method

Abstract

The present invention provides methods for selecting a bridging therapy for a patient having a B-cell malignancy who is about to receive treatment with CAR-T cells.

Description

[0001] METHOD

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods for selecting a bridging therapy for a patient having a B-cell malignancy who is about to receive treatment with CAR-T cells.

[0004] BACKGROUND TO THE INVENTION

[0005] Adoptive cell therapy for hematologic malignancies is a rapidly evolving field, with new iterations of novel constructs being developed at a rapid pace. Since the initial reports of chimeric antigen receptor T cell (CAR T cell) success in CD 19 B cell malignancies, multiple clinical trials of CAR T cell therapy directed to CD 19 have led to the approval of this therapy by the FDA and the European Medicines Agency for specific indications.

[0006] Tisagenlecleucel (Tisa-cel, Kymriah®) was the first CD 19 CAR T therapy to be approved for use in children and young adults with relapsed / refractory (r / r) ALL by the U.S. Food and Drug Administration (FDA) in 2017, with approvals in adult r / r diffuse large B-cell lymphoma (DLBCL) and in adult r / r follicular lymphoma (FL) later on. Axicabtagene ciloleucel (axi-cel; Yescarta®) was approved for adult patients with r / r DLBCL, adult patients with r / r large B-cell lymphoma (LBCL), and adult r / r follicular lymphoma (FL). Lisocabtagene maraleucel (liso-cel; Breyanzi®) was approved for adult patients with r / r DLBCL and r / r LBCL after one prior therapy. Brexucabtagene autoleucel (brexu-cel, Tecartus®) were approved for use in r / r MCL in and r / r B-ALL.

[0007] Despite these advances, however, eligible patients face several challenges to receiving CAR T cell treatment, one of which is progressive malignancy prior to administration of CAR T cells. Bridging therapy serves two purposes: to control disease while waiting for CAR T cell infusion and to reduce the risk of CAR T cell-associated toxicities by debulking the disease.

[0008] Bridging therapy may be indicated in patients during the period between T cell apheresis and initiation of lymphodepleting chemotherapy because of the proliferative nature of relapsed and refractory B-cell malignancies. However, this is not necessarily the case for isolated extramedullary leukaemias when local control may be sufficient. There is no consensus on the optimal bridging therapy regimen, and no randomized data comparing regimens exists. However, the impact of bridging therapy is not trivial, because multiple groups have shown that a low leukemic burden at the time of CAR-T infusion is associated with improved outcomes in children and young adults having B-cell acute lymphoblastic leukaemia (B-ALL) (Curran et al., 2019, Blood 134:2361-8; Schultz et al., 2022, J Clin Oncol. 40:945-55; Myers et al., 2022, J Clin Oncol. 40:932-44).

[0009] Several clinical trials that served as the basis for approval for various CAR T products allowed the use of bridging therapy but bridging therapy in these trials was diverse and reported with a varying degree of detail.

[0010] The ELIANA trial (NCT02435849), which led to the approval of tisa-cel for patients with B-cell ALL allowed the use of bridging therapy. Of the total 92 patients who were enrolled in the trial 65 received bridging therapy although details of regimens used for bridging were not specified median time to manufacture in this trial was reported at 23 days. The product was cryopreserved, and the median time from enrolment to infusion was 45 days (range 30- 105 days) (Maude et al., 2018, N Engl J Med 378:439-48).

[0011] Bridging therapy was not allowed in ZUMA-1 (NCT02348216) trial evaluating axi-cel in DLBCL. Of note, the median time to manufacture in this trial was 17 days and of the 10 patients who did not receive treatment one of them died due to disease progression (Neelapu et al., 2017, N Engl J Med 377:2531-44). The JULIET (NCT02445248) trial evaluating tisa- cel in DLBCL allowed bridging therapy, which was given in 92% of enrolled patients (number of patients receiving bridging was not reported) (Schuster et al., 2019, N Engl J Med 380:45-56). The choice of bridging was diverse, including combinations of rituximab, gemcitabine, etoposide, steroids, cisplatin, cytarabine, ibrutinib, and lenalidomide. The median time to manufacture in this trial was 23 (range not available) days. Though the exact numbers were not included, it was reported that the majority of the 50 patients who enrolled in the study but discontinued participation before receiving CAR T did so due to progressive disease or death. A subgroup analysis of seven patients from the JULIET trial who were excluded from the original efficacy data who had a complete response to bridging therapy and subsequently received CAR T infusion has also been published (Bishop et al., 2019, Blood Adv 3:2230-6). In this analysis, the duration of bridging therapy ranged from 2 days to 129 days, and a variety of regimens were used. Of these patients, five remained progression-free for more than 12 months, and this group of patients was found to have low rates of cytokine release syndrome (CRS) and neurotoxicity. The TRANSCEND (NCT02631044) study evaluated the use of liso-cel in patients with DLBCL and bridging therapy was allowed. Of the 344 patients enrolled, 159 (59%) received bridging therapy which included combinations of rituximab, gemcitabine, oxaliplatin, steroids, bendamustine, lenalidomide, brentuximab vedotin, and ibrutinib. Fourty-eight of 344 patients who underwent leukapheresis had complications of disease or died before receiving CAR T infusion. In most patients, bridging therapy did not result in lower disease burden and a higher tumor burden and receipt of bridging therapy were associated with higher rates of CRS and neurotoxicity. The median time to manufacture was the highest in this trial, reported at 24 (17-51) days (Abramson et al., 2020, Lancet 396:839-52).

[0012] The ZUMA-2 trial (NCT02601313) evaluating brexu-cel in MCL included 74 patients of which 25 (35%) received bridging therapy. Bridging for these patients included steroids, ibrutinib, acalabrutinib, or a combination. Of the 25 patients who received bridging, 17 had imaging assessments before and after, and the majority of those patients were found to have an increase in tumour burden. Three patients died of progressive disease before receiving CAR T treatment. The median time to manufacture was reported as 16 (11-28) days (Wang et al . , 2020, N Engl J Med 382: 1331 -42) .

[0013] Some data are also available regarding the use of bridging therapy with CAR T cells in commercial use. A study published by the U.S. Lymphoma CAR T Consortium evaluated the use of axi-cel for DLBCL in 298 patients across 17 centers (Nastoupil et al., 2020, J Clin Oncol 38:3119-28). Unlike the ZUMA-1 trial, bridging therapy was used in 158 (53%) of patients and consistent of combinations of steroids, chemotherapy, radiation, or targeted therapies such as lenalidomide or ibrutinib. A multivariate analysis from this study demonstrated worse overall survival at 12 months in patients who received bridging therapy (56% vs. 81% in patients who did not receive bridging therapy, p < 0.001). Another study evaluated the influence of bridging therapy on outcomes in 148 patients with DLBCL who were being treated with axi-cel. Eighty-one patients (55%) received bridging, whether in the form of systemic therapy, radiation therapy, or combined-modality therapy (Pinnix et al., 2020, Blood Adv 4:2871-83). The patients receiving bridging were more likely to have an elevated international prognostic index score, bulky disease, and an elevated lactate dehydrogenase (LDH). Overall survival at 1 year was significantly different in patients receiving bridging therapy, reported at 48% (vs. 65% in patients who did not receive bridging, p = 0.05). Of note, in this study radiation therapy alone was shown to be a safe and effective bridging strategy in a small cohort (n = 11) of patients. These patients who were bridged with radiation therapy had similar rates of toxicity as patients who received systemic bridging or no bridging and had an improved progression-free survival compared to patients who were bridged with systemic therapy. In a small study, 12 patients were treated with radiation therapy as bridging before receiving axi-cel and radiation was shown to be safe and response rates and complication rates after axi-cel were similar to those in the original study (Sim et al., 2019, Int J Radiat Oncol Biol Phys 105: 1012-1021).

[0014] Two retrospective studies have also evaluated the use of bridging therapy. The first evaluated 46 patients who were receiving commercial CAR T products and included 30 (65%) who received high-intensity bridging therapy, defined as chemotherapy with or without immunotherapy, and 16 (35%) who received low intensity or no bridging therapy (Paillassa et al., 2019, Blood 134(Suppl 1):2886). In this study, the intensity of bridging treatment was closely related to tumour burden at enrolment and while there was no difference in efficacy of CAR T infusion between the high-intensity and low-intensity groups, similar to other studies the high-intensity group did have a higher frequency of CRS and neurotoxicity. Another single-institution retrospective study evaluated 64 patients with non-Hodgkin lymphoma, 49 of whom received commercial CAR T (Dwivedy Nasta, 2019, Blood 134(Suppl l):4108). Thirty-four (69%) of these 49 patients received bridging therapy to reduce tumour burden or palliate symptoms. Bridging therapies included combination chemoimmunotherapy, radiation alone, systemic therapy with radiation therapy, targeted treatments, or combination treatment. Of the patients who received chemoimmunotherapy, three of 12 had progressive disease at the time of CAR T infusion and of five patients who received radiation alone, one had progressive disease at the time of CAR T infusion.

[0015] In clinical trials, data regarding the effects of bridging on disease burden are mixed, and several studies have demonstrated that the use of bridging therapy is associated with higher rates of toxicity and poorer overall survival. This is likely reflective of the fact that bridging therapy is often used in patients with more aggressive disease, a higher burden of disease at baseline, or disease that is refractory to chemotherapy.

[0016] Therefore, there is a need in the art for a bridging therapy that is suitable and effective for use with CAR T cell therapies. SUMMARY OF ASPECTS OF THE INVENTION

[0017] The present inventors have designed a bridging therapy protocol using an anti-CD22 antibody-drug conjugate which worked well in patients with B cell malignancies. Surprisingly, this bridging therapy was extremely effective in patients with high disease burden.

[0018] Thus, in a first aspect, the present invention provides a method for selecting a bridging therapy for a patient having a B-cell malignancy who is about to receive treatment with CAR-T cells, which comprises the following steps:

[0019] (i) determining the bone marrow (BM) blast percentage of the patient, and

[0020] (ii) selecting the use of an anti-CD22 antibody-drug conjugate as bridging therapy if the patient presents more than or equal to 5% blasts in the BM at screening.

[0021] The patient may undergo leukapheresis to prepare the population of CAR T cells specific for a B-cell malignancy.

[0022] The patient may have previously received chemotherapy but the BM blast percentage is still > 5%.

[0023] The BM blast percentage at screening may be more than or equal to 20%.

[0024] The BM blast percentage at screening may be more than or equal to 50%.

[0025] The BM blast percentage at screening may be more than or equal to 75%.

[0026] The anti-CD22 antibody-drug conjugate may be administered at least 1 week before the administration of the lymphodepleting therapy.

[0027] The patient may receive a lymphodepleting pre-conditioning treatment before CD 19 CAR- T cell infusion.

[0028] The population of CAR T cells specific for a B-cell malignancy may be autologous.

[0029] The anti-CD22 antibody-drug conjugate may be administered after leukapheresis.

[0030] The anti-CD22 antibody-drug conjugate may be selected from Inotuzumab ozogamicin (Besponsa), inotuzumab, and epratuzumab. The anti-CD22 antibody-drug conjugate may be Inotuzumab ozogamicin. The B-cell malignancy may be selected from the group consisting of acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia or WM), cutaneous B-cell lymphoma (CBCL), diffuse large B-cell lymphoma (DLBCL), and anaplastic large cell lymphoma (ALCL).

[0031] The CAR T cells may be specific to CD 19 or CD20.

[0032] The CAR-T cells may be specific to CD 19.

[0033] The anti-CD19 CAR may comprise: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

[0034] CDR1 - GYAFSSS (SEQ ID No. 1);

[0035] CDR2 - YPGDED (SEQ ID No. 2)

[0036] CDR3 - SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID No. 4);

[0037] CDR2 - DTSKLAS (SEQ ID No. 5)

[0038] CDR3 - QQWNINPLT (SEQ ID No. 6).

[0039] In another aspect, the present invention provides a method for improving the outcome of treatment with CAR-T cells for a patient having a B-cell malignancy, wherein the patient presents > 5% blasts in the BM at screening, which method comprises the step of administering an anti-CD22 antibody-drug conjugate to the patient after leukapheresis but prior to pre-conditioning the patient for CAR-T cell treatment.

[0040] The patient may present > 20% blasts in the BM at screening.

[0041] The patient may present > 50% blasts in the BM at screening.

[0042] The patient may present > 75% blasts in the BM at screening.

[0043] In another aspect, the present invention provides a method for treating a B-cell malignancy in a subject which comprises the step of using anti-CD22 antibody-drug conjugate as bridging therapy prior to treatment with anti-CD19 CAR-T cells, wherein the CAR comprises: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

[0044] CDR1 - GYAFSSS (SEQ ID No. 1);

[0045] CDR2 - YPGDED (SEQ ID No. 2)

[0046] CDR3 - SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID No. 4);

[0047] CDR2 - DTSKLAS (SEQ ID No. 5) CDR3 - QQWNINPLT (SEQ ID No. 6).

[0048] The method for treating a B-cell malignancy may comprise the following steps: i) administering the anti-CD22 antibody-drug conjugate to the patient after leukapheresis; ii) pre-conditioning the patient; and iii) administering anti CD- 19 CAR-T cells to the patient.

[0049] The method for treating a B-cell malignancy may comprise the following steps: i) leukapheresis of the patient to obtain a T cell composition for preparation of the anti-CD19 CAR-T cells; ii) administering the anti-CD22 antibody-drug conjugate to the patient; iii) pre-conditioning the patient; and iv) administering the anti CD-19 CAR-T cells to the patient.

[0050] DESCRIPTION OF THE FIGURES

[0051] Figure 1. CD19 CATCAR (AUTO1). A) Cartoon of CD19 CATCAR (which is AUTO1). This CAR is a type I transmembrane protein. The scFv (anti-CD19 CAT19) at the aminoterminus is linked to a CD8 stalk and transmembrane domain which is linked to an endodomain comprised of a fusion between 4-1BB and CD3(^. B) Lentiviral vectors encoding CATCAR.

[0052] Figure 2. Proliferation of T Cells Transduced with CD19 (CAT) CAR and CD19 (FMC63) CAR. CAR=chimeric antigen receptor; CD19=cluster of differentiation 19; CPM=counts per minute; NT=Non-transduced; SEM=standard error of the mean. Data: mean SEM, n=4; * p<0.05, ** p<0.01, 2-tailed paired Student t-test

[0053] Figure 3. Cytokine Production by Transduced CD19 CAR T Cells. CAR=chimeric antigen receptor; CD19=cluster of differentiation 19; IFNy=interferon y; IL=interleukin; ns=not significant; NT=non-transduced; SEM=standard error of the mean; TNFa=tumour necrosis factor a. Production of cytokines in response to 1 : 1 co-culture with irradiated Raji cells measured by Cytokine Bead Array of culture supernatants taken at 48 hours. Data: mean SEM, n=4; * p<0.05; 2-tailed paired Student t-test.

[0054] Figure 4. Antigen-specific Killing of CD19-positive Tumour Cells by CD19 CAR T Cells. CAR=chimeric antigen receptor; CD19=cluster of differentiation 19; E=effector cells; NT=non-transduced; SEM= standard error of the mean; SupTl= Human T cell lymphoblastic lymphoma cell line; T=target cells. The cytotoxic activity of CAR T cells was measured by standard 4 hour 51 -chromium release assay against a SupTl engineered to express CD19. Data: mean ± SEM, n = 5; * p<0.05, 2-way analysis of variance.

[0055] Figure 5. Tumour Growth in a Mouse Xenograft Model Injected with CD19 CAR T Cells. CAR=chimeric antigen receptor; CD19=cluster of differentiation 19; FLuc=firefly luciferase; p=photon; NT=non-transduced; SEM=standard error of the mean; sr=steradian. Photon emissions from FLuc-positive tumour cells were quantified and measured as maximum p / s / cm2 / sr. Lines represent cumulative results of light emission values ± SEM. Bioluminescence was determined in 2 separate experiments, n=18, **p<0.01, *** p<0.001, Student t-test.

[0056] Figure 6: Residual NALM-6 Tumour Cells in the Bone Marrow of Mice 2 Weeks Post CAR T Cell Infusion. CAR=chimeric antigen receptor; CD19=cluster of differentiation; NT=non-transduced. After termination of the experiment at 16 days following infusion of CAR T cells, absolute numbers of NALM-6 cells were assessed in bone marrow by flow cytometry, n=18; ** p<0.01, **** p<0.0001; 2-sided Student t-test.

[0057] Figure 7. Overview of the different stages of the clinical study. CRS, cytokine release syndrome; Cy, cyclophosphamide; Flu, fludarabine; ICANS, immune effector cell associated neurotoxicity syndrome Figure 8. Eligibility, Endpoints, and Disposition of the clinical study. Eighty-four percent of enrolled patients were infused with AUTO1. * R / R B-ALL: Primary refractory; First relapse if first remission <12 months; R / R disease after >2 lines of systemic therapy; R / R disease after allogeneic transplant; R / R Philadelphia chromosome-positive ALL if intolerant to / failed two lines of any TKI or one line of second-generation TKI, or if TKI therapy is contraindicated. Enrolment: all eligibility criteria met and the leukapheresate accepted for manufacturing. BM = bone marrow; CR = complete response; CRi = complete response with incomplete recovery of counts; DoR = duration of response; EFS = event free survival; MRD = minimal residual disease; OS = overall survival.

[0058] Figure 9. AUTO1 Manufacturing. Manufacturing quality and logistics were reliable and consistent.

[0059] Figure 10. Disease Response per IRRC Assessment. Seventy-six percent of infused patients achieved CR / CRi. Ninety-seven percent of responders with evaluable samples were MRD negative at 10-4 level by flow cytometry *One-sided p-value from the exact test on HO: ORR <40% vs Hl : ORR >40%. CR, complete remission, CRi, CR with incomplete blood count recovery; IRRC, independent response review committee; MRD, minimal residual disease; ORR, overall remission rate.

[0060] Figure 11. Duration of Remission. Sixty-one percent of responders in ongoing remission without subsequent anti-cancer therapies. Thirteen percent of responders who proceeded to SCT while in remission were censored at the time of SCT. NE, not estimable.

[0061] Figure 12. Subgroup Analysis of CR / CRi (IRRC Assessment). High risk subgroups include EMD and high BM blasts at pre-conditioning. CR, complete remission; CRi, CR with incomplete blood count recovery; EMD, extramedullary disease; IRRC, independent response review committee; ORR, overall remission rate.

[0062] Figure 13. AUTO1 Expansion and Persistence. CD 19 CAR-Positive T Cell Expansion and Persistence in the Peripheral Blood of Adult B ALL Patients Measured by qPCR. CAR-T cellular kinetics are consistent with the ALLCAR19 study (Roddie C et al., 2021. J Clin Oncol 39:3352-63). AUC, area under the curve; CV, coefficient of variation; Geo, geometric; PCR, polymerase chain reaction; SE, standard error. Figure 14. Annotated amino acid sequence (SEQ ID NO: 10) of the CD19 CATCAR (AUTO 1).

[0063] Figure 15. Phase Ib / II study - All cohorts: Patient eligibility and selected endpoints. *R / R B-ALL: primary refractory; first relapse if first remission <12 months; R / R disease after >2 lines of systemic therapy; R / R disease after allogeneic transplant; R / R Philadelphia chromosome-positive ALL if intolerant to / failed two lines of any TKI or one line of second-generation TKI, or if TKI therapy is contraindicated. {Primary endpoints: Cohorts A and C, ORR defined as the proportion of patients achieving CR or CRi; Cohort IIB, the proportion of patients achieving MRD-negative remission (<10- 4 leukemic cells). §EFS: the time from date of first infusion to the earliest of treatment failure, relapse, or death from any cause. ALL, acute lymphoblastic leukemia; B-ALL, B- cell acute lymphoblastic leukemia; BM, bone marrow; CAR-T, chimeric antigen receptor T-cell; CR, complete remission; CRi, CR with incomplete hematologic recovery; DoR, duration of remission; EFS, event-free survival; EMD, extramedullary disease; IRRC, Independent Response Review Committee; MRD, measurable residual disease; ORR, overall remission rate; OS, overall survival; R / R, relapsed / refractory; TKI, tyrosine kinase inhibitor

[0064] Figure 16. Phase Ib / II study - All cohorts: Patient disposition. *Seven patients received Dose 1 only. {All eligibility criteria met and the leukapheresate accepted for manufacturing AUTO1.

[0065] Figure 17. Phase Ib / II study - All cohorts: AUTO1 manufacturing. *Unless otherwise stated. BM, bone marrow; EMD, extramedullary disease; SCT, stem cell transplant.

[0066] Figure 18. Phase Ib / II study - All cohorts: Remission rate and MRD by status at lymphodepletion. *Morphologic disease defined as >5% BM blasts or presence of EMD regardless of BM blast status. {MRD status available for 64 / 73 patients, as assessed by NGS or flow cytometry. §MRD status available for 27 / 29 patients, as assessed by NGS or flow cytometry. BM, bone marrow; CR, complete remission; CRi, CR with incomplete hematologic recovery; EMD, extramedullary disease; MRD, measurable residual disease; NGS, next-generation sequencing.

[0067] Figure 19. Phase Ib / II study - All cohorts: CR / CRi subgroup analysis per IRRC.

[0068] *The red dashed line denotes the Phase IIA null hypothesis (40%). {The black dashed line denotes the ORR among all treated patients (ORR=CR+CRi). BM, bone marrow; CR, complete remission; CRi, CR with incomplete hematologic recovery; EMD, extramedullary disease; IRRC, Independent Response Review Committee; ORR, overall remission rate; SCT, stem cell transplant.

[0069] Figure 20. Phase Ib / II study - All cohorts: Event-free survival (EFS) in all treated patients. Censoring new non-protocol anti-cancer therapies including SCT with disease assessment by IRRC (data cut-off date: September 13, 2023). Median EFS: ITT population - 9.8 months (95% CI: 5.9, 12.9). CI, confidence interval; EFS, event-free survival; IRRC, Independent Response Review Committee; ITT, intent-to-treat; NE, not evaluable; SCT, stem cell transplant.

[0070] Figure 21. Phase Ib / II study - All cohorts: AUTO1 persistence in responders.

[0071] Figure 22. Phase Ib / II study - All cohorts: Leukemic burden in all treated patients.

[0072] Bridging therapy per physician’s choice, including inotuzumab ozogamicin. BM, bone marrow.

[0073] Figure 23. Phase Ib / II study - All cohorts: Event-free survival (EFS) by leukemic burden prior to lymphodepletion. Censoring new non-protocol anti-cancer therapies including SCT with disease assessment by IRRC (data cut-off date: September 13, 2023). BM, bone marrow; CI, confidence interval; EFS, event-free survival; IRRC, Independent Response Review Committee; NE, not evaluable; SCT, stem cell transplant.

[0074] Figure 24. Phase Ib / II study - All cohorts: CRS and ICANS.

[0075] Figure 25. Kaplan-Meier plot of EFS in patients who received bridging therapy with inotuzumab ozogamicin (INO) vs those who received bridging therapy without INO vs those who received no bridging therapy.

[0076] Figure 26. Leukemic burden in patients receiving inotuzumab as bridging therapy.

[0077] BM, bone marrow. The single patient who presented <5% blasts in the BM prior to leukapheresis evolved to 5% blasts in the BM prior to lymphodepletion.

[0078] Figure 27. Median BM blast percentage at screening and lymphodepletion per type of BT. Figure 28. Response outcomes by bridging therapy (BT) group. A and B. Kaplan-Meier analysis of outcomes by type of BT; A) event-free survival and B) overall survival. C) Overall remission rate (ORR) by BT group. Median follow-up: 21.5 months (range: 8.6- 41.4); data cut-off date: February 7, 2024. Error bars represent the 95% CI. BT, bridging therapy; CI, confidence interval; CR, complete remission; CRi, complete remission with incomplete haematological recovery; DoR, duration of remission; INO, inotuzumab ozogamicin; NE, not estimable; ORR, overall remission rate; w / o, without.

[0079] Figure 29. CAR T-cell persistence by bridging therapy (BT) group. *Time to loss of CAR T-cell persistence was defined as the days between the first AUTO1 infusion and the first time at which the AUTO1 transgene level, as measured by ddPCR in peripheral blood, dropped to zero from the last positive value. BT, bridging therapy; CAR, chimeric antigen receptor; ddPCR, droplet digital polymerase chain reaction; INO, inotuzumab ozogamicin.

[0080] DETAILED DESCRIPTION OF THE INVENTION

[0081] 1. Chimeric

[0082] A classical chimeric antigen receptor (CAR) is a chimeric type I trans-membrane protein which connects an extracellular antigen-binding domain to an intracellular signaling 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. Other examples 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 IgGl. More compact spacers can suffice, e.g., the stalk from CD8a and even just the IgGl hinge alone, depending on the antigen. A transmembrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

[0083] Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FcsRl or CD3(^ (Figure la). 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 4 IBB which transmit survival signals (Figure lb). Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals (Figure 1c).

[0084] 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. CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral or lentiviral vectors to generate antigen-specific T cells for adoptive cell transfer. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus, the CAR directs the specificity and cytotoxicity of the T cell towards cells expressing the targeted antigen.

[0085] 1.1. Antigen binding domain

[0086] The antigen binding domain is the portion of CAR which recognizes antigen. Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the antigenbinding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain antibody; an artificial single binder such as a Darpin (designed ankyrin repeat protein); or a single-chain derived from a T-cell receptor. In a classical CAR, the antigen-binding domain comprises: a single-chain variable fragment (scFv) derived from a monoclonal antibody (Figure 2a).

[0087] 1.2. Target antigens

[0088] A ‘target antigen’ is an entity which is specifically recognized and bound by the antigenbinding domains of a chimeric receptor provided herein.

[0089] The target antigen may be an antigen present on a malignant B cell, for example CD 19.

[0090] 1.3. Binding domains specific for CD 19 target antigen

[0091] The human CD 19 antigen is a 95 kd transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD 19 is classified as a type I transmembrane protein, with a single transmembrane domain, a cytoplasmic C-terminus, and extracellular N-terminus. CD 19 is expressed very early in B-cell differentiation and is only lost at terminal B-cell differentiation into plasma cells. CD 19 is a biomarker for normal B cells as well as follicular dendritic cells. CD 19 primarily acts as a B cell co-receptor in conjunction with CD21 and CD81. Upon activation, the cytoplasmic tail of CD 19 becomes phosphorylated, which leads to binding by Src-family kinases and recruitment of PI-3 kinase.

[0092] CD 19 is also expressed on all B-cells but not plasma 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 CD 19 CAR T cell therapy will result in B cell aplasia, the consequent hypogammaglobulinaemia can be treated with pooled immunoglobulin.

[0093] Different designs of CARs have been tested against CD 19 in various clinical trials, as outlined in the following Table 1.

[0094] Table 1

[0095] 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 CD 19.

[0096] The antigen-binding domain of a CAR which binds to CD 19 (referred to as a CD 19 CAR herein) may be any domain which is capable of binding CD 19.

[0097] For example, the antigen-binding domain may comprise a CD 19 antigen-binding domain as described in Table 2.

[0098] Table 2 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 CD 19 CAR herein may bind an epitope of CD 19 encoded by exon 1 of the CD 19 gene. The antigen-binding domain of a CD 19 CAR herein may bind an epitope of CD 19 encoded by exon 2 of the CD 19 gene. The antigen-binding domain of a CD 19 CAR herein may bind an epitope of CD 19 encoded by exon 3 of the CD 19 gene. The antigen-binding domain of a CD 19 CAR herein may bind an epitope of CD 19 encoded by exon 4 of the CD 19 gene.

[0099] A CD19-binding domain exemplified herein comprises variable regions with complementarity determining regions (CDRs) from an antibody referred to as CAT 19, a) a heavy chain variable region (VH) having CAT 19 CDRs with the following sequences:

[0100] CDR1 - GYAFSSS (SEQ ID NO: 1);

[0101] CDR2 - YPGDED (SEQ ID NO: 2)

[0102] CDR3 - SLLYGDYLDY (SEQ ID NO: 3); and b) a light chain variable region (VL) having CAT 19 CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID NO: 4);

[0103] CDR2 - DTSKLAS (SEQ ID NO: 5)

[0104] CDR3 - QQWNINPLT (SEQ ID NO: 6).

[0105] The CAT19 antibody is described in WO2016 / 139487.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] The CD 19 CAR may comprise the following VH sequence.

[0110] SEQ ID NO: 7 - VH sequence from CAT 19 murine monoclonal antibody QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPG

[0111] DEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDY WGQGTTLTVSS

[0112] The CD 19 CAR may comprise the following VL sequence.

[0113] SEQ ID NO: 8 - VL sequence from CAT 19 murine monoclonal antibody

[0114] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLAS GVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

[0115] The CD 19 CAR may comprise the following scFv sequence.

[0116] SEQ ID NO: 9 - VH-VL scFv sequence from murine monoclonal antibody

[0117] QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPG DEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDY WGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSV SYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAAT YYCQQWNINPLTFGAGTKLELKR

[0118] The CAR may consist of or comprise one of the following sequences.

[0119] SEQ ID NO: 10 - CAT19 CAR

[0120] MGTSLLCWMALCLLGADHAD AQ VQLQQSGPELVKPGAS VKISCKASGYAF S S SW MNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKS STTAYMQLS SLT SEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSP AIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSG SGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYCKRGRKKLL YIFKQPFMRP VQTTQEEDGC SCRFPEEEEGGCELRVKF SRS AD AP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0121] The CAT19 CAR has a CD8a spacer and transmembrane domain, 4-1BB endodomain and TCR CD3z endodomain. The CAR provided herein may comprise a variant of the polypeptide of SEQ ID NO: 1-10 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD 19 (when in conjunction with a complementary VL or VH domain, if appropriate).

[0122] 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.

[0123] The CD 19 CAR exemplified herein ( / .< ., the CAT 19 CAR, SEQ ID NO: 10) has properties which result in lower toxicity and better efficacy in treated patients. When compared with an fmc63 CAR having the same spacer and endodomains, the CAT 19 CAR exemplified herein effects killing of target cells expressing CD 19 and proliferates in response to CD 19 expressing targets, but releases less Interferon-gamma. 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.

[0124] The CAT 19 CAR 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.

[0125] The CAT 19 CAR provided herein may result in a smaller proportion of CAR T cells becoming exhausted than equivalent 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.

[0126] Another exemplary CD 19 antigen-binding domain contemplated herein is based on the CD19 antigen-binding domain CD19ALAb (described in WO2016 / 102965) and comprises: a) a heavy chain variable region (VH) having CDRs with the following sequences:

[0127] CDR1 - SYWMN (SEQ ID NO: 11);

[0128] CDR2 - QIWPGDGDTNYNGKFK (SEQ ID NO: 12)

[0129] CDR3 - RETTTVGRYYYAMDY (SEQ ID NO: 13); and b) a light chain variable region (VL) having CDRs with the following sequences:

[0130] CDR1 - KASQSVDYDGDSYLN (SEQ ID NO: 14);

[0131] CDR2 - DASNLVS (SEQ ID NO: 15) CDR3 - QQSTEDPWT (SEQ ID NO: 16).

[0132] 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.

[0133] The CAR may comprise one of the following amino acid sequences.

[0134] SEQ ID NO: 17 - Murine CD19ALAb scFv sequence

[0135] Q VQLQQSGAELVRPGS S VKISCKASGYAF S S YWMNWVKQRPGQGLEWIGQIWPG DGDTNYNGKFKGK ATLT ADES S ST A YMQL S SL ASED S AVYFC ARRETTT VGRYY YAMDYWGQGTTVTVSSDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLN WYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQS TEDPWTFGGGTKLEIK

[0136] SEQ ID NO: 18 - Humanized CD19ALAb scFv sequence - Heavy 19, Kappa 16

[0137] QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWP GDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRY YYAMDYWGKGTLVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLN WYQQKPGQPPKLLIYD ASNLVSGVPDRF SGSGSGTDFTLTIS SLQ AAD VAVYHCQ QSTEDPWTFGQGTKVEIKR

[0138] SEQ ID NO: 19 (Humanized CD19ALAb scFv sequence - Heavy 19, Kappa 7)

[0139] QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWP GDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRY YYAMDYWGKGTLVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLN WYQQKPGQPPKVLIYD ASNLVSGVPDRF SGSGSGTDFTLTIS SLQ AAD VAVYYCQ QSTEDPWTFGQGTKVEIKR

[0140] The scFv may be in a VH-VL orientation or a VL-VH orientation.

[0141] The CAR may comprise one of the following VH sequences:

[0142] SEQ ID NO: 20 - Murine CD 19 AL Ab VH sequence Q VQLQQSGAELVRPGS S VKISCKASGYAF S S YWMNWVKQRPGQGLEWIGQIWPG DGDTNYNGKFKGK ATLT ADES S ST A YMQL S SL ASED S AVYFC ARRETTT VGRYY YAMD YWGQGTT VT VS S

[0143] SEQ ID NO: 21 - Humanized CD19ALAb VH sequence

[0144] QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWP GDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRY YYAMDYWGKGTLVTVSS

[0145] The CAR may comprise one of the following VL sequences:

[0146] SEQ ID NO: 22 - Murine CD19ALAb VL sequence

[0147] DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDAS NLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK

[0148] SEQ ID NO: 23 (Humanized CD19ALAb VL sequence, Kappa 16)

[0149] DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDAS NLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPWTFGQGTKVEIKR

[0150] SEQ ID NO: 24 - Humanized CD19ALAb VL sequence, Kappa 7

[0151] DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYDA SNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPWTFGQGTKVEIK R

[0152] Another exemplary CD 19 antigen-binding domain contemplated herein is based on the CD 19 antigen-binding domain of monoclonal antibody fmc63 (FMC63) (described in Nicholson et al., 1997, Mol. Immunol., 34: 1157-65) and comprises: a) a heavy chain variable region (VH) having CDRs with the following sequences:

[0153] CDR1 - GVSLPDYG (SEQ ID NO: 25);

[0154] CDR2 - IWGSETT (SEQ ID NO: 26)

[0155] CDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 27); and b) a light chain variable region (VL) having CDRs with the following sequences:

[0156] CDR1 - QDISKY (SEQ ID NO: 28);

[0157] CDR2 - HTS

[0158] CDR3 - QQGNTLPYT (SEQ ID NO: 29). The CD 19 FMC63 CAR may comprise the following VH sequence.

[0159] SEQ ID NO: 30 - VH sequence from FMC63 murine monoclonal antibody

[0160] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSET TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYW GQGTSVTVSS

[0161] The CD 19 CAR may comprise the following VL sequence.

[0162] SEQ ID NO: 31 - VL sequence from FMC63 murine monoclonal antibody

[0163] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITK

[0164] The CD 19 FMC63 CAR may comprise the following scFv sequence.

[0165] SEQ ID NO: 32 - VH-VL scFv sequence from FMC63 murine monoclonal antibody

[0166] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSET TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYW GQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISK YLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYF CQQGNTLPYTFGGGTKLEITK

[0167] The CAR may consist of or comprise the following sequence.

[0168] SEQ ID NO: 33 - FMC63 CAR

[0169] MGTSLLCWMALCLLGADHADAEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQT TSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSG SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKSDPTTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY CKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY

[0170] QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR The CAR provided herein may comprise a variant of the sequence shown as any of SEQ ID NO: 25 to 33 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD 19 (when in conjunction with a complementary VL or VH domain, if appropriate).

[0171] 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.

[0172] 1.4. Signal peptides

[0173] 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.

[0174] 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 stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases.

[0175] The signal peptide may be at the amino terminus of the molecule.

[0176] The signal peptide may comprise the amino acid sequence of any of SEQ ID NO: 34-39 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.

[0177] The signal peptide of SEQ ID NO: 62 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.

[0178] SEQ ID NO: 34 MGTSLLCWMALCLLGADHADA

[0179] The signal peptide of SEQ ID NO: 35 follows.

[0180] METDTLLLWVLLLLVPGSTG

[0181] The signal peptide of SEQ ID NO: 36 follows. METDTLILWVLLLLVPGSTG

[0182] The signal peptide of SEQ ID NO: 37 follows.

[0183] MGWSCIILFLVATATGVHS

[0184] The signal peptide of SEQ ID NO: 38 is derived from IgGl.

[0185] SEQ ID NO: 38: MSLPVTALLLPLALLLHAARP

[0186] The signal peptide of SEQ ID NO: 39 is derived from CD8.

[0187] SEQ ID NO: 39: MAVPTQVLGLLLLWLTDARC

[0188] The signal peptide for the first CAR may have a different sequence from the signal peptide of the second CAR.

[0189] 1.5. Spacers

[0190] 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.

[0191] The spacer may, for example, comprise an IgGl Fc region, an IgGl 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 IgGl Fc region, an IgGl hinge or a CD8 stalk.

[0192] In the cells provided herein, the first and second CARs may comprise different spacer molecules. For example, the spacer may, for example, comprise an IgGl Fc region, an IgGl 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 IgGl Fc region, an IgGl hinge or a CD8 stalk. A human IgGl spacer may be altered to remove Fc binding motifs.

[0193] The spacer for the CD 19 CAR may comprise a CD8 stalk spacer, or a spacer having a length equivalent to a CD8 stalk spacer. The spacer for the CD 19 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. Examples of amino acid sequences for these spacers are given below:

[0194] SEQ ID NO: 40 (hinge-CH2CH3 of human IgGl)

[0195] AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPE

[0196] VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKKD

[0197] SEQ ID NO: 41 (human CD8 stalk):

[0198] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0199] SEQ ID NO: 42 (human IgGl hinge):

[0200] AEPKSPDKTHTCPPCPKDPK

[0201] SEQ ID NO: 43 (human IgGl hinge variation)

[0202] EPKSCDKTHTCPPCP

[0203] SEQ ID NO: 44 (IgGl Hinge-Fc)

[0204] AEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP

[0205] EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGKKDPK

[0206] SEQ ID NO: 45 (IgGl Hinge - Fc modified to remove Fc receptor recognition motifs)

[0207] AEPKSPDKTHTCPPCPAPPVA*GPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDP

[0208] EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGKKDPK

[0209] Modified residues are underlined; * denotes a deletion.

[0210] SEQ ID NO: 46 (CD2 ectodomain) KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKE KDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKIS WTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNK VSKESSVEPVSCPEKGLD

[0211] SEQ ID NO: 47 (CD34 ectodomain)

[0212] SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITE TTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDL STTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKD RGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLM KKHQSDLKKLGILDFTEQDVASHQSYSQKT

[0213] / .6. Transmembrane domains

[0214] The transmembrane domain is the domain of the CAR that spans the membrane.

[0215] 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 the transmembrane domain of a protein is a relatively simple structure, z.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 Bl describes synthetic transmembrane components).

[0216] The transmembrane domain may be derived from CD28, which gives good receptor stability. The CD28 transmembrane domain sequence is shown as SEQ ID NO: 48

[0217] SEQ ID NO: 48 FWVLVVVGGVLACYSLLVTVAFIIFWV

[0218] The transmembrane domain may be derived from human Tyrp-1. The tyrp-1 transmembrane domain sequence is shown as SEQ ID NO: 49.

[0219] SEQ ID NO: 49 IIAIAVVGALLLVALIFGTASYLI The transmembrane domain may be derived from CD8 A. The CD8 A transmembrane domain sequence is shown as SEQ ID NO: 50.

[0220] SEQ ID NO: 50 IYIWAPLAGTCGVLLLSLVITLYC

[0221] / . 7. Endodomains

[0222] As noted above, the endodomain is the signal-transmission portion of the CAR. After antigen recognition, receptors cluster, native CD45 and CD 148 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 / survival signal, or all three can be used together.

[0223] The cells provided herein comprise two CARs, each with an endodomain.

[0224] 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 co-stimulatory domain, such as the endodomain from CD28; and / or (iii) a domain which transmits a survival signal, for example a TNF receptor family endodomain such as OX-40 or 4-1BB.

[0225] Thus, the endodomain of the CAR of the present disclosure may comprise combinations of one or more of the CD3-Zeta endodomain, the 4 IBB endodomain, the 0X40 endodomain or the CD28 endodomain.

[0226] The intracellular T-cell signalling domain (endodomain) of the CAR of the present disclosure may comprise the sequence shown as any of SEQ ID NO: 51-58 or a variant thereof having at least 80% sequence identity.

[0227] SEQ ID NO: 51 (CD3 zeta endodomain)

[0228] RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR

[0229] SEQ ID NO: 52 (41BB endodomain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0230] SEQ ID NO: 53 (0X40 endodomain)

[0231] RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0232] SEQ ID NO: 54 (CD28 endodomain)

[0233] KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY

[0234] Examples of combinations of such endodomains include 41BB-Zeta, OX40-Zeta, CD28- Zeta and CD28-OX40-Zeta.

[0235] SEQ ID NO: 55 (41BB-Zeta endodomain fusion)

[0236] KRGRKKLL YIFKQPFMRP VQTTQEEDGC SCRFPEEEEGGCELRVKF SRS AD AP AYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0237] SEQ ID NO: 56 (OX40-Zeta endodomain fusion)

[0238] RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0239] SEQ ID NO: 57 (CD28Zeta endodomain fusion)

[0240] KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0241] SEQ ID NO: 58 (CD28OXZeta)

[0242] KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGG GSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD

[0243] GLYQGLSTATKDTYDALHMQALPPR

[0244] A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any of SEQ ID NO: 51-58 provided that the sequence provides an effective transmembrane domain / intracellular T cell signaling domain. 2. Nucleic acid

[0245] A nucleic acid provided herein encodes a CD 19 CAR of the disclosure. As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.

[0246] 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.

[0247] 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.

[0248] The alternative RNA codons which may be used to encode each amino acid are summarised in Table 3.

[0249] Table 3

[0250]

[0251] Alternative codons may be used in one or more nucleic acids which encode co-stimulatory domains, such as the CD28 endodomain.

[0252] Alternative codons may be used in one or more domains which transmit survival signals, such as 0X40 and 4 IBB endodomains.

[0253] 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. 3. Vector

[0254] The present disclosure also provides a vector, or kit of vectors which comprises one or more CAR-encoding nucleic acid. Such a vector may be used to introduce the nucleic acid into a host cell so that it expresses the CAR.

[0255] 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.

[0256] The vector may be capable of transfecting or transducing a T cell.

[0257] 4. Cell

[0258] A cell is provided herein which comprises a CD 19 CAR of the present disclosure. It will be understood that this cell expresses the CAR, wherein the CAR binds CD 19, such that the cell recognises a target cell expressing CD 19. Populations of cells which comprise a CD 19 CAR, also termed herein CD 19 CAR engineered cells or engineered cells, are also provided.

[0259] The cell may be any eukaryotic cell capable of expressing a CAR at the cell surface, such as an immunological cell.

[0260] In particular, the cell may be an immune effector cell such as a T cell or a natural killer (NK) cell.

[0261] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarized below.

[0262] Helper T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses. 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.

[0263] 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.

[0264] 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.

[0265] Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive Treg cells.

[0266] 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 (CD1 lc+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.

[0267] Adaptive Treg cells (also known as Tri cells or Th3 cells) may originate during a normal immune response.

[0268] The T cell provided herein may be any of the T cell types mentioned above, in particular a CTL. 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

[0269] 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.

[0270] The CAR-expressing cells provided herein may be any of the cell types mentioned above.

[0271] 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).

[0272] The present disclosure also provides a cell composition comprising CAR-expressing T cells and / or CAR-expressing NK cells, which cells express a CAR that binds CD 19, such that the cells can recognise a target cell expressing CD 19. The cell composition may be made by transducing a blood-sample ex vivo with a nucleic acid according to the present disclosure.

[0273] 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.

[0274] 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.

[0275] The T or NK cells provided herein may be made by: (i) isolation of a T or NK cellcontaining sample from a subject or other sources listed above, and (ii) transduction or transfection of the T or NK cells with a nucleic acid or a vector encoding the CD 19 CARs as described in the present disclosure.

[0276] 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.

[0277] The present invention also provides CD 19 CAR engineered cells, wherein the engineered cells are manufactured using a method with a manufacturing success rate of at least 80%. The manufacturing success rate can be defined as the percentage of patient samples that give rise to a usable drug product at the end of the manufacturing process. It may also be referred to as the degree of manufacturability. Preferably, the manufacturing success rate is 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%. Patient samples may be leukapheresis products, which may be used fresh or may be frozen and thawed before use.

[0278] A high manufacturing success rate is particularly advantageous when treating relap sed / refractory conditions as described herein, since patients with these conditions are least likely to be able to survive treatment delays caused by manufacturing failures. It will also be clear to those of skill in the art that the degree of manufacturability can be discussed in terms of a manufacturing failure rate, which is preferably 20% or lower, 19% or lower, 18% or lower, 17% or lower, 16% or lower, 15% or lower, 14% or lower, 13% or lower, 12% or lower, 11% or lower, 10% or lower, 9% or lower, 8% or lower, 7% or lower, 6% or lower, 5% or lower, 4% or lower, 3% or lower, 2% or lower, 1% or lower, or 0%.

[0279] Successful CAR T therapy relies on a rapid and effective end-to-end process. The challenge for product manufacturing is twofold: first, patients with high tumor burden can have T cells that are highly differentiated and exhausted; second, patients with leukemic cells in circulation have apheresis containing a substantial proportion of leukemic cells that require removal before manufacture can start. Timely vein-to-certification / vein-to-delivery (V2C / V2D) targets are thus important.

[0280] V2C (time from leukapheresis to quality release) may be about 23 days. V2D (time from leukapheresis to delivery of product to the hospital) may be about 25 days. Removal of leukemic cells can be achieved by a number of methods known to the skilled person, such as T cell enrichment.

[0281] 5. Pharmaceutical composition

[0282] 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 CD 19 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.

[0283] 6. Method of treatment

[0284] The cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, are capable of killing cancer cells recognizable by expression of CD 19, 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.

[0285] Examples of cancers which express CD 19 are B-cell lymphomas, including Hodgkin's lymphoma and non-Hodgkin’s lymphoma; and B-cell leukaemias.

[0286] 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 lymphocytic leukemia), 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.

[0287] 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.

[0288] The B-cell leukaemia may be acute lymphoblastic leukaemia (B-ALL or ALL).

[0289] The B-ALL may be adult ALL (aALL).

[0290] The patient may be 18 years of age or older.

[0291] Standard treatment for patients with aALL is typically divided into three different phases: induction, consolidation, and maintenance.

[0292] During the induction phase, different combinations of chemotherapeutic drugs may be used, such as vincristine, dexamethasone, prednisone, and an anthracy cline drug such as doxorubicin or daunorubicin. Some induction regimens may also include cyclophosphamide, L-asparaginase (or pegaspargase), and / or high doses of methotrexate or cytarabine (ara-C). For ALL patients whose leukaemia cells have the Philadelphia chromosome, a targeted drug such a tyrosine kinase inhibitor (TKI) (e.g., imatinib) and / or a second generation TKI (e.g., dasatinib) or is often included.

[0293] If the leukaemia goes into remission, the next phase consolidation (intensification) often consists of another fairly short course of chemotherapy, using many of the same drugs that were used for induction therapy in high doses. TKI or second generation TKI is also continued for Ph+-ALL patients. Response in patients often require consolidation an allogeneic haematopoietic stem cell transplant (HSCT).

[0294] After consolidation, the patient is generally put on a maintenance chemotherapy program of methotrexate and 6-mercaptopurine (6-MP). In some cases, this may be combined with other drugs such as vincristine and prednisone. For Ph+-ALL patients, a TKI or a second generation TKI is often included as well.

[0295] One or more of targeted immunotherapies, for example inotuzumab ozogamicin, blinatumomab and / or approved CD 19 CAR-T therapies, and low dose radiation therapy may be also administered. Treatment with the CD 19 CAR-expressing T cells provided herein is contemplated to help prevent the need of a subsequent allogeneic HSCT. Furthermore, these CAR-T cells have been shown to not cause severe toxicities, such as severe CRS and ICANS, and to persist longer.

[0296] 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 without causing severe toxicities.

[0297] 6.1. Method for selecting a bridging therapy

[0298] The present invention provides a method for selecting a bridging therapy for a patient having a B-cell malignancy who is about to receive treatment with CAR-T cells, which comprises the following steps:

[0299] (i) determining the bone marrow (BM) blast percentage of the patient, and

[0300] (ii) selecting the use of an anti-CD22 antibody-drug conjugate as bridging therapy if the patient presents more than or equal to 5% blasts in the BM at screening.

[0301] The patient may be of Caucasian, Black, Latino or Hispanic, Asian or any other ethnicity.

[0302] The patient may be Latino or Hispanic.

[0303] The patient may be 18 years of age or older.

[0304] The patient may have a complex karyotype. Complex karyotype refers to cytogenetic risks of hematologic malignancies associated with a poor prognosis that are well known in the art.

[0305] The patient may have received at least one prior line of therapy. The patient may have received at least two prior lines of therapy. The patient may have received at least three prior lines of therapy. The patient may have received at least four prior lines of therapy. The patient may have received at least five prior lines of therapy. The patient may have received at least six prior lines of therapy.

[0306] The at least one prior line of therapy may have been inotuzumab ozogamicin and / or blinatumomab.

[0307] The patient may have received an allogeneic hematopoietic stem cell transplant (allo- HSCT or allo-SCT). The patient may present extramedullary disease at screening.

[0308] The patient may undergo leukapheresis to prepare the population of CAR T cells specific for a B-cell malignancy.

[0309] The patient may have previously received chemotherapy but the BM blast percentage is still > 5%.

[0310] The BM blast percentage at screening may be more than or equal to 20%.

[0311] The BM blast percentage at screening may be more than or equal to 50%.

[0312] The BM blast percentage at screening may be more than or equal to 75%.

[0313] The patient having a B-cell malignancy who is about to receive treatment with CAR-T cells is understood to be a patient who has been leukapheresed and is waiting for the CAR- T cells to be produced and administered.

[0314] Bridging therapy, as used herein, refers to a therapeutic treatment or tool to stabilize or debulk disease between leukapheresis and CAR T cell administration.

[0315] The anti-CD22 antibody-drug conjugate may be administered at least 1 week before the administration of the lymphodepleting therapy. The anti-CD22 antibody-drug conjugate may be administered at least 2 weeks before the administration of the lymphodepleting or pre-conditioning therapy.

[0316] If 1 cycle of anti-CD22 antibody-drug conjugate is to be administered, at least 7 days washout may be required prior to the start of preconditioning chemotherapy. If 2 cycles of anti-CD22 antibody-drug conjugate were administered, at least 2 weeks washout may be required.

[0317] The patient may receive a lymphodepleting pre-conditioning treatment before CD 19 CAR- T cell infusion. The patient may be administered a preconditioning regimen comprising 120 mg / m2fludarabine (Flu) and 1000 mg / m2cyclophosphamide (Cy). This preconditioning regimen may be administered prior to CAR-T cell administration.

[0318] Fludarabine may be administered at 30 mg / m2 on Day -6, Day -5, Day -4, and Day -3 prior to being administered the first dose of CAR-T cells (i.e. Day 0). Fludarabine may be administered intravenously. Fludarabine may be administered over 30 min. Cyclophosphamide may be administered at 500 mg / m2on Day -6, and Day -5 prior to being administered the first dose of CAR-T cells (i.e. Day 0). Cyclophosphamide may be administered intravenously. Cyclophosphamide may be administered over 30 min.

[0319] The population of CAR T cells specific for a B-cell malignancy may be autologous.

[0320] The anti-CD22 antibody-drug conjugate may be administered after leukapheresis.

[0321] The anti-CD22 antibody-drug conjugate may be selected from Inotuzumab ozogamicin (Besponsa), inotuzumab, and epratuzumab. The anti-CD22 antibody-drug conjugate may be Inotuzumab ozogamicin.

[0322] The B-cell malignancy may be selected from the group consisting of acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia or WM), cutaneous B-cell lymphoma (CBCL), diffuse large B-cell lymphoma (DLBCL), and anaplastic large cell lymphoma (ALCL).

[0323] The CAR T cells may be specific to CD 19 or CD20.

[0324] The CAR-T cells may be specific to CD 19.

[0325] The anti-CD19 CAR may be a CAR as described in the context of chimeric antigen receptors (CARs) in previous sections, and its definitions and embodiments apply equally to this aspect of the invention.

[0326] The anti-CD19 CAR may comprise: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

[0327] CDR1 - GYAFSSS (SEQ ID No. 1);

[0328] CDR2 - YPGDED (SEQ ID No. 2)

[0329] CDR3 - SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID No. 4);

[0330] CDR2 - DTSKLAS (SEQ ID No. 5)

[0331] CDR3 - QQWNINPLT (SEQ ID No. 6). The anti-CD19 CAR may comprise: a) a heavy chain variable region (VH) having CDRs with the following sequences: CDR1 - GVSLPDYG (SEQ ID NO: 25);

[0332] CDR2 - IWGSETT (SEQ ID NO: 26)

[0333] CDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 27); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - QDISKY (SEQ ID NO: 28);

[0334] CDR2 - HTS

[0335] CDR3 - QQGNTLPYT (SEQ ID NO: 29).

[0336] 6.2. Method for improving the outcome of treatment with CAR-T cells for a patient having a B-cell malignancy

[0337] The present invention also provides a method for improving the outcome of treatment with CAR-T cells for a patient having a B-cell malignancy, wherein the patient presents > 5% blasts in the BM at screening, which method comprises the step of administering an anti- CD22 antibody-drug conjugate to the patient after leukapheresis but prior to preconditioning the patient for CAR-T cell treatment.

[0338] The patient may be of Caucasian, Black, Latino or Hispanic, Asian or any other ethnicity.

[0339] The patient may be Latino or Hispanic.

[0340] The patient may have a complex karyotype. Complex karyotype refers to cytogenetic risks of hematologic malignancies associated with a poor prognosis that are well known in the art.

[0341] The patient may have received at least one prior line of therapy. The patient may have received at least two prior lines of therapy. The patient may have received at least three prior lines of therapy. The patient may have received at least four prior lines of therapy. The patient may have received at least five prior lines of therapy. The patient may have received at least six prior lines of therapy.

[0342] The at least one prior line of therapy may have been inotuzumab ozogamicin and / or blinatumomab. The patient may have received an allogeneic hematopoietic stem cell transplant (allo- HSCT or allo-SCT).

[0343] The patient may present extramedullary disease at screening.

[0344] The patient may present > 20% blasts in the BM at screening.

[0345] The patient may present > 50% blasts in the BM at screening.

[0346] The patient may present > 75% blasts in the BM at screening.

[0347] The anti-CD22 antibody-drug conjugate may be administered at least 1 week before the administration of the lymphodepleting therapy. The anti-CD22 antibody-drug conjugate may be administered at least 2 weeks before the administration of the lymphodepleting or pre-conditioning therapy.

[0348] If 1 cycle of anti-CD22 antibody-drug conjugate is to be administered, at least 7 days washout may be required prior to the start of preconditioning chemotherapy. If 2 cycles of anti-CD22 antibody-drug conjugate were administered, at least 2 weeks washout may be required.

[0349] The patient may receive a lymphodepleting pre-conditioning treatment before CD 19 CAR- T cell infusion. The patient may be administered a preconditioning regimen comprising 120 mg / m2fludarabine (Flu) and 1000 mg / m2cyclophosphamide (Cy). This preconditioning regimen may be administered prior to CAR-T cell administration.

[0350] Fludarabine may be administered at 30 mg / m2 on Day -6, Day -5, Day -4, and Day -3 prior to being administered the first dose of CAR-T cells (i.e. Day 0). Fludarabine may be administered intravenously. Fludarabine may be administered over 30 min.

[0351] Cyclophosphamide may be administered at 500 mg / m2on Day -6, and Day -5 prior to being administered the first dose of CAR-T cells (i.e. Day 0). Cyclophosphamide may be administered intravenously. Cyclophosphamide may be administered over 30 min.

[0352] The population of CAR T cells specific for a B-cell malignancy may be autologous.

[0353] The anti-CD22 antibody-drug conjugate may be administered after leukapheresis. The anti-CD22 antibody-drug conjugate may be selected from Inotuzumab ozogamicin (Besponsa), inotuzumab, and epratuzumab. The anti-CD22 antibody-drug conjugate may be Inotuzumab ozogamicin.

[0354] The B-cell malignancy may be selected from the group consisting of acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia or WM), cutaneous B-cell lymphoma (CBCL), diffuse large B-cell lymphoma (DLBCL), and anaplastic large cell lymphoma (ALCL).

[0355] The CAR T cells may be specific to CD 19 or CD20.

[0356] The CAR-T cells may be specific to CD 19.

[0357] The anti-CD19 CAR may be a CAR as described in the context of chimeric antigen receptors (CARs) in previous sections, and its definitions and embodiments apply equally to this aspect of the invention.

[0358] The anti-CD19 CAR may comprise: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

[0359] CDR1 - GYAFSSS (SEQ ID No. 1);

[0360] CDR2 - YPGDED (SEQ ID No. 2)

[0361] CDR3 - SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID No. 4);

[0362] CDR2 - DTSKLAS (SEQ ID No. 5)

[0363] CDR3 - QQWNINPLT (SEQ ID No. 6).

[0364] The anti-CD19 CAR may comprise: a) a heavy chain variable region (VH) having CDRs with the following sequences: CDR1 - GVSLPDYG (SEQ ID NO: 25);

[0365] CDR2 - IWGSETT (SEQ ID NO: 26) CDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 27); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - QDISKY (SEQ ID NO: 28);

[0366] CDR2 - HTS

[0367] CDR3 - QQGNTLPYT (SEQ ID NO: 29).

[0368] 6.3. Method for treating a B-cell malignancy

[0369] The present invention also provides a method for treating a B-cell malignancy in a subject which comprises the step of using anti-CD22 antibody-drug conjugate as bridging therapy prior to treatment with anti-CD19 CAR-T cells, wherein the CAR comprises: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

[0370] CDR1 - GYAFSSS (SEQ ID No. 1);

[0371] CDR2 - YPGDED (SEQ ID No. 2)

[0372] CDR3 - SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID No. 4);

[0373] CDR2 - DTSKLAS (SEQ ID No. 5) CDR3 - QQWNINPLT (SEQ ID No. 6).

[0374] This aspect of the invention may be formulated as an anti-CD22 antibody-drug conjugate for use in the treatment of a B-cell malignancy in a subject, wherein the anti-CD22 antibody-drug conjugate is used as bridging therapy prior to treatment with anti-CD19 CAR-T cells, wherein the CAR comprises: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

[0375] CDR1 - GYAFSSS (SEQ ID No. 1);

[0376] CDR2 - YPGDED (SEQ ID No. 2)

[0377] CDR3 - SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID No. 4);

[0378] CDR2 - DTSKLAS (SEQ ID No. 5)

[0379] CDR3 - QQWNINPLT (SEQ ID No. 6).

[0380] This aspect of the invention may be formulated as use of an anti-CD22 antibody-drug conjugate in the manufacture of a medicament for the treatment of a B-cell malignancy in a subject, wherein the anti-CD22 antibody-drug conjugate is used as bridging therapy prior to treatment with anti-CD19 CAR-T cells, wherein the CAR comprises: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

[0381] CDR1 - GYAFSSS (SEQ ID No. 1);

[0382] CDR2 - YPGDED (SEQ ID No. 2)

[0383] CDR3 - SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID No. 4);

[0384] CDR2 - DTSKLAS (SEQ ID No. 5)

[0385] CDR3 - QQWNINPLT (SEQ ID No. 6).

[0386] The following definitions and embodiments apply equally to the aspects relating to the method for treating a B-cell malignancy, the anti-CD22 antibody-drug conjugate for use in the treatment of a B-cell malignancy, and the use of an anti-CD22 antibody-drug conjugate in the manufacture of a medicament for the treatment of a B-cell malignancy.

[0387] The method for treating a B-cell malignancy may comprise the following steps: i) administering the anti-CD22 antibody-drug conjugate to the patient after leukapheresis; ii) pre-conditioning the patient; and iii) administering anti CD- 19 CAR-T cells to the patient.

[0388] The method for treating a B-cell malignancy may comprise the following steps: i) leukapheresis of the patient to obtain a T cell composition for preparation of the anti-CD19 CAR-T cells; ii) administering the anti-CD22 antibody-drug conjugate to the patient; iii) pre-conditioning the patient; and iv) administering the anti CD-19 CAR-T cells to the patient.

[0389] The anti-CD19 CAR may be a CAR as described in the context of chimeric antigen receptors (CARs) in previous sections, and its definitions and embodiments apply equally to this aspect of the invention.

[0390] The anti-CD19 CAR may comprise: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

[0391] CDR1 - GYAFSSS (SEQ ID No. 1);

[0392] CDR2 - YPGDED (SEQ ID No. 2)

[0393] CDR3 - SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID No. 4);

[0394] CDR2 - DTSKLAS (SEQ ID No. 5) CDR3 - QQWNINPLT (SEQ ID No. 6).

[0395] The patient may be of Caucasian, Black, Latino or Hispanic, Asian or any other ethnicity.

[0396] The patient may be Latino or Hispanic.

[0397] The patient may have a complex karyotype. Complex karyotype refers to cytogenetic risks of hematologic malignancies associated with a poor prognosis that are well known in the art.

[0398] The patient may have received at least one prior line of therapy. The patient may have received at least two prior lines of therapy. The patient may have received at least three prior lines of therapy. The patient may have received at least four prior lines of therapy. The patient may have received at least five prior lines of therapy. The patient may have received at least six prior lines of therapy.

[0399] The at least one prior line of therapy may have been inotuzumab ozogamicin and / or blinatumomab.

[0400] The patient may have received an allogeneic hematopoietic stem cell transplant (allo- HSCT or allo-SCT).

[0401] The patient may present extramedullary disease at screening.

[0402] The patient may present > 20% blasts in the BM at screening.

[0403] The patient may present > 50% blasts in the BM at screening.

[0404] The patient may present > 75% blasts in the BM at screening. The anti-CD22 antibody-drug conjugate may be administered at least 1 week before the administration of the lymphodepleting therapy. The anti-CD22 antibody-drug conjugate may be administered at least 2 weeks before the administration of the lymphodepleting or pre-conditioning therapy.

[0405] If 1 cycle of anti-CD22 antibody-drug conjugate is to be administered, at least 7 days washout may be required prior to the start of preconditioning chemotherapy. If 2 cycles of anti-CD22 antibody-drug conjugate were administered, at least 2 weeks washout may be required.

[0406] The patient may receive a lymphodepleting pre-conditioning treatment before CD 19 CAR- T cell infusion. The patient may be administered a preconditioning regimen comprising 120 mg / m2fludarabine (Flu) and 1000 mg / m2cyclophosphamide (Cy). This preconditioning regimen may be administered prior to CAR-T cell administration.

[0407] Fludarabine may be administered at 30 mg / m2 on Day -6, Day -5, Day -4, and Day -3 prior to being administered the first dose of CAR-T cells (i.e. Day 0). Fludarabine may be administered intravenously. Fludarabine may be administered over 30 min.

[0408] Cyclophosphamide may be administered at 500 mg / m2on Day -6, and Day -5 prior to being administered the first dose of CAR-T cells (i.e. Day 0). Cyclophosphamide may be administered intravenously. Cyclophosphamide may be administered over 30 min.

[0409] The population of CAR T cells specific for a B-cell malignancy may be autologous.

[0410] The anti-CD22 antibody-drug conjugate may be administered after leukapheresis.

[0411] The anti-CD22 antibody-drug conjugate may be selected from Inotuzumab ozogamicin (Besponsa), inotuzumab, and epratuzumab. The anti-CD22 antibody-drug conjugate may be Inotuzumab ozogamicin.

[0412] The B-cell malignancy may be selected from the group consisting of acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia or WM), cutaneous B-cell lymphoma (CBCL), diffuse large B-cell lymphoma (DLBCL), and anaplastic large cell lymphoma (ALCL). The invention also contemplates a method for treating a B-cell malignancy in a subject which comprises the step of using anti-CD22 antibody-drug conjugate as bridging therapy prior to treatment with anti-CD19 CAR-T cells, wherein the CAR comprises: a) a heavy chain variable region (VH) having CDRs with the following sequences: CDR1 - GVSLPDYG (SEQ ID NO: 25);

[0413] CDR2 - IWGSETT (SEQ ID NO: 26)

[0414] CDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 27); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - QDISKY (SEQ ID NO: 28);

[0415] CDR2 - HTS

[0416] CDR3 - QQGNTLPYT (SEQ ID NO: 29).

[0417] This aspect of the invention may be formulated as an anti-CD22 antibody-drug conjugate for use in the treatment of a B-cell malignancy in a subject, wherein the anti-CD22 antibody-drug conjugate is used as bridging therapy prior to treatment with anti-CD19 CAR-T cells, wherein the CAR comprises: a) a heavy chain variable region (VH) having CDRs with the following sequences: CDR1 - GVSLPDYG (SEQ ID NO: 25);

[0418] CDR2 - IWGSETT (SEQ ID NO: 26)

[0419] CDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 27); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - QDISKY (SEQ ID NO: 28);

[0420] CDR2 - HTS

[0421] CDR3 - QQGNTLPYT (SEQ ID NO: 29).

[0422] This aspect of the invention may be formulated as use of an anti-CD22 antibody-drug conjugate in the manufacture of a medicament for the treatment of a B-cell malignancy in a subject, wherein the anti-CD22 antibody-drug conjugate is used as bridging therapy prior to treatment with anti-CD19 CAR-T cells, wherein the CAR comprises: a) a heavy chain variable region (VH) having CDRs with the following sequences: CDR1 - GVSLPDYG (SEQ ID NO: 25);

[0423] CDR2 - IWGSETT (SEQ ID NO: 26)

[0424] CDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 27); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - QDISKY (SEQ ID NO: 28);

[0425] CDR2 - HTS

[0426] CDR3 - QQGNTLPYT (SEQ ID NO: 29).

[0427] The following definitions and embodiments apply equally to the aspects relating to the method for treating a B-cell malignancy, the anti-CD22 antibody-drug conjugate for use in the treatment of a B-cell malignancy, and the use of an anti-CD22 antibody-drug conjugate in the manufacture of a medicament for the treatment of a B-cell malignancy.

[0428] The method for treating a B-cell malignancy may comprise the following steps: i) administering the anti-CD22 antibody-drug conjugate to the patient after leukapheresis; ii) pre-conditioning the patient; and iii) administering anti CD- 19 CAR-T cells to the patient.

[0429] The method for treating a B-cell malignancy may comprise the following steps: i) leukapheresis of the patient to obtain a T cell composition for preparation of the anti-CD19 CAR-T cells; ii) administering the anti-CD22 antibody-drug conjugate to the patient; iii) pre-conditioning the patient; and iv) administering the anti CD-19 CAR-T cells to the patient.

[0430] The anti-CD19 CAR may be a CAR as described in the context of chimeric antigen receptors (CARs) in previous sections, and its definitions and embodiments apply equally to this aspect of the invention.

[0431] The anti-CD19 CAR may comprise: a) a heavy chain variable region (VH) having CDRs with the following sequences: CDR1 - GVSLPDYG (SEQ ID NO: 25);

[0432] CDR2 - IWGSETT (SEQ ID NO: 26)

[0433] CDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 27); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - QDISKY (SEQ ID NO: 28);

[0434] CDR2 - HTS

[0435] CDR3 - QQGNTLPYT (SEQ ID NO: 29). The patient may be of Caucasian, Black, Latino or Hispanic, Asian or any other ethnicity.

[0436] The patient may be Latino or Hispanic.

[0437] The patient may have a complex karyotype. Complex karyotype refers to cytogenetic risks of hematologic malignancies associated with a poor prognosis that are well known in the art.

[0438] The patient may have received at least one prior line of therapy. The patient may have received at least two prior lines of therapy. The patient may have received at least three prior lines of therapy. The patient may have received at least four prior lines of therapy. The patient may have received at least five prior lines of therapy. The patient may have received at least six prior lines of therapy.

[0439] The at least one prior line of therapy may have been inotuzumab ozogamicin and / or blinatumomab.

[0440] The patient may have received an allogeneic hematopoietic stem cell transplant (allo- HSCT or allo-SCT).

[0441] The patient may present extramedullary disease at screening.

[0442] The patient may present > 20% blasts in the BM at screening.

[0443] The patient may present > 50% blasts in the BM at screening.

[0444] The patient may present > 75% blasts in the BM at screening.

[0445] The anti-CD22 antibody-drug conjugate may be administered at least 1 week before the administration of the lymphodepleting therapy. The anti-CD22 antibody-drug conjugate may be administered at least 2 weeks before the administration of the lymphodepleting or pre-conditioning therapy.

[0446] If 1 cycle of anti-CD22 antibody-drug conjugate is to be administered, at least 7 days washout may be required prior to the start of preconditioning chemotherapy. If 2 cycles of anti-CD22 antibody-drug conjugate were administered, at least 2 weeks washout may be required.

[0447] The patient may receive a lymphodepleting pre-conditioning treatment before CD 19 CAR- T cell infusion. The patient may be administered a preconditioning regimen comprising 120 mg / m2fludarabine (Flu) and 1000 mg / m2cyclophosphamide (Cy). This preconditioning regimen may be administered prior to CAR-T cell administration.

[0448] Fludarabine may be administered at 30 mg / m2 on Day -6, Day -5, Day -4, and Day -3 prior to being administered the first dose of CAR-T cells (i.e. Day 0). Fludarabine may be administered intravenously. Fludarabine may be administered over 30 min.

[0449] Cyclophosphamide may be administered at 500 mg / m2on Day -6, and Day -5 prior to being administered the first dose of CAR-T cells (i.e. Day 0). Cyclophosphamide may be administered intravenously. Cyclophosphamide may be administered over 30 min.

[0450] The population of CAR T cells specific for a B-cell malignancy may be autologous.

[0451] The anti-CD22 antibody-drug conjugate may be administered after leukapheresis.

[0452] The anti-CD22 antibody-drug conjugate may be selected from Inotuzumab ozogamicin (Besponsa), inotuzumab, and epratuzumab. The anti-CD22 antibody-drug conjugate may be Inotuzumab ozogamicin.

[0453] The B-cell malignancy may be selected from the group consisting of acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia or WM), cutaneous B-cell lymphoma (CBCL), diffuse large B-cell lymphoma (DLBCL), and anaplastic large cell lymphoma (ALCL).

[0454] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0455] EXAMPLES

[0456] 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. Example 1: Generation of a CD19 CAR-T cell composition

[0457] CAT was chosen as a binding domain as it showed a substantially lower affinity to CD 19 (>40-fold) than FMC63 scFv, a binder used in already marketed CAR T-cell therapies

[0458] Lentiviral vectors were generated expressing a second-generation CD 19 CAR (SEQ ID NO: 51) (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. See Figure 1 A- B and Figure 14.

[0459] AUTO1 was generated by ex vivo transduction of activated peripheral blood mononuclear cells (PBMCs) using an engineered HIV derived lentiviral vector containing the CD 19 CAR expression cassette. The lentiviral vector was produced under Good Manufacturing Practice (GMP) conditions by four-plasmid co-transfection of HEK293T cells and subsequent harvest and purification of the culture supernatant.

[0460] In brief, cells from the leukapheresate starting material were stimulated with mitogenic ligands and cytokines. One day after activation, cells were transduced with the lentiviral vector. Post-transduction, cells were expanded (drug substance) to produce the desired dose. The cells were then washed and formulated with a phosphate buffered saline (PBS) / ethylenediaminetetraacetic acid (EDTA) / human serum albumin (HSA) / dimethyl sulfoxide (DMSO) buffer and filled into final packaging and cryopreserved (drug product). The AUTO1 product consisted of transduced and non-transduced T cells. The dose given to patients was expressed as the total number of CD 19 CAR-positive T cells (the active substance).

[0461] Example 2: Non-clinical studies

[0462] The non-clinical results provided in the subsequent sections demonstrate that targeting of CD 19 by CD 19 (CAT) CAR is specific and efficacious in vitro and in vivo.

[0463] Binding Kinetics of the CAT Binder in AUTO1

[0464] Binding kinetics of the CAR binding domains CAT scFv (used in AUTO1) and FMC63 scFv (used in axicabtagene ciloleucel and tisagenlecleucel) with recombinant CD 19 were investigated using surface plasmon resonance. Equilibrium dissociation constants (KD) of 14.4 nM for CAT and 0.328 nM for FMC63 were determined, when the data were fitted to a 1 : 1 Langmuir binding model.

[0465] The > 40-fold lower KD with the CAT scFv was the result of a much faster off-rate (CAT: 3.1 x 10'3s'1versus FMC63: 6.8 x 10'5s'1), while the on-rate was equivalent (CAT: 2.2 x 105M'1versus FMC63: 2.1 x 105M' ) (Table 4).

[0466] Table 4: Summary of Binding Kinetics of the CAR Binding Domains CAT scFv and FMC63 scFv.

[0467] Abbreviations: CAR=chimeric antigen receptor; ka=association constant; kd=dissociation constant; KD=affinity, equilibrium dissociation constant; scFv=small chain variable fragment.

[0468] Proliferation of CD 19 CAR T Cells in Vitro

[0469] CD 19 (CAT) CAR T cell proliferation was greater than CD 19 (FMC63) CAR T cells when co-cultured with Raji and NALM-6 cells (Figure 2). The enhanced proliferation was not a result of increased IL-2 production suggesting an IL-2 independent mechanism.

[0470] Cytokine Production and Cytotoxicity of CD19 CAR T Cells

[0471] Normal donor T cells were transduced to express CD 19 (CAT) CAR or CD 19 (FMC63) CAR, challenged with Raji cells (a Burkitt’s lymphoma cell line that expresses CD19), and pro-inflammatory cytokines were analysed in the supernatant at 48 hours. The cytokine release profiles were similar for both CAR expressing T cell lines with the exception of a higher tumour necrosis factor a secretion from the CD 19 (CAT) CAR T cells than from the CD19 (FMC63) CAR T cells (mean 750.7 ± 103.3 pg / mL, and 292.1 ± 36.51 pg / mL, respectively; n=4, p<0.01) (Figure 3).

[0472] In a standard51Cr release assay, T cells transduced to express CD19 (CAT) CAR or CD19 (FMC63) CAR were incubated with CD 19-negative and CD 19 expressing targets (Figure 4A and Figure 4B, respectively). Cell killing was significantly greater for CD 19 (CAT) CAR T cells than for CD 19 (FMC63) CAR T cells, particularly at low effectortarget ratios.

[0473] In Vivo Pharmacology

[0474] In vivo testing of CD 19 CAR T cells often relies on xenograft models since (human) CD 19 CARs do not typically cross-react with mouse CD 19. The most commonly used model is the NALM-6 non-obese diabetic / severe combined immunodeficiency / gamma (NSG)- mouse xenograft model. NALM-6 is a CD19 expressing, human B-cell line established from the peripheral blood of a 19-year old man with ALL in relapse. The NSG mice are immunodeficient, lack mature T cells, B cells and natural killer cells, and are deficient in multiple cytokine signalling pathways. They permit the engraftment of a wide variety of primary human cells.

[0475] Anti-tumour efficacies of CD 19 (CAT) CAR T cells and CD 19 (FMC63) CAR T cells were assessed in the NALM-6 NSG-mouse xenograft model and compared. In control mice receiving non-transduced T cells, rapid, disseminated tumour infiltration was observed (Figure 5). CD 19 (FMC63) CAR T cells slowed but did not prevent growth of the tumour. In contrast, equivalent numbers of CD 19 (CAT) CAR T cells resulted in tumour regression. On Day 12 post-T cell injection, substantial differences were seen in tumour burden; mean CD 19 (CAT) CAR T cells: 1.1 x 108to 9.3 x 107photons / second / cm2 / steradian; CD19 (FMC63) CAR T cells 3.2 x 109to 7.7 x 108photons / second / cm2 / steradian, n=18, p<0.001.

[0476] Two weeks after the infusion of NALM-6 NSG mice with CAR T cells, blood and bone marrow (BM) were analysed for residual tumour cells and persisting CAR T cells. A significantly higher number of residual NALM-6 tumour cells were observed in the BM of the CD 19 (FMC63) CAR T cell cohort compared with the CD 19 (CAT) CAR T cell cohort (mean 2.8 x 105versus 3 x 102NALM-6 cells / mL, p<0.0001) (Figure 6).

[0477] Conversely, a significantly greater absolute number of CD 19 (CAT) CAR T cells than CD19 (FMC63) CAR T cells persisted in the BM of NALM-6 NSG mice (mean 3.4 x 104CD19 (CAT) CAR T cells / mL versus 1.3 x 104CD19 (FMC63) CAR T cells / mL, n=18, p<0.05) and in the peripheral blood (mean: 1.9 x l()4CD 19 (CAT) CAR T cells / mL versus 2.8 x 103CD19 (FMC63) CAR T cells / mL, n=9, p<0.001) (Figure 6). In summary, the CAT scFv binding domain used in AUTO1 had a > 40-fold lower affinity to recombinant CD 19 than the FMC63 scFv binding domain used in other CAR T products in vitro. CD 19 (CAT) CAR enables transduced T cells to proliferate, secrete cytokines in response to CD 19-positive targets and specifically lyse CD 19-positive cell lines in vitro with greater cytotoxicity compared to CD 19 (FMC63) CAR T cells. CD 19 (CAT) CAR T cells also showed better anti -tumour efficacy and engraftment versus CD 19 (FMC63) CAR T cells in an NSG NALM-6 mouse model of leukaemia.

[0478] Due to the restrictions of testing CAR T cells in conventional toxicological, safety and efficacy studies, the non-clinical programme focuses on verifying specificity and potency of anti-tumour activity.

[0479] It was demonstrated both in vitro and in vivo that CD 19 (CAT) CAR T cells can effectively eliminate CD19-expressing tumour cells with greater efficacy compared to CD 19 (FMC63) CAR T cells. Non-clinical studies suggested that no off-target toxicity was anticipated.

[0480] Example 3: Clinical study

[0481] A study of the safety and clinical efficacy to the CD19CAR T-cell product (also identified herein as AUTO1) was initiated in adult patients with relap sed / refractory B-ALL. This was an open-label, multi-centre, single arm Phase Ib / II, pivotal study. The enrolled patients had R / R B-ALL at screening with either morphological disease >5% bone marrow (BM) blasts (Cohort A), or in >2ndcomplete remission (CR) / CR with incomplete hematologic recovery (CRi) with measurable residual disease (MRD) (Cohort B), or with isolated extramedullary disease (EMD) (Cohort C). The Phase lb part of the study enrolled Cohorts A and B; the Phase II part enrolled Cohorts A, B, and C.

[0482] The results shown herein are as of 16thMarch 2023, in the morphological Phase II Cohort A of the clinical study.

[0483] A total of 112 patients were enrolled. Eighteen of the 112 enrolled patients discontinued before receiving pre-conditioning therapy and AUTO1 infusion due to death (11 patients), manufacturing related issues (5 patients), adverse events (AE; 1 patient) and physician’s decision (1 patient). A total of 94 patients received AUT01 (Figure 8, right panel). Therefore, 84% of enrolled patients were infused with AUT01.

[0484] The study design is summarised in Figure 7. Briefly, all 94 treated patients received preconditioning chemotherapy with fludarabine 30 mg / m2 / day over 4 days (Days -6 to -3) and cyclophosphamide 500 mg / m2 / day over 2 days (Days -6 to -5) prior to receiving a split dose of AUTO1 infusion. The dosing schedule is based on disease burden determined prior to the start of pre-conditioning on Day -6.

[0485] Patients received a total target dose of 410 x 106CD 19 CAR-positive T cells (±25%) on a schedule as illustrated below (Table 5).

[0486] Table 5: AUTO1 Dosing Regimen

[0487] BM = bone marrow; CAR = chimeric antigen receptor; CD = cluster of differentiation.

[0488] For the avoidance of doubt, the dosing schedule shown in Table 5 defines the day on which the first dose of CD 19 CAR-T cells is administered as Day 1 and the second dose as Day 10. The number of days passed between the first dose and the second dose was 9 days ±2 days and there is no Day 0. Therefore, the time of administration of the first dose can be considered as Day 0 and the time of administration of the second dose can be considered as Day 9 ±2 days.

[0489] Patients who developed AUTOl-related >Grade 3 CRS and / or >Grade 2 ICANS or > Grade 3 pulmonary or cardiac toxicities following the first split dose did not receive the second split dose. Patients with Grade 2 CRS and / or Grade 1 ICANS following the first split dose could receive the second dose on Day 10 (±2 days) only if CRS had resolved to Grade 1 or less and ICANS had completely resolved.

[0490] The eligibility criteria were as follows (Figure 8, top left panel):

[0491] Inclusion Criteria: • Age 18 years or older Age 18 years or older;

[0492] • ECOG performance status of 0 or 1;

[0493] • Relapsed or refractory B cell ALL;

[0494] • Patients with Ph+ ALL are eligible if intolerant to TKI, failed two lines of any TKI, or failed one line of second-generation TKI, or if TKI is contraindicated;

[0495] • Documented CD 19 positivity within 1 month of screening;

[0496] • Phase lb: Primary Cohort IA: Presence of >5% blasts in BM at screening;

[0497] • Phase lb: Exploratory Cohort IB: MRD-positive defined as > IE-4 and <5% blasts in the BM at screening;

[0498] • Phase IL Primary Cohort IIA: Presence of >5% blasts in BM at screening;

[0499] • Phase IL Cohort IIB: >2nd CR or CRi with MRD-positive defined as >lE-3 by central ClonoSEQ® NGS testing and <5% blasts in the BM at screening;

[0500] • Adequate renal, hepatic, pulmonary, and cardiac function.

[0501] Exclusion Criteria:

[0502] • Phase lb (Cohort IA and Cohort IB) and Phase II (Cohort IIA and Cohort IIB) B- ALL with isolated EM disease;

[0503] • Diagnosis of Burkitt's leukaemia / lymphoma or CML lymphoid in blast crisis;

[0504] • History or presence of clinically relevant CNS pathology;

[0505] • Presence of CNS-3 disease or CNS-2 disease with neurological changes;

[0506] • Presence of active or uncontrolled fungal, bacterial, viral, or other infection requiring systemic antimicrobials for management;

[0507] • Active or latent Hepatitis B virus or active Hepatitis C virus;

[0508] • Human Immunodeficiency Virus (HIV), HTLV-1, HTLV-2, syphilis positive test;

[0509] • Prior CD 19 targeted therapy other than blinatumomab. Patients who have experienced Grade 3 or higher neurotoxicity following blinatumomab.

[0510] A summary of the primary and secondary endpoints for the study is shown in Figure 8, bottom left panel.

[0511] The primary endpoints for the study were as follows:

[0512] 1. ORR defined as proportion of patients achieving CR or CRi by central assessment.

[0513] [Time Frame: Up to 24 months]

[0514] The secondary endpoints for the study were as follows: 1. Phase II - Proportion of patients achieving MRD-negative CR by NGS [Time Frame: Up to 24 months]

[0515] 2. Phase II - Complete remission rate [Time Frame: Up to 24 months]

[0516] 3. Phase II - Response to AUT01 treatment measured as duration of remission (DOR) [Time Frame: Up to 24 months]

[0517] 4. Phase II - Response to AUT01 measured as progression-free survival (PFS). [Time Frame: Up to 24 months]

[0518] 5. Phase II -Response to AUT01 treatment measured as overall survival (OS) [Time Frame: Up to 24 months]

[0519] 6. Phase II - Frequency and severity of AEs and SAEs [Time Frame: Up to 24 months]

[0520] 7. Phase II - Incidence of severe hypogammaglobulinaemia [Time Frame: Up to 24 months]

[0521] 8. Phase II - Duration of severe hypogammaglobulinaemia [Time Frame: Up to 24 months]

[0522] 9. Phase II - Detection of CAR T cells measured by PCR following AUT01 infusion [Time Frame: Up to 24 months]

[0523] The baseline characteristics of the patient population is shown in Table 6. These characteristics revealed that these were heavily pre-treated patients with high disease burden.

[0524] Table 6. Baseline characteristics.

[0525] Manufacturability of AUT01

[0526] Patients’ leukapheresates were sent for AUT01 product manufacturing.

[0527] Results are shown in Figure 9. Ninety-six percent of products reached the target dose (Figure 9A). The median transduction efficiency was 72% (Figure 9B). The median time from vein to release was 21 days. In conclusion, AUT01 was successfully manufactured.

[0528] Disease response per Independent Response Review Committee (IRRC) assessment Patient response was assessed centrally by an Independent Response Review Committee (IRRC). The overall remission rate (ORR) was defined as the proportion of patients achieving complete remission (CR) or complete remission with incomplete count recovery (CRi).

[0529] The ORR was achieved in 76% of infused patients, with 54.3% achieving CR and 21.3% achieving CRi (Figure 10). Furthermore, 97% of responders with evaluable samples were MRD negative at 10'4level by flow cytometry.

[0530] The duration of remission was followed up (Figure 11). With a median follow-up of 9.5 months, 61% responders in ongoing remission without subsequent anti-cancer therapies. Response to AUTO1 treatment measured as the median of the duration of remission (DOR) was 14.1 months. The number of events was 18. Of note, 13% of responders who proceeded to stem cell transplant (SCT) while in remission were censored at the time of SCT.

[0531] Subgroup Analysis of CR / CRi (IRRC Assessment)

[0532] Figure 12 shows the analysis of ORR by patient subgroup. Of note, the patient population includes high-risk subgroups, such as extramedullary disease (EMD) and high bone marrow (BM) blasts at pre-conditioning. Safety: CRS and ICANS

[0533] The safety results are shown in Table 7.

[0534] Table 7. Overall, low rates of Grade >3 CRS and / or ICANS were observed. Tocilizumab and steroid were used to treat CRS in 53 / 94 (56%) and 16 / 94 (17%) patients, respectively. Three out of 94 (3%) patients required vasopressor for treatment of CRS. Six out of 7 (86%) Grade >3 ICANS were observed among patients with >75% BM blasts at preconditioning. Safety: Treatment emergent adverse events (TEAEs)

[0535] The safety results regarding treatment emergent adverse events (TEAEs) are shown in Table 8.

[0536] Table 8.

[0537] The most common Grade >3 TEAEs were neutropenia (36.2%), thrombocytopenia (25.5%), febrile neutropenia (25.5%), and anaemia (19.1%). Only 1 out of 94 (1%) death was considered AUTO 1 -related per investigator assessment (due to HLH and neutropenic sepsis).

[0538] AUTO1 expansion and persistence

[0539] AUTO1 expansion and persistence results are shown in Figure 13 and Table 9.

[0540] Table 9.

[0541] AUC, area under the curve; CV, coefficient of variation; Geo, geometric; PCR, polymerase chain reaction; SE, standard error.

[0542] Results obtained from the peripheral blood of all 94 patients with r / r B ALL infused with AUTO1 measured by qPCR showed expansion and persistence of CD 19 CAR-positive T cells (Figure 13) consistent with cellular kinetics data of AUTO1 in the ALLCAR19 study (Roddie C et al., 2021. J Clin Oncol 39:3352-63).

[0543] Example 4: Pooled analysis of all r / r B-ALL patients treated with CD19 CAT CAR T- cell product (AUTO1) in the Phase Ib / II study

[0544] Pooled analysis of data from all patients across all cohorts in the Phase Ib / II study (Cohort A: morphological disease*, Cohort B: minimal residual disease, and Cohort C: isolated extramedullary disease) treated to date is presented below, with a median follow-up time from first AUT01 infusion to data cut-off at 16.6 months. Methods

[0545] The clinical study methods were described in Example 3. Patients aged 18 years and over with r / r B-ALL were eligible for the study. The study was designed to recruit patients in three cohorts at screening: patients with morphologic disease, patients with MRD disease and patients with isolated extramedullary disease (Figure 15). A list of selected endpoints is shown in Figure 15. 153 patients were enrolled. Of these, 127 (83%) received an AUT01 infusion. Only seven patients did not receive AUT01 due to manufacturing failure. The patient disposition across all cohorts is shown in Figure 16.

[0546] The baseline characteristics of the patients revealed that this was a heavily pre-treated population, many with post-allogeneic SCT, and a high proportion of Hispanic or Latinos

[0547] (Table 10).

[0548] Table 10. *Unless otherwise stated; BM, bone marrow; EMD, extramedullary disease; SCT, stem cell transplant

[0549] Results

[0550] The data cut-off date is 13thSeptember 2023.

[0551] The results shown in Figure 17 demonstrated a robust and rapid manufacturing, despite variable and challenging starting material. The starting material for manufacture - the patient pheresis - was of variable quality with many pheresis having low or very low T cell content, which is unsurprisingly given the complex treatment history and disease burden in these patients. AUTO1 was released for 95% of patients, with a median time from vein-to- release of 22 days. Consistent manufacturing was observed, despite leukapheresis from patients with multiple lines of prior therapy (many with prior allogeneic SCT) and high leukemic burden. Despite this, the closed system manufacture resulted in highly consistent manufacture (e.g. see transduction efficiency and cell viability). Importantly median vein- to-release time was 22 days trending towards 20 days towards the end of the study.

[0552] Data from all 127 treated patients continued to demonstrate high remission rates (CR / CRi; 78%) and a favorable safety profile. CR / CRi rates in patients with morphological disease, defined as bone marrow (BM) blasts >5% or BM blasts <5% but with extramedullary disease, was 74% (n=73 / 98), with 95% of evaluated responders MRD negative. Of the remaining 29 patients who did not have morphologic disease at lymphodepletion, 100% were MRD-negative following AUTO1 treatment (n=29) (Figure 18).

[0553] The Forest plot in Figure 19 shows that AUTO1 demonstrated high CR / Cri rates across all subgroups, including in expected predictors of poor outcome including young adults, Hispanic and Latino patients and patients exhibiting EMD at lymphodepletion. It is notable that even patients with a high disease burden still had a high response rate.

[0554] The event free survival estimate (EFS) at 12-months was 50% across all patients (Figure 20). The median follow-up time was 16.6 months (range: 3.7-36.6 months). 17 / 99 (17%) responders proceeded to SCT while in remission.

[0555] Cellular kinetic data and Kaplan-Meier EFS curves (Table 11 and Figure 21, respectively) provided evidence for high expansion and persistence of CAR T population in responders and long durability of responses, with longest follow-up extending beyond 36 months, respectively. AUT01 had excellent expansion with a mean Cmax of 110,000 copies / pg with an AUCO-28 of 1.1 million copies (Table 11). In terms of persistence, Figure 21 shows a Kaplan-Meier EFS curve not of disease response but rather where loss of persistence detection is an event. CAR T cells can still be detected in about half of patients at the one year mark, and notably, among responders CAR T cells can be detected in 70% at the last follow-up.

[0556] Table 11 : AUTO pharmacokinetics.

[0557] AUC, area under the curve; Cmax, maximum concentration; CV, coefficient of variation; d, day; D, day; Geo, geometric; M, month; PB, peripheral blood; Tmax, time to maximum concentration.

[0558] Since bone-marrow burden was assessed at screening and at enrolment, it was demonstrated that leukemic burden at screening was not predictive of leukemic burden prior to lymphodepletion (Figure 22). However, lower leukemic burden at lymphodepletion is associated with better outcomes (Figure 23 and Table 12).

[0559] Table 12. Data from all 127 treated patients continued to demonstrate a favorable safety profile (Table 13). Two deaths were considered treatment-related per investigator assessment: neutropenic sepsis (n = 1); acute respiratory distress syndrome and ICANS (n = 1).

[0560] Table 13 CRS, cytokine release syndrome; ICANS, immune effector cell-associated neurotoxicity syndrome;

[0561] TEAE, treatment emergent adverse event.

[0562] Low rates of Grade >3 CRS and / or ICANS were observed, as shown in Figure 24. No grade >3 CRS and / or ICANS were observed in patients with <5% BM blasts at lymphodepletion. Vasopressors were used to treat CRS in 2.4% of patients. Only 15% of patients were admitted to the ICU. Overall, Gr >3 CRS was 2% and Gr >3 ICANS was 7%, with most severe cases of immunotoxicity occurring in patients with higher leukemic burden. Subgroup analysis demonstrated that EFS and safety, particularly rate of CRS and ICANS, were better in patients with lower disease burden at lymphodepletion (Table 14). Table 14: Summary of data overall and by bone-marrow blasts prior to lymphodepletion

[0563] *Morphological disease (defined as >5% BM blasts or presence of EMD regardless of BM blast status); -Event free survival (EFS), Bone marrow (BM); Extramedullary disease (EMD); complete remission (CR); Complete remission incomplete (CRi).

[0564] In conclusion:

[0565] AUTO 1 was successfully manufactured in 95% of leukapheresed patients;

[0566] High remission rates independent of leukemic burden at lymphodepletion;

[0567] 50% EFS estimate at 12 months, with only 17% of responders proceeding to SCT while in remission;

[0568] Favorable safety profile: 2% grade >3 CRS and 7% grade >3 ICANS;

[0569] Severe toxicity mostly limited to patients with high leukemic burden at lymphodepletion;

[0570] Durable remission rates and toxicity inversely correlated with leukemic burden at lymphodepletion; and

[0571] Assessment of leukemic burden at lymphodepletion is essential for risk / benefit stratification.

[0572] AUTO1 is effective treatment for R / R adult B-ALL, with better outcomes observed in patients with lower leukemic burden at lymphodepletion.

[0573] Example 5: AUTO1 for R / R B-ALL: The Impact of Bridging Therapy on Treatment Outcomes

[0574] To analyze the impact of bridging therapy with and without inotuzumab ozogamicin (INO) on clinical outcomes with AUTO1 in the Phase Ib / II study described in Examples 3 and 4. Methods

[0575] Patients aged >18 years with R / R B-ALL were enrolled; written informed consent was obtained. Patients received bridging therapy (chemotherapy with or without INO or tyrosine kinase inhibitors [TKI], single-agent INO, single-agent TKI, steroids, or rituximab) per investigator decision. Patients then underwent lymphodepletion (fludarabine, 4><30mg / m2; cyclophosphamide, 2><500mg / m2), followed by AUTO1 split dose infusions on Days 1 and 10 based on pre-lymphodepletion leukemic burden, to a target dose of 410* 106CAR-T cells. In this analysis, outcomes in patients who received bridging therapy with INO (with or without chemotherapy), bridging therapy without INO (including chemotherapy, TKI, chemotherapy with TKI, steroids, or rituximab), or no bridging therapy were described.

[0576] When inotuzumab was used as a bridging therapy:

[0577] If 1 cycle of Inotuzumab was administered, 7 days washout were required prior to the start of preconditioning chemotherapy.

[0578] If 2 cycles of Inotuzumab were administered, 2 weeks washout were required.

[0579] Results

[0580] A total of 127 patients were infused with AUTO1, and 118 (93%) received any bridging therapy. At data cut-off (13 September 2023), median follow-up was 16.6 mos (range 3.7-36.6). Overall, 18 (14%) patients received bridging therapy with INO, 100 (79%) patients received bridging therapy without INO, and 9 (7%) received no bridging therapy. Overall, 5 / 18 (28%), 27 / 100 (27%), and 4 / 9 (44%) had Philadelphia chromosome-positive B-ALL, 5 / 18 (28%), 34 / 100 (34%), and 1 / 9 (11%) had received INO prior to screening, and 3 / 18 (17%), 16 / 100 (16%), and 0 / 9 (0%) had received >4 lines of therapy prior to screening, in patients who received bridging with INO, bridging without INO, and no bridging therapy, respectively.

[0581] Median bone marrow (BM) blast percentage at screening was numerically higher in patients who received bridging with INO vs bridging without INO vs no bridging therapy (73%, 34%, and 20%, respectively), and INO bridging led to an effective reduction of BM blasts at pre-conditioning (2%, 51%, and 30%, respectively). Following AUTO1 infusion, the number of patients achieving complete response (CR) or CR with incomplete hematologic recovery (CRi) to AUTO1 was 15 / 18 (83%), 75 / 100 (75%), and 9 / 9 (100%) in patients who received bridging with INO vs bridging without INO vs no bridging therapy, respectively. Amongst responders, median duration of response (DOR; 95% confidence interval [CI]) was 21.2 months (5.1, not estimable [NE]) vs 14.1 months (8.1, NE) vs NE, respectively. Median event-free survival (EFS; 95% CI) was 22.1 months (4.1, NE) vs 9.0 months (6.0, NE) vs NE (Figure 25), and median overall survival (95% CI) was 23.8 months (4.8, NE) vs 14.1 months (10.7, NE) vs NE, in patients who received bridging with INO vs bridging without INO vs no bridging therapy, respectively. The 18-month EFS probability estimate (95% CI) was 56% (27.6, 76.3), 38% (26.4, 50.1), and 75% (12.8, 96.1) amongst the three groups, respectively. These results are shown in Table 15.

[0582] Table 15.

[0583] Table 16 shows data from the 94 patients treated with AUTO1 in Cohort A. Patients with a numerical higher blast % at screening used bridging with INO and these patients experienced a large decrease of blast % after INO bridging therapy.

[0584] Table 16

[0585]

[0586] Conclusion

[0587] INO-containing bridging therapies were effective in reducing disease burden and, despite higher baseline BM blast percentage, did not appear to have any negative efficacy impacts.

[0588] Thus, choice of bridging therapy prior to CD 19 CAR-T cell treatment, though influenced by clinical care variables, may impact outcomes.

[0589] 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. Example 6: AUTO1 for treating R / R B-ALL in a Phase Ib / II study: The Impact of Bridging Therapy on Treatment Outcomes. Longer follow-up

[0590] To analyse the impact of bridging therapy with and without inotuzumab ozogamicin (INO) on clinical outcomes with AUTO1 in the Phase Ib / II study described in Examples 3 and 4. This Example describes results with a longer follow-up than Example 5.

[0591] Methods

[0592] The clinical study is an open-label, multi-center, global, single-arm, phase Ib / II study evaluating the safety and efficacy of AUTO1 in patients aged >18 years with R / R B-ALL.

[0593] AUTO1 was administered according to a tumour burden-guided split dosing schedule to a target dose of 410* 106CAR T-cells (Figure 7).

[0594] Prior to AUTO1 administration, some patients received bridging therapy (BT) as necessary between leukapheresis and 1 week prior to fludarabine / cyclophosphamide lymphodepletion. The choice of BT was at the discretion of the investigator; however, use of blinatumomab as a BT agent was not permitted. In this analysis, outcomes in patients who received bridging therapy with inotuzumab (INO) (with or without chemotherapy), bridging therapy without INO (including chemotherapy, TKI, chemotherapy with TKI, steroids, or rituximab), or no bridging therapy were described.

[0595] When inotuzumab was used as a bridging therapy:

[0596] If 1 cycle of Inotuzumab was administered, 7 days washout were required prior to the start of preconditioning chemotherapy.

[0597] If 2 cycles of Inotuzumab were administered, 14 days washout were required.

[0598] Results

[0599] Patients and bridging therapy:

[0600] A total of 127 patients were infused with AUTO1, with 120 / 127 (94.5%) receiving both doses and 7 / 127 (5.5%) receiving only one dose.

[0601] A total of 118 / 127 (92.9%) patients received any type of BT.

[0602] At data cut-off (07 February 2024), median follow-up was 21.5 months (range: 8.6-41.4).

[0603] - Overall, 18 / 127 (14.2%) patients received BT with INO, 100 / 127 (78.7%) received without INO, and 9 / 127 (7.1%) received no BT. Baseline characteristics at screening were generally well balanced across the groups (Table 17); however, disease status differed widely across groups.

[0604] Table 17. Baseline characteristics by type of bridging therapy (BT).

[0605] * Classification of cytogenetic risks of hematologic malignancies associated with a poor prognosis. Allo-SCT, allogeneic stem cell transplant; BM, bone marrow; BT, bridging therapy; INO, inotuzumab ozogamicin; w / o, without.

[0606] Disease status

[0607] Surprisingly, INO BT led to a notable reduction in BM blasts (BM blast %) at lymphodepletion while BT without INO or no BT did not (Figure 27). Median BM blast percentage at lymphodepletion after BT with INO, BT without INO, and no BT, was 2.0%, 52.0%, and 30.0%, respectively (Figure 27). INO BT also led to a greater reduction in extramedullary disease (EMD) status at lymphodepletion (38.9% to 33.3%) compared with BT without INO (20.0% to 19.0%), while there was no change for the no BT group (22.2%).

[0608] Efficacy

[0609] In the BT with INO, BT without INO, and no BT groups, respectively:

[0610] Overall complete remission (CR) or CR with incomplete hematologic recovery (CRi) were 15 / 18 (83.3%), 75 / 100 (75.0%), and 9 / 9 (100%).

[0611] Longer EFS was observed in patients who received bridging therapy with INO compared with those who received bridging therapy without INO. Median event- free survival (EFS; 95% CI) was 22.1 months (4.1-NE) vs 9.0 months (6.0-15.0) vs NE (Figure 28A).

[0612] The 18-month EFS probability estimates (95% CI) were 57.4% (30.2-77.3), 34.4% (23.2-45.9), and 80.0% (20.4-96.9).

[0613] Median overall survival was 23.8 months (4.8-NE) vs 14.1 months

[0614] (11.5-NE) vs NE (Figure 28B).

[0615] Median duration of remission (DoR) (95% confidence interval [CI]) was

[0616] 21.2 months (5.1-not estimable [NE]) vs 14.2 months (8.2-NE) vs NE (Figure 28C).

[0617] Thus, longer EFS was observed in patients who received bridging therapy with INO compared with those who received bridging therapy without INO.

[0618] Safety

[0619] In the BT with INO, BT without INO, and no BT groups, respectively:

[0620] Rates of Grade >3 cytokine release syndrome (CRS) were 5.6%, 2.0%, and 0% (Table 18).

[0621] Rates of Grade >3 immune effector cell-associated neurotoxicity syndrome (ICANS) were 11.1%, 7.0%, and 0% (Table 18).

[0622] Rates of Grade >3 hepatobiliary disorders were 5.6%, 6.0%, and 11.1% (Table 18).

[0623] Table 18. Summary of TEAEs by type of BT.

[0624] BT, bridging therapy; CRS, cytokine release syndrome; ICANS, immune effector cell- associated neurotoxicity syndrome; INO, inotuzumab ozogamicin; TEAE, treatment- emergent adverse event; w / o, without.

[0625] Conclusions

[0626] • INO-containing BTs were effective in reducing BM disease prior to lymphodepletion and administration of AUTO1.

[0627] • Data suggest that reducing BM blasts as much as possible prior to lymphodepletion predicts EFS and OS outcomes; however, patients with high disease status at screening are still at higher risk overall.

[0628] • BT with INO was utilized in patients with higher risk disease (median 81.5% blasts at screening vs 40% in BT w / o INO group) and helped minimize the risk of CRS and ICANS without increasing liver toxicity.

[0629] • Choice of BT prior to AUTO1 treatment, though influenced by clinical care variables, may impact outcomes for patients with R / R B-ALL.

[0630] Example 7: AUTO1 for treating R / R B-ALL in a Phase Ib / II study: The Impact of Bridging Therapy on CAR T-cell expansion and persistence. Longer follow-up

[0631] At data cut-off (07 February 2024), CAR T-cell expansion was high in all groups, including BT with INO despite the substantial reduction in disease burden in this group at lymphodepletion (Table 19). Compared with the BT with INO group, both the BT without INO and no BT groups had notably higher post-infusion Cmax and AUCo-28d values, potentially due to higher disease burden at lymphodepletion. Table 19. CAR T-cell expansion by bridging therapy group.

[0632] AUC, area under the curve; BT, bridging therapy; CAR, chimeric antigen receptor; Cmax, maximal expansion of transgene / CAR positive T-cells; CV%, coefficient of variation; INO, inotuzumab ozogamicin; Tmax, time to maximal expansion.

[0633] When looking at CAR T-cell persistence by BT group, CAR T-cell persistence was observed in all groups (Figure 29).

[0634] Conclusions

[0635] • In the AUT01 for treating R / R B-ALL in a Phase Ib / II study, 93% of patients receiving AUT01 were bridged prior to lymphodepletion as per investigator choice.

[0636] • Bridging with inotuzumab ozogamicin was employed in patients with the highest disease burden and had the greatest reduction in pre-lymphodepletion BM blast percentage.

[0637] • High expansion and long-term persistence of AUT01 was observed in all the bridging therapy groups evaluated, with excellent outcomes.

[0638] This application claims the benefit of United Kingdom application No. 2405050.2 filed 9thApril 2024 and United Kingdom application No. 2408341.2 filed 11thJune 2024. This application is incorporated herein by reference in its entirety.

[0639] 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 method for selecting a bridging therapy for a patient having a B-cell malignancy who is about to receive treatment with CAR-T cells, which comprises the following steps:(i) determining the bone marrow (BM) blast percentage of the patient, and(ii) selecting the use of an anti-CD22 antibody-drug conjugate as bridging therapy if the patient presents more than or equal to 5% blasts in the BM at screening.

2. The method according to claim 1, where the patient has previously received chemotherapy but the BM blast percentage is still > 5%.

3. The method according to claim 1 or 2, wherein the BM blast percentage at screening is more than or equal to 20%.

4. The method according to any of claims 1 to 3, wherein the BM blast percentage at screening is more than 50%.

5. The method according to any of claims 1 to 4, wherein the BM blast percentage at screening is more than 75%.

6. The method according to any of claims 1 to 5, wherein the anti-CD22 antibodydrug conjugate is administered at least 1 week before the administration of the lymphodepleting therapy.

7. The method according to any of claims 1 to 6, wherein the population of CAR T cells specific for a B-cell malignancy is autologous.

8. The method according to any of claims 1 to 7, wherein the patient undergoes leukapheresis to prepare the population of CAR T cells specific for a B-cell malignancy.

9. The method according to any of claims 1 to 8, wherein the anti-CD22 antibodydrug conjugate is administered after leukapheresis.

10. The method according to any of claims 1 to 9, wherein the anti-CD22 antibodydrug conjugate is selected from Inotuzumab ozogamicin (Besponsa), inotuzumab, and epratuzumab.

11. The method according to any of claims 1 to 10, wherein the B-cell malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma(SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia or WM), cutaneous B-cell lymphoma (CBCL), diffuse large B-cell lymphoma (DLBCL), and anaplastic large cell lymphoma (ALCL).

12. The method according to any of claims 1 to 11, wherein CAR T cells are specific to CD 19 or CD20.

13. The method according to any of claims 1 to 12, wherein the CAR-T cells are specific to CD 19, wherein the CAR comprises a) 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); and b) 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).

14. The method according to any of claims 1 to 13, wherein the anti-CD22 antibodydrug conjugate is administered at least 1 week before the administration of the lymphodepleting therapy.

15. A method for improving the outcome of treatment with CAR-T cells for a patient having a B-cell malignancy, wherein the patient presents > 5% blasts in the BM at screening, which method comprises the step of administering an anti-CD22 antibody-drug conjugate to the patient after leukapheresis but prior to pre-conditioning the patient for CAR-T cell treatment.

16. A method according to claim 15, wherein the patient presents > 50% blasts in the BM at screening.

17. A method according to claim 16, wherein the patient presents > 75% blasts in the BM at screening.

18. A method for treating a B-cell malignancy in a subject which comprises the step of using anti-CD22 antibody-drug conjugate as bridging therapy prior to treatment with antiCD 19 CAR-T cells, wherein the CAR comprises a) 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); and b) 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).

19. A method according to claim 18, which comprises the following steps: i) administering the anti-CD22 antibody-drug conjugate to the patient after leukapheresis; ii) pre-conditioning the patient; and iii) administering anti CD- 19 CAR-T cells to the patient.

20. A method according to claim 18 or 19, which comprises the following steps: i) leukapheresis of the patient to obtain a T cell composition for preparation of the anti-CD19 CAR-T cells; ii) administering the anti-CD22 antibody-drug conjugate to the patient; iii) pre-conditioning the patient; and iv) administering the anti-CD-19 CAR-T cells to the patient.

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