding domain of the second CAR may bind to an epitope located between amino acids 419-676 of CD22, such as between 505-676 of CD22. A CD22-binding domain exemplified herein comprises variable regions with CDRs from an antibody referred to as 9A8-1 (described in WO2019 / 220109):

[0164] a heavy chain variable region (VH) having 9A8-1 CDRs with the following sequences:

[0165] CDR1 - NFAMA (SEQ ID NO: 58);

[0166] CDR2 - SISTGGGNTYYRDSVKG (SEQ ID NO: 59)

[0167] CDR3 - QRNYYDGSYDYEGYTMDA (SEQ ID NO: 60); and

[0168] b) a light chain variable region (VL) having 9A8-1 CDRs with the following sequences:

[0169] CDR1 - RSSQDIGNYLT (SEQ ID NO: 61);

[0170] CDR2 - GAIKLED (SEQ ID NO: 62)

[0171] CDR3 - LQSIQYP (SEQ ID NO: 63).

[0172] The CD22 CAR may comprise the following VH sequence.

[0173] SEQ ID NO: 64 - VH sequence from 9A8-1 antibodyEVQLVESGGGLVQPGRSLKLSCAASGFTFSNFAMAWVRQPPTKGLEWVASISTGGGNTYY RDSVKGRFTISRDDAKNTQYLQMDSLRSEDTATYYCARQRNYYDGSYDYEGYTMDAWGQ GTSVTVSS

[0174] The CD22 CAR may comprise the following VL sequence.

[0175] SEQ ID NO: 65 - VL sequence from 9A8-1 antibody DIQMTQSPSSLSASLGDRVTITCRSSQDIGNYLTWFQQKVGRSPRRMIYGAIKLEDGVPSRF SGSRSGSDYSLTISSLESEDVADYQCLQSIQYPFTFGSGTKLEIK

[0176] The CD22 CAR may comprise the following scFv sequence.

[0177] SEQ ID NO: 66 - VL-VH scFv sequence based on 9A8-1 VH and VL sequences DIQMTQSPSSLSASLGDRVTITCRSSQDIGNYLTWFQQKVGRSPRRMIYGAIKLEDGVPSRF SGSRSGSDYSLTISSLESEDVADYQCLQSIQYPFTFGSGTKLEIKRSGGGGSGGGGSGGG GSEVQLVESGGGLVQPGRSLKLSCAASGFTFSNFAMAWVRQPPTKGLEWVASISTGGGN TYYRDSVKGRFTISRDDAKNTQYLQMDSLRSEDTATYYCARQRNYYDGSYDYEGYTMDA WGQGTSVTVS

[0178] The antigen-binding domain of the 9A8-1 antibody exhibits particularly good efficacy in a CAR. For example, 9A8-1 in a FabCAR format showed improved target cell killing and cytokine release that an equivalent CAR with an alternative CD22 binder, 3B4 (WQ2019 / 220109).

[0179] An antigen-binding domain of a CAR which binds to CD22 may comprise the VH and / or VL sequence from any of the CD22 antibodies listed above, or a variant thereof which has at least 70, 80, 90 or 90% sequence identity, which variant retains the capacity to bind CD22. The antigen-binding domain of a CD22 CAR may bind CD22 with a KD in the range 30- 50nM, for example 30-40nM. The KD may be about 32nM.

[0180]

[0181] The CARs of the cell may comprise a signal peptide so that when the CAR is expressed inside a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.

[0182] The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency 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 stretchof 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.

[0183] The signal peptide may be at the amino terminus of the molecule.

[0184] The signal peptide may comprise the amino acid sequence of any of SEQ ID NO: 67-69 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR.

[0185] The signal peptide of SEQ ID NO: 67 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase. SEQ ID NO: 67 MGTSLLCWMALCLLGADHADA

[0186] The signal peptide of SEQ ID NO: 68 follows.

[0187] M ETDTLLLWVLLLLVPGSTG

[0188] The signal peptide of SEQ ID NO: 68 is derived from lgG1.

[0189] SEQ ID NO: 68: MSLPVTALLLPLALLLHAARP

[0190] The signal peptide of SEQ ID NO: 69 is derived from CD8.

[0191] SEQ ID NO: 69: MAVPTQVLGLLLLWLTDARC

[0192] The signal peptide for the first CAR may have a different sequence from the signal peptide of the second CAR.

[0193] 6. Spacers

[0194] CARs comprise a spacer to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding. The spacer may, for example, comprise an IgG 1 Fc region, an IgG 1 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.In the cells provided herein, the first and second CARs may comprise different spacer molecules. For example, the spacer may, for example, comprise an IgG 1 Fc region, an lgG1 hinge or a human CD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprise an alternative linker which has similar length and / or domain spacing properties as an lgG1 Fc region, an IgG 1 hinge or a CD8 stalk. A human lgG1 spacer may be altered to remove Fc binding motifs.

[0195] The spacer for the CD19 CAR may comprise a CD8 stalk spacer, or a spacer having a length equivalent to a CD8 stalk spacer. The spacer for the CD19 CAR may have at least 30 amino acids or at least 40 amino acids. It may have between 35-55 amino acids, for example between 40-50 amino acids. It may have about 46 amino acids.

[0196] The spacer for the CD22 CAR may comprise an I gG 1 hinge spacer, or a spacer having a length equivalent to an lgG1 hinge spacer. The spacer for the CD22 CAR may have fewer than 30 amino acids or fewer than 25 amino acids. It may have between 15-25 amino acids, for example between 18-22 amino acids. It may have about 20 amino acids.

[0197] Examples of amino acid sequences for these spacers are given below:

[0198] SEQ ID NO: 71 (hinge-CH2CH3 of human lgG1) AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD SEQ ID NO: 72 (human CD8 stalk):

[0199] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 73 (human lgG1 hinge):

[0200] AEPKSPDKTHTCPPCPKDPK SEQ ID NO: 74 (lgG1 Hinge-Fc) AEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK SEQ ID NO: 75 (lgG1 Hinge - Fc modified to remove Fc receptor recognition motifs)AEPKSPDKTHTCPPCPAPPVA*GPSVFLFPPKPKDTLMIARTPEVTCWVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK

[0201] Modified residues are underlined; * denotes a deletion.

[0202] SEQ ID NO: 76 (CD2 ectodomain) KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLF KNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNG TDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD SEQ ID NO: 77 (CD34 ectodomain) SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKF TSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATS PTKPYTSSSPI LSDI KAEI KCSGI REVKLTQGICLEQN KTSSCAEFKKDRGEGLARVLCGEEQ ADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVA SHQSYSQKT

[0203] Since CARs are typically homodimers (see Figure 1A), cross-pairing may result in a heterodimeric chimeric antigen receptor. This is undesirable for various reasons, for example: (1) the epitope may not be at the same "level" on the target cell so that a crosspaired CAR may only be able to bind to one antigen; (2) the VH and VL from the two different scFv could swap over and either fail to recognize target or worse recognize an unexpected and unpredicted antigen. The spacer of the first CAR may be sufficiently different from the spacer of the second CAR in order to avoid cross-pairing. The amino acid sequence of the first spacer may share less that 50%, 40%, 30% or 20% identity at the amino acid level with the second spacer.

[0204] 7. Transmembrane domains

[0205] The transmembrane domain is the domain of the CAR that spans the membrane.

[0206] A transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion provided herein. The presence and span of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs. dtu.dk / services / TMHMM-2.0 / ). Further, given that thetransmembrane domain of a protein is a relatively simple structure, / .e, a polypeptide predicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed transmembrane domain may also be used (US 7052906 B1 describes synthetic transmembrane components).

[0207] The transmembrane domain may be derived from CD28, which gives good receptor stability. The transmembrane domain may be derived from human Tyrp-1. The tyrp-1 transmembrane domain sequence is shown as SEQ ID NO: 78.

[0208] SEQ ID NO: 78 IIAIAVVGALLLVALIFGTASYLI

[0209] The transmembrane domain may be derived from CD8A. The CD8A transmembrane domain sequence is shown as SEQ ID NO: 79.

[0210] SEQ ID NO: 79 IYIWAPLAGTCGVLLLSLVITLYC

[0211] 8. Endodomains

[0212] As noted above, the endodomain is the signal-transmission portion of the CAR. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta which contains three 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 I survival signal, or all three can be used together.

[0213] The cells provided herein comprise two CARs, each with an endodomain.

[0214] The endodomain of the first CAR and the endodomain of the second CAR may comprise: (i) an ITAM-containing endodomain, such as the endodomain from CD3 zeta; and / or (ii) a costimulatory 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 4-1BB.

[0215] Thus, the endodomain of the CAR of the present invention may comprise combinations of one or more of the CD3-Zeta endodomain, the 41 BB endodomain, the 0X40 endodomain or the CD28 endodomain.The intracellular T-cell signalling domain (endodomain) of the CAR of the present invention may comprise the sequence shown as any of SEQ ID NO: 80-87 or a variant thereof having at least 80% sequence identity.

[0216] SEQ ID NO: 80 (CD3 zeta endodomain) RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 81 (41 BB endodomain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 82 (0X40 endodomain) RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO: 83 (CD28 endodomain) KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY

[0217] Examples of combinations of such endodomains include 41 BB-Zeta, OX40-Zeta, CD28-Zeta and CD28-OX40-Zeta.

[0218] SEQ ID NO: 84 (41 BB-Zeta endodomain fusion) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 85 (OX40-Zeta endodomain fusion) RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 86 (CD28Zeta endodomain fusion) KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 87 (CD28OXZeta)KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRT PIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR

[0219] A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any of SEQ ID NO: 80-87 provided that the sequence provides an effective transmembrane domain / intracellular T cell signaling domain.

[0220] 9. Nucleic acids

[0221] One or more nucleic acid(s) provided herein encode a CD19 CAR and a CD22 CAR of the disclosure. As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.

[0222] The nucleic acid may be, for example, an RNA, a DNA or a cDNA. Nucleic acids may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

[0223] Due to the degeneracy of the genetic code, it is possible to use alternative codons which encode the same amino acid sequence. For example, the codons “ccg” and “cca” both encode the amino acid proline, so using “ccg” may be exchanged for “cca” without affecting the amino acid in this position in the sequence of the translated protein.

[0224] The alternative RNA codons which may be used to encode each amino acid are summarized in Table 6.

[0225] Table 6

[0226] " >

[0227]

[0228] Alternative codons may be used in the portions of nucleic acid which encode the spacer of the first CAR and the spacer of the second CAR, especially if the same or similar spacers are used in the first and second CARs. Figure 4 shows two sequences encoding the spacer HCH2CH3 - hinge, in one of which alternative codons have been used.

[0229] Alternative codons may be used in the portions of nucleic acid which encode the transmembrane domain of the first CAR and the transmembrane of the second CAR, especially if the same or similar transmembrane domains are used in the first and second CARs.

[0230] Alternative codons may be used in one or more nucleic acids which encode co-stimulatory domains, such as the CD28 endodomain.

[0231] Alternative codons may be used in one or more domains which transmit survival signals, such as 0X40 and 41 BB endodomains.Alternative codons may be used in the portions of nucleic acid encoding a CD3zeta endodomain and / or the portions of nucleic acid encoding one or more costimulatory domain(s) and / or the portions of nucleic acid encoding one or more domain(s) which transmit survival signals.

[0232] 10. Vectors

[0233] The present disclosure also provides a vector, or kit of vectors which comprises one or more CAR-encoding nucleic acid(s). Such a vector may be used to introduce the nucleic acid(s) into a host cell so that it expresses the one of the CD19 CAR or the CD22 CAR.

[0234] The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.

[0235] The vector may be capable of transfecting or transducing a T cell.

[0236] 11. Cells

[0237] Cells are provided herein which express a CD19 CAR, such that the cells recognise a target cell expressing CD19. Cells are also provided herein which express a CD22 CAR, such that the cells recognise a target cell expressing CD22.

[0238] Populations of cells which comprise cells which express the CD19 CAR and cells which express the CD22 CAR are also provided.

[0239] In double transduction, which results of a population of dual transduced CAR T cells as well as cells transduced with only one of the vectors, T cells expressing the two CARs seem to be more susceptible to activation induced cell death after repetitive antigenic exposure (AICD) compared to the T cells expressing one CAR. Co-infusion of separately transduced CAR-T cells has the advantage of preventing AICD while ensuring that a fixed cell dose of CD19CAR and CD22CAR T cells are infused without the heterogeneity introduced by having single and double-positive populations.

[0240] The cell may be any eukaryotic cell capable of expressing a CAR at the cell surface, such as an immunological cell.

[0241] In particular, the cell may be an immune effector cell such as a T cell or a natural killer (NK) cell.

[0242] 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 naturalkiller cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarized below.

[0243] 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, orTFH, which secrete different cytokines to facilitate different types of immune responses.

[0244] Cytotoxic 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.

[0245] 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 reexposure 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.

[0246] 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.

[0247] Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive Treg cells.

[0248] 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 anintracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.

[0249] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.

[0250] The T cell provided herein may be any of the T cell types mentioned above, in particular a CTL.

[0251] Natural killer (NK) cells are a type of cytolytic cell which forms part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner

[0252] 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.

[0253] The CAR-expressing cells provided herein may be any of the cell types mentioned above. CAR-expressing cells, such as CAR-expressing T or NK 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).

[0254] The present disclosure also provides a cell population comprising T and / or NK cells expressing a CD19 CAR and T and / or NK cells expressing a CD22 CAR. The cell population may be made by transducing ex vivo a blood sample with a nucleic acid encoding a CD19 CAR according to the present disclosure and another blood sample with a nucleic acid encoding a CD22 CAR according to the present disclosure.

[0255] The term “CD19+22 CAR T-cells“ refers herein to a cell population comprising a mixture of separately transduced cells, i.e. cells expressing a CD19 CAR alone and cells expressing a CD22 CAR alone. The cell population may be pooled at a 1 :1 ratio prior to cryopreservation. Alternatively, T or NK cells provided herein may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to 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 CAR cells are generated by introducing DNA or RNA coding for the CARs by one of many means including, but not limited to, transduction with a viral vector, transfection with DNA or RNA. Cells may be activated and / or expanded prior to being transduced with CAR-encoding nucleic acid, for example by treatment with an anti-CD3 monoclonal antibody. The T or NK cells provided herein may be made by: (i) isolation of a T or NK cell-containing sample from a subject or other sources listed above, and (ii) transduction or transfection of the T or NK cells with one nucleic acid encoding the CD19 or CD22 CARs.

[0256] The T or NK cells may then by purified, for example, selected on the basis of expression of the antigen-binding domain of the antigen-binding polypeptide.

[0257] 12. Pharmaceutical compositions

[0258] The present disclosure also relates to a pharmaceutical composition containing a plurality of CAR-expressing cells, such as T cells or NK cells provided herein. Pharmaceutical compositions comprising the CD19 / 22 CAR T-cell product described in Example 1 are provided. 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.

[0259] 13. Methods of treatment

[0260] The cell populations of the present disclosure, for example the CD19+CD22 CAR T-cell population described in Example 1, are capable of killing cancer cells recognizable by expression of CD19 or CD22, such as B-cell lymphoma cells. CAR-expressing cells, such as T 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, CAR T-cells may be derived from ex-vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T-cells. In these instances, CAR T-cells are generated by introducing DNA or RNA coding for the CAR by one of many means including transduction with a viral vector, transfection with DNA or RNA.

[0261] Examples of cancers which express CD19 or CD22 are B-cell lymphomas, including Hodgkin's lymphoma and non-Hodgkins lymphoma; and B-cell leukaemias.

[0262] For example the B-cell lymphoma may be Diffuse large B cell lymphoma (DLBCL), Follicular lymphoma, Marginal zone lymphoma (MZL) or Mucosa-Associated Lymphatic Tissue lymphoma (MALT), Small cell lymphocytic lymphoma (overlaps with Chronic lymphocyticleukemia), Mantle cell lymphoma (MCL), Burkitt lymphoma, Primary mediastinal (thymic) large B-cell lymphoma, Lymphoplasmacytic lymphoma (may manifest as Waldenstrom macroglobulinemia), Nodal marginal zone B cell lymphoma (NMZL), Splenic marginal zone lymphoma (SMZL), Intravascular large B-cell lymphoma, Primary effusion lymphoma, Lymphomatoid granulomatosis, T cell / histiocyte-rich large B-cell lymphoma or Primary central nervous system lymphoma.

[0263] The B-cell leukaemia may be acute lymphoblastic leukaemia, B-cell chronic lymphocytic leukaemia, B-cell prolymphocytic leukaemia, precursor B lymphoblastic leukaemia or hairy cell leukaemia.

[0264] The B-cell leukaemia may be acute lymphoblastic leukaemia (B-ALL or ALL).

[0265] The B-ALL may be pediatric ALL (pALL).

[0266] Standard treatment for relapsed pALL includes the following treatment phases: Induction, Consolidation, Interim Maintenance, Delayed Intensification, and Maintenance.

[0267] Patients are stratified according to risk levels. Several criteria are available to stratify patients, for example the National Cancer Institute (NCI) criteria, which differentiates patients between NCI Standard Risk and NCI High Risk: NCI Standard Risk patients are patients aged >1year and <10 years old at diagnosis and with a highest white cell count (WCC) before starting treatment of <50x10A9 / L; NCI High Risk patients are patients aged >10 years old at diagnosis, and / or with a diagnostic WCC of >50x10A9 / L.

[0268] There are national guidelines describing the standard treatment regimen. An example of these guidelines is the UKALL 2019 Interim Guidelines, which is the guideline for the management of ALL in children and young adults used in the UK (also referred to as UKALL 2019 Guidelines). Induction therapy according to the UKALL 2019 Guidelines is as follows NCI Standard Risk: Patients in this group receive a 3-drug (dexamethasone, vincristine and asparaginase) induction (Regimen A Induction, Table 7).

[0269] NCI High Risk: Patients in this group receive a 4-drug (dexamethasone, vincristine, asparaginase and daunorubicin) induction (Regimen B Induction, Table 8).

[0270] Regimen C Induction (Table 9): Patients with NCI Standard Risk BCP ALL who are subsequently found to have high risk cytogenetics, or Down syndrome patients with a slow early response.Table 7: Regimen A Induction

[0271] <

[0272] < >

[0273] > >

[0274]

[0275]

[0276]

[0277]

[0278]

[0279] Table 8: Regimen B Induction

[0280] <

[0281] >

[0282]

[0283] < >

[0284] > >

[0285]

[0286] Table 9: Regimen C Induction

[0287] >

[0288] < >

[0289] > >

[0290]

[0291]

[0292] Details of the treatment regiments of the Consolidation, Interim Maintenance, Delayed Intensification, and Maintenance phases can be found on the LIKALL 2019 Guidelines (docplayer.net / 170103468-Clcn-ukall-2019-interim-guidelines.html, which are included herein by reference).

[0293] Treatment with the T cells provided herein is contemplated to help prevent the escape or release of tumour cells which often occurs with standard care approaches.

[0294] The methods provided herein slow or prevent progression of the cancer, diminish the extent of the cancer, result in remission (partial or total) of the cancer, and / or prolong survival of the patient.

[0295] In the provided methods, the patient treated has a high risk / relapsed or resistant CD19+ or CD22+ haematological malignancy.

[0296] Where the CD19+ or CD22+ haematological malignancy is pALL, there are several parameters that may be used to define high risk / relapsed or resistant pALL:

[0297] a. Resistant disease;

[0298] b. ALL with persisting disease high level MRD at second time point of frontline national protocol;

[0299] c. High risk infant;

[0300] d. Any patient with t(17,19) TCF3-HLF rearrangement;

[0301] e. High risk first relapse, early relapses, and any relapse with high risk genetics; f. Any patient aged 16 to 24 on therapy relapse;

[0302] g. Any relapse of infant ALL;

[0303] h. ALL post > 2nd relapse;i. Any refractory relapse of ALL;

[0304] j. ALL with MRD >10'4prior to planned stem cell transplant

[0305] k. Any relapse of ALL eligible for stem cell transplant (SCT) but with no available HLA matched donor or other contraindication to transplant;

[0306] l. Any relapse of ALL after SCT as long as planned time of infusion of anti- CD19 CAR-T cells and anti-CD22 CAR T cells is longer than 4 months posttransplant; or

[0307] m. Early loss of B cell aplasia or any CD19+CD22+ relapse following CD19CAR T cell therapy with Tisagenlecleucel.

[0308] The patient may have an isolated CNS relapse meeting one or more of a) - m).

[0309] Resistant disease is defined as the presence of >5% blasts at the end of the induction phase at end of ALLTogether-1 protocol (NCT03911128) or equivalent induction . This defines the primary refractory population.

[0310] ALL with persisting high level minimal residual disease (MRD) at 2nd time point of frontline national protocol is defined as MRD >10'4at week 9 ALLTogether-1 Protocol or equivalent. High risk infant ALL is defined as an infant of age < 6 months at diagnosis with MLL gene rearrangement and either presenting white cell count > 300 x 109 / L or poor steroid early response. Poor steroid early response is defined as the presence of circulating blast count >1x109 / L following 7 day steroid pre-phase of induction as per national guidelines or equivalent.

[0311] High risk first relapse is defined as very early relapse, i.e. within 18 months of diagnosis. Early relapse is defined as any patient relapsing on therapy or within 6 months of completing treatment.

[0312] High risk genetics is defined as (KMT2A (MLL) rearrangements, low hypodiploidy / near haploidy, t(17;19)(q22;p13) / TCF3-HLF, iAMP21 ort(1;19)(q21;p13) / TCF3- PBX1, t(9;22)(34.1 q11.2) / BCR-ABL1.

[0313] Any refractory relapse of ALL is defined as the presence of > 1% blasts by flow cytometry after at least one cycle of standard chemotherapy.

[0314] Early loss of B cell aplasia is defined as < 6 months post-infusion.

[0315] The patient may be administered a cell population comprising anti-CD19 CAR T-cells (CD19 CAR-T cells) and anti-CD22 CAR-T cells (CD22 CAR-T cells).The CD19 CARs and CD22 CARs have been described in detail previously.

[0316] The cell population may comprise a 1:1 ratio of CD19 CAR-T cells and CD22 CAR-T cells. The patient may be administered a total dose of:

[0317] 4 x105CD19 and CD CAR T-cells / kg, composed of 2 x105CD19 CAR T-cells / kg and 2 x105CD22 CAR T-cells / kg;

[0318] 5x105CD19 and CD22 CAR T-cells / kg, composed of 2.5x105CD19 CAR T-cells / kg and 2.5 x105CD22 CAR T-cells / kg;

[0319] 8x105CD19 and CD22 CAR T-cells / kg, composed of 4x105CD19 CAR T-cells / kg and 4 x105CD22 CAR T-cells / kg;

[0320] 1 x106CD19 and CD22 CAR T-cells / kg, composed of 5 x105CD19 CAR T-cells / kg and 5 x105CD22 CAR T-cells / kg;

[0321] - 1.5 x 106CD19 and CD22 CAR T-cells / kg, composed of 7.5 x105CD19 CAR T- cells / kg and 7.5 x105CD22 CAR T-cells / kg; or

[0322] 2 x 106CD19 and CD22 CAR T-cells / kg composed of 1 x106CD19 CAR T-cells / kg and 1 x106CD22 CAR T-cells / kg.

[0323] The method of treating the high risk / relapsed CD19+ or CD22+ haematological malignancy in the patient may comprise administering a first dose and a second dose of the cell population comprising CD19 CAR T-cells and CD22 CAR-T cells, wherein the second dose is administered between about 12 days and about 21 days after the administration of the first dose.

[0324] The second dose may be preferably administered about 14 days after the administration of the first dose.

[0325] Alternatively, the method of treating the high risk / relapsed CD19+ or CD22+ haematological malignancy in the patient may comprise administering a single dose of the cell population comprising CD19 CAR T-cells and CD22 CAR-T cells to the patient.

[0326] The method of treating the high risk / relapsed CD19+ or CD22+ haematological malignancy in the patient may comprise administering a single dose of the cell population comprising 4 x 105CD19 and CD CAR T-cells / kg, which may comprise 2 x 105CD19 CAR T-cells / kg and 2 x 105CD22 CAR T-cells / kg, to the patient.The patient may receive lymphodepletion comprising fludarabine, cyclophosphamide and low dose total body irradiation prior to receiving the cell population comprising CD19 CAR T-cells and CD22 CAR-T cells.

[0327] The lymphodepletion may comprise:

[0328] a) fludarabine 30 mg / m2once daily on day -6 to -3;

[0329] b) cyclophosphamide 0.5 g / m2once daily on day -6 to -5; and

[0330] c) low dose Total Body Irradiation 2Gy on day -2.

[0331] The administration of the cell population may be an intravenous injection through a Hickman line or peripherally inserted central catheter (PICC line).

[0332] Other terminoloav and disclosure

[0333] 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.

[0334] 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.

[0335] 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.

[0336] 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 theother 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.

[0337] 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.

[0338] EXAMPLES

[0339] While the following examples describe specific embodiments, variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.

[0340] Example 1 : Generation of a cell population comprising CD19 CAR T cells and CD22 CAR T cells

[0341] Lentiviral vectors were generated expressing either a) a second-generation CD19 CAR (SEQ ID NO: 89) (CD19CAT CAR described in W02016 / 139487, otherwise referred to herein as CAT CAR or AUTO1) which comprises an anti-CD19 antigen-binding domain, a CD8 stalk spacer and transmembrane domain, and a compound 4-1BB-CD3 endodomain, under the control of a PGK promoter (pCCL.PGK.aCD19cat-CD8STK-41BBZ); or b) a CD22 CAR (SEQ ID NO: 91) (9A8-1 based CAR described in WO2019 / 220109) (9A8 CAR) which comprises an anti-CD22 antigen-binding domain, a CD8 stalk spacer and a second generation endodomain comprising CD3 and a 4-1 BB costimulatory domain, under the control of an EF1a promoter (pCCL.EF1a.aCD22_9A8-1-64_LH_scFv-CD8STK-41BBz). See Figure 1A-B.

[0342] To characterise the expression of both CAT and 9A8 CARs in transduced T cells, patient-derived T cells were separately transduced with either CAT CAR lentiviral vector or 9A8 CAR lentiviral vector at an MOI of 1.8 - 2.3 and following an abbreviated manufacture (total 5 days) were pooled 1:1 prior to cryopreservation. CD19+CD22CAR T cells were stained before cryopreservation with anti-CAT idiotype (to detect CD19 CAR) and anti-9A8 idiotype (to detect CD22 CAR). As shown in Figure 2, lower panel, CD19+CD22CAR T cells showed very similar levels of expression of CATCAR and 9A8CAR on separate populations of cells in 3 different patients. The total transduction efficiency (mean 80.2%, range 71.2-86.4%) was similar to that observed using the co-transduction methodology previously used in cohort 3on the same donors (mean 84.9%, range 80.7-92.6%) though because of separate transduction no significant population of dual CAR+ve cells was seen.

[0343] The resulting mixed population is referred to as “CD 19+22 CAR T-cell product,” “CD19CAT+CD229A8 CAR T-cell product” or “cell population comprising CD19 CAR T cells and CD22 cells” herein.

[0344] These experiments demonstrate that it was possible to generate a mixed population of T-cells which include T cells which are single positive for each CAR with similar levels of expression of both CARs.

[0345] Example 2: Pre-clinical assessment

[0346] In vitro functionality of CD19+CD22C AR T cells

[0347] The in vitro functionality of CD19+CD22CAR T cells generated using separate transduction and abbreviated manufacture was compared with CD19 / 22CAR co-transduced T cells from the same donors manufactured using the previous co-transduction methodology used in cohort 3. Cryopreserved CD19 / 22 CAR T cells dose generated using the 2 different methodologies were tested in cytotoxicity, proliferation and cytokine secretion when cultured with CD19 / 22 expressing cell lines (Nalm6, Raji). As shown in Figure 3, CD19+22 CAR T cells generated using separate transduction and abbreviated manufacture killed CD19+CD22+ Nalm6 and Raji cells highly effectively but not CD19-CD22- SLIPT1 cells. Moreover, CD19+CD22CAR T cells showed high level, specific proliferation and IFN-y secretion in response to CD19+CD22+ targets but not CD19-CD22- cells. Of note, CD19+CD22CAR T cells generated using separate transduction and abbreviated manufacture showed equivalent cytotoxicity, proliferation and cytokine secretion in response to co-transduced CD19 / CD22 expressing targets to CAR T cells from the same donors generated using the previous cohort 3 methodology.

[0348] In vivo functionality of CD19+CD22CAR T cells

[0349] The activity of CD19+CD22 CAR T cells generated by the two manufacturing methods was compared in vivo using an NSG-NALM6 xenogeneic mouse of ALL (Figure 4A).

[0350] GFP+ / Luciferase+NALM6 cells were injected into NSG mice one day before treatment with PBS or CD19+CD22 CAR T cells from patient CPL-27. PBS treated mice rapidly developed leukaemia and were all culled by day 22 (Figure 4B and 4E) with GFP+ / Luc+NALM6 cells detected in the blood, bone marrow, spleen and liver (Figure 4C). Leukaemia development was transiently delayed in mice treated with CD19 / 22 CAR T cells generated using the cohort 3 methodology, which correlated with prolonged survival compared to PBS treatedmice (Figure 4B and 4E). Most animals in this cohort showed increasing leukaemic burden by day 28, correlating with detection of GFP+ / Luc+NALM6 cells in the blood and liver with no / low levels of CAR T cells in the tissues analysed and 40% of mice were alive at day 40. In contrast, mice treated with CD19+CD22 CAR T cells generated from the same donor using the methodology to be used in cohort 4 showed better control of leukaemia burden, with low bioluminescence signals throughout, which correlated with undtectable levels of GFP+leukaemic cells and better persistence of of CAR T cells in the tissues analysed, in a sample of animals analysed on day 28 (Figure 4B-4E). All Cohort 4 CD19+CD22 CAR T cell treated mice were alive at day 40. These data show that Cohort 4 CD19+CD22 CAR T cells function better than Cohort 3 CD19+CD22 CAR T cells and appear to have better persistence in vivo.

[0351]

[0352] Full scale manufacturing also produced a therapeutic CD19+CD22 CAR T-cell population. The manufacturing process is described in Figure 5. Briefly, CD4 / 8+ T cells from autologous patient leukapheresate were collected by positive selection using the CliniMACS Prodigy®. Enriched CD4 / 8+ T cells were harvested from the Prodigy and cultured overnight in X-VIVO 15 medium (5% human AB serum) with CTS™ Dynabeads (anti-CD3 / CD28 coated beads) in G-rex flasks. The activated cells were transduced separately in G-rex flasks with lentiviral vectors encoding either CD19 CATCAR (pCCL.PGK.aCD19CAT-41BBz) or CD229A8CAR (pCCL.EF1.aCD229A8-41BBz) for 3 days. Following transduction, Dynabeads were magnetically removed and the T cells were combined at a 1:1 ratio (CD19:CD22 CAR+ T cells) prior to cryopreservation. The final product was cryopreserved in bags or vials in CryoStor® CS10 (cryopreservation medium containing 10% DMSO).

[0353] Validation of the manufacture procedure was completed by undertaking 3 scale-up manufacture runs. All runs used cryopreserved leucapheresis products from patients with Acute Lymphoblastic Leukaemia (ALL) as starting material. All runs met the release criteria proposed for cohort 4 of the CARPALL study. In each, the product was sterile with no Mycoplasma or endotoxin detectable and a cell dose adequate for that proposed in cohort 4 of the study was achieved.

[0354] The release criteria for the CD19CAT41BBZ + CD229A8-41BBZ CAR T cells in CARPALL cohort 4 are:

[0355] a) Bacterial I Fungal sterility

[0356] b) Absence of Mycoplasma (PCR)

[0357] c) Absence of endotoxin (< 2EU / ml in Limulus Amoebocyte Lysate assay)d) Viability of >70% (flow cytometry)

[0358] e) Transduction efficiency: >7.5% CD19CAT CAR+ cells and >7.5% CD229A8 CAR+ cells

[0359] f) Cell dose:

[0360] Dose level 1: 2 doses of 4x105CAR+ T-cells / kg

[0361] Dose level 2: 2 doses of 1x106CD19+CD22CAR+ T-cells / kg

[0362] g) < 30 activating beads / 106cells

[0363] In addition, the following assays may have been performed, though they do not constitute release criteria: flow cytometry to determine the immunophenotype and the percentage of non-T cell immune sub-sets in the ATIMP, viral copy number assessment by qPCR.

[0364] In addition, the memory phenotype of the products from each scale up run using the cohort 4 methodology was compared with the corresponding Cohort 3 CAR T cell products generated from the same patient starting material. These data demonstrate a 14-fold increase in the proportion of TSCM (CCR7+, CD45RA+, CD95+) in cohort 4 products when compared with cohort 3 (Figure 6).

[0365] Example 4: Clinical study

[0366] A study of the safety, efficacy and duration of response to the CD19+CD22 CAR T-cell population was initiated in children and young adults with high risk, relapsed CD19+ and / or CD22+ haematological malignancies (Acute Lymphoblastic Leukemia). This is Cohort 4 of a multi-centre, non-randomised, open label Phase I clinical trial.

[0367] The study design for Cohort 4 is summarized in the following Table 10.

[0368] Table 10

[0369]

[0370] >

[0371] >

[0372]

[0373] The primary outcome measures for the study are as follows:

[0374] a) Incidence of dose limiting toxicity following CD19+CD22 CAR T-cell infusion

[0375] b) Proportion of patients achieving molecular remission at D28 post-CD19+CD22 CAR T- cell infusion.

[0376] Toxicity evaluation following CD19+CD22CAR T-cell infusion: The incidence of grade 3-5 toxicity occurring within 60 days of CD19+CD22CAR T-cell infusion. In particular, the incidence of Severe Cytokine Release Syndrome and Grade 3-5 neurotoxicity occurring within 30 days of CD19+CD22CAR T-cell infusion. [Time Frame: 1 month]

[0377] Molecular remission: Efficacy will be assessed by determining Minimal Residual Disease in the bone marrow aspirate using immunoglobulin heavy chain (IgH) quantitative polymerase chain reaction (qPCR) and / or Next Generation Sequencing in all patients. The proportion of patients achieving molecular remission at 1 month post CD19 / 22CAR T-cell infusion will be determined. [Time Frame: 1 month]

[0378] The secondary outcome measures for the study are as follows.

[0379] 1. Proportion of patients achieving molecular remission (IgH qPCR / NGS) and / or flow MRD negativity (flow cytometry) at 3 months post CAR T-cell infusion2. Proportion of patients in molecular remission and / or are flow MRD negative without further therapy at 1 and 2 years

[0380] 3. Duration of response

[0381] 4. Safety and tolerability of CAR T

[0382] 5. Incidence and duration of B cell aplasia and hypogammaglobulinaemia

[0383] 6. Feasibility of generation of CAR T cells

[0384] 7. Changes in the MRD levels at each time point post CAR T cell infusion as compared to timepoint pre-CAR T cell infusion

[0385] 8. Persistence and frequency of circulating CAR transduced T-cells in the peripheral blood of recipients after adoptive transfer as assessed by flow cytometry and qPCR 9. Time to relapse, Time to antigen negative relapse, Time to treatment failure, Event- Free Survival and Overall Survival after immunotherapy with CAR transduced T-cells. The inclusion criteria for the study are as follows.

[0386] a) Children and young adults (age 24 years or younger) with high risk / relapsed CD19+ and CD22+ acute lymphoblastic leukaemia:

[0387] b) Resistant disease (>5% blasts) at end of Altogether- 1 protocol or equivalent induction

[0388] c) ALL with persistent high level MRD at 2ndtime point of frontline national protocol (currently >10-4at week 9 ALLTogether-1 protocol or equivalent)

[0389] d) High risk infant ALL (age < 6 months at diagnosis with MLL gene rearrangement and either presenting white cell count > 300 x 109 / L or poor steroid early response (i.e. circulating blast count >1x109 / L following 7 day steroid pre-phase induction as per national guidelines or equivalent)

[0390] e) Any patient with t(17, 19) TCF3-HLF rearrangement

[0391] f) High risk 1strelapse (defined as very early (relapse within 18 months of diagnosis) and early relapses (any patient relapsing on therapy or within 6 months of completing treatment) and any relapse with high risk genetics, namely (KMT2A (MLL) rearrangements, low hypodiploidy / near haploidy, t(17;19)(q22;p13) / TCF3-HLF, iAMP21 and t(1;19)(q21;p13) / TCF3- PBX1, t(9;22)(34.1 q11.2) / BCR-ABL1 g) Any on therapy relapse of ALL in patients age 16-24

[0392] h) Any relapse of infant ALL

[0393] i) ALL post > 2ndrelapse

[0394] j) Any refractory relapse of ALL (defined as > 1% blasts by flow cytometry after a at least 1 cycle of standard chemotherapy)

[0395] k) ALL with MRD >10'4prior to planned stem cell transplantl) Any relapse of ALL eligible for stem cell transplant but no available HLA matched donor or other contraindication to transplant

[0396] m) Any relapse of ALL after stem cell transplant as long as planned time of CD19+CD22CAR T cell infusion is > 4 months post-transplant

[0397] n) Early (defined as < 6 months post-infusion) loss of B cell aplasia or any CD19+CD22+relapse following CD19CAR T cell therapy with Tisagenlecleucel

[0398] Note: patients with isolated CNS relapse meeting one or more of the criteria above are eligible for the study

[0399] Exclusion Criteria for registration are as follows.

[0400] 1. Active hepatitis B, C or HIV infection

[0401] 2. Oxygen saturation < 90% on air

[0402] 3. Bilirubin > 3 x upper limit of normal

[0403] 4. Creatinine > 3 x upper limit of normal

[0404] 5. Women who are pregnant or breastfeeding

[0405] 6. Stem Cell Transplant patients only: active significant acute GVHD (overall Grade > II, Seattle criteria) or moderate / severe chronic GVHD (NIH consensus criteria) requiring systemic steroids

[0406] 7. Inability to tolerate leucapheresis

[0407] 8. Karnofsky (age > 10 years) or Lansky (age < 10) score < 50%

[0408] 9. Pre-existing significant neurological disorder (other than CNS involvement of underlying haematological malignancy)

[0409] 10. CD19 negative or CD22 negative disease

[0410] Exclusion criteria for CD19+CD22 CAR T-cell infusion are as follows:

[0411] 1. Severe intercurrent infection at the time of scheduled CD19+CD22 CAR T-cell infusion

[0412] 2. Requirement for supplementary oxygen or active pulmonary infiltrates at the time of scheduled CD19+CD22 CAR T-cell infusion

[0413] 3. Allogeneic transplant recipients with active significant acute GVHD overall grade >2 or moderate / severe chronic GVHD requiring systemic steroids at the time of scheduled CD19+CD22 CAR T-cell infusion

[0414] In addition, for cohort 4 patients will not be eligible for the 2nd CAR T cells infusion on D14 if they have developed CRS>Gr2 or ICANS>Gr2 following the dose on DO.

[0415] The study design is a multi-center, non-randomized, open label phase I clinical trial of an Advanced Therapy Investigational Medicinal Product (ATIMP) in children and young adultswith high risk, relapsed CD19+ and / or CD22+ hematological malignancies (B-ALL). The ATIMP tested in cohort 1 and 2 of this study is CD19CAT-41BB CAR T-cells (referred to as CD19CAR T-cells). The ATIMP tested in cohort 3 is CD19CAT-CD229A8-41BBZ CAR T-cells. The ATIMP tested in cohort 4 of this study is a population of CD19CAT CAR T-cells and CD229A8 CAR-T cells (as described in the Examples above). A total of 45 patients will be treated:

[0416] 14 patients in cohort 1 (manual manufacture with CD19 CAR transduction) - completed

[0417] 7 patients in cohort 2 (Prodigy manufacture with CD19 CAR transduction) - completed

[0418] 12 patients in cohort 3 (manual manufacture with dual CD19 and CD22 CAR cotransduction) - completed

[0419] up to 12 patients will be treated in cohort 4 (abbreviated manufacture with separate CD19 and CD22 transduction, optimised lymphodepletion with therapeutic drug monitoring of fludarabine levels and low dose TBI, split dosing)

[0420] The trial schema is depicted in Figure 7.

[0421] Patients were / will be followed up regularly (with daily, weekly and monthly visits) until two years post-CD19 / 22CAR T-cell infusion. After two years, patients will continue to be followed up annually for up to 15 years post ATIMP infusion.

[0422] Example 5: Clinical study. Dose level

[0423] The study design for Cohort 4 was revised as shown in the following Table 11 and Figure 12. Table 11

[0424]

[0425]

[0426] This application claims the benefit of United Kingdom application No. 2501677.5 filed on 5 February 2025. This application is incorporated herein by reference in its entirety.

[0427] All publications mentioned in the above specification are herein incorporated by reference. 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

CLAIMS1. A cell population comprising anti-CD19 CAR T-cells and anti-CD22 CAR-T cells for use in a method of treating a high risk / relapsed CD19+ or CD22+ haematological malignancy in a patient.

2. Use of a cell population comprising anti-CD19 CAR T-cells and anti-CD22 CAR-T cells for the manufacture of a medicament to use in a method of treating a high risk / relapsed CD19+ or CD22+ haematological malignancy in a patient.

3. A method of treating a high risk / relapsed CD19+ or CD22+ haematological malignancy in a patient comprising administering a cell population comprising anti-CD19 CAR T- cells and anti-CD22 CAR-T cells to the patient.

4. The cell population for use according to claim 1 , or the use according to claim 2, or the method according to claim 3, wherein the age of the patient is twenty-four years or younger.

5. The cell population for use according to claim 1 or claim 4, or the use according to claim 2 or claim 4, or the method according to claim 3 or claim 4, wherein the haematological malignancy is acute lymphoblastic leukemia (ALL), or a CD19+ and / or CD22+ lymphoma.

6. The cell population for use according to any of claims 1 , 4 or 5, or the use according to any of claims 2, 4 or 5, or the method according to any of claims 3-5, wherein the lymphoma is Burkitt’s lymphoma.

7. The cell population for use according to any of claims 1 or 4-6, or the use according to any of claims 2 or 4-6, or the method according to any of claims 3-6, wherein the patient has:a. Resistant disease (>5% blasts) at end of ALLTogether-1 protocol (NCT03911128) or equivalent induction;b. ALL with persisting high level MRD at second time point of frontline national protocol (currently MRD >10-4at week 9 ALLTogether-1 Protocol or equivalent); c. High risk infant ALL (age < 6 months at diagnosis with MLL gene rearrangement and either presenting white cell count > 300 x 109 / L or poor steroid early response (i.e. circulating blast count >1x109 / L following 7 day steroid pre-phase of induction as per national guidelines or equivalent);d. Any patient with t(17, 19) TCF3-HLF rearrangement;e. High risk first relapse (defined as very early (relapse within 18 months of diagnosis) and early relapses (any patient relapsing on therapy or within 6 months of completing treatment) and any relapse with high risk genetics, namely (KMT2A (MLL) rearrangements, low hypodiploidy / near haploidy, t(17;19)(q22;p13) / TCF3- HLF, iAMP21 and t(1;19)(q21;p13) / TCF3- PBX1, t(9;22)(34.1 q11.2) / BCR-ABL1); f. Any patient aged 16 to 24 on therapy relapse;g. Any relapse of infant ALL;h. ALL post > 2nd relapse;i. Any refractory relapse of ALL (defined as > 1 % blasts by flow cytometry after at least one cycle of standard chemotherapy)j. ALL with MRD >10-4 prior to planned stem cell transplantk. Any relapse of ALL eligible for stem cell transplant (SCT) but with no available HLA matched donor or other contraindication to transplant;l. Any relapse of ALL after SCT as long as planned time of infusion of anti-CD19 CAR-T cells and anti-CD22 CAR T cells is longer than 4 months post-transplant; orm. Early (defined as < 6 months post-infusion) loss of B cell aplasia or any CD19+CD22+ relapse following CD19CAR T cell therapy with Tisagenlecleuc.

8. The cell population for use according to any of claims 1 or 4-7, or the use according to any of claims 2 or 4-7, or the method according to any of claims 3-7, wherein the patient has an isolated CNS relapse meeting one or more of a) - m).

9. The cell population for use according to any of claims 1 or 4-8, or the use according to any of claims 2 or 4-8, or the method according to any of claims 3-8, wherein the cell population comprises a 1:1 ratio of anti-CD19 CAR-T cells and anti-CD22 CAR-T cells.

10. The cell population for use according to any of claims 1 or 4-9, or the use according to any of claims 2 or 4-9, or the method according to any of claims 3-9, wherein the patient is administered a total dose of 4 x105anti-CD19 and anti-CD22 CAR T-cells / kg, 5 x105anti-CD19 and anti-CD22 CAR T-cells / kg, 8 x105anti-CD19 and anti-CD22 CAR T- cells / kg, 1 x106anti-CD19 and anti-CD22 CAR T-cells / kg, 1.5 x 106anti-CD19 and anti- CD22 CAR T-cells / kg, or 2 x 106anti-CD19 and anti-CD22 CAR T-cells / kg.

11. The cell population for use according to any of claims 1 or 4-10, or the use according to any of claims 2 or 4-10, or the method according to any of claims 3-10, wherein the method of treating the high risk / relapsed CD19+ or CD22+ haematological malignancyin the patient comprises administering a total dose of 4 x105anti-CD19 and anti-CD22 CAR T-cells / kg,wherein the total dose comprises 2 x105CD19 CAR T-cells / kg and 2 x105CD22 CAR T- cells / kg.

12. The cell population for use according to any of claims 1 or 4-11, or the use according to any of claims 2 or 4-11 , or the method according to any of claims 3-11 , wherein the anti- CD19 CAR-T cells express a chimeric antigen receptor (CAR) comprising a CD19- binding domain which comprises:i) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:CDR1 - GYAFSSS (SEQ ID NO: 1);CDR2 - YPGDED (SEQ ID NO: 2)CDR3 - SLLYGDYLDY (SEQ ID NO: 3); andii) a light chain variable region (VL) having CDRs with the following sequences:CDR1 - SASSSVSYMH (SEQ ID NO: 4);CDR2 - DTSKLAS (SEQ ID NO: 5)CDR3 - QQWNINPLT (SEQ ID NO: 6).

13. The cell population for use, or the use, or the method according to claim 12, wherein the CD19-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 or a variant thereof having at least 95% sequence identity.

14. The cell population for use, or the use, or the method according to claim 13, wherein the CD19-binding domain comprises the sequence shown as SEQ ID NO: 9 or a variant thereof having at least 90% sequence identity.

15. The cell population for use according to any of claims 1 or 4-4, or the use according to any of claims 2 or 4-14, or the method according to any of claims 3-14, wherein the anti- CD22 CAR T-cells express a chimeric antigen receptor (CAR) comprising a CD22- binding domain which comprises:i) a heavy chain variable region (VH) having CDRs with the following sequences:CDR1 - NFAMA (SEQ ID NO: 58);CDR2 - SISTGGGNTYYRDSVKG (SEQ ID NO: 59)CDR3 - QRNYYDGSYDYEGYTMDA (SEQ ID NO: 60); andii) a light chain variable region (VL) having CDRs with the following sequences:CDR1 - RSSQDIGNYLT (SEQ ID NO: 61);CDR2 - GAIKLED (SEQ ID NO: 62)CDR3 - LQSIQYP (SEQ ID NO: 63).

16. The cell population for use, or the use, or the method according to claim 15, wherein the CD22-binding domain comprises a VH domain having the sequence shown as SEQ ID NO: 64 and / or or a VL domain having the sequence shown as SEQ ID NO: 65 or a variant thereof having at least 95% sequence identity.

17. The cell population for use, or the use, or the method according to claim 16, wherein the CD22-binding scFv comprises the sequence shown as SEQ ID NO: 66 or a variant thereof having at least 90% sequence identity.

18. The cell population for use according to any of claims 1 or 4-17, or the use according to any of claims 2 or 4-17, or the method according to any of claims 3-17, wherein the patient has received lymphodepletion comprising fludarabine, cyclophosphamide and low dose total body irradiation prior to receiving the cell population comprising anti- CD19 CAR T-cells and anti-CD22 CAR-T cells.

19. The cell population for use according to any of claims 1 or 4-18, or the use according to any of claims 2 or 4-18, or the method according to any of claims 3-18, wherein the lymphodepletion comprises:a) fludarabine 30 mg / m2once daily on day -6 to -3;b) cyclophosphamide 0.5 g / m2once daily on day -6 to -5; andc) low dose Total Body Irradiation 2Gy on day -2.

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