CD19 / 22 CAR T Cell Therapy for High-Risk or Relapsed Pediatric Acute Lymphoblastic Leukemia

Abstract

The present disclosure relates to CD19 / 22 CAR T cell products and methods for treating high-risk or recurrent CD19+ or CD22+ hematological malignancies. The present disclosure addresses the need for an immunotherapeutic agent capable of targeting two or more cell surface structures to reflect the complex pattern of marker expression associated with many cancers, including CD19-positive cancers, and provides a means of solving the prior art problem of the need for an alternative CAR-T cell approach capable of killing target cells that express large or bulky target antigens.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 340,857, filed May 11, 2022; U.S. Provisional Application No. 63 / 350,861, filed Jun. 9, 2022; U.S. Provisional Application No. 63 / 386,742, filed Dec. 9, 2022; and U.S. Provisional Application No. 63 / 498,458, filed Apr. 26, 2023. These applications are hereby incorporated by reference in their entirety.

[0002] Field The present disclosure relates to CD19 / 22 CAR T - cell products and methods for treating high - risk or relapsed CD19+ or CD22+ hematological malignancies.

[0003] Incorporation by Reference of Sequence Listing This application includes, as another part of the present disclosure, a computer - readable form sequence listing (file name: 57768_SeqListing.txt; 121,399 bytes - XML file dated Dec. 8, 2022), which is hereby incorporated by reference in its entirety.

Background Art

[0004] Background Pediatric B-cell acute lymphoblastic leukemia (ALL) accounts for approximately 30% of childhood cancer diagnoses. It is a serious life-threatening disease that progresses rapidly if left untreated. B-ALL is characterized by the rapid proliferation of poorly differentiated lymphoid progenitor cells within the bone marrow. Standard treatment is combination chemotherapy. Typically, the treatment procedure is divided into several stages: pre-steroid stage, induction, consolidation, intensification, and maintenance, including administration of steroids, chemotherapy, cancer-targeted drugs, and / or bone marrow or stem cell transplantation (SCT). The overall survival (OS) rate for pediatric B-cell ALL patients is 90%, however, 10-20% of pediatric B-cell ALL patients relapse with chemotherapy-resistant lesions. The clinical outcome for relapsed pediatric patients has not changed over the past 20 years. The long-term OS for children suffering from ALL relapse remains at 40-50% despite considerable efforts to optimize the standard approach. Current treatment strategies for relapsed ALL include either further intensive chemotherapy and often consolidation with allogeneic SCT, but these patients have suboptimal outcomes. Most of these patients often already have the maximum tolerance to chemotherapy / radiotherapy, so new therapies are urgently needed for such patients.

[0005] Several immunotherapeutic agents, including therapeutic monoclonal antibodies (mAbs), immunoconjugated mAbs, radiolabeled conjugated mAbs, and bispecific T cell engagers, have been described for use in cancer treatment. Typically, these immunotherapeutic agents target a single antigen: for example, rituximab targets CD20, mylotarg targets CD33, and alemtuzumab targets CD52.

[0006] Chimeric antigen receptors are proteins in which the specificity of monoclonal antibodies (mAbs) is grafted onto the effector functions of T cells. Their normal form is that of a type I transmembrane domain protein with an amino-terminal (binder) that recognizes an antigen, and a transmembrane domain connected to an endodomain that transmits T cell activation signals. The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody that recognizes a target antigen, fused to a signal transduction endodomain via a transmembrane domain. Such molecules result in the activation of T cells in response to recognition by their target scFv. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. CARs have been developed against various tumor-associated antigens, and many are currently in clinical trials.

[0007] Human CD19 antigen is a 95kd transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is expressed very early in B cell differentiation and is lost only in the final B cell differentiation into plasma cells. Therefore, CD19 is expressed in all B cell malignancies except multiple myeloma. Since the loss of the normal B cell compartment is an acceptable toxicity, CD19 is a CAR target, and clinical studies targeting CD19 with CARs are being conducted.

[0008] CD19-directed CAR therapy has shown efficacy in treating ALL. The first studies in ALL were published in the spring of 2013 by groups from Memorial Sloane Kettering [Brentjens et al., Leukemia. Sci. Transl. Med., 5: 177ra38) (2013)] and the University of Pennsylvania. The latest version of the University of Pennsylvania study was reported [Maude et al., N. Engl. J. Med., 371: 1507-1517 (2014)]. In the latter study, 25 patients under 25 years of age and 5 patients over that age were treated. Ninety percent achieved complete remission at 1 month, 22 of 28 evaluable cases achieved minimal residual disease (MRD)-negative status, and the 6-month event-free survival rate was 67%. Fifteen patients did not receive further therapy after the study.

[0009] The Memorial Sloan Kettering study, in the adult setting, treated five ALL patients (two with refractory relapses, two with MRD-positive lesions, and one who was MRD negative) with autologous T cells transduced with a retrovirus to express a CD19 CAR incorporating an scFv derived from the SJ25C1 hybridoma and the CD28 costimulatory domain. All of these patients achieved a molecularly deep remission, and four of these patients became eligible for allogeneic SCT. This precluded assessment of the durability of the response, but the CAR T cells were only detectable in blood or bone marrow 3 - 8 weeks after infusion. Patients who were not transplanted relapsed at 90 days with CD19+ disease. Subsequently, Davila et al., Sci. Transl. Med. 6: 224:ra25 (2014) provided an update on this cohort, and 14 of 16 adult patients had detectable lesions at the point of CAR T cell infusion despite salvage chemotherapy and cyclophosphamide conditioning. Fourteen of 16, including seven of nine patients with morphological evidence of residual disease detectable after salvage chemotherapy, achieved a complete remission regardless of the presence or absence of a numerical recovery. Twelve of 16 patients achieved MRD negativity, which, as of the time of publication, enabled seven to undergo allogeneic transplantation. The response was durable in some patients, with four of eight non-transplanted patients maintaining a morphological remission for up to 24 months of follow-up, but the survival curve for this cohort has not yet stabilized.

[0010] Another published study of a cohort of pediatric and young adult patients mainly with ALL provides an analysis by initial treatment intent of its outcomes. This can help remove the inherent bias in excluding patients who did not receive the expected dose of CAR-T cells [Lee et al., Lancet (2014) doi:10.1016 / S0140-6736(14)61403-3]. Twenty-one patients were treated with a second-generation CAR containing the CD28 domain. All patients except two received the expected T cell dose, highlighting the feasibility of delivering this treatment to those with refractory or multiply relapsed ALL. This study showed that 67% achieved complete remission and 60% of those with ALL achieved a MRD-negative status.

[0011] The first clinical trial demonstrating the clinical activity of CD22 CAR T cells in pediatric and adult patients with B-ALL was reported in Fry et al., Nature Med., 24:20-28 (2018). Twenty-one pediatric and adult patients, including 17 who had previously been treated with CD19-directed immunotherapy, received CD22 CAR T cells. Complete remission was achieved in 73% (11 / 15) of patients who received ≥1×10 6 CAR T cells per kg of body weight, including 5 of 5 patients enrolled with CD19-lower / negative relapse. The median duration of remission was 6 months. Relapse was associated with a decrease in the density of the CD22 site, which may have enabled CD22+ cells to escape death by CD22 CAR T cells.

[0012] A particular problem in the field of oncology is provided by the Goldie-Coldman hypothesis: this describes that single antigen targeting alone can lead to tumor escape by modulation of said antigen due to the high mutation rate inherent to most cancers. This modulation of antigen expression can reduce the effectiveness of known immunotherapeutic agents, including those targeting CD19. Despite excellent clinical responses to CD19-directed T cell therapy, a significant number of patients still relapse. The major cause of disease relapse is either the emergence of CD19-negative leukemia clones or non-persistence of CAR-T cells [Sotillo et al., Cancer Discov., 5:1282-1295 (2015); Gardner et al., Blood 127:2406-2410 (2016)].

[0013] Therefore, the problem with immunotherapeutic agents targeted against CD19 is that B cell malignancies can mutate and become CD19 negative. This can lead to recurrence due to CD19-negative cancers that do not respond to CD19-targeting therapeutics. The emergence of CD19-negative escape clones has been reported in all major studies of ALL and may be related to the selection of leukemia clones that have somatic mutations or express alternatively spliced CD19 mRNA lacking exon 2, which prevents recognition by the CD19 CAR (Sotillo et al., supra). In a pediatric B cell ALL study at the University of Pennsylvania, two-thirds of the patients relapsed due to CD19-negative lesions, while the remaining one-third relapsed due to inadequate engraftment of CAR-T cells [Grupp et al., Blood 128(22):221 (2016)]. CD19-negative relapses have also been reported by the NCI in lymphoma patients treated with a fully human anti-CD19 CAR (HuCAR-19) [Brudno et al., Blood, 128(22): 999(2016)]. Another study recently reported that CD19-negative relapses were more frequently observed after tisagenlecleucel infusion in patients with high tumor burdens [Dourthe et al., Leukaemia, 35:3383-3393 (2021)].

[0014] Therefore, there is a need for immunotherapeutic agents that can target two or more cell surface structures to reflect the complex patterns of marker expression associated with many cancers, including CD19-positive cancers.

[0015] CAR-T cell-mediated treatments have shown success against compact target antigens such as CD19 or GD2, but chimeric antigen receptors have been unable to signal in response to antigens with bulky extracellular domains.

[0016] Optimal synaptic distance is required for efficient induction of downstream signaling after antigen encounter. When a T cell encounters an antigen-presenting cell (via TCR interaction with peptide-MHC), proteins at the interface are passively separated based on size. Phosphatases such as CD45 and CD148 with large ectodomains are excluded from the region of close contact between the T cell and APC. The synapse formed through the interaction of peptide-MHC and TCR is optimal for CD45 occlusion. In the case of CAR-T cells targeting smaller antigens such as CD19, there is no barrier to synapse formation, and such antigens can be efficiently targeted with multiple epitopes. Giant proteins such as CD22 have unique problems. Targeting of membrane-distal epitopes in such proteins may provide a suboptimal synaptic length, allowing phosphatases to enter the synapse and inhibit tyrosine phosphorylation. Targeting of the membrane-proximal region can improve synapse formation; however, steric occlusion of the epitope can result in suboptimal ligation of the target that allows the presence of phosphatases within the synapse, weakening tyrosine phosphorylation and kinase activity, and thus weakening CAR signaling.

[0017] Therefore, there is a need for alternative CAR-T cell approaches that can kill target cells expressing large or bulky target antigens.

[0018] T cell exhaustion is a state of T cell dysfunction that occurs during many chronic infections and cancers. It is defined by insufficient effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infections and tumors. Recently, a clearer picture of the functional and phenotypic profiles of exhausted T cells has emerged with the expression of the inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells that is a key feature [Day et al., Nature, 443: 350-354 (2006)].

[0019] Responses in CD19 CAR studies suggest that sustained high levels of T cells over a long period are important for generating durable responses [Mueller et al., Blood, 130, 2317-2325 (2017)]. There remains a need in the art for effective CAR therapies against CD19+ or CD22+ hematological malignancies that are not associated with the aforementioned drawbacks.

Prior Art Documents

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Summary of the Invention

[0021] Abstract The present disclosure provides a method for treating high-risk / relapsed CD19+ or CD22+ hematological malignancies in a patient, comprising administering to the patient autologous CD19 / 22 CAR T cells (e.g., autologous CD19 / 22 CAR T cell products comprising the CARs of CAT19CAR and 9A8CAR described in Example 1 herein). The present disclosure also provides autologous CD19 / 22 CAR T cells (e.g., autologous CD19 / 22 CAR T cell products comprising the CARs of CAT19CAR and 9A8CAR described in Example 1 herein), or pharmaceutical compositions comprising these cells, for use in the treatment of high-risk / relapsed CD19+ or CD22+ hematological malignancies. The present disclosure further provides the use of autologous CD19 / 22 CAR T cells (e.g., autologous CD19 / 22 CAR T cell products comprising the CARs of CAT19CAR and 9A8CAR described in Example 1 herein), or pharmaceutical compositions comprising these cells, in the manufacture of a medicament for the treatment of high-risk / relapsed CD19+ or CD22+ hematological malignancies.

[0022] A method, autologous CD19 / 22 CAR T cells, or use is provided, wherein the age of the patient is 24 years or younger.

[0023] A method, autologous CD19 / 22 CAR T cells, or use is provided, wherein the hematological malignancy is acute lymphoblastic leukemia (ALL), or a CD19+ or CD22+ lymphoma. A method is provided wherein the lymphoma is Burkitt lymphoma.

[0024] In particular, the patient has a) resistant disease (>5% blasts) at the end of induction of the UKALL 2019 guidelines or equivalent, b) high-level persistent MRD at the second timepoint of frontline national protocols (currently, MRD > 10 -4 ) in ALL at week 14 according to the UKALL2019 guidelines or equivalent, c) high-risk infant ALL (age <6 months at diagnosis due to MLL gene rearrangement, and white blood cell count > 300×10 9 / L or inadequate steroid initial response (i.e., >1×10 circulating blasts after 7-day steroid pre-phase of induction according to national guidelines or equivalent), d) MRD > 10 9 in intermediate-risk infant ALL at the end of induction according to national guidelines or equivalent, e) high-risk first relapse (as defined by the latest IntreALL 2019 classification: bone marrow or composite relapse within 30 months of diagnosis), f) standard-risk relapse in patients with high-risk cytogenetics (defined as BCR-ABL, KMT2A rearrangement, near-haploidy (<30 chromosomes) and low hypodiploidy (30 - 39 chromosomes), iAMP21 and TCF3-HLF translocation), g) bone marrow minimal residual disease (MRD) > 10 -3 at the end of re-induction in standard-risk relapse, h) any refractory relapse of ALL (defined as >1% blasts by flow cytometry after at least 1 cycle of standard chemotherapy), or i) any relapse of CD22+ lymphoma. The patient may have isolated CNS relapse fulfilling one or more of a) - o). -3

[0025] ​The provided method, autologous CD19 / 22 CAR T cells, or use thereof involves the patient receiving a single dose of 0.75×10 6 CAR T cells / kg body weight, 1×10 6 CAR T cells / kg body weight, or 1.2×10 6 CAR T cells / kg body weight. The administration may be by intravenous injection, preferably by intravenous injection through a Hickman line or a central catheter inserted peripherally.

[0026] The CD19 / 22 CAR T cells have a) a heavy chain variable region (VH) having complementarity-determining regions (CDRs) with the following sequences: CDR1 - GYAFSSS (SEQ ID NO: 1); CDR2 - YPGDED (SEQ ID NO: 2) CDR3 - SLLYGDYLDY (SEQ ID NO: 3) ; and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID NO: 4); CDR2 - DTSKLAS (SEQ ID NO: 5) CDR3 - QQWNINPLT (SEQ ID NO: 6) ; and express a chimeric antigen receptor (CAR) comprising a CD19-binding domain. A method, autologous CD19 / 22 CAR T cells, or use thereof is provided. The CDRs may be grafted into a human antibody framework.

[0027] In the provided method, autologous CD19 / 22 CAR T cells, or use thereof, the CD19-binding domain may comprise a VH domain having the sequence shown in SEQ ID NO: 7 and / or a VL domain having the sequence shown in SEQ ID NO: 8, or any variant thereof having at least 95% sequence identity.

[0028] The CD19 binding domain may comprise a scFv with the VH-VL orientation. The CD19 binding domain may comprise the sequence shown in SEQ ID NO: 9 or a variant thereof having at least 90% sequence identity. The CD19 binding domain and the transmembrane domain may be connected in the CAR by a spacer such as a CD8 stalk. The CAR may comprise an intracellular T cell signaling domain, for example, an intracellular T cell signaling domain comprising a 41BB endodomain and a CD3-zeta endodomain.

[0029] CD19 / 22 CAR T cells are a) the following sequences: CDR1 - NFAMA (SEQ ID NO: 58); CDR2 - SISTGGGNTYYRDSVKG (SEQ ID NO: 59) CDR3 - QRNYYDGSYDYEGYTMDA (SEQ ID NO: 60) having a heavy chain variable region (VH) with CDRs; and b) the following sequences: CDR1 - RSSQDIGNYLT (SEQ ID NO: 61); CDR2 - GAIKLED (SEQ ID NO: 62) CDR3 - LQSIQYP (SEQ ID NO: 63) having a light chain variable region (VL) with CDRs A method, autologous CD19 / 22 CAR T cells, or use is provided that expresses a CAR comprising a CD22 binding domain. The CDRs may be grafted into a human antibody framework.

[0030] In the method, autologous CD19 / 22 CAR T cells, or use provided, the CD22 binding domain may comprise a VH domain having the sequence shown in SEQ ID NO: 64 and / or a VL domain having the sequence shown in SEQ ID NO: 65, or a variant thereof having at least 95% sequence identity.

[0031] The CD22 binding domain may comprise an scFv with the VH-VL orientation. The CD22 binding domain may comprise the sequence shown in SEQ ID NO: 966 or a variant thereof having at least 90% sequence identity. The CD22 binding domain and the transmembrane domain may be connected in the CAR by a spacer such as a CD8 stalk. The CAR may comprise an intracellular T cell signaling domain, for example, an intracellular T cell signaling domain comprising a 4-1BB end domain and a CD3-zeta end domain.

[0032] This patent or application file contains at least one drawing created in color. Copies of this patent or patent application publication with color drawings are provided by the United States Patent and Trademark Office upon request and payment of the necessary fees.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0047] Detailed Description The CARPALL clinical study [Ghorashian et al., J. Haematol., 169: 463-478 (2015); Ghorashian et al., Nature Med., 25:1408-14 (2019)] that tested the efficacy of CD19 CATCAR (also sometimes referred to herein as CAT19CAR or AUTO1) showed that AUTO1, a rapid off-rate CAR of its own in pediatric and young adult patients with relapsed / refractory (r / r) B-ALL, was effective in 12 out of 14 patients with molecular genetic remission. However, among the 7 patients who relapsed, 5 relapses were due to CD19 antigen-negative escape. To address antigen escape relapses in AUTO1, additional ligands can be targeted.

[0048] CD22 is contemplated herein as another target for B-ALL malignancies, but creating a CAR effective against CD22 is difficult due to the size, density, and rigidity of this ligand. Furthermore, in the context of B-ALL, CD22 expression levels are known to be downregulated in response to selective CAR pressure. In the clinical trial of CD22 CAR2, this resulted in patients relapsing with low CD22 density (2,839 epitopes / cell) after treatment, presumably due to the target density falling below the sensitivity threshold for CD22 CAR2 (Fry et al., supra).

[0049] The methods provided herein can improve the treatment of r / r B-ALL by combining a highly sensitive CD22 CAR that can target cells expressing less than 1,000 CD22 molecules per cell with AUTO1 to create a dual-targeting CD19 and CD22 product (AUTO1 / 22) via co-transduction for the treatment of pediatric r / r B-ALL. The results described in the examples below were obtained as part of an expanded cohort of the CARPALL clinical trial (NCT02443831).

[0050] Chimeric antigen receptor (CAR)

[0051] Classical chimeric antigen receptors (CARs) are chimeric type I transmembrane proteins that connect an extracellular antigen-binding domain to an intracellular signaling domain (endodomain). The antigen-binding domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), although this can be based on other formats that include antibody fragments or antibody-like antigen-binding sites. Other examples include, but are not limited to, the natural ligand of the target antigen, a peptide with sufficient affinity for the target, an F(ab) fragment, an F(ab’)2 fragment, an F(ab’) fragment, a single-domain antibody (sdAb), a domain antibody (dAb), a VHH antigen-binding domain or nanobody, an artificial single binder, e.g., a DARPin (designed ankyrin repeat protein), an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a VNAR, an iBody, an affimer, a finomer, an avdulin / nanobody, a centyrin, an alphabody, a nanofitin, or a single chain derived from a T cell receptor that can bind to the target antigen. A spacer is usually required to isolate the antigen-binding domain from the membrane and to allow for the proper orientation. A common spacer used is the Fc of IgG1. More compact spacers can be, for example, the stalk derived from CD8α, and furthermore, depending on the antigen, the IgG1 hinge alone may be sufficient. The transmembrane domain anchors the protein to the cell membrane and connects the spacer to the endodomain.

[0052] Initial CAR designs had an endodomain derived from the intracellular portion of either the γ chain of FcεR1 or CD3ζ. Therefore, these first-generation receptors transmitted immunological signal 1, which was sufficient to induce T cell killing of cognate target cells but could not fully activate T cells to proliferate and survive. To overcome this limitation, compound endodomains were constructed: the fusion of the intracellular portion of a T cell costimulatory molecule and the intracellular portion of CD3ζ results in a second-generation receptor that can transmit both activation and costimulatory signals simultaneously upon antigen recognition. One common costimulatory domain is that of CD28. This provides the most potent costimulatory signal, i.e., immunological signal 2 that induces T cell proliferation. Several receptors, including TNF receptor family endodomains such as the closely related OX40 and 41BB that transmit survival signals, have also been described. Even more potent third-generation CARs are currently described, which have endodomains that can transmit activation, proliferation, and survival signals.

[0053] When the CAR binds to the target antigen, an activation signal is transmitted to the T cell in which the CAR is expressed, thereby directing the specificity and cytotoxicity of the T cell towards cells expressing the target antigen.

[0054] Target antigen

[0055] A "target antigen" is an entity that is specifically recognized and bound by the antigen-binding domain of the chimeric receptor provided herein.

[0056] The target antigen may be an antigen present on cancer cells, such as a tumor-associated antigen. CD19 and CD22 are target antigens contemplated herein.

[0057] Binding domain specific for the CD19 target antigen

[0058] Human CD19 antigen is a 95kd transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is classified as a type I transmembrane protein with a single transmembrane domain, a cytoplasmic C-terminus, and an extracellular N-terminus. CD19 is expressed very early in B cell differentiation and is lost only in the final B cell differentiation into plasma cells. CD19 is a biomarker for normal B cells and follicular dendritic cells. CD19 acts mainly as a B cell co-receptor together with CD21 and CD81. Upon activation, the cytoplasmic tail of CD19 is phosphorylated, which leads to binding by Src family kinases and recruitment of PI-3 kinase.

[0059] CD19 is expressed in all B cell malignancies but not in multiple myeloma cells. It is not expressed in other hematopoietic populations or non-hematopoietic cells, and thus targeting of this antigen should not result in toxicity to the bone marrow or non-hematopoietic organs. Loss of the normal B cell compartment results in B cell aplasia from effective CD19 CAR T cell therapy, but the resulting hypogammaglobulinemia can be treated with pooled immunoglobulin and is considered an acceptable toxicity when treating lymphoid malignancies.

[0060] Different designs of CARs have been tested against CD19 in various clinical trials, as outlined in Table 1 below. Table 1

Table 1

[0061] As shown above, most of the studies conducted so far have used the scFv derived from the hybridoma fmc63 as part of the binding domain that recognizes CD19.

[0062] The antigen-binding domain of a CAR that binds to CD19 (referred to herein as CD19 CAR) can be any domain that can bind to CD19.

[0063] For example, the antigen-binding domain may include the CD19 antigen-binding domain described in Table 2. Table 2

Table 2-1

Table 2-2

[0064] The gene encoding CD19 contains 10 exons: exons 1-4 encode the extracellular domain; exon 5 encodes the transmembrane domain; exons 6-10 encode the cytoplasmic domain. The antigen-binding domain of the CD19 CAR herein may bind to the epitope of CD19 encoded by exon 1 of the CD19 gene. The antigen-binding domain of the CD19 CAR herein may bind to the epitope of CD19 encoded by exon 2 of the CD19 gene. The antigen-binding domain of the CD19 CAR herein may bind to the epitope of CD19 encoded by exon 3 of the CD19 gene. The antigen-binding domain of the CD19 CAR herein may bind to the epitope of CD19 encoded by exon 4 of the CD19 gene.

[0065] The CD19 binding domain exemplified herein is a variable region having complementarity-determining regions (CDRs) derived from an antibody designated CAT19. a) The following sequences: CDR1 - GYAFSSS (SEQ ID NO: 1); CDR2 - YPGDED (SEQ ID NO: 2) CDR3 - SLLYGDYLDY (SEQ ID NO: 3); A heavy chain variable region (VH) having the CAT19 CDR having: and b) The following sequences: CDR1 - SASSSVSYMH (SEQ ID NO: 4); CDR2 - DTSKLAS (SEQ ID NO: 5) CDR3 - QQWNINPLT (SEQ ID NO: 6) A light chain variable region (VL) having a CAT 19 CDR having is included. The CAT19 antibody is described in WO2016 / 139487.

[0066] It is contemplated that one or more mutations (substitutions, additions or deletions) can be introduced into one or more CDRs without negatively affecting the CD19 binding activity. Each CDR may have, for example, one, two or three amino acid mutations.

[0067] The CDR may be a fusion protein of the heavy variable region (VH) and the light chain variable region (VL) of the antibody and may be in the form of a single-chain variable fragment (scFv) connected by a short linker peptide of 10 to about 25 amino acids. The scFv may be in the VH-VL orientation, i.e., VH is at the amino terminus of the CAR molecule, the VL domain is linked to the spacer and then to the transmembrane domain and the end domain.

[0068] The CDR may be grafted into the framework of a human antibody or scFv. For example, the CAR may comprise a CD19 binding domain consisting of or comprising one of the following sequences.

[0069] The CD19 CAR may comprise the following VH sequence. VH sequence derived from SEQ ID NO: 7 - CAT19 mouse monoclonal antibody

Chemical Structure

[0070] The CD19 CAR may comprise the following VL sequence. VL sequence derived from SEQ ID NO: 8 - CAT19 mouse monoclonal antibody

Chemical Structure

[0071] The CD19 CAR may contain the following scFv sequences. SEQ ID NO: 9 - VH-VL scFv sequence derived from a murine monoclonal antibody

Chem.

[0072] The CAR may consist of or contain one of the following sequences. SEQ ID NO: 10 - CAT19 CAR using a "bell-shaped" structure

Chem.

Chem.

Chem.

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Chem.

[0073] The CARs provided herein may include variants of the polypeptides of SEQ ID NOs: 1-15 having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity, provided that (when combined with a suitable complementary VL or VH domain, if appropriate) the variant sequence retains the ability to bind to CD19.

[0074] The percentage identity between two polypeptide sequences can be readily determined by programs such as BLAST, which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0075] The CD19 CARs exemplified herein (i.e., CAT19 CAR using the "bell-shaped" structure, SEQ ID NO: 10) have the properties contemplated by this disclosure to provide lower toxicity and good efficacy in treated patients. When compared to the fmc63-bell-shaped CAR, the CAT19 CARs exemplified herein cause the death of target cells expressing CD19 and proliferate in response to targets expressing CD19, but had low interferon gamma release. Furthermore, a murine model of aggressive B cell lymphoma showed comparable efficacy and comparable engraftment between fmc63- and CAT19-based CAR-T cells, but surprisingly, CAT19 CAR T cells were less depleted than fmc63 CAR T cells. See Examples 2 and 3 of U.S. Publication No. 2018-0044417.

[0076] The CAT19 CARs provided herein may cause 25%, 50%, 70% or 90% lower IFNγ release in a competitive assay that includes contacting CAR T cells with target cells.

[0077] The CAT19CAR provided herein can result in a smaller percentage of CAR T cells that are more exhausted than fmc63 CAR T cells. T cell exhaustion can be evaluated using methods known in the art such as analysis of PD-1 expression. The CAR can cause CAR T cells to express 20, 30, 40, 50, 60 or 70% less PD-1 than fmc63 CAR T cells in a competitive assay that includes contacting the CAR T cells with target cells.

[0078] Another exemplary CD19 antigen-binding domain contemplated by the present disclosure is based on the CD19 antigen-binding domain CD19ALAb (described in WO2016 / 102965), a) the following sequences: CDR1 - SYWMN (SEQ ID NO: 16); CDR2 - QIWPGDGDTNYNGKFK (SEQ ID NO: 17) CDR3 - RETTTVGRYYYAMDY (SEQ ID NO: 18) a heavy chain variable region (VH) having CDRs having; and b) the following sequences: CDR1 - KASQSVDYDGDSYLN (SEQ ID NO: 19); CDR2 - DASNLVS (SEQ ID NO: 20) CDR3 - QQSTEDPWT (SEQ ID NO: 21) a light chain variable region (VL) having CDRs having including.

[0079] 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 have, for example, one, two or three amino acid mutations.

[0080] The CAR may include one of the following amino acid sequences. SEQ ID NO: 22 - mouse CD19ALAb scFv sequence

Chemical formula

Chem.

Chem.

[0081] The scFv may be in the VH - VL orientation (as shown in SEQ ID NOs: 9, 22, 23 and 24) or the VL - VH orientation.

[0082] The CAR may optionally include one of the following VH sequences: SEQ ID NO: 25 - Mouse CD19ALAb VH Sequence

Chem.

Chem.

[0083] The CAR may optionally include one of the following VL sequences: SEQ ID NO: 27 - Mouse CD19ALAb VL Sequence

Chem.

Chem.

Chem.

[0084] The CARs provided herein may include variants of the sequences set forth in any of SEQ ID NOs: 16-29 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that (when combined with a suitable complementary VL or VH domain, if appropriate) the variant sequence retains the ability to bind to CD19.

[0085] The percentage of identity between two polypeptide sequences can be readily determined by programs such as BLAST, which is freely available at blast.ncbi.nlm.nih.gov.

[0086] Binding domain specific for the CD22 target antigen

[0087] The human CD22 antigen is a molecule belonging to the SIGLEC family of lectins. It is found on the surface of mature B cells and some immature B cells. Generally speaking, CD22 is a regulatory molecule that prevents overactivation of the immune system and the development of autoimmune diseases.

[0088] CD22 is a glycoconjugate membrane-spanning protein that specifically binds sialic acid to its immunoglobulin (Ig) domain located at the N-terminus. The presence of the Ig domain makes CD22 a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling.

[0089] CD22 is a molecule of the IgSF that can exist in two isoforms: one with seven domains and a cytoplasmic tail composed of three ITIMs (immunoreceptor tyrosine-based inhibitory motifs) and ITAMs, and another splicing variant composed of five extracellular domains and a cytoplasmic tail with one ITIM. CD22 is thought to be an inhibitory receptor involved in the regulation of B cell responses to antigens. Like CD19, CD22 is widely considered to be a pan-B antigen, although its expression in some non-lymphoid tissues has been described. Monoclonal antibodies and immunoconjugates for CD22 targeting therapy are in clinical trials.

[0090] The antigen-binding domain of a CAR that binds to CD22 can be any domain that can bind to CD22. For example, the antigen-binding domain may include the CD22 binders described in Table 3. Table 3

Table 3-1

Table 3-2

[0091] Other anti-CD22 antibody-binding domains are known, such as the mouse anti-human CD22 antibodies 1D9-3, 3B4-13, 7G6-6, 6C4-6, 4D9-12, 5H4-9, 10C1-D9, 15G7-2, 2B12-8, 2C4-4, and 3E10-7; and the humanized anti-human CD22 antibodies LT22 and inotuzumab (G5_44). Table 4 shows the VH, VL, and CDR sequences (bold and underlined), and the positions of the target epitopes on CD22 for each antibody. Table 4

Table 4-1

Table 4-2

Table 4-3

[0092] Examples of CD22 CARs are described by Haso et al., Blood, 121(7): 1165-1174 (2013). Specifically, CD22 CARs having antigen-binding domains derived from m971, HA22, and BL22 scFv are described.

[0093] CD22 has seven extracellular IgG-like domains, which are generally identified as Ig domain 7 to Ig domain 1, with Ig domain 1 being the most proximal to the B cell membrane and Ig domain 7 being the most distal from the Ig cell membrane.

[0094] The positions of the Ig domains with respect to the amino acid sequence of CD22 (UniProt accession number P20273, entry version 224, http: / / www.uniprot.org / uniprot / P20273) are summarized in Table 5 below. Table 5

Table 5

[0095] The antigen-binding domain of the second CAR may bind to a membrane-distal epitope on CD22, such as Ig domain 7. The antigen-binding domain of the second CAR may bind to an epitope on Ig domain 7, 6, 5, or 4 of CD22, such as an epitope on Ig domain 5 of CD22. The antigen-binding domain of the second CAR may bind to an epitope located between amino acids 20 - 416 of CD22, such as between amino acids 242 - 326 of CD22.

[0096] The antigen-binding domain of the second CAR may bind to a membrane-proximal epitope on CD22. The antigen-binding domain of the second CAR may bind to an epitope on Ig domain 3, 2, or 1 of CD22. The antigen-binding domain of the second CAR may bind to an epitope located between amino acids 419-676 of CD22, for example, between 505-676 of CD22.

[0097] The CD22-binding domains exemplified herein have variable regions having CDRs from an antibody designated 9A8-1 (described in WO2019 / 220109): a) The following sequences: CDR1 - NFAMA (SEQ ID NO: 58); CDR2 - SISTGGGNTYYRDSVKG (SEQ ID NO: 59) CDR3 - QRNYYDGSYDYEGYTMDA (SEQ ID NO: 60) A heavy chain variable region (VH) having the CDRs of 9A8-1 having: and b) The following sequences: CDR1 - RSSQDIGNYLT (SEQ ID NO: 61); CDR2 - GAIKLED (SEQ ID NO: 62) CDR3 - LQSIQYP (SEQ ID NO: 63) A light chain variable region (VL) having the CDRs of 9A8-1 having including.

[0098] The CD22 CAR may include the following VH sequences. VH sequence from the 9A8-1 antibody of SEQ ID NO: 64

Chemical formula

[0099] The CD22 CAR may include the following VL sequences. VL sequence from the 9A8-1 antibody of SEQ ID NO: 65

Chemical formula

[0100] The CD22 CAR may comprise the following scFv sequences. A VL-VH scFv sequence based on the VH and VL sequences of SEQ ID NO: 66-9A8-1

Chemical formula

[0101] The antigen-binding domain of the 9A8-1 antibody exhibits particularly good efficacy in the CAR. For example, 9A8-1 in the FabCAR format showed improved target cell killing and cytokine release equivalent to that of 3B4 (WO2019 / 220109), a CAR with an alternative CD22 binder.

[0102] The antigen-binding domain of a CAR that binds to CD22 may comprise VH and / or VL sequences derived from any of the CD22 antibodies listed above, or variants thereof having at least 70%, 80%, 90% or 90% sequence identity, which variants retain the ability to bind to CD22.

[0103] The antigen-binding domain of the CD22 CAR may bind to CD22 with a KD in the range of 30-50 nM, for example 30-40 nM. The KD may be 32 nM.

[0104] OR gate

[0105] The CAR may be used in combination with one or more other activating or inhibitory chimeric antigen receptors. For example, they may be used in combination with one or more other CARs in a "logic gate", and the combination of CARs can detect a specific pattern of expression of at least two target antigens when expressed by cells such as T cells. If at least two target antigens are arbitrarily designated as antigen A and antigen B, the three possible options are as follows: "OR gate" - T cells are induced when either antigen A or antigen B is present on the target cell "AND gate"-T cells are induced only when both antigen A and B are present on the target cell. "AND NOT gate"-T cells are induced when antigen A alone is present on the target cell, but not when both antigen A and B are present on the target cell.

[0106] Engineered T cells expressing combinations of these CARs can be tuned to be exquisitely specific for cancer cells based on the specific expression (or lack thereof) of two or more markers.

[0107] Such "logic gates" are described, for example, in WO2015 / 075469, WO2015 / 075470, and WO2015 / 075470.

[0108] The "OR gate" contains two or more activating CARs each directed to a distinct target antigen expressed by the target cell. The advantage of the OR gate is that the effective targetable antigen is effectively antigen A + antigen B, which is increased on the target cell. This is particularly important for antigens expressed at variable or low density on the target cell, as the level of a single antigen may fall below the threshold required for effective targeting by CAR-T cells. Also, this avoids the phenomenon of antigen escape. For example, some lymphomas and leukemias become CD19-negative after targeting CD19, and using an OR gate targeting CD19 in combination with another antigen provides a "backup" antigen if this occurs.

[0109] The OR gate may include a CAR against a second antigen expressed in B cells, such as CD22.

[0110] Therefore, the antigen-binding domains of the first and second CARs bind to different antigens, and both CARs may include an activation endodomain. The two CARs may be the same or may include spacer domains that are different enough to prevent cross-pairing of the two different receptors. As contemplated herein, cells can therefore be engineered to be activated upon recognition of either or both of CD19 and CD22. This is useful in the field of oncology as shown by the Goldie-Coldman hypothesis: targeting a single antigen alone can lead to tumor escape by modulation of said antigen due to the high mutation rate inherent in most cancers. By targeting two antigens simultaneously, such escape is likely to be exponentially reduced.

[0111] It is important that the two CARs do not heterodimerize.

[0112] The first and second CARs of the T cell may be produced as a polypeptide containing both CARs, together with the cleavage site.

[0113] Signal peptide

[0114] The CAR of the cell may include a signal peptide, such that when the CAR is expressed inside a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and then to the cell surface, where it is expressed.

[0115] The core of the signal peptide may contain a long stretch of hydrophobic amino acids that tend to form a single alpha-helix. The signal peptide may start with a short stretch of positively charged amino acids, which helps to enforce the proper topology of the polypeptide during translocation. At the end of the signal peptide, typically, there is a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase can be cleaved either during or after translocation to create a free signal peptide and a mature protein. The free signal peptide is then digested by specific proteases.

[0116] The signal peptide may be at the amino terminus of the molecule.

[0117] The signal peptide may include the amino acid sequence of any of SEQ ID NOs: 68-70, 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.

[0118] The signal peptide of SEQ ID NO: 67 is compact and very efficient. It is predicted to give about 95% cleavage after the terminal glycine, providing efficient removal by signal peptidase.

[0119] SEQ ID NO: 67 MGTSLLCWMALCLLGADHADA

[0120] The signal peptide of SEQ ID NO: 68 is as follows.

[0121] METDTLLLWVLLLLVPGSTG

[0122] The signal peptide of SEQ ID NO: 2 is derived from IgG1. SEQ ID NO: 2: MSLPVTALLLPLALLLHAARP

[0123] The signal peptide of SEQ ID NO: 3 is derived from CD8.

[0124] SEQ ID NO: 3: MAVPTQVLGLLLLWLTDARC

[0125] The signal peptide for the first CAR may have a sequence different from that of the signal peptide of the second CAR.

[0126] Spacer

[0127] The CAR includes a spacer that connects the antigen-binding domain to the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. A flexible spacer enables the antigen-binding domain to be oriented in different directions, facilitating binding.

[0128] The spacer may include, for example, the IgG1 Fc region, the IgG1 hinge or the CD8 stalk, or a combination thereof. Alternatively, the spacer may include an alternative sequence having a length and / or domain spatial characteristics similar to the IgG1 Fc region, the IgG1 hinge or the CD8 stalk.

[0129] In the cells provided herein, the first and second CARs may include different spacer molecules. For example, the spacer may include, for example, the IgG1 Fc region, the IgG1 hinge, or the human or mouse CD8 stalk. Alternatively, the spacer may include an alternative linker having a length and / or domain spatial characteristics similar to the IgG1 Fc region, the IgG1 hinge or the CD8 stalk. The human IgG1 spacer may be modified to remove the Fc binding motif.

[0130] The spacer for the CD19 CAR may include a CD8 stalk spacer or a spacer having a length equivalent to that of the CD8 stalk spacer. The spacer for the CD19 CAR may have at least 30 amino acids or at least 40 amino acids. It may have 35 to 55 amino acids, for example, 40 to 50 amino acids. It may have about 46 amino acids.

[0131] The spacer for the CD22 CAR may include an IgG1 hinge spacer or a spacer having a length equivalent to that of the IgG1 hinge spacer. The spacer for the CD22 CAR may have fewer than 30 amino acids or fewer than 25 amino acids. It may have 15 to 25 amino acids, for example, 18 to 22 amino acids. It may have about 20 amino acids.

[0132] Examples of the amino acid sequences for these spacers are shown below: SEQ ID NO: 71 (hinge-CH2CH3 of human IgG1)

Chemical formula

Chemical formula

Chemical formula

[0133] Since CARs are typically homodimers (see Figure 1A), cross-pairing can result in heterodimeric chimeric antigen receptors. This can occur for a variety of reasons, e.g., (1) the epitopes may not be at the same "level" on the target cell, and as a result, the cross-paired CAR may only be able to bind to one antigen; (2) the VH and VL from two different scFvs are exchanged and may not recognize the target or, even worse, may recognize an unexpected and unpredictable antigen, which is undesirable. The spacer of the first CAR may be sufficiently different from the spacer of the second CAR to avoid cross-pairing. The amino acid sequence of the first spacer may share less than 50%, 40%, 30%, or 20% identity at the amino acid level with the second spacer.

[0134] Transmembrane domain

[0135] The transmembrane domain is the domain of the CAR that spans the membrane.

[0136] A transmembrane domain can be any protein structure that is thermodynamically stable in a membrane. This is typically an alpha helix composed of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion provided herein. The presence and span of the transmembrane domain of a protein can be determined by one of ordinary skill in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Additionally, considering that the transmembrane domain of a protein is a polypeptide predicted to form a hydrophobic alpha helix long enough to span the membrane, which has a relatively simple structure, an artificially designed transmembrane domain can also be used (US7052906B1 describes synthetic transmembrane components).

[0137] The transmembrane domain may be derived from CD28, which confers good receptor stability.

[0138] The transmembrane domain may be derived from human Tyrp-1. The tyrp-1 transmembrane domain sequence is shown as SEQ ID NO: 78.

[0139] SEQ ID NO: 78 IIAIAVVGALLLVALIFGTASYLI

[0140] The transmembrane domain may be derived from CD8A. The CD8A transmembrane domain sequence is shown as SEQ ID NO: 79. SEQ ID NO: 79 IYIWAPLAGTCGVLLLSLVITLYC

[0141] End domain

[0142] As described above, the end domain is the signaling portion of the CAR. After antigen recognition, the receptor clusters, native CD45 and CD148 are excluded from the synapse, and signals are transmitted to the cell. The most commonly used end domain component is that of CD3-zeta, which contains three ITAMs. This transmits an activation signal to the T cell after antigen binding. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling may be required. For example, chimeric CD28 and OX40 can be used together with CD3-zeta to transmit proliferation / survival signals, or all three can be used together.

[0143] The cells provided herein contain two CARs, each having an end domain.

[0144] The end domain of the first CAR and the end domain of the second CAR may include (i) an ITAM-containing end domain, such as an end domain derived from CD3 zeta; and / or (ii) a co-stimulatory domain, such as an end domain derived from CD28; and / or (iii) a domain that transmits a survival signal, such as a TNF receptor family end domain such as OX-40 or 4-1BB.

[0145] Therefore, the end domain of the CAR of the present invention may include one or a combination of multiple of the CD3-zeta end domain, 41BB end domain, OX40 end domain, or CD28 end domain.

[0146] The intracellular T cell signaling domain (end domain) of the CAR of the present invention may include a sequence shown in any of SEQ ID NOs: 80-87, or a variant thereof having at least 80% sequence identity. SEQ ID NO: 80 (CD3 zeta end domain)

Chemical formula

[0147] Examples of such combinations of end domains include 41BB-zeta, OX40-zeta, CD28-zeta, and CD28-OX40-zeta. SEQ ID NO: 84 (41BB-zeta end domain fusion) [Chemical formula] SEQ ID NO: 85 (OX40-zeta end domain fusion) [Chemical formula] SEQ ID NO: 86 (CD28 zeta end domain fusion) [Chemical formula] SEQ ID NO: 87 (CD28OX zeta) [Chemical formula]

[0148] Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with any of SEQ ID NOs: 80 to 87, provided that the sequence provides a functional transmembrane domain / intracellular T cell signaling domain.

[0149] Nucleic acid

[0150] One or more nucleic acids provided herein encode the CD19 CAR and CD22 CAR of the present disclosure. As used herein, the terms “polynucleotide,” “nucleotide,” and “nucleic acid” are intended to be synonymous with one another.

[0151] The nucleic acid may be, for example, RNA, DNA or cDNA. The nucleic acid may contain DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides containing synthetic or modified nucleotides within the polynucleotide. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that for the purposes of use as described herein, the polynucleotide may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0152] Alternative codons may be used in regions of the sequence that encode the same or similar amino acid sequences to avoid homologous recombination when both CARs are encoded by the same vector.

[0153] Due to the degeneracy of the genetic code, it is possible to use alternative codons that encode the same amino acid sequence. For example, the codons “ccg” and “cca” both encode the amino acid proline, and thus “ccg” can be exchanged with “cca” without affecting the amino acid at this position in the translated protein sequence.

[0154] Alternative RNA codons that can be used to encode each amino acid are summarized in Table 6. Table 6 [Table 6]

[0155] Alternative codons may be used, in particular, in the portions of the nucleic acids encoding the spacer of the first CAR and the spacer of the second CAR when the same or similar spacers are used in the first and second CARs. Figure 4 shows two sequences encoding the spacer HCH2CH3 - hinge, and in one of them, alternative codons are used.

[0156] Alternative codons may be used, in particular, in the portions of the nucleic acids encoding the transmembrane domain of the first CAR and the transmembrane domain of the second CAR when the same or similar transmembrane domains are used in the first and second CARs.

[0157] Alternative codons may be used in one or more nucleic acids encoding a co - stimulatory domain such as the CD28 endodomain.

[0158] Alternative codons may be used in one or more domains that transmit survival signals such as the OX40 and 41BB endodomains.

[0159] Alternative codons may be used in the portion of the nucleic acid encoding the CD3 zeta endodomain and / or in the portion of the nucleic acid encoding one or more co - stimulatory domains and / or in the portion of the nucleic acid encoding one or more domains that transmit survival signals.

[0160] Vector

[0161] The present disclosure also provides a vector or a kit of vectors comprising one or more CAR - encoding nucleic acids. Such vectors may be used to introduce the nucleic acids into host cells, and as a result, the host cells express the first and second CARs.

[0162] The vector may be, for example, a plasmid, or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.

[0163] The vector may be capable of transfecting or transducing T cells.

[0164] Cell

[0165] Cells co-expressing a first CAR and a second CAR are provided herein, where one CAR binds to CD19 and the other CAR binds to CD22, such that the cells recognize target cells expressing any of these markers. Also provided are populations of cells comprising cells co-expressing a CD19 CAR and a CD22 CAR, as well as cells expressing a CD19 CAR and cells expressing a CD22 CAR. Dual transduction has several advantages. 1. The relative effect on persistence can be studied. The persistence of CAT19 CAR T cells is well documented. The reported persistence of CD22 CAR T cells is typically short-lived. This may be due to the short linker used, for example, in the M971 CAR, an inherent property of the CD22 CAR currently under clinical evaluation. Alternatively, this may also be due to a decrease in target density of CD22 or reduced signaling due to other factors. Studying the long-term engraftment of single / double positive populations can help elucidate this. For example, the long-term engraftment of single positive \CD19 CAR T cells only may suggest an inherent effect of the CAR, and the long-term engraftment of CD19 CAR T cells (both single and double) only may suggest that higher antigen targeting is required for persistence. 2. The effect of different expression or stoichiometry can be studied. If different relative expressions of CD19 vs CD22 CAR are required for optimal persistence, the optimal ratio of expression or co-expression can be elucidated by measuring CAR expression in long-term engrafted cells. 3. The immune response to the introduced gene product may be reduced. When two potentially immunogenic binders are encoded in the same expression cassette, the probability of induction and immune response is doubled. In double transduction, the probability that at least one population persists is increased. This is observed in patient 3 in whom CD22 CAR-expressing cells are lost (Figure 6C).

[0166] In some embodiments, a population of cells comprising cells expressing a CD19 CAR and cells expressing a CD22 CAR is also provided.

[0167] The cells may be any eukaryotic cell capable of expressing a CAR on its cell surface, such as an immunological cell.

[0168] In particular, the cells may be immune effector cells such as T cells or natural killer (NK) cells.

[0169] 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 their cell surface. As summarized below, there are various types of T cells.

[0170] Helper T cells (TH cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when a peptide antigen is presented to them by MHC class II molecules on the surface of an antigen-presenting cell (APC). These cells can differentiate into one of several subtypes including TH1, TH2, TH3, TH17, Th9 or TFH, which secrete different cytokines to promote different types of immune responses.

[0171] Cytotoxic T cells (TC cells, or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection reactions. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens 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, CD8+ cells can be inactivated into an anergic state, which prevents autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0172] Memory T cells are a subset of antigen-specific T cells that persist for a long time after an infection has been resolved. Upon re-exposure to their cognate antigen, they rapidly expand and proliferate into a large number of effector T cells, thus providing the immune system with a "memory" of past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0173] Regulatory T cells (Treg cells), previously known as suppressor T cells, are important for maintaining immune tolerance. Their main roles are to shut down T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus.

[0174] Two major classes of CD4+ Treg cells - naturally occurring Treg cells and adaptive Treg cells - have been described.

[0175] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are associated with the interaction during T cell development by both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells activated by TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells and cause IPEX, a fatal autoimmune disease.

[0176] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0177] The T cells provided herein can be any of the T cell types listed above, particularly CTLs.

[0178] Natural killer (NK) cells are a type of cytotoxic cell that forms part of the innate immune system. NK cells provide a rapid response to natural signals from virus-infected cells in an MHC-independent manner.

[0179] NK cells (which belong to the group of natural lymphocytes) are defined as large granular lymphocytes (LGL) and constitute a third type of cell differentiated from common lymphoid progenitor cells that give rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus and then enter the circulation.

[0180] The CAR-expressing cells provided herein can be any of the cell types listed above.

[0181] CAR-expressing cells, e.g., CAR-expressing T or NK cells, can be generated ex vivo from a patient's own peripheral blood (first degree relative), or in the context of a hematopoietic stem cell transplant from a donor's peripheral blood (second degree relative), or from a peripheral blood of an unrelated donor (third degree relative).

[0182] The present disclosure also provides a cell composition comprising CAR-expressing T cells and / or CAR-expressing NK cells, which cells co-express a CAR that binds to CD19 and another CAR that binds to CD22, such that the cells are capable of recognizing target cells that express any of these markers. In some embodiments, the cell composition comprises cells that express only a CAR that binds to CD19, and cells that express only another CAR that binds to CD22. The cell composition may be made ex vivo, according to the present disclosure, by transducing a blood sample with nucleic acid.

[0183] The term "CD19 / 22 CAR T cells" as used herein refers to a cell composition comprising untransduced cells, cells that express only a CD19 CAR, cells that express only a CD22 CAR, and a mixture of cells that express both CD19 and CD22 CARs. In some embodiments, the cell composition comprises a mixture of untransduced cells, cells that express only a CD19 CAR, and cells that express only a CD22 CAR.

[0184] Alternatively, the T or NK cells provided herein may be derived from the ex vivo differentiation of inducible or embryonic progenitor cells into T or NK cells. Alternatively, an immortalized T cell line that retains its lytic function and can act as a therapeutic agent may be used.

[0185] CAR cells are created by introducing DNA or RNA encoding the CAR by one of many means including, but not limited to, transduction with a viral vector, transfection with DNA or RNA. The cells may be activated and / or expanded, for example, by treatment with an anti-CD3 monoclonal antibody, prior to transduction with the CAR-encoding nucleic acid.

[0186] The T or NK cells provided herein may be produced by (i) isolation of T or NK cell-containing samples from a subject or other origins listed above, and (ii) transduction or transfection of T or NK cells with one or more nucleic acids encoding CD19 and CD22 CARs.

[0187] The T or NK cells may then be purified and selected, for example, based on the expression of the antigen-binding domain of the antigen-binding polypeptide.

[0188] Pharmaceutical composition

[0189] The present disclosure also relates to pharmaceutical compositions containing a plurality of CAR-expressing cells such as the T cells or NK cells provided herein. A pharmaceutical composition containing the CD19 / 22 CAR T cell product described in Example 1 is provided. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active polypeptides and / or compounds. Such formulations may be in a form suitable for, for example, intravenous infusion.

[0190] Methods of treatment

[0191] The present disclosure relates to a method for treating high-risk / relapsed CD19+ or CD22+ hematological malignancies in a patient, the method comprising administering to the patient autologous CD19 / 22 CAR T cells (e.g., autologous CD19 / 22 CAR T cell products comprising the CARs of CAT19CAR and 9A8CAR described in Example 1 herein). The present disclosure also relates to autologous CD19 / 22 CAR T cells (e.g., autologous CD19 / 22 CAR T cell products comprising the CARs of CAT19CAR and 9A8CAR described in Example 1 herein), or pharmaceutical compositions containing such cells, for use in the treatment of high-risk / relapsed CD19+ or CD22+ hematological malignancies. The present disclosure also relates to the use of autologous CD19 / 22 CAR T cells (e.g., autologous CD19 / 22 CAR T cell products comprising the CARs of CAT19CAR and 9A8CAR described in Example 1 herein), or pharmaceutical compositions containing such cells, in the manufacture of a medicament for the treatment of high-risk / relapsed CD19+ or CD22+ hematological malignancies.

[0192] The cell compositions of the present disclosure, e.g., the CD19 / 22 CAR T cell product compositions described in Example 1, can kill cancer cells recognizable by the expression of CD19 or CD22, e.g., B cell lymphoma cells. CAR-expressing cells, e.g., T cells, can be generated ex vivo from the patient's own peripheral blood (first-degree relative), or in the context of a hematopoietic stem cell transplant from a donor's peripheral blood (second-degree relative), or from a peripheral blood of an unrelated donor (third-degree relative). Alternatively, the CAR T cells may be derived from the ex vivo differentiation of inducible or embryonic progenitor cells into T cells. In these cases, the CAR T cells are generated by introducing DNA or RNA encoding the CAR by one of many means including transduction with a viral vector, transfection with DNA or RNA.

[0193] Examples of cancers expressing CD19 or CD22 include B cell lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma; and B cell leukemia.

[0194] For example, B-cell lymphoma may be diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma (MZL) or mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (overlapping with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal primary (thymic) B-cell large cell lymphoma, lymphoplasmacytic lymphoma (which may present as Waldenström macroglobulinemia), nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large cell type B-cell lymphoma, primary humoral lymphoma, lymphomatoid granulomatosis, T cell / histiocyte-rich large cell type B-cell lymphoma or primary central nervous system lymphoma.

[0195] B-cell leukemia may be acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, precursor B-lymphoblastic leukemia or hairy cell leukemia.

[0196] B-cell leukemia may be acute lymphoblastic leukemia (B-ALL or ALL).

[0197] B-ALL may be pediatric ALL (pALL).

[0198] pALL may express CD19 or CD22. pALL may express CD19 and CD22.

[0199] The standard treatment for relapsed pALL includes the following treatment phases: induction, consolidation, interim maintenance, late intensification, and maintenance.

[0200] Patients are stratified according to risk level. Several criteria, such as the NCI criteria that distinguish patients between National Cancer Institute (NCI) standard risk and NCI high risk, are available for stratifying patients: NCI standard risk patients are those who are ≧1 year and <10 years of age at diagnosis and have a maximum white cell count (WCC) of <50×10^9 / L before treatment is initiated; NCI high risk patients are those who are ≧10 years of age at diagnosis and / or have a diagnostic WCC of ≧50×10^9 / L.

[0201] There are national guidelines that describe standard treatment regimens. An example of these guidelines is the UKALL 2019 provisional guidelines, which are guidelines for the management of ALL in children and young adults used in the UK (also referred to as the UKALL 2019 guidelines). The induction therapy according to the UKALL 2019 guidelines is as follows. - NCI standard risk: Patients in this group receive induction with three drugs (dexamethasone, vincristine, and asparaginase) (Induction Regimen A, Table 7). - NCI high risk: Patients in this group receive induction with four drugs (dexamethasone, vincristine, asparaginase, and daunorubicin) (Induction Regimen B, Table 8). - Induction Regimen C (Table 9): Patients with NCI standard risk BCP ALL who are subsequently found to have high risk cytogenetics, or patients with Down syndrome who have a slow initial response. Table 7: Induction Regimen A

Table 7-1

Table 7-2

Table 8-1

Table 8-2

Table 9-1

Table 9-2

[0202] Details of the regimens for land consolidation, provisional maintenance, late intensification, and maintenance phases can be found in the UKALL 2019 guidelines (docplayer.net / 170103468-Clcn-ukall-2019-interim-guidelines.html, which is incorporated herein by reference).

[0203] The treatment with T cells provided herein is intended to help prevent tumor cell escape or shedding that often occurs with standard treatment approaches.

[0204] The methods, autologous CD19 / 22 CAR T cells, or uses provided herein may slow or prevent cancer progression, reduce the extent of cancer, result in cancer remission (partial or complete), and / or extend the survival of the patient.

[0205] In the methods, autologous CD19 / 22 CAR T cells, or uses provided, the patient to be treated has high-risk / relapsed or refractory CD19+ or CD22+ hematological malignancies.

[0206] When the CD19+ or CD22+ hematological malignancy is pALL, there are several parameters that can be used to define high-risk / relapsed or refractory pALL: a) Resistant disease, b) ALL with high-level persistent minimal residual disease (MRD) at the second time point of frontline national protocols, c) High-risk infant ALL, d) Intermediate-risk infant ALL, e) High-risk first relapse, f) Standard-risk relapse in patients with high-risk cytogenetics, g) Minimal residual disease (MRD) of bone marrow > 10 at the end of reintroduction -3 for standard-risk relapse, h) Any refractory relapse of ALL, or i) Any relapse of CD22+ lymphoma.

[0207] Resistant disease is defined as the presence of > 5% blast cells at the end of the induction phase by the introduction of UKALL 2019 guidelines or equivalents. This defines the primary refractory population.

[0208] ALL with persistent high-level minimal residual disease (MRD) at the second time point of frontline national protocols is defined as MRD > 10 at week 14 according to UKALL 2019 guidelines or equivalents. -4 as defined.

[0209] High-risk infant ALL is defined as infants < 6 months of age at diagnosis due to MLL gene rearrangement, and white blood cell count > 300 × 10 9 / L or showing either insufficient steroid initial response. Insufficient steroid initial response is defined as the presence of > 1 × 10 circulating blast cells after 7 days of steroid pre-phase of induction according to national guidelines (e.g., UKALL 2019 provisional guidelines or equivalents). 9 / L.

[0210] Intermediate-risk infants with ALL are defined as infants showing MRD > 10-3 at the end of induction according to national guidelines (e.g., UKALL 2019 provisional guidelines or equivalents).

[0211] High-risk first relapse is defined as bone marrow relapse or isolated / mixed extramedullary relapse within 30 months of diagnosis according to the latest version of the INTREALL 2010 classification [International Study for Treatment of High Risk Childhood Relapsed ALL (IntReALL) HR 2010 study; NCT03590171].

[0212] High-risk cytogenetics is defined as cytogenetic abnormalities that correlate with poor outcome and include the following: - Philadelphia chromosome or Philadelphia translocation (Ph), also known as BCR-ABL1 or t(9;22)(q34;q11), - BCR-ABL1-like or Philadelphia-like (Ph-like), e.g., ABL-class fusions, which include fusions of a variety of "activating genes" (e.g., ETV6, PAX5, EBF1, NUP214, ZMIZ1, FLIPL1, etc.) with kinase genes (e.g., ABL1, ABL2, CSF1R, PDGFRA or PDGFRB), - MLL (KMT2A) rearrangement, - Near-haploid (<30 chromosomes) and low hypodiploidy (30 - 39 chromosomes), - Intrachromosomal amplification of chromosome 21 (iAMP21), and - TCF3-HLF translocation, i.e., t(17;19)(q22;p13) / TCF3(E2A)-HLF), which is a variant of t(1;19)(E2A-PBX), t(17;19)(E2A-HLF).

[0213] Standard-risk relapse is defined as having minimal residual disease (MRD) > 10 -3 in the bone marrow at the end of reinduction.

[0214] Any refractory relapse of ALL is defined as > 1% blasts by flow cytometry after at least 1 cycle of standard chemotherapy.

[0215] The patient may be ineligible for treatment with other CD19 CAR T cell products, such as Kymriah™ (tisagenlecleucel).

[0216] The patient may have one or more treatment histories. The patient may have two or more, three or more, four or more, five or more, or six or more treatment histories.

[0217] The patient may have received prior treatment with an anti-CD19 immunotherapy agent. Examples of anti-CD19 immunotherapy agents include, but are not limited to, inotuzumab ozogamicin (Besponsa®), blinatumomab (Blincyto®), and tisagenlecleucel (Kymriah™). The patient may have been previously administered one or more of inotuzumab ozogamicin (Besponsa®), blinatumomab (Blincyto®), and tisagenlecleucel (Kymriah™). The patient may have been previously administered one or more of inotuzumab ozogamicin (Besponsa®), blinatumomab (Blincyto®), and tisagenlecleucel (Kymriah™).

[0218] The patient may exhibit an extramedullary lesion.

[0219] The patient may have received an allogeneic stem cell transplant.

[0220] The patient may be administered a single dose of 1×10 6 CAR T cells / kg, such as the CD19 / 22 CAR T cell product described in Example 1. The patient may be administered a single dose of 0.5×10 6 CAR T cells / kg, such as the CD19 / 22 CAR T cell product described in Example 1. The patient may be administered a single dose of 0.75×10 6A single dose of CAR T cells / kg may be administered. The patient may be administered a single dose of 1.2×10 6 CAR T cells / kg. The administration may be intravenous injection, for example, intravenous injection through a Hickman line or a peripherally inserted central catheter (PICC line).

[0221] The patient may show progression-free survival for at least 6 months after the administration, or at least 12 months after the administration.

[0222] Other technical terms and disclosures

[0223] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any element, e.g., any optional element. Therefore, this description is intended to serve as a preamble for the use of such exclusive terms as "solely", "only", etc., or the use of "negative" limitations in connection with the recitation of elements of the claims.

[0224] When ranges of values are provided herein, each intervening value, between the upper and lower limits of that range and any other stated value or intervening value in that stated range, is included within the disclosure to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and also be included within the disclosure, subject to any specifically excluded limitation in the stated range. Ranges that include any one or both of the recited limitations, excluding either or both of those included limitations, are also included in the disclosure.

[0225] Unless otherwise defined, 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 to or equivalent to those described herein can be used in the practice or testing of this disclosure.

[0226] All publications cited herein are hereby incorporated by reference herein for the purpose for which they are cited, to disclose and describe the methods and / or materials.

[0227] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein can be readily separated from or combined with any of the features of some other embodiments without departing from the scope or spirit of the disclosure. Any of the recited methods can be performed in the order of recited events or any other order that is logically possible. The disclosure is intended to provide support for all such combinations.

[0228] As used herein, "may", "may comprise", "may be", "can", "can comprise", and "can be" all function as part of the subject matter provided, indicating what is contemplated by the inventors as being available and functional.

Examples

[0229] The following examples describe specific embodiments, but variations and modifications will occur to those skilled in the art. Accordingly, only the limitations as appear in the claims should be recognized in this invention. (Example 1) Preparation of CAR-T cell compositions transfected with multiple vectors a) A second-generation CD19 CAR (SEQ ID NO: 89) (the CD19 CAT CAR described in WO2016 / 139487, otherwise referred to herein as CAT CAR or AUTO1) containing an anti-CD19 antigen-binding domain, a CD8 stalk spacer, a transmembrane domain, and a compound 4-1BB-CD3 endodomain, under the control of the PGK promoter (pCCL.PGK.aCD19cat-CD8STK-41BBZ); or b) A CD22 CAR (SEQ ID NO: 91) (a CAR based on 9A8-1 described in WO2019 / 220109) (9A8 CAR) containing an anti-CD22 antigen-binding domain, a CD8 stalk spacer, and a second-generation endodomain containing CD3 and 4-1BB co-stimulatory domains, under the control of the EF1a promoter (pCCL.EF1a.aCD22_9A8-1-64_LH_scFv-CD8STK-41BBz). A lentiviral vector expressing either was generated. See FIGS. 1A - B.

[0230] Two separate lentiviral supernatants were generated and mixed 1:1 at an MOI of 2.5 + 2.5.

[0231] Normal donor T cells were transduced with either the CAT CAR lentiviral vector, the 9A8 CAR lentiviral vector, or double transduced with both. T cells were stained with anti-CAT idiotypic (to detect CAT CAR) and recombinant soluble CD22 (to detect 9A8 CAR). Single positive and double positive populations were observed with each vector at an MOI of 2.5 / 2.5.

[0232] After transduction with the lentiviral composition, the cells are a mixture of untransduced cells (46.5%); cells expressing only CD19 CAR (23.1%); cells expressing only CD22 CAR (11.1%), and cells expressing both CD19 and CD22 CAR (19.3%). The resulting mixed population is referred to herein as the "CD19 / 22 CAR T cell product", the "CD19CAT-CD22 9A8-41BBZ CAR T cell product" or the "AUTO1 / 22 product".

[0233] These experiments demonstrated that it was possible to create a mixed population of T cells containing T cells that are single and double positive for each CAR, and that CAR expression was not affected by double transduction. (Example 2) Preclinical evaluation

[0234] The in vitro functional performance of the CD19 / 22 CAR T cell product of Example 1 was determined. T cells were challenged with SupT1 cells (negative for CD19 and CD22), and SupT1 cells engineered to express either or both of CD19 and CD22 [high or low (<1000 copies of CD22) surface antigen density]. Target cell killing, as well as the release of IFN-gamma and IL-2 cytokines, were measured. The CD19 / 22 CAR T cell product maintained cytolytic capacity against all tested targets compared to single CAR-expressing T cells (Figures 2A-E). Both CD19 CAR T cells and CD19 / 22 CAR T cells were able to efficiently kill and secrete cytokines in response to targets expressing low levels of CD22.

[0235] Raji cells are a B cell line derived from Burkitt lymphoma and originally express both CD19 and CD22. To determine the performance of CD19 / 22 CAR T cells against target cells with native expression of CD19 / CD22, a functional assay was performed using normal donor T cells. To simulate the scenario of CD19 negative escape, the functional assay was further performed using Raji cells with CD19 gene disruption. The CD19 / 22 CAR T cell product maintained cytolytic function in the CD19 knockout Raji cell line and CD19 negative primary human B-ALL target cells (Figures 3A - D).

[0236] Next, the in vivo function of CD19 CAR T cells (AUTO1) and CD19 / 22 co-transduced CAR T cells (AUTO1 / 22) was determined. NALM6 cells (a cell line derived from B-ALL, which expresses both CD19 and CD22) were engineered to express HA-tagged firefly luciferase. To simulate the scenario of CD19 negative escape, the HA-FLuc NALM6 cell line was further engineered by genome editing to disrupt CD19 expression (NALM6 CD19ko). NALM6 cells were first engrafted into NSG mice by tail vein injection, and their engraftment was determined by bioluminescence imaging (BLI). Thereafter, an equal number of either non-transduced, AUTO1 cells or AUTO1 / 22 CAR T cells were administered by tail vein injection. NALM6 burden was measured sequentially using BLI. On day 14 after CAR T cell administration, the mice were sacrificed and necropsied. NALM6 burden and CAR T cell engraftment were determined by flow cytometry.

[0237] At a sub-optimal dose for in vivo tumor clearance, the CD19 / 22 CAR T cell product was significantly better than CAT19CAR in controlling the growth of double positive (Nalm-6 WT) tumors. Only the CD19 / 22 CAR T cell product was able to control the growth of CD19 negative (Nalm-6 CD19KO) tumors. See Figures 4A - E.

[0238] Finally, the percentages of engrafted AUTO1, CD22CAR, and AUTO1 / 22 CAR T cells in mice at the time of sacrifice were determined by flow cytometry of bone marrow aspirates. As shown in Figure 4F, AUTO1 / 22 CAR T cells maintained their relative percentages after challenge with NALM6 cells. When CAR T cells were challenged with NALM6 CD19 ko cells, CD22CAR+ and AUTO1 / 22 (CD19CAR / CD22CAR) T cells expanded, while AUTO1 (CD19CAR) single-positive T cells did not expand (Figure 4F). When challenged in the Nalm-6 CD19KO model, there was selective expansion and engraftment of CAR T cells expressing anti-CD22 9A8 CAR in the recipient's bone marrow, accompanied by the loss of single anti-CD19 CAR T cells. (Example 3) Phenotype of clinical products

[0239] Full-scale manufacturing also generated a therapeutic CD19 / 22 CAR T cell product having a mixture of single and double CAR-positive T cells (Figures 5A, 5B). Briefly, PBMCs were obtained from fresh or thawed apheresis of unstimulated leukocytes (when the absolute lymphocyte count > 0.5 × 10 9 / L, 2 volumes of apheresis were performed according to the practice of local facilities, and for patients with an absolute lymphocyte count < 0.5 × 10 9 / L, 2.5 volumes of apheresis were performed). PBMCs were activated with CD3 / CD28 in X-VIVO 15 medium. Absolute lymphocyte count < 0.3 × 10 9For patients with / L, interleukin-2 (IL-2) was supplemented to the culture medium from this point. Then, transduction was performed by exposing activated PBMCs to a lentiviral vector at a defined multiplicity of infection (MOI) in a RetroNectin-coated differentiation bag. The MOI is 3.5 for the CD19CAR vector and 1.5 - 2.5 for the CD22CAR vector. These MOIs were selected to give both high-level transduction efficiency and a balanced population of CD19- and CD22CAR single-positive populations. On day 4, the lentiviral vector was removed by centrifugation, and the cells were transferred to a WAVE bioreactor cell culture bag in fresh X-VIVO 15 medium + / - IL-2 (depending on the starting absolute lymphocyte count). Then, the cells were expanded in the WAVE bioreactor for up to an additional 3 days. After that, the CAR-transduced T cells were cryopreserved in injectable cryomedium (CryoStor® CS10, a cryopreservation medium containing 10% USP-grade DMSO). The aliquots taken at this time were then subjected to quality control assays to ensure a transduced T cell product that meets the release criteria further listed below.

[0240] The release criteria for CAR T cells are as follows: a) Bacterial / fungal sterility b) Absence of mycoplasma (PCR) c) Absence of endotoxin (<2 EU / ml in the Limulus Amoebocyte Lysate assay) d) >70% viability (flow cytometry) e) Transduction efficiency: ≥10% CD19CAT-CD22 9A8 CAR+ cells, ≥2.5% CD19CAT CAR+ cells, ≥2.5% CD22 9A8 CAR+ cells f) Cell dose at cryopreservation of 1.2×10 6 CAR+ T cells / kg (manual count) (= 10 6 CAR+ T cells / kg considering 20% cell loss during thawing) * * Advanced Therapy Medicinal Products (ATMPs) that meet the release criteria but where it is not possible to produce this target dose and it is not feasible to produce another dose may be administered at a reduced dose (minimally 0.6 × 10 6 CAR+T cells / kg taking into account a 20% cell loss during thawing of 5 × 10 5 CAR+T cells / kg).

[0241] In addition, the following assays may be performed but they do not constitute release criteria: flow cytometry to determine the immunophenotype and percentage of non-T cell immune subsets in ATMPs, viral copy number assessment by qPCR.

[0242] The results shown in Figures 5A and 5B revealed that the dual-transduced population was dominant and that, in general, the CD19 and CD22 CAR single CAR populations were balanced in ATMPs. Table 20 shows the CAR-T cell dose, transduction (Td) efficiency (expressed as the percentage of T cells expressing single CD19, single CD22 or CD19 / 22 dual CAR), composition between single CD19, single CD22 and dual CD19 / 22 transduced products across the cohort of pts, and vector copy number. Median, range and interquartile range (IQR) were reported. Table 20

Table 20

[0243] There was no difference in the proportion of naive T cells, central memory or effector memory T cells and terminally differentiated T cells in the different expressed CAR populations of the product. All three CAR T cell populations showed preservation of the initial memory T cell phenotype. See Tables 10 and 11 below. Table 10

Table 10

Table 11

[0244] The characteristics of ATIMP from 11 patients (n = 11) are as follows. - Median transduced cell dose achieved: 664×10 6 (range 66 - 1597) - Median transduction efficiency was 83% (range 60.8 - 92.6): CD19 / 22 DP > CD19SP = CD22SP in the product - Median vector copy number (VCN) was 5.5 (range 3.39 - 8.00) - The percentages of naive T cells, central or effector memory T cells, and terminally differentiated T cells were T CM 87.7%, T N / SCM 0.56%, T EM 11.61%, T EMRA 0.17%. (Example 4) Method

[0245] Clinical test evaluation

[0246] Serum cytokine measurements were evaluated by ISO - certified methods using a cytometric bead array assay (BD Biosciences) for IL - 2, IL - 4, IL - 6, IL - 10, TNF, IFNγ at 0, 2, 5, 7, 9, 12, 14 days after CAR T - cell infusion. The validated lower limit of this assay was 50 pg / ml and the upper limit was 5000 pg / ml.

[0247] The expansion / proliferation / survival of CAR T cells was evaluated in peripheral blood (PB) at 0, 2, 7, 14, 28 days post-injection, monthly up to 6 months, every 6 weeks up to 1 year, and then every 3 months up to 2 years. Bone marrow (BM) was evaluated monthly for the first 6 months and then at the same intervals as for blood. CAR T cells were detected using a validated qPCR assay that detects the transgene-specific sequence. Mononuclear cells were isolated from peripheral blood and bone marrow and DNA was extracted. Primers and probes specific for the transgene for the CD19CAR and CD22CAR coding sequences (Integrated DNA Technologies) were incorporated with a parallel control gene (albumin) and utilized in the qPCR reaction plate. Known standards for the target were run for both the target and control genes. This provided quantitative data for the target and control genes that could be used to quantify CAR copies per μg / gDNA at a detection limit of 100 copies / μg DNA. Circulating CAR T cells in blood and bone marrow were also analyzed by flow cytometry using anti-CAT and anti-9A8 CAR anti-idiotype antibodies. Absolute T cell counts were obtained using the Trucount method (BD Biosciences) and staining for viable CD45+CD3+ cells. The reagents used were 7-AAD, Fc gamma block, CD45 FITC, CD3 APC-Cy). The percentage of CAR+ T cells was evaluated using co-staining to enable detection of viable CAR+CD45+CD3+ cells, using anti-CAT CAR anti-idiotype and secondary anti-rat IgG PE antibodies and anti-9A8 CAR anti-idiotype and secondary anti-rabbit IgG BV421. From this, the absolute CAR T cell count was established. Normal donor PBMCs were used as negative controls. The threshold for detection was 0.1% CAR T cells.

[0248] Analysis of cell kinetics

[0249] The analysis of CAR T cell kinetics was performed starting from the CAR transgene. The area under the curve analysis (AUC 0-28) of CAR T cell levels up to 28 days was estimated by the trapezoidal algorithm and represented the initial CAR T cell expansion. Cmax was the peak concentration of the confirmed CAR T cells, Tmax was the time (days) from injection to the maximum CAR T cell concentration, and Tlast was the time from injection to the detection of the last confirmed CAR T cells. T1 / 2 was the half-life of CAR T cell persistence over the systolic period measured in patients at the lowest three data points confirmed after Tmax. Clinical study

[0250] A study on the safety, efficacy, and duration of response of the CD19 / 22 CAR T cell product was initiated in pediatric and young adult patients with high-risk relapsed CD19+ and / or CD22+ hematological malignancies (acute lymphoblastic leukemia and Burkitt lymphoma). This is cohort 3 of a multi-center, non-randomized, non-blinded phase I clinical trial.

[0251] The study design for cohort 3 is summarized in Table 12 below. Table 12

Table 12

[0252] The primary evaluation items for the study were as follows. a) Toxicity evaluation after CD19 / 22 CAR T cell injection The occurrence of grade 3-5 toxicity within 60 days after CD19 / 22 CAR T cell injection. In particular, the occurrence of severe cytokine release syndrome and grade 3-5 neurotoxicity within 30 days after CD19 / 22 CAR T cell injection. [Timeframe: 1 month] b) Molecular genetic remission Efficacy was evaluated by determining minimal residual disease in bone marrow aspirates using immunoglobulin heavy chain (IgH) quantitative polymerase chain reaction (qPCR) and / or next-generation sequencing in all patients. The percentage of patients achieving molecular genetic remission 1 month after CD19 / 22 CAR T cell infusion. [Timeframe: 1 month]

[0253] Secondary evaluation items for the study were as follows. a) Long-term molecular genetic remission The number of patients in molecular genetic remission without further therapy at 2 years. [Timeframe: 2 years] b) Frequency of circulating CD19 / 22 CAR T cells Persistence and frequency of circulating CD19 / 22 CAR T cells in peripheral blood by flow cytometry and qPCR analysis. [Timeframe: 2 years] c) Incidence of hypogammaglobulinemia Incidence and duration of hypogammaglobulinemia [Timeframe: 2 years] d) Recurrence rate The recurrence rate was monitored for a total of 10 years after cell infusion during the intervention phase and long-term follow-up. The number of patients who relapsed can be summarized as a percentage or rate (for all patients enrolled in the clinical trial and for patients who received cell infusion only). [Timeframe: 10 years] e) Duration of response. The duration of response was measured from the time of confirmed response to either molecular genetic or morphological relapse or death, whichever came first. Patients who did not experience a disease failure event were censored at their last follow-up date. f) Event-free survival (EFS) at 1 year and 2 years after infusion. Define the event-free survival period as reported in the ELIANA study, where the events of interest included any of the following: no response, morphological relapse before response maintained for at least 28 days, incomplete hematological recovery, or death, with or without morphological relapse after complete remission. Patients were censored if they received further therapy or on the date they were last seen alive. Define the event-free survival period more strictly by including, as events, either failure to achieve remission, morphological or molecular genetic relapse after remission, or death, whichever occurred first. g) Overall survival period Measure OS from the time of CAR T cell infusion until death, and censor patients who did not experience the event of interest on the date they were last seen alive. The overall survival period is monitored for 10 years after CD19 / 22 CAR T cell infusion during the intervention phase and long-term follow-up. The number of patients alive can be summarized as a percentage (for all patients registered in the trial and for patients who received cell infusion only). [Timeframe: 10 years]

[0254] Eligible patients were pediatric and young adult (age ≤ 24 years) with high-risk relapsed CD19+ and / or CD22+ B-lineage ALL who were ineligible for Kymriah in the UK National Access Program.

[0255] The inclusion criteria for the study were as follows. Pediatric and young adult (age 24 years or younger) with high-risk / relapsed CD19+ and / or CD22+ hematological malignancies having the following: a) Resistant disease (> 5% blasts) at the end of induction of UKALL 2019 guidelines or equivalent b) Persistent high-level MRD at the second timepoint of frontline national protocol (currently, MRD > 10 -4 ) at week 14 in UKALL 2019 guidelines or equivalent) in ALL. c) High-risk infant ALL (at diagnosis due to MLL gene rearrangement, age < 6 months, and white blood cell count > 300×10 9 / L or inadequate steroid initial response (i.e., > 1×10 9 / L of circulating blast cells after 7-day steroid pre-phase of induction according to national guidelines or equivalents) d) Intermediate-risk infant ALL with MRD > 10 -3 at the end of induction according to national guidelines or equivalents e) High-risk first relapse (as defined by the latest IntreALL 2019 classification: bone marrow or composite relapse within 30 months of diagnosis, or any relapse within 18 months of diagnosis) f) Standard-risk relapse in patients with high-risk cytogenetics (defined as BCR-ABL, KMT2A rearrangement, near-haploid (< 30 chromosomes) and low hypodiploidy (30 - 39 chromosomes), iAMP21, and TCF3-HLF translocation). g) Bone marrow minimal residual disease (MRD) > 10 -3 at the end of re-induction for standard-risk relapse h) Relapse during any therapy in patients aged 16 - 24 years i) Any relapse of infant ALL j) ALL after ≥ 2nd relapse k) Any refractory relapse of ALL (defined as > 1% blasts by flow cytometry after at least 1 cycle of standard chemotherapy) l) ALL with MRD > 10 -4 before planned stem cell transplantation m) Any relapse of ALL that is eligible for stem cell transplantation but for which an HLA-matched donor is not available or has other contraindications to transplantation n) Any relapse of ALL after stem cell transplantation o) Any relapse of Burkitt lymphoma or other CD19+ and / or CD22+ lymphomas Note that patients with isolated CNS relapse fulfilling one or more of the above criteria were eligible for the study.

[0256] The exclusion criteria for registration were as follows. a) Active hepatitis B, C or HIV infection b) Oxygen saturation in air ≤ 90% c) Bilirubin > 3 × upper limit of normal d) Creatinine > 3 × upper limit of normal e) Women who are pregnant or breastfeeding f) For stem cell transplant patients only: Active severe (overall grade ≥ II, Seattle criteria) acute GVHD or moderate / severe chronic GVHD (NIH consensus criteria) requiring systemic steroids g) Unable to tolerate leukapheresis h) Karnofsky (age ≥ 10 years) or Lansky (age < 10) score ≤ 50% i) Existing severe neuropathy (other than CNS involvement of underlying hematological malignancies)

[0257] The exclusion criteria for CD19 / 22 CAR T cell infusion were as follows. a) Severe concurrent infections at the time of scheduled CD19 / 22 CAR T cell infusion b) Requirement for oxygen supplementation or active pulmonary infiltrates at the time of scheduled CD19 / 22 CAR T cell infusion c) Allogeneic transplant recipients with overall grade ≥ II active severe acute GVHD or moderate / severe chronic GVHD requiring systemic steroids at the time of scheduled CD19 / 22 CAR T cell infusion. Note: Such patients are excluded until the patient is GVHD-free and steroid-free.

[0258] The study design was a multi-center, non-randomized, open-label, phase I clinical trial of an advanced therapy medicinal product (ATIMP) in children and young adults with high-risk relapsed CD19+ and / or CD22+ hematological malignancies (mainly ALL and Burkitt lymphoma). The ATIMP tested in cohorts 1 and 2 of this study was CD19CAT-41BBζ CAR T cells (referred to as CD19CAR T cells). The ATIMP tested in cohort 3 of this study was CD19CAT-CD22 9A8-41BBZ CAR T cells (as described in the above examples). A total of 33 patients were treated at three participating sites. The expected recruitment was over 5.5 years.

[0259] Thirteen patients were screened and enrolled, and one withdrew due to progressive viral infection that precluded lymphodepletion. Twelve patients were treated. Patients received apheresis of unstimulated leukocytes, which was sent to the Gene and Cell Therapy facility, Great Ormond Street Hospital (GCT-GOSH, London, UK) for the manufacture of CD19 / 22CAR T cells. ATIMP manufacture took approximately 15 days. During this period, patients received "hold" chemotherapy to maintain disease control.

[0260] Prior to CD19 / 22CAR T cell infusion, patients received lymphodepleting chemotherapy: fludarabine 30 mg / m2 i.v. on days -7 to -3 and cyclophosphamide 0.5 g / m2 i.v. on days -4 to -2. A single dose of 10 6 / kg of cryopreserved CD19 / 22CAR T cells was administered intravenously. Patients were followed regularly (daily, weekly, and monthly visits) until 2 years after CD19 / 22CAR T cell infusion. After 2 years, patients continued to be followed once a year until 10 years after ATIMP infusion.

[0261] Results

[0262] The characteristics of the patients in cohort 3 are summarized in Tables 13, 14, and 21 below. The median age was 12 years (range 3.7 - 20.5 years). This was a highly pre-treated cohort, with a median of three lines of therapy (range 2 - 6). Half (6 / 12) had relapsed after allogeneic stem cell transplantation (SCT). Six patients had previously received blinatumomab, two of whom had also received inotuzumab. Four patients had relapsed after tisagenlecleucel therapy. Three had detectable CD19-negative lesions at registration. The leukemia was completely CD19-negative, and in addition, in one case, had a 5% CD22-negative population. Otherwise, there were significant proportions (>5%) of lesions that did not express CD19. Six patients had isolated extramedullary (EM) relapse (two of whom had non-CNS EM lesions). For the reasons of the above characteristics, all patients were ineligible for tisagenlecleucel therapy at the time of registration. The pre-lymphodepletion bone marrow lesion burden was >5% blasts in 4 / 12 patients, <5% blasts (or positive for measurable residual disease - MRD) in 5 / 12 patients, and MRD-negative in 3 / 12 patients.

[0263] Figure 13 shows the consort diagram for cohort 3. Table 13

Table 13

Table 14-1

Table 14-2

Table 21

[0264] All patients except one received a target dose of 1×10 6 / kg total CAR T cells at a median total CAR transduction efficiency of 83.2% (range 60.8 - 92.6%). One patient received a total of 0.9×10 6 / kg CART cells per ideal body weight.

[0265] The details of toxicity for each patient are shown in Table 15 below. Table 15

Table 15

[0266] The toxicity in n = 12 patients is summarized as follows (Table 16): - 11 / 12 patients showed CRS: Grade 1 (G1) n = 5, G2 n = 6, with a median of 5 days (range 1 - 19) and persisted. In most cases, CRS occurred early after CAR T cell infusion (median 9 days, range 4 - 85 days), however, in one case, delayed CRS occurred after infusion of additional selected donor stem cells of CD34 + and was presumed to be related to CAR T expansion after infusion of CD19 + B cells / progenitor cells. No patient developed severe (≥ Grade 3) CRS and no patient was admitted to the pediatric intensive care unit (PICU) due to CRS. Tocilizumab was given to 5 patients. - 6 / 12 patients developed immune effector cell-associated neurotoxicity syndrome (ICANS) (G1 n = 4, G2 n = 1, G4 n = 1). Grade 1 - 2 ICANS was noticed at a median time of 10 days (range 2 - 13) from CAR-T cell infusion and all resolved spontaneously; - One patient had G4 ICANS which was associated with leukoencephalopathy indistinguishable radiologically, clinically and pathologically from the neurotoxicity of fludarabine; - Cytopenia, as for cohort 1 (treated with CD19 CAT T cells), was severe, with 10 / 12 having long-term grade 3-4 cytopenia >D28, 8 / 10 resolving by the last follow-up (FU), and 1 patient requiring a subsequent CD34+ selected donor stem cell boost 2.5 months after infusion. Nevertheless, only 4 episodes of grade 4 infections were present and no grade 5 toxicities were recorded; - The grade 3-4 infection rate was as expected for a highly pre-treated cohort. - Importantly, there was no grade 5 toxicity (death) and no evidence of haemophagocytic lymphohistiocytosis as described in other CD22 CAR studies [Lichtenstein, et al. (2021) Blood 138:2469-84]; - Other grade 3-4 toxicities are listed in Table 16. Table 16

Table 16-1

Table 16-2

[0267] CAR T cell kinetics in peripheral blood and bone marrow were measured by flow cytometry using anti-idiotype antibodies to detect CAR T cell populations with either or both CARs, and by qPCR for CD19CAR and CD22CAR. By flow cytometry, the inventors noted a rapid expansion of all CAR T cell populations at the peak 14 days after infusion. The median time to loss of both singly transduced CD19 and doubly transduced CD19 / 22 CART populations by flow cytometry in peripheral blood was 5 months, while the median time to loss of CD22 singly transduced CART cells was 7 months.

[0268] In six exemplary patients infused with the CD19 / 22 CAR T cell product, expression of both CAT19CAR and 9A8 CAR by flow cytometry using specific anti-idiotype antibodies was detected in the blood of all patients 28 days after CD19 / 22 CAR T cell product administration (Figure 6). For CD19 CAR on the y-axis and CD22 CAR on the x-axis, data from a representative patient by flow cytometry using staining revealed unusual early expansion and proliferation (Figure 11C).

[0269] The expansion and persistence of CAR-T cells determined by qPCR for the CAR transgene are shown in Figures 11A and 11B. The kinetics of expansion and persistence generally coincided with very high levels of early expansion and proliferation with a peak 14 days after infusion followed by contraction. The persistence of CD19 and CD22 CAR-T cells was generally correlated. The median period of B cell aplasia did not reach the data cut-off date.

[0270] The pharmacokinetic analysis is summarized in Table 17 below. The pharmacokinetic analysis showed similar early expansion and proliferation of both CARs (Cmax for CD19 CAR: 937,947.80 copies / μg DNA; Cmax for CD22 CAR: 270,171.20 copies / μg DNA; median time to Cmax of 14 days (range 13 - 28 days) and very high cumulative CAR T cell exposure (AUC0 - 28) in the first 28 days for both CARS (CD19CAR: 9,492,498.00 copies / μg DNA; CD22CAR: 2,586,767.00 copies / μg DNA). Using qPCR, CD19 CAR T cells were detectable in 7 / 12 patients and CD22 CAR T cells were detectable in 5 / 12 patients at the last follow-up. The median half-life of CAR T cells in 10 evaluable patients was 15.4 days (range: 2.2 - 34.4) for CD19 and 17.7 days (range: 1.2 - 40.2) for CD22 (determined in 10 evaluable patients). 7 out of 12 patients had ongoing B cell aplasia and the median duration of B cell aplasia for the entire cohort has not yet been reached. Pharmacokinetic data comparing the maximum concentration (Cmax) and area under the curve (AUC) in the first 28 days were compared between the first cohort and the current cohort. Peak concentrations were 1 log higher for the CD19CAR component compared to cohort 1 when comparing geometric means and approximately 4-fold higher than for CD22 CAR. AUC exposure within the first 28 days was also approximately 1 log higher for CD19 and approximately 5-fold for CD22 CAR compared to cohort 1. Table 17

Table 17

[0271] The patient responses of cohort 3 are summarized in Table 18 below. One month after CAR T cell infusion, 10 / 12 (83%) patients had complete remission with or without hematological recovery (CR / CRi). Three of these patients had reached this status prior to lymphodepletion and CAR T infusion and continued in CR / CRi. Nine of these 10 patients with CR / CRi had no MRD detectable by flow cytometry or PCR. One patient with MRD below the quantification range at 1 month had this disappear by 2 months after infusion, resulting in a 100% MRD-negative CR rate among responders at this time. Importantly, of the three patients with CD19-negative lesions at screening, two reached MRD-negative CR / CRi, supporting the therapeutic efficacy of CAR T cells with CD22 CAR in our product. Two patients, one with CD19+ / CD22+ lesions and one with progression of double CD19- / CD22- lesions that were present as a minority (5%) population prior to CAR T cell infusion, did not respond. Both patients subsequently died of disease at 3 months and 15 days after infusion, respectively.

[0272] Of the 10 patients who reached MRD-negative CR / CRi, three subsequently had recurrence with CD19 + / CD22 + lesions at 3, 7.5, and 9 months after infusion, and in 2 / 3 of the cases, this was associated with early loss of CAR T cell persistence prior to 6 months after infusion. In two further cases, MRD-level lesions (CD19 + CD22 +) occurred, and as a result, the patient received further therapy including SCT in one case and maintenance chemotherapy in the second case. Both of them are in remission. Due to the early loss of CAR T cell persistence 3 and 4 months after infusion, two patients continued further therapy during MRD-negative remission. This included allogeneic SCT in one case and maintenance chemotherapy in the other case (Figure 12, Table 22). At a median follow-up of 8.7 months (95% CI: 3.9 - 12.2), 5 out of 10 responding patients were alive and disease-free, and there has been no recurrence due to antigen loss so far. The overall survival was 75% (95% CI: 41 - 91%) at 6 and 12 months (Figure 14). Using the same EFS definition as in the ELIANA study, the EFS at 6 and 12 months was 75% (95% CI: 41 - 91%) and 60% (95% CI: 17 - 84%), respectively (Figure 14). The inventors also applied a more stringent event definition including the events as described above, importantly including the need for further therapy for either the appearance of MRD or the early loss of CAR-T cells. The stringent EFS at 6 and 12 months was 75% (95% CI: 41 - 91%) and 38% (95% CI: 9 - 67%), respectively (Figure 14). The median duration of remission in responding patients was 9.9 months. Table 18

Table 18-1

Table 18-2

[0273] A summary of patient outcomes is shown in the swim plot of Figure 12, the survival curve of Figure 14, Table 19 which combines patient characteristics and outcome data, and Table 22 which shows a summary of responses and recurrences. At a median follow-up of 8.7 months, there were no cases of antigen-negative recurrence, and 5 / 10 responding patients had a complete response (CR) with MRD negative.

[0274] The 12-month event-free survival (EFS) defined in the ELIANA study was 60%, comparable to that seen in a pivotal study of tisagenlecleucel (Kymriah), despite one-third of the cohort already failing that therapy. A stringent EFS, with event definition including MRD appearance or further therapy, was also confirmed. The 12-month stringent EFS was 38%, again comparable to UK real-world data on stringent EFS after tisagenlecleucel (Kymriah™) therapy. Table 19 [Table 19] Table 22 [Table 22-1] [Table 22-2]

[0275] The provisional conclusions of this study are as follows.

[0276] - AUTO1 / 22 ATIMP: well-balanced between CD19 CAR and CD22 CAR, enabling reproducible generation of products with mainly central memory phenotype; - Favorable safety profile: no severe CRS, one patient with G4 ICANS, although atypical, clinically resembled fludarabine neurotoxicity due to the presence of leukoencephalopathy; - Excellent CAR-T cell expansion, including in the CD22 CAR population. - Median persistence of CAR-T cells in bone marrow by flow was 6 months for CD19 and CD19 / 22. - Low-risk patient cohort: no patients eligible for Kymriah (4 patients had failed Kymriah); 3 patients had CD19-negative lesions; 3 patients had non-CNS extramedullary lesions. - 10 / 12 were two non-responders who demonstrated complete responses (MRD-negative CR / CRi / CCR) despite the low-risk characteristics of the patient cohort. - 2 / 3 of patients with prior CD19-negative lesions achieved CR, demonstrating the efficacy of CD22 CAR. - Despite the low-risk characteristics of the patient cohort (4 patients had failed Kymriah and 3 patients had CD19-negative lesions), the 1-year EFS was 60%, equivalent to that of the ELIANA study (tisagenlecleucel / Kymriah), and the 38% "stringent EFS" was similar to UK real-world data with Kymriah. - Three patients had CD19+CD22+ relapses and two patients had CD19+CD22+ MRD emergence. All were associated with non-sustainability. - Antigen-positive relapses were associated with loss of CAR-T cells, the major cause of treatment failure. - Antigen-negative relapses were not seen in responding patients, suggesting effective dual targeting. - At a median follow-up of 8.7 months, 5 / 10 responding patients who were MRD-negative had CR (4 - 12 months), and two later received additional therapy due to early loss of CAR T cell persistence.

[0277] Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications in the described modes for carrying out the invention will be apparent to those skilled in the molecular biology or related fields and are intended to be within the scope of the following claims.

Claims

1. A method for treating high-risk / relapsed CD19+ or CD22+ hematological malignancies in a patient, the method comprising administering autologous CD19 / 22 CAR T cells to the patient.

2. Autologous CD19 / 22 CAR T cells for use in the treatment of high-risk / relapsed CD19+ or CD22+ hematological malignancies.

3. Use of autologous CD19 / 22 CAR T cells in the manufacture of a medicament for the treatment of high-risk / relapsed CD19+ or CD22+ hematological malignancies.

4. The method according to claim 1, the autologous CD19 / 22 CAR T cells for use according to claim 2, or the use according to claim 3, wherein the age of the patient is 24 years or younger.

5. The method according to any one of claims 1 or 4, the autologous CD19 / 22 CAR T cells for use according to any one of claims 2 or 4, or the use according to any one of claims 3 or 4, wherein the hematological malignancy is acute lymphoblastic leukemia (ALL), or CD19+ or CD22+ lymphoma.

6. The method according to claim 5, the autologous CD19 / 22 CAR T cells for use, or the use, wherein the lymphoma is Burkitt lymphoma.

7. The patient is a) resistant disease (>5% blast cells) at the end of the introduction of UKALL 2019 guidelines or equivalents, b) High-level MRD at the second time point of the frontline national protocol (currently, in the UKALL 2019 guidelines or equivalent, MRD > 10 －４ ) in ALL, c) High-risk infant ALL (diagnosed by MLL gene rearrangement, age < 6 months at diagnosis, and white blood cell count > 300 × 10 ９ / L or inadequate steroid initial response (i.e., > 1 × 10 ９ / L of circulating blast cells after 7-day steroid prephase of induction according to national guidelines or equivalents)), d) Intermediate-risk infant ALL with MRD > 10 at the end of induction according to national guidelines or equivalents －３ having e) high-risk first relapse (as defined by the latest IntreALL 2019 classification: bone marrow or composite relapse within 30 months of diagnosis), f) standard-risk relapse in patients with high-risk cytogenetics (defined as BCR-ABL, KMT2A rearrangement, near-haploidy (<30 chromosomes) and low hypodiploidy (30-39 chromosomes), iAMP21 and TCF3-HLF translocation), g) At the end of reintroduction, minimal residual disease (MRD) of the bone marrow > 10 －３ with standard-risk recurrence h) any refractory relapse of ALL (defined as >1% blast cells by flow cytometry after at least 1 cycle of standard chemotherapy), or i) any relapse of CD22+ lymphoma and has the method according to any one of claims 1 or 4-6, the autologous CD19 / 22 CAR T cells for use according to any one of claims 2 or 4-6, or the use according to any one of claims 3-6.

8. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 7, wherein the patient has a single CNS relapse satisfying one or more of a) to i).

9. The method according to any one of claims 1 or 4 to 8, the autologous CD19 / 22 CAR T cells for use according to claims 2 or 4 to 8, or the use according to claims 3 to 8, wherein the patient has one or more treatment histories.

10. The method according to claim 9, the autologous CD19 / 22 CAR T cells for use, or the use, wherein the patient has received one or more of inotuzumab ozogamicin, blinatumomab, and tisagenlecleucel.

11. wherein the patient is administered a single dose of 0.5×10 ６ CAR T cells / kg, 0.75×10 ６ CAR T cells / kg, 1×10 ６ CAR T cells / kg, or 1.2×10 ６ CAR T cells / kg, the method according to any one of claims 1 or 4 to 10, autologous CD19 / 22 CAR T cells for use according to claim 2 or 4 to 10, or use according to claims 3 to 10.

12. The method according to claim 11, the autologous CD19 / 22 CAR T cells for use, or the use, wherein the administration is by intravenous injection, preferably by intravenous injection through a Hickman line or a central catheter inserted peripherally.

13. The CD19 / 22 CAR T cell product is a) the following sequences: CDR1 - GYAFSSS (SEQ ID NO: 1); CDR2 - YPGDED (SEQ ID NO: 2) CDR3 - SLLYGDYLDY (SEQ ID NO: 3) having a heavy chain variable region (VH) having complementarity determining regions (CDRs); and b) the following sequences: CDR1 - SASSSVSYMH (SEQ ID NO: 4); CDR2 - DTSKLAS (SEQ ID NO: 5) CDR3 - QQWNINPLT (SEQ ID NO: 6) having a light chain variable region (VL) having CDRs expressing a chimeric antigen receptor (CAR) comprising a CD19 binding domain, the method according to any one of claims 1 or 4 to 12, the autologous CD19 / 22 CAR T cells for use according to claims 2 or 4 to 12, or the use according to claims 3 to 12.

14. The method according to claim 13, the autologous CD19 / 22 CAR T cells for use, or the use, wherein the CD19 binding domain comprises a VH domain having the sequence shown in SEQ ID NO: 7 and / or a VL domain having the sequence shown in SEQ ID NO: 8, or a variant thereof having at least 95% sequence identity.

15. The method according to claim 13 or claim 14, the autologous CD19 / 22 CAR T cells for use, or the use, wherein the CD19 binding domain comprises a scFv in the VH-VL orientation.

16. The method, autologous CD19 / 22 CAR T cells for use, or use according to claims 13 to 15, wherein the CD19 binding domain comprises the sequence shown in SEQ ID NO: 9 or a variant thereof having at least 90% sequence identity.

17. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 13, wherein the CD19 binding domain comprises the six CDRs defined in claim 13 grafted into a human antibody framework.

18. The method according to any one of claims 1 or 4 to 17, the autologous CD19 / 22 CAR T cells for use according to claim 2 or 4 to 17, or the use according to claims 3 to 17, wherein the CD19 binding domain and the transmembrane domain are connected by a spacer.

19. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 18, wherein the spacer comprises a CD8 stalk.

20. The method according to any one of claims 1 or 4 to 19, the autologous CD19 / 22 CAR T cells for use according to claim 2 or 4 to 19, or the use according to claims 3 to 19, comprising an intracellular T cell signaling domain.

21. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 20, wherein the intracellular T cell signaling domain comprises a 41BB endodomain and a CD3-zeta endodomain.

22. The CD19 / 22 CAR T cell product is a) the following sequences: CDR1 - NFAMA (SEQ ID NO: 58); CDR2 - SISTGGGNYYRDSVKGG (SEQ ID NO: 59) CDR3 - QRNYYDGSYDYEGYTMDA (SEQ ID NO: 60) having a heavy chain variable region (VH) having CDRs; and b) the following sequences: CDR1 - RSSQDIGNYLTL (SEQ ID NO: 61); CDR2 - GAIKLED (SEQ ID NO: 62) CDR3 - LQSIQYP (SEQ ID NO: 63) having a light chain variable region (VL) having CDRs The method according to any one of claims 1 or 4 to 21, the autologous CD19 / 22 CAR T cells for use according to claim 2 or 4 to 21, or the use according to claims 3 to 21, expressing a chimeric antigen receptor (CAR) comprising a CD22 binding domain.

23. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 22, wherein the CD22 binding domain comprises a VH domain having the sequence shown in SEQ ID NO: 64 and / or a VL domain having the sequence shown in SEQ ID NO: 65, or a variant thereof having at least 95% sequence identity.

24. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 22 or claim 23, wherein the CD22 binding domain comprises a scFv in VH-VL orientation.

25. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 24, wherein the CD22 binding scFv comprises the sequence shown in SEQ ID NO: 66, or a variant thereof having at least 90% sequence identity.

26. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 25, wherein the CD22 binding domain comprises the six CDRs defined in claim 22 grafted into a human antibody framework.

27. The method according to any one of claims 1 or 4 to 26, the autologous CD19 / 22 CAR T cells for use according to claim 2 or 4 to 26, or the use according to claims 3 to 26, wherein the CD22 binding domain and the transmembrane domain are connected by a spacer.

28. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 27, wherein the spacer comprises a CD8 stalk.

29. The method according to any one of claims 1 or 4 to 28, the autologous CD19 / 22 CAR T cells for use according to claim 2 or 4 to 28, or the use according to claims 3 to 28, wherein the CAR comprises an intracellular T cell signaling domain.

30. The method, autologous CD19 / 22 CAR T cells for use, or use according to claim 29, wherein the intracellular T cell signaling domain comprises a 41BB end domain and a CD3-zeta end domain.

31. The method according to any one of claims 1 or 4 to 31, the autologous CD19 / 22 CAR T cells for use according to claim 2 or 4 to 31, or the use according to claims 3 to 31, wherein the autologous CD19 / 22 CAR T cells comprise a CD19 / 22 CAR T cell product comprising the CARs of CAT19CAR and 9A8CAR.

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