WO2024246526A1

WO2024246526A1 - Cd19car t-cell treatment of relapsed/refractory b-cell acute lymphoblastic leukaemia

Info

Publication number: WO2024246526A1
Application number: PCT/GB2024/051396
Authority: WO
Inventors: Wolfram Brugger; Martin PULÉ; Yiyun Zhang; Vijay PEDDAREDDIGARI; Edgar Braendle
Original assignee: Autolus Ltd
Current assignee: Autolus Ltd
Priority date: 2023-05-30
Filing date: 2024-05-30
Publication date: 2024-12-05
Anticipated expiration: 2025-11-30

Abstract

The present disclosure relates to CD19 CAR-T cell products and methods of treating relapsed or refractory CD19+ haematological malignancies.

Description

CD19CAR T-CELL TREATMENT OF RELAPSED/REFRACTORY B-CELL ACUTE LYMPHOBLASTIC LEUKAEMIA

FIELD OF THE INVENTION

The disclosure relates to CD19 CAR T-cell products and methods for treating high risk or relapsed, CD 19+ haematological malignancies.

BACKGROUND TO THE INVENTION

B- cell acute lymphoblastic leukaemia (B-ALL) is a disease that occurs in both children and adults. As an acute leukaemia, it is a serious and life-threatening disease, and will progress rapidly if left untreated. In the United States (US), there will be an estimated 5,482 new cases of ALL and an estimated 1,500 related deaths in 2019. The overall prevalence of ALL in the US is estimated to be 49,415. In Europe, there will be an estimated 5,649 new cases of ALL and an estimated 1,700 deaths in 2019. The overall prevalence of the disease in Europe is estimated to be 51,099. Patients are predominantly children; approximately 60% of cases occur at age <20 years. The incidence of ALL peaks between ages 2 and 5 years and another peak occur in patients older than 50 years of age.

In adults, B-ALL chemotherapy enables 90% of adult patients to enter complete response (CR), but despite this, and in contrast to paediatric B-ALL, the prognosis of adult ALL is still poor and has not changed significantly during the last two to three decades with long-term remission rates limited to approximately 40%. Approximately 50% of all adult patients will relapse, and UKALL12 data shows that 5-year overall survival (OS) in adults who relapse following standard multi-agent chemotherapy is 7%. The only curative option for r/r ALL consists of achieving a second CR by salvage therapy followed by an allogeneic haematopoietic stem cell transplant (HSCT), since without consolidation by allogeneic HSCT, a subsequent relapse occurs in nearly all patients. However, less than half of patients achieve a second CR and thus only a subset will be eligible for this procedure. The main objective of salvage therapy currently is to try to attain second CR (ideally with minimal residual disease [MRD] negativity) and to offer allogeneic HSCT to potential candidates. Even then, for the fraction of patients who are candidates for allogeneic HSCT, less than one third are expected to sustain long-term disease-free survival. Furthermore, allogeneic HSCT is associated with severe morbidity and significant mortality.

A number of immunotherapeutic agents have been described for use in cancer treatment, including therapeutic monoclonal antibodies (mAbs), immunoconjugated mAbs, radioconjugated mAbs and bi-specific T-cell engagers. Immunotherapies such as blinatumomab, a bispecific T cell engager, have been recently approved in a Phase III study reporting that 44% of the treated patients achieved a CR/complete response with partial haematologic recovery (CRh)/CRi of which 76% also achieved MRD-negative remission (Blincyto SmPC 2019; https://www.ema.europa.eu/en/documents/product- information/blincyto-epar-product-information en.pdf). Clinical data suggest that the role of blinatumomab in r/r B-ALL is as a bridge to allogeneic HSCT. Furthermore, patients who achieve a response with blinatumomab but not proceed to HSCT have a worse prognosis than those who undergo HSCT. In a single institution study of 65 patients, event free survival (EFS) and OS was significantly improved in those patients who underwent consolidation with HSCT after response to blinatumomab compared to those who did not (Aldoss, et al., 2017, Am J Hematol 92:858-65). Thus, there is a need for more efficient therapies to treat ALL patients that do not require consolidation with HSCT.

Similarly, Philadelphia positive (Ph+) ALL patients with resistance or intolerance to one TKI may respond to another TKI typically administered with combination chemotherapy. However, the second response is likely to be short-lived, without allogeneic HSCT. Treatment with blinatumomab is an acceptable alternative therapy as a bridge to HSCT. In the single arm Alcantara trial, 36% of the patients achieved CR/CRh, 88% of which were MRD-negative, and 44% proceeded to allogeneic HSCT (Martinelli, et al., 2017, J Clin Oncol 35: 1795-1802). Thus, there is a need for standalone therapies to treat patients with Ph+ ALL without the need of a subsequent allogeneic HSCT.

Isolated extramedullary (EM) relapse of B-ALL is a rare event. In a review of 381 adult ALL patients who received HSCT at a single centre, 123 (32%) subsequently relapsed and 24 (20%) experienced isolated EM relapse. The survival outcomes after isolated EM relapse compared with systemic relapse did not show significant differences (Poon, et al., 2013, Bone Marrow Transplant 48:666-70). Thus, there is a need for innovative therapies to treat patients with isolated EM relapse. Hence, r/r ALL continues to remain a leading cause of cancer death in adults suffering from this haematological cancer. Since most of these patients have frequently had the maximal tolerable dose of chemotherapy, it would be highly desirable to develop safe and effective new therapies such as a CAR T cell therapy that could also avoid the mortality, and morbidity associated with HSCT.

Despite treatment advances, many patients with r/r B-ALL remain incurable with the currently established therapeutic modalities, including allogeneic haematopoietic stem cell transplant (HSCT). During the course of their treatment, most patients received the maximal tolerated dose of chemotherapy/radiotherapy so that novel treatment strategies are needed to address and overcome the limitations of current therapeutic modalities.

Chimeric antigen receptors (CARs) are proteins which graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus (binder), and a transmembrane domain connected to an endodomain which transmits T-cell activation signals. The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, which recognize a target antigen, fused via a trans-membrane domain to a signalling endodomain. Such molecules result in activation of the T-cell in response to recognition by the scFv of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. CARs have been developed against various tumour-associated antigens and many are currently undergoing clinical trials.

CD 19 is an ideal target for a CAR T cell therapy as it is a cell surface marker for lymphocytes that is present on most B cell malignancies, including >95% of B ALL cases. Its selective expression on the B cell lineage makes it an excellent target for immunotherapy approaches avoiding toxicity to the non-lymphoid tissues. Recently tisagenlecleucel (Kymriah®), a CD 19 targeted CAR T cell therapy has been approved in the US, European Union (EU), Switzerland, Canada and Australia for paediatric and young adult patients with B-ALL that is refractory, in relapse post-transplant or in second or later relapse. However, the treatment is associated with significant toxicities although manageable, such as cytokine release syndrome (CRS) and neurological toxicities. Grade 3 or higher CRS occurred in 49% of patients following treatment with tisagenlecleucel. Grade 3 or higher neurological toxicities were reported in 21% of patients following treatment with tisagenlecleucel (Kymriah US Prescribing Information 2018).

Another CD19 CAR, axicabtagene ciloleucel (Yescarta®), is approved in r/r diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma, DLBCL arising from follicular lymphoma and high-grade B-cell lymphoma (YESCARTA Prescribing Information 2017). In the ZUMA-3 study (NCT02614066; EudraCT 2015-005009-35; Shah et al., 2021, Blood 138: 11-22), KTE XI 9 (previously called KTE-C19) was used for the treatment of adult patients with r/r B ALL. As of 1^st Apr 2019, 45 patients received KTE-X19 in the Phase I portion and were included in the safety and efficacy analyses. In the dose escalation phase of the study, 6 patients received the dose of 2 x 10⁶ KTE-X19 cells/kg. Based on the recommendation of the Safety Review Team, additional patients were treated at lower doses; 16 patients received 0.5 x 10⁶KTE-X19 cells/kg and 14 patients received 1 x 10⁶ KTE-X19 cells/kg. To mitigate the risk of CRS and neurologic events (NE), adverse events (AE) management guidelines were revised to limit tocilizumab to the treatment of CRS (and not isolated neurotoxicity), and to initiate corticosteroid treatment for Grade 2 rather than Grade 3 NE. Nine patients received 1 x 10⁶ KTE-X19 cells/kg under the revised AE management guidelines. The median age of the patients treated in the safety population was 46 (range 18 to 77) years. Eight of 45 patients (18%) presented with Ph+ chromosome, 21 of 45 patients (47%) received prior blinatumomab and 6 of 45 (13%) received prior inotuzumab ozogamicin. The CR rate (CR+CRi) was 69% (31 of 45 patients) in all cohorts and 83% (19 of 23 patients) in the 1 x 10⁶ KTE-X19 cells/kg cohort. Of note, 6 of 9 (67%) patients who received 1 x 10⁶ KTE-X19 cells/kg and were managed under the revised AE management guidelines achieved CR/CRi (4 CR, 2 CRi). Two patients (4%) experienced a Grade 5 KTE- XI 9 related AE, 1 patient died due to a multi organ failure secondary to CRS on Study Day 6 and 1 patient died due to stroke after infusion in the context of Grade 2 CRS and Grade 4 neurologic event on Day 7. These observations prompted the study of lower doses and revision of AE management. CRS of any grade was reported for 93% of the patients and CRS Grade 3 or above was described for 31% of the patients. No changes were observed in the frequency of CRS of any grade or Grade 3 with the revised AE management guidelines. Approximately a third of the patients required vasopressors for the treatment of the CRS, while tocilizumab was used in 53% of the patients and steroids in 36%. Neurologic events (NE) of any grades have been reported for 78% of the patients and neurologic events Grade 3 or above were reported for 38% of the patients. No changes were observed in the frequency of NE of any grade while a reduction of Grade 3 NE (to 11%) was observed with the revised AE management guidelines. Tocilizumab was used in 31% of the patients and steroids in 44%. Overall, the administration of KTE-X19 in adult patients with r/r B ALL resulted in high rates of morphological remission (69%, 31 of 45 patients) but with significant rates of toxicity: Grade >3 treatment emergent neurologic events occurred in 17 of 45 (38%) patients and Grade >3 CRS occurred in 14 of 45 (31%) patients. The dose of 1 x 10⁶ KTE-X19 cells/kg was selected for the Phase II part of the study.

Frey et al., (Frey et al., 2021, J Clin Oncol 38:415-22) reported the data about the use of tisagenlecleucel (CTL019) in adults with r/r B ALL. Thirty -five adults with r/r B ALL received CTL019 through 3 dosing cohorts and were included in the study analysis. Nine patients received 5x 10⁷ CTL019 in the low-dose (LD) cohort (n = 9) as single or fractionated dosing and had manageable toxicity with a 33% CR rate. Six patients received 5x 10⁸ CTL019 as a single dose in the high-dose single infusion (HDS) cohort, 3 patients died before disease response assessment as a result of complications of CRS and infections; the surviving 3 (50%) patients achieved CR. Twenty patients received 5x 10⁸ CTL019 in the high-dose fractionated (HDF) cohort with manageable toxicity and a 87% CR rate. The HDF cohort had the highest survival, with a 2-year OS of 73% and a 2-year EFS of 49.5%. Neurocognitive toxicity of any grade affected 14 (40%) patients. Grade 1-2 toxicity occurred in 13 (37%) patients and grade 3 in 2 (6%), and no patient had Grade 4 neurotoxicity. CRS of any grade graded with the Penn grading scale occurred in 33 (94%) patients. Grade 1-2 CRS occurred in 8 (23%) patients, Grade 3 in 19 (54%), Grade 4 in 3 (9%), and Grade 5 in 3 (9%) patients.

Park et al (Park et al., 2018, N Engl J Med 378:449-59) reported the data about the use of autologous T cells expressing the 19-28z CAR in adults with r/r B-cell ALL. The study included 3 stages. In Stage 1 of the study, all patients received cyclophosphamide (Cy) conditioning chemotherapy followed by 3xl0⁶ 19-28z CAR T cells/kg. In Stage 2 of the study, patients were divided into two cohorts based on pre-treatment disease burden. High disease patients included patients with > 5% blasts in bone marrow (BM) or radiographically evident disease; low disease patients included patients with <5% BM blasts and with no evident extramedullary disease. High disease patients (n=12) received lx 10⁶ and low disease patients (n=l 1) received 3x 10⁶ 19-28z CAR T cells/kg. In Stage 3, patients received Fludarabine + Cy chemotherapy while keeping the same two T-cell dosing scheme used in Stage 2. Overall, 44 (83%) patients experienced a CR. At a median follow-up of 29 months, the median EFS was 6.1 months, and the median OS was 12.9 months. Patients with a low disease burden (<5% bone marrow blasts) had a median EFS of 10.6 months and a median OS of 20.1 months. Patients with a higher burden of disease (>5% bone marrow blasts or extramedullary disease) had a greater incidence of CRS and neurotoxic events and shorter long-term survival than did patients with a low disease burden. Overall, CRS of any grade occurred in 45 (85%) of 53 patients, CRS Grade >3 in 14 (26%). One patient died from CRS and multi organ failure on day 5. Grade 2 neurotoxic effects were observed in 1 patient (2%), Grade 3 in 19 (36%), and Grade 4 in 3 (6%). No case of Grade 5 neurotoxic effect or cerebral oedema was observed.

Overall CD 19 CAR T cell therapies are active in r/r B-ALL. However, two key challenges remain, severe CRS which in adults is likely to be less tolerable due to age and co morbidities and severe neurotoxicity is yet another safety concern. The overall rate of severe CRS has decreased overtime due to intensive oversight, early management guidelines and the use of tocilizumab. However, management of neurotoxicity still needs further improvement, but close monitoring, use of appropriate dose and early use of steroids are likely to improve the management of this toxicity. Such intense patient management adds significant burden to the healthcare system. However, adult patients who suffer from additional co-morbidities are less likely to tolerate treatments with significant toxicities, so novel approaches to generate CAR therapies with a lower propensity to cause severe toxicities such as severe CRS or ICANS are needed.

Furthermore, one of the most critical determinants of achieving a durable response in B-ALL is the persistence of CAR T-cells. Responses in CD 19 CAR studies suggest that persistence of T-cells for a protracted period at high levels seems to be important in effecting durable responses (Mueller et al., 2017, Blood 130:2317-25). Therefore, recurrence of disease may, in part, be due to non-persistence of CAR T-cells. Indeed, a lack of CAR T-cell persistence leading to CD 19+ relapse is the main cause of therapy failure following licensed CAR T-cell therapy for B-ALL. Therefore, there also remains a need in the art to promote persistence of CAR T cells, in order to improve anti-cancer CAR T cell responses and promote long-term remission of disease.

SUMMARY OF ASPECTS OF THE INVENTION

The disclosure provides methods for treating a relapsed or refractory CD 19+ haematological malignancy in a patient comprising administering to the patient autologous CD19 CAR T-cells (for example, the autologous CD19 CAR T-cell product comprising CAT19 CAR described in Example 1 herein).

In particular, an autologous CD 19 CAR-T cell for use in a method of treating a relapsed or refractory CD 19+ haematological malignancy in a patient is provided, comprising administering an autologous CD19 CAR T-cell to the patient, wherein: if the patient has a presence of less than or equal to 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 100 x 10⁶ CAR T-cells and a second dose comprising about 310 x 10⁶ CAR T-cells; and/or if the patient has a presence of more than 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 10 x 10⁶ CAR T- cells and a second dose comprising about 400 x 10⁶ CAR T-cells.

The second dose may be administered at between about 7 days and about 11 days after the administration of the first dose.

The haematological malignancy may be B-cell acute lymphoblastic leukaemia (B-ALL).

The relapsed or refractory B-ALL: may be primary refractory disease; may have had a first relapse within 12 months of first remission; may be relapsed or refractory after two or more lines of (systemic) therapy, or wherein the B-ALL may be after stem cell transplant (SCT).

The patient may not subsequently receive a stem cell transplant (SCT), preferably an allogeneic SCT.

The method of treating a relapsed or refractory CD 19+ haematological malignancy in a patient further comprises a step of determining CAR-T cell persistence in a sample comprising peripheral blood mononuclear cells (PBMCs) from the patient: wherein if CAR-T cells are detected, then the patient may not receive a stem cell transplant (SCT), and wherein if CAR-T cells are not detected, then the patient may receive a SCT.

At least 50% of patients treated may achieve an overall remission (OR) or may be identified as responders. At least 50% of patients treated may achieve a complete response (CR).

At least 20% of patients treated may achieve a CR with incomplete blood count recovery (CRi).

At least 90% of responding patients may have minimal residual disease (MRD)-negative status.

Less than 80% of the patients treated may exhibit any grade of cytokine release syndrome (CRS).

Less than 20% of the patients treated may exhibit a grade 3 or greater CRS.

The CRS may be treated with an anti-IL-6 antibody, an anti-IL-6R antibody or a steroid.

The anti-IL-6R antibody may be tocilizumab.

Less than 30% of the patients treated may exhibit any grade of neurotoxicity or immune effector cell-associated neurotoxicity syndrome (ICANS).

Less than 15% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS.

The age of the patient may be eighteen years or older.

The age of the patient may be between eighteen and thirty-nine years. At least 50% of patients treated may achieve an overall remission (OR) or may be identified as responders.

The age of the patient may be between forty and sixty-four years. At least 60% of patients treated may achieve an overall remission (OR) or may be identified as responders.

The age of the patient may be sixty-five years or older. At least 80% of patients treated may achieve an overall remission (OR) or may be identified as responders.

A step of lymphodepletion may be done prior to the administration of an autologous CD 19 CAR T-cell or autologous CD 19 CAR T-cell population to the patient.

The patient may have less than or equal to 20% blasts in the bone marrow (BM) at lymphodepletion. At least 70% of patients treated may achieve an overall remission (OR) or may be identified as responders. Less than 70% of the patients treated may exhibit any grade of CRS. Less than 10% of the patients treated may exhibit a grade 3 or greater CRS. Less than 10% of the patients treated may exhibit any grade of ICANS. Less than 5% of the patients treated may exhibit a grade 3 or greater ICANS.

The patient has between more than 20% and less than or equal to 75% blasts in the BM at lymphodepletion. At least 70% of patients treated may achieve an overall remission (OR) or may be identified as responders. Less than 80% of the patients treated may exhibit any grade of CRS. Less than 10% of the patients treated may exhibit a grade 3 or greater CRS. Less than 20% of the patients treated may exhibit any grade of ICANS. Less than 10% of the patients treated may exhibit a grade 3 or greater ICANS.

The patient may have more than or equal to 75% blasts in the BM at lymphodepletion. At least 40% of patients treated achieve an overall remission (OR) or may be identified as responders. Less than 80% of the patients treated may exhibit any grade of CRS. Less than 10% of the patients treated may exhibit a grade 3 or greater CRS. Less than 30% of the patients treated may exhibit any grade of ICANS. Less than 10% of the patients treated may exhibit a grade 3 or greater ICANS.

The patient may have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

The patient may have Philadelphia chromosome positive ALL (Ph+ ALL). The patient may be intolerant to a tyrosine kinase inhibitor or a second generation tyrosine kinase inhibitor is contraindicated. The patient may have previously been administered one or more tyrosine kinase inhibitor. At least 75% of patients treated may achieve an OR or may be identified as responders.

The patient may have been previously administered one or more lines of therapy.

The patient may have been administered one prior line of therapy. At least 60% of patients treated may achieve an overall remission (OR) or may be identified as responders.

The patient may have been administered two prior lines of therapy. At least 60% of patients treated may achieve an overall remission (OR) or may be identified as responders.

The patient may have been administered three prior lines of therapy. At least 70% of patients treated may achieve an overall remission (OR) or may be identified as responders.

The patient may have been administered four or more prior lines of therapy. At least 40% of patients treated may achieve an overall remission (OR) or may be identified as responders. The patient may have been previously administered one or more of inotuzumab ozogamicin and blinatumomab.

The patient may have been previously administered inotuzumab ozogamicin. At least 50% of patients treated may achieve an overall remission (OR) or may be identified as responders.

The patient may have been previously administered blinatumomab. At least 50% of patients treated may achieve an overall remission (OR) or may be identified as responders.

The patient may have previously received a stem cell transplant (SCT), preferably allogeneic SCT. At least 70% of patients treated may achieve an overall remission (OR) or may be identified as responders.

The patient may have extramedullary disease at preconditioning. At least 50% of patients treated may achieve an overall remission (OR) or may be identified as responders.

The patient may be administered a preconditioning or lymphodepletion regimen comprising fludarabine and cyclophosphamide. The preconditioning or lymphodepletion regimen may comprise 120 mg/m² fludarabine (Flu) and 1000 mg/m² cyclophosphamide (Cy). The preconditioning or lymphodepletion regimen may be initiated up to 6 days prior to CAR-T cell administration.

The autologous CD 19 CAR-T cell may be manufactured using a method with a manufacturing success rate of at least 80%.

The autologous CD 19 CAR-T cell may expand at a high-level following administration to the patient.

The autologous CD 19 CAR-T cell may persist for at least 3 months in the patient’s peripheral blood or bone marrow.

The disclosure provides methods of selecting a patient for receiving a second therapy following a treatment with CD 19 CAR engineered cells, which comprises determining CD 19 CAR engineered cell persistence in a sample comprising peripheral blood mononuclear cells (PBMCs) from the patient: wherein if CD 19 CAR engineered cells are detected, then the patient may not receive the second therapy; and wherein if CD 19 CAR engineered cells are not detected, then the patient may receive the second therapy, wherein the patient has a relapsed or refractory CD 19+ haematological malignancy prior to receiving the CD 19 CAR engineered cells.

The second therapy is selected from stem cell transplant (SCT) and a tyrosine inhibitor (TKI).

The second therapy is a tyrosine inhibitor (TKI) and the patient has Ph+ B-ALL.

FURTHER ASPECTS OF THE INVENTION

1. An autologous CD 19 CAR-T cell for use in a method of treating a relapsed or refractory CD 19+ haematological malignancy in a patient comprising administering an autologous CD 19 CAR T-cell to the patient, wherein: if the patient has a presence of less than or equal to 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 100 x 10⁶ CAR T-cells and a second dose comprising about 310 x 10⁶ CAR T-cells; and/or if the patient has a presence of more than 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 10 x 10⁶ CAR T- cells and a second dose comprising about 400 x 10⁶ CAR T-cells.

2. The autologous CD 19 CAR-T cell for use according to paragraph 1, wherein the second dose is administered at between about 7 days and about 11 days after the administration of the first dose.

3. The autologous CD 19 CAR-T cell for use according to paragraph 1 or paragraph 2, wherein the haematological malignancy is B-cell acute lymphoblastic leukaemia (B- ALL).

4. The autologous CD19 CAR-T cell for use according to any of paragraphs 1 to 3, wherein the relapsed or refractory B-ALL: is primary refractory disease; has had a first relapse within 12 months of first remission; is relapsed or refractory after two or more lines of (systemic) therapy, or wherein the B-ALL is after stem cell transplant (SCT). The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 4, wherein at least 50% of patients treated achieve an overall remission (OR) or are identified as responders. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 5, wherein at least 50% of patients treated achieve a complete response (CR). The autologous CD 19 CAR-T cell for use according to paragraph 6, wherein at least 20% of patients treated achieve a CR with incomplete blood count recovery (CRi). The autologous CD 19 CAR-T cell for use according to paragraph 6, wherein at least 90% of responding patients have minimal residual disease (MRD)-negative status. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 8, wherein less than 80% of the patients treated exhibit any grade of cytokine release syndrome (CRS). The autologous CD 19 CAR-T cell for use according paragraph 9, wherein less than 20% of the patients treated exhibit a grade 3 or greater CRS. The autologous CD 19 CAR-T cell for use according to paragraph 9 or paragraph 10, wherein the CRS is treated with an anti-IL-6 antibody, an anti-IL-6R antibody or a steroid. The autologous CD19 CAR-T cell for use according to paragraph 11, wherein the anti- IL-6R antibody is tocilizumab. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 12, wherein less than 30% of the patients treated exhibit any grade of neurotoxicity or immune effector cell-associated neurotoxicity syndrome (ICANS). The autologous CD19 CAR-T cell for use according to paragraph 13, wherein less than 15% of the patients treated exhibit a grade 3 or greater neurotoxicity or ICANS. 15. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 14, wherein the age of the patient is eighteen years or older.

16. The autologous CD 19 CAR-T cell for use according to paragraph 15, wherein the age of the patient is between eighteen and thirty-nine years.

17. The autologous CD 19 CAR-T cell for use according to paragraph 16, wherein at least 50% of patients treated achieve an overall remission (OR) or are identified as responders.

18. The autologous CD19 CAR-T cell for use according to paragraph 15, wherein the age of the patient is between forty and sixty-four years.

19. The autologous CD 19 CAR-T cell for use according to paragraph 18, wherein at least 60% of patients treated achieve an overall remission (OR) or are identified as responders.

20. The autologous CD 19 CAR-T cell for use according to paragraph 15, wherein the age of the patient is sixty-five years or older.

21. The autologous CD 19 CAR-T cell for use according to paragraph 20, wherein at least 80% of patients treated achieve an overall remission (OR) or are identified as responders.

22. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 21, wherein the patient has less than or equal to 20% blasts in the bone marrow (BM).

23. The autologous CD 19 CAR-T cell for use according to paragraph 22, wherein at least 70% of patients treated achieve an overall remission (OR) or are identified as responders.

24. The autologous CD19 CAR-T cell for use according to any of paragraphs 22 or 23, wherein less than 70% of the patients treated exhibit any grade of CRS.

25. The autologous CD 19 CAR-T cell for use according to paragraph 24, wherein less than 10% of the patients treated exhibit a grade 3 or greater CRS. 26. The autologous CD 19 CAR-T cell for use according to any of paragraphs 22 to 25, wherein less than 10% of the patients treated exhibit any grade of ICANS.

27. The autologous CD 19 CAR-T cell for use according to paragraph 26, wherein less than 5% of the patients treated exhibit a grade 3 or greater ICANS.

28. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 21, wherein the patient has between more than 20% and less than or equal to 75% blasts in the BM at lymphodepletion.

29. The autologous CD 19 CAR-T cell for use according to paragraph 26, wherein at least 70% of patients treated achieve an overall remission (OR) or are identified as responders.

30. The autologous CD19 CAR-T cell for use according to any of paragraphs 26 or 27, wherein less than 80% of the patients treated exhibit any grade of CRS.

31. The autologous CD 19 CAR-T cell for use according to paragraph 28, wherein less than 10% of the patients treated exhibit a grade 3 or greater CRS.

32. The autologous CD 19 CAR-T cell for use according to any of paragraphs 28 to 31, wherein less than 20% of the patients treated exhibit any grade of ICANS.

33. The autologous CD19 CAR-T cell for use according to paragraph 32, wherein less than 10% of the patients treated exhibit a grade 3 or greater ICANS.

34. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 21, wherein the patient has more than or equal to 75% blasts in the BM at lymphodepletion.

35. The autologous CD 19 CAR-T cell for use according to paragraph 26, wherein at least 40% of patients treated achieve an overall remission (OR) or are identified as responders.

36. The autologous CD19 CAR-T cell for use according to any of paragraphs 34 or 35, wherein less than 80% of the patients treated exhibit any grade of CRS. 37. The autologous CD19 CAR-T cell for use according to paragraph 28, wherein less than 10% of the patients treated exhibit a grade 3 or greater CRS.

38. The autologous CD 19 CAR-T cell for use according to any of paragraphs 28 to 31, wherein less than 30% of the patients treated exhibit any grade of ICANS.

39. The autologous CD19 CAR-T cell for use according to paragraph 32, wherein less than 10% of the patients treated exhibit a grade 3 or greater ICANS.

40. The autologous CD19 CAR-T cell for use according to any of paragraphs 1 to 39, wherein the patient has an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

41. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 40, wherein the patient has Philadelphia chromosome positive ALL (Ph+ ALL).

42. The autologous CD 19 CAR-T cell for use according to paragraph 42, wherein the patient is intolerant to a tyrosine kinase inhibitor or a second generation tyrosine kinase inhibitor is contraindicated.

43. The autologous CD 19 CAR-T cell for use according to paragraph 42, wherein the patient has previously been administered one or more tyrosine kinase inhibitor.

44. The autologous CD19 CAR-T cell for use according to any of paragraphs 41 to 43, wherein at least 75% of patients treated achieve an OR or are identified as responders.

45. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 44, wherein the patient has been previously administered one or more lines of therapy.

46. The autologous CD 19 CAR-T cell for use according to paragraph 45, wherein the patient has been administered one prior line of therapy.

47. The autologous CD 19 CAR-T cell for use according to paragraph 46, wherein at least 60% of patients treated achieve an overall remission (OR) or are identified as responders. The autologous CD19 CAR-T cell for use according to paragraph 45, wherein the patient has been administered two prior lines of therapy. The autologous CD 19 CAR-T cell for use according to paragraph 48, wherein at least 60% of patients treated achieve an overall remission (OR) or are identified as responders. The autologous CD 19 CAR-T cell for use according to paragraph 45, wherein the patient has been administered three prior lines of therapy. The autologous CD 19 CAR-T cell for use according to paragraph 50, wherein at least 70% of patients treated achieve an overall remission (OR) or are identified as responders. The autologous CD 19 CAR-T cell for use according to paragraph 45, wherein the patient has been administered four or more prior lines of therapy. The autologous CD 19 CAR-T cell for use according to paragraph 52, wherein at least 40% of patients treated achieve an overall remission (OR) or are identified as responders. The autologous CD19 CAR-T cell for use according to any of paragraphs 1 to 53, wherein the patient has been previously administered one or more of inotuzumab ozogamicin and blinatumomab. The autologous CD 19 CAR-T cell for use according to paragraph 54, wherein the patient has been previously administered inotuzumab ozogamicin. The autologous CD19 CAR-T cell for use according to paragraph 55, wherein at least 50% of patients treated achieve an overall remission (OR) or are identified as responders. The autologous CD 19 CAR-T cell for use according to paragraph 54, wherein the patient has been previously administered blinatumomab. 58. The autologous CD19 CAR-T cell for use according to paragraph 55, wherein at least 50% of patients treated achieve an overall remission (OR) or are identified as responders.

59. The autologous CD19 CAR-T cell for use according to any of paragraphs 1 to 58, wherein the patient has previously received a stem cell transplant (SCT), preferably allogeneic SCT.

60. The autologous CD 19 CAR-T cell for use according to paragraph 59, wherein at least 70% of patients treated achieve an overall remission (OR) or are identified as responders.

61. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 60, wherein the patient has extramedullary disease at preconditioning.

62. The autologous CD 19 CAR-T cell for use according to paragraphs 61, wherein at least 50% of patients treated achieve an overall remission (OR) or are identified as responders.

63. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 62, wherein the patient is administered a preconditioning regimen comprising 120 mg/m² fludarabine (Flu) and 1000 mg/m² cyclophosphamide (Cy).

64. The autologous CD19 CAR-T cell for use according to any of paragraphs 1 to 63, wherein the autologous CD 19 CAR-T cell is manufactured using a method with a manufacturing success rate of at least 80%.

65. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 64, wherein the autologous CD 19 CAR-T cell expands at a high level following administration to the patient.

66. The autologous CD 19 CAR-T cell for use according to any of paragraphs 1 to 65, wherein the autologous CD 19 CAR-T cell persists for at least 3 months in the patient’s peripheral blood or bone marrow. 67. A method of selecting a patient for receiving a second therapy following a treatment with CD 19 CAR engineered cells, which comprises determining CAR-T cell persistence in a sample comprising peripheral blood mononuclear cells from the patient: wherein if CAR-T cells are detected, then the patient may not receive a second therapy; and wherein if CAR-T cells are not detected, then the patient may receive a second therapy, wherein the patient has a relapsed or refractory CD 19+ haematological malignancy as described herein.

DESCRIPTION OF THE FIGURES

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 CAT 19) 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 CATC AR.

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

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.

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.

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.

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.

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.

Figure 9. AUTO1 Manufacturing. Manufacturing quality and logistics were reliable and consistent.

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.

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.

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.

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: 51) of the CD19 CATCAR (AUTO 1).

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 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.

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

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.

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

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.

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

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

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

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.

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

Figure 25. Patient disposition - R/R B-ALL. Investigator-assessed disease evaluations were performed locally by CT and BM biopsy for B-ALL. Allo-HSCT, allogeneic hematopoietic stem cell transplant; B-ALL, B-cell acute lymphoblastic leukemia; BM, bone marrow; CR/CRi, complete remission/complete remission with incomplete hematologic recovery; CT, computed tomography; N/A, not available; R/R, relapsed/refractory.

Figure 26. Swimmer plot.

Figure 27. Event free survival (EFS) and overall survival (OS) in R/R B-ALL. A) EFS.

B) OS. *Censored for allo-HSCT and other anti-cancer treatment. Investigator-assessed disease evaluations were performed locally by CT and BM biopsy for B-ALL. Allo-HSCT, allogeneic hematopoietic stem cell transplant; B-ALL, B-cell acute lymphoblastic leukemia; BM, bone marrow; CI, confidence interval; CT, computed tomography; EFS, event-free survival; N/A, not available; OS, overall survival; R/R, relapsed/refractory.

Figure 28. AUTO1 persistence in patients with R/R B-ALL.

Figure 29. Durable responses in patients with R/R B-CLL or B-NHL. *Patient death unrelated to AUTO1 and without relapse or disease progression (COVID-19 [DLBCL and FL, n = 1 each]; grade 5 appendicitis on a background of myelodysplastic syndrome [FL, n = 1]; unrelated esophageal cancer [CLL, n = 1]). Investigator-assessed disease evaluations were performed locally by CT and BM biopsy for B-CLL, and by FDG-PET-CT imaging per Lugano criterial for B-NHL. MRD status was determined using flow cytometry or IgH PCR/NGS (MRD-negative: <10-4 [<0.01%]). BM, bone marrow; CLL, chronic lymphocytic leukemia; CMR, complete metabolic remission; CT, computed tomography; FDG-PET-CT, fluorodeoxyglucose-positron emission tomography-computed tomography; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; MCL, mantle cell lymphoma; MRD, measurable residual disease; NGS, next-generation sequencing; PCR, polymerase chain reaction; PR, partial remission; R/R, relapsed/refractory; SD, stable disease.

Figure 30. AUTO1 persistence in patients with R/R B-CLL/B-NHL. Figure 31. Manufacturing, testing, and logistics implemented in Phase Ib/II study with supported global AUTO1 delivery. *Measuring biological activity and sterility testing. '''Key targets: V2C - time from leukapheresis to quality release: ~23 days, V2D - time from leukapheresis to delivery of product to the hospital: ~25 days. B-ALL, B-cell acute lymphoblastic leukemia; DP, drug product; DV, dry vapor; ERP, enterprise resource planning; LN2, liquid nitrogen; QC, quality control; QP, qualified person; r/r, relapsed/refractory; TAT, turnaround time; UK, United Kingdom; V2C, vein-to-certification; V2D, vein-to-delivery.

Figure 32. Target V2C and V2D times were met during CAR T-cell manufacturing of AUTO1.

Figure 33. Heterogeneous leukapheresis material to homogeneous drug product.

Figure 34. Leukapheresis collections and product deliveries were executed successfully despite the COVID-19 pandemic. COVID-19, coronavirus disease 2019; US, United States; V2D, vein-to-delivery. United States Department of Transportation, Bureau of Transportation Statistics 2021 [online]. Available at: https://www.bts.gov/data- spotlight/commercial-aviation-2020-downtum-airline-passengers-employment-profits-and- flights Accessed October 2023; ²World Health Organization COVID-19 dashboard [online]. Available at: https://covidl9.who.int/ Accessed October 2023.

Figure 35. Phase Ib/II clinical study design with patient-reported outcomes (PROs) collection.

Figure 36. Median change from baseline in (A) EQ-5D-5L VAS, and (B) EORTC QLQ- C30 GHS scores for infused patients with observed data. EORTC QLQ-C30, European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire; EQ-5D- 5L VAS, EQ-5D-5L visual analog scale; Q, quartile.

Figure 37. Screening, enrolment, and treatment. Consort study for the entire Phase Ib/II population. Enrolment = All inclusion/exclusion criteria have been fulfilled and leukapheresate has been accepted for manufacturing. Cohort A: Adults aged >18 years with B ALL who have >5% blasts in the bone marrow at screening. Cohort B: Adults aged >18 years with B ALL in morphological remission with minimal residual disease-positive disease and <5% blasts in the bone marrow at screening. Cohort C: Adults aged >18 years with B ALL with isolated extramedullary disease at screening. Figure 38. Manufacturing overview.

Figure 39. Figure 2: AUTO1 starting material, manufacture, and product characteristics, (a) CD3+ T-cell content of the Phase Ib/II patient leukapheresis starting material, median CD3+% was 13.4% (0.8-83.1); (b) AUTO1 transduction efficiency, median transduction efficiency was 69.3% (11.7 - 86.7); (c) AUTO1 viability post thawing, median was 88.9% (77 - 96); (d) AUTO1 vein (leukapheresis) to release time in days, median was median 21.2 days (17.9 - 50.4).

Figure 40. Extended product characteristics.

Figure 41. Subgroup analysis for of overall response rate for key baseline characteristics and clinical covariates. Response according to subgroup is shown for the entire study population. Response is defined as CR/CRi. The dashed vertical line represents the median response rate for the entire study population.

Figure 42. Response rates and survival for all patients and grouped by disease burden, (a) Overall EFS and (b) overall EFS by leukemic burden prior to lymphodepletion for the entire study population; (c) Overall OS and (d) overall OS by leukemic burden prior to lymphodepletion for the entire study population.

Figure 43. EFS and OS by leukemic burden prior to enrolment.

Figure 44. SCT consolidation following AUTO1. A. EFS with or without censoring for consolidative SCT or new therapies. B. OS with or without censoring for consolidative SCT.

Figure 45. Serum biomarkers and cytokines. A. Peak biomarker and cytokine concentrations in patients with vs without CRS. B. Peak biomarker cytokine concentrations in patients with vs without ICANS.

Figure 46. Prolonged cytopenia. Time to recovery in responders by count at lymphodepletion for (A) neutrophil count (>0.5* 10⁹/L) and (B) platelet count (>50* 10⁹/L).

Figure 47. Cmax and AUC28 by ddPCR vs safety. A. Cmax vs CRS. B. AUC28 vs CRS. C. Cmax vs ICANS. Figure 9d: AUC28 vs ICANS.

Figure 48. CAR T-cell engraftment and persistence on PHASE IB/II. A. Mean (SE, black line) and individual copy numbers of AUTO1 transgene level (copies/pg DNA) by ddPCR in peripheral blood. B. Kaplan-Meir curve for patients with CR/CRi beyond 6 months without consolidative SCT or new therapies who had ongoing persistence versus patients who lost persistence.

Figure 49. B-cell recovery and persistence, (a) B-cell recovery, (b) CAR T cell persistence.

Figure 50. CAR T persistence and predicted relapse. Ongoing CAR T persistence correlates with long-term EFS. A) CAR-T cell persistence. B) B-cell recovery

Figure 51. Correlation between A) surface and intracellular FC, B) intracellular FC and VCN, C) surface FC and VCN, and D) Spearman correlation coefficients for all data versus for data above the LLoQ.

Figure 52. Landmark analysis of EFS at Month 6 post AUTO1 infusion by CAR T persistence measured by ddPCR and intracellular FC among patients with ongoing remission without new anti-cancer therapies. A) Number of patients included in the landmark analysis, B) and C) Kaplan-Meier analysis of EFS using CAR T persistence by ddPCT (B) and intracellular flow cytometry (C).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have designed CAT 19 CAR T cells (sometimes also referred to as CAT 19 or AUTO1 herein) to improve the safety profile of the CAR T therapy using a CD 19 antigen recognition domain that has fast off rate binding kinetics. AUTO1 has a similar structure to tisagenlecleucel with the incorporation of a survival 4-1 BB-CD3-^ co-stimulatory signal, with the main difference between the products being the scFv used as the CD 19 binder. In pre-clinical studies, CD 19 (CAT) CAR T cells have shown better proliferation and cytotoxicity, compared to CD 19 CARs using an FMC63 scFv format (Example 2). The faster off rate of the CD 19 (CAT) CAR confers an opportunity for a more physiological T cell interaction and surprisingly results in a better safety profile. All these improvements lead to a decrease in the need for an allogenic HSCT and an increase in the chance of success as a standalone therapy. The improved safety profile (Example 3) also enables CAT 19 CAR T cells to be well-suited for the treatment of adult ALL, particularly for older patients who have more co-morbidities and are less likely to tolerate toxicity. Unexpectedly, the CD 19 (CAT) CAR T cells have shown high expansion and long persistence in treated patients (Example 3). These characteristics help to improve anti-cancer CAR T cell responses and promote long-term remission of disease.

1. Chimeric antigen receptors (CARs)

A classical chimeric antigen receptor (CAR) is a chimeric type I trans-membrane protein which connects an extracellular antigen-binding domain to an intracellular signalling domain (endodomain). The antigen-binding domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody fragment or an antibody-like antigen-binding site. 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.

Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FcsRl or CD3(^. Consequently, these first generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell costimulatory 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 costimulatory 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. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.

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.

2. Target antigen

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

The target antigen may be an antigen present on a cancer cell, for example, a tumour- associated antigen. CD 19 is a target antigen contemplated herein.

3. Binding domains specific for CD19 target antigen

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.

CD 19 is also expressed on all B-cell malignancies but not multiple myeloma cells. It is not expressed on other haematopoietic populations or non-haematopoietic cells and therefore targeting this antigen should not lead to toxicity to the bone marrow or non-haematopoietic organs. Loss of the normal B-cell compartment is considered an acceptable toxicity when treating lymphoid malignancies, because although effective CD 19 CAR T cell therapy will result in B cell aplasia, the consequent hypogammaglobulinaemia can be treated with pooled immunoglobulin.

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

Table 1

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.

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.

For example, the antigen-binding domain may comprise a CD19 antigen-binding domain as described in Table 2.

Table 2

The gene encoding CD 19 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.

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: 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 CAT 19 CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID NO: 4);

CDR2 - DTSKLAS (SEQ ID NO: 5)

CDR3 - QQWNINPLT (SEQ ID NO: 6).

The CAT19 antibody is described in WO2016/139487.

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.

The CDRs may be in the format of a single-chain variable fragment (scFv), which is a fusion protein of the heavy variable region (VH) and light chain variable region (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The scFv may be in the orientation VH-VL, i.e., the VH is at the amino-terminus of the CAR molecule and the VL domain is linked to the spacer and, in turn the transmembrane domain and endodomain.

The CDRs may be grafted on to the framework of a human antibody or scFv. For example, the CAR may comprise a CD19-binding domain consisting or comprising one of the following sequences.

The CD 19 CAR may comprise the following VH sequence.

SEQ ID NO: 7 - VH sequence from CAT 19 murine monoclonal antibody QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDE DTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQ GTTLTVSS

The CD 19 CAR may comprise the following VL sequence. SEQ ID NO: 8 - VL sequence from CAT 19 murine monoclonal antibody

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGV

PDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

The CD 19 CAR may comprise the following scFv sequence.

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

QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDE DTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQ GTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHW YQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQW NINPLTFGAGTKLELKR

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

SEQ ID NO: 10 - CAT 19 CAR using “Campana” architecture

MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWM NWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKS STT AYMQLS SLTSED SAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMS ASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGT SYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

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

SEQ ID NO: 11 - CAT 19 CAR with an OX40-Zeta endodomain

MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWM NWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKS STT AYMQLS SLTSED SAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMS ASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGT SYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRDQ RLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE

RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 12 - CAT 19 CAR with a CD28-Zeta endodomain

MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWM NWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKS STT AYMQLS SLTSED SAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMS ASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGT SYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRS RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 13 - Third generation CD 19 CAR

MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWM NWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKS STT AYMQLS SLTSED SAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMS ASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGT SYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGG GSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR

SEQ ID NO: 14 - CD 19 CAR with IgGl hinge spacer

MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWM NWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKS STT AYMQLS SLTSED SAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMS ASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGT SYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPAEPKSPDKTHTCPPC PKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG

LSTATKDTYDALHMQALPPR

SEQ ID NO: 15 - CD 19 CAR with hinge-CH2-CH3 of human IgGl with FcR binding sites mutated out

MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWM NWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKS STT AYMQLS SLTSED SAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMS ASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGT SYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPAEPKSPDKTHTCPPC PAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVL VVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR

The CAR provided herein may comprise a variant of the polypeptide of SEQ ID NO: 1-15 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD 19 (when in conjunction with a complementary VL or VH domain, if appropriate).

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.

The CD19 CAR exemplified herein (i.e., the CAT19CAR using “Campana” architecture, SEQ ID NO: 10) has properties contemplated by the disclosure to result in lower toxicity and better efficacy in treated patients. When compared with an fmc63 -Campana CAR, the CAT19CAR exemplified herein effected killing of target cells expressing CD 19 and proliferated in response to CD 19 expressing targets, but Interferon-gamma release was less. Further, a small animal model of an aggressive B-cell lymphoma showed equal efficacy and equal engraftment between the fmc63- and CAT19-based CAR-T cells, but surprisingly, less of the CAT19 CAR T-cells were exhausted than fmc63 CAR T-cells. See, Examples 2 and 3 of US Publication No.: 2018-0044417. The CAT19CAR provided herein may cause 25, 50, 70 or 90% lower IFNy release in a comparative assay involving bringing CAR T cells into contact with target cells.

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

Another exemplary CD 19 antigen-binding domain contemplated by the disclosure 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: CDR1 - SYWMN (SEQ ID NO: 16);

CDR2 - QIWPGDGDTNYNGKFK (SEQ ID NO: 17)

CDR3 - RETTTVGRYYYAMDY (SEQ ID NO: 18); and b) a light chain variable region (VL) having CDRs with the following sequences:

CDR1 - KASQSVDYDGDSYLN (SEQ ID NO: 19);

CDR2 - DASNLVS (SEQ ID NO: 20) CDR3 - QQSTEDPWT (SEQ ID NO: 21).

It is contemplated that it is possible to introduce one or more mutations (substitutions, additions or deletions) into one or more CDRs without negatively affecting CD19-binding activity. Each CDR may, for example, have one, two or three amino acid mutations.

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

SEQ ID NO: 22 - Murine CD19ALAb scFv sequence QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGD GDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYA MDYWGQGTTVTVSSDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQ IPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWT FGGGTKLEIK

SEQ ID NO: 23 - Humanized CD19ALAb scFv sequence - Heavy 19, Kappa 16 QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGD GDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYA MDYWGKGTLVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQ

KPGQPPKLLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDP WTFGQGTKVEIKR

SEQ ID NO: 24 (Humanized CD19ALAb scFv sequence - Heavy 19, Kappa 7)

QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGD

GDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYA

MDYWGKGTLVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQ KPGQPPKVLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDP WTFGQGTKVEIKR

The scFv may be in a VH-VL orientation (as shown in SEQ ID Nos: 9, 22, 23 and 24) or a VL-VH orientation.

The CAR may comprise one of the following VH sequences:

SEQ ID NO: 25 - Murine CD19ALAb VH sequence

QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGD

GDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYA MDYWGQGTTVTVSS

SEQ ID NO: 26 - Humanized CD19ALAb VH sequence

QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGD

GDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYA MDYWGKGTLVTVSS

The CAR may comprise one of the following VL sequences:

SEQ ID NO: 27 - Murine CD19ALAb VL sequence

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNL

VSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK

SEQ ID NO: 28 (Humanized CD19ALAb VL sequence, Kappa 16)

DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDASN

LVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPWTFGQGTKVEIKR SEQ ID NO: 29 - Humanized CD19ALAb VL sequence, Kappa 7 DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYDASN LVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPWTFGQGTKVEIKR

The CAR provided herein may comprise a variant of the sequence shown as any of SEQ ID NO: 16-29 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD 19 (when in conjunction with a complementary VL or VH domain, if appropriate).

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.

4. Signal peptide

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.

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.

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

The signal peptide may comprise the amino acid sequence of any of SEQ ID NO: 30-33 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.

The signal peptide of SEQ ID NO: 30 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.

SEQ ID NO: 30 MGTSLLCWMALCLLGADHADA

The signal peptide of SEQ ID NO: 31 follows. METDTLLLWVLLLLVPGSTG

The signal peptide of SEQ ID NO: 32 is derived from IgGl.

SEQ ID NO: 2: MSLPVTALLLPLALLLHAARP

The signal peptide of SEQ ID NO: 33 is derived from CD8.

SEQ ID NO: 3: MAVPTQVLGLLLLWLTDARC

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

5. Spacer

CARs comprise a spacer to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.

The spacer may, for example, comprise an 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.

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.

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.

The spacer for the CD22 CAR may comprise an IgGl hinge spacer, or a spacer having a length equivalent to an IgGl hinge spacer. The spacer for the CD22 CAR may have fewer than 30 amino acids or fewer than 25 amino acids. It may have between 15-25 amino acids, for example between 18-22 amino acids. It may have about 20 amino acids. Examples of amino acid sequences for these spacers are given below:

SEQ ID NO: 34 (hinge-CH2CH3 of human IgGl)

AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEV I<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<A LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKKD

SEQ ID NO: 35 (human CD8 stalk):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

SEQ ID NO: 36 (human IgGl hinge):

AEPKSPDKTHTCPPCPKDPK

SEQ ID NO: 37 (IgGl Hinge-Fc)

AEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI< ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPGKKDPK

SEQ ID NO: 38 (IgGl Hinge - Fc modified to remove Fc receptor recognition motifs)

AEPKSPDKTHTCPPCPAPPVA*GPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI< ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPGKKDPK

Modified residues are underlined; * denotes a deletion.

SEQ ID NO: 39 (CD2 ectodomain)

KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKD TYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCI NTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKES SVEPVSCPEKGLD SEQ ID NO: 40 (CD34 ectodomain)

SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETT VKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTS TSL ATSPTKP YTS S SPILSDIKAEIKC SGIREVKLTQGICLEQNKTS SC AEFKKDRGEGL ARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSD LKKLGILDFTEQDVASHQSYSQKT

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

6. Transmembrane domain

The transmembrane domain is the domain of the CAR that spans the membrane.

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).

The transmembrane domain may be derived from CD28, which gives good receptor stability.

The transmembrane domain may be derived from human Tyrp-1. The tyrp-1 transmembrane domain sequence is shown as SEQ ID NO: 41. SEQ ID NO: 41 IIAIAVVGALLLVALIFGTASYLI

The transmembrane domain may be derived from CD8A. The CD8A transmembrane domain sequence is shown as SEQ ID NO: 42.

SEQ ID NO: 42 IYIWAPLAGTCGVLLLSLVITLYC

7. Endodomain

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.

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

The endodomain of the first CAR and the endodomain of the second CAR may comprise: (i) an ITAM-containing endodomain, such as the endodomain from CD3 zeta; and/or (ii) a costimulatory domain, such as the endodomain from CD28; and/or (iii) a domain which transmits a survival signal, for example a TNF receptor family endodomain such as OX-40 or 4-1BB.

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

The intracellular T-cell signalling domain (endodomain) of the CAR of the present invention may comprise the sequence shown as any of SEQ ID NO: 43-50 or a variant thereof having at least 80% sequence identity.

SEQ ID NO: 43 (CD3 zeta endodomain) RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN

PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

ALPPR

SEQ ID NO: 44 (41BB endodomain)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

SEQ ID NO: 45 (0X40 endodomain)

RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

SEQ ID NO: 46 (CD28 endodomain)

KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY

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

SEQ ID NO: 47 (41BB-Zeta endodomain fusion)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ

GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 48 (OX40-Zeta endodomain fusion)

RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQL

YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 49 (CD28Zeta endodomain fusion)

KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQ

NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 50 (CD28OXZeta)

KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGS

FRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR

RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG L ST ATKDTYDALHMQ ALPPR A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any of SEQ ID NO: 43-50 provided that the sequence provides an effective transmembrane domain/intracellular T cell signaling domain.

8. Nucleic acid

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.

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.

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.

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

Table 3

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

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

Alternative codons may be used in the portions of nucleic acid encoding a CD3zeta endodomain and/or the portions of nucleic acid encoding one or more costimulatory domain(s) and/or the portions of nucleic acid encoding one or more domain(s) which transmit survival signals. 9. Vector

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.

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.

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

10. Cell

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.

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

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

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.

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.

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.

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

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

Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (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.

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

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

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

CAR-expressing cells, such as CAR-expressing T or NK cells may either be created ex vivo either from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).

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.

Alternatively, T or NK cells provided herein may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T or NK cells. Alternatively, an immortalized T-cell line which retains its lytic function and could act as a therapeutic may be used.

The CAR cells are generated by introducing DNA or RNA coding for the CARs by one of many means including, but not limited to, transduction with a viral vector, transfection with DNA or RNA. Cells may be activated and/or expanded prior to being transduced with CAR- encoding nucleic acid, for example by treatment with an anti-CD3 monoclonal antibody.

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

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.

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.

A high manufacturing success rate is particularly advantageous when treating relapsed/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%.

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.

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.

11. Pharmaceutical composition

The present disclosure also relates to a pharmaceutical composition containing a plurality of CAR-expressing cells, such as T cells or NK cells provided herein. Pharmaceutical compositions comprising the CD19 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.

12. Method of treatment

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.

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

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.

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.

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

The B-ALL may be adult ALL (aALL). Standard treatment for patients with aALL is typically divided into three different phases: induction, consolidation, and maintenance.

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.

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).

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.

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 cells have been shown to not cause severe toxi cities, such as severe CRS and ICANS, and to persist longer.

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.

The present disclosure provides an autologous CD 19 CAR-T cell or an autologous CD 19 CAR-T cell population for use in a method of treating a relapsed or refractory CD 19+ haematological malignancy in a patient comprising administering an autologous CD 19 CAR T-cell or an autologous CD19 CAR-T cell population to the patient, wherein: if the patient has a presence of less than or equal to 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 100 x 10⁶ CAR T-cells and a second dose comprising about 310 x 10⁶ CAR T-cells; and/or if the patient has a presence of more than 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 10 x 10⁶ CAR T-cells and a second dose comprising about 400 x 10⁶ CAR T-cells.

Alternatively, this aspect may be reformulated as a method of treating a relapsed or refractory CD 19+ haematological malignancy in a patient comprising administering and autologous CD19 CAR T-cell or an autologous CD19 CAR-T cell population to the patient, wherein: if the patient has a presence of less than or equal to 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 100 x 10⁶ CAR T-cells and a second dose comprising about 310 x 10⁶ CAR T-cells; and/or if the patient has a presence of more than 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 10 x 10⁶ CAR T-cells and a second dose comprising about 400 x 10⁶ CAR T-cells.

Alternatively, this aspect may be reformulated as a use of an autologous CD 19 CAR-T cell or an autologous CD 19 CAR-T cell population in the manufacturing of a medicament for the treatment of a relapsed or refractory CD 19+ haematological malignancy in a patient, wherein: if the patient has a presence of less than or equal to 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 100 x 10⁶ CAR T-cells and a second dose comprising about 310 x 10⁶ CAR T-cells; and/or if the patient has a presence of more than 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 10 x 10⁶ CAR T-cells and a second dose comprising about 400 x 10⁶ CAR T-cells.

The second dose may be administered at a time between about 7 days and about 11 days after the administration of the first dose.

The second dose may be administered at about 7 days and 10 days, or about 7 days and 9 days, or about 8 days and 11 days, or about 8 days and 10 days, or about 8 days and 9 days, or about 9 days and 11 days, or about 9 days and 10 days after the administration of the first dose. The second dose may be administered at about 7 days, about 8 days, about 9 days, about 10 days, or about 11 days after the administration of the first dose. The second dose may be administered after about 9 days after the administration of the first dose.

The patient may receive a lymphodepleting pre-conditioning treatment before CD 19 CAR-T cell infusion. Lymphodepleting pre-conditioning treatment, lymphodepletion and preconditioning are intended to be used as synonyms in the present invention. The patient may be administered a preconditioning regimen comprising 120 mg/m² fludarabine (Flu) and 1000 mg/m² cyclophosphamide (Cy). This preconditioning regimen may be administered prior to CAR-T cell administration.

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/m² on 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.

The relapsed or refractory CD 19+ haematological malignancy may be B-cell acute lymphoblastic leukemia (B-ALL).

The relapsed or refractory B-ALL may be primary refractory disease. Primary refractory is defined as not achieving complete response (CR) after two cycles of induction chemotherapy.

The relapsed or refractory B-ALL may have had a first relapse within 12 months, i.e. less than or equal to 12 months, of first remission.

The B-ALL may be relapsed or refractory after two or more lines of therapy. The therapy may be systemic therapy.

The B-ALL may be in a patient following haematopoietic stem cell transplant (HSCT). The CD 19 CAR-T cells provided in this disclosure may be infused at least 3 months after HSCT.

The determination of the percentage of blasts present in the bone marrow may be determined at screening.

The determination of the percentage of blasts present in the bone marrow may be determined prior to the start of pre-conditioning or lymphodepletion. The start of pre-conditioning or lymphodepletion may be on Day 6 prior to the administration of the CD19 CAR T cells, i.e. Day -6. The determination of the percentage of blasts present in the bone marrow may be determined at infusion or administration of the CD 19 CAR-T cells.

Overall disease response criteria include complete remission or response (CR), CR with incomplete recovery of counts (CRi). Details are shown in Table 4. Table 4. Overall Disease Response Criteria.

ALL = acute lymphoblastic leukaemia; ANC = absolute neutrophil count; BM = bone marrow; CNS = central nervous system; CR = complete response; CRi = complete response with incomplete recovery of counts.

*No recurrence of the disease should be observed for at least 4 weeks to confirm CR/CRi. This is defined as no clinical evidence of relapse as assessed by peripheral blood (% of blasts) and extramedullary disease assessment (physical exam and CNS symptom assessment) at a minimum of 4 weeks (28 days) after the initial achievement of CR or CRi.

Overall remission is defined herein as complete response (CR) or complete response with incomplete recovery of counts (CRi) following B-ALL treatment. The B-ALL treatment may involve the administration of CD 19 CAR T cells as described herein. The patients having overall remission, i.e. either CR or CRi, are identified as responders, The skilled person will understand that counts refers to the blood cell counts.

Following treatment according to the invention, at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 76%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated may achieve an overall remission (OR) or are identified as responders.

Following treatment according to the invention, at least 35%, or at least 40%, or at least 50%, or at least 54%, or at least 55%, or at least 57%, or at least 60%, or at least 65%, or more patients treated may achieve a complete response (CR). At least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of patients achieving a CR by one month and/or by three months may remain in response, or may remain in CR, and/or may survive or may survive without progression, for at least 3 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 15 months, or at least 18 months, or at least 21 months, or at least 24 months, or at least 36 months, or at least 48 months, or at least 60 months, or longer after achieving CR.

Remission may be in absence of subsequent anti-cancer therapies.

Following treatment according to the invention, at least 10%, or at least 15%, or at least 20%, or at least 21.3%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or more of patients treated may achieve a CR with incomplete blood count recovery (CRi). At least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or more of patients achieving a CRi by one month and/or by three months may remain in response, may remain in CRi, and/or may survive or may survive without progression, for at least 3 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 15 months, or at least 18 months, or at least 21 months, or at least 24 months, or at least 36 months, or at least 48 months, or at least 60 months, or longer after achieving the CRi.

The duration of overall remission may be at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 15 months, or at least 18 months, or at least 21 months, or at least 24 months, or at least 36 months, or at least 48 months, or at least 60 months, or longer.

Following treatment according to the invention, at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or responding patients are minimal residual disease (MRD)-negative or have MRD-negative status. The MRD-positive disease is defined as having between equal to or higher than 10'⁴ and lower than 5% blasts in the bone marrow (BM).

The MRD-negative status may be at 10'⁴ level of blasts in the BM. The MRD-negative status may be lower than 10'⁴ level of blasts in the BM.

The MRD-negative status may be determined by flow cytometry of by polymerase chain reaction (PCR).

Following treatment according to the invention, at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or more of patients achieving an OR may remain in response or may survive for at least 3 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 15 months after achieving the OR.

Following treatment according to the invention, the event-free survival rate may be at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% at 1 month.

Following treatment according to the invention, the event-free survival rate may be at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% at 3 months. Following treatment according to the invention, the event-free survival rate may be at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 64%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% at 6 months.

Following treatment according to the invention, the event-free survival rate may be at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 49%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% at 12 months.

Following treatment according to the invention, the event-free survival rate may be at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% at 18 months.

Following treatment according to the invention, the event-free survival rate may be at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%or at least 80%, or at least 85%, or at least 90%, or at least 95% at 24 months.

Following treatment according to the invention, the overall survival (OS) rate may be at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% at 1 month.

Following treatment according to the invention, the overall survival (OS) rate may be at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% at 3 months.

Following treatment according to the invention, the overall survival (OS) rate may be at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% at 6 months.

Following treatment according to the invention, the overall survival (OS) rate may be at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% at 12 months. Following treatment according to the invention, the overall survival (OS) rate may be at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% at 18 months.

Following treatment according to the invention, the overall survival (OS) rate may be at least 30%, or at least 35%, or at least 40%, or at least 44%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% at 24 months.

Following treatment according to the invention, the overall survival (OS) rate may be at least 30%, or at least 35%, or at least 39%, or at least 40%, or at least 44%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% at 36 months.

Following treatment according to the invention, the overall survival (OS) rate may be at least 30%, or at least 35%, or at least 39%, or at least 40%, or at least 44%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% at 48 months.

The patient may be in remission without consolidation by allogeneic haematopoietic stem cell transplant (HSCT).

Cytokine Release Syndrome (CRS) is a recognised toxicity with CAR T cell therapies and for some CAR T therapies it can be severe (Grade 3 or 4). Clinical symptoms indicative of CRS includes culture negative fever, but may also include myalgia, nausea/vomiting, tachycardia, hypoxia, hypotension, headache, confusion, tremor, and delirium. Potentially life-threatening complications of CRS may include cardiac dysfunction, acute respiratory distress syndrome, renal and/or hepatic failure, and disseminated intravascular coagulation (DIC).

Macrophage activation syndrome (MAS) and haemophagocytic lymphohistiocytosis (HLH) may occur in some for whom CAR-mediated inflammatory responses continue to evolve. The clinical syndrome of MAS is characterised by high-grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with biochemical abnormalities, such as high circulating levels of serum ferritin, soluble IL-2 receptor (sCD25), and triglycerides, together with a decrease of circulating natural killer activity. Other findings include variable levels of transaminases up to signs of acute liver failure and coagulopathy with findings consistent with DIC.

Following treatment according to the invention, less than 80%, or less than 75%, or less than 70%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than

20%, or less than 15%, or less than 10%, or less than 5%, or less than 1% of the patients treated may exhibit any grade of cytokine release syndrome (CRS).

Grading for CRS is provided in Table 5 with recommendations regarding treatment provided in Table 6.

Table 5. Severity Grading of CRS (ASCTT/ASBMT CRS Consensus Grading and NCI CTCAE Version 5.0) (Lee et al., 2019, Biol Blood Marrow Transplant 25:625-38).

ASCTT/ASBMT = American Society for Transplantation and Cellular Therapy /American Society for Blood and Marrow Transplantation; CPAP = Continuous positive airway pressure; CRS = cytokine release syndrome; CTCAE = Common Terminology Criteria for Adverse Events; BiPAP = Bilevel positive airway pressure. Organ toxicides associated with CRS may be graded according to NCI CTCAE version 5.0 but they do not influence CRS grading. f Fever is defined as temperature > 38°C not attributable to any other cause. In patients who have CRS and then receive antipyretics or anti-cytokine therapy such as tocilizumab or steroids, fever is no longer required to grade subsequent CRS severity. In this case, CRS grading is driven by hypotension and/or hypoxia.

J CRS grade is determined by the more severe event: hypotension or hypoxia not attributable to any other cause. For example, a patient with temperature of 39.5°C, hypotension requiring one vasopressor and hypoxia requiring low-flow nasal cannula is classified as having Grade 3 CRS.

^A Low-flow nasal cannula is defined as oxygen delivered at <6 L/minute. Low flow also includes blow-by oxygen delivery, sometimes used in paediatrics. High-flow nasal cannula is defined as oxygen delivered at >6 L/minute.

Table 6. Management of CRS

CRS = cytokine release syndrome; CTCAE = Common Terminology Criteria for Adverse Events;

IL = interleukin; MAS = macrophage activation syndrome; NCI = National Cancer Institute; TNF = tumour necrosis factor.

Following treatment according to the invention, less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit a grade 3 or greater CRS.

The CRS may be treated with an anti-IL-6 antibody, an anti-IL-6R antibody or a steroid. The anti-IL-6R antibody may be tocilizumab or siltuximab. The steroid may be dexamethasone or methylprednisolone.

Where the CRS is associated with macrophage activation syndrome (MAS) or haemophagocytoses, the CRS may be additionally treated with an IL-1 receptor antagonist, such as anakinra. The CRS may be additionally treated with a vasopressor. The vasopressor may be selected from one or more of noradrenaline/norepinephrine, dopamine, adrenaline, and vasopressin.

Neurotoxicity has been seen in patients with leukaemia and lymphoma after treatment with CAR T cell therapy and it is referred to as “Immune Effector Cell-associated Neurotoxicity Syndrome (ICANS)”. Although symptoms can vary the early manifestations of ICANS are often tremor, dysgraphia, mild difficulty with expressive speech especially naming objects, impaired attention, apraxia, and mild lethargy. Other symptoms can include confusion, depressed level of consciousness/encephalopathy, hallucinations, dysphasia, ataxia, apraxia, cranial nerve palsies, and seizures. Headache is a non-specific symptom, frequently occurring during fever or after chemotherapy, thus, headache alone is not a useful marker of ICANS. Expressive aphasia, on the other hand, appears to be a very specific symptom of ICANS. In addition to more common neurotoxicity symptoms, rare cases of rapid-onset and lethal diffuse cerebral oedema have occurred with some CAR T cell therapies.

Following treatment according to the invention, less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit any grade of neurotoxicity or immune effector cell-associated neurotoxicity syndrome (ICANS).

Grading for CRS is provided in Table 7 with recommendations regarding treatment provided in Table 9.

Table 7. Assessment and Grading of ICANS

CAPD = Cornell Assessment of Paediatric Delirium; CTCAE = Common Terminology Criteria for Adverse Events; EEG = electroencephalogram; ICANS = Immune effector Cell-Associated Neurotoxicity Syndrome; ICE = immune effector cell-associated encephalopathy; ICP = intracranial pressure; N/A = Not applicable; NCI = National Cancer Institute.

Adapted from Lee 2018, ASCTT/ASBMT ICANS Consensus (Lee et al 2019)

ICANS grade is determined by the most severe event (ICE score, level of consciousness, seizure, motor findings, raised ICP/cerebral oedema) not attributable to any other cause.

^A A patient with an ICE score of 0 may be classified as having Grade 3 ICANS if the patient is awake with global aphasia. But a patient with an ICE score of 0 may be classified as having Grade 4 ICANS if the patient is unarousable.

* Depressed level of consciousness should be attributable to no other cause (e.g. no sedating medication)

§ Tremors and myoclonus associated with immune effector cell therapies may be graded according to NCI CTCAE version 5.0 but they do not influence ICANS grading.

# Intracranial haemorrhage with or without associated oedema is not considered a neurotoxicity feature and is excluded from ICANS grading. It may be graded according to NCI CTCAE version 5.0.

Table 8. Immune Effector Cell-associated Encephalopathy (ICE) Scale

ICANS = Immune effector Cell-Associated Neurotoxicity Syndrome; ICE = Immune effector Cell-Associated Encephalopathy.

Table 9. Management of ICANS.

AE = adverse event; CRS = cytokine release syndrome; CSF = cerebrospinal fluid; CT = computed tomography; EEG = electroencephalogram; ICU = intensive care unit; IL = interleukin; i.v. = intravenous; MRI = magnetic resonance imaging. Following treatment according to the invention, less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit a grade 3 or greater neurotoxicity or immune effector cell-associated neurotoxicity syndrome (ICANS).

Following treatment according to the invention, less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit a grade 3 or greater CRS, and less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. Preferably, following treatment according to the invention, less than 5% of the patients treated may exhibit a grade 3 or greater CRS, and less than 8% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. More preferably, following treatment according to the invention, less than about 3% of the patients treated may exhibit a grade 3 or greater CRS, and less than about 7% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS.

The age of the patient may be eighteen years or older.

The age of the patient may be between eighteen and thirty -nine years, or between forty and sixty-four years, or sixty -five years or older.

The age of the patient may be between eighteen and thirty-nine years and at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The age of the patient may be between forty and sixty-four years and at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The age of the patient may be sixty-five years or older and wherein at least at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

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

The patient may be Latino or Hispanic and at least 40%, or at least 45%, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The patient may have less than or equal to 20% blasts in the BM at lymphodepletion.

The patient may have less than or equal to 20% blasts in the BM at lymphodepletion and at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated may achieve an overall remission (OR) or are identified as responders.

The patient may have less than or equal to 20% blasts in the BM at lymphodepletion and less than 70%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 1% of the patients treated may exhibit any grade of CRS. Among this patient cohort, less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of patients treated may exhibit a grade 3 or greater CRS.

The patient may have less than or equal to 20% blasts in the BM at lymphodepletion and less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit any grade of neurotoxicity or ICANS. Among this patient cohort, less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS.

The patient may have less than or equal to 20% blasts in the BM at lymphodepletion, and less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit a grade 3 or greater CRS, and less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. Preferably, the patient may have less than or equal to 20% blasts in the BM at lymphodepletion. and less than 5% of the patients treated may exhibit a grade 3 or greater CRS, and less than 5% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. More preferably, the patient may have less than or equal to 20% blasts in the BM at lymphodepletion, and about 4% of the patients treated may exhibit a grade 3 or greater CRS, and about 4% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. More preferably, the patient may have less than or equal to 20% blasts in the BM at lymphodepletion, and about 3% of the patients treated may exhibit a grade 3 or greater CRS, and about 3% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. More preferably, the patient may have less than or equal to 20% blasts in the BM at lymphodepletion, and about 2% of the patients treated may exhibit a grade 3 or greater CRS, and about 2% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. More preferably, the patient may have less than or equal to 20% blasts in the BM at lymphodepletion, and about 1% of the patients treated may exhibit a grade 3 or greater CRS, and about 1% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS.

The patient may have between more than 20% and less than or equal to 75% blasts in the BM at lymphodepletion.

The patient may have between more than 20% and less than or equal to 75% blasts in the BM at lymphodepletion, and at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated may achieve an overall remission (OR) or are identified as responders.

The patient may have between more than 20% and less than or equal to 75% blasts in the BM at lymphodepletion, and less than 80%, or less than 70%, or less than 60%, or less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10%, or less of the patients treated may exhibit any grade of CRS. Among this patient cohort, 20%, or less than 15%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of patients treated may exhibit a grade 3 or greater CRS.

The patient may have between more than 20% and less than or equal to 75% blasts in the BM at lymphodepletion, and less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 1% of the patients treated may exhibit any grade of neurotoxicity or ICANS. Among this patient cohort, less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. The patient may have more than or equal to 75% blasts in the BM at lymphodepletion.

The patient may have more than or equal to 75% blasts in the BM at lymphodepletion and at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated may achieve an overall remission (OR) or may be identified as responders.

The patient may have more than or equal to 75% blasts in the BM at lymphodepletion and less than 95%, or less than 90%, or less than 85%, or less than 80%, or less than 75%, or less than 70%, or less of the patients treated may exhibit any grade of CRS. Among this patient cohort, 20%, or less than 15%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of patients treated may exhibit a grade 3 or greater CRS.

The patient may have more than or equal to 75% blasts in the BM at lymphodepletion and less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 1% of the patients treated may exhibit any grade of neurotoxicity or ICANS. Among this patient cohort, less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS.

The patient may have more than 20% blasts in the BM at lymphodepletion, and less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit a grade 3 or greater CRS, and less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. Preferably, the patient may have more than 20% blasts in the BM at lymphodepletion, and less than 5% of the patients treated may exhibit a grade 3 or greater CRS, and less than 10% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. More preferably, the patient may have more than 20% blasts in the BM at lymphodepletion, and about 4% of the patients treated may exhibit a grade 3 or greater CRS, and about 9% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. More preferably, the patient may have more than 20% blasts in the BM at lymphodepletion, and about 3% of the patients treated may exhibit a grade 3 or greater CRS, and about 8% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS. More preferably, the patient may have more than 20% blasts in the BM at lymphodepletion, and about 2% of the patients treated may exhibit a grade 3 or greater CRS, and about 6% of the patients treated may exhibit a grade 3 or greater neurotoxicity or ICANS.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may result in an event free survival (EFS) rate of at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or more at 6 months in the treated patient population. EFS is defined as the time from date of first infusion to the earliest of treatment failure, relapse, or death from any cause.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may result in an EFS rate of at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or more at 12 months in the treated patient population.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may result in an event free survival (EFS) rate of at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or higher at 6 months in patients having fewer than 5% blasts in the BM at lymphodepletion.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may result in an event free survival (EFS) rate of at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or higher at 12 months in patients having fewer than 5% blasts in the BM at lymphodepletion.

The median EFS may not be reached in patients having less than 5% blasts in the BM at lymphodepletion.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may result in an event free survival (EFS) rate of at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or higher at 6 months in patients having between greater than or equal to 5% blasts and fewer than or equal to 75% blasts in the BM at lymphodepletion.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may result in an event free survival (EFS) rate of at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or higher at 12 months in patients having between greater than or equal to 5% blasts and fewer than or equal to 75% blasts in the BM at lymphodepletion.

The median EFS may be 15 months in patients having less than 5% blasts in the BM at lymphodepletion.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may result in an event free survival (EFS) rate of at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or higher at 6 months in patients having greater than 75% blasts in the BM at lymphodepletion.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may result in an event free survival (EFS) rate of at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or higher at 12 months in patients having greater than 75% blasts in the BM at lymphodepletion.

The median EFS may be 4.5 months in patients having less than 5% blasts in the BM at lymphodepletion.

The patient may present morphological disease with >5% bone marrow (BM) blasts at screening.

The patient may be in >2^nd complete remission (CR)/CR with incomplete hematologic recovery (CRi) with measurable residual disease (MRD) at screening. MRD may be determined by any method known to the skilled person, such as flow cytometry or polymerase chain reaction (PCR).

The patient may be in >2^nd CR/CRi with MRD at screening and at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients may achieve CR/CRi or may be identified as responders. The patient may be in >2^nd CR/CRi with MRD at screening, may achieve CR/CRi or may be identified as responder, and may achieve MRD negative status. The patient may be in >2^nd CR/CRi with MRD at screening and less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or 0% of patients may exhibit a grade 3 or greater CRS. The patient may be in >2^nd CR/CRi with MRD at screening and less than 20%, or less than 15%, or less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of patients may exhibit a grade 3 or greater neurotoxicity or ICANS.

The patient may be in morphological remission at the time of lymphodepletion.

The patient may be in morphological remission at the time of lymphodepletion and at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of response evaluable patients may achieve CR/CRi.

The patient may be in morphological remission at the time of lymphodepletion and at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% of MRD evaluable responders may achieve MRD negative CR/CRi.

The patient may be in morphological remission at the time of lymphodepletion and may not exhibit a grade 3 or greater CRS. The patient may be in morphological remission at the time of lymphodepletion and may not exhibit a grade 3 or greater ICANS. The patient may be in morphological remission at the time of lymphodepletion and may not exhibit a grade 3 or greater CRS, and may not exhibit a grade 3 or greater neurotoxicity or ICANS.

The patient may present with isolated extramedullary disease (EMD).

The ECOG performance status determines the ability of patient to tolerate therapies in serious illness. Details of the ECOG performance status score are provided in Table 10.

Table 10. Eastern Cooperative Oncology Group (ECOG) Performance Status Score.

The patient may have Philadelphia chromosome positive ALL (Ph+ ALL). The leukaemia cells of these patients have the Philadelphia chromosome, which is formed by a translocation between parts of chromosomes 9 and 22. This chromosomal alteration creates a fusion gene called BCR- ABL1.

The Ph+ ALL patient may be intolerant to a tyrosine kinase inhibitor (TKI) or a second generation TKI is contraindicated.

The Ph+ ALL patient may have had previously been administered one or more TKIs. The Ph+ ALL patient may have had previously been administered two lines of a TKI. The Ph+ ALL patient may have had previously been administered one line of a second generation TKI. Non-limiting examples of TKIs or second generation TKIs include imatinib (Gleevec®), dasatinib (Sprycel®), ponatinib (Iclusig®), bosutinib (Bosulif®), and nilotinib (Tasigna®).

The patient may be a Ph+ ALL patient and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of Ph+ ALL patients treated achieve an overall remission (OR) or are identified as responders.

The patient may have been previously administered one or more prior lines of therapy. The patient may have been previously administered one prior line of therapy. The patient may have been previously administered two prior lines of therapy. The patient may have been previously administered three prior lines of therapy. The patient may have been previously administered four or more prior lines of therapy.

The B-ALL may be refractory to the last prior line of therapy.

The patient may have been previously administered one prior line of therapy and at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated achieve an OR or are identified as responders.

The patient may have been previously administered two prior lines of therapy. The patient may have been previously administered three prior lines of therapy. The patient may have been previously administered four or more prior lines of therapy.

The patient may have been previously administered one or more lines of therapy. The patient may have been administered two prior lines of therapy. The patient may have been administered three prior lines of therapy. The patient may have been administered four or more prior lines of therapy.

The patient may have been administered two prior lines of therapy and at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The patient may have been administered three prior lines of therapy and at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The patient may have been administered four or more prior lines of therapy and at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The patient may have been previously administered one or more lines of therapy and the B- ALL may be refractory to the last prior line of therapy.

The patient may have been previously administered one or more of an immunotherapeutic agent. Examples of immunotherapeutic agents include inotuzumab ozogamicin or blinatumomab.

The patient may have been previously administered blinatumomab.

The patient may have been previously administered blinatumomab and at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85 %, or at least 90%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The patient may have been previously administered inotuzumab ozogamicin. The patient may have been previously administered inotuzumab ozogamicin and at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85 %, or at least 90%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The patient may have been previously administered an approved CAR-T cell therapy. Examples of approved CAR-T cell therapies include tisagenlecleucel (Kymriah®) and brexucabtagene autoleucel (Tecartus®). Approved CAR-T cell therapies that may be approved in the future are also contemplated.

The patient may have previously received a stem cell transplant (SCT). SCT or haematopoietic stem cell transplant (HSCT) are intended to be synonyms herein.

The SCT may be allogeneic SCT.

The SCT may have been received at least 3 months prior to administration of the CAR-T cells.

The patient may have previously received an SCT and at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The patient may have extramedullary disease at preconditioning. Preconditioning is used herein as an alternative term for lymphodepletion.

The patient may have extramedullary disease at preconditioning and at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or more of patients treated achieve an overall remission (OR) or are identified as responders.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may be useful as a standalone therapy, i.e. without a consolidating stem cell transplant (SCT). Thus, the patient may not subsequently receive a SCT, preferably an allogeneic SCT. This may be reformulated as the patient may not receive a SCT, preferably an allogeneic SCT, after the administration of the CD 19 CAR T cell composition.

The treatment with the cell compositions of the present disclosure, for example the CD 19 CAR T-cell product composition described in Example 1, may be followed by a consolidating stem cell transplant (SCT) at a point when the patient experiences loss of CAR- T cell persistence. Thus, the patient may not subsequently receive a SCT, preferably an allogeneic SCT. Alternatively, the patient may not receive a SCT, preferably an allogeneic SCT, after the administration of the CD 19 CAR T cell composition.

The method of treating a relapsed or refractory CD 19+ haematological malignancy in a patient may further comprise a step of determining CAR-T cell persistence in a sample comprising peripheral blood mononuclear cells from the patient: wherein if CAR-T cells are detected, then the patient may not receive a SCT; and wherein if CAR-T cells are not detected, then the patient may receive a SCT.

The SCT may be an allogeneic SCT.

Stem cell transplant (SCT) or haematopoietic stem cell transplant (HSCT) are intended to be synonyms herein.

The sample may be taken at least 1 month, or at least 2 months, or at least 3 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months, or at least 36 months, or at least 42 months, or at least 48 months, or at least 54 months, or at least 60 months, or longer after administration of the CAR-T cells.

The SCT may be administered up to 60 months, or up to 54 months, or up to 48 months, or up to 42 months, or up to 36 months, or up to 30 months, or up to 24 months, or up to 24 months, or up to 18 months, or up to 15 months, or up to 12 months, or up to 9 months, or up to 6 months, or up to 3 months, or up to 1.5 months, or up to 1 month after administration of the CAR-T cells.

The SCT may be administered between 1 month and 60 months, or between 1 month and 54 months, or between 1 month and 48 months, or between 1 month and 42 months, or between 1 month and 36 months, or between 1 month and 30 months, or between 1 month and 24 months, or between 1 month and 24 months, or between 1 month and 18 months, or between 1 month and 15 months, or between 1 month and 12 months, or between 1 month and 9 months, or between 1 month and 6 months, or between 1 month and 3 months, or between 1 month and 1.5 months after administration of the CAR-T cells.

CAR-T cell persistence, or the presence of CAR-T cells, may be determined by detecting or determining the number of copies of the nucleic acid encoding the, or number of CAR transgene copies, measured by any means involving DNA amplification of the CAR transgene, such as PCR, qPCR, ddPCR, or rtPCR, in a peripheral blood sample taken from the patient. Alternatively, CAR-T cell persistence, or the presence of CAR-T cells, may be determined by detecting the intracellular domain(s) or extracellular domain(s) of the CAR protein by any means known by the skilled person, such as flow cytometry.

Less than 50%, 45%, 40%, 35%, 30%, 25%, or 20% of the patients treated may exhibit any grade of neutropenia or decreased neutrophil count. Less than 50%, 45%, 40%, 35%, 30%, 25%, or 20% of the patients treated may exhibit a grade 3 or greater neutropenia or decreased neutrophil count.

Less than 35%, 30%, 25%, or 20% of the patients treated may exhibit any grade of thrombocytopenia or decreased platelet count. Less than 35%, 30%, 25%, or 20% of the patients treated may exhibit a grade 3 or greater thrombocytopenia or decreased platelet count.

Less than 35%, 30%, 25%, or 20% of the patients treated may exhibit any grade of nausea. Less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the patients treated may exhibit a grade 3 or greater nausea.

Less than 35%, 30%, 25%, or 20% of the patients treated may exhibit any grade of pyrexia. Less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the patients treated may exhibit a grade 3 or greater pyrexia.

Less than 50%, 45%, 40%, 35%, 30%, 25%, or 20% of the patients treated may exhibit any grade of febrile neutropenia. Less than 50%, 45%, 40%, 35%, 30%, 25%, or 20% of the patients treated may exhibit a grade 3 or greater febrile neutropenia.

Less than 35%, 30%, 25%, or 20% of the patients treated may exhibit any grade of headache. Less than 5%, 4%, 3%, 2%, or 1% of the patients treated may exhibit a grade 3 or greater headache.

Less than 35%, 30%, 25%, or 20% of the patients treated may exhibit any grade of diarrhoea. Less than 5%, 4%, 3%, 2%, or 1% of the patients treated may exhibit a grade 3 or greater diarrhoea.

Less than 35%, 30%, 25%, or 20% of the patients treated may exhibit any grade of anaemia. Less than 35%, 30%, 25%, or 20% of the patients treated may exhibit a grade 3 or greater anaemia. Less than 35%, 30%, 25%, or 20% of the patients treated may exhibit any grade of hypotension. Less than 10%, 5%, 4%, 3%, 2%, or 1% of the patients treated may exhibit a grade 3 or greater hypotension.

The patient may have a presence of between about 0% blasts and 100% blasts in the BM at preconditioning.

The present disclosure also provides a method for reducing the risk of toxicity resulting from administration of an autologous CD 19 CAR-T cell or an autologous CD 19 CAR-T cell population to a patient having a relapsed or refractory CD 19+ haematological malignancy, comprising administering an autologous CD 19 CAR T-cell or an autologous CD 19 CAR-T cell population to the patient, wherein: if the patient has a presence of less than or equal to 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 100 x 10⁶ CAR T-cells and a second dose comprising about 310 x 10⁶ CAR T-cells; and/or if the patient has a presence of more than 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 10 x 10⁶ CAR T-cells and a second dose comprising about 400 x 10⁶ CAR T-cells.

The toxicity resulting from administration of an autologous CD 19 CAR-T cell or an autologous CD 19 CAR-T cell population to a patient having a relapsed or refractory CD 19+ haematological malignancy may be CRS or ICANS.

The definitions and particular embodiments of the autologous CD 19 CAR-T cell or autologous CD 19 CAR-T cell population for use in a method of treating a relapsed or refractory CD 19+ haematological malignancy in a patient of the invention apply equally to the method for reducing the risk of toxicity resulting from administration of an autologous CD 19 CAR-T cell or an autologous CD 19 CAR-T cell population to a patient described herein.

The present invention also provides a method of predicting the severity of toxicity resulting from administration of an autologous CD 19 CAR-T cell or an autologous CD 19 CAR-T cell population to a patient having a relapsed or refractory CD 19+ haematological malignancy, and/or deterring or decreasing such toxicity, comprising determining the percentage of blasts in the BM at lymphodepletion, wherein if the BM has more than 20% blasts, or more than 50% blasts, or more than 75% blasts, then the treated patient may exhibit a grade 3 or greater toxicity.

The BM may have more than 75% blasts at lymphodepletion.

The toxicity may be CRS or ICANS.

The present invention also provides CD 19 CAR engineered cells for use in a method of treatment of a hematological malignancy as described herein, 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 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.

The administration may be an intravenous injection. The intravenous injection may be through a Hickman line or peripherally inserted central catheter (PICC line).

The present invention also provides CD 19 CAR engineered cells, e.g. autologous CD 19 CAR T cells, for use in a method of treatment of a hematological malignancy as described herein, wherein the engineered cells may expand at a high level upon administration to the patient.

The CAR engineered cells may be monitored over time in the blood of the patient to determine the cellular kinetics. This may be done by detecting the number of copies of the nucleic acid encoding the, or number of CAR transgene copies, measured by any means involving DNA amplification of the CAR transgene, such as PCR, qPCR, ddPCR, or rtPCR, in a peripheral blood sample taken from the patient. The maximum serum or blood concentration of CD19 CAR engineered cells (Cmax) may be about 80,000 copies/pg DNA or higher, about 85,000 copies/pg DNA or higher, about 90,000 copies/pg DNA or higher, about 95,000 copies/pg DNA or higher, about 100,000 copies/pg DNA or higher, about 105,000 copies/pg DNA or higher, about 110,000 copies/pg DNA or higher, about 115,000 copies/pg DNA or higher, about 120,000 copies/pg DNA or higher, about 125,000 copies/pg DNA or higher, or about 130,000 copies/pg DNA or higher. Alternatively, this may be done by detecting the intracellular domain(s) or extracellular domain(s) of the CAR protein by any means known by the skilled person, such as flow cytometry.

The time to reach maximum serum or blood concentration (Tmax) of CD 19 CAR engineered cells may be between about 2 and about 55 days, or between about 3 and about 40 days, or between about 4 and about 30 days, or between about 5 and about 25 days, or between about 6 and about 23 days, or between about 7 and about 21 days, or between about 8 and about 20 days following administration of the engineered cells to the patient. The Tmax may be at 8 days, or at 9 days, or at 10 days, or at 11 days, or at 12 days, or at 13 days, or at 14 days, or at 15 days, or at 16 days, or at 17 days, or at 18 days, or at 19 days, or at 20 days, or at 21 days, or at 22 days, or at 23 days, or at 24 days, or at 25 days, or longer following administration of the engineered cells to the patient.

The area under the curve at day 28 (AUCo-28d) may be at least 1,000,000 copies/pg *d, or at least 1,000,000 copies/pg *d, or at least 1,050,000 copies/pg *d, or at least 1,100,000 copies/pg xd, or at least 1,150,000 copies/pg xd, or at least 1,200,000 copies/pg xd, or at least 1,250,000 copies/pg xd, or more.

The CD 19 CAR engineered cells may persist or be detectable in the patient at at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 36 months, or at least 48 months, or at least 60 months, or longer, following administration.

The present invention also provides CD 19 CAR engineered cells for use in a method of treatment of a hematological malignancy as described herein, wherein the CD 19 CAR engineered cells may persist in the patient’s peripheral blood or bone marrow for a prolonged period. A long persistence of CAR engineered cell is particularly advantageous when treating relapsed/refractory conditions as described herein, since high levels are important in effecting durable responses and prevent CD 19+ relapse. The CD 19 CAR engineered cells may persist for at least 28 days, or at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 15 months, or at least 18 months, or at least 21 months, or at least 24 months, or at least 36 months, or at least 48 months, or at least 60 months, or longer in the patient’s peripheral blood or bone marrow.

The present invention also provides a method of selecting a patient for receiving a second therapy following a treatment with CD 19 CAR engineered cells, which comprises determining CAR-T cell persistence in a sample comprising peripheral blood mononuclear cells from the patient: wherein if CAR-T cells are detected, then the patient may not receive a second therapy; and wherein if CAR-T cells are not detected, then the patient may receive a second therapy, wherein the patient has a relapsed or refractory CD 19+ haematological malignancy as described herein prior to receiving the CD 19 CAR engineered cells.

The second therapy may be a stem cell transplant (SCT) or another suitable therapy. The SCT may be an allogeneic SCT. The other suitable therapy may be a tyrosine kinase inhibitor (TKI) if the patient has Ph+ ALL.

Stem cell transplant (SCT) and haematopoietic stem cell transplant (HSCT) are intended to be synonyms herein.

The CD 19 CAR engineered cells may be the cell composition of the present disclosure, for example the CD19 CAR T-cell product composition described in Example 1.

The patient may have relapsed.

The patient may have measurable residual disease (MRD).

The sample may be taken at least 1 month, or at least 2 months, or at least 3 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months, or at least 36 months, or at least 42 months, or at least 48 months, or at least 54 months, or at least 60 months, or longer after administration of the CAR-T cells. The second therapy (e.g. SCT or TKI) may be administered up to 60 months, or up to 54 months, or up to 48 months, or up to 42 months, or up to 36 months, or up to 30 months, or up to 24 months, or up to 24 months, or up to 18 months, or up to 15 months, or up to 12 months, or up to 9 months, or up to 6 months, or up to 3 months, or up to 1.5 months, or up to 1 month after administration of the CAR-T cells.

The second therapy (e.g. SCT or TKI) may be administered between 1 month and 60 months, or between 1 month and 54 months, or between 1 month and 48 months, or between 1 month and 42 months, or between 1 month and 36 months, or between 1 month and 30 months, or between 1 month and 24 months, or between 1 month and 24 months, or between 1 month and 18 months, or between 1 month and 15 months, or between 1 month and 12 months, or between 1 month and 9 months, or between 1 month and 6 months, or between 1 month and 3 months, or between 1 month and 1.5 months after administration of the CAR-T cells.

Patient-reported outcomes (PROs) are an important tool for assessing the impact of CAR-T therapy on symptom burden and health-related quality of life (HRQoL), such as changes over time in symptoms, functioning, and overall QoL. PRO measures may be collected using the EuroQol EQ-5D-5L instrument and the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30; https://www.eortc.Org/app/uploads/sites/2/2018/08/Specimen-QLQ-C30-English.pdf). PRO measures may be collected prior to CAR-T cell infusion to determine the baseline and at several times post-infusion, for example at 28 days and 3, 6, 9, 12, and 18 months postinfusion.

The patient may report PROs which exceed baseline status at 28 days, or at 3 months, or at 6 months, or at 9 months, or at 12 months, or at 18 months post-infusion, for examples using the EuroQol EQ-5D-5L instrument and the EORTC QLQ-C30. Other terminology and disclosure

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

When a range of values is provided herein, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.

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

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

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

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

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

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 AUT01) 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.

AUT01 was generated by ex vivo transduction of activated peripheral blood mononuclear cells (PBMCs) using an engineered HIV derived lentiviral vector (LV18970) 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.

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 AUT01 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).

Example 2: Non-clinical studies

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. Binding Kinetics of the CA T Binder in A UT01

Binding kinetics of the CAR binding domains CAT scFv (used in AUT01) 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.

The > 40-fold lower KD with the CAT scFv was the result of a much faster off-rate (CAT : 3.1 x 10'³ s'¹ versus FMC63: 6.8 * 10'⁵ s'¹), while the on-rate was equivalent (CAT: 2.2 * 10⁵ M' ¹ versus FMC63: 2.1 x io⁵ M' ) (Table 11).

Table 11 : Summary of Binding Kinetics of the CAR Binding Domains CAT scFv and FMC63 scFv.

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

Proliferation of CD 19 CAR T Cells in Vitro

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.

Cytokine Production and Cytotoxicity of CD19 CAR T Cells

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). In a standard ⁵¹Cr release assay, T cells transduced to express CD 19 (CAT) CAR or CD 19 (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.

In Vivo Pharmacology

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.

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 CD19 (CAT) CAR T cells: 1.1 x 10⁸ to 9.3 x 10⁷ photons/second/cm2/steradian; CD19 (FMC63) CAR T cells 3.2 x 10⁹ to 7.7 x 10⁸ photons/second/cm2/steradian, n=18, p<0.001.

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 10⁵ versus 3 x 10² NALM-6 cells/mL, p<0.0001) (Figure 6).

Conversely, a significantly greater absolute number of CD 19 (CAT) CAR T cells than CD 19 (FMC63) CAR T cells persisted in the BM of NALM-6 NSG mice (mean 3.4 x 10⁴ CD19 (CAT) CAR T cells/mL versus 1.3 x 10⁴ CD19 (FMC63) CAR T cells/mL, n=18, p<0.05) and in the peripheral blood (mean: 1.9 x l()⁴ CD19 (CAT) CAR T cells/mL versus 2.8 x 10³ CD19 (FMC63) CAR T cells/mL, n=9, p<0.001) (Figure 7). 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.

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.

It has been 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 suggest that no off-target toxicity is anticipated.

Example 3: Clinical study

A study of the safety and clinical efficacy to the CD19CAR T-cell product (also identified herein as AUT01) 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 >2^nd complete 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.

The results shown herein are as of 16^th March 2023, in the morphological Phase II Cohort A of the clinical study.

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 AUTO1 (Figure 8, right panel). Therefore, 84% of enrolled patients were infused with AUTO1.

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.

Patients received a total target dose of 410 x 10⁶ CD 19 CAR-positive T cells (±25%) on a schedule as illustrated below (Table 12).

Table 12: AUTO1 Dosing Regimen

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

For the avoidance of doubt, the dosing schedule shown in Table 12 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.

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.

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

Inclusion Criteria:

• Age 18 years or older Age 18 years or older;

• ECOG performance status of 0 or 1 ;

• Relapsed or refractory B cell ALL;

• 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; • Documented CD 19 positivity within 1 month of screening;

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

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

• Phase II: Primary Cohort IIA: Presence of >5% blasts in BM at screening;

• Phase II: 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;

• Adequate renal, hepatic, pulmonary, and cardiac function.

Exclusion Criteria:

• Phase lb (Cohort I A and Cohort IB) and Phase II (Cohort IIA and Cohort IIB) B-ALL with isolated EM disease;

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

• History or presence of clinically relevant CNS pathology;

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

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

• Active or latent Hepatitis B virus or active Hepatitis C virus;

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

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

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

The primary endpoints for the study were as follows:

1. ORR defined as proportion of patients achieving CR or CRi by central assessment. [Time Frame: Up to 24 months]

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]

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

3. Phase II - Response to AUT01 treatment measured as duration of remission (DOR) [Time Frame: Up to 24 months] 4. Phase II - Response to AUT01 measured as progression-free survival (PFS).

[Time Frame: Up to 24 months]

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

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

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

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

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

The baseline characteristics of the patient population is shown in Table 13. These characteristics revealed that these were heavily pre-treated patients with high disease burden.

Table 13. Baseline characteristics.

Manufacturability of AUT01

Patients’ leukapheresates were sent for AUT01 product manufacturing. 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.

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).

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 10'⁴ level by flow cytometry.

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.

Subgroup Analysis of CR/CRi (IRRC Assessment)

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

The safety results are shown in Table 14.

Table 14.

Overall, low rates of Grade >3 CRS and/or ICANS were observed. Tocilizumab and steroid was 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 pre-conditioning.

Safety: Treatment emergent adverse events (TEAEs)

The safety results regarding treatment emergent adverse events (TEAEs) are shown in Table 15.

Table 15.

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).

AUT01 expansion and persistence

AUT01 expansion and persistence results are shown in Figure 13 and Table 16.

Table 16.

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

Results obtained from the peripheral blood of all 94 patients with r/r B ALL infused with AUT01 measured by qPCR showed expansion and persistence of CD 19 CAR-positive T cells (Figure 13) consistent with cellular kinetics data of AUT01 in the ALLCAR19 study (Roddie C et al., 2021. J Clin Oncol 39:3352-63).

Example 4: Clinical study. Pooled analysis of all treated patients

The CD19CAR T-cell product (AUTO1) is an autologous chimeric antigen receptor (CAR) T cell product with a novel CD 19 binding domain CAT conferring a fast antigen off-rate designed to mitigate safety concerns and improve persistence over approved CD 19 CAR T therapies. Early results in B-ALL from the pivotal clinical study Phase IIA cohort (N=94) are described in Example 3. Example 4 contains the findings from a pooled analysis of all patients treated to date with AUTO1 in the Phase Ib/II study, with a focus on patients with low leukaemia burden prior to AUTO1 infusion.

Methods

The clinical study enrolled adults with R/R B-ALL at screening with either morphological disease >5% bone marrow (BM) blasts (Cohort A), or in >2^nd complete 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.

As already described in Example 3, CD19CAR T-cell products were generated from leukapheresis material using an automated process. Patients received bridging therapy as needed and lymphodepletion with fludarabine (4 x 30mg/m²) and cyclophosphamide (2 x 500mg/m²). A target dose of 410 x io⁶ CAR T cells was infused as a split dose on Days 1 and 10 based on pre-lymphodepletion BM blast burden.

The primary endpoint was overall remission rate (best response of CR/CRi by independent review). Secondary endpoints included: duration of remission (DoR),

MRD negative remission rate, safety, and

CAR T expansion/persistence. This pooled analysis included data from patients treated with AUT01 across all cohorts in the Phase Ib/II parts of the study. Low leukaemia burden was defined as morphological remission per investigator assessment (<5% BM blasts without EMD) as measured at screening or at the start of lymphodepletion.

Results

Between September 2020 and December 2022, 152 pts were enrolled and underwent leukapheresis. As of 16 March 2023, AUTO1 was successfully administered to 126/152 (83%) patients. The distribution of patients across the three cohorts is shown in Table 17. The baseline characteristics at screening of the patient population are shown in Table 18.

Table 17.

Table 18. Baseline characteristics at screening.

At a median follow up of 11.0 (range 0.9-30.6) months, the CR/CRi rate was 77% (95/124 response evaluable pts) with CR rate 57% (71/124). Among MRD evaluable responders, 96% achieved MRD negative status by central flow cytometry analysis. Median DoR was not reached at the current follow up. Low rates of Grade (Gr) >3 cytokine release syndrome (CRS; 2.4%) and/or Gr >3 immune effector cell associated neurotoxicity syndrome (ICANS; 7.1%) were observed. CAR T expansion was similar across the Phase Ib/II cohorts and CAR T persistence was ongoing in the majority of responders at the current follow up.

Preliminary data indicated favourable efficacy and safety in patients with low leukaemia burden prior to AUT01 infusion. Among 12 patients with minimal residual disease (MRD) at screening (Cohort B), two patients were not evaluable and 9/10 evaluable patients achieved CR/CRi, with 100% achieving MRD negative status by central flow cytometry analysis post AUTO1. Median DoR was not reached at the current follow up. In this subset, no Gr >3 CRS was observed; one patient had Gr >3 ICANS.

In a subset of 28 patients (across all cohorts) in morphological remission at the time of lymphodepletion, 24/27 (89%) response evaluable patients achieved CR/CRi and 100% of MRD evaluable responders achieved MRD negative CR/CRi by central flow cytometry analysis post AUTO1. Median DoR was not reached at the current follow up. In this subset, no patients experienced Gr >3 CRS/ICANS.

This pooled analysis of data from all patients treated in the Phase Ib/II clinical study demonstrated high rates of CR/CRi after AUTO1 treatment, durable responses (median DoR not reached), and a favourable safety profile. Preliminary data indicate better outcomes in patients with low leukaemia burden at screening/lymphodepletion, with higher rates of deep MRD negative complete remission, median DoR not reached at the current follow up, no Gr >3 CRS and one Gr >3 ICANS.

Example 5: Long term efficacy of CD19 CAT CAR T-cell product (AUTO1)

The clinical activity of AUTO1 has been explored in adults with R/R B-ALL in a Phase I study (ALLCAR19; Roddie C et al. J Clin Oncol 2021) and a Phase Ib/II study. Additionally, AUTO1 has been tested in patients with R/R B-cell chronic lymphocytic leukemia (B-CLL) and R/R B-cell non-Hodgkin lymphoma (B-NHL) (ALLCAR19 extension; Roddie C et al. Blood 2022;140[l Suppl]:7452-3). Patients from the ALLCAR19 and Phase lb studies are in long-term follow up (>22 mos), and the ALLCAR19 extension has been recruiting for 3 years. Below is an analysis of long-term efficacy and safety data from the ALLCAR19 and Phase lb studies, as well as data from the ALLCAR19 extension. Methods

ALLCAR19 was a multicenter, non-randomized, open-label Phase I study in patients aged >16 years with B-cell malignancies. ALLCAR19 initially patients with R/R B-ALL but was then amended (extension study) to also include patients with R/R B-CLL and R/R B-NHL. Additionally, a global, single-arm Phase Ib/II study was enrolling patients aged >18 years with R/R B-ALL. Study designs are shown in Table 19.

Table 19: ALLCAR19 and Phase Ib/II clinical studies with AUT01.

AUT01 was administered as a split dose in patients with B-ALL (target dose 410 x io⁶ CAR T cells) and patients with CLL (target dose 230 x io⁶ CAR T cells), and as a single infusion in patients with B-NHL (target dose 200 x 10⁶ CAR T cells); the patient populations in the two studies were similar. Patients with B-ALL from the ALLCAR19 and Phase lb studies are being followed long term for disease progression and survival. For this analysis, data in patients with B-ALL from the ALLCAR19 and Phase lb studies were pooled. Data in patients with CLL or B-NHL were taken from the ALLCAR19 extension study.

Results

Outcomes in patients with R/R B-ALL:

Data in patients with B-ALL were pooled (20 patients from ALLCAR19 [data cut-off Jun 26, 2023] and 16 from Phase lb [data cut-off Mar 16, 2023]). The median age of the pooled cohort was 41.5 (range 18 to 74) years and patients had received a median of 3 (range 2 to 6) prior lines of treatment. Twenty-nine of the 36 patients (81%) achieved complete remission (CR)/CR with incomplete hematologic recovery (CRi) post AUT01 infusions, per investigator assessment. The event-free survival rate was 64% at 6 months (mos) and 49% at 12 mos. With a median follow up of 43 (range 19 to 62) months, 13/36 patients (36%) remain in remission (8 from ALLCAR19; 5 from Phase lb). Among these 13 ongoing responders, 2 (15%) had consolidation with allogeneic hematopoietic stem cell transplantation (allo- HSCT). Ten of the 11 ongoing responders (91%) who did not receive allo-HSCT still had detectable CAR T cells at the last follow up. All ongoing remissions were measurable residual disease (MRD) negative at last available assessment. The estimated 2-, 3- and 4-year overall survival rates were 44%, 39% and 39%, respectively.

Outcomes in patients with R/R B-CLL/B-NHL:

The extension phase of the ALLCAR19 study enrolled 35 patients with B-CLL or B-NHL, of which 26 (B-CLL n=5; B-NHL n=21) received AUT01 (data cut-off Jun 26, 2023). The median age of this combined cohort was 61 (range 39 to 79) years and patients had received a median of 3 (range 2 to 8) prior lines of treatment. At a median follow up of 24 mos, the overall response rate for this cohort was 92% (n=24), and 58% of responders (n=14) were alive without disease progression at last follow up.

Late toxicity:

Of the 11 long-term R/R B-ALL responders who had not received consolidation allo-HSCT, 10 have ongoing B-cell aplasia. Of the 14 ongoing responders in the R/R B-CLL/B-NHL cohort, 12 have ongoing B-cell aplasia (<20 B cells/pl). Of note, ongoing B-cell aplasia did not correlate with an increased risk of late serious infection. No other long-term toxicity ascribed to AUTO1 was reported.

The combined analysis of data from the ALLCAR19 and Phase lb studies shows long-term efficacy and safety of AUTO1 in patients with R/R B-ALL, with approximately one-third of patients still in remission without consolidative allo-HSCT after a median follow up of >3 years. Durable responses of >2 years were also seen in patients with R/R B-CLL and R/R B- NHL. B-cell aplasia was commonly found in long-term follow up of AUTO1 recipients, but without a corresponding rise in serious late infections. AUTO1 can effect durable long-term remissions in B-cell malignancies. Example 6: Vein-to-vein delivery of CD19 CAT CAR T-cell product (AUTO1)

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. Having robust, reliable manufacturing, testing, logistics processes, and teams that are able to provide a consistent CAR T product is key to achieving timely vein-to-certification/vein-to-delivery (V2C/V2D) targets. Below is a report on the manufacturing, quality control (QC) and logistics processes set up for the Phase Ib/II clinical study with AUT01 in B-ALL patients, and the impact of on-study optimization for scale up of production.

Methods

The Phase Ib/II clinical study design required manufacturing, testing and logistics processes that could support a global, multicenter study with a target infusion of ~90 pts (Cohort A of Phase II) (Figure 31). The key elements were: 1) manufacturing and testing location: AUT01 was manufactured in Stevenage, UK, chosen because of proximity to a major international airport (London Heathrow) that provides logistics access to Europe and the US, and the ability to leverage existing knowledge and talent; 2) manufacturing process: manufacturing was carried out 7 days/week, was highly automated with mainly closed- system processing, using a Miltenyi Biotec Prodigy® (https://www.miltenyibiotec.com), 3) analytical procedures: QC procedures, including measuring biological activity and sterility testing, were carried out in-house to reduce turnaround time (TAT); 4) logistics: TAT was minimized and reliability improved by establishing primary and secondary routes plus turnkey contingency plans with courier and charter flight options 5) targets: V2C (time from leukapheresis to quality release) and V2D (time from leukapheresis to delivery of product to the hospital) of ~23 and ~25 days, respectively, for the Phase Ib/II clinical study.

Results

Between Jul 2021 and Dec 2022 (Phase II Cohort A), the 32 clinical sites for the Phase Ib/II clinical study in the US, UK and Spain were all supplied AUT01 from the UK. Median V2C and V2D times were 21 and 24 days, respectively (Figure 32A and 32B, respectively).

The manufacturing process drove towards a less differentiated T-cell phenotype in the final product (Table 42). Despite the median leukemic B-cell content in apheresis being 21% (range 0-97%) and the median CD3+ T cell content being 13 % (range 1-91%), 96% of manufactured AUTO1 batches reached their target dose of 410 x 10⁶ CAR T cells. From apheresis to product the median (range) percentage of naive T cells increased from 20% (1- 73%) to 31% (2-74%) while the medians for TCM and TEM remained unchanged at 13% to 13% and 19% to 17%, respectively, and the median TEMRA content decreased from 37% (range 4-93%) to 31% (range 5-90%), indicating that the manufacturing process managed to drive towards a less differentiated T cell phenotype in the final drug product.

Table 42:

n/a: not applicable.

Transduction rates were consistently high despite the heterogeneous leukapheresis product (Figure 33). Transduction rates were consistently high with a median of 70% (range 14- 87%).

In the morphological Phase II Cohort A of the clinical study, 112 pts were leukapheresed and 94 (84%) were dosed.

All leukapheresis collections and cell product deliveries were executed successfully and with the expected quality, despite the challenges posed by the COVID-19 pandemic. In addition, US international airline flights decreased by 41 % (US Dept of Transportation, Bureau of Transportation Statistics; July 2023) compared with pre COVID-19 pandemic, but sample collection and product delivery were successfully maintained, with no batches impacted (Figure 34). Maintaining consistently short QC and release times were driven by streamlined analytical strategy and batch records. The Phase Ib/II clinical study with AUT01 successfully demonstrated the robust operability of AUT01 manufacturing, QC and logistics processes, meeting target V2C and V2D. All leukapheresis starting material was successfully processed despite the multitude of constraints posed by patient health and the COVID-19 pandemic. Further optimisation and improvements made during the study increased reliability, consistency and precision of the manufacturing process.

Example 7: Pooled analysis of all r/r B-ALL patients treated with CD19 CAT CAR T-cell product (AUTO1) in the Phase Ib/II study

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

The clinical study methods were described in Example 4. 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 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 (Table 20).

Table 20.

*Unless otherwise stated; BM, bone marrow; EMD, extramedullary disease; SCT, stem cell transplant

Results

The data cut-off date is 13^th September 2023.

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.

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). The Forest plot in Figure 19 shows that AUT01 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.

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.

Cellular kinetic data and Kaplan-Meier EFS curves (Table 21 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. AUTO1 had excellent expansion with a mean Cmax of 110,000 copies/pg with an AUCO-28 of 1.1 million copies (Table 21). 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.

Table 21 : AUTO pharmacokinetics.

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.

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 22). Table 22.

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

Table 23

CRS, cytokine release syndrome; ICANS, immune effector cell-associated neurotoxicity syndrome;

TEAE, treatment emergent adverse event. 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 24).

Table 24: Summary of data overall and by bone-marrow blasts prior to lymphodepletion

*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).

Other TEAEs are listed in Table 23.

In conclusion:

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

High remission rates independent of leukemic burden at lymphodepletion;

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

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

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

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

Assessment of leukemic burden at lymphodepletion is essential for risk/benefit stratification. AUT01 is effective treatment for R/R adult B-ALL, with better outcomes observed in patients with lower leukemic burden at lymphodepletion.

Example 8: Long term efficacy and safety of CD19 CAT CAR T-cell product (AUTO1) in Adult Patients (pts) with Relapsed/Refractory B-cell Acute Lymphoblastic Leukemia ([R/R B-ALL]; Pooled Analysis from ALLCAR19 and Phase lb Studies) or Other B-cell Malignancies (ALLCAR19 Extension Study)

The clinical activity of AUT01 was explored in adults with R/R B-ALL in a Phase I study (ALLCAR19), and a Phase Ib/II study. Additionally, AUT01 was tested in patients with R/R B-cell chronic lymphocytic leukemia (B-CLL) and R/R B-cell non-Hodgkin lymphoma (B- NHL).

Methods

ALLCAR19 is a multicenter, non-randomized, open-label Phase I study in patients aged >16 years with B-cell malignancies (data cut-off: November 01, 2023).

The Phase Ib/II study is a global, open-label, single-arm study enrolling patients aged >18 years with R/R B-ALL (data cut-off: September 13, 2023).

AUTO1 was administered as a split dose in patients with B-ALL (target dose: 410 x 10⁶ CAR T-cells) and B-CLL (target dose: 230 x 10⁶ CAR T-cells) and as a single infusion (200 x 10⁶ CAR T-cells) in patients with B-NHL.

Results

Outcomes in patients with R/R B-ALL

The baseline characteristics of the 36 patients with r/r B-ALL included in this analysis is shown in Table 25.

Table 25:

*N = 36 patients with R/R B-ALL comprised n = 20 from ALLCAR19 and n = 16 from the Phase lb study. Allo-HSCT, allogeneic hematopoietic stem cell transplant; B-ALL, B-cell acute lymphoblastic leukemia; BM, bone marrow; R/R, relapsed/refractory.

ALLCAR19 The patient disposition is shown in Figure 25.

Data from the pooled analysis of r/r ALL patients (n=36) treated with AUTO1 in the ALLCAR19 and Phase lb studies demonstrate high remission rates of 81% (29/36) (Figure 26. After a median follow-up of 3 years and without subsequent transplant 41% of patients continue in complete remission (Figure 27B). The estimated EFS rate with censoring of subsequent transplant or new treatment was 45% at 36 months (Figure 27A); all patients in ongoing remission were MRD negative at last assessment and median duration of response was not reached (Figure 26). Results shown in Figure 28 demonstrated a long persistence of the CAR-T cells. There were no new safety signals or deaths related to AUTO1 in R/R B- ALL, as shown in Table 26.

Table 26:

*Patients with ongoing CR/CRi without subsequent allogeneic hematopoietic stem cell transplant. 'Grade 3 was the highest reported grade. Safety assessments were conducted according to NCI-CTCAE 4.03/5.0 and ASTCT/ASBMT criteria.¹ B-cell aplasia was assessed using flow cytometry and defined as <20 B-cells/pL. ASBMT, American Society for Blood and Marrow Transplantation; ASTC, American Society for Transplantation and Cellular Therapy; B-ALL, B-cell acute lymphoblastic leukemia; CR/CRi, complete remission/complete remission with incomplete hematologic recovery; CRS, cytokine release syndrome; ICANS, immune effector cell-associated neurotoxicity syndrome; IVIG, intravenous immunoglobulin; NCI-CTCAE, National Cancer Institute common terminology criteria for adverse events; R/R, relapsed/refractory. Outcomes in patients with CLL and NHL

In the CLL and NHL cohorts of the ALLCAR19 study and with >2 years follow up, high response rates and durable responses were observed (Figure 29, Table 27).

Table 27:

Low grade or low frequency grade >3 CRS/ICANS was observed across all indications and all dosing regimens (Table 28).

Table 28:

*Patients currently alive without progression. Safely assessments were conducted according to NCI-CTCAE 4.03/5.0 and ASTCT/ASBMT criteria. B-cell aplasia was assessed using flow cytometry and defined as <20 B- cells/pL. ASBMT, American Society for Blood and Marrow Transplantation; ASTCT, American Society for Transplantation and Cellular Therapy; CRS, cytokine release syndrome; ICANS, immune effector cell- associated neurotoxicity syndrome; IVIG, intravenous immunoglobulin; NCI-CTCAE, National Cancer Institute common terminology criteria for adverse events.

Excellent expansion and persistence of CAR T cells was evident across the studies (Figure 30). In summary, AUT01 shows durable remissions in a range of B-cell malignancies with a consistent safety profile.

Conclusions:

AUT01 can result in durable remissions in adults with R/R B-ALL, and prolonged AUT01 persistence is seen in most long-term responders;

High response rates with durable remissions were observed in patients with R/R B- CLL/B-NHL;

- Notably, in patients with DLBCL, 8/9 evaluable patients entered CMR; 6 patients are in ongoing CMR with one relapse at 12 months and one unrelated death;

As expected, B-cell aplasia was common among patients in long-term ongoing remission following AUT01 therapy but without a corresponding increase in serious late infections; and

AUT01 is consistently associated with low levels of grade >3 CRS and ICANS across indications and dosing regimens.

Example 9: Quality of life in adult patients with R/R B-ALL treated with CD19 CAT CAR T-cell product (AUTO1) in the pivotal Phase 2 clinical study

Patient-reported outcomes (PROs) are an important tool for assessing the impact of CAR-T therapy on symptom burden and health-related quality of life (HRQoL).

The efficacy and safety of AUT01 in adults with R/R B-ALL enrolled in the Phase Ib/II clinical study has been reported in the previous Examples.

A secondary objective of the Phase Ib/II clinical study was the evaluation of HRQoL (changes over time in symptoms, functioning, and overall QoL) using PROs in patients enrolled to Phase II Cohort A (Cohort IIA; patients with morphological disease defined as >5% leukemic B blast cells in the bone marrow at screening).

Methods

Patients were treated with AUT01 according to the study design shown in Figure 7 and Figure 35.

PRO measures were collected prior to AUT01 infusion (at lymphodepletion; baseline) and at 28 days and 3, 6, 9, 12, and 18 months post-infusion, using the EuroQol EQ-5D-5L instrument and the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30); questionnaires were available in multiple languages. PRO data were not collected for individual patients after treatment failure or relapse.

Results for the EQ-5D-5L visual analog scale (VAS) and EORTC QLQ-C30 Global Health Scores (GHS) scores in patients receiving AUT01 are described as follows.

Both PRO measures were scaled with scores ranging 0-100, with higher scores indicating higher QoL.

Changes in PRO scores were assessed descriptively by analysing median changes from baseline for EQ-5D-5L VAS and EORTC QLQ-C30 GHS scores.

Changes from baseline were interpreted using pre-specified thresholds defining improvement or deterioration, with a >10-point increase or decrease used to distinguish meaningful improvement or deterioration, respectively.

Results

Overall, 112 patients were enrolled to the Phaselb/II study Cohort IIA (Table 1), of whom 94 received an infusion of AUTO1. The baseline characteristics of the 112 enrolled patients is shown in Table 29.

Table 29: Baseline characteristics of patients enrolled to Phaselb/II study Cohort IIA.

*Missing: n=l. Allo-HSCT, allogeneic hematopoietic stem cell transplant; BM, bone marrow; ECOG PS, Eastern Cooperative Oncology Group performance status; R/R B-ALL, relapsed/refractory B-cell acute lymphoblastic leukemia. The main results related to Cohort IIA have been described in detail previously. Briefly:

Discontinuation prior to AUT01 infusion occurred due to: death (n=l 1), manufacturing issues (n=5), adverse events (n=l), and physician decision (n=l). Overall response rate in the 94 infused patients was 76.6% (95% confidence interval, 66.7-84.7).

Among the 94 infused patients, 7.4% and 3.2% of patients experienced Grade >3 immune effector cell-associated neurotoxicity syndrome and cytokine release syndrome, respectively, and 12.8% of patients were admitted to the intensive care unit; mean total duration of hospitalization was 39.7 days.

PROs and HRQoL

Of the 94 patients who received an infusion of AUT01, baseline data for the EQ-5D-5L and EORTC QLQ-C30 were available for 70 and 71 patients, respectively.

The median score at baseline for EQ-5D-5L VAS and EORTC QLQ-C30 GHS was 67.00 and 58.33, respectively.

There was an observed decrease in median EQ-5D-5L VAS score at Day 28, but this was not considered clinically meaningful (Figure 36).

Starting at Month 3 for EQ-5D-5L VAS and Month 6 for EORTC QLQ-C30 GHS, median measured HRQoL for both scores exceeded baseline status to levels indicative of meaningful improvement and remained at those levels throughout the 18 months of follow up.

Maximum change from baseline occurred at Month 18 for EQ-5D-5L VAS (median change from baseline 17.50) and at Month 12 for EORTC QLQ-C30 GHS (median change from baseline 29.17).

The median baseline score and longitudinal trajectory of EQ-5D-5L VAS and EORTC QLQ- C30 GHS scores was similar between responders and all treated patients.

Conclusions

After an initial deterioration or plateau post- ALITO 1 infusion, patients recovered to and later exceeded baseline status in overall HRQoL. HRQoL was similar in responders and all treated patients.

EORTC QLQ-C30 GHS scores provided HRQoL data that are not routinely collected in CAR-T trials for ALL. These data demonstrate that AUT01 therapy is associated with a meaningful improvement in HRQoL by 3 months post-infusion and provide a basis for further analysis as additional Phaselb/II trial data become available.

Example 10: Updated analysis of all r/r B-ALL patients treated with CD19 CAT CAR T- cell product (AUTO1) in the Phase Ib/II study

METHODS'.

Study design and conduct.

The Phase Ib/II study was conducted at 34 sites in the United States, United Kingdom, and Spain. Data cut-off date was 7^th February 2024.

As described in previous examples, eligible patients in the Phase Ib/II study were at least 18 years of age, and had relapsed or refractory CD 19-positive B-ALL defined as one of the following (with refractory defined as less than CR/CRi):

• primary refractory disease (not achieving CR after two cycles of induction chemotherapy),

• first relapse if remission <12 months,

• relapsed or refractory disease after two or more lines of systemic therapy, or

• relapsed or refractory disease after allogeneic transplant.

Patients with CD 19+ Philadelphia chromosome positive ALL (Ph+ ALL) were eligible if they were intolerant to or had failed two lines of any TKI or one line of second-generation TKI, or if TKI therapy was contraindicated.

The Phase Ib/II study was buildt on findings from the phase I study. The present study had a phase lb and phase II component. Phase lb comprised of 2 cohorts: Cohort IA, patients with morphological disease (>5% bone marrow (BM) blasts); and Cohort B, for patients with MRD level disease (<5% BM blasts).

The phase II component was designed to assess the clinical efficacy of AUTO1 in adults with r/r B- ALL with >5% BM blasts at screening. These patients were recruited to a pivotal cohort IIA. The phase II study also had two exploratory cohorts: IIB for patients with MRD- level disease, and IIC for patients with isolated EM disease.

The inclusion and exclusion criteria are shown in Tables 30 and 31.

Table 30: Inclusion criteria Key inclusion criteria

• Age >18 years

• ECOG PS of O or l

• R/R B-ALL defined as one of the following: a. Primary refractory disease (not achieving CR after two cycles of induction chemotherapy) b. First relapse if first remission <12 months (Phase lb Cohort IA and Phase II Cohort II A) c. R/R disease after two or more lines of systemic therapy d. R/R disease after allogeneic transplant provided AUT01 infusion occurs >3 months after SCT

• Patients with Philadelphia chromosome positive ALL are eligible if they are intolerant to or have failed two lines of any TKI or one line of second-generation TKI, or if TKI therapy is contraindicated

• Documentation of CD 19 expression on leukemic blasts in the BM, peripheral blood, or CSF by flow cytometry within 1 month of screening. In patients treated with blinatumomab, testing should be undertaken after blinatumomab therapy has been stopped

• Phase lb: o Primary Cohort IA: Presence of >5% blasts in BM at screening o Exploratory Cohort IB: MRD-positive defined as >10'⁴ and <5% blasts in the BM at screening

• Phase II: o Primary Cohort IIA: Presence of >5% blasts in BM at screening o Cohort IIB: Adults aged >18 years with B ALL in >2nd complete remission (CR) or complete remission with incomplete recovery of counts (CRi) with MRD-positive disease (>10-3 by central ClonoSEQ NGS testing§ and <5% blasts) in the BM at screening. o Cohort IIC (Exploratory): Adults aged >18 years with B ALL with isolated extramedullary disease (EMD) (including isolated CNS disease), with or without MRD.

• Adequate renal, hepatic, pulmonary, and cardiac function defined as: a. Serum alanine aminotransferase/aspartate aminotransferase <2.5 x ULN b. Creatinine clearance (as estimated by Cockcroft Gault) >50 cc/min c. Total bilirubin <1.5 x ULN, except in patients with Gilbert’s syndrome who must have normal direct bilirubin d. LVEF >45% (or ^institutes lower limit of normal) confirmed by ECHO or MUGA in patients with history of coronary artery disease or cardiovascular disease or those with history of low LVEF. e. Baseline oxygen saturation >92% on room air ALL = acute lymphoblastic leukemia; B-ALL = B-cell acute lymphoblastic leukemia; BM = bone marrow; CD 19 = cluster of differentiation 19; CNS = central nervous system; CR = complete remission; CSF = cerebrospinal fluid; ECHO = echocardiogram; ECOG PS = Eastern Cooperative Oncology Group performance status; EMD = extramedullary disease; LVEF = left ventricular ejection fraction; MRD = minimal residual disease; MUGA = multigated acquisition; R/R = relapsed or refractory; SCT = stem cell transplant; TKI = tyrosine kinase inhibitor; ULN = upper limit of normal. § The threshold for MRD-positive disease was changed to >10~³ per FDA request.

Table 31 : Exclusion criteria

Key exclusion criteria

• B-ALL with isolated EMD for Phase lb (Cohort IA and IB) and Phase II Cohort IIA

• Diagnosis of Burkitt’s leukemia/lymphoma according to WHO classification or chronic myelogenous leukemia lymphoid in blast crisis

• History or presence of clinically relevant CNS pathology such as epilepsy, paresis, aphasia, stroke within 3 months prior to consent, severe brain injuries, dementia, Parkinson’s disease, cerebellar disease, organic brain syndrome, uncontrolled mental illness, or psychosis

• Presence of CNS 3 disease or CNS 2 disease with neurological changes. Patients developing CNS 3 disease or symptomatic CNS 2 disease at any time after consent will also be excluded until they no longer meet these criteria

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

• Active or latent Hepatitis B virus or active Hepatitis C virus

• HIV, HTLV-1, HTLV-2, or syphilis positive test

• Patients who have received a prior SCT <3 months prior to AUT01 infusion. Active significant (overall Grade >11, Seattle criteria) acute GVHD or moderate/severe chronic GVHD (NIH consensus criteria) requiring systemic steroids or other immunosuppressants within 4 weeks of consent

• Prior CD 19 targeted therapy other than blinatumomab. Patients who have experienced Grade >3 neurotoxicity following blinatumomab

• The following medications are excluded: a. Steroids: Therapeutic doses of corticosteroids (greater than 10 mg daily of prednisone or its equivalent) within 7 days of leukapheresis or 72 hours prior to AUTO1 administration (physiological replacement, topical, and inhaled steroids are permitted) b. Immunosuppression: Immunosuppressive medication must be stopped >2 weeks prior to leukapheresis and AUTO1 infusion c. Allogeneic cellular therapy: any donor lymphocyte infusions must be completed >2 weeks prior to leukapheresis and not repeated thereafter d. GVHD therapies: any drug used for GVHD must be stopped >2 weeks prior to leukapheresis and not reinitiated thereafter e. Chemotherapy (including TKIs for patients with Philadelphia chromosome positive ALL): should be stopped 1 week prior to leukapheresis or starting preconditioning chemotherapy f. Treatment with any T cell lytic or toxic antibody (e.g. alemtuzumab) within 6 months prior to leukapheresis, or treatment with clofarabine or cladribine within 3 months prior to leukapheresis g. Live vaccine <4 weeks prior to leukapheresis h. Intrathecal therapy within 2 weeks prior to starting pre-conditioning chemotherapy i. Use of blinatumomab after leukapheresis

ALL = acute lymphoblastic leukemia; B-ALL = B-cell acute lymphoblastic leukemia; CD 19 = cluster of differentiation 19; CNS = central nervous system; EMD = extramedullary disease; GVHD = graft versus host disease; HIV = human immunodeficiency virus; HTLV = human T-cell lymphotropic virus; NIH = National Institutes of Health; SCT = stem cell transplant; TKI = tyrosine kinase inhibitor; WHO = World Health Organization

At screening, all patients underwent BM assessment and CSF examination to exclude CNS disease. Patients were eligible to enter cohort IIA with <CNS2 if asymptomatic. Imaging for non-CNS extramedullary disease (EMD) was conducted in patients with known or clinically suspected EMD.

Eligible patients underwent leukapheresis. After leukapheresis, patients could receive bridging therapy at investigator’s discretion, with the exception of blinatumomab.

Immediately before lymphodepletion/AUTOl infusion, a second bone marrow examination was mandated in all patients. Lymphodepletion consisted of intravenous (i.v.) fludarabine 30mg/m² for 4 days (total dose

120mg/m²) and cyclophosphamide i.v. 500mg/m² for 2 days (total dose 1000mg/m²).

AUTO1 was administered in a leukaemia burden-adjusted split-dose schedule (Figure 7). On

Day 1, patients >20% BM blasts (as assessed just before LD) received 10 x 10⁶ AUTO1

CAR-T cells, while patients with BM blasts <20% received 100 x 10⁶ CAR-T cells. At an interval of 9 days, a second dose was administered to a total target dose of 410 x 10⁶ CAR-T cells. Patients who developed grade >3 cytokine release syndrome (CRS) and/or Grade >2 immune effector cell-associated neurotoxicity syndrome (ICANS) or Grade >3 pulmonary or cardiac toxicities following dose 1 were not eligible to receive dose 2. Patients with Grade 2 I l l

CRS and/or Grade 1 ICANS received dose 2 only if CRS had resolved to Grade <1 and ICANS had resolved. If necessary, dose 2 could be postponed up to day 21.

Management guidelines for CAR T-cell-related adverse events were included in the protocol. Toxicity was graded using the NCI CTCAE version 5.0 criteria, except for neurotoxicity, which was graded using the ASTCT/ASBMT consensus guidelines (Lee et al., 2019. ASTCT Consensus Grading for Cytokine Release Syndrome and Neurologic Toxicity Associated with Immune Effector Cells. Biology of Blood and Marrow Transplantation 25, 625-38).

Haemophagocytic lymphohistiocytosis (HLH) was defined as per CARTOX criteria (Neelapu et al., 2018. Chimeric antigen receptor T-cell therapy — assessment and management of toxicities. Nature Reviews Clinical Oncology 15, 47). Patients were observed as an in-patient for a minimum of 10 days following CAR-T cell administration.

In the phase II part of the study, tyrosine kinase inhibitors (TKI) were permitted from month 3 post- AUTO1 infusion in Ph+ ALL patients who achieved CR/CRi after AUTO1 infusion per investigator discretion. Intrathecal CNS prophylaxis could be given post- AUTO1 infusion per investigator discretion in accordance with institutional guidelines.

Assessments and study end points

The Enrolled Set comprises all patients who are enrolled in the study. Enrolment was defined as the point at which the patient met all inclusion/exclusion criteria, and the patients’ leukapheresate was accepted for manufacturing. The infused set comprises all patients who received at least one infusion of AUTO1 treatment. The safety set comprises all patients who received at least one infusion of AUTO1. For this study, the safety set was the same as the infused set, and was the main analysis set for safety.

The primary endpoint for Cohort IIA was ORR (Overall Response Rate) and CR/CRi. Response evaluations were performed by an independent response review committee (IRRC), which used the response criteria for ALL according to the adapted NCCN guidelines version 2.2019. Patients with CNS disease at enrolment had CSF re-examined on day 28. Patients with EMD were re-imaged after AUTO1 infusion at day 28 (Table 32).

Table 32: Phase Ib/II endpoints

(a) Primary objectives and endpoints Phase Primary objectives Primary endpoints

Phase lb To evaluate the safety of AUT01. Frequency and severity of AEs and SAEs occurring after AUT01 infusion.

Phase II To evaluate the clinical efficacy Cohort IIA: ORR defined as proportion of patients achieving of AUTO 1. CR or CRi as assessed by an Independent Response Review

Committee.

Cohort IIB: Proportion of patients achieving MRD -negative remission by central ClonoSEQ NGS testing (<10⁴ leukemic cells).

(b) Secondary objectives and endpoints

Phase Secondary objectives Secondary endpoints

Phase lb To evaluate the feasibility of Proportion of enrolled patients for whom an AUT01 manufacturing and administering product can be manufactured and administered as per AUTO1. protocol.

To evaluate the clinical efficacy • ORR defined as proportion of patients achieving CR or of AUTO 1. CRi

• Proportion of patients achieving MRD-negative remission in BM by PCR and/or flow cytometry

To evaluate the expansion and Detection of CAR-T cells measured by PCR in the persistence of AUTO 1. peripheral blood and BM following AUTO 1 infusion.

Phase II To evaluate the clinical efficacy • Proportion of patients achieving MRD-negative CR by of AUTOi. NGS (<10-4 leukemic cells) (key secondary endpoint for

Cohort IIA), with results also including PCR/flow cytometry as supportive analysis

• CR rate

• Duration of remission

• Relapse free survival

• Event free survival

• Progression free survival

• Overall survival

• ORR [CR+CRi] as assessed by the Investigator

• Proportion of patient undergoing stem cell transplantation prior to leukemia relapse

• Proportion of patients in CR/CRi without stem cell transplants or other subsequent therapies at 6, 12 and 24 months following AUTOI infusion

• Incidence of CD 19-negative relapse

To assess the safety and • Frequency and severity of AEs and SAEs tolerability of AUTOI. • Incidence and duration of severe hypogammaglobulinemia

To evaluate the feasibility of Proportion of enrolled patients for whom an AUTOI manufacturing and administering product can be manufactured and administered. AUTOI. To evaluate the expansion and Detection of CAR-T cells measured by PCR in the persistence of AUTO1. peripheral blood and BM following AUTO1 infusion.

To evaluate the duration of B cell Depletion of circulating B cells assessed by flow cytometry aplasia. in the peripheral blood.

To evaluate Patient Reported Changes over time in symptom, functioning and quality of Outcome and Quality of life. life scores of the EQ-5D and the EORTC instruments

To evaluate health care resource Frequency and duration of hospitalization and/or critical utilisation for the management of care support to manage AUTO 1 -related toxicity. AUTO 1 -related toxicity.

AE = adverse event; BM = bone marrow; CAR = chimeric antigen receptor; CD 19 = cluster of differentiation 19; CR = complete remission; CRi = complete remission with incomplete recovery of counts; EORTC = European Organisation for Research and Treatment of Cancer; EQ-5D-5L = EuroQol; MRD = minimal residual disease; NGS = next generation sequencing; ORR = overall complete remission rate; PCR = polymerase chain reaction; SAE = serious adverse event

Following establishment of response, patients were assessed for relapse at least 4 weeks after onset of CR/CRi. Patients were considered to be in continuing CR/CRi if there was no clinical evidence of relapse as assessed by peripheral blood and extramedullary disease assessment. Bone marrow assessments were not mandated after the initial achievement of CR or CRi unless clinical indicated. Efficacy was assessed by determining the ORR (CR/CRi) defined as the proportion of patients achieving CR or CRi based on the IRRC evaluation.

Secondary end points included the proportion of patients achieving MRD-negative remission, duration of remission, event-free survival, overall survival, safety, AUT01 kinetics data as assessed by PCR in the peripheral blood and BM, duration of B-cell aplasia (defined as <20 CD 19+ lymphocytes/pL in peripheral blood), proportion of patients undergoing stem cell transplantation in remission post AUTO1 treatment, proportion of patients in CR/CRi without stem cell transplant or other subsequent therapies at 6, 12 and 24 months following AUTO1 infusion, and incidence of CD 19 negative relapse. The evaluation of biomarkers was an exploratory analysis.

Statistical analysis

The primary endpoint of ORR in Cohort IIA was first tested against the null hypothesis of < 40% for the Infused population. If it was achieved, the key secondary endpoint of CR at any time post-infusion was tested against the null hypothesis of < 20%. These two endpoints were first tested in the pre-specified efficacy interim analysis when 50 patients from Cohort IIA had been treated with AUTO1 and had been followed for 3 months or discontinued before the Month 3 visit, according to an alpha spending approach according to Lan-DeMets (O’Brien- Fleming). If any of the primary or key secondary endpoints were not rejected at the efficacy interim analysis, they were to be tested again at the primary analysis according to the prespecified alpha spending approach, so that the family-wise Type I error at the one-sided 2.5% level is controlled throughout the testing sequence. All other endpoints and analyses for other cohorts were summarized descriptively.

Vector and vector manufacture.

Lentiviral vector manufacture was subcontracted to AGC bio. Lentiviral vector was generated by transient transfection of 293T cells with plasmids encoding HIV1 gagpol, HIV1 rev, VSVG enveloped and a transfer vector encoding the AUTO1 CAR. Harvested supernatant was purified and concentrated using anion exchange chromatography and tangential flow filtration.

CAR T cell manufacture.

AUTO1 manufacturing was performed on the Miltenyi CliniMACS Prodigy® using a semiautomated closed process from autologous non-mobilized patient leukapheresis starting material. T cells were enriched using anti-CD4 and anti-CD8 immunomagnetic beads (Miltenyi Biotec) followed by activation via CD3/CD28 with MACS GMP TransAct® (Miltenyi Biotec) in TexsMACSTM GMP media (Miltenyi Biotec) supplemented with human AB serum (BioIVT) and cytokines (Miltenyi Biotec). Lentiviral vector was added at predefined MOI. The process lasted up to 10 days. On the harvest day, the cells were harvested from the CliniMACS Prodigy®, washed and concentrated using the Sepax C-Pro (Cytiva) system and formulated in one or more CryoMACS® bag(s) prior to cry opreservation in a controlled rate freezer (Cytiva), and stored in a vapour-phase liquid nitrogen environment prior to administration.

Flow cytometry for % CD19 CAR Expression and % CD3+

For the measurement of cell phenotype in the leukapheresis starting material and the transduction efficiency (% CD 19 CAR Expression) in the drug product, a multi-color flow cytometry panel was used with BD TruCOUNT™ Absolute Counting Tubes. The tube contains a known concentration of lyophilized fluorescent beads. Upon addition of the sample the lyophilized pellet dissolved, releasing a known number of fluorescent beads. A cocktail of fluorescent monoclonal antibodies/reagent were added to the samples and incubated for 15 minutes at room temperature (RT). Samples were acquired on the BD FACSLyric™ Flow Cytometer.

% Cell Viability upon Thaw

Cell viability was measured from cryopreserved drug product upon thaw using an automated cell counter (Chemometec) by acridine orange/ 4',6-diamidino-2-phenylindole (DAPI) staining.

Flow cytometry Leukapheresis and drug product characterisation.

Patients’ leukapheresis starting material and drug product lots were characterised using an 18-parameter flow cytometry assay. The data produced from the assay were used to provide insight into the immunophenotype, memory and exhaustion status of the drug product and apheresis starting material. Memory phenotype was defined by CCR7 and CD45RA markers while PD-1, LAG3, PD1-LAG3 (dual positive) and TIGIT defined exhaustion.

CAR T persistence by PCR

A validated droplet digital dPCR duplex assay (CellCarta, Montreal, Quebec) was used to quantify the presence of AUTO1 transgene in genomic DNA extracted from peripheral whole blood of infused patients. AUTO1 transgene was detected and quantified using Lentiviral Psi (L-Psi) primers to detect the L-Psi sequence encoded in AUTO1 transduced T cells. Ribonuclease P protein subunit p30 (RPP30) primers were used as a reference gene and together with L-Psi, the number of copies of the CAR construct integrated into the genome was assessed and quantified using the Quantasoft software (Bio-Rad, CA, USA). Samples below limit of quantification (LOQ) of 21 copies per reaction (corresponding mathematically to 76.9 copies/pg of DNA) were reported as zero. AUTO1 transgene was measured on days - 6 pre-infusion and days 1, 3, 6, 9, 12, 15, 22, 28, and months 2, 3, 4, 6, 9, 12, 15, 18, 21, 24 and then monthly until end of study post-infusion.

Serum cytokines

Two Meso Scale Discovery (MSD, Rockville, USA) multiplex sandwich immunoassay panels (10- V Plex and Cytokine Human Panel 1), performed at Q2 Solutions (Edinburgh, UK) according to manufacturer’s instructions, were used to determine serum cytokines (IFN- y, GM-CSF, IL-2, IL-5, IL-6, IL-8, IL-10, IL-15, IL-7 and TNF-a) concentrations in serum samples.

CD19 and CD22 assessment peripheral blood

CD 19 and CD22 status at morphological relapse was assessed amongst the B cell population (CD45 positive/CD3 negative/CD14 negative/CD16 negative) in peripheral blood using flow cytometry. The number of CD 19 and/or CD22 cells was used to attribute the CD 19 status at morphological relapse, if a sample was available within ±14 days of the date of morphological relapse. A cut-off of > 50/pL CD 19 positive cells was used for CD 19 positive status. Cases could be called CD 19 mixed if >50/pL each of CD 19 positive/CD22 positive cells and CD 19 negative/CD22 positive were identified. If no suitable sample was available for flow cytometry, CD 19 data from the electronic case report form was used if it was generated from a sample collected within ±14 days of the date of morphological relapse.

Statistical analysis of manufacturing and translational data

Clinical data are captured in the clinical database via the Encapsia electronic data capture (EDC) system vl.O. SAS 9.4 was used for clinical data analysis. All data are summarized descriptively due to the Phase I exploratory nature of the study. Categorical variables are reported in terms of frequency and percentage, and continuous variables in terms of median and range unless otherwise specified. Time-to-event outcomes were summarized using the Kaplan-Meier method. Toxicity events are reported at the maximum grade experienced according to the CTCAE.

Methodology to determine MRD status

Evaluation of MRD to 10'⁴ sensitivity was performed by a variety of methods: ClonoSEQ NGS, qPCR and flow cytometry: NGS results were used if available, if no NGS results were available then qPCR results were used, if neither NGS results nor qPCR results were available, then flow cytometry results were used. If no results source were available, then the MRD status was unknown.

NGS-MRD was measured using MRD ClonoSEQ® B-cell Clonality (Adaptive Biotechnologies, Seatie USA), an NGS-based assay designed for tumors pecific Ig sequence rearrangement detection. It identifies and tracks rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences, as well as translocated BCLl/IgH (J) and BCL2/IgH (J) sequences. The clonoSEQ® Assay has a sensitivity of 10-6 and requires a calibration archival sample to enable identification of leukemic clones of interest. For more information on methodology and validation please refer to the manufacturer website.

Real-time quantitative PCR (RQ-PCR) MRD analysis was performed at Bristol Genetics Laboratory (Bristol, UK) on DNA extracted from bone marrow using real-time quantitative (RQ-PCR) analysis of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements following guidelines from the EuroMRD group (https://euromrd.org/). The RQ-PCR assay has a 10-4 MRD detection level. The assay requires a calibration sample (at least 3-5% blasts) at screen to enable identification of Ig/TCR rearrangements.

MRD by flow cytometry was assessed in bone marrow aspirate by a validated assay performed at a CAP/CLIA certified central laboratory (NeoGenomics, California, USA). The B-ALL MRD flow cytometry assay contained the following markers for assessment in fresh bone marrow: CD3, CD9, CD10, CD13/33, CD19, CD20, CD34, CD38, CD45, CD58, CD71 and Sytol6. The assay was suitable for detection of malignant B-ALL blasts at KT⁴ sensitivity at both testing sites. The MRD flow panel measures leukemic-associated immunophenotype with a 10'⁴.

RESULTS:

Patient enrolment and treatment

A consort diagram is shown in Figure 37. 153 patients with r/r B-ALL were enrolled in the combined Phase Ib/II study; 127/153 (83.0%) patients received at least one AUTO1 infusion and were evaluable. The remaining 26 patients were not infused due to manufacturing failure in 7 and death / uncontrolled disease in 19.

Manufacturing feasibility

AUTO1 was manufactured from patient pheresis using a closed, semi-automated process (summarized in Figure 38). Patient pheresis median T-cell content was 13.4% (range 0.8- 83.1), but this was highly variable including pheresates with <1% T cell content (Figure 39a). Despite this, AUTO1 was successfully released for 146/153 (95%) of patients with a median release time of 21 days from leukapheresis (range, 18-50; Figure 39b). Median transduction efficiency was 69% (range, 12-87; Figure 39c), and median viability upon thaw was 89% (range, 77-96; Figure 39d). Extended product characteristics are presented in Figure 40.

Table 33. Patient demographics and disease features for enrolled and infused patients at screening.

Key: *All inclusion/exclusion criteria have been fulfilled and leukapheresate has been accepted for manufacturing. f4.ll patients who have received at least one infusion of AUTO 1. fl'he highest value from BM aspirate and trephine at screening. §No lymphoblasts in cerebrospinal fluid regardless of white blood cell count. ^White blood cell < 5/pL in cerebrospinal fluid with presence of lymphoblasts. ALL = acute lymphoblastic leukaemia; SCT = allogeneic stem cell transplantation; BM = bone marrow; CD 19 = cluster of differentiation 19; CNS = Central nervous system; ECOG = Eastern Cooperative Oncology Group. Patient Characteristics

Patient characteristics at enrolment for cohort IIA and for the total infused cohort (n=127 patients) are summarised in Table 33. Median age at enrolment was 47 years (range, 20-81) and 25/127 (20%) were >65 years old. Patients received a median of 2 prior lines of therapy (range, 1-6), and 52% were refractory to their last therapeutic line. Notably, 41.7% and

31.5% had previously received either blinatomumab, or inotuzumab ozogamicin respectively, and 16.5% had received both. Forty-four percent of patients had a prior allogeneic stem cell transplant (allo-SCT). Twenty-eight percent of patients had Ph+ B-ALL and 23% had EMD. Patients had a median of 40% BM blasts at screening (range, 0-100%). A breakdown of patient characteristics in all cohorts is shown in Table 34. Therefore, these were heavily pretreated patients.

Table 34: Infused patient demographics and disease features at screening by cohort.

Key: *All patients who have received at least one infusion of AUT01. ‘The highest value from BM aspirate and trephine at screening. §No lymphoblasts in cerebrospinal fluid regardless of white blood cell count. ' While blood cell < 5 u in cerebrospinal fluid with presence of lymphoblasts. ALL = acute lymphoblastic leukemia; SCT = allogeneic stem cell transplantation; BM = bone marrow;

CD 19 = cluster of differentiation 19; CNS = Central nervous system; ECOG = Eastern Cooperative Oncology Group.

Bridging therapy (Bl) and baseline disease burden.

Bridging therapy (BT, additional therapy given after enrolment, but before lymphodepletion/AUTOl administration) was administered to 118/127 (93%) patients: 80/118 (68%) received chemotherapy alone; 10/118 (8.5%) received chemotherapy with tyrosine kinase inhibitors (TKI); 7/118 (6%) received TKI alone; 9/118 (8%) received chemotherapy with inotuzumab; and 9/118 (8%) received inotuzumab alone (Table 35). Prior to LD, all patients had a repeat BM assessment to guide dosing; amongst the entire study population prior to LD, the median BM blast percentage was 40% (range 0%-100%) with <5% blasts in 28.3%, 5-20% blasts in 12.6% and >20 blasts in 59.1% of patients. Twentyseven patients (21%) had EMD. BM disease burden changed substantially following BT in many patients (Figure 22). For instance, at BM assessment prior to LD, 24.5% (23/94) of cohort IIA patients had <5% BM blasts after bridging.

Table 35: Bridging therapy.

Key: ^Bridging therapies were coded using WHO Drug Global B3 202303. Multiple bridging therapies were counted only once per patient for each preferred term. Chemotherapies included vincristine, methotrexate, cytarabine, cyclophosphamide, fludarabine, mercaptopurine, doxorubicin, etoposide, clorafarabine, daunorubicin, hydroxycarbamide, vincristine sulfate, dexamethasone, mesna, rituximab, bortezomib, idarubicin and vinblastine sulfate. Tyrosine kinase inhibitors included ponatinib, dasatinib, bosutinib and imatinib. Steroids included prednisone, hydrocortisone, hydrocortisone sodium succinate and prednisolone. Other included calcium folinate and cytosine.

AUT01 administration

AUTO1 was administered as a split dose, titrated to BM disease burden assessed just prior to LD (Figure 7); 60% (76/127) and 40% (51/127) received 10xl0⁶ CAR and lOOxlO⁶ CAR T cells respectively, as the first dose. Ninety-four percent of the patients (120/127) received both doses of AUTO1 (Table 36). Seven patients (5.5%) only received a first dose. In 3/7 of these patients, this was due to immunotoxicity (grade 3 CRS in one patient and grade 3 ICANS in two); in 2/7 this was product related; in 1/7 this was due to rapid disease progression and death and in 1/7 due to rapid progression and a cerebrovascular incident. Eighty-eight of the 94 patients (93.6%) in cohort IIA received both doses.

Table 36: AUT01 exposure in all infused patients and by cohort.

Key: * Target dose is 410x 10⁶ CD 19 CAR-positive T cells (±25%). {Eleven patients did not receive the target dose. Among those, 7 patients received only the first dose of AUT01. Four patients received 2 infusions of AUTOlbut did not receive the full target dose, including 3 patients with less than target dose manufactured, and 1 patient with a CAR-T infusion bag damaged at the time of CAR- T dose administration. CAR = chimeric antigen receptor; CD19 = cluster of differentiation 19. ±Patients who did not receive the 2^nd dose: 3/7 due to immunotoxicity after the first dose (2 ICANS grade 3, and 1 CRS Grade 3), 2/7 product related, 1 / 7 due to rapid disease progression and death 1/7 due to AE [cerebrovascular incident] in the context refractory disease; 5 patients required product remanufacture. Response rates and survival

Cohort II A (n = 94): The ORR (CR/CRi) in all patients in Cohort IIA who received at least one infusion of AUTO1 was 76.6% (95% CI: 66.7, 84.7), of whom 55% (95% CI: 44.7, 65.6) achieved CR and 21% CRi. Both the ORR and CR endpoints met the pre-specified hypothesis testing threshold with p-value <0.0001. The median event-free survival (EFS) in Cohort IIA was 9.03 months (95% CL 6.14, 14.98). Responses in all Phase Ib/II cohorts are summarized in Table 37.

Table 37. Overall response for all infused patients and by cohort.

ORR = overall response rate; CI = confidence interval; CR = complete remission; CRi = complete remission with incomplete recovery of counts.

Notably, for most patients, BM disease burden changed between screening and LD, mainly due to the effect of BT (Figure 22). Consequently, a post-hoc analysis of the entire AUTO1 study was performed and is described below.

All treated patients (n = 127): Of the 91 /127 who had >5% BM blasts at LD, 68/91 (74.7% with 95% CI: 64.5%, 83.3%) had a morphological response (CR, CRi). Of these 68 responding patients, 62 had MRD data available and 58/62 (93.5%) were MRD-negative after AUTO1 . In the cohort of 29 patients with <5% BM blasts without EMD at LD, 25/26 (96.2%) of patients who had MRD data available, were MRD-negative. Seven patients had <5% blasts and EMD at LD; EMD was no longer detectable following AUTO1 in 5 patients (71.4%). High CR/CRi rates were observed across all subgroups of patients who received AUTO1 including older patients, Ph+ patients, and those with prior allo-SCT (Figure 41). High disease burden prior to LD (>75% BM blasts) correlated with worse ORR: the ORR for patients with intermediate (>5% to <75% BM blasts) and high (>75% BM blasts) burden disease at LD were 82.4% (95% CI: 69.1, 91.6) versus 65.0% (95% CI: 48.3, 79.4), respectively.

An intention to treat analysis of all cohorts is shown in Table 38.

Table 38: Intention to treat analysis of responses in all cohorts.

Event free and overall survival in all treated patients: The median duration of follow-up from first AUTO1 infusion to data cut-off (7^th February 2024) for all patients was 21.45 months (range, 8.6-41.4). Median event-free survival (EFS) was 11.9 months (95% CI: 8.0, 22.1); the estimated 6- and 12-month EFS rates were 65.4% and 49.5%, respectively. Disease burden at LD correlated with median EFS: for instance, patients with low (<5% BM blasts), intermediate (5-75% blasts), and high burden disease (>75% blasts) had median EFS which was not reached, 15.0 months (95% CL 6.14, NE), and 5.1 (95% CI: 1.6, 9.0) months respectively. This is illustrated in Figure 42a and 42b.

Median overall survival (OS) was 15.6 months (95% CL 12.9, NE); the estimated 6- and 12- month OS rates were 80.3% and 61.1%, respectively. Similarly to EFS, disease burden at LD correlated with median OS - patients with low, intermediate, and high burden disease had median OS which was not reached, 15.6 months (95% CI: 10.6, NE), and 12.8 months (95% CL 6.9, 15.3) respectively. This is illustrated in Figure 42c and 42d. BM burden at enrolment also influenced EFS and OS (Figure 43).

The potential of long-term plateau is evident for OS, as well as EFS.

Consolidation with allo-SCT: Among the 99 responding patients on the Phase Ib/II study, 40% (40%) were in ongoing remission without subsequent allo-SCT or other therapy, while 18 (18%) proceeded to allo-SCT while in remission at a median of 101 days post-AUTOl infusion (range, 38 - 421). In 5/17 (29.4%), this was a 2^nd allo-SCT. Those patients receiving a subsequent allo-SCT were MRD-negative prior to allo-SCT.

No benefit to EFS and OS of allo-SCT was observed (Figure 44A and 44B, respectively). Ten out of 18 (55.6%) patients had ongoing CAR T persistency prior to SCT, 80% of which experienced relapse/death, showing that consolidative SCT may not further protect patients from relapse. Eight out of 18 (44.4%) patients lost CAR T persistency prior to SCT, and 62.5% of those patients experienced relapse/death. This suggests a higher risk for those moving on to consolidative SCT post-AUTOl. Other characteristics were similar between patients who did and did not undergo consolidative SCT. These results indicate that a subset of patients benefitted from standalone treatment with AUTO1.

Median overall survival was 23.8 months; the estimated 12-month OS rate was 61.1%. As seen with EFS, consolidative SCT for patients post-AUTOl does not improve OS.

Consolidation with TKI: Nineteen patients with Ph+ B-ALL who achieved CR/CRi following AUTO1 received consolidation with TKI per investigator discretion.

Toxicity

Immunotoxicities are summarised in Table 39a, incidence of febrile cytopenia and infection in Table 39b.

CRS: Overall, 87 of the 127 (68.5%) patients developed CRS, with grade > 3 events in 3 (2.4%). Median time to CRS onset was 8 days (range, 1-23), median duration was 5 days (range, 1-21). Tocilizumab was administered to 66 (52.0%) patients and corticosteroids to 20 (15.7%) patients. Three patients (2.4%) required vasopressors, and 15 (11.8%) required supplemental oxygen. Notably, grade 3 and higher events occurred only in patients with >75% blasts at LD (Table 39a). Immune effector cell associated hemophagocytic lymphohistiocytosis (IEC-HLH) was observed in 2 patients and both were grade > 3 events. ICANS: Overall, 29/127 (22.8%) patients developed ICANS, with grade > 3 events in 9 (7%) patients. Median time to ICANS onset was 12 days post-infusion (range, 1-31) with a median duration of 8 days (range, 1-53). 24/29 patients received corticosteroids. Severe (grade > 3) events were mostly seen in patients with high disease burden pre-LD. ICANS of any grade was not observed in those with <5% BM blasts pre-LD.

Peak serum biomarkers were generally low, with median IL-6, IFNy and ferritin of 99.2 ug/L, 358 ug/L and 4570 ug/L, respectively (Figure 45A and 45B). Compared to patients with no CRS and/or no ICANS, peak IL-6, IFNg, CRP and ferritin were higher in patients with grade > 3 events (Figure 45A and 45B).

Cytopenias: grade > 3 thrombocytopenia and grade > 3 neutropenia occurred in 74.8% and 98.4% patients respectively. Among the 99 patients who were in CR/CRi after AUTO1, median (95% CI) time to neutrophil recovery to >0.5xl0⁹/L and platelet count recovery to >50X10⁹/L was 0.7 (0.5, 0.9) and 0.7 (0.3, 1.7) months respectively (Table 39b). Notably, many patients had pre-existing cytopenia(s) and pre-existing cytopenia was associated with longer duration of cytopenia (Figure 46A and 46B). All patients had neutrophil count recovery to >0.5xl0⁹/L before month 6, and only 1 patient had platelet count recovery to >50X10⁹/L which took longer than 6 months. Allo-SCT donor haematopoietic stem cells were administered to 3 patients for prolonged cytopenia.

Infections: Within the first 60 days following AUTO1 infusion, febrile neutropenia was reported in 31/127 (24.4%) patients. Infectious deaths occurred in 5 patients: 2 from neutropenic sepsis (one related; one not related to AUTO1); 2 from sepsis (both not related) and 1 from abdominal infection (not related). Fifty/127 (39.4%) of all infused patients received IV immunoglobulins (IVIG) post AUTO1 infusion.

Intensive Care Unit (ICU) admissions: Twenty of the 127 (15.7%) patients were admitted to the ICU for a median of 5.5 days (range, 1-37). Seven of these 19 patients were admitted for management of immunotoxicity (5 for ICANS, 2 for CRS).

Deaths are detailed in Table 40. Two patients’ deaths were attributable to AUTO1 : one patient died of respiratory distress syndrome with ongoing ICANS, and a second patient died of neutropenic sepsis.

Table 39. Summary of adverse events of particular interest. (a) CRS and ICANS for all infused patients and by leukemic burden prior to lymphodepletion.

(b) Cytopenias and infections for all infused patients.

Key: Cytopenias were defined as reduced neutrophil or platelet count since B lymphocyte depletion. B cell aplasia was defined as <20 B-cells/L assessed from day 28 onwards post CAR-T cell infusion. * Cytopenias were defined as reduced neutrophil or platelet count since B lymphocyte depletion. BM = bone marrow; CAR-T = chimeric antigen receptor-T cell therapy; CRS = cytokine release syndrome; ICANS = immune effector cell-associated neurotoxicity syndrome.

Table 40. Deaths on study.

Patient deaths post AUTO 1 treatment as of data cutoff date 13 \

CAR-T expansion, persistence, and relationship with relapse CAR T expansion: Geometric mean peak CAR-T concentration for all treated patients was 110,896 copies/ug genomic DNA (gDNA) (range, 129-600,000) at a median of 14 days (range, 2-55) post infusion. Mean area under the curve from day 0 to 28 (AUCdo-2s) was 1. IxlO⁶ day copies/ug gDNA. While Cmax and AUCdo-28 did not correlate with response, both correlated with high disease burden. Higher Cmax and AUCdo-28 were associated with more frequent CRS and ICANS (Figure 47).

CAR T persistence and relapse: Persistence is shown in Figure 48A. The median (95% CI) duration of CAR T persistence by ddPCR in peripheral blood was 17.8 (6.21, NE) months (Figure 49a). The probability of ongoing B-cell aplasia at 6 and 12 months was 80.7% (70.3%, 87.8%) and 66.7% (53.9%, 76.7%), respectively (Figure 49b). In total, 38 patients had morphological relapse. Of these, 8 (21.1%) were CD 19-positive, 18 (47.4%) were CD19- negative, 3 (7.9%) were mixed and 9 (23.7%) had unknown CD19 status. Loss of CAR-T cell persistence was associated with relapse: e.g. for patients in ongoing remission beyond month 6, the estimated 12-month EFS rates was 87.3% (95% CI: 72.0%, 94.5%) for those with ongoing persistence, compared to 59.3% (95% CI: 33.0, 78.1) for those without persistence (Figure 48B).

Among patients with CR/CRi beyond 6 months without SCT or new therapies, patients with ongoing CAR T persistence are associated with improved EFS vs. those with a loss of CAR T persistence.

CAR T persistence and B-cell aplasia were both associated with improved EFS compared with loss of persistency and B-cell recovery. Patients with loss of CAR T persistence had hazard risk of relapse or death 2.7 times compared with patients with ongoing CAR T persistence (Figure 50A). Patients who experienced B-cell recovery had a hazard risk of relapse or death 1.7 times compared with patients without B-cell recovery (Figure 50B).

CONCLUSIONS:

At this follow-up, 40% of patients were in ongoing remission without allo-SCT or other therapy. At median 21.45 months’ follow-up, the median EFS was 11.9 months and the median OS was 23.8 months. The 12-month EFS and OS rates were 49.5% and 61.1%. The survival outcomes showed potential of long-term plateau.

These data also showed that allo-SCT consolidation for remission following AUTO1 did not improve EFS or OS and that ongoing CAR T-cell persistence and B-cell aplasia were associated with improved EFS. AUTO1 should be considered as standard of care for adult R/R B-ALL DISCUSSION:

In the Phase Ib/II study, AUTO1 was administered to 127 patients with r/r B-ALL. Of the 94 patients in the pivotal IIA cohort, the CR/CRi rate was 76.6%. Since disease burden frequently changed between enrolment and re-assessment prior to LD, a post-hoc analysis of the entire study was performed. Of the 91 patients who had >5% BM blasts at LD, 74.7% had CR/CRi, and 93.5% of evaluated responders were MRD negative. In 29 patients with <5% blasts at LD, 96.2% were MRD negative post AUTO1. In all patients, median EFS was 11.7 months with the estimated 6- and 12-month EFS rates of 65.2% and 49.6%. Median OS was 15.6 months with estimated 6- and 12-month OS rates of 80.3% and 58.6%. AUTO1 was associated with minimal immunotoxicity, with only three patients (2.4%) developing grade >3 CRS, and 9 (7.0%) grade > 3 ICANS despite high disease burden pre-LD in a large proportion of patients.

Targeted therapies such as blinatumomab and inotuzumab ozogamicin have improved response rates in r/r B-ALL, but consolidation with allo-SCT is still needed for durable response. Recently, the CD28-(^ CD 19 CAR brexu-cel was licensed for use in adults with r/r B-ALL (Shah et al., 2021. The Lancet 398, 491-502). Brexu-cel was associated with a CR/CRi rate of 71%, comparable to that of AUTO1 (Shah et al., 2021). In contrast to AUTO1 however, brexu-cel was associated with much more frequent grade > 3 CRS (24- 26% vs 2.4% for AUTO1) and grade > 3 ICANS (25-35% vs 7% for AUTO1) (Shah et aL, 2021; Bouchkoujc/ aL, 2022. Oncologist 27, 892-9). In addition, brexu-cel was associated with frequent requirement for vasopressor support (40% vs 2.4% for AUTO1) (Shah et aL, 2021). Notably, median time to CRS/ICANs was longer in AUTO1 compared with brexu-cel (CRS - day 5 vs 8 for AUTO1, ICANS - day 7-9 vs 12 for AUTO1). Severe ICANS following AUTO1 was largely limited to patients with high disease burden pre-LD and no grade >3 CRS and/or ICANS was observed in patients with <5% BM blasts pre-LD. Together, these data suggest that AUTO1 may be administered in an ambulatory setting, particularly in patients with low disease burden.

Amongst the entire Phase Ib/II population, the 12-month OS and EFS estimates were 61.1% and 49.5% respectively. Patients with MRD-level disease and those with intermediate disease burden (5-75%) pre-LD had excellent EFS, better than for patients with high disease burden (<75% BM blasts) pre-LD. An association between ongoing CAR-T persistence and durable response was observed in this Phase Ib/II study, echoing the findings of Pulsipher et al. which describes the association between ongoing B-cell aplasia (a surrogate of persistence) and better EFS following tisa-cel (Pulsipher et al., 2022. Blood Cancer Discov 3, 66-81). Allo-SCT is frequently used to consolidate responses to salvage therapy in r/r adult B-ALL, including CAR-T therapy, but may be a double-edged sword here where allo-SCT conditioning is likely to eradicate AUTO1 engraftment/persistence in the blood, reducing immunosurveillance for CD 19+ relapse. Notably, in this Phase Ib/II study, allo-SCT did not improve outcome, although patient numbers are small. Strategies which combine assessment of MRD by next-generation sequencing e.g. ClonoSeq and CAR-T persistence may in the future predict risk of relapse and guide the appropriate use of allo-SCT consolidation. The Phase Ib/II data presented in the present document suggests that durable responses from AUTO1 as a stand-alone therapy can be achieved in some patients.

In summary, the present Phase Ib/II study showed that AUTO1 therapy resulted in high response rates and a favorable safety profile in adults with r/r B-ALL, with toxicity mostly limited to patients with high leukemic burden. Furthermore, AUTO1 was associated with durable responses, particularly in patients with low/intermediate leukemic burden, including patients who did not receive consolidative allo-SCT. AUTO1 represents a new option for adults with r/r B-ALL.

Example 11: CAR-T cell kinetics in adult patients with r/r B-ALL treated with treated with CD19 CAT CAR T-cell product (AUTO1) in the Phase Ib/II study

METHODS

Longitudinal monitoring of CAR T-cell pharmacokinetics and persistence is valuable for assessing clinical outcomes. Flow cytometry has traditionally been used to monitor CAR T- cell kinetics; however, droplet digital PCR (ddPCR) may offer higher sensitivity.

Flow cytometry and ddPCR were compared using 512 matched samples collected between Day 6 and Month 24 post-AUTOl infusion from 127 patients with R/R B-ALL in the Phase Ib/II study.

CAR T enumeration by flow cytometry

Immunophenotypic numeration was assessed by surface and intracellular flow cytometry, using an anti-CD19 CAR antibody. Samples were run in BD Trucount™ tubes for absolute counting. CAR T enumeration by ddPCR

The ddPCR duplex assay used a lentiviral Psi sequence encoded by the CAR T vector to assess copy number and an internal reference gene, RPP30. Vector copy number (VCN) per pg of genomic DNA was subsequently derived.

Technical considerations for comparing ddPCR and flow cytometry ddPCR was the primary assay used to measure CAR T levels in the Phase Ib/II trial and was designed and optimized to provide the highest sensitivity possible.

The flow cytometry assays were comprehensive 12-color assays primarily designed for assessing CAR immunophenotyping, differentiation state, and exhaustion status rather than CAR T marking.

The lower limit of quantification (LLoQ) for both the surface and intracellular flow cytometry assays was assessed by spiking known quantities of CAR Ts into whole blood (surface flow cytometry LLoQ: 20.22 cells/pL and intracellular flow cytometry LLoQ: 3.11 cells/pL).

The sampling for the two technologies reflects this prioritization with an increased number of timepoints collected for ddPCR (up to 19 in 24 months post-infusion) compared with flow cytometry (up to 9 in 24 months post-infusion).

Statistical methods

The Spearman correlation coefficient was used to measure the correlation between the number of CAR T-positive cells by flow cytometry and VCN by ddPCR.

A landmark analysis for event-free survival was conducted among patients with ongoing remission without new anti-cancer therapies at Month 6 post- AUTO 1 infusion, to evaluate the impact of loss of CAR T persistence prior to Month 6, as measured by flow cytometry or ddPCR, on event-free survival.

All statistical analyses were descriptive in nature for these secondary analyses of the study.

RESULTS

Intracellular and surface flow cytometry assays demonstrated a very strong correlation for all data and for data above the LLoQ (Figure 51). For all data, both flow cytometry assays correlated strongly with the VCN data from ddPCR. Surface flow cytometry data demonstrated a moderate correlation with VCN when data above the LLoQ were used:

Most likely due to the reduced number of matched pairs available.

Given the variation in the number of insertion sites within a CAR T population, VCN measures will have a greater variation compared with an absolute measure such as cells/pL. ddPCR can detect non-expressing integrants and therefore some ddPCR-positive/flow cytometry-negative results could be false positive reads.

Due to a high LLoQ, the surface flow cytometry assay used in the clinical study had a low overall percent agreement and negative percent agreement with ddPCR (Table 41). This particular assay would likely need further development to be suitable to monitor CAR T levels. When a sample was positive by surface and/or intracellular flow cytometry, it tended to be positive by ddPCR (positive percent agreement >95%). When a sample was negative by surface and/or intracellular flow cytometry, -60% and -40% of samples were positive for ddPCR, respectively, thus reflecting a higher sensitivity of ddPCR for detecting CAR T- positive samples. The intracellular flow cytometry dataset demonstrated improved correlation and concordance compared with the surface flow cytometry dataset; therefore, this dataset was chosen to take forward for clinical comparison.

Table 41. Analysis of concordance between FC data and ddPCR data.

ddPCR, droplet digital polymerase chain reaction; FC, flow cytometry. Overall percent agreement (%) = number of samples with ddPCR and FC both positive or both negative/total number of matched samples *100; positive percent agreement (%) = number of samples with ddPCR and FC both positive/number of samples with FC positive among matched samples *100; negative percent agreement (%) = number of samples with ddPCR and FC both negative/number of samples with FC negative among matched samples *100. A matched sample is one with both ddPCR and FC results available, and a total of 512 matched samples were available. Continued CAR T persistence was observed by ddPCR in 70% (42/60) and by flow cytometry in 63% (38/60) of patients with ongoing remission (complete remission or complete remission with incomplete hematologic recovery) (Figure 52).

Loss of CAR T persistence at Month 6, measured by either ddPCR or intracellular flow cytometry, was associated with shorter event-free survival compared with patients who had ongoing CAR T persistence at Month 6. This association was less marked for the intracellular flow cytometry assay used in the Phase Ib/II study compared to ddPCR.

CONCLUSIONS

• Strong correlation was observed between flow cytometry and ddPCR assays for assessment of CAR T levels.

• ddPCR is a more sensitive technology than flow cytometry for monitoring CAR T persistence.

• Flow cytometry assays developed specifically for CAR T monitoring may be sufficiently sensitive to be of clinical relevance.

Claims

1. An autologous CD 19 CAR-T cell for use in a method of treating a relapsed or refractory CD 19+ haematological malignancy in a patient comprising administering an autologous CD19 CAR T-cell to the patient, wherein: if the patient has a presence of less than or equal to 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 100 x 10⁶ CAR T-cells and a second dose comprising about 310 x 10⁶ CAR T-cells; and/or if the patient has a presence of more than 20% blasts in the bone marrow (BM), then the patient is administered a first dose comprising about 10 x 10⁶ CAR T- cells and a second dose comprising about 400 x 10⁶ CAR T-cells, preferably wherein the haematological malignancy is B-cell acute lymphoblastic leukaemia (B-ALL).

2. The autologous CD 19 CAR-T cell for use according to claim 1, wherein the second dose is administered at between about 7 days and about 11 days after the administration of the first dose.

3. The autologous CD 19 CAR-T cell for use according to claim 1 or claim 2, wherein the patient does not subsequently receive a stem cell transplant (SCT), preferably an allogeneic SCT.

4. The autologous CD19 CAR-T cell for use according to any of claims 1 to 3, wherein the method of treating a relapsed or refractory CD 19+ haematological malignancy in a patient further comprises a step of determining CAR-T cell persistence in a sample comprising peripheral blood mononuclear cells (PBMCs) from the patient: wherein if CAR-T cells are detected, then the patient may not receive a stem cell transplant (SCT), and wherein if CAR-T cells are not detected, then the patient may receive a SCT.

5. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 4, wherein at least 50% of patients treated achieve an overall remission (OR) or are identified as responders.

6. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 5, wherein less than 20% of the patients treated exhibit a grade 3 or greater CRS.

7. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 6, wherein less than 15% of the patients treated exhibit a grade 3 or greater neurotoxicity or ICANS.

8. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 7, wherein the age of the patient is eighteen years or older.

9. The autologous CD 19 CAR-T cell for use according to claim 8, wherein:

■ the age of the patient is between eighteen and thirty-nine years and at least 50% of patients treated achieve an overall remission (OR) or are identified as responders; or

■ the age of the patient is between forty and sixty-four years and at least 60% of patients treated achieve an overall remission (OR) or are identified as responders; or

■ the age of the patient is sixty-five years or older and at least 80% of patients treated achieve an overall remission (OR) or are identified as responders.

10. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 9, wherein the patient has less than or equal to 20% blasts in the bone marrow (BM), and wherein:

■ at least 70% of patients treated achieve an overall remission (OR) or are identified as responders; and/or

■ less than 10% of the patients treated exhibit a grade 3 or greater CRS; and/or

■ less than 5% of the patients treated exhibit a grade 3 or greater ICANS.

11. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 9, wherein the patient has between more than 20% and less than or equal to 75% blasts in the BM and wherein: ■ at least 70% of patients treated achieve an overall remission (OR) or are identified as responders; and/or

■ less than 10% of the patients treated exhibit a grade 3 or greater ICANS.

12. The autologous CD19 CAR-T cell for use according to any of claims 1 to 9, wherein the patient has more than or equal to 75% blasts in the BM, and wherein:

■ at least 40% of patients treated achieve an overall remission (OR) or are identified as responders; and/or

■ less than 10% of the patients treated exhibit a grade 3 or greater ICANS.

13. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 12, wherein the patient has Philadelphia chromosome positive ALL (Ph+ ALL).

14. The autologous CD19 CAR-T cell for use according to claim 13, wherein at least 75% of patients treated achieve an OR or are identified as responders.

15. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 14, wherein the patient has been previously administered one or more lines of therapy, and wherein:

■ the patient has been administered one prior line of therapy and at least 60% of patients treated achieve an overall remission (OR) or are identified as responders; or

■ the patient has been administered two prior lines of therapy and at least 60% of patients treated achieve an overall remission (OR) or are identified as responders; or

■ the patient has been administered three prior lines of therapy and at least 70% of patients treated achieve an overall remission (OR) or are identified as responders; or

■ the patient has been administered four or more prior lines of therapy and at least 40% of patients treated achieve an overall remission (OR) or are identified as responders.

16. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 15, wherein the patient has been previously administered one or more of inotuzumab ozogamicin and blinatumomab.

17. The autologous CD 19 CAR-T cell for use according to claim 16, wherein:

■ the patient has been previously administered inotuzumab ozogamicin and at least 50% of patients treated achieve an overall remission (OR) or are identified as responders; or

■ the patient has been previously administered blinatumomab and at least 50% of patients treated achieve an overall remission (OR) or are identified as responders.

18. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 17, wherein the patient has previously received a stem cell transplant (SCT), preferably allogeneic SCT.

19. The autologous CD 19 CAR-T cell for use according to claim 18, wherein at least 70% of patients treated achieve an overall remission (OR) or are identified as responders.

20. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 19, wherein the patient has extramedullary disease at preconditioning.

21. The autologous CD 19 CAR-T cell for use according to claim 20, wherein at least 50% of patients treated achieve an overall remission (OR) or are identified as responders.

22. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 21, wherein the patient is administered a preconditioning regimen comprising 120 mg/m² fludarabine (Flu) and 1000 mg/m² cyclophosphamide (Cy).

23. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 22, wherein the autologous CD 19 CAR-T cell is manufactured using a method with a manufacturing success rate of at least 80%.

24. The autologous CD19 CAR-T cell for use according to any of claims 1 to 23, wherein the autologous CD 19 CAR-T cell expands at a high-level following administration to the patient.

25. The autologous CD 19 CAR-T cell for use according to any of claims 1 to 24, wherein the autologous CD 19 CAR-T cell persists for at least 3 months in the patient’s peripheral blood or bone marrow.

26. A method of selecting a patient for receiving a second therapy following a treatment with CD 19 CAR engineered cells, which comprises determining CD 19 CAR engineered cell persistence in a sample comprising peripheral blood mononuclear cells (PBMCs) from the patient: wherein if CD 19 CAR engineered cells are detected, then the patient may not receive the second therapy; and wherein if CD 19 CAR engineered cells are not detected, then the patient may receive the second therapy, wherein the patient has a relapsed or refractory CD 19+ haematological malignancy prior to receiving the CD 19 CAR engineered cells.

27. The method according to claim 26, wherein the second therapy is selected from stem cell transplant (SCT) and a tyrosine inhibitor (TKI).

28. The method according to claim 27, wherein the second therapy is a tyrosine inhibitor (TKI) and the patient has Ph+ B-ALL.