Products and methods for treating autoimmune diseases

Abstract

The present invention relates to the use of chimeric antigen receptors (CARs) that bind to the B lymphocyte antigen CD19 (cluster of differentiation 19) and T cells expressing such CARs for the treatment of autoimmune diseases, such as systemic lupus erythematosus (SLE).

Description

Technical Field

[0001] This invention relates to a chimeric antigen receptor (CAR) that binds to the B lymphocyte antigen CD19 (differentiation cluster 19), and to the use of CAR-expressing cytolytic cells for the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE). Background Technology

[0002] B cells, together with T cells, form the core of the adaptive arm of the immune system and play a crucial role in regulating the immune response under both normal and pathological conditions. B cells are identified pharmacological targets in B-cell malignancies and autoimmune diseases.

[0003] Autoimmune diseases

[0004] Many diseases are believed to be caused by autoimmune mechanisms. Common examples include rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, type 1 diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, scleroderma, vasculitis, and pemphigus vulgaris. Autoimmune diseases affect millions of individuals worldwide, and their costs are measured in billions of dollars annually in terms of actual treatment expenditures and lost productivity.

[0005] Systemic lupus erythematosus

[0006] Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the formation of autoantibodies and immune complex-mediated inflammation and organ damage, including the skin, joints, central nervous system, heart, lungs, and kidneys (Tsokos GC. (2011). Systemic lupus erythematosus. N Engl J Med 365(22):2110-21; Kaul A, Gordon C, Crow M, et al. (2016). Systemic lupus erythematosus. Nature Reviews 2:1-21; Mackensen A, Müller F, Mouglakakos D, et al. (2022). Anti-CD19 CAR T celltherapy for refractory lupus erythematosus. Nature Medicine 28:2124-2132). It is a genetically complex disease exhibiting genetic heterogeneity (Kaul et al., ibid.). The vast differences in clinical and serological presentations make SLE a highly heterogeneous disease. The severity of the disease also varies over time, with alternating periods of disease-free activity and flare / relapse periods. In addition to this heterogeneity, SLE can be complicated by infections, premature atherosclerosis, and chronic organ damage, as well as subjective manifestations including reduced quality of life. The age of onset is generally between 20 and 40 years, and it primarily affects young women. SLE patients have approximately three times the mortality risk of the general population.

[0007] Current treatments aim to reduce inflammation, control symptoms, and prevent organ damage, but they have significant side effects, raising safety concerns. Durable, complete remission without serological or clinical disease activity, as well as glucocorticoid and immunosuppressant states, are rarely achieved. Therefore, there is an unmet medical need for treatments with potential curative intent, better safety profiles, and convenient, treatment-interval-free approaches, particularly for severely refractory patients.

[0008] Autoreactive B cells with the ability to form autoantibodies play a key role in the pathogenesis of SLE (Mackensen et al., ibid.). However, in randomized studies of SLE and lupus nephritis (LN), B-cell depletion agents (such as the anti-CD20 antibody rituximab) did not improve clinical outcomes compared to placebo (Merrill J, Neuwelt CM, Wallace DJ, Shanahan JC, et al. (2010). Efficacy and safety of rituximab in moderately-to-severely active systemic lupus erythematosus: the randomized, double-blind, phase II / III systemic lupus erythematosus evaluation of rituximab trial. Arthritis Rheum 62(1):222-33; Rovin et al., (2012). Efficacy and safety of rituximab in patients with active proliferative lupus nephritis: the Lupus Nephritis Assessment with Rituximab study ArthritisRheum 64(4): 1215-26), and two different biologics have recently been approved for SLE: belinumab (an anti-BAFF / BLyS monoclonal antibody), approved as adjunctive therapy for adult patients with active, autoantibody-positive SLE who have high disease activity despite standard therapy (Benlysta (belinumab) USPI. 2023. Available at: gskpro.com / content / dam / global / hcpportal / en_US / Prescribing_Information / Benlysta / pdf / BENLYSTA-PI-MG-IFU.PDF. Last accessed August 25, 2023; Benlysta (belinumab) SmPC. 2023. Available at: www.ema.europa.eu / en / documents / product-information / benlysta-epar-product-information_en.pdf. Last accessed August 30, 2023).Avolimumab is a type I interferon (IFN) receptor antagonist that has also been approved in the US and EU and shown to be an adjunct therapy for the treatment of adult patients with moderate to severe SLE who are receiving standard therapy (Saphnelo (avolimumab-fnia) USPI. 2022.). Available at: www.den8dhaj6zs0e.cloudfront.net / 50fd68b9-106b-4550-b5d0-12b045f8b184 / 44b6985c-8268-46b1-ba3e-2bb43bfd4d4c / 44b6985c-8268-46b1-ba3e-2bb43bfd4d4c_viewable_rendition__v.pdf. Last accessed August 25, 2023; Saphnelo (Avolimumab-fnia) SmPC. 2022. Available at: www.ema.europa.eu / en / documents / product-information / saphnelo-epar-product-information_en.pdf. Last accessed 25 August 2023). In addition, based on the AURORA1 trial (Rovin et al., Lancet 2021) (www.doi.org / 10.1016 / S0140-6736(21)00578-X), vortexporin (Lupkynis USPI, 2021) was approved for lupus nephritis. Compared with MMF and low-dose steroids alone, vortexporin in combination with MMF and low-dose steroids resulted in clinically and statistically better complete renal response rates with a comparable safety profile. Despite these approvals, some patients have inadequate responses, lack of response, or lack of sustained response despite standard therapy and are at risk of further organ damage. Therefore, treating drug-resistant diseases remains a challenge.

[0009] Another strategy for inducing deeper exhaustion of B-cell compartments stems from the highly effective treatment of patients with B-cell malignancies using CD19-targeting chimeric antigen receptor (CAR) T cells (Cappell KM, Kochenderfer JN. (2023) Long-term outcomes following CAR T cell therapy: what we know so far. Nat Review 20:359-371). Several CD19-targeting CAR T cells are now approved for relapsed and / or refractory B-cell lymphomas and B-cell acute lymphoblastic leukemia (B-ALL) (Acilanc, Tisagenlecleucel, Lisocabtagene Maraleucel, and Brexucabtagene Autoleucel). Long-term follow-up data suggest that CD19-targeted CAR T-cell therapy may be curative for a subset of patients with B-cell lymphoma and potential B-ALL (Cappell and Kochenderfer, ibid.; Westin J, Davis RE, Feng L, Hagemeister F, et al. (2023). Smart start: rituximab, lenalidomide, and ibrutinib in patients with newly diagnosed large B-cell lymphoma. J Clin Oncol 41(4):745-55).

[0010] Two preclinical studies in lupus-prone mice support the efficacy of CD19 CAR T cells in SLE. Following administration of CD19-targeting CAR T cells (1D3 CAR and FMC63 CAR), these mice experienced B cell depletion, cessation of autoantibody production, and reversal of glomerulonephritis and other organ manifestations.

[0011] Using an autoimmune New Zealand mixed mouse (NZB × NZW) F1 and a lupus-infected Murphys Roths Large (MRL) fas / fas mouse model, CD8 CARs targeting CD19 were expressed. +T cells [1D3 CAR (Kochenderfer JN, Yu Z, Frasheri D, et al. (2010), Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 caneradicate lymphoma and normal B cells. Blood 116(19), 3875–3886] showed persistent CD19 depletion. + B cells eliminate autoantibody production, reverse disease manifestations in target organs, and extend lifespan far beyond the normal range in autoimmune mouse models (Kansal R, Richardson N, Neeli I, et al. (2019). Sustained B cell depletion by CD19-targeted CAR T cells is a highly effective treatment for murine lupus. Sci Transl Med 11(482): eaav1648-eaav1648). CAR T cells persist in vivo for one year and are associated with CD44. + CD62L + Central Memory CD8 + Significant enrichment in T cell subsets suggests that this population may carry CAR and contribute to persistent B cell exhaustion. Kansel et al. demonstrated that adoptive transfer of spleen T cells from CAR-treated mice depleted CD19. + B cells reduced disease in naive autoimmune mice, suggesting that disease control is cell-mediated.

[0012] The therapeutic efficacy of constructed mouse anti-CD19 CARs with CD28 or 4-1BB as intracellular co-stimulatory motifs [1D3 CAR (Kochenderfer et al., 2010, ibid.)] and Jin et al. (Jin X, Xu Q, Pu C, Zhu K, et al. (2021). Therapeutic efficacy of anti-CD19 CAR-T cells in a mouse model of systemiclupus erythematosus. Cell Mol Immunol 18(8):1896-1903) was evaluated by infusing the corresponding CAR-T cells into a mouse model of lupus erythematosus (MRL-lpr). The anti-CD19 CAR T cells transferred to MRL-lpr mice at disease onset were evaluated to determine their role in SLE prevention. Compared with antibody therapy, the constructed anti-CD19 CAR T cells showed efficacy of infusion into the mouse model of lupus erythematosus (MRL-lpr). Adoptive transfer of CAR-T cells showed a more durable B-cell depletion effect in MRL-lpr mice. Transfer of homologous anti-CD19 CAR-T cells not only prevented disease onset before symptoms appeared but also showed therapeutic benefits late in the disease progression. CAR-T cells with the 4-1BB co-stimulatory motif showed better therapeutic efficacy in the absence of cell enrichment compared to CAR-T cells with the CD28 co-stimulatory motif.

[0013] In summary, the results from these two preclinical studies demonstrate that anti-CD19 CAR-T cell therapy is effective in both prevention and treatment, albeit in a mouse model of SLE.

[0014] Recently, belimumab, a monoclonal antibody against B cell activating factor (BAFF) / B lymphocyte stimulator (BlyS), has been approved as adjunctive therapy for adult patients with active, autoantibody-positive SLE who are receiving standard therapy (Benlysta USPI, ibid.).

[0015] Tolerability and efficacy were initially tested in a single SLE patient treated with CD19 CAR (FMC63 CAR) (Mougiakakos D, Krönke G, Völkl S, Kretschmann S, et al. (2021). CD-19-targetedCAR T cells in refractory systemic lupus erythematosus. N Engl J Med 385(6):567-9). The same research group further evaluated the tolerability and efficacy of CD19 CAR T cell therapy (FMC63 CAR) in a small consortium of severely ill and treatment-resistant SLE patients (Mackensen et al., ibid.). In this consortium, autologous T cells from five SLE patients were transduced with a lentiviral anti-CD19 CAR vector, expanded, and then fed at 1 × 10⁻⁶ cells per cell line after lymphocyte depletion with fludarabine and cyclophosphamide. 6 A dose of CAR T cells / kg body weight was infused into the patients. CAR T cells expanded in vivo and led to deep B cell depletion, with clinical symptoms improving and laboratory parameters normalizing, including seroconversion of anti-dsDNA antibodies. According to DORIS criteria, all 5 patients achieved SLE remission after 3 months and maintained drug-free remission during a longer follow-up period following CAR T cell administration.

[0016] Recent anecdotal evidence has emerged regarding CD19CAR T-cell therapy (FMC63 CAR) in another autoimmune disease in patients with refractory idiopathic inflammatory myopathy associated with antisynthetase syndrome (Müller F, Boeltz S, Knitza J, Aigner M, et al. (2023). CD19-targeted CAR T cells inrefractory antisynthetase syndrome. Lancet 401(10379):815-8; Percher AC, Hensen L, Klein R, et al. (2023). CD19-targeting CAR T cells for myositis and interstitial lung disease associated with antisynthetase syndrome. JAMA 329(24):2154-62).

[0017] Chimeric antigen receptor

[0018] Traditionally, antigen-specific T cells have been generated through the selective expansion of peripheral blood T cells that are naturally specific to the target antigen. However, selecting and expanding large numbers of T cells specific to most cancer antigens is difficult, and often impossible. Gene therapy using integrative vectors offers a solution to this problem: transgenic expression of chimeric antigen receptors (CARs) allows for the easy generation of large numbers of T cells specific to any surface antigen via ex vivo viral vector transduction from a large number of peripheral blood T cells.

[0019] The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody that recognizes the target antigen, which is fused to the intracellular signaling domain via a spacer and a transmembrane domain. These molecules cause T cells to activate in response to the scFv's recognition of its homologous target. When T cells express such CARs, they recognize and kill target cells expressing the target antigen. Several CARs targeting tumor-associated antigens have been developed, and adoptive transfer of T cells expressing such CARs is currently in clinical trials for the treatment of various cancers. However, to date, the primary clinical exploration and potential application of CAR therapy has been for the treatment of B-cell malignancies.

[0020] CAR targeting CD19

[0021] CD19 is a B-cell antigen that is expressed very early in B-cell differentiation and is lost only at the terminal lobes of B cells as they differentiate into plasma cells. Therefore, CD19 is expressed in all B-cell malignancies except multiple myeloma. It is not expressed in other hematopoietic populations or non-hematopoietic cells, and therefore targeting this antigen should not lead to toxicity to the bone marrow or non-hematopoietic organs. In the treatment of lymphomas, the loss of normal B-cell compartments is considered acceptable toxicity because, although effective CD19 CAR T-cell therapy will result in impaired B-cell regeneration, subsequent hypogammaglobulinemia can be treated with concomitant immunoglobulin therapy.

[0022] Therefore, CD19 is an attractive CAR target. To date, the main clinical focus in the CAR field has been research on targeting CD19 for refractory B-cell carcinoma, as summarized in Table 1.

[0023] Different CAR designs have been tested for CD19 at different centers, as outlined in Table 1.

[0024] Table 1. Summary of CAR-T therapy targeting CD19

[0025]

[0026] Most studies have tested CD19 CARs based on scFv derived from hybridoma fmc63 (i.e., CARs targeting CD19). The most promising application is in the treatment of acute lymphoblastic leukemia (ALL).

[0027] Immunotoxicity of CD19 CAR therapy

[0028] Cytokine release syndrome (CRS) encompasses a range of inflammatory symptoms, from mild to multiple organ failure with hypotension and respiratory failure. Some degree of CRS typically occurs in patients treated with CD19 CAR T-cell therapy. In a recent cohort, approximately 30% (21 / 73) of treated patients showed some degree of CRS (Davila et al. ((2014). Sci. Transl. Med. 6, 224ra25; Lee et al. (2014) Lancet. doi:10.1016 / S0140-6736(14)61403-3; Kochenderfer et al. (2014) J. Clin. Oncol. Off. J. Am. Soc. Clin.Oncol., doi:10.1200 / JCO.2014.56.2025). CRS has also been observed in patients treated with belintolimumab, a bispecific recombinant single-chain antibody that recognizes both CD19 and CD3. CRS typically occurs 5–21 days after CAR T-cell infusion.

[0029] CRS can be life-threatening and requires treatment in an intensive care setting. CRS is associated with elevated serum cytokine levels. The most significantly elevated cytokines are IL-6, IL-10, and interferon-γ (IFNγ). The clinical presentation of severe CRS (fever, hepatosplenomegaly, coagulopathy, and ferritinemia) is similar to macrophage activation syndrome (MAS), for example, found in patients with congenital T-cell defects. This suggests a shared immunopathological process is involved. It is unclear which cell type (CAR T cells, dying tumor cells, or locally activated macrophages) is responsible for producing key cytokines, particularly IL-6. However, a key initiating factor in MAS is the release of large amounts of interferon-γ (López-Alvarez et al. (2009). Clin. Vaccine Immunol. CVI 16, 142–145).

[0030] Neurotoxicity

[0031] In multi-institutional CD19 CAR studies, many patients developed transient neurotoxicity ranging in severity from aphasia to lethargy, delirium, and seizures (Davilia et al., ibid.). This appears to be limited to ALL patients, and similar syndromes have been documented following belintoometab therapy. Brain imaging was normal. Neurotoxicity may reflect high levels of systemic cytokines that cross the blood-brain barrier.

[0032] Persistence, relapse and T cell exhaustion

[0033] Durable responses appear to be associated with higher peak levels of circulating CAR-transduced T cells and duration of impaired B cell regeneration. Relapse, except in patients with CD19 disease relapse, is typically associated with loss of circulating CAR T cells and recovery of normal B cells.

[0034] T cell exhaustion is a state of T cell dysfunction that occurs during many chronic infections and cancers. It is defined by the following: poor effector function, persistent expression of inhibitory receptors, and a transcriptional state that differs from that of functional effector T cells or memory T cells.

[0035] Exhaustion prevents optimal control of infection and tumors. Recently, a clearer picture of the function and phenotype of exhausted T cells has emerged as the expression of the inhibitory receptor programmed death 1 (PD-1; also known as PDCD1) (a negative regulator of activated T cells) has become a key feature (Day et al. (2006) Nature 443, 350–354).

[0036] Responses in CD19 CAR studies suggest that sustained high T cell levels for an extended period appear to be important for achieving durable responses. Reducing T cell depletion in CD19 CARs can lead to improved clinical responses.

[0037] Therefore, alternative CARs targeting CD19 are needed in the treatment of autoimmune diseases, regardless of the aforementioned drawbacks. Summary of the Invention

[0038] This disclosure provides a method for treating autoimmune diseases with CD19 CAR T cells. Treatment with CD19 CAR T cells is expected to more completely remove B cell compartments and more effectively eliminate autoreactive B cells than rituximab treatment. CD19 is a superior target for CD20-targeted therapy in autoimmune diseases because CD19 is more widely expressed on B cells, including very early B progenitor cells and immature plasmablasts.

[0039] Therefore, in a first aspect, the present invention provides a method for treating an autoimmune disease in a patient, wherein cells comprising a chimeric antigen receptor (CAR) are administered to the patient, the chimeric antigen receptor (CAR) comprising a CD19 binding domain, the CD19 binding domain comprising a) a heavy chain variable region (VH) having a complementarity-determining region (CDR), the CDR having the following sequence:

[0040] CDR1 – GYAFSSS (SEQ ID No. 1);

[0041] CDR2 – YPGDED (SEQ ID No. 2)

[0042] CDR3 – SLLYGDYLDY (SEQ ID No. 3); and

[0043] b) Light chain variable regions (VLs) with CDRs having the following sequence:

[0044] CDR1 – SASSSVSYMH (SEQ ID No. 4);

[0045] CDR2 – DTSKLAS (SEQ ID No. 5)

[0046] CDR3 – QQWNINPLT (SEQ ID No. 6).

[0047] Autoimmune diseases can include systemic lupus erythematosus (SLE). Autoimmune diseases can be severe SLE or refractory SLE.

[0048] The cells can be T cells or NK cells. The cells can be autologous peripheral blood T cells transduced in vitro to express CAR. The cells can have been isolated from a patient's peripheral blood mononuclear cell (PBMC) sample.

[0049] 50 × 10 can be administered to the patient. 6 A dose of (±20%) CD19 CAR-positive live T cells. Alternatively, the dose could be 100 x 10 6 (±20%) CD19 CAR-positive live T cells or 30 × 10 6 (±20%) CD19 CAR-positive live T cells.

[0050] Administration can be via intravenous injection through a Hickman catheter or a peripherally inserted central catheter.

[0051] The CD19 binding domain may contain six CDRs (SEQ ID No. 1-6) transplanted onto the human antibody framework.

[0052] The CD19 binding domain may include 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 of either having at least 95% sequence identity.

[0053] The CD19 binding domain can contain scFv in the VH-VL direction.

[0054] The CD19 binding domain may contain the sequence shown in SEQ ID No. 9 or a variant thereof having at least 90% sequence identity.

[0055] CARs can contain spacers that act as CD8 stems.

[0056] CARs can contain an intracellular T cell signaling domain, which includes the 41BB intracellular domain and the CD3-ζ intracellular domain.

[0057] In a second aspect, the present invention provides a method for treating an autoimmune disease in a patient, comprising administering 50 × 10 6 A dose of (±20%) CD19 CAR-positive live T cells. Alternatively, the patient can be given 30 × 10⁻⁶ cells. 6 A dose of (±20%) CD19 CAR-positive live T cells. Alternatively, 100 × 10⁻⁶ cells can be administered to the patient. 6 (±20%) dose of CD19 CAR-positive live T cells.

[0058] According to the second aspect, autoimmune diseases can include SLE. Autoimmune diseases can be severe SLE or refractory SLE.

[0059] In either the first or second aspect, the CAR may comprise a sequence as shown in any one of SEQ ID Nos. 10 to 15 or a variant thereof, which has at least 80% sequence identity but retains the ability to i) bind CD19 and ii) induce T cell signaling. This CAR may possess advantageous properties compared to the fmc63-based CAR used in the UPENN study. For example, the CD19 (CAT) CAR described herein has a lower affinity for a CD19 binder and a faster dissociation rate, which more closely reflects physiological interactions and therefore more physiological T cell activation. This is expected to reduce the toxicity of CAR-expressing cells and make those cells less prone to T cell exhaustion, thereby enhancing persistence and improving the ability of programmed T cells to engage in continuous killing of target cancer cells. As another example, when the CAR is expressed by T cells and used to target CD19-expressing cells, it induces lower IFNγ release in target cells compared to IFNγ release induced by T cells expressing CARs containing a CD19-binding domain comprising: a) a heavy chain variable region (VH) with a complementarity-determining region (CDR) having the following sequences: CDR1 – GVSLPDY (SEQ ID No. 16); CDR2 – WGSET (SEQ ID No. 17); CDR3 – HYYYGGSYAMDY (SEQ ID No. 18); and b) a light chain variable region (VL) with a CDR having the following sequences: CDR1 – RASQDISKYLN (SEQ ID No. 19); CDR2 – HTSRLHS (SEQ ID No. 20); CDR3 – QQGNTLPYT (SEQ ID No. 21)). The CDR provided in this article can be ported to human or humanized frameworks. Attached Figure Description

[0060] Figure 1 The SLEDAI-2K score of the top SLE patient treated with obe-cel.

[0061] Figure 2 CAR-T cell expansion in the first SLE patient treated with obe-cel. A) Flow cytometry of CD3+CAT19+ cells. B) ddPCR. Detailed Implementation

[0062] Chimeric antigen receptor (CAR)

[0063] Chimeric antigen receptors (CARs), also known as chimeric T-cell receptors, artificial T-cell receptors, and chimeric immune receptors, are engineered receptors that can be specifically transplanted onto immune effector cells. In classic CARs, a monoclonal antibody is specifically transplanted onto T cells. Nucleic acid encoding the CAR can be transferred to T cells using vectors such as retroviruses. In this way, a large number of CD19-specific T cells can be generated for adoptive cell transfer. Phase I clinical studies of this method have shown efficacy.

[0064] The target antigen-binding domain of a CAR typically fuses with an intracellular domain via a spacer and a transmembrane domain. The intracellular domain may contain or associate with an intracellular T cell signaling domain. When a CAR binds to a target antigen, this results in activation signals being transmitted to T cells expressing that CAR.

[0065] Binding domain specific to CD19 target antigen

[0066] Human CD19 antigen is a 95 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily. Classified as a type I transmembrane protein, CD19 possesses 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 at the terminal portion of B cells as they differentiate into plasma cells. CD19 is a biomarker for normal B cells and follicular dendritic cells. CD19 primarily functions as a B cell co-receptor along with CD21 and CD81. Upon activation, the cytoplasmic tail of CD19 becomes phosphorylated, leading to binding by Src family kinases and recruitment by PI-3 kinase.

[0067] CD19 is also expressed in all B-cell malignancies except for multiple myeloma cells. It is not expressed in other hematopoietic populations or non-hematopoietic cells, and therefore targeting this antigen should not lead to toxicity to the bone marrow or non-hematopoietic organs. In the treatment of lymphomas, the loss of normal B-cell compartments is considered acceptable toxicity because, although effective CD19CAR T-cell therapy will result in impaired B-cell regeneration, subsequent hypogammaglobulinemia can be treated with concomitant immunoglobulin therapy.

[0068] Different designs of CARs have been tested for CD19 in various clinical trials, as summarized in Table 2 below.

[0069] Table 2

[0070]

[0071] As shown above, most studies to date have used scFv derived from hybridoma fmc63 as part of the binding domain that recognizes CD19.

[0072] The antigen-binding domain of a CD19-binding CAR (referred to as CD19 CAR in this paper) can be any domain capable of binding CD19.

[0073] For example, the antigen-binding domain may include the CD19 antigen-binding domain as described in Table 3.

[0074] Table 3

[0075]

[0076] The CD19 gene contains ten exons: exons 1-4 encode the extracellular domain; exon 5 encodes the transmembrane domain; and exons 6-10 encode the cytoplasmic domain. The antigen-binding domain of the CD19 CAR described in this paper can bind to the CD19 epitope encoded by exon 1 of the CD19 gene. The antigen-binding domain of the CD19 CAR described in this paper can bind to the CD19 epitope encoded by exon 2 of the CD19 gene. The antigen-binding domain of the CD19 CAR described in this paper can bind to the CD19 epitope encoded by exon 3 of the CD19 gene. The antigen-binding domain of the CD19 CAR described in this paper can bind to the CD19 epitope encoded by exon 4 of the CD19 gene.

[0077] The CD19 binding domain illustrated in this article includes a variable region having a complementarity-determining region (CDR) from an antibody called CAT19.

[0078] a) Heavy chain variable region (VH) with a CAT19 CDR containing the following sequence:

[0079] CDR1 – GYAFSSS (SEQ ID No. 1),

[0080] CDR2 – YPGDED (SEQ ID No. 2) and

[0081] CDR3 – SLLYGDYLDY (SEQ ID No. 3), and

[0082] b) Light chain variable region (VL) having a CAT 19 CDR containing the following sequence:

[0083] CDR1 – SASSSVSYMH (SEQ ID No. 4),

[0084] CDR2 – DTSKLAS (SEQ ID No. 5) and

[0085] CDR3 – QQWNINPLT (SEQ ID No. 6).

[0086] The CAT19 antibody is described in WO 2016 / 139487.

[0087] It is anticipated that one or more mutations (substitution, addition, or deletion) can be introduced into one or more CDRs without negatively affecting CD19 binding activity. Each CDR may, for example, have one, two, or three amino acid mutations.

[0088] CDRs can exist in the form of single-chain variable fragments (scFvs), which are fusion proteins of the heavy variable region (VH) and light variable region (VL) of an antibody, linked by short linker peptides of 10 to 25 amino acids. scFvs can be VH-VL oriented, meaning the VH is at the amino terminus of the CAR molecule, and the VL domain is linked to the spacer region, which in turn links to the transmembrane and intracellular domains.

[0089] CDRs can be grafted onto the framework of human antibodies or scFvs. For example, a CAR can contain a CD19 binding domain consisting of one of the following sequences or containing one of the following sequences.

[0090] CD19 CAR can contain the following VH sequences.

[0091] SEQ ID No. 7 – VH sequence from CAT19 mouse monoclonal antibody

[0092] QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSS

[0093] CD19 CAR can contain the following VL sequences.

[0094] SEQ ID No. 8 - VL sequence from CAT19 mouse monoclonal antibody

[0095] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

[0096] CD19 CAR can contain the following scFv sequences.

[0097] SEQ ID No. 9 - VH-VL scFv sequence from a mouse monoclonal antibody

[0098] QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSG GGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

[0099] A CAR can consist of or contain one of the following sequences.

[0100] SEQ ID No. 10 - CAT19 CAR using the "Campana" architecture

[0101] MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLL YGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNNMEAEDAATYYCQQ WNINPLTFGAGTKLELKRSDPPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0102] The “Campana” architecture refers to a CAR with a CD8a spacer region and transmembrane domain, a 4-1BB intracellular domain, and a TCR CD3z intracellular domain.

[0103] SEQ ID No. 11 - CAT19 CAR with OX40-ζ intracellular domain

[0104] MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0105] SEQ ID No. 12 - CAT19 CAR with CD28-ζ intracellular domain

[0106] MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0107] SEQ ID No. 13 - Third-generation CD19 CAR

[0108] MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0109] SEQ ID No. 14 - CD19 CAR, with IgG1 hinge spacer

[0110] MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPAEPKSPDKTHTCPPCPKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0111] SEQ ID No. 15 - CD19 CAR, hinge-CH2-CH3 of human IgG1 with FcR binding site mutation eliminated

[0112] MGTSLLCWMALCLLGADHADAQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASP GEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGG VLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0113] The CARs provided herein may comprise variants of peptides of SEQ ID Nos. 1-15 having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity, provided that the variant sequence retains the ability to bind CD19 (if applicable, when binding to complementary VL or VH domains). The percentage of identity between two peptide sequences can be readily determined by a procedure such as BLAST, which is freely available at blast.ncbi.nlm.nih.gov.

[0114] The CD19 CAR illustrated herein (i.e., CAT19CAR using the “Campana” architecture, SEQ ID No. 10) possesses the properties contemplated in this disclosure, resulting in lower toxicity and better efficacy in treated patients. When compared to the fmc63-Campana CAR, the CAT19CAR illustrated herein achieves the killing of CD19-expressing target cells and proliferates in response to CD19-expressing targets, but with less interferon-γ release. Furthermore, small animal models of aggressive B-cell lymphoma showed equivalent efficacy and equivalent engraftment between fmc63- and CAT19-based CAR-T cells, but surprisingly, fewer CAT19 CAR T cells were depleted compared to fmc63 CAR T cells. See Examples 2 and 3 of U.S. Publication No. 2018-0044417.

[0115] In comparative assays involving contacting CAR T cells with target cells, the CAT19CAR described herein resulted in a 25%, 50%, 70%, or 90% reduction in IFN-γ release. Compared to fmc63 CAR T cells, the CAT19CAR described herein resulted in a smaller proportion of CAR T cells becoming exhausted. T cell exhaustion can be assessed using methods known in the art, such as analyses of PD-1 expression. In comparative assays involving contacting CAR T cells with target cells, CAR resulted in 20%, 30%, 40%, 50%, 60%, or 70% reduction in PD-1 expression in CAR T cells compared to fmc63 CAR cells.

[0116] Another exemplary CD19 antigen-binding domain covered by this disclosure is based on the CD19 antigen-binding domain CD19ALAb (described in WO2016 / 102965) and includes:

[0117] a) Heavy chain variable region (VH) with a CDR containing the following sequence:

[0118] CDR1 – SYWMN (SEQ ID No. 44);

[0119] CDR2 – QIWPGDGDTNYNGKFK (SEQ ID No. 45)

[0120] CDR3 – RETTTVGRYYYAMDY (SEQ ID No. 46); and

[0121] b) Light chain variable region (VL) having a CDR containing the following sequence:

[0122] CDR1 – KASQSVDYDGDSYLN (SEQ ID No. 47);

[0123] CDR2 – DASNLVS (SEQ ID No. 48)

[0124] CDR3 – QQSTEDPWT (SEQ ID No. 49).

[0125] It is anticipated that one or more mutations (substitution, addition, or deletion) can be introduced into one or more CDRs without negatively affecting CD19 binding activity. Each CDR may, for example, have one, two, or three amino acid mutations.

[0126] CAR can contain one of the following amino acid sequences.

[0127] SEQ ID No. 22 - Mouse CD19ALAb scFv sequence

[0128] QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQG TTVTVSSDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK

[0129] SEQ ID No. 23 - Humanized CD19ALAb scFv sequence - Heavy chain 19, Kappa 16

[0130] QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKGT LVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPWTFGQGTKVEIKR

[0131] SEQ ID No. 24 (Humanized CD19ALAb scFv sequence - heavy chain 19, Kappa 7)

[0132] QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKGT LVTVSSDIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPWTFGQGTKVEIKR

[0133] scFv can be in the VH-VL direction (as shown in SEQ ID NO: 9, 22, 23 and 24) or the VL-VH direction.

[0134] CAR can contain one of the following VH sequences:

[0135] SEQ ID No. 25 - Mouse CD19ALAb VH sequence

[0136] QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS

[0137] SEQ ID No. 26 - Humanized CD19ALAb VH sequence

[0138] QVQLVQSGAEVKKPGASVKLSCKASGYAFSSYWMNWVRQAPGQSLEWIGQIWPGDGDTNYNGKFKGRATLTADESARTAYMELSSLRSGDTAVYFCARRETTTVGRYYYAMDYWGKGTLVTVSS

[0139] CAR can contain one of the following VL sequences:

[0140] SEQ ID No. 27 - Mouse CD19ALAb VL sequence

[0141] DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK

[0142] SEQ ID No. 28 (humanized CD19ALAb VL sequence, κ16)

[0143] DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKLLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYHCQQSTEDPWTFGQGTKVEIKR

[0144] SEQ ID No. 29 - Humanized CD19ALAb VL sequence, κ7

[0145] DIQLTQSPDSLAVSLGERATINCKASQSVDYDGDSYLNWYQQKPGQPPKVLIYDASNLVSGVPDRFSGSGSGTDFTLTISSLQAADVAVYYCQQSTEDPWTFGQGTKVEIKR

[0146] The CARs provided herein may comprise variants of sequences as shown in any one of SEQ ID NO: 16-29 having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity, provided that the variant sequence retains the ability to bind CD19 (if applicable, when binding to complementary VL or VH domains). The CARs provided herein may comprise variants of sequences as shown in any one of SEQ ID NO: 22-29 or 44-49 having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity, provided that the variant sequence retains the ability to bind CD19 (if applicable, when binding to complementary VL or VH domains). The percentage of identity between two polypeptide sequences can be readily determined by a procedure such as BLAST, which is freely available at blast.ncbi.nlm.nih.gov.

[0147] Transmembrane domain

[0148] The transmembrane domain is the domain of the CAR that crosses the membrane.

[0149] A transmembrane domain can be any protein structure that is thermodynamically stable in the membrane. This is typically an α-helix containing several hydrophobic residues. A transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion described herein. The presence and span of a protein's transmembrane domain can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Furthermore, given that protein transmembrane domains are relatively simple structures, i.e., polypeptides predicted to form hydrophobic α-helices of sufficient length to span the membrane, artificially designed transmembrane domains can also be used (US 7052906 B1 describes synthetic transmembrane components).

[0150] The transmembrane domain can be derived from CD28, which provides good receptor stability. The CD28 transmembrane domain is shown in SEQ ID No. 50.

[0151] SEQ ID No. 50

[0152] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0153] The transmembrane domain can be derived from human Tyrp-1. The tyrp-1 transmembrane domain sequence is shown in SEQ ID No. 51.

[0154] SEQ ID No. 51IIAIAVVGALLLVALIFGTASYLI

[0155] The transmembrane domain can be derived from CD8A. The CD8A transmembrane domain sequence is shown in SEQ ID No. 52.

[0156] SEQ ID No. 52 IYIWAPLAGTCGVLLLSLVITLYC

[0157] Intracellular T cell signaling domain (intramocyte domain)

[0158] The intracellular domain is the signaling component of CARs. Upon antigen recognition, receptors cluster and signals are transmitted to the cell. The most commonly used intracellular domain component is a component of CD3-ζ, which contains three ITAMs. This transmits an activation signal to T cells after antigen binding. CD3-ζ may not provide a fully competent activation signal and may require additional co-stimulatory signaling. For example, intracellular domains from CD28, OX40, or 41BB can be used in conjunction with CD3-ζ to transmit proliferation / survival signals, or all three can be used together.

[0159] Early CAR designs featured intracellular domains derived from the intracellular portion of the γ chain of FcεR1 or CD3ζ. Therefore, these first-generation receptors delivered an immune signal¹ sufficient to trigger T cell killing of homologous target cells, but failed to fully activate T cells for proliferation and survival. To overcome this limitation, composite intracellular domains were constructed. The fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ yielded a second-generation receptor that could simultaneously deliver activation and costimulatory signals upon antigen recognition. The most commonly used costimulatory domain is the CD28 costimulatory domain. This provides the most potent costimulatory signal, i.e., the immune signal², which triggers T cell proliferation. Several receptors, including intracellular domains of the TNF receptor family, such as OX40 and 41BB, which deliver survival signals, have also been described. Finally, even more potent third-generation CARs have been described, possessing intracellular domains capable of delivering activation, proliferation, and survival signals.

[0160] The intracellular domain of the CAR of the present invention may include a combination of one or more of the CD3-ζ intracellular domain, 41BB intracellular domain, OX40 intracellular domain, or CD28 intracellular domain.

[0161] The intracellular T-cell signaling domain (intracellular domain) of the CAR of the present invention may contain sequences as shown in SEQ ID No. 53, 54, 55, 56, 57, 30, 31 or 32 or variants thereof having at least 80% sequence identity.

[0162] SEQ ID No. 53 (CD3ζ intracellular domain)

[0163] RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0164] SEQ ID No. 54 (41BB intracellular domain)

[0165] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0166] SEQ ID No. 55 (OX40 intracellular domain)

[0167] RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0168] SEQ ID No. 56 (CD28 intracellular domain)

[0169] KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY

[0170] Examples of such combinations of intracellular domains include 41BB-Z, OX40-Z, CD28-Z, and CD28-OX40-ζ.

[0171] SEQ ID No. 57 (41BB-Z intracellular domain fusion)

[0172] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0173] SEQ ID No. 30 (OX40-Z intracellular domain fusion)

[0174] RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0175] SEQ ID No. 31 (CD28Z intracellular domain fusion)

[0176] KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0177] SEQ ID No. 32 (CD28OXZ)

[0178] KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0179] The variant sequence may have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID No. 25, 26, 27, 28, 29, 30, 31, or 32, provided that the sequence provides an effective transmembrane domain / intracellular T cell signaling domain.

[0180] signal peptide

[0181] The CAR of the present invention may contain a signal peptide such that when the CAR is expressed in a cell (such as a T cell), the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface where it is expressed.

[0182] The core of a signal peptide can contain a long, hydrophobic amino acid segment with a tendency to form a single α-helix. Signal peptides can begin with a short, positively charged amino acid segment, which helps to reinforce the appropriate topology of the polypeptide during translocation. At the end of the signal peptide, there is usually a segment of amino acids that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave the peptide during or after translocation to produce a free signal peptide and the mature protein. The free signal peptide is then digested by a specific protease.

[0183] Signal peptides can be located at the amino terminus of a molecule.

[0184] The CAR of the present invention can have the following general formula:

[0185] Signal peptide - CD19 binding domain - spacer domain - transmembrane domain / intracellular T cell signal transduction domain.

[0186] Signal peptides can contain variants with 5, 4, 3, 2, or 1 amino acid mutations (insertion, substitution, or addition), provided that the signal peptide still functions to induce CAR expression on the cell surface.

[0187] The signal of SEQ ID No. 33 is a compact and highly efficient peptide. Approximately 95% cleavage is expected after the terminal glycine, allowing for efficient removal by the signal peptidase.

[0188] SEQ ID No. 33: METDTLLLWVLLLWVPGSTG

[0189] The signal of SEQ ID No. 34 is a compact and highly efficient peptide. Approximately 95% cleavage is expected after the terminal glycine, allowing for efficient removal by the signal peptidase.

[0190] SEQ ID No. 34MGTSLLCWMALCLLGADHADA

[0191] The signal peptide of SEQ ID NO: 35 is derived from IgG1.

[0192] SEQ ID No. 35: MSLPVTALLLPLALLLHAARP

[0193] The signal peptide of SEQ ID NO: 36 is derived from CD8.

[0194] SEQ ID No. 36: MAVPTQVLGLLLLWLTDARC

[0195] spacers

[0196] The CAR of the present invention may include a spacer sequence to connect the CD19 binding domain to the transmembrane domain and spatially separate the CD19 binding domain from the intracellular domain. The flexible spacer region allows the CD19 binding domain to be oriented in different directions to achieve CD19 binding.

[0197] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a CD8 stem, or a combination thereof. The spacer may alternatively comprise a surrogate sequence having similar length and / or domain-spacer characteristics to the IgG1 Fc region, IgG1 hinge, or CD8 stem.

[0198] It can alter the human IgG1 spacer to remove the Fc binding motif.

[0199] The following are examples of the amino acid sequences of these spacers:

[0200] SEQ ID No. 37 (Hinge of Human IgG1 - CH2CH3)

[0201] AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD

[0202] SEQ ID No. 38 (Human CD8 Stem):

[0203] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

[0204] SEQ ID No. 39 (Human IgG1 Hinge):

[0205] AEPKSPDKTHTCPPCPKDPK

[0206] SEQ ID No. 40 (IgG1 Hinge - Fc)

[0207] AEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK

[0208] SEQ ID No. 41 (IgG1 Hinge - Fc Modified to Remove Fc Receptor Recognition Motif)

[0209] AEPKSPDKTHTCPPCPAPPVA GPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK

[0210] Modified residues are underlined; Indicates something is missing.

[0211] SEQ ID NO: 42 (CD2 extracellular domain)

[0212] KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD

[0213] SEQ ID NO: 43 (CD34 extracellular domain)

[0214] SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTTSLATSPTKP YTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT

[0215] Interferon release and CAR T cell exhaustion

[0216] The inventors have discovered that CD19 CARs based on CAT19 scFv have properties that can lead to lower toxicity and better efficacy.

[0217] Given that the primary experience with CD19 CAR therapy is based on fmc63 scFv CARs, and that the oldest, largest, and arguably most important clinical dataset is based on the fmc63-based Campana CAR, the inventors have designated the Campana CAR as the "gold standard." Therefore, comparisons were made between the fmc63-Campana CAR and similar CARs (but in which CAT19 scFv replaces fmc63). Surprisingly, the inventors found that CAT19 CAR T cells achieve killing of CD19-expressing target cells and proliferate in response to CD19-expressing targets, while releasing less interferon-γ. Furthermore, small animal models of aggressive B-cell lymphoma showed equivalent efficacy and engraftment between fmc63-based and CAT19-based CARs, but surprisingly, fewer CAT19 CAR T cells were depleted compared to fmc63 CAR T cells.

[0218] In comparative assays involving contact between CAR T cells and target cells, the CAR of the present invention can induce 25%, 50%, 70%, or 90% or lower IFNγ release.

[0219] Compared to fmc63 CAR T cells, the CAR of the present invention can lead to a smaller proportion of CAR T cell exhaustion. T cell exhaustion can be assessed using methods known in the art, such as analysis of PD-1 expression. In comparative assays involving contact between CAR T cells and target cells, the CAR of the present invention can induce 20%, 30%, 40%, 50%, 60%, or 70% fewer CAR T cells to express PD-1 compared to fmc63 CAR T cells.

[0220] Nucleic acid sequence

[0221] A second aspect of the invention relates to a nucleic acid sequence encoding a CAR of the method described herein.

[0222] The nucleic acid sequence can encode a CAR having an amino acid sequence shown in any of SEQ ID No. 10-15.

[0223] As used in this article, the terms “polynucleotide,” “nucleotide,” and “nucleic acid” are intended to be synonymous with each other.

[0224] Nucleic acids can be, for example, RNA, DNA, or cDNA. They can include DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides that include synthetic or modified nucleotides.

[0225] Many different types of modifications to oligonucleotides are known in the art. These include the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule and the use of methylphosphonates and thiophosphates. For the purposes described herein, it should be understood that polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifetime of the polynucleotide of interest.

[0226] When two CARs are encoded by the same vector, alternative codons can be used in sequence regions that encode the same or similar amino acid sequences to avoid homologous recombination.

[0227] Due to the degeneracy of the genetic code, alternative codons encoding the same amino acid sequence can be used. For example, the codons "ccg" and "cca" both encode the amino acid proline, so "ccg" can be replaced with "cca" without affecting the amino acid at that position in the translated protein sequence.

[0228] Table 4 summarizes the alternative RNA codons that can be used to encode each amino acid.

[0229] Table 4

[0230]

[0231] Alternative codons can be used in portions of the nucleic acids encoding the spacers of the first CAR and the second CAR, especially if the same or similar spacers are used in the first and second CARs.

[0232] Alternative codons can be used to encode the transmembrane domain of the first CAR and the transmembrane portion of the nucleic acid of the second CAR, especially if the same or similar transmembrane domains are used in the first and second CARs.

[0233] Alternative codons can be used to encode one or more nucleic acids that encode co-stimulatory domains (such as the CD28 intracellular domain).

[0234] Alternate codons can be used to transmit survival signals in one or more domains, such as the OX40 and 41BB intracellular domains.

[0235] Alternative codons can be used in portions of nucleic acids encoding the CD3ζ intracellular domain and / or portions of nucleic acids encoding one or more co-stimulatory domains and / or portions of nucleic acids encoding one or more domains that transmit survival signals.

[0236] carrier

[0237] The present invention also provides vectors comprising nucleic acid sequences according to the invention. Such vectors can be used to introduce nucleic acid sequences into host cells, causing them to express and produce molecules according to the first aspect of the invention.

[0238] The vector can be, for example, a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector.

[0239] Vectors can be used to transfect or transduce cells, such as T cells.

[0240] cell

[0241] The present invention also provides cells comprising nucleic acids according to the present invention. The present invention provides cells expressing the CARs provided herein on their cell surface.

[0242] The cell can be any eukaryotic cell capable of expressing CAR on its cell surface, such as immune cells.

[0243] Specifically, the cells can be immune effector cells, such as T cells or natural killer (NK) cells.

[0244] T cells, or T lymphocytes, are lymphocytes 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 the T cell receptor (TCR) on their cell surface. Various types of T cells exist, summarized below.

[0245] Helper T cells (TH cells) assist other leukocytes in the immune process, 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 become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to promote different types of immune responses.

[0246] Cytotoxic T cells (TC cells, or CTLs) destroy virus-infected and tumor cells and are also associated with graft rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to MHC class I-associated antigens, which are present on the surface of all nucleated cells. CD8+ cells can be inactivated to an allergic state through IL-10, adenosine, and other molecules secreted by regulatory T cells, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0247] Memory T cells are a subset of antigen-specific T cells that persist long after infection has subsided. They rapidly proliferate into a large number of effector T cells upon re-exposure to their homologous antigens, 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 CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0248] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity at the end of the immune response and to suppress autoreactive T cells that escape negative selection processes in the thymus. Two main classes of CD4+ Treg cells have been described—naturally occurring Treg cells and adaptive Treg cells.

[0249] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) are present in the thymus and are associated with interactions between developing T cells and myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells, leading to the fatal autoimmune disease IPEX.

[0250] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can originate during normal immune responses.

[0251] The T cells described in this article can be any of the above-mentioned T cell types, especially CTLs.

[0252] Natural killer (NK) cells are cytolytic cells that form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0253] NK cells (belonging to the innate lymphoid cell group) are defined as large granular lymphocytes (LGLs) and constitute the third type of cell that differentiates from common lymphoprogenitor cells that produce B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus before entering circulation.

[0254] The CAR-expressing cells provided in this article can be any of the cell types mentioned above.

[0255] The CAR-expressing cells of this invention can be generated in vitro. The cells can be derived from cell samples, such as peripheral blood mononuclear cell (PBMC) samples from a patient or donor. The cells can be activated and / or expanded prior to transduction with nucleic acids encoding the CAR, for example, by treatment with an anti-CD3 monoclonal antibody.

[0256] Alternatively, the T cells or NK cells described in this article can be derived from induced progenitor cells or embryonic progenitor cells and differentiate into T cells or NK cells in vitro.

[0257] Alternatively, immortalized T cell lines that retain their lysis function and can act as therapeutic agents can be used.

[0258] obecabtagene autoleucel (referred to as "obe-cel" in this paper) consists of autologous peripheral blood T cells transduced in vitro using a lentiviral vector encoding the CD19 CAR "CD19 (CAT) CAR" disclosed herein, similar to the approved CAR T-cell therapy Kymriah. ® (tisagenlecleucel), Yescarta ® (Achrenset) and Tecartus ® Compared to the CD19 (FMC63) CAR used in (brexucabtagene autoleucel), this CD19 CAR has lower affinity and faster dissociation from CD19.

[0259] Surface plasmon resonances were used to determine the binding kinetics of CAT scFv (for obe-cel) and FMC63 scFv (for acylencephalosporin and tisagenlecleucel) with recombinant CD19. When the data were fitted to a 1:1 Langmuir binding model, the equilibrium dissociation constant (KD) for CAT was determined to be 14.4 nM, and for FMC63, it was 0.328 nM. The KD of CAT scFv being greater than 40-fold lower is due to its much faster dissociation rate (CAT: 3.1 × 10⁻⁶). 3 s -1 Compared to FMC63: 6.8 × 10 -5 s -1 The binding rate was comparable (CAT: 2.2 × 10⁻⁶). 5 M -1 s -1 Compared to FMC63: 2.1 × 10 5 M -1 s -1 ).

[0260] When co-cultured with Raji and NALM-6 cells, CD19 (CAT) CAR T cells proliferated more than CD19 (FMC63) CAR T cells. This enhanced proliferation was not a result of increased IL-2 production, suggesting an IL-2-independent mechanism.

[0261] Normal donor T cells were transduced to express either CD19 (CAT) CAR or CD19 (FMC63) CAR, challenged with Raji cells (a CD19-expressing Burkitt lymphoma cell line), and pro-inflammatory cytokines in the supernatant were analyzed at 48 hours. The cytokine release profiles of the two CAR-expressing T cell lines were similar, except that CD19 (CAT) CAR T cells secreted higher levels of tumor necrosis factor-α than CD19 (FMC63) CAR T cells (mean values ​​were 750.7 ± 103.3 pg / mL and 292.1 ± 36.51 pg / mL, respectively; n=4, p<0.01).

[0262] In standard 51 In the Cr release assay, T cells transduced to express CD19 (CAT) CAR or CD19 (FMC63) CAR were incubated with CD19-negative and CD19-expressing targets. CD19 (CAT) CAR T cells showed significantly greater cytotoxicity than CD19 (FMC63) CAR T cells, especially at low effector:target ratios.

[0263] The antitumor efficacy of CD19 (CAT) CAR T cells and CD19 (FMC63) CAR T cells was evaluated and compared in a NALM-6 NSG mouse xenograft model. Rapid, disseminated tumor infiltration was observed in control mice receiving untransduced T cells. CD19 (FMC63) CAR T cells slowed but did not stop tumor growth. In contrast, an equivalent number of CD19 (CAT) CAR T cells led to tumor regression. A substantial difference in tumor burden was observed on day 12 post-T cell injection; mean CD19 (CAT) CAR T cells: 1.1 × 10⁻⁶. 8 Up to 9.3 × 10 7 photons / second / cm 2 / Sphericity; CD19(FMC63) CAR T cells 3.2 × 10 9 Up to 7.7 × 10 8 photons / second / cm 2 / Sphericity, n=18, p<0.001. Two weeks after CAR T cell infusion into NALM-6 NSG mice, residual tumor cells and persistent CAR T cells in the blood and bone marrow (BM) were analyzed. A significantly higher number of residual NALM-6 tumor cells (mean 2.8 × 10⁻⁶) were observed in the BM of the CD19 (FMC63) CAR T cell cohort compared to the CD19 (CAT) CAR T cell cohort. 5 With 3 × 10 2 NALM-6 cells / mL, p<0.0001). Conversely, in the BM of NALM-6 NSG mice (mean 3.4 x 10⁻⁶ cells / mL), 4 CD19 (CAT) CAR T cells / mL with 1.3 × 10⁻⁶ cells / mL 4 CD19 (FMC63) CAR T cells / mL, n=18, p<0.05) and in peripheral blood (mean 1.9 × 10⁻⁶). 4 CD19 (CAT) CAR T cells / mL with 2.8 x 10⁻⁶ cells / mL 3 (CD19 (FMC63) CAR T cells / mL, n=9, p<0.001) There was a significantly greater absolute number of CD19 (CAT) CAR T cells than CD19 (FMC63) CAR T cells.

[0264] In summary, in vitro, the CAT scFv binding domain used in obe-cel exhibits a >40-fold lower affinity for recombinant CD19 than the FMC63 scFv binding domain used in other CAR T products. Compared to CD19 (FMC63) CAR T cells, CD19 (CAT) CAR enables transduced T cells to proliferate, respond to CD19-positive targets by secreting cytokines, and lyse CD19-positive cell lines in vitro with greater cytotoxic specificity. In the NSG NALM-6 mouse model of leukemia, CD19 (CAT) CAR T cells also demonstrate better antitumor efficacy and engraftment compared to CD19 (FMC63) CAR T cells.

[0265] Clinical experience with obe-cel CAR T cells includes clinical studies of various hematologic malignancies. Currently, four ongoing studies are evaluating the use of obe-cel in pediatric and adult patients with B-cell acute lymphoblastic leukemia (BALL) and other B-cell malignancies: three investigator-initiated phase I studies, CARPALL (EudraCT2015-001144-10 2019, NCT02443831 2016) (Ghorashian S, Kramer AM, Onuoha S, Wright G, et al. (2019). Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nat Med.25(9):1408-14), ALLCAR19 (EudraCT 2016-004027-22, NCT02935257 2017) (Roddie C, Dias J, O'Reiilly MA, et al. (2021). Durable Responses and Low Toxicity AfterFast Off-Rate CD19 Chimeric Antigen Receptor-T Therapy in Adults WithRelapsed or Refractory B-Cell Acute Lymphoblastic Leukemia. J Clin Oncol; 39(30):3352-63; Roddie C, Dias J, O'Reilly M, Mitsikakou M, et al (2022a). Safety, efficacy and long-term follow-up of AUTO1, a foster-off rate CD19 CARin relapsed / refractory B-cell acute lymphoblastic leukaemia and other B-cellmalignancies. American Society of Hematology Annual Meeting Poster 3318; Roddie C, Dias J, O'Reilly M, Mitsikakou M, et al (2023b).长期随访快速解离速率CD19嵌合抗原受体(CAR)AUTO1在复发 / 难治性B细胞急性淋巴细胞白血病中的情况以及与持久缓解相关的因素。美国移植与细胞治疗学会年会海报277),CAROUSEL(NCT04443829)(Roddie C、Dias J、O’Reilly M、Green L.(2022c)。快速解离速率CD19 CAR AUTO1在复发 / 难治性原发性中枢神经系统淋巴瘤中的安全性和疗效发现。欧洲血液学协会年会海报P1460)以及1项由Autolus赞助的针对复发 / 难治性B-ALL成年患者的Ib / II期AUTO1-AL1(FELIX)研究(EudraCT 2019-001937-16,NCT04404660)。FELIX研究的II期部分是获得obe-cel上市许可的关键研究(Roddie C、Sandhu KS、Tholouli E、Shaughnessy P等人(2023a)。Safety and efficiency of obecabtagene autoleucel (obe-cel, AUTO1), a fast-off rate CD19 CAR, in relapsed / refractory adult B-cell acute lymphoblastic leukemia (r / r B-ALL): top line result of the pivotal FELIX study. (American Society of Clinical Oncology Annual Meeting Oral abstract #7000). As of August 1, 2023, a total of 201 patients with various B-cell malignancies have been treated with obe-cel in these four studies. Based on the novel mode of action of the CAT CAR construct with increased physiological T cell activation and reduced cytokine release, obe-cel has demonstrated a favorable safety and tolerability profile, with very low rates of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) in various indications in both pediatric and adult patients (Ghorashian et al., ibid.; Roddie et al. (2021), ibid.; Roddie et al. (2022a), ibid.; Roddie et al. (2023a), ibid.). No clinical studies of obe-cel have been conducted in patients with autoimmune diseases.

[0266] Table 5. Ongoing OBE-CEL Clinical Studies

[0267]

[0268]

[0269]

[0270] Abbreviations: AE = Adverse Event; ATIMP = Advanced Therapy Product; BALL = B-cell Acute Lymphoblastic Leukemia; BCLL = B-cell Chronic Lymphoblastic Leukemia; BNHL = B-cell Non-Hodgkin Lymphoma; BM = Bone Marrow; B SLL = Small B-cell lymphoma; CAR = Chimeric antigen receptor; CD = Differentiation cluster; CNS = Central nervous system; CR = Complete remission; CRi = Complete remission with incomplete recovery of blood counts; DLBCL = Diffuse large B-cell lymphoma; EudraCT = European Medicines Agency clinical trial; ICANS = Immune effector cell-related neurotoxicity syndrome; IRRC = Independent response review committee; IV = Intravenous; min = Minimal; MRD = Minimal residual disease; NCT = National clinical trial; NGS = Next-generation sequencing; ORR = Overall response rate; PCNSL = Primary central nervous system lymphoma; r / r = Relapsed or refractory; qPCR = Quantitative polymerase chain reaction; PD = Progressive disease; SAE = Serious adverse event; SD = Stable disease.

[0271] Available clinical data from the Phase I CARPALL and ALLCAR19 studies and the Phase Ib / II FELIX study of obe-cel in treating patients with B ALL support the non-clinical data and demonstrate promising efficacy with manageable toxicity in patients with r / r B ALL.

[0272] Of the 13 pediatric or young adult patients treated with obe-cel using an open-process manufacturing method in CARPALL (cohort 1), ten achieved MRD-negative CR after obe-cel infusion, resulting in an ORR of 77%. All seven patients treated with obe-cel using a closed-process manufacturing method (cohort 2) achieved MRD-negative CR after obe-cel infusion (ORR = 100%).

[0273] In the ALLCAR19 study, 17 out of 20 evaluable adult patients achieved MRD-negative CR after obe-cel infusion, resulting in an ORR of 85%.

[0274] As of March 16, 2023, in FELIX, among 94 patients with ≥5% blasts in bone marrow (BM) at screening and who received obe-cel infusion, 71 patients achieved CR+CRi, resulting in an ORR of 75.5%.

[0275] CRS of any grade occurred in 95% of pediatric and young adult BALL patients in CARPALL, 55% of adult BALL patients in ALLCAR19, and 69% of adult BALL patients who received all infusions in FELIX (N=126). Three patients (2.4%) experienced ≥ grade 3 CRS. Severe neurotoxicity was limited, with 1 patient (5%) in the CARPALL study, 3 patients (15%) in the ALLCAR19 study, and 9 patients (7.1%) in the FELIX study reporting ≥ grade 3 neurological AEs / ICANS.

[0276] Preliminary results obtained in indolent B NHL appear promising, with all 10 patients treated with obe-cel achieving a complete molecular response (data cutoff date: November 18, 2021). At this small sample size, the safety profile remains favorable, with four patients experiencing Grade 1 CRS, two patients experiencing Grade 2 CRS, and no ICANS reported.

[0277] Pharmaceutical Composition

[0278] The present invention also relates to pharmaceutical compositions comprising one or more CAR-expressing cells of the present invention, a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more other pharmaceutically active peptides and / or compounds. For example, such formulations may be in a form suitable for intravenous infusion.

[0279] Treatment methods

[0280] Cells expressing the CAR molecules described herein, such as T cells or NK cells, can be used to treat autoimmune diseases, particularly those mediated by CD19-positive B lymphocytes or B cells.

[0281] This disclosure provides a method for treating a patient with a CD19-positive B lymphocyte-mediated autoimmune disease, comprising administering CD19 CAR T cells or a population of CD19 CAR T cells to the patient.

[0282] This disclosure provides CD19 CAR T cells or CD19 CAR T cell populations for the treatment of autoimmune diseases mediated by CD19-positive B lymphocytes.

[0283] This disclosure provides the use of CD19 CAR T cells or CD19 CAR T cell populations in the preparation of medicaments for the treatment of autoimmune diseases mediated by CD19-positive B lymphocytes.

[0284] Examples of autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, idiopathic inflammatory myopathy (IIM, myositis), systemic sclerosis, ANCA-associated vasculitis, inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes mellitus, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Graves' disease, Hashimoto's thyroiditis or Hashimoto's disease, myasthenia gravis, neuromyelitis optica, N-methyl-D-aspartate receptor (NMDAR) encephalitis, Lambert-Eton syndrome, scleroderma, vasculitis, pemphigus vulgaris, pemphigus foliaceus, acquired epidermolysis bullosa, bullous pemphigoid, lupus nephritis, membranous nephropathy, Goodpasture's syndrome, immune thrombocytopenic purpura, thrombotic thrombocytopenic purpura, antiphospholipid syndrome, autoimmune hemolytic anemia, and acquired hemophilia.

[0285] Autoimmune diseases can include SLE.

[0286] Autoimmune diseases can be severe SLE.

[0287] Autoimmune diseases can be refractory SLE.

[0288] Autoimmune diseases can include severe, refractory SLE.

[0289] SLE can manifest as lupus nephritis.

[0290] Lupus nephritis can be severe.

[0291] Lupus nephritis can be active.

[0292] SLE can manifest as active, severe lupus nephritis.

[0293] The methods for treating autoimmune diseases described herein relate to the therapeutic use of the cells or cell populations described herein. In this regard, the cells can be administered to a subject suffering from an existing disease or condition to alleviate, reduce, or improve at least one symptom associated with the disease and / or slow, reduce, or halt the progression of the disease. The method can induce or promote cell-mediated killing of CD19-expressing cells, such as B cells.

[0294] SLE treatment

[0295] Given the important role of B cells in the pathogenesis of SLE and the evidence of the safety and activity of CD19 CAR T-cell therapy, this article anticipates that infusion of patients with CD19 CAR T-cell products (e.g., obe-cel) will eliminate dysfunctional autoreactive B cells and subsequently generate normal nonreactive B cells.

[0296] Patients awaiting treatment include, but are not limited to, patients with severe, refractory SLE who are expected to have poor outcomes if treated with standard care due to the severity of their disease, involvement of one or more areas of the SLE-related organ system, and prior exposure to multiple SLE treatment strategies.

[0297] Patients awaiting treatment: Those with a diagnosis of SLE meeting the 2019 European League Against Rheumatism (EULAR) / American College of Rheumatology (ACR) classification criteria for systemic lupus erythematosus (SLE); those who are positive for at least one of the following autoantibodies: antinuclear antibody (ANA) at a titer ≥1:80, or anti-dsDNA (≥30 IU / mL), or anti-Sm (>ULN), anti-histone, or anti-chromatin (>ULN); and those who may have severe SLE, defined as:

[0298] a) Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score ≥ 8 (of which 4 are non-laboratory scoring items, and scores of neurological related items [SLEDAI 2K items 1-7] are excluded) and

[0299] b) At least one of the following significant SLE-related organ involvements:

[0300] i) Kidneys (persistently active, biopsy-confirmed lupus nephritis),

[0301] ii) Moderate or severe pericarditis / myocarditis,

[0302] iii) Moderate or severe pleurisy or other lung involvement,

[0303] iv) Vasculitis, and

[0304] v) Severe hematological manifestations (e.g., severe thrombocytopenia or severe autoimmune hemolytic anemia);

[0305] and / or

[0306] Patients may have treatment-resistant SLE (defined as no response, inadequate response, or lack of sustained response) or be intolerant to the following treatments:

[0307] a) Combined treatment with hydroxychloroquine and corticosteroids, and

[0308] b) ≥2 or fewer treatment groups, each lasting at least 3 months, or a shorter duration if intolerance occurs:

[0309] i) Immunosuppressive drugs (such as methotrexate, azathioprine, mycophenolate mofetil, mycophenolic acid, tacrolimus, leflunomide, cyclosporine, vorciclosporine, or cyclophosphamide).

[0310] ii) Agents targeting B cells (e.g., belimumab, anti-CD20 mAb), and

[0311] iii) Cytokine inhibitors (e.g., avolimumab).

[0312] Patients can undergo pre-treatment conditioning chemotherapy, such as lymphopenic chemotherapy, prior to CD19 CAR therapy. Lymphopenic chemotherapy may include treatment with fludarabine and cyclophosphamide. In contrast to CD19 CAR therapy, patients can receive three doses of fludarabine 25 mg / m² on days -5, -4, and -3. 2 Administered intravenously on day 3, and in contrast to CD19CAR treatment, received a single dose of cyclophosphamide 1000 mg / m² on day 3. 2 Administer intravenously.

[0313] In the treatment methods for autoimmune diseases such as SLE described in this article, a single dose of 50 x 10 mmol / L can be administered to the patient. 6 (±20%) CD19 CAR-positive live T cells. The expected replacement dose is incremental (100 × 10⁻⁶). 6 [±20%]) or decreasing (30 × 10 6 [±20%]) doses of CAR-positive T cells. Administration may be via intravenous injection, for example, through a Hickman catheter or a peripherally inserted central catheter (PICC line).

[0314] It will be understood that variations may exist in, for example, the starting materials (leukocyte apheresis) and / or preparation of CD19 CAR-positive T cells. These variations may result in dose variability, i.e., variability in the amount of CD19 CAR-positive T cells administered to a patient. The dose is expected to have variability of ±5%, ±10%, ±15%, ±20%, ±25%, or ±30%.

[0315] Treated patients will show improvement in one or more of the following assessments:

[0316] SLE remission (Doris) definition: improvement may include SLEDAI=0 and / or PGA<0.5 on the visual analog scale of 0-3;

[0317] Low disease activity status of lupus (LLDAS), improvement may include SLEDAI-2K < 4 (no activity in major organ systems (renal, central nervous system, cardiopulmonary, vasculitis, fever and no hemolytic anemia or gastrointestinal activity), no new features of lupus disease activity compared to previous assessment, PGA < 1 on the visual analog scale of 0-3, current prednisolone (or equivalent) dose ≤ 7.5 mg daily, and / or well-tolerated standard maintenance doses of immunosuppressive drugs and approved biologics, excluding investigational drugs;

[0318] Systemic lupus erythematosus Disease Activity Index-2000 (SLEDAI-2K) score;

[0319] Physician Overall Assessment (PGA);

[0320] Health Assessment Questionnaire - Disability Index (HAQ-DI);

[0321] Functional assessment of chronic disease therapy - Fatigue Scale (FACIT) score;

[0322] National assessment of estrogen safety in lupus erythematosus - SLEDAI (SELENA-SLEDAI) outbreak index (SFI) score;

[0323] Urinary protein-to-creatinine ratio (UPCR);

[0324] Autoantibody group (antinuclear antibody [ANA], anti-double-stranded DNA [anti-dsDNA], anti-Smith, anti-RNA binding protein [anti-RBP]);

[0325] Overview of antiphospholipids (lupus anticoagulants, anticardiolipin antibodies, and β-2 glycoprotein 1)

[0326] Complement group (CH50, C3, C4);

[0327] The duration of B-cell regeneration impairment;

[0328] Determination of T and B cell phenotypes in peripheral blood over time by flow cytometry assessment

[0329] Determination of the dynamic concentration of BlyS / BAFF in peripheral blood; and

[0330] Determining the dynamics of cytokine concentrations in peripheral blood.

[0331] Other terms and publications

[0332] As used herein and in the appended claims, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural indicators. It should also be noted that claims may be drafted to exclude any element, such as any optional element. Therefore, this statement is intended to serve as a precondition for the use of exclusive terms such as “solely,” “only,” or the use of negative limitations in relation to the recitation of claim elements.

[0333] When numerical ranges are provided herein, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range and any other value or intermediate value within the range, up to one-tenth of the lower limit unit, is covered within the present invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and also covered within this disclosure, but are subject to any specifically excluded limitations within the ranges. Where the range includes one or both limits, the range excluding any or both of these included limits is also included in this disclosure.

[0334] 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 pertains. Any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this disclosure.

[0335] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials used for the purposes of citing those publications.

[0336] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of this disclosure. Any method described may be performed in the order of the events described or in any other logically possible order. This disclosure is intended to support all such combinations.

[0337] As used herein, “can,” “may contain,” “may be,” “can,” “may contain,” and “may be” all indicate that the inventor envisions something functional and available as part of the subject matter provided.

[0338] The following are various abbreviations.

[0339]

[0340]

[0341] The invention will now be further described by way of examples, which are intended to help those skilled in the art to implement the invention, and are not intended to limit the scope of the invention in any way.

[0342] Example

[0343] The examples describe studies designed to demonstrate the safety, tolerability, and preliminary efficacy of obe-cel CAR T cells in patients with severe refractory SLE.

[0344] Conceptually, CAR T cell-based therapies such as obe-cel offer a further advantage over antibodies because CAR T cells require only a single administration, expansion, and have the potential to persist in vivo, migrate to multiple lymphoid tissues and organs in the recipient, and develop into effector and memory cell populations. When indirectly compared to approved FMC63 CD19 CAR T cells, the longer persistence of obe-cel, with its CAT CD19 binder, provides another advantage.

[0345] As described above, obe-cel is prepared from enriched autologous T cells transduced with a lentiviral vector to express a novel second-generation CAR that targets CD19 and has 4-1BB and CD3-ζ intracellular domains.

[0346] The obe-cel CAR T-cell CAR contains a single-chain variable fragment (scFv) called CAT, which has a lower affinity for CD19 and a faster dissociation rate compared to the FMC63 scFv used in other approved CD19 CAR T-therapies. This rapid dissociation binding kinetics is expected to offer opportunities for physiological T-cell activation, reduced immunotoxicity, and improved durability. See WO2016 / 139487.

[0347] obe-cel is classified as an Advanced Therapeutic Investigational Drug (ATIMP). It is an autologous cell therapy product in which the active ingredient is the patient's own T cells transduced using a lentiviral vector to express a novel second-generation CAR targeting CD19 (sometimes referred to in this article as "CD19CAT CAR"). obe-cel also contains untransduced autologous T cells and non-T cells.

[0348] Once administered to a patient, obe-cel CAR T cells circulate throughout the body. CAR T cells recognize the CD19 surface antigen on all B cells and B progenitor cells, leading to CAR T cell activation and proliferation. The activated CAR T cells then subsequently cause the destruction of these cells through several physiological effector mechanisms.

[0349] Example 1 - Qualification Criteria

[0350] The patients treated were those with severe, refractory SLE, who were expected to have poor outcomes if treated with standard care due to the severity of their disease, involvement of one or more areas of the SLE-related organ system, and prior exposure to multiple SLE treatment strategies.

[0351] Patients must meet all eligibility criteria.

[0352] Key inclusion criteria

[0353] The diagnosis of SLE meets the 2019 European League Against Rheumatism (EULAR) / American College of Rheumatology (ACR) classification criteria for systemic lupus erythematosus.

[0354] The autoantibody is positive for at least one of the following: antinuclear antibody (ANA) titer ≥1:80, or anti-dsDNA (≥30 IU / mL) or anti-Smith (> ULN), anti-histone or anti-chromatin (> ULN).

[0355] Severe SLE is defined as

[0356] - Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score ≥ 8 (4 points for non-laboratory scoring items, excluding neurological related items [SLEDAI 2K items 1-7]) and

[0357] - At least one of the following significant SLE-related organ involvements:

[0358] Kidney (persistent active, biopsy-confirmed lupus nephritis). Note: A biopsy should be performed 12 months prior to the screening visit or during the screening period (unless contraindicated) to confirm ongoing active, biopsy-confirmed lupus nephritis. A histopathological report is required as the source.

[0359] Moderate or severe pericarditis / myocarditis

[0360] Moderate or severe pleurisy or other lung involvement

[0361] vasculitis

[0362] Severe hematological manifestations (e.g., severe thrombocytopenia or severe autoimmune hemolytic anemia)

[0363] Treatment-resistant SLE (defined as no response, inadequate response, or lack of sustained response) or intolerance to the following treatments:

[0364] a. Hydroxychloroquine combined with corticosteroids, and

[0365] b. ≥ Two or fewer treatment groups, each lasting at least 3 months, or for a shorter period if intolerable:

[0366] i. Immunosuppressive drugs (such as methotrexate, azathioprine, mycophenolate mofetil, mycophenolic acid, tacrolimus, leflunomide, cyclosporine, vorciclosporine, or cyclophosphamide)

[0367] ii. Agents targeting B cells (e.g., belimumab, anti-CD20 mAb)

[0368] iii. Cytokine inhibitors (e.g., avolimumab)

[0369] Key Exclusion Criteria

[0370] SLE and Autoimmunity

[0371] Candidates must have a history of recurrent neuropsychiatric lupus at any point prior to the screening visit, or have had active, severe, or unstable neuropsychiatric lupus within the past two years.

[0372] Diagnosis of drug-induced SLE rather than idiopathic SLE.

[0373] The screening period requires immediate treatment and / or results in any acute, severe lupus-related flare-ups that prevent immunosuppressive washout; thus, as determined by the investigator or sponsor, the patient is ineligible for CD19 CAR T therapy.

[0374] Researchers believe that CD19 CAR T-cell therapy is unlikely to benefit patients with significant, potentially irreversible organ damage associated with SLE (e.g., end-stage renal disease).

[0375] Diagnosis of primary antiphospholipid syndrome.

[0376] Diagnosis of clinically significant uveitis.

[0377] Diagnosis of other non-SLE autoimmune diseases (e.g., dermatomyositis, polymyositis, scleroderma, rheumatoid arthritis) or other non-SLE autoimmune diseases or overlap syndromes.

[0378] Medical history

[0379] 1. The following medical history or conditions exist:

[0380] a. Within 3 months prior to the screening visit

[0381] Clinically relevant CNS pathologies, such as epilepsy, hemiparesis, aphasia, or stroke.

[0382] Evidence of deep vein thrombosis or pulmonary embolism

[0383] b. Any time prior to screening visit: severe brain injury, dementia, Parkinson's disease, cerebellar disease, organic brain syndrome, uncontrolled mental illness or psychosis.

[0384] 2. Clinically significant uncontrolled heart disease that is not due to SLE (New York Heart Association class III or IV heart failure, uncontrolled angina, severe uncontrolled arrhythmia or acute ischemia or ECG evidence of a grade 3 conduction system abnormality, unless the patient has a pacemaker) or a recent (within the 12 months of screening) cardiac event.

[0385] 3. Active or uncontrolled fungal, bacterial, viral (including COVID-19) or other infections requiring systemic antimicrobial management.

[0386] 4. Active or latent hepatitis B or active hepatitis C.

[0387] 5. Screening results for positive human immunodeficiency virus, HTLV-1, HTLV-2, or syphilis.

[0388] 6. History of malignant tumors, unless disease-free for at least 24 months (allowing for basal cell or squamous cell carcinoma in situ, or breast cancer in situ currently undergoing hormone therapy).

[0389] 7. History of heart, lung, kidney, liver transplantation or hematopoietic stem cell transplantation.

[0390] 8. Pregnancy and breastfeeding.

[0391] Laboratory and organ function

[0392] 9. Patients are not eligible if they meet the following laboratory criteria.

[0393] a. Neutrophil count <1,000 cells / µL

[0394] b. Platelet count <50,000 / µL

[0395] c. Hemoglobin < 7 g / dL in SLE-related hemolytic anemia or < 8 g / dL in all other patients.

[0396] d. B cell regeneration disorder

[0397] e. Serum alanine aminotransferase / aspartate aminotransferase ratio > 2.5 x ULN

[0398] f. Total bilirubin >1.5 x ULN in patients without Gilbert's syndrome or direct bilirubin >1 x ULN in patients with Gilbert's syndrome.

[0399] g. Creatinine clearance (estimated by Cockcroft Gault) < 30 mL / min / 1.73m 2

[0400] h.INR and PTT > 1.5 ULN.

[0401] 10. Left ventricular ejection fraction < 45% (or < lower limit of normal) confirmed by ECHO.

[0402] 11. SpO2 < 90% under conditions where oxygen support is absent.

[0403] Drug treatment

[0404] Previous treatment with anti-CD19 therapy (including bispecific), adoptive T-cell therapy, or any prior gene therapy product (such as CAR T-cell therapy).

[0405] Within 2 months of leukapheresis: use anti-CD20 therapy.

[0406] Immunize with live or attenuated vaccines within 2 months of leukapheresis.

[0407] The following conditions must be ruled out within 14 days prior to apheresis:

[0408] a. Use of standard antimalarial drugs, immunosuppressive therapy, or immunomodulatory therapy, including biologics.

[0409] b. Systemic corticosteroids, more than 10 mg prednisone or its equivalent daily.

[0410] Within 2 months or 5 half-lives of leukapheresis: for any investigational drug, the longer of the two-month or five-half-life period shall prevail.

[0411] Note: If the half-life is unknown, please consider that a 2-month elution period may be required.

[0412] Overview

[0413] The presence of any indwelling catheters or drainage tubes (e.g., percutaneous nephrostomy tube, indwelling Foley catheter, biliary drainage tube, or pleural / peritoneal / pericardial catheter). Note: Central venous access catheters, such as Port-a-Cath or Hickman catheters, are permitted.

[0414] Cannot tolerate leukapheresis.

[0415] Unable to understand or comply with the safety monitoring requirements of the study, or unlikely to complete all study visits or procedures required by the protocol, including follow-up visits.

[0416] There are any contraindications to cyclophosphamide or fludarabine.

[0417] Example 2 - Leukapheresis and Lymphocyte Reduction Therapy

[0418] Leukapheresis

[0419] Prior to apheresis, from day -6 to day -8, patients will undergo clinical and laboratory assessments to confirm that they meet all eligibility criteria.

[0420] Ensure adequate elution and gradual reduction of SLE medication.

[0421] After the required elution is completed, the patient will undergo unstimulated apheresis to generate obe-cel. This may require central venous access and is a day case procedure for collecting only PBMCs.

[0422] Insufficient leukapheresis

[0423] If there are any problems with the procedure (i.e., insufficient cell collection for dose production, contamination of apheresis materials), patients may undergo repeat apheresis if clinically appropriate.

[0424] Bridging therapy

[0425] If it is necessary to control the disease / symptoms, patients can receive bridging therapy with permitted standard of care (SOC) medications to treat SLE.

[0426] Lymphocyte reduction chemotherapy

[0427] Lymphopenic chemotherapy with fludarabine and cyclophosphamide is considered standard care prior to CAR T-cell infusion. Clinical CAR T-cell data collectively indicate that CAR T-cell engraftment requires fludarabine / cyclophosphamide, and that CAR T-cell engraftment requires activity against B cells.

[0428] The patient will receive three doses of fludarabine and one dose of cyclophosphamide, as follows:

[0429] Fludarabine: Administered intravenously (IV) at 25 mg / m² on days -5, -4, and -3. 2 / d

[0430] Cyclophosphamide: 1000 mg / m² IV on day -3 2

[0431] This lymphoreduction protocol is consistent with that published by Mackensen et al., 2022. If obe-cel infusion cannot be performed within 10 days of lymphoreduction, the lymphoreduction therapy can be repeated.

[0432] Example 3 – obe-cel

[0433] The starting material used to generate obe-cel is mononuclear cells from a non-mobilized apheresis collection of leukocytes from the patient. Therefore, obe-cel is an autologous product and is thus specific to each patient.

[0434] Autologous T cells were enriched and transduced in vitro using a lentiviral vector to express a novel CD19-targeting (i.e., anti-CD19) CAR called “CD19 (CAT) CAR.” The “CAT” binding domain was chosen as the binding domain because it showed a significantly lower affinity for CD19 (>40-fold) than FMC63 scFv (a binder used in already marketed CAR T-cell therapies).

[0435] More specifically, the CD19 (CAT) CAR construct is derived from mouse CAT13.1E10 hybridoma, wherein the CAR contains an intracellular domain fusion of a compound containing anti-CD19 scFv, CD8-derived stem and transmembrane domains, and 4-1BB-CD3ζ intracellular domain.

[0436] obe-cel was generated by in vitro transduction of activated peripheral blood mononuclear cells using an engineered HIV-derived lentiviral vector containing a CD19 CAR expression cassette. The lentiviral vector was produced under Good Manufacturing Practice (GMP) conditions via co-transfection of HEK293T cells with four plasmids, followed by harvesting and purification of the culture supernatant.

[0437] Cells from apheresis starting material are enriched by positively selecting CD4+ / CD8+ cells using magnetic beads on the Miltenyi CliniMACS® Prodigy bioreactor. Cells are then washed and stimulated with mitotic ligands and cytokines. One day after activation, cells are transduced using a lentiviral vector. Following transduction, cells (drug substance) are expanded to produce the desired dose. Cells are then washed and formulated with phosphate-buffered saline (PBS) / ethylenediaminetetraacetic acid (EDTA) / human serum albumin (HSA) / dimethyl sulfoxide (DMSO) buffer, filled into final packaging, and cryopreserved (drug product). The obe-cel product contains both transduced and untransduced T cells. The dose given to a patient is expressed as the total number of CD19 CAR-positive T cells (active substance).

[0438] obe-cel is provided as a cryopreserved cell suspension, stored in an infusion bag compatible with cryopreservation, and will thaw at 37°C.

[0439] obe-cel dosage

[0440] The choice and rationale for the obe-cel dosage are based in part on the previous use of obe-cel in patients with B-ALL and other B-cell malignancies.

[0441] To date, obe-cel has been administered to over 200 patients with B-cell malignancies. Different dosing strategies have been evaluated.

[0442] In targeting relapsed / refractory CD19 + In the pivotal FELIX study of B-ALL patients, 410 x 10⁻⁶ cells were administered as a fractionated dose on days 1 and 10, based on the percentage of blast cells in the bone marrow, prior to the initiation of lymphocyte-reducing chemotherapy. 6The planned total target dose of CAR T cells (Roddie et al. (2021), ibid.; Roddie et al. (2023a), ibid.). This dosing regimen was safe and effective in these difficult-to-treat relapsed / refractory B-ALL patients. Importantly, no grade 3 or higher CRS and / or ICANS events were observed in B-ALL patients with complete remission (myeloblasts <5% at the start of lymphocyte-reducing chemotherapy). Any grade of CRS or ICANS was observed in 48% and 7% of patients, respectively.

[0443] However, this study anticipates that, in contrast to relapsed / refractory B-ALL patients carrying significantly higher numbers of CD19-positive B lymphoblasts, the total number of B cells (including autoreactive B cells) in refractory SLE patients is limited. Therefore, this study anticipates the need for much lower doses of obe-cel to reduce and reset B-cell compartments in refractory SLE patients.

[0444] In the first clinical evaluation of CD19 CAR T therapy in a limited number of SLE patients (Mackensen et al., ibid.), a single dose of 1 x 10 6 The CAR T cells / kg body weight were well tolerated and showed promising initial signs of efficacy.

[0445] In this article, a single dose of 50 × 10 6 (±20%) CD19 CAR-positive live T cells will be infused into the patient on day 1 following lymphocyte depletion chemotherapy. Other expected doses are escalating (100 × 10⁻⁶). 6 [±20%]) or decreasing (30 × 10 6 [±20%]) CAR-positive T cell dose.

[0446] Among the currently evaluated dosing strategies, obe-cel has demonstrated a tolerable safety profile and significant CAR T amplification across various patient weights. Compared to the FMC63 CAR T construct used by Mackensen et al., 2022, obe-cel exhibits a different mode of action, a greater AUC tendency, and improved amplification. Based on these product characteristics and considering the impact of CAR T amplification, changes in patient weight or body surface area are expected to have minimal impact on safety and efficacy. 50 x 10 6The starting dose of (±20%) CD19 CAR-positive live T cells was 8-fold lower than the total obe-cel target dose used in adult B-ALL patients, demonstrating a tolerable safety profile in the B-ALL patient population. In two large randomized phase 3 belimumab studies involving a total of 865 SLE patients, the mean (standard deviation) body weight was 61 ± 13 kg, with the lowest weight recorded in these studies being 35 kg (US Food and Drug Administration Arthritis Advisory Committee Briefing Document BLA 125370. October 2010), indicating a 50 × 10⁻⁶ dose. 6 A fixed dose of [±20%] CAR-positive T cells is indeed expected to be safe and effective.

[0447] Example 4 - Exclusion criteria for obe-cel infusion

[0448] The exclusion criteria for obe-cel infusion are as follows.

[0449] 1. Use of any systemic corticosteroid greater than 10 mg daily prednisone or its equivalent within 72 hours prior to obe-cel infusion.

[0450] 2. Body temperature >38℃ unrelated to SLE.

[0451] 3. Severe complication of obe-cel infusion requiring systemic antibiotics. (If the severe complication is considered to be under control, continuing systemic antibiotics to complete the course of treatment does not constitute a criterion for blocking obe-cel infusion.)

[0452] 4. During planned obe-cel infusion, SpO2 < 90% in the absence of oxygen support.

[0453] 5. Persistent non-hematological adverse events (AEs) that have not recovered to baseline or are ≤ grade 2 for any reason.

[0454] 6. Ongoing therapeutic anticoagulation.

[0455] 7. Uncontrolled cardiac arrhythmia.

[0456] Example 5: Assessment of OBE-CEL infusion and disease activity and response to treatment

[0457] After a 2-day rest period following lymphatic depletion (days -2 and -1), the patient received a single intravenous (IV) dose of obe-cel.

[0458] Infuse obe-cel on day 1. Briefly, obe-cel is thawed under sterile conditions in a 37°C water bath. Obe-cel is administered via intravenous infusion (IV) for a short period (up to 30 minutes after thawing to maintain cell viability) through a central or peripheral venous line using a syringe or gravity-assisted infusion via an 18-gauge blood device.

[0459] The assessment of disease activity and response to treatment includes the following.

[0460] Table 6. Endpoint

[0461]

[0462]

[0463]

[0464]

[0465] Table 7 Other endpoints

[0466]

[0467] Example 6 - A single-arm, open-label, phase I study to determine the safety, tolerability, and preliminary efficacy of obecabtagene autoleucel (obe-cel) in patients with severe, refractory systemic lupus erythematosus (SLE).

[0468] Research Design / Description

[0469] Participants are expected to receive a target dose of 50 × 10⁻⁶. 6 (±25%) of CD19 CAR-positive T cells were infused via a single Obe-cel infusion.

[0470] The primary endpoint was the percentage of participants who achieved the CRR at 12 months following the obe-cel infusion, as determined by the Independent Response Review Committee (IRRC).

[0471] Following Day 1 (infusion day), at least 10 days of post-infusion safety monitoring is required, with particular attention to identify signs and symptoms indicating potential adverse events (AEs) specific to CAR T cells of particular interest. This includes daily contact with the on-site clinical team and on-site visits according to the SoA (Self-Assessment of Action). Once the 10-day post-infusion safety monitoring period has ended, participants will be followed up every 1 to 3 days, with contact with the on-site clinical research team and on-site outpatient visits on days 15, 22, 25, and 28. If fever ≥38°C or neurobehavioral changes occur, the investigator must be contacted immediately, and the participant may require readmission.

[0472] Based on emerging data, the study may be discontinued due to inadequate response or excessive treatment toxicity.

[0473] The study included three time periods:

[0474] 1. Screening period: From day -30 to the registration date

[0475] a. Screening: After obtaining informed consent, participants will undergo a screening process to confirm their eligibility to participate in the study and their ability to undergo apheresis.

[0476] b. Apheresis: Eligible participants will undergo apheresis to collect mononuclear cells for the production of obe-cel.

[0477] c. Enrollment: Participants will be enrolled in the study once they meet all eligibility criteria and their apheresis material has been accepted for production.

[0478] 2. Treatment period: From the day of registration until day 1 until the end of treatment (EOT) / day 1

[0479] a. Lymphopenia (LD) assessment: Participants will undergo an assessment from day -8 to day -6 to confirm eligibility to begin LD.

[0480] b. LD chemotherapy: Participants will receive intravenous (iv) fludarabine and cyclophosphamide for LD, thereby enhancing treatment efficacy and CD19 CAR T cell survival.

[0481] Fludarabine: 25 mg / m² on days -5, -4, and -3. 2 / dayiv

[0482] Cyclophosphamide: 1,000 mg / m² on day -3 2 iv

[0483] Day -2, Day -1 = 48 hours of washing. Note: There is no Day 0. After Day -1, the next step is infusion, defined as Day 1.

[0484] c. Administration: If a participant's eligibility to receive an infusion is confirmed, the participant will receive a single infusion on Day 1. Note: EOT is defined as the day of infusion (Day 1) or the day if the study is terminated before infusion.

[0485] 3. Post-treatment period: Day 1 to end of study (EOS)

[0486] Follow-up on efficacy and safety:

[0487] a. Safety and efficacy will be closely monitored in all participants following infusion. The first efficacy assessment will be conducted on day 28 post-infusion.

[0488] b. Subsequently, participants will be assessed for safety, tolerability, and efficacy monthly or every 3 months until EOS.

[0489] c. Following the 24-month visit, participants will be followed up every 3 months until the last participant of the study completes the final visit (LPLV) (i.e., when the last enrolled participant reaches 24 months). The final 3-month follow-up assessment prior to the LPLV is to determine the EOS date for each individual participant.

[0490] d.EOS is defined as the LPLV that has completed the study.

[0491] Key inclusion criteria

[0492] Participants must be between 16 and 65 years old when signing the informed consent form.

[0493] SLE diagnosis meeting the 2019 European League Against Rheumatism / American College of Rheumatology SLE classification criteria.

[0494] The autoantibody is positive for at least one of the following: ANA titer ≥1:80, or anti-dsDNA (≥30 IU / mL) or anti-Smith (>Upper Limit of Normal [ULN]), anti-histone or anti-chromatin (>ULN).

[0495] Severe active SLE is defined as:

[0496] An ASLEDAI-2K score ≥ 8 (with 4 points for non-laboratory scoring items, and excluding scores for neurological related items [SLEDAI-2K items 1-7]), and

[0497] Severely active LN is defined as:

[0498] SLE-related kidney involvement. A biopsy must be performed 6 months prior to or during the screening visit to confirm active, grade III, IV, or V (grade V coexists only with grade III or IV) active or active / chronic LN based on renal biopsy evidence according to the International Society of Nephrology / Society for Nephrology 2019.

[0499] UPCR levels in 24-hour urine samples >1 mg / mg, and eGFR ≥ 30 mL / min / 1.73 m 2

[0500] Treatment-resistant SLE (defined as no response, inadequate response, lack of sustained response, or intolerance associated with this type of drug that precludes its further use):

[0501] a. Hydroxychloroquine combined with corticosteroids, and

[0502] b. Use ≥2 of the following treatment groups for at least 6 months:

[0503] i. Immunosuppressive drugs (e.g., methotrexate, azathioprine, mycophenolate mofetil, mycophenolic acid, leflunomide or cyclophosphamide).

[0504] ii. Calcineurin inhibitors (e.g., cyclosporine, vorticol, tacrolimus).

[0505] iii. Agents targeting B cells (e.g., belimumab, anti-CD20 mAb) or cytokine inhibitors (e.g., avolimumab).

[0506] Key Exclusion Criteria

[0507] Those with recurrent neuropsychiatric lupus at any point before screening, or those with active, severe, or unstable neuropsychiatric lupus within one year of screening.

[0508] Participants who require immediate treatment during screening and / or experience more than one acute, severe lupus-related flare-up that prevents immunosuppressive washout, thus disqualifying them from receiving CD19 CAR T therapy, are excluded. (Treatment for one flare-up is permitted, but the participant must be completely rescreened; such cases should be discussed with the medical monitor.)

[0509] Researchers believe that significant, potentially irreversible organ damage associated with SLE (e.g., ESRD) is unlikely to benefit participants from CD19 CAR T cell-based gene therapy.

[0510] Diagnosis of clinically significant uveitis

[0511] Participants are ineligible if any of the following laboratory criteria are met.

[0512] Note: If one or more laboratory parameters are not up to standard, the screening can be repeated once within the 30-day screening period.

[0513] - Neutrophil count <1,000 cells / µL.

[0514] - Platelet count <50,000 / µL. - Hemoglobin <7 g / dL in SLE-associated hemolytic anemia or <8 g / dL in all other participants.

[0515] - B cell regeneration disorder.

[0516] - Serum alanine aminotransferase / aspartate aminotransferase >2.5 × ULN.

[0517] - Total bilirubin >1.5 × ULN in participants without Gilbert's syndrome, or direct bilirubin >1 × ULN in participants with Gilbert's syndrome.

[0518] - International normalized ratio and activated partial thromboplastin clotting time >1.5 ULN.

[0519] Prohibited and permitted drugs are listed in Table 8.

[0520] Table 8: Prohibited / Permitted Drugs

[0521]

[0522]

[0523] Table 9: Evaluation Timeline

[0524]

[0525]

[0526]

[0527] Example 7 - A single-arm, open-label, phase I study to determine the safety, tolerability, and preliminary efficacy of obecabtagene autoleucel (obe-cel) in patients with severe, refractory systemic lupus erythematosus (SLE): Results from the first treated patient.

[0528] A 32-year-old female SLE patient was treated with obe-cel. The patient, a Black or African American, was diagnosed with SLE in 2021 with a history of kidney (LN IV / V), musculoskeletal, dermal, and immune system involvement, and hypertension. At screening, she had ANA, anti-dsDNA, anti-Smith, anti-RBP, anti-chromatin autoantibodies, and a SLEDAI-2K score of 24. Previous SLE treatments included hydroxychloroquine, corticosteroids, mycophenolate mofetil, rituximab, and oxotuzumab. After tapering off immunosuppressive drugs, the patient received 50 × 10⁵ mmol / L on day 1 of lymphopenic chemoradiotherapy. 6 A single dose of treatment was administered to (±20%) CD19 CAR-positive live T cells. Methylprednisolone was used as a bridging therapy.

[0529] therapeutic effect

[0530] At the 2-month follow-up, the patient showed clear signs of efficacy with obe-cel treatment:

[0531] • Anti-dsDNA: decreased from >300 at screening to 40.6;

[0532] • Improvement in proteinuria;

[0533] • Complement returned to normal;

[0534] • Serum creatinine was 4.6 mg / dL;

[0535] · IgG levels decreased from 6.07 at screening to 2.39 on day 28. Specifically, IgA decreased from 1.84 to 0.46, and IgM decreased from 0.53 to 0.12.

[0536] • The SLEDAI-2K score dropped from 24 at screening to less than 2 in the second month. Figure 1 );and

[0537] • Impaired B cell regeneration.

[0538] Security

[0539] Prior to infusion, the patient experienced a grade 3 serious adverse reaction (SAE), namely an infected abscess on the left sternocleidomastoid process.

[0540] No CRS or ICANS was observed after infusion.

[0541] Following obe-cel infusion, the patient exhibited the following SAE:

[0542] • Grade 2 CMV reactivation treated with valganciclovir;

[0543] • Grade 3 malignant hypertension;

[0544] • Suspected grade 3 neutropenia secondary to valganciclovir. Newer drugs administered include acyclovir and G-CSF.

[0545] CAR-T cell expansion

[0546] CD19 CAR-T cells (obe-cel) showed good expansion using flow cytometry and ddPCR, reaching a peak on day 10 after CAR-T cell infusion, as shown below. Figure 2 As shown in a and 2b.

[0547] This patent application claims the benefit of UK application No. 2316183.9, filed October 23, 2023; UK application No. 2404412.5, filed March 27, 2024; UK application No. 2412689.8, filed August 29, 2024; and UK application No. 2414148.3, filed September 26, 2024. These applications are incorporated herein by reference in their entirety.

[0548] All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations to the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications to the described modes of carrying out the invention that will be apparent to those skilled in molecular biology, CAR technology, or related fields are intended to fall within the scope of the appended claims.

Claims

1. A method of treating an autoimmune disease in a patient, comprising administering to the patient cells containing a chimeric antigen receptor (CAR), wherein the CAR contains a CD19 binding domain, the CD19 binding domain comprising: a) Heavy chain variable regions (VH) having complementarity-determining regions (CDRs) having the following sequence: CDR1-GYAFSSS (SEQ ID NO: 1); CDR2 – YPGDED (SEQ ID NO: 2) and CDR3 – SLLYGDYLDY (SEQ ID NO: 3), and b) A light chain variable region (VL) having a CDR having the following sequence: CDR1 – SASSSVSYMH (SEQ ID NO: 4), CDR2 – DTSKLAS (SEQ ID NO: 5) and CDR3-QQWNINPLT (SEQ ID NO: 6).

2. The method according to claim 1, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

3. The method according to claim 2, wherein the autoimmune disease is severe SLE.

4. The method according to claim 2, wherein the autoimmune disease is refractory SLE.

5. The method according to any one of the preceding claims, wherein the cells are autologous peripheral blood T cells transduced in vitro to express the CAR.

6. The method according to any one of the preceding claims, wherein 50 × 10 6 (±20%) dose of CD19 CAR-positive live T cells.

7. The method according to any one of the preceding claims, wherein 100 × 10 6 (±20%) dose of CD19 CAR-positive live T cells.

8. The method according to any one of the preceding claims, wherein 30 × 10 6 (±20%) dose of CD19 CAR-positive live T cells.

9. The method according to any one of the preceding claims, wherein the administration is intravenous injection.

10. The method according to any one of the preceding claims, 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 of either having at least 95% sequence identity.

11. The method of claim 10, wherein the CD19 binding domain comprises scFv in the VH-VL direction.

12. The method of claim 11, wherein the CD19 binding domain comprises the scFv sequence as shown in SEQ ID NO: 9 or a variant thereof having at least 90% sequence identity.

13. The method according to any one of the preceding claims, wherein the CAR comprises a CD8 stem spacer.

14. The method according to any one of the preceding claims, wherein the CAR comprises an intracellular T cell signaling domain, the intracellular T cell signaling domain comprising a 41BB intracellular domain and a CD3-ζ intracellular domain.

15. A method for treating an autoimmune disease in a patient, comprising administering 50 × 10 to the patient. 6 (±20%) doses of CD19CAR-positive live T cells.

16. A method for treating an autoimmune disease in a patient, comprising administering 30 × 10 to the patient. 6 (±20%) doses of CD19CAR-positive live T cells.

17. A method for treating an autoimmune disease in a patient, comprising administering 100 × 10 6 (±20%) doses of CD19CAR-positive live T cells.

18. The method according to claims 15 to 17, wherein the autoimmune disease is SLE.

19. The method of claim 18, wherein the autoimmune disease is severe SLE.

20. The method of claim 18, wherein the autoimmune disease is refractory SLE.

21. The method according to claims 18 to 20, wherein the SLE exhibits active lupus nephritis.

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