Method for treating autoimmune disease

Abstract

Provided in the present disclosure is a chimeric antigen receptor targeting CD20, CD19 and CD22. Further provided is an engineered immune effector cell (e.g. a T cell) containing the chimeric antigen receptor. Further provided are a pharmaceutical composition and a method for treating an autoimmune disease (e.g. systemic lupus erythematosus).

Description

Methods for treating autoimmune diseases

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of International Application No. PCT / CN2024 / 070936, filed on January 5, 2024, and International Application No. PCT / CN2024 / 134941, filed on November 27, 2024, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Declaration

[0004] This application contains a sequence listing submitted electronically in the form of an XML file named "IEC240761PCT-seq1.xml". This XML file was created on January 2, 2025 and is 102,665 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to methods for treating autoimmune diseases, comprising administering to a subject an effective amount of a CD20, CD19, and CD22 antagonist. The present disclosure also relates to multispecific chimeric antigen receptors, combinations of chimeric antigen receptors, engineered immune effector cells, and methods of using the same. Background Art

[0006] Autoimmune disease (AID) refers to a disease caused by the body's immune response to its own antigens, resulting in damage to its own tissues. The exact pathogenesis of autoimmune diseases is unclear and is closely related to multiple immune abnormalities. Recent studies have found that B cells capable of producing autoantibodies play a key role in the pathogenesis of some autoimmune diseases, including systemic lupus erythematosus (SLE), systemic sclerosis (SSc), ANCA-associated vasculitis (AAV), idiopathic inflammatory myopathies (IIM), multiple sclerosis (MS), neuromyelitis optica spectrum disease (NMOSD) / anti-myelinoligodendrocyte glycoprotein-IgG associated disorders (MOGAD), and myasthenia gravis (MG).

[0007] Current treatments for autoimmune diseases have limited efficacy. Traditional treatments rely on glucocorticoids, immunosuppressants, and biologics. However, these treatments are often empirical and ineffective, with widespread, nonspecific effects on immune and inflammatory pathways, causing short-term and long-term adverse reactions and potentially exacerbating organ damage. Therefore, there is a need for a drug and method that is effective in treating autoimmune diseases with minimal side effects.

[0008] Because B cells play a key role in the pathogenesis of some autoimmune diseases, B-cell-targeted therapies have become a mainstream approach in novel drug development. For example, belimumab, a fully humanized monoclonal antibody targeting B-cell activating factor (BAFF) / B lymphocyte stimulator (BLyS), and tetasipumab, a recombinant fusion protein targeting both BAFF and proliferation-inducing ligand (APRIL), have been approved for the treatment of SLE patients. However, the efficacy of these biologics remains limited, necessitating an urgent need for new treatments. Immune cell therapies (such as CAR-T and CAR-NK) offer significant advantages, including strong specificity, virtually no toxic side effects, and long-lasting efficacy in vivo, potentially addressing the shortcomings of traditional therapies. Although no CAR-T products have yet been approved for the treatment of autoimmune diseases, B-cell-targeted chimeric receptor T-cell therapy offers promising potential for more thorough B-cell depletion, further deepening disease remission, and potentially offering a cure.

[0009] Overview

[0010] The present disclosure provides a method of treating an autoimmune disease (AID) in a subject in need thereof, comprising administering to the subject an effective amount of a CD20, CD19, and CD22 antagonist.

[0011] In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE).

[0012] In some embodiments, the autoimmune disease is systemic sclerosis (SSc), ANCA-associated vasculitis (AAV), idiopathic inflammatory myopathies (IIM), or an autoimmune disease of the nervous system.

[0013] In some embodiments, the autoimmune disease of the nervous system is multiple sclerosis (MS), neuromyelitis optica spectrum disease (NMOSD) / anti-myelinoligodendrocyte glycoprotein-IgG associated disorders (MOGAD), or myasthenia gravis (MG).

[0014] In some embodiments, the autoimmune disease is a relapsed / refractory autoimmune disease.

[0015] In some embodiments, the subject has had an inadequate response to at least one drug for treating systemic lupus erythematosus selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tetanusip, rituximab, anirumab, chloroquine, hydroxychloroquine, stem cell therapy, and immune cell therapy.

[0016] In some embodiments, the subject has had an inadequate response to at least two drugs for treating systemic lupus erythematosus selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tetasip, rituximab, anirumab, chloroquine, hydroxychloroquine, stem cell therapy, and immune cell therapy.

[0017] In some embodiments, the subject has had an inadequate response to a combination of glucocorticoids and at least one drug for treating systemic lupus erythematosus selected from mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tetanusip, rituximab, anirumab, chloroquine, hydroxychloroquine, stem cell therapy, and immune cell therapy.

[0018] In some embodiments, the subject has had an inadequate response to a combination of glucocorticoids and at least two drugs for treating systemic lupus erythematosus, selected from mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tebucentrum, rituximab, anirumab, chloroquine, hydroxychloroquine, stem cell therapy, and immune cell therapy.

[0019] In some embodiments, the immune cell therapy is CAR-T or CAR-NK targeting at least one of CD19, CD20, and BCMA.

[0020] In some embodiments, the subject has concurrent lupus nephritis (LN).

[0021] In some embodiments, the autoimmune disease is systemic sclerosis.

[0022] In some embodiments, the subject has had an inadequate response to at least one drug for treating systemic sclerosis, selected from antifibrotic drugs (such as nintedanib, pirfenidone), vascular protective agents (such as PDE5 inhibitors, endothelin receptor inhibitors, calcium channel blockers), glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, belimumab, stem cell therapy, and immune cell therapy.

[0023] In some embodiments, the subject has had an inadequate response to an anti-fibrotic drug (such as nintedanib, pirfenidone) and / or a vascular protective agent (such as a PDE5 inhibitor, an endothelin receptor inhibitor, a calcium channel blocker) combined with at least one drug for treating systemic sclerosis, the drug being selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, belimumab, stem cell therapy, and immune cell therapy.

[0024] In some embodiments, the immune cell therapy is CAR-T or CAR-NK targeting at least one of CD19, CD20, and BCMA.

[0025] In some embodiments, the autoimmune disease is ANCA-associated vasculitis.

[0026] In some embodiments, the subject has had an inadequate response to at least one drug for treating ANCA-associated vasculitis selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tetasip, rituximab, mepolizumab, tocilizumab, avacopan, and a JAK inhibitor.

[0027] In some embodiments, the subject has had an inadequate response to a glucocorticoid combined with at least one drug for ANCA-associated vasculitis selected from mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tetasip, rituximab, mepolizumab, tocilizumab, avacopan, and a JAK inhibitor.

[0028] In some embodiments, the autoimmune disease is idiopathic inflammatory myopathy.

[0029] In some embodiments, the subject has had an inadequate response to at least one drug for treating idiopathic inflammatory myopathy selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, and a JAK inhibitor.

[0030] In some embodiments, the subject has had an inadequate response to a glucocorticoid combined with at least one drug for an idiopathic inflammatory myopathy selected from mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, and a JAK inhibitor.

[0031] In some embodiments, the autoimmune disease of the nervous system is multiple sclerosis.

[0032] In some embodiments, the subject has had an inadequate response to at least one drug for treating multiple sclerosis selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, alemtuzumab, cladribine, natalizumab, ocrelizumab, rituximab, ofatumumab, and a sphingosine 1-phoshate receptor (S1PR) modulator.

[0033] In some embodiments, the autoimmune disease of the nervous system is neuromyelitis optica spectrum disorder / anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-associated disease.

[0034] In some embodiments, the subject has had an inadequate response to at least one drug for treating neuromyelitis optica spectrum disorder / anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-associated disease selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, satelizumab, rituximab, inelizumab, ravelizumab, eculizumab, and tocilizumab.

[0035] In some embodiments, the autoimmune disease of the nervous system is myasthenia gravis.

[0036] In some embodiments, the subject has had an inadequate response to at least one drug for treating myasthenia gravis selected from acetylcholinesterase inhibitors, glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, eculizumab, ravelizumab, belimumab, igatimod, rozelixizumab, or requires repeated administration of intravenous immunoglobulin (IVIG) or plasma exchange to alleviate the disease.

[0037] In some embodiments, the subject has had an inadequate response to at least two drugs for treating myasthenia gravis selected from acetylcholinesterase inhibitors, glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, eculizumab, ravulizumab, belimumab, igatimod, and rozelixizumab.

[0038] In some embodiments, the CD20, CD19 and CD22 antagonist is selected from engineered immune effector cells, engineered receptors, antibodies, antibody-drug conjugates (ADCs), aptamers and small RNAs.

[0039] In some embodiments, the CD20, CD19, and CD22 antagonist is a single antagonist, a combination comprising two antagonists, or a combination comprising three antagonists.

[0040] In some embodiments, the CD20, CD19 and CD22 antagonists are a combination of three antagonists, namely a CD20 antagonist, a CD19 antagonist and a CD22 antagonist, wherein: (1) the CD20 antagonist is a first group of engineered immune effector cells comprising an engineered receptor that specifically targets CD20, which comprises: an extracellular antigen binding domain comprising at least one anti-CD20 binding portion, a transmembrane domain and an intracellular signaling domain; (2) the CD19 antagonist is a second group of engineered immune effector cells comprising an engineered receptor that specifically targets CD19, which comprises: an extracellular antigen binding domain comprising at least one anti-CD19 binding portion, a transmembrane domain and an intracellular signaling domain; and (3) the CD22 antagonist is a third group of engineered immune effector cells comprising an engineered receptor that specifically targets CD22, which comprises: an extracellular antigen binding domain comprising at least one anti-CD22 binding portion, a transmembrane domain and an intracellular signaling domain.

[0041] In some embodiments, the CD20, CD19, and CD22 antagonist is an engineered immune effector cell comprising a first engineered receptor that specifically targets CD20, a second engineered receptor that specifically targets CD19, and a third engineered receptor that specifically targets CD22, wherein (1) the first engineered receptor that specifically targets CD20 comprises: a first extracellular antigen binding domain comprising at least one anti-CD20 binding portion, a first transmembrane domain, and a first intracellular signaling domain; (2) the second engineered receptor that specifically targets CD19 comprises: a second extracellular antigen binding domain comprising at least one anti-CD19 binding portion, a second transmembrane domain, and a second intracellular signaling domain; and (3) the third engineered receptor that specifically targets CD22 comprises: a third extracellular antigen binding domain comprising at least one anti-CD22 binding portion, a third transmembrane domain, and a third intracellular signaling domain.

[0042] In some embodiments, the CD20, CD19, and CD22 antagonist is an engineered immune effector cell comprising an engineered receptor that simultaneously targets CD20, CD19, and CD22, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD20 binding portion, at least one anti-CD19 binding portion, and at least one anti-CD22 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0043] In some embodiments, the engineered receptor is selected from the group consisting of an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a T cell antigen conjugate (TAC), or a portion thereof.

[0044] In some embodiments, the CD20, CD19, and CD22 antagonist is an engineered immune effector cell comprising a chimeric antigen receptor (CAR) that simultaneously targets CD20, CD19, and CD22, the CAR comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD20 binding portion, at least one anti-CD19 binding portion, and at least one anti-CD22 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD20 binding portion is more proximal to the transmembrane domain than the anti-CD19 binding portion or the anti-CD22 binding portion is.

[0045] In some embodiments, the anti-CD19 binding moiety is at the N-terminus or C-terminus of the anti-CD22 binding moiety.

[0046] In some embodiments, the CD20, CD19 and CD22 antagonist is an engineered immune effector cell comprising a chimeric antigen receptor that simultaneously targets CD20, CD19 and CD22, the CAR comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD20 binding portion, at least one anti-CD19 binding portion and at least one anti-CD22 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD20 binding portion, the anti-CD19 binding portion and the anti-CD22 binding portion are fused to each other directly or via one or more peptide linkers; and wherein the one or more peptide linkers comprise no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids.

[0047] In some embodiments, the one or more peptide linkers are (GGGGS)n, where n is 1, 2, 3, or 4.

[0048] In some embodiments, the anti-CD20 binding portion, the anti-CD19 binding portion, and the anti-CD22 binding portion are selected from Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), minibody, diabody, single domain antibody (sdAb), or VHH domain.

[0049] In some embodiments, the anti-CD20 binding portion is an anti-CD20 single domain antibody (sdAb) comprising: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (iii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (v) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or 18; a CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20. NO:13; and CDR3 comprising the amino acid sequence of SEQ ID NO:14; or (vi) CDR1 comprising the amino acid sequence of SEQ ID NO:15; CDR2 comprising the amino acid sequence of SEQ ID NO:16; and CDR3 comprising the amino acid sequence of SEQ ID NO:17.

[0050] In some embodiments, the anti-CD19 binding portion is an anti-CD19 single domain antibody (sdAb) comprising: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or 27; a CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23; or (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 24; a CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0051] In some embodiments, the anti-CD22 binding portion is an anti-CD22 single domain antibody (sdAb) comprising: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 30 or 36; a CDR2 comprising the amino acid sequence of SEQ ID NO: 31; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 32; or (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR2 comprising the amino acid sequence of SEQ ID NO: 34; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 35.

[0052] In some embodiments, the CAR comprises: (a) an extracellular antigen binding domain comprising an anti-CD20 sdAb, an anti-CD19 sdAb, and an anti-CD22 sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein: (i) the anti-CD20 sdAb comprises: (i) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 10; (ii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 11; (iii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: 19; or (iv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO: NO:20; (II) the anti-CD19 sdAb comprises: (i) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO:28; or (ii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO:29; (III) the anti-CD22 sdAb comprises: (i) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO:37; or (ii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3, respectively, as shown in SEQ ID NO:38.

[0053] In some embodiments, the anti-CD20 sdAb, the anti-CD19 sdAb, and the anti-CD22 sdAb are each independently a camelid sdAb or a humanized sdAb.

[0054] In some embodiments, the CAR comprises: (I) the anti-CD20 sdAb comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 19, or SEQ ID NO: 20; (II) the anti-CD19 sdAb comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29; and (III) the anti-CD22 sdAb comprises the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.

[0055] In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of: CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0056] In some embodiments, the transmembrane domain is derived from CD8α.

[0057] In some embodiments, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0058] In some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0059] In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain.

[0060] In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of a ligand for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof.

[0061] In some embodiments, the costimulatory signaling domain is derived from CD137.

[0062] In some embodiments, the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

[0063] In some embodiments, the hinge domain is derived from CD8α.

[0064] In some embodiments, the CAR comprises (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-85; or (ii) an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 81-85.

[0065] In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide.

[0066] In some embodiments, the signal peptide is derived from CD8α.

[0067] In some embodiments, the CAR comprises (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-71; or (ii) an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 67-71.

[0068] In some embodiments, the engineered immune effector cells are selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0069] In some embodiments, the amount of the engineered immune effector cells administered to the subject is 0.1 to 1×10 6 Pieces / kg.

[0070] In some embodiments, the subject is lymphodepleted prior to or concurrently with administration of the antagonist.

[0071] In some embodiments, wherein the lymphodepletion comprises administration of cyclophosphamide and fludarabine.

[0072] In some embodiments, lymphodepletion comprises: (1) administering cyclophosphamide 500-1500 mg / m 2 1 dose and fludarabine 15-35 mg / m 2 Once daily for up to 3 days; or (2) cyclophosphamide 200-400 mg / m 2 Once daily for up to 3 days, and fludarabine 15–35 mg / m 2 Once daily, up to 3 days.

[0073] In some embodiments, lymphodepletion comprises intravenous infusion of cyclophosphamide 1000 mg / m 2 1 dose and fludarabine 25 mg / m 2Once daily, up to 3 days.

[0074] In some embodiments, lymphodepletion comprises intravenous cyclophosphamide 300 mg / m 2 Once daily for up to 3 days, and fludarabine 25 mg / m 2 Once daily, up to 3 days.

[0075] In some embodiments, lymphodepletion is initiated at least 5 days prior to administration of the antagonist.

[0076] In some embodiments, the subject is not lymphodepleted prior to administration of the antagonist.

[0077] In another aspect, provided is a use of the CD20, CD19 and CD22 antagonists of the present disclosure in the preparation of a medicament for treating an autoimmune disease in a subject in need thereof.

[0078] In another aspect, provided are the CD20, CD19, and CD22 antagonists of the present disclosure for use in treating an autoimmune disease in a subject in need thereof.

[0079] In another aspect, provided is a use of the CD20, CD19 and CD22 antagonists of the present disclosure in the preparation of a medicament for treating systemic lupus erythematosus in a subject in need thereof.

[0080] In another aspect, provided are the CD20, CD19, and CD22 antagonists of the present disclosure for use in treating systemic lupus erythematosus in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 shows the structure of the trispecific AIO CAR-T.

[0082] Figure 2 shows the in vitro cytotoxicity of mono / bi / trispecific CAR-T cells against the Nalm.6.CD20.Luc cell line. Untransduced T (UnT) cells served as a negative control.

[0083] Figure 3 shows the diversity of major cell subsets and B cell subsets and the expression of CD19, CD20, and CD22 receptors in PBMCs of SLE patients.

[0084] Figures 4A-B show the cytotoxicity of monospecific CAR-T and trispecific AIO CAR-T cells against B cells in PBMCs from SLE patients at E:T ratios of 3:1, 1:3, 1:27, 1:54, or 1:81. UnT served as a negative control group.

[0085] Figures 4C-D show the statistical and flow cytometric analysis of the percentage of CD19+, CD20+, or CD22+ B cells remaining in PBMCs from SLE patients after 24 hours of incubation with single- and trispecific AIO CAR-T cells at an E:T ratio of 3:1. UnT served as a negative control group.

[0086] Figure 5 shows the cytotoxicity of single-, bi-, and tri-specific CAR-T cells against B cells in SLE PBMCs at E:T ratios of 3:1, 1:3, and 1:27 after 72 hours of incubation. UnT served as a negative control group.

[0087] Figure 6 shows flow cytometry analysis of the cytotoxicity of trispecific AIO CAR-T cells against B cells from apheresis (APH) of the same donor at an E:T ratio of 1:3 after 2 hours of incubation. UnT served as a negative control group.

[0088] FIG7 shows a schematic flow diagram of an exemplary AIO CAR-T in vivo efficacy study conducted in a Raji.Luc xenograft NCG mouse model.

[0089] Figure 8A-B shows data analysis of tumor reduction and CAR-T expansion and persistence in response to AIO CAR-T treatment in the Raji.Luc xenograft mouse model. UnT cells were used as a negative control.

[0090] Figure 9 shows the AIO CAR-T infusion and monitoring schedule for the in vivo mouse study.

[0091] Figures 10A-E show the observation results of long-term efficacy and safety-related indicators of mice after AIO CAR-T treatment. Figure 10A shows the changes in tumor photon number, showing the tumor control results, Figure 10B shows the changes in body weight, and Figures 10C and 10D show the expression of human CD45 in various tissues. + Cell infiltration.

[0092] Figures 10E-G show the HE abnormality score chart for various tissues in mice after AIO CAR-T treatment, used to evaluate the safety of AIO CAR-T. ( / : non-target tissue; N: no obvious abnormality; 1+: mild lesions, 2+: mild lesions, 3+: moderate lesions, 4+: severe lesions, 5+: severe lesions).

[0093] Figure 11 shows the cycle of AIO CAR-T cell preparations in clearing B cells in patients, alleviating or curing relapsed / refractory acute B lymphoblastic leukemia (ALL), and restoring normal B cells.

[0094] Figure 12A-B shows the results of the pharmacokinetic analysis of the AIO CAR-T cell formulation, analyzed by flow cytometry (CAR+T cell counts / mL) and qPCR (CAR+copies / μg DNA).

[0095] Details

[0096] The present disclosure is based on novel multispecific chimeric antigen receptors that bind to CD20, CD19, and CD22 or engineered cells comprising the novel multispecific chimeric antigen receptors and their improved properties.

[0097] The human CD19 antigen is a 95 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD20 is a 33-37 kDa non-glycosylated phosphoprotein with four transmembrane regions and intracellular amino and carboxyl termini. Both CD19 and CD20 are widely expressed during B cell development, from the early pre-B cell stage to the mature B cell stage, but are lost during differentiation into plasma cells. CD22 is also a 130-150 kDa B cell lineage-restricted cell surface phosphoglycoprotein. Cytoplasmic CD22 is expressed alongside CD19 at the earliest stages of B cell differentiation and precedes the expression of CD20. With the increasing understanding of the role of B cells in the pathogenesis of autoimmune diseases, targeting B cells has become a common approach for treating these diseases.

[0098] Systemic lupus erythematosus (SLE) is a common chronic autoimmune disease characterized by the presence of large amounts of autoantibodies in the patient's blood and multi-organ involvement (TSOKOS G CN Engl J Med, 2011, 365(22):2110-21.). The incidence of SLE varies by race and region. Currently, the global prevalence of SLE is 0-241 / 100,000, approximately 3.2-97.5 / 100,000 in Asia and the Pacific, and approximately 30-70 / 100,000 in China. SLE is more common in women of childbearing age, with the onset age of women typically being 15-40 years old, and the female-to-male ratio being approximately 7-9:1 (SHEN N et al. Chinese Journal of Internal Medicine, 2023, 62(7):775-84.). With the improvement of disease awareness and the extension of patient survival, the prevalence of SLE has also increased. Lupus nephritis (Lupus nephritis, LN) is kidney damage caused by systemic lupus erythematosus (SLE). Immune complexes are deposited in the glomerular capillary loops, activating complement and causing immune complex glomerulonephritis. Lupus nephritis is one of the common causes of end-stage renal disease in my country (Zhang Hui et al. Chinese Journal of Internal Medicine, 2021, 60(09):784-790.). In my country, the complete remission rate of existing induction therapy for proliferative lupus nephritis is low, and lupus nephritis is prone to relapse.

[0099] Over the past few decades, although improvements in medical care and treatment options have reduced overall morbidity and mortality, and the 5-year survival rate (95%) and 10-year survival rate (91%) of SLE patients have further increased, many patients still have inadequate disease control and develop end-stage organ damage, and patients still die prematurely, with a 20-year survival rate of less than 80% (KASITANON N et al. Medicine (Baltimore), 2006, 85(3):147-56.).

[0100] The current standard of care (SOC) for SLE includes traditional drugs such as glucocorticoids, antimalarial drugs, immunosuppressants, and biologics. However, relatively few drugs are approved for the treatment of SLE. Generally, treatment plans are roughly the same around the world, and are formulated based on the severity of the disease and the specific organs involved. However, traditional treatments are often empirical and ineffective, with nonspecific and widespread effects on immune and inflammatory pathways, causing short-term and long-term adverse reactions, and can aggravate organ damage, further endangering the prognosis and long-term survival of SLE patients, especially those with moderate to severe active SLE. B cells play a crucial role in the pathogenesis of SLE (MERRILL JT et al. Arthritis Rheum, 2010, 62(1):222-33.), and targeted B cell therapy has become the mainstream of new drug development for SLE. However, in the past 60 years, only belimumab, a fully humanized monoclonal antibody targeting B-cell activating factor (BAFF) / B lymphocyte stimulator (BLyS), and tetasipimab, a recombinant fusion protein targeting both BAFF and proliferation-inducing ligand (APRIL), have been approved for the treatment of SLE patients. However, both biologics have limited efficacy, with placebo-corrected SLE responder index (SRI)-4 rates of only 7% or 14% to 44.5% at 52 weeks of treatment (NAVARRA SV et al. Lancet, 2011, 377(9767):721-31 and Wu D et al. Arthritis Rheumatol. 2022;74(suppl 9).[Z].). Therefore, SLE patients urgently need new treatment options with a reasonable safety profile, the ability to reduce disease activity, achieve long-term remission, reduce relapses, prevent or delay organ damage, and reduce the use of hormones and immunosuppressants.

[0101] Heterogeneous antigen expression has been observed in pathological B cells of patients with systemic lupus erythematosus (SLE) and lupus nephritis (LN). CD19, CD20, and CD22 antigens are expressed at different levels on B cells, and B cell signatures expressing single, double, triple, or multiple antigens have been observed in individual patients, with B cells of different subtypes or phenotypes coexisting simultaneously.

[0102] Systemic sclerosis (SSc) is a rare, chronic autoimmune disease characterized by localized or diffuse skin thickening and fibrosis, which can affect internal organs (such as the lungs, kidneys, and cardiovascular system). The pathogenesis has not been fully elucidated, but pathophysiological features include autoimmune dysregulation, inflammation, microangiopathy, and fibrosis (GABRIELLI A, AVVEDIMENTO EV, KRIEG TN Engl J Med, 2009, 360(19):1989-2003). In the United States, the prevalence of SSc is approximately 500,000 to 300,000 per million, while in Asia, it is approximately 200,000 to 50,000 per million (BARNES J, MAYES MD. Curr Opin Rheumatol, 2012, 24(2):165-70). Currently, there is no approved causal treatment for SSc. Conventional treatment is primarily based on empirical medication, lacking high-quality evidence and exhibiting limited efficacy. Treatment options include low- and medium-dose glucocorticoids, immunosuppressants (cyclophosphamide, methotrexate, etc.), antifibrotic therapies, and vascular protective agents (calcium channel blockers, PDE5 inhibitors, endothelin receptor antagonists, etc.). B cells participate in the development and progression of SSc by secreting cytokines, inducing the differentiation of M2 macrophages and Th2 cells, promoting fibrosis, and secreting autoantibodies. Autoantibodies such as anti-centromere antibodies, anti-topoisomerase I antibodies, and anti-RNA polymerase III antibodies are detected in the peripheral blood of 95% of SSc patients. Specific autoantibodies have a predictive effect on disease prognosis and organ involvement (ROSENDAHL AH, K, KRIEG T. Kaohsiung J Med Sci, 2022, 38(3): 187-95.). New treatments are urgently needed for SSc patients, especially for those with refractory disease and major organ involvement, as there is still a huge unmet clinical need.

[0103] ANCA-associated vasculitis (AAV) is a group of systemic autoimmune diseases characterized by the involvement of small blood vessels (arterioles, arterioles, venules, and capillaries), including but not limited to microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) (ROBSON JC, GRAYSON PC, PONTE C, et al. Arthritis Rheumatol, 2022, 74(3):393-9.). The pathological manifestations of GPA are granulomas, necrosis, and vasculitis, while MPA is mainly fibrinoid necrotizing vasculitis with no or minimal immune complex deposition. Studies from Europe suggest that the annual incidence of GPA is approximately 2 to 10 cases per million. Conventional treatment drugs include glucocorticoids and immunosuppressants (cyclophosphamide, mycophenolate mofetil, etc.).

[0104] In ANCA-associated vasculitis, B cell-derived ANCAs lead to neutrophil activation and endothelial damage, which are closely related to the pathogenesis of AAV. It is currently believed that ANCA activates neutrophils after binding to their target antigens. On the one hand, they directly release proinflammatory granules and reactive oxygen species, enhancing adhesion to and damage to the vascular endothelium. On the other hand, activated neutrophils bind to C5a through their surface receptors, activating the alternative complement pathway and further amplifying the damage. Rituximab, which targets B cells, and avacopan, which targets the complement pathway, have both been used in the treatment of AAV. However, in a phase III RCT study (JAYNE DRW, MERKEL PA, SCHALL TJ, et al. N Engl J Med, 2021, 384(7): 599-609.), the disease remission rate in the avacopan group (background treatment: GC combined with rituximab) and the standard treatment group at week 26 was similar (72.3% VS 70.1%), and rituximab did not show superiority to cyclophosphamide in the RITUXVAS study (JONES RB, FURUTAS, TERVAERT JW, et al. Ann Rheum Dis, 2015, 74(6): 1178-82.). Therefore, AAV patients still lack new treatments that can effectively and permanently reduce disease activity, reduce or stop hormone use, and protect long-term organ function.

[0105] Idiopathic inflammatory myopathies (IIMs) are a group of systemic autoimmune diseases that primarily affect the skin and skeletal muscle, including but not limited to dermatomyositis (DM), polymyositis (PM), amyopathic dermatomyositis (ADM), antisynthetase syndrome (ASS), and immune-mediated necrotizing myopathy (IMNM). Due to the rarity of IIMs and evolving classification criteria, accurate epidemiological data are lacking. IIMs are divided into subtypes based on clinical manifestations and histopathology. No treatments validated by high-quality studies have been approved for the treatment of IIMs. Glucocorticoids are still the first-line treatment for IIM. Other treatments include immunosuppressants (azathioprine, methotrexate, cyclophosphamide, etc.), biological agents (such as CD20 monoclonal antibody, IL-6 monoclonal antibody, IFN-α monoclonal antibody), JAK inhibitors, and intravenous immunoglobulin (IVIG). Due to the lack of evidence-based medicine, current diagnosis and treatment are mainly individualized based on myositis subtype, clinical experience, and disease severity. However, these treatments have challenges such as poor response and long-term side effects. A Chinese study reported that myositis-specific antibodies were detected in 88% of IIM patients, suggesting that B cells are involved in the pathogenesis of IIM (CONTICINI E, DOURADO E, BOTTAZZI F, et al. Clin Exp Rheumatol, 2024, 42(2): 213-24.). Recent studies have explored the mechanism of immune abnormalities in IIM patients (SUGIMORI Y, IWASAKI Y, TAKESHIMA Y, et al. ACR Open Rheumatol, 2023, 5(2):93-102.) and found a significant decrease in peripheral blood memory B cells and an increase in plasmablasts, suggesting that B cell activation is involved in the development of IIM. Targeting B cells may be an important development direction for IIM.

[0106] Multiple sclerosis (MS) is an immune-mediated inflammatory demyelinating disease affecting the central nervous system (CNS). The specific etiology is unknown, and the typical pathological features are focal demyelination in the CNS accompanied by varying degrees of inflammation and gliosis, with some axonal preservation (FRISCHER JM, WEIGAND SD, GUO Y, et al. Ann Neurol, 2015, 78(5):710-21.). The treatment of MS includes rapid induction recovery therapy in the acute phase and disease-modifying therapy (DMT) in the remission phase to prevent relapses and prevent the continued progression of disability. Acute treatment is usually first-line high-dose glucocorticoids and second-line plasma exchange. Immunoadsorption and intravenous immunoglobulin (IVIG) injection can also be used as one of the treatment options in the acute phase. Although DMT can reduce the risk of relapse and disability progression in MS patients, they are not a cure. Currently, there is still insufficient evidence to show that the above DMT can reduce the risk of long-term (more than 3 years) relapse and disability progression compared with the placebo group (GONZALEZ-LORENZO M, RIDLEY B, MINOZZI S, et al. Cochrane Database Syst Rev, 2024, 1(1): Cd011381.). As the current research deepens, more and more studies have found that B cells play a vital role in the pathogenesis of MS (GHARIBI T, BABALOO Z, HOSSEINI A, et al. Immunology, 2020, 160(4): 325-35.), and several CD20 monoclonal antibodies have also confirmed their efficacy in large-sample clinical trials (ocrelizumab, rituximab, ofatumumab). Targeted B cell CAR-T therapy can more thoroughly eliminate B cells, further deepen remission and potentially cure the disease, and has good application prospects (MULLARD A. Nature, 2024.).

[0107] Neuromyelitis optica spectrum disease (NMOSD) and anti-myelinoligodendrocyte glycoprotein-IgG associated disorders (MOGAD) are similar to MS in that they are also autoimmune-mediated demyelinating diseases of the CNS, but NMOSD and MOGAD primarily affect the optic nerve and spinal cord. The pathogenesis of NMOSD is primarily related to antibodies against aquaporin-4 (AQP4), while MOGAD is associated with antibodies against MOG. AQP4 and MOG antibodies can cross the blood-brain barrier and bind to AQP4 on astrocytes and MOG on oligodendrocytes in the CNS, respectively, activating the classical complement pathway and causing varying degrees of target cell damage (CACCIAGUERRA L, FLANAGAN EP. Neurol Clin, 2024, 42(1):77-114.). During acute attacks, treatment for NMOSD and MOGAD is similar to that for MS, including high-dose glucocorticoids, plasma exchange, and human immunoglobulin infusions. The goal is to alleviate acute symptoms, improve functional disability, and prevent complications. Because 70% of NMOSD patients relapse within 1 year of initial treatment, and the risk of disability is closely correlated with relapse, clinical practice necessitates the initiation of subsequent preventive sequential therapy as soon as possible after the completion of acute treatment, and long-term adherence to treatment is essential to minimize the accumulation of functional disability caused by recurrent attacks. Both diseases lack effective cures, and the appropriate time to discontinue long-term preventive medications remains controversial. The cumulative functional disability caused by recurrent relapses severely impacts patients' quality of life, and there remains a significant unmet need for treatment for those who fail existing drug treatments. The pathogenesis of both NMOSD and MOGAD is closely linked to antibodies. B-cell-targeted therapies, inelizumab (CD19 monoclonal antibody) and rituximab (CD20 monoclonal antibody), have both demonstrated preliminary efficacy in NMOSD, significantly reducing the overall relapse rate compared with placebo (12% vs 39% and 21.2% vs 45.7%, respectively). The inability of CD19 / 20 monoclonal antibody drugs to completely eliminate B cells and antibodies may be the main reason for the final relapse. Based on this, CAR-T therapy may become a potential cure for these two types of diseases.

[0108] Myasthenia gravis (MG) is an autoimmune disease characterized by an acquired neuromuscular junction disorder mediated by autoantibodies. It is associated with autoantibody formation during adolescence or adulthood that attacks acetylcholine receptors (AChRs), fixes complement, and reduces AChR numbers. The most common antibodies are AChR antibodies, followed by antibodies to muscle-specific receptor tyrosine kinase (MuSK). The global annual incidence of MG is approximately 0.4 to 1 per 100,000 people, with a prevalence of approximately 15 to 25 per 100,000. MG can occur in all age groups, with a slightly higher incidence in women than in men, and a bimodal distribution at ages 30 and 50. Myasthenia gravis is primarily characterized by fluctuating motor weakness affecting the eye muscles, bulbar muscles, limb muscles, and / or respiratory muscles. Clinical manifestations vary greatly, but the risk of myasthenic crisis increases year by year as the disease progresses, seriously threatening the life and health of MG patients (BERROUSCHOT J, BAUMANN I, KALISCHEWSKIP, et al. Crit Care Med, 1997, 25(7): 1228-35.). Generally, acetylcholinesterase inhibitors and immunosuppressive therapy can effectively control or improve clinical myasthenia symptoms, but approximately 10% of MG patients still do not benefit from current standard treatments (MANTEGAZZA R, ANTOZZI C. Ther Adv Neurol Disord, 2018, 11: 1756285617749134.). Currently, limited options exist for this subset of refractory MG. Targeted biologics currently used clinically for the treatment of MG include eculizumab, which targets complement and has been approved by the US Food and Drug Administration (FDA), and rituximab (RTX), which targets B cells and is used off-label. These agents may offer some efficacy, but the overall benefits are limited. Therefore, there is currently no definitive cure for MG, especially refractory MG, and new treatment options are urgently needed. Reports have shown that chimeric antigen receptor (CAR) T cell therapy is feasible in patients with myasthenia gravis.

[0109] Although targeted antibodies have been used to treat autoimmune diseases, antigen escape after targeted therapy can occur through multiple mechanisms, such as decreased gene and protein expression, mutation, splicing, lineage switching, epitope masking, and trygocytosis. CAR-T has been used in clinical trials to treat autoimmune diseases, but no CAR-T products have been approved for the treatment of autoimmune diseases (such as SLE and LN). There is an urgent need for a CAR-T therapy that can simultaneously achieve broad and deep B cell depletion to completely alleviate disease symptoms and improve clinical outcomes.

[0110] This article provides an engineered immune effector cell containing a CD20×CD19×CD22 trispecific CAR. The CAR contains three VHHs in series (one specific for CD19, one specific for CD20, and one specific for CD22). The triple targeting strategy can kill heterogeneous pathogenic B cells extensively and deeply, and minimize the risk of antigen escape, preventing the reduction in efficacy caused by antigen escape and thus reducing relapse. The engineered immune effector cell has the ability to expand persistently and can be used to treat subjects in need at low doses, ultimately achieving long-term drug-free remission or hormone withdrawal in patients with autoimmune diseases (such as systemic lupus erythematosus).

[0111] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, unless otherwise indicated, certain embodiments or features of certain embodiments of the present disclosure may be implemented using conventional techniques of molecular biology, microbiology, recombinant DNA, etc., which are within the capabilities of those skilled in the art. In the meantime, for a better understanding of the present disclosure, definitions and explanations of relevant terms are provided below.

[0112] As used herein, the term "antagonist" refers to a substance that can interfere with or inhibit the physiological effects of a target antigen or a signal transduction pathway mediated by a target antigen. Various types of antagonists are known in the art, including but not limited to engineered immune effector cells, engineered receptors such as engineered T cell receptors (TCRs), chimeric antigen receptors (CARs), T cell antigen conjugates (TACs) or a portion thereof, antibodies, antibody-drug conjugates (ADCs), aptamers, small RNAs, and compound inhibitors.

[0113] As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target antigen by at least one antigen recognition site located in the variable region of an immunoglobulin molecule. The term includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, scFv), nanobodies, and fusion proteins comprising antibodies, and immunoglobulin molecules comprising any other modified configuration of an antigen recognition site. The VH and VL regions of an antibody can also be subdivided into regions with high variability (referred to as complementary determining regions (CDRs)), interspersed with more conservative regions referred to as framework regions (FRs). The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. As used herein, the term "complementary determining regions" or "CDRs" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each variable region of a heavy chain and a light chain contains three CDRs, designated as CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system.

[0114] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, nanobodies, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0115] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0116] As used herein, "single domain antibody" or "sdAb" refers to a single monomeric variable antibody domain and is capable of antigen binding (e.g., a single domain antibody that binds to CD20, CD19, or CD22). Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies that naturally lack light chains (such as antibodies from Camelidae species (e.g., llamas)), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than single domain scaffolds derived from antibodies. Single domain antibodies can be derived from any species, including, but not limited to, mice, humans, camels, llamas, goats, rabbits, and cattle. For example, single domain antibodies can be derived from antibodies produced in Camelidae species, such as camels, llamas, dromedaries, alpacas, and guanacos, as described herein. Other species outside the Camelidae family can produce heavy chain antibodies that naturally lack light chains; VHHs derived from such other species are within the scope of the present disclosure. Single domain antibodies (e.g., VHHs) provided herein can have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As described herein, single domain antibodies can be genetically fused or chemically conjugated to another molecule (e.g., a pharmaceutical agent). Single domain antibodies can be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0117] "Percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after the candidate sequence and, if necessary, gaps have been introduced to achieve the maximum percentage of sequence identity and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be performed in various ways within the skill in the art (e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN). TM (DNASTAR) software). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0118] The term "specificity" refers to the selective recognition of a specific epitope of an antigen by an antigen-binding protein (such as CAR or sdAb). For example, natural antibodies are monospecific. As used herein, the term "multispecific" means that an antigen-binding protein (such as CAR or sdAb) has two or more antigen-binding sites, at least two of which bind to different antigens. As used herein, the term "bispecific" means that an antigen-binding protein (such as CAR or sdAb) has two different antigen-binding specificities. As used herein, the term "trispecific" means that an antigen-binding protein (such as CAR or sdAb) has three different antigen-binding specificities. As used herein, the term "monospecific" CAR represents an antigen-binding protein (such as CAR or sdAb) with one or more binding sites, each of which binds to the same antigen.

[0119] As used herein, the term "valence" refers to the presence of a specified number of binding sites in an antigen binding protein such as a CAR or sdAb. For example, a natural antibody or a full-length antibody has two binding sites and is bivalent. Therefore, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two binding sites, three binding sites, four binding sites, five binding sites, and six binding sites in an antigen binding protein such as a CAR or sdAb, respectively.

[0120] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens into immune effector cells such as T cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." CAR can include an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain of T cells and / or other receptors that are specific for one or more antigens (such as B cell antigens). "CAR-T cells" refer to T cells that express CAR.

[0121] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). Vectors can contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. "Retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, primarily derived from retroviruses. "Lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, primarily derived from lentiviruses (including LTRs).

[0122] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopoeia, the European Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0123] As used herein, the term "excipient" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, humectants, lubricants, fragrances, preservatives, propellants, release agents, bactericides, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" may also refer to a diluent, an adjuvant (e.g., Freund's adjuvant (complete or incomplete) or a vehicle. An excipient may be a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®. TM , polyethylene glycol (PEG) and PLURONICS TM Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th edition 1990). Pharmaceutically acceptable excipients are nontoxic to cells or mammals to which they are exposed at the dosages and concentrations employed. Pharmaceutically acceptable excipients can be aqueous pH buffered solutions.

[0124] Excipients can be sterile liquids such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary excipient. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol (propylene, glycol), water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifier or pH buffer. The composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc. Oral compositions including formulations can contain standard excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. The excipient may be sterile water, physiological saline, glucose, human serum albumin (HSA), dimethyl sulfoxide (DMSO), dextrose or its analogues (eg, dextran 40).

[0125] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (i.e., no longer worsening) of the state of the disease, delaying or slowing the progression of the disease, improvement or alleviation of the state of the disease, and relief of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0126] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a therapeutically effective amount is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the state of the patient's immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered treatments, among others.

[0127] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be a mammal, such as a non-primate or a primate (e.g., a human). A subject can be a human. A subject can be a mammal, such as a human, diagnosed with a disease or condition. A subject can be a mammal, such as a human, who is at risk of developing a disease or condition.

[0128] 1. CD20, CD19, and CD22 antagonists

[0129] CD20, CD19 and CD22 antagonists refer to substances with specific antagonistic CD20, CD19 and CD22 abilities. The CD20, CD19 and CD22 antagonists can have various categories, for example, CD20, CD19 and CD22 antagonists can be selected from engineered immune effector cells, engineered receptors, antibodies, antibody-drug conjugates (ADC), aptamers and small RNAs. CD20, CD19 and CD22 antagonists can be single antagonists, a combination comprising two antagonists, or a combination comprising three antagonists. CD20, CD19 and CD22 antagonists can be single antagonists, for example, a trispecific antibody that simultaneously targets CD20, CD19 and CD22. The CD20, CD19, and CD22 antagonists may be a combination comprising two antagonists, such as a combination comprising an antagonist that simultaneously targets CD20 and CD19 and an antagonist that specifically targets CD22, a combination comprising an antagonist that simultaneously targets CD20 and CD22 and an antagonist that specifically targets CD19, or a combination comprising an antagonist that simultaneously targets CD19 and CD22 and an antagonist that specifically targets CD20. For example, the combination may comprise a bispecific antibody that simultaneously targets CD20 and CD19 and an antibody that targets CD22, a combination comprising a bispecific antibody that simultaneously targets CD20 and CD22 and an antibody that targets CD19, or a combination comprising a bispecific antibody that simultaneously targets CD19 and CD22 and an antibody that targets CD20. The CD20, CD19, and CD22 antagonists may also be a combination comprising three antagonists, such as a combination comprising an antibody that targets CD20, an antibody that targets CD19, and an antibody that targets CD22.

[0130] 1.1. Single antagonist

[0131] The CD20, CD19 and CD22 antagonist may be a single antagonist that simultaneously targets CD20, CD19 and CD22.

[0132] The single antagonist can be an engineered immune effector cell comprising an engineered receptor that simultaneously targets CD20, CD19, and CD22, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD20 binding portion, at least one anti-CD19 binding portion, and at least one anti-CD22 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0133] The single antagonist can be an engineered immune effector cell comprising two engineered receptors, wherein the first engineered receptor simultaneously targets any two of CD20, CD19, and CD22, and the second engineered receptor specifically targets another one of CD20, CD19, and CD22.

[0134] The engineered immune effector cell comprising two engineered receptors can comprise a first engineered receptor that simultaneously targets CD20 and CD19 and a second engineered receptor that specifically targets CD22, wherein (1) the first engineered receptor that simultaneously targets CD20 and CD19 comprises: a first extracellular antigen binding domain comprising at least one anti-CD20 binding portion and at least one anti-CD19 binding portion, a first transmembrane domain, and a first intracellular signaling domain; and (2) the second engineered receptor that specifically targets CD22 comprises: a second extracellular antigen binding domain comprising at least one anti-CD22 binding portion, a second transmembrane domain, and a second intracellular signaling domain.

[0135] The engineered immune effector cell comprising two engineered receptors can comprise a first engineered receptor that simultaneously targets CD20 and CD22 and a second engineered receptor that specifically targets CD19, wherein (1) the first engineered receptor that simultaneously targets CD20 and CD22 comprises: a first extracellular antigen binding domain comprising at least one anti-CD20 binding portion and at least one anti-CD22 binding portion, a first transmembrane domain, and a first intracellular signaling domain; and (2) the second engineered receptor that specifically targets CD19 comprises: a second extracellular antigen binding domain comprising at least one anti-CD19 binding portion, a second transmembrane domain, and a second intracellular signaling domain.

[0136] The engineered immune effector cell comprising two engineered receptors can comprise a first engineered receptor that simultaneously targets CD19 and CD22 and a second engineered receptor that specifically targets CD20, wherein (1) the first engineered receptor that simultaneously targets CD19 and CD22 comprises: a first extracellular antigen binding domain comprising at least one anti-CD19 binding portion and at least one anti-CD22 binding portion, a first transmembrane domain, and a first intracellular signaling domain; and (2) the second engineered receptor that specifically targets CD20 comprises: a second extracellular antigen binding domain comprising at least one anti-CD20 binding portion, a second transmembrane domain, and a second intracellular signaling domain.

[0137] The single antagonist can be an engineered immune effector cell comprising a first engineered receptor that specifically targets CD20, a second engineered receptor that specifically targets CD19, and a third engineered receptor that specifically targets CD22, wherein (1) the first engineered receptor that specifically targets CD20 comprises: a first extracellular antigen binding domain comprising at least one anti-CD20 binding portion, a first transmembrane domain, and a first intracellular signaling domain; (2) the second engineered receptor that specifically targets CD19 comprises: a second extracellular antigen binding domain comprising at least one anti-CD19 binding portion, a second transmembrane domain, and a second intracellular signaling domain; and (3) the third engineered receptor that specifically targets CD22 comprises: a third extracellular antigen binding domain comprising at least one anti-CD22 binding portion, a third transmembrane domain, and a third intracellular signaling domain.

[0138] 1.2. Combinations containing two antagonists

[0139] The CD20, CD19 and CD22 antagonists can be a combination comprising two antagonists, wherein the first antagonist is a first engineered immune effector cell that simultaneously targets any two of CD20, CD19 and CD22, and the second antagonist is a second engineered immune effector cell that specifically targets another one of CD20, CD19 and CD22.

[0140] The combination comprising two antagonists can be a combination of a first engineered immune effector cell that simultaneously targets CD20 and CD19 and a second engineered immune effector cell that specifically targets CD22. The first engineered immune effector cell comprises an engineered immune receptor that simultaneously targets CD20 and CD19, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD20 binding portion and at least one anti-CD19 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain. The second engineered immune effector cell receptor comprises an engineered immune receptor that specifically targets CD22, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD22 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0141] The combination comprising two antagonists can be a combination of a first engineered immune effector cell that simultaneously targets CD20 and CD19 and a second engineered immune effector cell that specifically targets CD22. The first engineered immune effector cell comprises a first engineered receptor that specifically targets CD20 and a second engineered receptor that specifically targets CD19, wherein (1) the first engineered receptor that specifically targets CD20 comprises: a first extracellular antigen binding domain comprising at least one anti-CD20 binding portion, a first transmembrane domain, and a first intracellular signaling domain; (2) the second engineered receptor that specifically targets CD19 comprises: a second extracellular antigen binding domain comprising at least one anti-CD19 binding portion, a second transmembrane domain, and a second intracellular signaling domain. The second engineered immune effector cell receptor comprises an engineered immune receptor that specifically targets CD22, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD22 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0142] The combination comprising two antagonists can be a combination of a first engineered immune effector cell that simultaneously targets CD20 and CD22 and a second engineered immune effector cell that specifically targets CD19. The first engineered immune effector cell comprises an engineered immune receptor that simultaneously targets CD20 and CD22, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD20 binding portion and at least one anti-CD22 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain. The second engineered immune effector cell receptor comprises an engineered immune receptor that specifically targets CD19, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD19 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0143] The combination comprising two antagonists can be a combination of a first engineered immune effector cell that simultaneously targets CD20 and CD22 and a second engineered immune effector cell that specifically targets CD19. The first engineered immune effector cell comprises a first engineered receptor that specifically targets CD20 and a second engineered receptor that specifically targets CD22, wherein (1) the first engineered receptor that specifically targets CD20 comprises: a first extracellular antigen binding domain comprising at least one anti-CD20 binding portion, a first transmembrane domain, and a first intracellular signaling domain; (2) the second engineered receptor that specifically targets CD22 comprises: a second extracellular antigen binding domain comprising at least one anti-CD22 binding portion, a second transmembrane domain, and a second intracellular signaling domain. The second engineered immune effector cell receptor comprises an engineered immune receptor that specifically targets CD19, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD19 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0144] The combination comprising two antagonists can be a combination of a first engineered immune effector cell that simultaneously targets CD19 and CD22 and a second engineered immune effector cell that specifically targets CD20. The first engineered immune effector cell comprises an engineered immune receptor that simultaneously targets CD19 and CD22, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD19 binding portion and at least one anti-CD22 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain. The second engineered immune effector cell receptor comprises an engineered immune receptor that specifically targets CD20, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD20 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0145] The combination comprising two antagonists can be a combination of a first engineered immune effector cell that simultaneously targets CD19 and CD22 and a second engineered immune effector cell that specifically targets CD20. The first engineered immune effector cell comprises a first engineered receptor that specifically targets CD19 and a second engineered receptor that specifically targets CD22, wherein (1) the first engineered receptor that specifically targets CD19 comprises: a first extracellular antigen binding domain comprising at least one anti-CD19 binding portion, a first transmembrane domain, and a first intracellular signaling domain; (2) the second engineered receptor that specifically targets CD22 comprises: a second extracellular antigen binding domain comprising at least one anti-CD22 binding portion, a second transmembrane domain, and a second intracellular signaling domain. The second engineered immune effector cell receptor comprises an engineered immune receptor that specifically targets CD20, the receptor comprising: (a) an extracellular antigen binding domain comprising at least one anti-CD20 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0146] 1.3. Combinations containing three antagonists

[0147] The CD20, CD19 and CD22 antagonists can be a combination of three antagonists comprising a CD20 antagonist, a CD19 antagonist and a CD22 antagonist, wherein: (1) the CD20 antagonists are a first group of engineered immune effector cells comprising an engineered receptor that specifically targets CD20, which comprises: an extracellular antigen binding domain comprising at least one anti-CD20 binding portion, a transmembrane domain and an intracellular signaling domain; (2) the CD19 antagonists are a second group of engineered immune effector cells comprising an engineered receptor that specifically targets CD19, which comprises: an extracellular antigen binding domain comprising at least one anti-CD19 binding portion, a transmembrane domain and an intracellular signaling domain; and (3) the CD22 antagonists are a third group of engineered immune effector cells comprising an engineered receptor that specifically targets CD22, which comprises: an extracellular antigen binding domain comprising at least one anti-CD22 binding portion, a transmembrane domain and an intracellular signaling domain.

[0148] 2. Engineered receptors (e.g., CAR, TCR, and TAC)

[0149] One aspect of the present disclosure provides engineered cells (such as immune cells) expressing engineered receptors. Engineered receptors can include extracellular antigen binding domains and optional intracellular signaling domains. Exemplary engineered receptors include but are not limited to chimeric antigen receptors (CAR), engineered TCR (TCR) and T cell antigen conjugates (TAC) receptors. Engineered receptors can include extracellular antigen binding domains, transmembrane domains and intracellular signaling domains that specifically bind antigens (such as CD20, CD19 and / or CD22). The intracellular signaling domain can include primary intracellular signaling domains and / or costimulatory signaling domains. The intracellular signaling domain can include the intracellular signaling domains of TCR co-receptors. Engineered receptors can be encoded by heterologous polynucleotides operably connected to a promoter (such as a constitutive promoter or an inducible promoter).

[0150] The engineered receptor can comprise one or more specific binding domains that target at least one B cell antigen and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and / or co-stimulatory signaling domains.

[0151] The antigen binding domain can be selected from the group consisting of: Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), single domain antibody (sdAb) and a peptide ligand that specifically binds to a target molecule. The antigen binding domain can be an antibody portion. The antibody portion can be monospecific. The antibody portion can be multispecific, such as bispecific or trispecific. The term "multispecific" is used in a broader sense in this disclosure, that is, if the antigen binding domain can target more than one epitope on the same antigen or can target more than one antigen, the antigen binding domain is multispecific. The antibody portion can be bispecific. The antibody portion can be a tandem scFv, a diabody (Db), a single-chain diabody (scDb), a dual affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, a chemically cross-linked antibody, a heteromultimer antibody or a heterologous conjugate antibody. The antibody portion can be a scFv. The antibody portion can be a single domain antibody (sdAb). The antibody portion can be a VHH. The antibody portion may comprise a tandem VHH. The antibody portion may be camelid, shark, chimeric, fully human, semisynthesized using human antibody framework regions, or humanized. The engineered receptors disclosed herein may comprise an antigen-binding domain comprising one or more VHHs (e.g., any one of 1, 2, 3, 4, 5, 6, or more). The VHHs may be fused to each other directly via a peptide bond or via a peptide linker.

[0152] Chimeric Antigen Receptor (CAR)

[0153] In one aspect, the chimeric antigen receptor (CAR) provided by the present disclosure binds to one or more antigens (e.g., one or more of CD20, CD19, and CD22). CAR can be used as an antagonist of the antigen, or expressed by immune cells, and then used as an antagonist for treatment. CAR may comprise: (a) an extracellular antigen binding domain comprising one or more antigen binding portions (e.g., VHH) that bind to CD20, CD19, and CD22; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0154] Each component and additional region is described in more detail below.

[0155] 2.1.1. Extracellular antigen-binding domain

[0156] The extracellular antigen binding domain of CAR described herein comprises two, three or more single domain antibodies.Single domain antibodies can be fused to each other directly via a peptide bond or via a peptide linker.

[0157] 2.1.1.1. Single-domain antibodies

[0158] The CAR of the present disclosure comprises an extracellular antigen binding domain comprising a variety of single domain antibodies. sdAbs may be of the same or different origins and may be of the same or different sizes. Exemplary sdAbs include, but are not limited to, heavy chain variable domains (e.g., VHH or VHH) from antibodies having only heavy chains. NAR ), binding molecules naturally lacking light chains, single domains derived from conventional 4-chain antibodies (such as V H or V L ), humanized heavy chain-only antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than single domain scaffolds derived from antibodies. Any sdAb known in the art or developed by the present disclosure (including the single domain antibodies described above in the present disclosure) can be used to construct the CAR described herein. sdAb can be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, fish, sharks, goats, rabbits, and cattle. The single domain antibodies considered herein also include naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0159] sdAbs can be derived from naturally occurring single domain antigen binding molecules known as heavy chain antibodies lacking light chains (also referred to herein as "heavy chain-only antibodies"). Such single domain molecules are disclosed, for example, in WO 94 / 04678 and Hamers-Casterman, C. et al., Nature 363:446-448 (1993). For clarity, the variable domains derived from naturally occurring heavy chain molecules lacking light chains are referred to herein as VHH to distinguish them from the conventional VHH of four-chain immunoglobulins. H Such VHH molecules can be derived from antibodies produced in species of the Camelidae family (e.g., camels, llamas, vicunas, dromedaries, alpacas, and guanacos). Species other than the Camelidae family may produce heavy chain molecules that naturally lack light chains, and such VHHs are within the scope of the present disclosure. In addition, humanized forms of the VHHs and other modifications and variants are also contemplated and within the scope of the present disclosure.

[0160] The VHH molecules from camelids are about 10 times smaller than IgG molecules. They are single polypeptides and can be very stable and resistant to extreme pH and temperature conditions. In addition, they can resist the action of proteases, which is not the case with conventional 4-chain antibodies. In addition, the in vitro expression of VHH produces high-yield, correctly folded functional VHH. In addition, the antibodies produced in camelids can recognize epitopes other than those recognized by antibodies produced in vitro using antibody libraries or via immunization of mammals other than camelids (see, for example, WO9749805). Therefore, a multispecific or multivalent CAR comprising one or more VHH domains can interact with the target more efficiently than a multispecific or multivalent CAR comprising an antigen-binding fragment derived from a conventional 4-chain antibody. Since it is known that VHH binds to "unusual" epitopes (such as cavities or grooves), the affinity of the CAR comprising such VHH may be more suitable for therapeutic treatment than conventional multispecific polypeptides.

[0161] sdAbs can be derived from the variable regions of immunoglobulins found in cartilaginous fish. For example, sdAbs can be derived from an immunoglobulin isotype known as the novel antigen receptor (NAR) found in shark serum. Methods for generating single domain molecules derived from the variable regions of NARs ("IgNARs") are described in WO 03 / 014161 and Streltsov, Protein Sci. 14:2901-2909 (2005).

[0162] sdAbs can be recombinant, CDR-grafted, humanized, camelized, deimmunized, and / or produced in vitro (e.g., by phage display selection). The amino acid sequence of the framework region can be altered by "camelization" of specific amino acid residues in the framework region. Camelization refers to the substitution of (naturally occurring) V residues from conventional 4-chain antibodies with the V residues from conventional 4-chain antibodies. H One or more amino acid residues in the amino acid sequence of a VHH domain are replaced or substituted with one or more amino acid residues present at one or more corresponding positions in the VHH domain of a heavy chain antibody. This can be done in a manner known in the art, as will be clear to the skilled person. Such "camelizing" substitutions are preferably inserted between the positions forming the V H -V L interface and / or at amino acid positions present at said interface, and / or at so-called Camelid Hallmark residues, as defined herein (see, e.g., WO 94 / 04678; Davies and Riechmann FEBS Letters 339:285-290 (1994); Davies and Riechmann, Protein Engineering 9(6):531-537 (1996); Riechmann, J. Mol. Biol. 259:957-969 (1996); and Riechmann and Muyldermans, J. Immunol. Meth. 231:25-38 (1999)).

[0163] sdAbs can be human single domain antibodies produced by transgenic mice or rats expressing human heavy chain segments. See, for example, US20090307787, US Patent No. 8,754,287, US20150289489, US20100122358, and WO2004049794. sdAbs can be affinity matured.

[0164] Naturally occurring VHH domains directed against a particular antigen or target can be obtained from (original or immune) libraries of Camelidae VHH sequences. Such methods may or may not involve screening such libraries using the antigen or target or at least a portion, fragment, antigenic determinant or epitope thereof using one or more screening techniques known in the art. Such libraries and techniques are described in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semisynthetic libraries derived from (original or immune) VHH libraries can be used, such as VHH libraries obtained from (original or immune) VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, for example as described in WO 00 / 43507.

[0165] Single-domain antibodies can be generated from conventional four-chain antibodies. See, for example, EP 0 368 684; Ward et al., Nature, 341(6242):544-6 (1989); Holt et al., Trends Biotechnol., 21(11):484-490 (2003); WO 06 / 030220; and WO 06 / 003388.

[0166] Exemplary single domain antibodies in the CARs provided herein are listed in Table 1.

[0167] Single domain antibodies that bind to CD20

[0168] The anti-CD20 single domain antibodies (e.g., VHH) in the CAR provided herein can bind to human CD20. The anti-CD20 single domain antibodies provided herein can modulate one or more CD20 activities. The anti-CD20 single domain antibodies provided herein can be antagonist antibodies.

[0169] The anti-CD20 single domain antibodies in the CAR provided herein can be VHH domains. As shown in Table 1 above, these VHH domains are referred to as VHH-273, huVHH-253, VHH-496, and huVHH-750.

[0170] The anti-CD20 sdAb in the CAR of the present disclosure may comprise one or more CDR sequences of any one of VHH-273, huVHH-253, VHH-496, and / or huVHH-750. The anti-CD20 sdAb in the CAR of the present disclosure may comprise the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequences are selected from those of VHH-273, huVHH-253, VHH-496, and huVHH-750.

[0171] The anti-CD20 sdAb in the CAR of the present disclosure may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 10. The anti-CD20 sdAb in the CAR of the present disclosure may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 11. The anti-CD20 sdAb in the CAR of the present disclosure may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 19. The anti-CD20 sdAb in the CAR of the present disclosure may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 20. CDR sequences may be determined according to well-known numbering systems. CDRs may be numbered according to IMGT. CDRs may be numbered according to Kabat. CDRs may be numbered according to AbM. CDRs may be numbered according to Chothia. CDRs may be numbered according to Contact. The anti-CD22 single-domain antibody may be of Camelidae. The anti-CD22 single-domain antibody may be humanized. The anti-CD22 single-domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0172] The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. The anti-CD20 single domain antibody may be of camelid origin. The anti-CD20 single domain antibody may be humanized. The anti-CD20 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0173] The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6. The anti-CD20 single domain antibody may be of camelid origin. The anti-CD20 single domain antibody may be humanized. The anti-CD20 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0174] The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. The anti-CD20 single domain antibody may be of Camelidae. The anti-CD20 single domain antibody may be humanized. The anti-CD20 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0175] The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6. The anti-CD20 single domain antibody may be of camelid origin. The anti-CD20 single domain antibody may be humanized. The anti-CD20 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0176] The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or 18; a CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 14. The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 12; a CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 14. The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 18; a CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 14. The anti-CD20 single domain antibody may be of camelid origin. The anti-CD20 single domain antibody may be humanized. The anti-CD20 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0177] The anti-CD20 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 15; a CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17. The anti-CD20 single domain antibody may be of camelid origin. The anti-CD20 single domain antibody may be humanized. The anti-CD20 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0178] The anti-CD20 single domain antibody in the CAR of the present disclosure may further comprise one or more framework regions of VHH-273, huVHH-253, VHH-496, and huVHH-750. The single domain antibody may comprise one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 10. The single domain antibody may comprise one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 11. The single domain antibody may comprise one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 19. The single domain antibody may comprise one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 20.

[0179] The framework regions described herein are defined based on the boundaries of the CDR numbering system. In other words, if the CDRs are defined by, for example, Kabat, IMGT, or Chothia, the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residue N-terminal to the CDR1 amino acid residue as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residue between the CDR1 and CDR2 amino acid residues as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residue between the CDR2 and CDR3 amino acid residues as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residue C-terminal to the CDR3 amino acid residue as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0180] The anti-CD20 sdAb in the CAR of the present disclosure comprises an amino acid sequence having a certain percentage identity with respect to any one of antibodies VHH-273, VHH-496, huVHH-253, and / or huVHH-750.

[0181] The anti-CD20 sdAb in the CAR of the present disclosure comprises a VHH domain having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 10, 11, 19 and 20. In some embodiments, the VHH sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CD20 single domain antibody comprising the sequence retains the ability to bind to CD20. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in an amino acid sequence selected from SEQ ID NOs: 10, 11, 19, and 20. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). Optionally, the anti-CD20 single domain antibody comprises an amino acid sequence selected from SEQ ID NOs: 10, 11, 19, and 20, including post-translational modifications of that sequence.

[0182] The anti-CD20 sdAb in the CAR described in the present disclosure can be those anti-CD20 sdAbs in PCT / CN2020 / 102470, the disclosure of which is incorporated herein by reference.

[0183] Single domain antibodies that bind to CD19

[0184] The anti-CD19 single domain antibodies (e.g., VHH) in the CAR provided herein can be bound to human CD19. The anti-CD19 single domain antibodies provided herein can regulate one or more CD19 activities. The anti-CD19 single domain antibodies provided herein can be antagonist antibodies.

[0185] The anti-CD19 single domain antibodies in the CAR provided herein can be VHH domains. As shown in Table 1 above, these VHH domains are referred to as VHH-083 and huVHH-773.

[0186] The anti-CD19 sdAb in the CAR of the present disclosure may comprise one or more CDR sequences of any one of VHH-083 and / or huVHH-773. The anti-CD19 sdAb in the CAR of the present disclosure may comprise the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequences are selected from those in VHH-083 and huVHH-773.

[0187] The anti-CD19 sdAb in the CAR of the present disclosure may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 28. The anti-CD19 sdAb in the CAR of the present disclosure may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 29. The CDR sequences may be determined according to a well-known numbering system. The CDRs may be numbered according to IMGT. The CDRs may be numbered according to Kabat. The CDRs may be numbered according to AbM. The CDRs may be numbered according to Chothia. The CDRs may be numbered according to Contact. The anti-CD22 single domain antibody may be of camelid origin. The anti-CD22 single domain antibody may be humanized. The anti-CD22 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0188] The anti-CD19 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or 27; a CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23. The anti-CD19 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23. The anti-CD19 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 27; a CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23. The anti-CD19 single domain antibody may be of Camelidae. The anti-CD19 single domain antibody may be humanized. The anti-CD19 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0189] The anti-CD19 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 24; a CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26. The anti-CD19 single domain antibody may be of camelid origin. The anti-CD19 single domain antibody may be humanized. The anti-CD19 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0190] The anti-CD19 single domain antibody in the CAR of the present disclosure may further comprise one or more framework regions of VHH-083 and HuVHH-773. The single domain antibody may comprise one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 28. The single domain antibody may comprise one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 29.

[0191] The framework regions described herein are defined based on the boundaries of the CDR numbering system. In other words, if the CDRs are defined by, for example, Kabat, IMGT, or Chothia, the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residue N-terminal to the CDR1 amino acid residue as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residue between the CDR1 and CDR2 amino acid residues as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residue between the CDR2 and CDR3 amino acid residues as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residue C-terminal to the CDR3 amino acid residue as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0192] The anti-CD19 sdAb in the CAR of the present disclosure comprises an amino acid sequence having a certain percentage identity with respect to either of anti-VHH-083 and / or HuVHH-773.

[0193] The anti-CD19 sdAb in the CAR of the present disclosure comprises a VHH domain having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 28 and 29. In some embodiments, the VHH sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CD19 single domain antibody comprising the sequence retains the ability to bind to CD19. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in an amino acid sequence selected from SEQ ID NOs: 28 and 29. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). Optionally, the anti-CD19 single domain antibody comprises an amino acid sequence selected from SEQ ID NOs: 28 and 29, including post-translational modifications of that sequence.

[0194] The anti-CD19 sdAb in the CAR described in the present disclosure can be those anti-CD19 sdAbs in PCT / CN2020 / 102470, the disclosure of which is incorporated herein by reference.

[0195] Single domain antibodies that bind to CD22

[0196] The anti-CD22 single domain antibodies (e.g., VHH) in the CAR provided herein can bind to human CD22. The anti-CD22 single domain antibodies provided herein can modulate one or more CD22 activities. The anti-CD22 single domain antibodies provided herein can be antagonist antibodies.

[0197] The anti-CD22 single domain antibodies in the CAR provided herein can be VHH domains. As shown in Table 1 above, these VHH domains are referred to as VHH-66 and huVHH-077.

[0198] The anti-CD22 sdAb in the CAR of the present disclosure may comprise one or more CDR sequences of any one of VHH-66 and / or huVHH-077. The anti-CD22 sdAb in the CAR of the present disclosure may comprise the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequences are selected from those in VHH-66 and huVHH-077.

[0199] The anti-CD22 sdAb in the CAR of the present disclosure may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 37. The anti-CD22 sdAb in the CAR of the present disclosure may comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 38. The CDR sequences may be determined according to a well-known numbering system. The CDRs may be numbered according to IMGT. The CDRs may be numbered according to Kabat. The CDRs may be numbered according to AbM. The CDRs may be numbered according to Chothia. The CDRs may be numbered according to Contact. The anti-CD22 single domain antibody may be of Camelidae. The anti-CD22 single domain antibody may be humanized. The anti-CD22 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0200] The anti-CD22 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 30 or 36; a CDR2 comprising the amino acid sequence of SEQ ID NO: 31; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 32. The anti-CD22 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 30; a CDR2 comprising the amino acid sequence of SEQ ID NO: 31; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 32. The anti-CD22 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 36; a CDR2 comprising the amino acid sequence of SEQ ID NO: 31; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 32. The anti-CD22 single domain antibody may be of Camelidae. The anti-CD22 single domain antibody may be humanized. The anti-CD22 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0201] The anti-CD22 sdAb in the CAR of the present disclosure may comprise: a CDR1 comprising the amino acid sequence of SEQ ID NO: 33; a CDR2 comprising the amino acid sequence of SEQ ID NO: 34; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 35. The anti-CD22 single domain antibody may be of camelid origin. The anti-CD22 single domain antibody may be humanized. The anti-CD22 single domain antibody may comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0202] The anti-CD22 single-domain antibody in the CAR of the present disclosure may further comprise one or more framework regions of VHH-66 and huVHH-077. The single-domain antibody may comprise one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 37. The single-domain antibody may comprise one or more frameworks derived from a VHH domain comprising a sequence of SEQ ID NO: 38.

[0203] The framework regions described herein are defined based on the boundaries of the CDR numbering system. In other words, if the CDRs are defined by, for example, Kabat, IMGT, or Chothia, the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residue N-terminal to the CDR1 amino acid residue as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residue between the CDR1 and CDR2 amino acid residues as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residue between the CDR2 and CDR3 amino acid residues as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residue C-terminal to the CDR3 amino acid residue as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0204] The anti-CD22 sdAb in the CAR of the present disclosure comprises an amino acid sequence having a certain percentage identity relative to either of anti-VHH-66 and / or huVHH-077.

[0205] The anti-CD22 sdAb in the CAR of the present disclosure comprises a VHH domain having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 37 and 38. In some embodiments, the VHH sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CD22 single domain antibody comprising the sequence retains the ability to bind to CD22. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in an amino acid sequence selected from SEQ ID NOs: 37 and 38. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). Optionally, the anti-CD22 single domain antibody comprises an amino acid sequence selected from SEQ ID NOs: 37 and 38, including post-translational modifications of that sequence.

[0206] The anti-CD22 sdAb in the CAR described in the present disclosure can be those anti-CD22 sdAbs in PCT / CN2020 / 102470, the disclosure of which is incorporated herein by reference.

[0207] 2.1.1.2. Humanized single-domain antibodies

[0208] Single domain antibodies as described herein include humanized single domain antibodies. General strategies for humanizing single domain antibodies from Camelidae species have been described, for example, in Vincke et al., J. Biol. Chem., 284 (5): 3273-3284 (2009)), and can be used to generate humanized VHH domains as disclosed herein. The design of humanized single domain antibodies from Camelidae species can include hallmark residues in VHH, such as residues 11, 37, 44, 45, and 47 (residues are numbered according to Kabat) (Muyldermans, Reviews Mol Biotech 74: 277-302 (2001).

[0209] Humanized antibodies, such as the humanized single domain antibodies disclosed herein, can also be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); and Roguska et al., PNAS 91:969-973 (1994)), chain shuffling, and remodeling. shuffling) (U.S. Patent No. 5,565,332) and the techniques disclosed in, for example, U.S. Patent No. 6,407,213; U.S. Patent No. 5,766,886; WO 9317105; Tan et al., J. Immunol. 169: 1119 25 (2002); Caldas et al., Protein Eng. 13(5): 353-60 (2000); Morea et al., Methods 20(3): 267 79 (2000); Baca et al., J. Biol. Chem. 272(16): 10678-84 (1997); Roguska et al., Protein Eng. 9(10): 895 904 (1996); Couto et al., Cancer Res. 55(23 Suppl): 5973s-5977s (1995); Couto et al., Cancer Res. 55(8): 1717-22 (1995); Sandhu JS, Gene 150(2): 409-10 (1994); and Pedersen et al., J. Mol. Biol. 235(3): 959-73 (1994). See also U.S. Patent Publication No. US 2005 / 0042664 A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0210] The single domain antibodies provided herein can be humanized single domain antibodies that bind to CD20, CD19 or CD22. For example, the humanized single chain antibodies of the present disclosure can comprise one or more of the CDRs set forth in SEQ ID NOs: 10, 11, 19, 20, 28, 29, 37, and 38. Various methods for humanizing non-human antibodies are known in the art.

[0211] 2.1.2. CD20×CD19×CD22 Trispecific CAR

[0212] In yet another aspect, provided herein is a trispecific CAR targeting CD20, CD19, and CD22, the trispecific CAR comprising an anti-CD20 sdAb provided herein, an anti-CD19 sdAb provided herein, and an anti-CD22 sdAb provided herein located in the extracellular antigen binding domain.

[0213] The degree to which the anti-CD20 sdAb is close to the transmembrane domain may be higher than that of the anti-CD19 sdAb or anti-CD22 sdAb. The anti-CD19 sdAb may be at the N-terminus of the anti-CD22 sdAb. The anti-CD19 sdAb may be at the C-terminus of the anti-CD22 sdAb. The three sdAbs in the CAR provided herein may be in an order from N-terminus to C-terminus of anti-CD19 sdAb, anti-CD22 sdAb, and anti-CD20 sdAb. The three sdAbs in the CAR may be in an order from N-terminus to C-terminus of anti-CD22 sdAb, anti-CD19 sdAb, and anti-CD20 sdAb.

[0214] The degree to which the anti-CD19 sdAb is close to the transmembrane domain may be higher than the degree to which the anti-CD20 sdAb or anti-CD22 sdAb is close to the transmembrane domain. The anti-CD20 sdAb may be located at the N-terminus of the anti-CD22 sdAb. The anti-CD20 sdAb may be located at the C-terminus of the anti-CD22 sdAb. The three sdAbs in the CAR may be arranged in an order from N-terminus to C-terminus as anti-CD20 sdAb, anti-CD22 sdAb, and anti-CD19 sdAb. The three sdAbs in the CAR may be arranged in an order from N-terminus to C-terminus as anti-CD22 sdAb, anti-CD20 sdAb, and anti-CD19 sdAb.

[0215] The degree to which the anti-CD22 sdAb is close to the transmembrane domain may be higher than the degree to which the anti-CD19 sdAb or anti-CD20 sdAb is close to the transmembrane domain. The anti-CD19 sdAb may be located at the N-terminus of the anti-CD20 sdAb. The anti-CD19 sdAb may be located at the C-terminus of the anti-CD20 sdAb. The three sdAbs in the CAR may be arranged in an order from N-terminus to C-terminus as anti-CD19 sdAb, anti-CD20 sdAb, and anti-CD22 sdAb. The three sdAbs in the CAR may be arranged in an order from N-terminus to C-terminus as anti-CD20 sdAb, anti-CD19 sdAb, and anti-CD22 sdAb.

[0216] The trispecific CARs provided herein may comprise: (a) an extracellular antigen binding domain comprising an anti-CD20 sdAb, an anti-CD19 sdAb, and an anti-CD22 sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD20 sdAb comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 19, or SEQ ID NO: 20; wherein the anti-CD19 sdAb comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 28 or SEQ ID NO: 29; and wherein the anti-CD22 sdAb comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: The sequence of SEQ ID NO: 37 or SEQ ID NO: 38 has an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0217] The trispecific CARs provided herein may comprise a combination of anti-CD20 VHH, anti-CD19 VHH, and anti-CD22 VHH exemplified in Table 2 below. The trispecific CARs provided herein may comprise a combination of anti-CD20 VHH, anti-CD19 VHH, and anti-CD22 VHH exemplified in Table 2 below, CDRs of anti-CD20 VHH, CDRs of anti-CD19 VHH, and CDRs of anti-CD22 VHH.

[0218] Table 2. Exemplary CD20×CD19×CD22 trispecific CARs

[0219] The CARs provided herein may comprise VHH-273, VHH-083, and VHH-66. The CARs provided herein may comprise VHH-496, VHH-083, and VHH-66. The CARs provided herein may comprise huVHH-750, huVHH-773, and huVHH-077. The CARs provided herein may comprise huVHH-253, huVHH-773, and huVHH-077. The CARs provided herein may comprise VHH-273, huVHH-773, and huVHH-077. The CARs provided herein may comprise VHH-496, huVHH-773, and huVHH-077. The CARs provided herein may comprise VHH-273, huVHH-773, and VHH-66. The CARs provided herein may comprise VHH-496, huVHH-773, and VHH-66. The CARs provided herein may comprise VHH-273, VHH-083, and huVHH-077. The CARs provided herein may comprise VHH-496, VHH-083, and huVHH-077. The CARs provided herein may comprise huVHH-750, VHH-083, and VHH-66. The CARs provided herein may comprise huVHH-253, VHH-083, and VHH-66. The CARs provided herein may comprise huVHH-750, VHH-083, and huVHH-077. The CARs provided herein may comprise huVHH-253, VHH-083, and huVHH-077. The CARs provided herein may comprise huVHH-750, huVHH-773, and VHH-66. The CARs provided herein may comprise huVHH-253, huVHH-773, and VHH-66.

[0220] The order of the three VHHs contained in the CAR described herein from N-terminus to C-terminus is not limited. For example, for a CAR comprising VHH-273, VHH-083 and VHH-66, the order of the three VHHs in the CAR from N-terminus to C-terminus can be VHH-273, VHH-083 and VHH-66, and the order of the three VHHs in the CAR from N-terminus to C-terminus can be VHH-083, VHH-273 and VHH-66. The order of the three VHHs in the CAR from N-terminus to C-terminus can be VHH-273, VHH-66 and VHH-083, the order of the three VHHs in the CAR from N-terminus to C-terminus can be VHH-083, VHH-66 and VHH-273, the order of the three VHHs in the CAR from N-terminus to C-terminus can be VHH-66, VHH-273 and VHH-083, and the order of the three VHHs in the CAR from N-terminus to C-terminus can be VHH-66, VHH-083 and VHH-273.

[0221] The order of the three VHHs in the CAR described herein from N-terminus to C-terminus can be VHH-083, VHH-66 and VHH-273. The order of the three VHHs in the CAR described herein from N-terminus to C-terminus can be huVHH-773, huVHH-077 and huVHH-750. The order of the three VHHs in the CAR described herein from N-terminus to C-terminus can be huVHH-077, huVHH-773 and huVHH-750. The order of the three VHHs in the CAR described herein from N-terminus to C-terminus can be huVHH-773, huVHH-077 and huVHH-253. The order of the three VHHs in the CAR described herein from N-terminus to C-terminus can be huVHH-077, huVHH-773 and huVHH-253.

[0222] 2.1.3. Other multispecific CARs

[0223] The multispecific CAR provided herein may further include one or more additional binding domains (such as sdAb) bound to one or more additional antigens. One or more additional antigens targeted by the CAR of the present disclosure may be cell surface molecules. Single domain antibodies may be selected to identify antigens that act as cell surface markers on target cells associated with a specific disease state. The antigen may be a B cell antigen. The plasma cells (Plasma cells) antigens of the B cells and their differentiation may be selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD24, CD79a, CD79b, BAFF-R, CD123, CD38, CD138 or BCMA (CD269). The antigen targeted by CAR may be directly or indirectly involved in the disease.

[0224] In addition to the binding domain(s) provided herein, the CARs provided herein may further comprise one or more of the following: a linker (e.g., a peptide linker), a transmembrane domain, a hinge region, a signal peptide, an intracellular signaling domain, a co-stimulatory signaling domain, each of which is described in more detail below.

[0225] For example, the intracellular signaling domain may include a primary intracellular signaling domain of an immune effector cell (such as a T cell). The primary intracellular signaling domain may be derived from CD3ζ. The intracellular signaling domain may include a costimulatory signaling domain. The costimulatory signaling domain may be derived from a costimulatory molecule selected from the group consisting of a ligand of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and a combination thereof. The costimulatory signaling domain may be derived from CD137. The CAR may further include a hinge domain (such as a CD8α hinge domain) between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. The CAR may further include a signal peptide (such as a CD8α signal peptide) at the N-terminus of the polypeptide. The polypeptide may comprise, from N-terminus to C-terminus: a CD8α signal peptide, an extracellular antigen binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ.

[0226] 2.1.4. Peptide linkers

[0227] The various single domain antibodies in the multispecific CAR described herein can be fused to each other via peptide linkers. Single domain antibodies can be fused directly to each other without any peptide linkers. The peptide linkers connecting different single domain antibodies (e.g., VHH) can be the same or different. The different domains of CAR can also be fused to each other via peptide linkers.

[0228] Each peptide linker in CAR may have the same or different length and / or sequence, depending on the structural and / or functional characteristics of the single domain antibody and / or various domains. Each peptide linker can be selected and optimized independently. The length, flexibility and / or other properties of the one or more peptide linkers used in CAR may have some influence on properties including, but not limited to, affinity, specificity or avidity for one or more specific antigens or epitopes. For example, a longer peptide linker can be selected to ensure that two adjacent domains do not interfere with each other spatially. A short peptide linker can be provided between the transmembrane domain and the intracellular signaling domain of CAR. The peptide linker can include flexible residues (such as glycine and serine) so that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0229] The length of the peptide linker between sdAb has an effect on the effect of CAR-T cells, and shorter linkers produce better effects than longer linkers. Therefore, the peptide linker length may be no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less amino acids. The length of the peptide linker can be about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids. The length of the peptide linker may be no more than 30 amino acids. The length of the peptide linker may be no more than 25 amino acids. The length of the peptide linker may be no more than 20 amino acids (e.g., any of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids in length). The length of the peptide linker may be no more than 15 amino acids. The length of the peptide linker may be no more than 10 amino acids. The length of the peptide linker may be no more than 5 amino acids.

[0230] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO 1996 / 34103. The peptide linker may be a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G). n , glycine-serine polymers (including, for example, (GS) n 、(GSGGS) n 、(GGGS) n and (GGGGS) n , wherein n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Exemplary peptide linkers are listed in the table below. A peptide linker connecting two or more anti-CD20 VHH domains provided herein can be (GGGGS) n (SEQ ID NO: 147), wherein n is optionally 1, 2, 3, 4, 5 or 6.

[0231] Table 3. Exemplary peptide linkers

[0232] For example, other linkers known in the art as described in WO2016014789, WO2015158671, WO2016102965, US20150299317, WO2018067992, US7741465; Colcher et al., J. Nat. Cancer Inst. 82: 1191-1197 (1990) and Bird et al., Science 242: 423-426 (1988), the disclosures of each of which are incorporated herein by reference.

[0233] 2.1.5. Transmembrane domain

[0234] The CAR of the present disclosure comprises a transmembrane domain that can be fused directly or indirectly to an extracellular antigen binding domain. The transmembrane domain can be derived from a natural source or a synthetic source. As used herein, a "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain suitable for the CAR described herein can be obtained from naturally occurring proteins. Alternatively, it can be a synthetic non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0235] Membrane spaning domain is classified based on the three-dimensional structure of membrane spaning domain.For example, membrane spaning domain can form alpha helix, more than the complex of an alpha helix, β-barrel structure or any other stable structure that can cross the phospholipid bilayer of cell.In addition, membrane spaning domain can also or alternatively be classified based on membrane spaning domain topology (comprising the number of times that membrane spaning domain passes through film and the orientation of protein).For example, single-pass membrane protein passes cell membrane once, and multipass membrane protein passes cell membrane at least twice (for example, 2,3,4,5,6,7 or more times).Membrane protein can be defined as I type, II type or III type, and this depends on their end and one or more membrane-penetrating segments relative to cell interior and outside topology.Type I membrane protein has single membrane spaning region, and is oriented so that the N-terminal of protein is present in the cell extracellular of lipid bilayer of cell, and the C-terminal of protein is present in cytoplasm side.Type II membrane protein also has single membrane spaning region, but is oriented so that the C-terminal of protein is present in the cell extracellular of lipid bilayer of cell, and the N-terminal of protein is present in cytoplasm side. Type III membrane proteins have multiple transmembrane segments and can be further subdivided based on the number of transmembrane segments and the positions of the N- and C-termini.

[0236] The transmembrane domain of CAR described herein can be derived from type I single-pass membrane protein.The transmembrane domain from multi-pass membrane protein may also be applicable to CAR described herein.Multi-pass membrane protein may include a composite (at least 2, 3, 4, 5, 6, 7 or more) alpha helix or beta folded structure.The N-terminus and C-terminus of multi-pass membrane protein may be present on opposite sides of the lipid bilayer, for example, the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer, and the C-terminus of the protein is present on the extracellular side.

[0237] The transmembrane domain of the CAR may comprise a transmembrane domain selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160 , CD19, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C. The transmembrane domain can be derived from a molecule selected from the group consisting of: CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

[0238] The transmembrane domain may be derived from CD8α. The transmembrane domain may be the transmembrane domain of CD8α comprising the amino acid sequence of SEQ ID NO: 62.

[0239] The transmembrane domain may be derived from CD28. The transmembrane domain may be the transmembrane domain of CD28 comprising the amino acid sequence of SEQ ID NO: 63.

[0240] The membrane-spanning domain for CAR described herein can also include at least a portion of a synthetic non-natural protein segment. The membrane-spanning domain can be a synthetic non-natural alpha helix or beta fold. The protein segment can be at least approximately 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Examples of synthetic membrane-spanning domains are known in the art, for example, in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000 / 032776, and the relevant disclosures of the U.S. Patents and PCT publications are incorporated herein by reference.

[0241] The membrane-spanning domain provided herein can comprise a membrane-spanning region and a cytoplasmic region positioned at the C-terminal side of the membrane-spanning domain. The cytoplasmic region of the membrane-spanning domain can comprise three or more amino acids, which can contribute to orienting the membrane-spanning domain in the lipid bilayer. One or more cysteine ​​residues can be present in the membrane-spanning region of the membrane-spanning domain. One or more cysteine ​​residues can be present in the cytoplasmic region of the membrane-spanning domain. The cytoplasmic region of the membrane-spanning domain can comprise positively charged amino acids. The cytoplasmic region of the membrane-spanning domain can comprise amino acids arginine, serine and lysine.

[0242] The transmembrane region of the transmembrane domain may include hydrophobic amino acid residues. The transmembrane domain of the CAR provided herein may include an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan and valine may be present at the C-terminus of the transmembrane domain. The transmembrane region may mainly include hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine. The transmembrane region may be hydrophobic. The transmembrane region may include a polyleucine-alanine sequence. The hydrophilicity or hydrophobicity or hydrophilic characteristics of a protein or protein segment may be assessed by any method known in the art, such as Kyte and Doolittle hydrophilicity analysis.

[0243] 2.1.6. Intracellular signaling domain

[0244] The CAR of the present disclosure comprises an intracellular signaling domain. The intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune effector cells expressing the CAR. The term "effector function" refers to a special function of a cell. For example, the effector function of a T cell can be a cytolytic activity or an auxiliary activity including the secretion of cytokines. Therefore, the term "cytoplasmic signaling domain" refers to a protein portion that transduces effector function signals and instructs cells to perform special functions. Although the entire cytoplasmic signaling domain can generally be used, in many cases it is not necessary to use the entire chain. In terms of using a truncated portion of a cytoplasmic signaling domain, such a truncated portion can be used in place of the complete chain as long as it transduces the effector function signal. Therefore, the term cytoplasmic signaling domain is intended to include any truncated portion of the cytoplasmic signaling domain that is sufficient to transduce the effector function signal.

[0245] The intracellular signaling domain may comprise a primary intracellular signaling domain of an immune effector cell. CAR may comprise an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. "Primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector function. The primary intracellular signaling domain may contain a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. As used herein, "ITAM" is a conserved protein motif that is typically present in the tail of a signaling molecule expressed in many immune cells. The motif may comprise two repeats of an amino acid sequence YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid that produces a conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in ITAMs after activation of the signaling molecule. ITAMs can also be used as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0246] The primary intracellular signaling domain may be derived from CD3ζ. The intracellular signaling domain may consist of the cytoplasmic signaling domain of CD3ζ. The primary intracellular signaling domain may be the cytoplasmic signaling domain of wild-type CD3ζ. The primary intracellular signaling domain of CD3ζ may comprise the amino acid sequence of SEQ ID NO: 66. The primary intracellular signaling domain may be a functional mutant of the cytoplasmic signaling domain of CD3ζ containing one or more mutations (such as Q65K).

[0247] 2.1.7. Costimulatory signaling domain

[0248] Many immune effector cells also need co-stimulation in addition to stimulating antigen-specific signals to promote cell proliferation, differentiation and survival, and to activate the effector functions of cells. CAR can include at least one co-stimulatory signaling domain. As used herein, the term "co-stimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response such as an effector function. The co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein that transduces signals and regulates responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils." Co-stimulatory signaling domain" can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand to mediate the co-stimulatory response of an immune cell, such as, but not limited to, proliferation and survival.

[0249] The intracellular signaling domain may comprise a single costimulatory signaling domain. The intracellular signaling domain may comprise two or more (such as any one of about 2, 3, 4 or more) costimulatory signaling domains. The intracellular signaling domain may comprise two or more identical costimulatory signaling domains. The intracellular signaling domain may comprise two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. The intracellular signaling domain may comprise a primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ) and one or more costimulatory signaling domains. One or more costimulatory signaling domains and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ) may be fused to each other via an optional peptide linker. The primary intracellular signaling domain and the one or more costimulatory signaling domains may be arranged in any suitable order. One or more costimulatory signaling domains may be located between the transmembrane domain and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ). Multiple costimulatory signaling domains can provide additive or synergistic stimulation.

[0250] The activation of the costimulatory signal transduction domain in host cells (e.g., immune cells) can induce cells to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival and / or cytotoxicity. The costimulatory signal transduction domain of any costimulatory molecule is applicable to CAR as described herein. Based on factors such as the type of immune effector cells expressing effector molecules (e.g., T cells, NK cells, macrophages, neutrophils or eosinophils) and required immune effector functions (e.g., ADCC effects) One or more types of costimulatory signal transduction domains are selected.Examples of costimulatory signaling domains for CARs can be cytoplasmic signaling domains of costimulatory proteins, including but not limited to members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNF RII / TNFRSF1B); members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other costimulatory molecules, such as CD2, CD7, CD53, CD82 / KAI-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA Class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C.

[0251] The one or more co-stimulatory signaling domains can be selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0252] The intracellular signaling domain in the CAR of the present disclosure may include a costimulatory signaling domain (i.e., 4-1BB) derived from CD137. The intracellular signaling domain may include a cytoplasmic signaling domain of CD3 ζ and a costimulatory signaling domain of CD137. The intracellular signaling domain may include a costimulatory signaling domain of CD137 comprising an amino acid sequence of SEQ ID NO: 64.

[0253] Also within the scope of the present disclosure are variants of any co-stimulatory signaling domains described herein, such that the co-stimulatory signaling domains are capable of regulating the immune response of immune cells. Compared to the wild-type corresponding co-stimulatory signaling domain, the co-stimulatory signaling domain may comprise up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8). Such co-stimulatory signaling domains comprising one or more amino acid variations may be referred to as variants. Relative to co-stimulatory signaling domains that do not comprise mutations, mutations in the amino acid residues of the co-stimulatory signaling domain may result in increased signal transduction and enhanced immune response stimulation. Relative to co-stimulatory signaling domains that do not comprise mutations, mutations in the amino acid residues of the co-stimulatory signaling domain may result in reduced signal transduction and decreased immune response stimulation.

[0254] Hinge region

[0255] The CAR of the present disclosure may include a hinge domain located between the extracellular antigen binding domain and the transmembrane domain. The hinge domain is an amino acid segment typically found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen binding domain relative to the transmembrane domain of the effector molecule may be used.

[0256] The hinge domain can contain about 10-100 amino acids, such as about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. The hinge domain can be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0257] The hinge domain can be a hinge domain of a naturally occurring protein. The hinge domain of any protein comprising a hinge domain known in the art is suitable for use in chimeric receptors as described herein. The hinge domain can be at least a portion of a hinge domain of a naturally occurring protein and imparts flexibility to the chimeric receptor. The hinge domain can be derived from CD8α. The hinge domain can be a part of a hinge domain of CD8α, for example, a fragment of at least about 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of a hinge domain containing CD8α. The hinge domain of CD8α can comprise the amino acid sequence of SEQ ID NO:60.

[0258] Hinge domains of antibodies (such as IgG, IgA, IgM, IgE or IgD antibodies) are also suitable for use in the pH-dependent chimeric receptor systems described herein. The hinge domain can be a hinge domain that connects the constant domains C1 and C2 of an antibody. The hinge domain can be of an antibody and comprise the hinge domain of the antibody and one or more constant regions of the antibody. The hinge domain can comprise the hinge domain of the antibody and the C3 constant region of the antibody. The hinge domain can comprise the hinge domain of the antibody and the C2 and C3 constant regions of the antibody. The antibody can be an IgG, IgA, IgM, IgE or IgD antibody. The antibody can be an IgG antibody. The antibody can be an IgG1, IgG2, IgG3 or IgG4 antibody. The hinge region can comprise the hinge region and the C2 and C3 constant regions of an IgG1 antibody. The hinge region can comprise the hinge region and the C3 constant region of an IgG1 antibody.

[0259] Non-naturally occurring peptides can also be used as hinge domains of the chimeric receptors described herein. The hinge domain between the C-terminus of the extracellular ligand binding domain of the Fc receptor and the N-terminus of the transmembrane domain can be a peptide linker, such as a (GxS)n linker, wherein x and n independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more.

[0260] 2.1.9. Signal peptide

[0261] The CAR of the present disclosure may include a signal peptide (also referred to as a signal sequence) at the N-terminus of the polypeptide. In general, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. The signal peptide can target the secretory pathway of the effector molecule to the cell and will allow the effector molecule to be integrated and anchored into the lipid bilayer. It will be apparent to those skilled in the art that the signal peptides suitable for use in the CAR described herein include signal sequences of naturally occurring proteins or synthetic non-naturally occurring signal sequences. The signal peptide may be derived from a molecule selected from the group consisting of CD8 α, GM-CSF receptor α, and IgG1 heavy chains. The signal peptide may be derived from CD8 α. The signal peptide of CD8 α may include the amino acid sequence of SEQ ID NO: 59.

[0262] 2.1.10. Exemplary CAR

[0263] Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 67. Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 68. Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 69. Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 70. Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 71.

[0264] Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 81. Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 82. Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 83. Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 84. Provided herein are CARs that may comprise or consist of the amino acid sequence of SEQ ID NO: 85.

[0265] In some embodiments, the expression of CAR is achieved by introducing the nucleic acid encoding it into the body (in vivo cell therapy) or in vitro (including autologous cell therapy and allogeneic cell therapy) cells. In some embodiments, the cell is an immune cell.

[0266] 2.2. Engineering TCR

[0267] The engineered receptor can be an engineered T cell receptor. The engineered TCR can be specific for a B cell antigen. The B cell antigen can be selected from CD19, CD20, and CD22. The prior art has described many TCRs that are specific for B cell antigens. The TCR can have an enhanced affinity for the B cell antigen. For example, exemplary TCRs and methods for introducing TCRs into immune cells are described in PCT / EP2023 / 066121 and Kessels et al., Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001), which are incorporated herein by reference.

[0268] The TCR receptor complex is an octameric complex composed of the variable TCR receptor α and β chains (γ and δ chains in the case of γδ T cells) and three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247 (the T cell surface glycoprotein CD3ζ chain)ζ / ζ or ζ / η. The ionizable residues in the transmembrane domain of each subunit form a polar network of interactions that holds the complex together. The TCR complex has the function of activating the signaling cascade in T cells.

[0269] The engineered receptor can be an engineered TCR comprising one or more T cell receptor (TCR) fusion proteins (TFP).Exemplary TFPs are described, for example, in PCT / US2020 / 050503, which is incorporated herein by reference. TFP can include the extracellular domain of a TCR subunit, which includes the extracellular domain of a protein selected from the group consisting of: TCR α chains, TCR β chains, CD3εTCR subunits, CD3γTCR subunits, CD3δTCR subunits, their functional fragments, and their amino acid sequences with at least one but no more than 20 modifications. TFP can include a membrane spaning domain, which includes the membrane spaning domain of a protein selected from the group consisting of: TCR α chains, TCR β chains, CD3εTCR subunits, CD3γTCR subunits, CD3δTCR subunits, their functional fragments, and their amino acid sequences with at least one but no more than 20 modifications. The TFP may comprise a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: TCRα chain, TCRβ chain, TCRζ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0270] The TFP may comprise a TCR subunit and an antigen binding domain, the TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3ε; wherein the TCR subunit is operably linked to the antigen binding domain, and wherein when expressed in a T cell, the TFP is incorporated into the TCR.

[0271] 2.3. T cell antigen conjugate (TAC) receptor

[0272] The engineered receptor can be a T cell antigen conjugate (TAC) receptor. For example, an exemplary TAC receptor is described in PCT / US2019 / 042297, which is incorporated herein by reference. The TAC can include a targeting domain, a TCR binding domain that specifically binds to a protein associated with the TCR complex, and a T cell receptor signaling domain. The targeting domain can be an antibody fragment that specifically binds to a B cell antigen, such as an scFv or VHH. The targeting domain can be a designed ankyrin repeat (DARPin) polypeptide. The B cell antigen can be selected from CD19, CD20, and CD22. The protein associated with the TCR complex can be CD3, such as CD3ε. The TCR binding domain can be a single-chain antibody, such as an scFv or VHH. The TCR binding domain can be derived from UCHT1. The TAC receptor can include a cytoplasmic domain and a transmembrane domain. The T cell receptor signaling domain can include a cytoplasmic domain derived from a TCR co-receptor. Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. A TAC receptor may comprise a transmembrane domain and a cytoplasmic domain derived from CD4. A TAC receptor may comprise a transmembrane domain and a cytoplasmic domain derived from CD8 (e.g., CD8α).

[0273] T cell co-receptors are expressed as membrane proteins on T cells. They stabilize the TCR:peptide:MHC complex and facilitate signal transduction. Two subtypes of T cell co-receptors, CD4 and CD8, exhibit strong specificity for specific MHC classes. The CD4 co-receptor stabilizes only the TCR:MHC II complex, while the CD8 co-receptor stabilizes only the TCR:MHC I complex. Differential expression of CD4 and CD8 on different T cell types results in distinct functional T cell subsets. CD8+ T cells are cytotoxic T cells.

[0274] 3. Nucleic Acids and Vectors

[0275] The present disclosure also provides polynucleotides encoding the multi-specific CAR provided herein. The polynucleotides of the present disclosure can be in RNA form or DNA form. DNA includes cDNA, genomic DNA and synthetic DNA; and can be double-stranded or single-stranded (if the single-stranded can be a coding strand or a non-coding (antisense) strand). The polynucleotide is in the form of cDNA. The polynucleotide can be a synthetic polynucleotide.

[0276] The present disclosure further relates to variants of the polynucleotides described herein, wherein the variants encode, for example, fragments, analogs and / or derivatives of the single domain antibodies or CARs of the present disclosure. The present disclosure provides a polynucleotide that may comprise a polynucleotide having a nucleotide sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and at least about 96%, 97%, 98% or 99% identical to the polynucleotide encoding the single domain antibodies or CARs of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence" is intended to indicate that the nucleotide sequence of the polynucleotide is identical to the reference sequence, but the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or at any position between these terminal positions, interspersed individually between nucleotides in the reference sequence, or interspersed in one or more consecutive groups in the reference sequence.

[0277] Polynucleotide variants may contain changes that are located in the coding region, the non-coding region, or both. Polynucleotide variants may contain changes that produce silent substitutions, additions, or deletions but do not change the properties or activity of the encoded polypeptide. Polynucleotide variants may comprise silent substitutions that do not cause the amino acid sequence of the polypeptide to change (due to the degeneracy of the genetic code). Polynucleotide variants may be produced for a variety of reasons, for example, to optimize the codon expression of a specific host (i.e., changing the codons in human mRNA to codons preferred by bacterial hosts (such as E. coli). Polynucleotide variants may comprise at least one silent mutation in the non-coding region or the coding region of the sequence.

[0278] Polynucleotide variants can be produced to regulate or alter the expression (or expression level) of an encoded polypeptide. Polynucleotide variants can be produced to increase the expression of an encoded polypeptide. Polynucleotide variants can be produced to reduce the expression of an encoded polypeptide. Compared to the parent polynucleotide sequence, a polynucleotide variant can have increased expression of an encoded polypeptide. Compared to the parent polynucleotide sequence, a polynucleotide variant can have reduced expression of an encoded polypeptide.

[0279] Also provided are vectors comprising the nucleic acid molecules described herein. The nucleic acid molecules can be incorporated into recombinant expression vectors. The present disclosure provides recombinant expression vectors comprising any of the nucleic acids disclosed herein. As used herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is contacted with a host cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, allows the cell to express the mRNA, protein, polypeptide, or peptide. The vectors described herein as a whole are not naturally occurring; however, individual parts of the vector may be naturally occurring. The recombinant expression vectors described may comprise any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic, or partially obtained from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring internucleotide bonds, or both types of bonds. Non-naturally occurring or altered nucleotides or internucleotide bonds do not hinder transcription or replication of the vector.

[0280] The recombinant expression vector of the present disclosure can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host.Suitable vectors include those designed for breeding and amplification or for expression or for the vectors of both purposes, such as plasmids and viruses.The vector can be selected from the group consisting of: pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λEMBL4 and λNM1149, λZapII (Stratagene) can be used. The example of plant expression vector includes pBI01, pBI01.2, pBI121, pBI101.3 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, such as a retroviral vector, such as a gamma-retroviral vector.

[0281] Recombinant expression vectors can be prepared using standard recombinant DNA techniques, such as those described in Sambrook et al. and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain replication systems that function in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColE1, SV40, 2μ plasmids, lambda, bovine papilloma virus, and the like.

[0282] Depending on the circumstances and whether the vector is DNA-based or RNA-based, the recombinant expression vector may contain regulatory sequences (such as transcription and translation start and stop codons) that are specific for the type of host (e.g., bacteria, plants, fungi, or animals) into which the vector is to be introduced.

[0283] The recombinant expression vector can include one or more marker genes that allow selection of transformed or transfected hosts. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc.; complementation for providing prototrophy in auxotrophic hosts, and the like. Suitable marker genes for the described expression vectors include, for example, neomycin / G418 resistance genes, histidinol x resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0284] The recombinant expression vector can comprise a native or standard promoter operably connected to the nucleotide sequence of the present disclosure. The selection of promoters (such as strong promoters, weak promoters, tissue-specific promoters, inducible promoters and development-specific promoters) is within the ordinary skill range of the technician. Similarly, the combination of nucleotide sequence and promoter is also within the skill range of the technician. Promoter can be a non-viral promoter or a viral promoter, such as cytomegalovirus (CMV) promoter, RSV promoter, SV40 promoter or the promoter found in the long terminal repeat sequence of mouse stem cell virus.

[0285] The recombinant expression vector can be designed for transient expression, for stable expression, or for both purposes. In addition, the recombinant expression vector can be prepared for constitutive expression or for inducible expression.

[0286] Further, the recombinant expression vector can be prepared to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of cells expressing the suicide gene. The suicide gene can be a gene that confers sensitivity to an agent (e.g., a drug) on ​​cells expressing the gene, and causes the cell to die when the cell is in contact with or exposed to the agent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0287] A polynucleotide may be isolated. A polynucleotide may be substantially pure.

[0288] Also provided are host cells comprising the nucleic acid molecules described herein. The host cell can be any cell containing a heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). For example, the host cell can be a cell from any organism that is selected, modified, transformed, grown, used, or manipulated in any manner to produce a substance from the cell, such as a gene, DNA, or RNA sequence, protein, or enzyme expressed by the cell. An appropriate host can be determined. For example, a host cell can be selected based on the vector backbone and the desired results. For example, a plasmid or cosmid can be introduced into a prokaryotic host cell to replicate several types of vectors. Bacterial cells, such as, but not limited to, DH5α, JM109, and KCB, Competent cells and SOLOPACK Gold cells can be used as host cells for vector replication and / or expression. In addition, bacterial cells such as Escherichia coli LE392 can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include but are not limited to yeast (e.g., YPH499, YPH500, and YPH501), insects, and mammals. Examples of mammalian eukaryotic host cells for replication and / or expression of vectors include but are not limited to HeLa, NIH3T3, Jurkat, 293, COS, Saos, PC12, SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NSO (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61), or DG44.

[0289] 4. Engineered Immune Effector Cells

[0290] "Immune effector cells" are immune cells that can perform immune effector functions. Immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0291] The immune effector cell can be a T cell. The T cell can be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8- or a combination thereof. The T cell can produce IL-2, TFN and / or TNF after expressing CAR and binding to target cells such as CD20+, CD19+ and / or CD22+ tumor cells. CD8+ T cells can lyse antigen-specific target cells after expressing CAR and binding to target cells.

[0292] The immune effector cell may be a T cell, and wherein the T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a gamma delta T cell.

[0293] The immune effector cells may be NK cells. In other embodiments, the immune effector cells may be established cell lines, such as NK-92 cells.

[0294] Immune effector cells can be differentiated from stem cells such as hematopoietic stem cells, pluripotent stem cells, iPS cells, or embryonic stem cells.

[0295] Engineered immune effector cells are prepared by introducing CAR into immune effector cells (such as T cells). In some embodiments, the method for delivering the nucleic acid encoding CAR to immune effector cells (such as T cells) is an in vitro delivery method. In some embodiments, the method for delivering the nucleic acid encoding CAR to immune effector cells (such as T cells) is an in vivo delivery method.

[0296] CAR can be introduced into immune effector cells by transfecting any of the isolated nucleic acids described above or any of the vectors described above. CAR can be introduced into immune effector cells by inserting the protein into the cell membrane while passing the cell through a microfluidic system such as CELL. (See, e.g., U.S. Patent Application Publication No. 20140287509) to introduce CAR into immune effector cells.

[0297] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The vectors can be transferred into immune effector cells by physical, chemical or biological methods.

[0298] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Vectors can be introduced into cells by electroporation.

[0299] Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (eg, human) cells.

[0300] Chemical methods for introducing carriers into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0301] Any one of the CARs in CAR encoding described herein can be prepared by conventional methods (for example, in vitro transcription) RNA molecules, then introduced into immune effector cells via known methods such as mRNA electroporation.See, for example, Rabinovich et al., Human Gene Therapy 17: 1027-1035 (2006).

[0302] The immune effector cells of transduction or transfection can be propagated in vitro after introducing a vector or isolated nucleic acid. The immune effector cells of transduction or transfection can be cultured to breed at least about any one of the following: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days or 14 days. The immune effector cells of transduction or transfection can be further evaluated or screened to select engineered mammalian cells.

[0303] Reporter gene can be used to identify the cells of potential transfection and evaluate the functionality of regulatory sequences. In general, reporter gene is a gene that is not present in or is not expressed by the recipient organism or tissue and encodes a polypeptide whose expression can be shown by some easily detectable properties (such as enzyme activity). After DNA has been introduced into the recipient cell, the expression of the reporter gene is determined at the appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein genes (for example, Ui-Tei et al. FEBS Letters 479:79-82 (2000)). Suitable expression systems are well known and can be prepared or commercially available using known techniques. Other methods for confirming the presence of nucleic acids encoding CAR in engineered immune effector cells include, for example, molecular biological assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; Biochemical assays, such as, for example, detecting the presence or absence of specific peptides by immunological methods (such as ELISA and Western blotting).

[0304] 5. Pharmaceutical Compositions

[0305] In one aspect, the present disclosure further provides a pharmaceutical composition comprising the engineered immune effector cells of the present disclosure. The pharmaceutical composition may comprise a therapeutically effective amount of the engineered immune effector cells of the present disclosure and a pharmaceutically acceptable excipient.

[0306] Provided herein is a pharmaceutical composition that may comprise a therapeutically effective amount of a therapeutic molecule comprising a CAR provided herein and a pharmaceutically acceptable excipient.

[0307] Provided herein is a pharmaceutical composition that can include a therapeutically effective amount of a nucleic acid provided herein (e.g., a therapeutically effective amount of a nucleic acid provided herein in a vector) and a pharmaceutically acceptable excipient (e.g., a pharmaceutically acceptable excipient suitable for gene therapy).

[0308] The choice of excipient can be determined in part by specific cells, binding molecules and / or antibodies, and / or the method of administration. Therefore, there are a variety of suitable formulations. The pharmaceutical composition may comprise the engineered immune effector cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, dextran, mannitol and dextrose or its analogs (such as dextran 40); proteins such as human serum albumin (HSA); polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and cryoprotectants such as DMSO, glycerol, etc.

[0309] Various compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the single domain antibodies or therapeutic molecules provided herein, constructing the nucleic acid as part of a retroviral or other vector, etc.

[0310] The pharmaceutical composition provided herein can contain the binding molecule and / or the cell of the amount (such as therapeutically effective dose or preventive effective dose) of effective treatment or prevention disease or illness. Treatment or prevention efficacy can be monitored by regular assessment to the treated experimenter. For repeated administration (this depends on the illness) in a few days or longer, maintenance treatment is until the disease symptoms obtain required inhibition. However, other dosage regimens are available, and these dosage regimens can be determined.

[0311] 6. Treatment Methods

[0312] In another aspect, provided herein are methods and uses of chimeric antigen receptors (CARs) and / or engineered immune effector cells expressing the recombinant receptors.

[0313] Such methods and uses include, for example, methods and uses involving the following treatment methods: administering the molecules, cells, or compositions containing them to subjects suffering from autoimmune diseases. The autoimmune disease may be systemic lupus erythematosus. The autoimmune disease may be systemic sclerosis, ANCA-associated vasculitis, idiopathic inflammatory myopathy, multiple sclerosis, neuromyelitis optica spectrum disease / anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-related disease, or myasthenia gravis. The molecules, cells, and / or compositions may be administered in an amount effective to achieve the treatment of the disease or condition. Uses include the use of the CAR and cells in such methods and treatments, as well as the use in preparing drugs to implement such treatment methods. The method may be implemented by administering the CAR or cells or compositions containing them to subjects suffering from or suspected of having the disease or condition. The method may thereby treat the disease or condition of the subject.

[0314] The treatments provided herein can completely or partially improve or alleviate the disease or condition or the symptoms, side effects, consequences, or phenotypes associated therewith. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or slowing the disease state, and alleviating or improving prognosis. The term includes, but does not imply, complete cure of the disease or complete elimination of any symptom or effect on all symptoms or consequences.

[0315] As used herein, the treatment provided herein can delay the development of a disease or condition, for example, delay, hinder, slow down, block, stabilize, suppress and / or postpone the development of an autoimmune disease (such as systemic lupus erythematosus). This delay may have different time lengths, depending on the medical history and / or the individual being treated. As will be apparent to those skilled in the art, sufficient or significant delay can actually encompass prevention, as the individual does not develop a disease or condition. The methods or uses provided herein can prevent a disease or condition.

[0316] The method includes administering adoptive cell therapy, whereby genetically engineered cells expressing a provided multispecific CAR (CD20×CD19×CD22 trispecific CAR-T) are administered to the subject. This administration can promote cell activation (e.g., T cell activation) in a CD20, CD19, and CD22 targeting manner, such that the cells of the disease or disorder are targeted for destruction.

[0317] Methods for administering cells for adoptive cell therapy are known, for example, as described in U.S. Patent Application Publication No. 2003 / 0170238; U.S. Patent No. 4,690,915; Rosenberg, Nat Rev Clin Oncol. 8(10):577-85 (2011); Themeli et al., Nat Biotechnol. 31(10):928-933 (2013); Tsukahara et al., Biochem Biophys Res Commun 438(1):84-9 (2013); and Davila et al., PLoS ONE 8(4):e61338 (2013). These methods can be used in combination with the methods and compositions provided herein.

[0318] Cell therapy (for example, adoptive T cell therapy) can be carried out by autologous transfer, wherein from the experimenter to be accepted cell therapy or from the sample separation and / or otherwise prepare cell derived from such experimenter.Therefore, in some aspects, the cell derives from the experimenter in need of treatment, and the cell is administered to the same experimenter after separation and processing. Cell therapy (for example, adoptive T cell therapy) can also be carried out by allogeneic transfer, wherein from the experimenter (for example, the first experimenter) to be accepted or finally accept the experimenter other than cell therapy, separation and / or otherwise prepare cell.In this case, then the cell is applied to different experimenters (for example, the second experimenter) of the same species.The first and second experimenters can be identical in genetics.The first and second experimenters can be similar in genetics.The second experimenter can express the HLA category or supertype identical with the first experimenter.

[0319] The subject to which the cells, cell populations, or compositions are administered can be a primate, such as a human. The subject can be male or female and can be of any suitable age, including infants, adolescents, teenagers, adults, and elderly subjects. The subject can be a validated animal model for disease, adoptive cell therapy, and / or for evaluating toxicity outcomes.

[0320] Compositions provided herein can be by any suitable means, for example, by injection, for example intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, through septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection (subconjectval injection), subconjunctival injection, sub-Tenon's injection), injection behind the eyeball, periorbital injection or posterior sclera and send to use. They can also be by parenteral, intrapulmonary and intranasal use, and if local treatment is needed, then by intralesional use. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Compositions provided herein is preferably used by intravenous delivery.

[0321] Can determine the amount of the effective prevention and / or treatment disease or condition of disease of prevention and treatment or therapeutic agent provided herein by standard clinical techniques.Can be extrapolated effective dose by the dose-response curve that derives from in vitro or animal model test system.For the prevention or treatment of disease, the appropriate dosage of binding molecule or cell can depend on the type of disease to be treated or the type of illness, the type of binding molecule, the severity of disease or illness and the course of disease, administering therapeutic agent be for preventive purposes or therapeutic purposes, previous therapy, patient's clinical history and the reaction to agent and the judgment of attending physician.Can be once or through a series of treatments, described compositions, molecule and cell are suitably administered to patient.Can use multiple dosages intermittently.Can use initial higher loading dose, then one or more lower dosages.

[0322] In the context of genetically engineered cells containing the binding molecule, a subject may be administered approximately 0.1 to 1 × 10 6 In the case where the pharmaceutical composition comprises any of the engineered immune effector cells described herein, the pharmaceutical composition can be administered at a dose of at least about 10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 or 10 6 The dosage may vary depending on the disease or condition and / or the particular attributes of the patient and / or other treatments.

[0323] The pharmaceutical composition can be administered in a single dose. The pharmaceutical composition can be administered multiple times (e.g., any one of 2, 3, 4, 5, 6 or more times). The pharmaceutical composition can be administered once or multiple times in a dosing cycle. The dosing cycle can be, for example, 1, 2, 3, 4, 5 weeks or more, or 1, 2, 3, 4, 5 months or more. Those skilled in the art of medicine can determine the optimal dose and treatment regimen for a particular patient by monitoring the patient's disease signs and adjusting treatment accordingly.

[0324] The compositions provided herein can be administered as part of a combination therapy, such as simultaneously or sequentially in any order with another therapeutic intervention (such as another antibody or engineered cell or receptor or agent, such as a cytotoxic agent or therapeutic agent).

[0325] The compositions provided herein can be co-administered with one or more additional therapeutic agents together or in combination with another therapeutic intervention simultaneously or in any order. The cells and another therapy can be co-administered close enough in time so that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. The compositions provided herein can be administered before the one or more additional therapeutic agents. The compositions provided herein can be administered after the one or more additional therapeutic agents.

[0326] Once the cells are applied to mammals (e.g., humans), the bioactivity of the engineered cell population and / or binding molecules can be measured by any of a variety of known methods. Parameters for evaluation include specific binding of engineered or natural T cells or other immune cells to antigens in vivo (e.g., by imaging) or ex vivo (e.g., by ELISA or flow cytometry). Any suitable method known in the art (such as in, for example, Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009) and Herman et al. J. Immunological Methods, 285 (1): 25-40 (2004)) can be used to measure the ability of engineered cells to destroy target cells. The bioactivity of the cells can also be measured by measuring the expression and / or secretion of certain cytokines such as CD107a, IFNγ, IL-2, and TNF. In some aspects, bioactivity is measured by assessing the reduction of clinical outcomes such as tumor burden or load.

[0327] Provided herein is a method for treating a disease or illness, comprising administering to a subject an engineered immune effector cell (such as a T cell) as described herein, including, for example, a cell comprising CAR. The engineered immune effector cell administered to a subject can include a CAR, wherein the CAR includes an extracellular antigen binding domain comprising at least two (e.g., all three) of anti-CD20 sdAb, anti-CD19 sdAb, and anti-CD22 sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD20 sdAb, the anti-CD19 sdAb, and the anti-CD22 sdAb are as described above, including, for example, those with the CDRs in Table 1. The engineered immune effector cell administered to a subject can include the CAR listed in Table 4. The engineered immune effector cell administered to a subject can include the CAR with the CDRs of the CAR listed in Table 4. The engineered immune effector cells administered to the subject may comprise a CAR comprising an amino acid sequence selected from SEQ ID NOs: 67-71, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 67-71.

[0328] The disease or disorder may be an autoimmune disease. The autoimmune disease may be a relapsed / refractory autoimmune disease.

[0329] The disease or condition may be systemic lupus erythematosus.

[0330] The subject described herein may have an inadequate response to at least one drug for treating systemic lupus erythematosus selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tetanusip, rituximab, anirumab, chloroquine, hydroxychloroquine, stem cell therapy, and immune cell therapy.

[0331] The subject described herein may have an inadequate response to at least two drugs for treating systemic lupus erythematosus selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tetanusip, rituximab, anirumab, chloroquine, hydroxychloroquine, stem cell therapy, and immune cell therapy.

[0332] The subject described herein may have an inadequate response to a combination of glucocorticoids and at least one drug for treating systemic lupus erythematosus selected from mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tebucentrum, rituximab, anirumab, chloroquine, hydroxychloroquine, stem cell therapy, and immune cell therapy.

[0333] The subject described in this question may have an inadequate response to a combination of glucocorticoids and at least two drugs for treating systemic lupus erythematosus, wherein the drugs are selected from mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tebucentrazole, rituximab, anirumab, chloroquine, hydroxychloroquine, stem cell therapy and immune cell therapy.

[0334] The immune cell therapy may be CAR-T or CAR-NK targeting at least one of CD19, CD20, and BCMA.

[0335] The subjects described herein may also have concurrent lupus nephritis (LN).

[0336] The disease or condition may be systemic sclerosis.

[0337] The subject described herein may have an inadequate response to at least one drug for treating systemic sclerosis, selected from antifibrotic drugs (such as nintedanib, pirfenidone), vascular protective agents (such as PDE5 inhibitors, endothelin receptor inhibitors, calcium channel blockers), glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, belimumab, stem cell therapy and immune cell therapy.

[0338] The subjects described herein may have an inadequate response to an antifibrotic drug (such as nintedanib, pirfenidone) and / or a vascular protective agent (such as a PDE5 inhibitor, an endothelin receptor inhibitor, a calcium channel blocker) in combination with at least one drug for treating systemic sclerosis, wherein the drug is selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, belimumab, stem cell therapy, and immune cell therapy.

[0339] The immune cell therapy may be CAR-T or CAR-NK targeting at least one of CD19, CD20, and BCMA.

[0340] The disease or condition may be ANCA-associated vasculitis.

[0341] The subject described herein may have an inadequate response to at least one drug for treating ANCA-associated vasculitis selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tetasip, rituximab, mepolizumab, tocilizumab, avacopan, and a JAK inhibitor.

[0342] The subject described herein may have an inadequate response to a glucocorticoid combined with at least one drug for ANCA-associated vasculitis selected from mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tetasip, rituximab, mepolizumab, tocilizumab, avacopan, and a JAK inhibitor.

[0343] The disease or disorder may be an idiopathic inflammatory myopathy.

[0344] The subject described herein may have an inadequate response to at least one drug for treating idiopathic inflammatory myopathy selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, and a JAK inhibitor.

[0345] The subject described herein may have an inadequate response to a glucocorticoid in combination with at least one drug for an idiopathic inflammatory myopathy selected from mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, and a JAK inhibitor.

[0346] The disease or condition may be multiple sclerosis.

[0347] The subject described herein may have an inadequate response to at least one drug for treating multiple sclerosis selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, alemtuzumab, cladribine, natalizumab, ocrelizumab, rituximab, ofatumumab, and a sphingosine 1-phoshate receptor (S1PR) modulator.

[0348] The disease or disorder may be a neuromyelitis optica spectrum disorder / anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-associated disease.

[0349] The subject described herein may have an inadequate response to at least one drug for treating neuromyelitis optica spectrum disorder / anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-associated disease, selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, satelizumab, rituximab, inelizumab, ravelizumab, eculizumab, and tocilizumab.

[0350] The disease or condition may be myasthenia gravis.

[0351] The subject described herein may have an inadequate response to at least one drug for treating myasthenia gravis selected from acetylcholinesterase inhibitors, glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, eculizumab, ravulizumab, belimumab, igatimod, rozelixizumab, or may require repeated administration of intravenous immunoglobulin (IVIG) or plasma exchange to alleviate the condition.

[0352] The subject described herein may have had an inadequate response to at least two drugs for treating myasthenia gravis selected from acetylcholinesterase inhibitors, glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolate mofetil or a derivative thereof, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, eculizumab, ravulizumab, belimumab, igatimod, and rozelixizumab.

[0353] In the methods described herein, lymphocyte depletion can be performed on the subject prior to administering the engineered immune effector cells. In the methods described herein, lymphocyte depletion can be performed on the subject at the same time as the engineered immune effector cells are administered. Lymphocyte depletion can include administering cyclophosphamide and fludarabine. Cyclophosphamide and fludarabine can be administered by intravenous infusion. Lymphocyte depletion can include administering cyclophosphamide 500-1500 mg / m 2 1 dose and fludarabine 15-35 mg / m 2 Once daily for up to 3 days (e.g., 1, 2, or 3 days). Lymphodepletion may include administration of cyclophosphamide 200-400 mg / m 2 Once daily for up to 3 days, and fludarabine 15–35 mg / m 2 Once daily for up to 3 days (e.g., 1, 2, or 3 days). Lymphodepletion may include intravenous cyclophosphamide 1000 mg / m 2 1 dose and fludarabine 25 mg / m 2Once daily for up to 3 days. Lymphodepletion may include intravenous cyclophosphamide 300 mg / m 2 Once daily for up to 3 days, and fludarabine 25 mg / m 2 Once daily, up to 3 days.

[0354] Lymphocyte depletion can be initiated at least 5 days before the administration of the engineered immune effector cells (e.g., 5 days before, 6 days before, 7 days before, 8 days before, 9 days before, 10 days before, etc.). Lymphocyte depletion can include intravenous infusion of cyclophosphamide 1000 mg / m2 starting 7 days before the administration of the engineered immune effector cells. 2 1 dose and fludarabine 25 mg / m 2 Once daily for a total of 3 days. Lymphocyte depletion may include intravenous infusion of cyclophosphamide 1000 mg / m2 starting 6 days before administration of engineered immune effector cells. 2 1 dose and fludarabine 25 mg / m 2 Once daily for a total of 3 days. Lymphocyte depletion may include intravenous infusion of cyclophosphamide 1000 mg / m2 starting 5 days before administration of engineered immune effector cells. 2 1 dose and fludarabine 25 mg / m 2 Once daily for a total of 3 days. Lymphocyte depletion may include intravenous infusion of cyclophosphamide 300 mg / m 7 days before administration of engineered immune effector cells. 2 Once daily for 3 days, and fludarabine 25 mg / m 2 Once daily for a total of 3 days. Lymphocyte depletion may include intravenous infusion of cyclophosphamide 300 mg / m 6 days before administration of engineered immune effector cells. 2 Once daily for 3 days, and fludarabine 25 mg / m 2 Once daily for a total of 3 days. Lymphocyte depletion may include intravenous infusion of cyclophosphamide 300 mg / m 5 days before administration of engineered immune effector cells. 2 Once daily for 3 days, and fludarabine 25 mg / m 2 Once a day, for a total of 3 days.

[0355] In the methods described herein, the subject may not be lymphodepleted prior to administration of the engineered immune effector cells.

[0356] Failure to perform lymphodepletion on the subject means failure to eliminate lymphocytes in the subject's body. This includes not administering lymphodepleting agents, whole-body radiation therapy, or a combination thereof, or other means that cause lymphocyte depletion; and after administering lymphodepleting agents, whole-body radiation therapy, or a combination thereof, or other means that cause lymphocyte depletion, when the lymphocyte depletion rate in the subject is less than 60%.

[0357] Not performing lymphocyte clearance on the subject or reducing the concentration and / or application time of the lymphocyte clearance agent can reduce the toxic side effects caused by damage to normal tissues, especially severe suppression of bone marrow. Lymphocyte depletion can be avoided, reducing the risk of infection in the subject and increasing safety. The engineered immune effector cells described herein (for example) have a strong amplification capacity, so the engineered immune effector cells are expected to be able to expand in the subject without lymphocyte clearance to achieve the purpose of treatment. Example

[0358] The embodiments of the present disclosure will be described in detail below with reference to the examples. If no specific conditions are specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be obtained commercially. It is understood by those skilled in the art that the examples describe the present disclosure by way of example and are not intended to limit the scope of protection claimed by the present disclosure. Various modifications and changes can be made to the details based on all the teachings that have been published, and these changes are within the scope of protection of the present disclosure.

[0359] Example 1 In vitro cytotoxicity testing of single, bi, and trispecific CAR-T cells

[0360] 1.1 Construction of CAR-T cells

[0361] For the construction method of CAR-T cells, please refer to PCT / CN2020 / 102470. In order to construct a multi-target VHH CAR using a CAR vector backbone, nucleic acid sequences encoding multiple different VHH domains are interconnected by peptide linker sequences and connected to the 3' end of the nucleic acid sequence encoding the CD8α signal peptide. Bispecific VHH or scFv CAR constructs are prepared by fusing two VHHs or two scFvs or one VHH with one scFv domain with a series of glycine-serine peptide connectors ((G4S)n). Bispecific VHH×VHH CAR generally contains any two of the anti-CD19 VHH domain, anti-CD20 VHH domain, and anti-CD22 VHH domain, and CAR is constructed in different orders and combinations. Bispecific scFv×scFv CARs generally contain any two of the anti-CD19 scFv domain, anti-CD20 scFv domain, and anti-CD22 scFv domain, with the CARs constructed in different orders and combinations. Bispecific scFv×VHH CARs generally contain any one of the anti-CD19 scFv domain, anti-CD20 scFv domain, and anti-CD22 scFv domain, as well as any one of the anti-CD19 VHH domain, anti-CD20 VHH domain, and anti-CD22 VHH domain, with the bispecific CARs constructed in different VHH or scFv orders and combinations. The trispecific VHH CAR (AIO CAR) was constructed by fusing the anti-CD19 VHH domain, anti-CD20 VHH domain, and anti-CD22 VHH domain (including various VHH orders and combinations) with a series of glycine-serine peptide linkers ((G4S)n) (Figure 1). Monospecific CARs expressing a single VHH were also constructed using the same CAR framework. In addition, anti-CD19 scFv (murine FMC63 scFv), anti-CD20 scFv (murine Leu16 scFv), anti-CD22 scFv (murine m971 scFv), and anti-CD20 scFv × anti-CD19 scFv (Bi-CD19 × CD20 scFv CAR-T-1, Bi-CD19 × CD20 scFv CAR-T-2) were also cloned into the CAR framework as controls.

[0362] In trispecific AIO CAR-T design, the function of the AIO CAR is optimized by regulating its binding to the extracellular domains (ECDs) of three antigens. Another advantage of multispecific CAR-T cells is the potential for forming more stable immunological synapses. Compared to trispecific scFv CARs, the trispecific VHH CAR structure and production advantages are smaller DNA and amino acids (AAs), making it easier to integrate, more efficiently amplify, and easier to complete short-cycle production.

[0363] Exemplary mono-, bi-, and tri-specific CAR-Ts can be found in Tables 4-6 below.

[0364] Table 4 Exemplary trispecific CAR-T

[0365] 1 The linker used in the constructs in Table 4 was (GGGGS)1.

[0366] 2 CT SD stands for costimulatory signaling domain.

[0367] 3 PI SD stands for primary intracellular signaling domain.

[0368] Table 5 Exemplary bispecific CAR-T

[0369] 1 The linker used in the constructs in Table 5 was (GGGGS)1.

[0370] 2 CT SD stands for costimulatory signaling domain.

[0371] 3 PI SD stands for primary intracellular signaling domain.

[0372] Table 6 Exemplary monospecific CAR-T

[0373] 1 The linker used in the constructs in Table 6 was (GGGGS)1.

[0374] 2 CT SD stands for costimulatory signaling domain.

[0375] 3 PI SD stands for primary intracellular signaling domain.

[0376] 1.2 CAR-T in vitro cytotoxicity test

[0377] In order to quickly evaluate the in vitro anti-tumor activity of CAR-T cells, a luciferase cytotoxicity assay was performed. On the third day after viral transduction, CAR-T cells were harvested and CAR-T cell preparation was completed in a short cycle (6 days). CAR-T cells were incubated with target cell lines (Nalm.6.CD20.luc tumor cell lines that stably and highly express CD19, CD20, and CD22 receptors) at an E:T ratio (ratio of effector cell CAR-T to target cell) of 4:1, 1:1, or 1:4 at 37°C for 20 hours. Untransduced T cells (Un-transduced T cell, UnT) from the same batch were co-incubated with tumor cells as a negative control. Monospecific CD19 scFv CAR-T (mouse FMC63 scFv) was used as a benchmark CAR-T cell ("Benchmark CAR") and co-incubated with tumor cells as a positive control. Trispecific camelid AIO CAR-T cells (Cam AIO CAR) and humanized AIO CAR-T cells (Hu AIO CAR) were compared head-to-head with monospecific CAR-T cells (CD19 scFv CAR) or bispecific CAR-T cells (Bi-CD19×CD20 scFv CAR-T-1 and Bi-CD19×CD20 scFv CAR-T-2). Hu AIO CAR-T represents humanized AIO CAR-T cells generated using the pLenti vector backbone, with all three VHHs humanized; Hu AIO (Lenti-4X) CAR-T represents humanized AIO CAR-T cells generated using the optimized pLenti-4X vector backbone, with all three VHHs humanized. Luciferase assays were performed according to the luciferase assay operating system instructions (PROMEGA, Cat. #E6120) and detection standards.

[0378] All single / bi / trispecific CAR constructs can effectively transduce human T cells, with CAR expression rates ranging from 20% to 50%. Relative to untransduced T cells (UnT) in the same experimental batch, the cell growth and viability of the transduced cells were not affected (the experimental results are not shown here). As shown in Figure 2, all CAR-T cells showed in vitro cytotoxicity against Nalm.6.CD20.Luc tumor cells and showed dose dependence. At an E:T of 4:1, the cytotoxicity of all CAR-Ts was comparable, with the percentage of target cell lysis ranging from 80% to 100%. At E:T of 1:1 and 1:4, the cytotoxicity of trispecific camel-derived and humanized AIO CAR-T was significantly higher than that of monospecific and bispecific CAR-T (Figure 2). This result shows that at a lower E:T effect-target ratio (such as E:T = 1:4), trispecific camel-derived and humanized AIO CAR-T have stronger tumor cell recognition and killing efficacy.

[0379] Example 2 Detection of PBMC (Peripheral blood mononuclear cell) cell subsets and CD19, CD20, and CD22 expression on B cells in SLE patients

[0380] To evaluate the diversity of PBMC cell subsets and B cell subsets in SLE patients, flow cytometry was used to identify B cell subsets. In the construction of the multicolor scheme, biomarkers and fluorescent antibodies were rationally matched and color matching was carried out based on the experimental biological background, instrument configuration, marker expression level, and dye physical and chemical properties (such as brightness, spectral characteristics, and stability). After the cryopreserved PBMC cells of SLE patients were revived and cultured in a cell culture incubator at 37°C for 20 hours, 2×10 6Transfer cells to a 1.5 mL centrifuge tube and centrifuge at 800 g for 2 minutes. Discard the supernatant. Wash twice with BD stain buffer (BD, Cat. #554656) and discard the supernatant after centrifugation. FACS antibodies were diluted and mixed with BD stain buffer, including Perp cy5.5-CD45 (BioLegend, Cat.#304028), APC F750-CD3 (BioLegend, Cat.#300470), BV510-CD4 (BioLegend, Cat.#317444), BV570-CD8 (BioLegend, Cat.#301038), PE EF610-CD56 (Invitrogen, Cat.#61-0566-42), BV421-CD33 (BioLegend, Cat.#366622), APC-CD19 (BioLegend, Cat.#302212), PE Cy5.5-CD20 (Invitrogen, Cat.#35-0209-42), PE-CD22 (BioLegend, Cat.#302506), AF647-ADA FMC63 (BIOSWAN, Cat.#200101), or FITC-AIO ADA (Legend, Cat.#LGBUADAb-16) were added to 2.5 μL of each antibody per 100 μL of staining buffer. The antibody mixture was added to the cells, mixed thoroughly, and incubated at 4°C for 30 min. The cells were then washed twice with BD stain buffer, the supernatant discarded, and 7-AAD (BioLegend, Cat.#420404) staining solution, which can distinguish between live and dead cells, was added to resuspend the cells. Multi-color co-staining was detected using a Cytex full-spectrum flow cytometer. The target cell populations to be detected in the experiment, the lineage markers required to define the cell populations, and the co-expression of as many markers as possible were sorted out. Finally, the data were imported into FlowJo.v10 software for plotting and data analysis, which verified the diversity of B cell subsets and the expression and distribution of CD19, CD20, and CD22 targets in SLE peripheral blood (Figure 3).

[0381] Example 3 Detection of Cytotoxicity of Trispecific AIO CAR-T on B Cells in Peripheral Blood Mononuclear Cells (PBMC) of SLE Patients

[0382] In order to evaluate the killing ability of camel-derived and humanized AIO CAR-T on B cells in peripheral blood mononuclear cells (PBMC) of SLE patients, a cell killing test was performed and cytotoxicity was detected by flow cytometry. After the frozen PBMC of SLE patients was revived, they were cultured in a 37 ° C cell culture incubator for 20 hours and incubated with CAR-T cells for 24 hours at an E:T ratio (ratio of effector cell-CAR-T (effector) to target cell (target)) of 3:1, 1:3, 1:27, 1:54 or 1:81. Untransduced T cells (UnT) from the same batch were used as negative controls. CD19 scFv (mouse FMC63) CAR-T was used as a positive control. The co-incubated cells were stained using the staining method described in Example 2 and then flow cytometry was performed and analyzed. The in vitro B cell cytotoxicity test data showed that the killing efficacy of camel-derived AIO CAR-T (Cam AIO CAR-T) was stronger than that of humanized AIO CAR-T (Hu AIO CAT-T), and both were stronger than CD19 scFv (mouse FMC63) CAR-T. All CAR-Ts showed dose- and time-dependence (Figure 4A-B). Under the condition of E:T=3:1, humanized AIO CAR-T (Hu AIO CAT-T) and camel-derived AIO CAR-T (Cam AIO CAR-T) effectively eliminated B cells in SLE PBMCs and eliminated almost all B cells expressing three antigens: CD19, CD20, and CD22. However, CD19 scFv (mouse FMC63) CAR-T selectively eliminated its targeted CD19-positive B cells, and the remaining B cell population was mainly composed of CD20-positive and CD22-positive B cells (Figure 4C). In addition, CD19 antigen escape was observed within 20 hours of contact with CD19 scFv (mouse FMC63) CAR-T, and the CD19-negative B cell subset was mainly composed of CD20-positive and CD22-positive B cells. Based on the flow cytometry data showing that the CD19-negative cell population did not increase over time in the UnT and PBMC co-incubation group, it can be further inferred that CD19 antigen escape or downregulation occurred after contact with CD19 scFv (mouse FMC63) CAR-T, showing an increase in the CD19-negative population of B cells (Figure 4D).

[0383] Example 4 Detection of cytotoxicity of single, bi, and trispecific CAR-T cells against B cells in peripheral blood mononuclear cells (PBMC) of SLE patients

[0384] In order to evaluate the killing ability of single, double, and tri-specific CAR-T on B cells in PBMC of SLE patients, a cell killing test was performed and cytotoxicity was detected by flow cytometry. After the cryopreserved PBMC cells of SLE patients were revived, they were incubated with single, double, and tri-specific CAR-T cells at an E:T ratio (ratio of effector cell-CAR-T (effector) to target cell (target)) of 3:1, 1:3, and 1:27 for 72 hours. Untransduced T cells (UnT) from the same batch were used as negative controls. CD19 scFv (mouse FMC63) CAR-T was used as a positive control. The co-incubated cells were stained using the staining method described in Example 2 and then flow cytometry was performed and analyzed. The in vitro B cell cytotoxicity data showed that the killing efficacy of Luoyuan AIO CAR-T (Cam AIO CAR-T) was stronger than that of humanized AIO CAR-T (Hu AIO CAR-T), and both were stronger than bispecific CAR-T cells (Bi-CD19×CD20 scFv CAR-T-1, Bi-CD19×CD20 scFv CAR-T-2). All CAR-Ts showed dose-dependency (Figure 5).

[0385] Example 5 Detection of Cytotoxicity of Trispecific AIO CAR-T on B Cells in APH

[0386] To evaluate the rapid killing ability of Hu AIO CAR-T, Hu AIO CAR-T was co-incubated with apheresis cells (APH) from the same donor, and multicolor flow cytometry was performed to detect cytotoxicity. After the frozen apheresis cells were revived, they were co-incubated with Hu AIO CAR-T cells at an E:T ratio of 1:3 (ratio of effector cells-CAR-T (effector) to target cells (target)) for 2 hours. Untransduced T cells (unT) from the same batch were used as a negative control. Flow cytometry data showed that Hu AIO CAR-T could eliminate B cells in the same donor APH within 2 hours, and the percentage of B cell lysis reached 89.9% compared with UnT cells (Figure 6).

[0387] Example 6 In vivo efficacy of trispecific AIO CAR-T cells with different sequences or sequential combinations in tumor xenograft mice

[0388] According to the in vivo CAR-T transfusion and monitoring schedule for mice shown in Figure 7, the anti-tumor activity of trispecific AIO CAR-T cells was evaluated in the Raji.Luc xenograft NCG mouse model. Monospecific CD19 scFv (mouse FMC63 scFv) CAR-T, CD20 scFv (mouse Leu16 scFv) CAR-T, and CD22 scFv (mouse m971 scFv) CAR-T cells were used as positive controls, and UnT cells were used as negative controls. Four days in advance (Day-4), Raji.Luc cells expressing the firefly luciferase reporter gene were intravenously injected into NCG mice. Raji cells themselves have high levels of expression of CD19, CD20, and CD22, enabling them to be used to evaluate the therapeutic effects of AIO CAR-T cells targeting CD19, CD20, and CD22 in vivo. After inoculation of Raji.Luc tumor cells, bioluminescence imaging (BLI) was performed weekly or biweekly to monitor tumor progression. Animals were randomized based on BLI photon count and animal body weight. After randomization, a single dose (0.3×10 6 The mice were then injected with AIO CAR-T cells or UnT cells (cells), followed by weekly BLI imaging to record tumor growth. Control mice treated with UnT cells showed rapid progression of lymphoma, with most dying around day 14. In contrast, mice infused with AIO CAR-T cells showed a significant reduction in systemic lymphoma burden. In vivo experiments in mice demonstrated that AIO CAR-T cells exhibited a stronger tumor growth inhibitory effect compared to positive controls of CD19 scFv (murine FMC63 scFv) CAR-T and CD22 scFv (murine m971scFv) CAR-T cells (Figure 8A). Over 51 days, AIO-3 CAR-T demonstrated the strongest tumor suppression effect and significantly enhanced CAR-T cell expansion and persistence (Figure 8B), surpassing other trispecific AIO CAR-Ts and single CD19 scFv (murine FMC63 scFv) CAR-Ts, CD20 scFv (murine Leu16 scFv) CAR-Ts, and CD22 scFv (murine m971 scFv) CAR-Ts, extending overall survival in mice. Following CAR-T therapy, mice whose tumors were cleared maintained normal health and stable weight.

[0389] Example 7 In vivo safety evaluation of trispecific AIO CAR-T cell preparations in tumor xenograft mice

[0390] According to the in vivo CAR-T infusion and monitoring schedule for the mouse study shown in Figure 9, luciferase-labeled Nalm6 cells expressing human CD20 were inoculated into NCG mice to establish a Nalm6-hCD20-FFluc xenograft NCG mouse model. The antitumor activity and safety of trispecific Hu AIO CAR-T cells were then evaluated. AIO CAR-T cells served as treatment groups (G4–G7), the vehicle group (G2) and the UnT cell-treated group (G3) served as drug-negative controls, and a non-tumor-bearing group (Blank, G1) served as a model control. Eight days prior (Day 8), NCG mice were intravenously injected with Nalm6-hCD20-FFluc cells expressing the firefly luciferase reporter gene. Nalm6-hCD20-FFluc cells have high levels of CD19, CD20, and CD22 expression, enabling them to be used for in vivo evaluation of the therapeutic effects of AIO CAR-T (Hu AIO CAR-T) cells targeting CD19, CD20, and CD22. Animals were randomly divided into groups based on BLI photon counts and animal body weight. After randomization, high doses (3×10 6 cells) and low dose (1×10 6 cells) of AIO CAR-T cells, or UnT cells, and then BLI imaging was performed weekly to record tumor growth (Figure 9). In mice treated with UnT cells (G3) or Vehicle (G2), the tumor progressed rapidly, and most died around the 13th day; while mice that were infused with different doses of AIO CAR-T cells showed a significant tumor control effect (Figure 10A). In addition, after receiving low and high doses of AIO CAR-T treatment, the mice were in good condition and their weight was stable (Figure 10B). Some tissues were infiltrated with immune cells, mainly in the liver, spleen, lung, kidney, and femur. The rest of the tissues had no or less immune cell infiltration, and the T cell infiltration level in the high-dose group was higher than that in the low-dose group (Figures 10C-10D). However, no obvious tissue damage was observed in the corresponding HE staining (Figures 10E-10G). In addition, no obvious damage was observed in the HE staining of the reproductive system of female mice (Figure 10G), indicating that the AIO cell preparation is safe in mice and has a very low safety risk to the reproductive organs.

[0391] Example 8 Trispecific AIO CAR-T cell preparation for clinical treatment of relapsed / refractory acute B-lymphoblastic leukemia (ALL)

[0392] This clinical study is a prospective, single-arm, open-label Phase I exploratory clinical study designed to evaluate the safety, tolerability, pharmacokinetics, and anti-tumor efficacy of AIO CAR-T cell therapy in subjects with relapsed / refractory B-cell acute lymphoblastic leukemia (ALL).

[0393] This study design includes two phases: dose exploration and dose expansion. In the dose exploration phase, the accelerated titration and BOIN (Bayesian Optimal Interval design) design schemes were used. Based on the absolute value of CAR+T cells, the dose was increased from 0.1×10 6 CAR+T cells / kg is the starting dose, and 5 dose groups are planned. Among the 5 dose groups, the low dose group includes: 0.1×10 6 CAR+T cells / kg, 0.25×10 6 CAR+T cells / kg; these two dose levels are planned to be evaluated by accelerated titration. The medium and high dose groups include: 0.5×10 6 CAR+T cells / kg, 1.0×10 6 CAR+T cells / kg and 2.0×10 6 CAR+T cells / kg, the BOIN dose increase and decrease rule was directly used for dose assessment.

[0394] After completing the screening assessment, eligible subjects underwent apheresis to collect peripheral blood mononuclear cells (PBMCs). T cells from each subject were isolated from the PBMCs and used to prepare the AIO CAR-T cell preparation, which was then returned to the subject during treatment. The AIO CAR-T cell preparation infusion was scheduled 5 to 7 days after the start of the conditioning regimen. Following the completion of the cell preparation infusion, subjects were followed up for safety and efficacy as specified in the study protocol.

[0395] Preliminary clinical results show that AIO CAR-T achieves extensive and profound clearance of each B cell subset expressing CD19, CD20, or CD22. It has comparable efficacy to CD19 scFv (murine FMC63 scFv) CAR-T cells for B cell clearance, and its B cell clearance window is related to the dose level of reinfusion, which can be adjusted clinically based on disease progression and B cell load. Anti-tumor efficacy evaluation and B cell target clinical follow-up data show that within 6-11 days after reinfusion, AIO CAR-T cell preparations efficiently or completely eliminate B cells during their expansion phase, alleviating or curing relapsed / refractory acute B lymphoblastic leukemia (ALL), completing immune reconstitution, and normal B cell recovery occurs within 2-6 months (Figure 11). Pharmacokinetic data showed that after receiving the AIO CAR-T cell formulation, CAR-positive T cells underwent in vivo proliferation within the dose range of 0.1 M / kg to 1.0 M / kg, undergoing rapid expansion followed by a slow decline. AIO CAR-T PK analysis was performed using flow cytometry (CAR+T cell counts / mL) and qPCR (CAR+copies / μg DNA) (Figures 12A and 12B). Pharmacokinetic data demonstrated the ability of AIO CAR-T cells to rapidly expand in subjects. Safety and tolerability observations showed no non-targeted killing. All CRS were low (most were grade 1 or 2). Among the 13 patients, the incidence of ICANS was low (one patient was grade 1). The incidence of infection was also low in patients with long-term B cell deficiency, and immunoglobulin therapy was not required. Due to the use of a humanized AIO CAR, the incidence of ADA before and after treatment was low and late (detectable after ~180 days), which did not affect the efficacy and expansion of CAR-T cells.

[0396] Example 9 Trispecific AIO CAR-T cell preparation for clinical treatment of relapsed / refractory systemic lupus erythematosus (SLE)

[0397] This prospective, single-arm, open-label Phase I study will evaluate the safety, tolerability, pharmacokinetics, and efficacy of AIO CAR-T in subjects with relapsed / refractory SLE. All subjects will receive a single intravenous infusion of the AIO CAR-T cell formulation. The study will be divided into two phases: dose-finding and dose-expansion. The dose-finding phase will utilize a BOIN design, with four dose cohorts planned. The planned sample size for the dose-finding phase is approximately 25 subjects. Following the dose-finding phase, a dose-expansion phase will be conducted based on the results, with a planned enrollment of 10 subjects.

[0398] Enrolled subjects must have had an inadequate response to glucocorticoids combined with at least two of the following treatments (each for at least 3 months): cyclophosphamide, mycophenolate mofetil or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, and biologics including belimumab, tetanusip, rituximab, and aniluomab, unless contraindications limit continued use of the above drugs.

[0399] After the screening evaluation is completed, eligible subjects will undergo apheresis to collect peripheral blood mononuclear cells (PBMC). T cells from each subject will be separated from the PBMC and used to prepare the AIO CAR-T cell preparation, which will then be returned to the subject during the treatment period. If clinically indicated, subjects can receive bridging therapy. After meeting the pretreatment criteria for treatment, the subject will first receive a pretreatment regimen (intravenous infusion of cyclophosphamide 1000mg / m 2 1 dose and fludarabine 25 mg / m 2 Once a day for 3 days); researchers can adjust the regimen based on the subject's creatinine clearance, disease status, and major organ function, referring to the drug instructions and previous clinical experience. Depending on the specific clinical protocol design, subjects may not receive the conditioning regimen. The first infusion is scheduled 5 to 7 days after the start of the conditioning regimen, and the day of infusion is counted as D1. AIO CAR-positive T cells 0.1×10 6 cells / kg was the starting dose, and the BOIN dose-escalation rule was used for dose assessment. Four dose groups of 0.1×10 6 cells / kg, 0.25×10 6 cells / kg, 0.5×10 6 cells / kg, 1.0×10 6 cells / kg to evaluate the safety, tolerability, pharmacokinetic characteristics, and immunogenicity of AIO CAR cell therapy; determine the RP2D (Phase II recommended dose) of AIO CAR-T cell therapy and preliminarily observe the efficacy.

[0400] Example 10: Clinical Treatment of Relapsed / Refractory Autoimmune Disease (r / rAID) with Trispecific AIO CAR-T Cell Preparations

[0401] This project is a prospective, single-arm, open-label clinical study to evaluate the safety, tolerability, pharmacokinetics, and efficacy of AIO CAR-T in subjects with relapsed / refractory autoimmune diseases (SLE / SSc / AAV / IIM). All subjects will receive a single intravenous injection of the AIO CAR-T cell preparation. The study will be divided into two phases: dose exploration and dose expansion. The dose exploration phase will adopt a BF-BOIN design and plans to conduct a study in three dose groups. The planned sample size of the study is approximately 25 subjects in the dose exploration phase. After the dose exploration phase, the dose expansion phase will be conducted based on the results, and 12 subjects are planned to be enrolled in this phase.

[0402] The enrolled subjects with relapsed / refractory systemic lupus erythematosus (r / r SLE) had previously responded poorly to or were intolerant to hormones and at least one immunosuppressant or one biological agent (such as belimumab, temetasipu, rituximab, etc.) or one cell therapy (such as stem cell therapy, other CAR-T therapy with different targets), or had relapsed after remission.

[0403] The enrolled subjects with relapsing / refractory systemic sclerosis (r / r SSc) had previously received conventional treatment (an immunosuppressant or a biological agent or a cell therapy (such as stem cell therapy, other different target CAR-T therapy), with or without anti-fibrotic therapy and / or vascular protective agents), and the investigators judged that the efficacy was poor or the subject was intolerant; or had achieved remission with previous treatment and then relapsed during maintenance therapy or after drug reduction or discontinuation.

[0404] The enrolled subjects with relapsed / refractory ANCA-associated vasculitis (r / r AAV) had previously been treated with glucocorticoids and at least one immunosuppressant or a biological agent (such as rituximab, belimumab, tetanusip, tocilizumab, etc.) or a JAK inhibitor, and the investigators judged that the treatment had failed or had poor efficacy or intolerance; or had previously achieved remission with induction therapy and then relapsed during maintenance therapy or after drug reduction or discontinuation.

[0405] The enrolled subjects with relapsed / refractory inflammatory myopathy (r / r IIM) had previously been treated with glucocorticoids and at least one immunosuppressant or a biologic or a JAK inhibitor, and the investigator judged that the treatment had failed, had poor efficacy, or was intolerant; or had achieved remission with previous treatment and then had disease relapse during maintenance or after drug reduction or discontinuation.

[0406] Eligible subjects will undergo apheresis to collect peripheral blood mononuclear cells (PBMCs). Study enrollment is defined as the date of apheresis. AIO CAR-T cell preparations will be prepared using the subject's own apheresis. A second apheresis is permitted for subjects whose apheresis or preparation fails.

[0407] During the preparation of the AIO CAT-T cell preparation (i.e., after apheresis and before lymphoablation), AID treatment may be allowed to control the disease if necessary. The bridging therapy regimen is a short-term treatment regimen (hormone monotherapy or hormones + immunosuppressants) that the subject has previously received to at least achieve disease stability. During the bridging phase, hormones are ≤40 mg / day of prednisolone or other hormones at an equivalent dose. The use of biological agents (such as belimumab), JAK inhibitors, cyclophosphamide, or experimental drugs is prohibited during the bridging therapy phase, and bridging therapy must be stopped at least 7 days before starting lymphoablation (the researcher may retain low-dose hormones, ≤20 mg / day of prednisone equivalent, based on the patient's condition).

[0408] After meeting the pretreatment criteria for treatment, the subjects will first receive the pretreatment regimen (intravenous infusion of cyclophosphamide 300mg / m 2 and fludarabine 25 mg / m 2 , once a day for 3 days [-5, -4, -3 days]); researchers can make adjustments based on the subject's eGFR, disease status, and major organ function status, referring to the drug instructions and previous clinical experience. In addition, researchers can also decide the specific amount of fluid replacement, fluid replacement speed, and other treatment measures according to the subject's age and condition. Depending on the specific clinical protocol design, the subject may not receive the conditioning regimen. The first infusion is scheduled 5 to 7 days after the start of the conditioning regimen, and the day of infusion is counted as D1. The starting dose is selected as 0.25×10 viable AIO CAR+T cells 6 The dosage of AIO CAR-T cell preparation was divided into three groups. The dose increase and decrease rule designed by BF-BOIN was used for dose evaluation. It was planned to conduct three dose groups with 0.25×10 6 / kg (range: 0.175-0.325×10 6 / kg), 0.5×10 6 / kg (range: 0.35-0.65×10 6 / kg), 1.0×10 6 / kg (range: 0.7-1.3×10 6 / kg) to evaluate the safety, tolerability, pharmacokinetic characteristics, and immunogenicity of AIO CAR-T cell therapy; determine the RP2D of AIO CAR-T cell therapy and preliminarily observe the efficacy.

[0409] Example 11 Trispecific AIO CAR-T cell preparation for clinical treatment of relapsed / refractory nervous system autoimmune diseases

[0410] This project is a prospective, single-arm, open-label clinical study to evaluate the safety, tolerability, pharmacokinetics, and efficacy of AIO CAR-T in subjects with relapsed / refractory neurological autoimmune diseases (MS / NMOSD / MOGAD / MG). All subjects will receive a single intravenous injection of the AIO CAR-T cell formulation. The study will be divided into two phases: dose-finding and dose-expansion. The dose-finding phase will adopt a BF-BOIN design, and three dose groups are planned. The planned sample size of the study is approximately 25 subjects in the dose-finding phase. After the dose-finding phase, a dose-expansion phase will be conducted based on the results. This phase plans to enroll 12 subjects to ensure that at least one subject is enrolled in each indication (MS / NMOSD / MOGAD / MG) at this dose level for verification.

[0411] The enrolled subjects had relapsing / refractory multiple sclerosis (r / r MS) and had an inadequate response to, intolerance to, or contraindications to at least one highly effective DMT (disease-modifying therapy, such as alemtuzumab, cladribine, natalizumab, ocrelizumab, rituximab, ofatumumab, or an S1PR modulator).

[0412] The enrolled subjects with relapsed / refractory neuromyelitis optica spectrum disorder / anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-associated disease (r / r NMOSD / MOGAD) have had an inadequate response, intolerance, or contraindications to at least one DMT (such as IST, rituximab, inelizumab, etc.), and IST treatment must last for at least 6 months.

[0413] The enrolled subjects with relapsed / refractory myasthenia gravis (r / r MG) had an inadequate response to at least two traditional immunosuppressants and / or biologics, with no significant improvement in myasthenia symptoms during treatment and affecting their living conditions; could not tolerate the side effects of immunosuppressants / biologics or had contraindications to immunosuppressants and required repeated IVIG or plasma exchange to alleviate the condition; or had repeated myasthenic crises during the course of the disease.

[0414] Eligible subjects will undergo apheresis to collect peripheral blood mononuclear cells (PBMCs). Study enrollment is defined as the date of apheresis. AIO CAR-T cell preparations will be prepared using the subject's own apheresis. Subjects whose apheresis or preparation fails are allowed to undergo a second apheresis. During the preparation of AIO CAR-T cell preparations (i.e., after apheresis and before lympholysis treatment), treatment for autoimmune diseases of the nervous system (MS / NMOSD / MOGAD / MG) may be allowed to control the disease if necessary. A bridging therapy is a short-term treatment regimen in which the subject has achieved at least disease stability after previous treatment (it is recommended to use hormone monotherapy or hormone + immunosuppressant, and glucocorticoids should not exceed 40 mg / day of prednisone or equivalent dose). During the bridging treatment phase, the use of previously unused disease-modifying treatment regimens or experimental drugs is prohibited, as is the use of drugs targeting CD19 or CD20 (such as rituximab, inerizumab, etc.), and bridging treatment must be stopped at least 7 days before starting lymphoproliferative disorder (the researcher may retain low-dose hormones, ≤20 mg / day prednisone equivalent, based on the patient's condition).

[0415] After meeting the pretreatment criteria for treatment, the subjects will first receive the pretreatment regimen (intravenous infusion of cyclophosphamide 300mg / m 2 and fludarabine 25 mg / m 2 , once daily for 3 days [-5, -4, -3 days]); researchers may adjust the dosage based on the subject's eGFR, disease status, and major organ function, referring to the drug instructions and previous clinical experience. In addition, researchers may also determine the specific amount of fluid replacement, the rate of fluid replacement, and other treatment measures based on the subject's age and condition. Depending on the specific clinical design, subjects may not receive the conditioning regimen. The first infusion is scheduled 5 to 7 days after the start of the conditioning regimen, and the day of infusion is counted as D1. The dosage of AIO CAR-T cell preparations is divided into 3 groups.

[0416] The starting dose of this study was selected as 0.25×10 viable AIO CAR+T cells. 6 / kg (range: 0.175-0.325×10 6 The selection of single infusion and starting dose was based on safety considerations, with 0.25×10 viable AIO CAR+ T cells. 6 / kg (range: 0.175-0.325×10 6 / kg) was used as the starting dose, and the dose increase and decrease rule designed by BF-BOIN was used for dose evaluation. Three dose groups of 0.25×10 6 / kg (range: 0.175-0.325×10 6 / kg), 0.5×10 6 / kg (range: 0.35-0.65×10 6 / kg), 1.0×10 6 / kg (range: 0.7-1.3×10 6 / kg) to evaluate the safety, tolerability, pharmacokinetic characteristics, and immunogenicity of AIO CAR-T cell therapy; determine the RP2D (Recommended Phase II dose level, Recommended Phase II dose) of AIO CAR-T cell therapy and preliminarily observe the efficacy.

[0417] sequence

[0418] Table 7

Claims

1. A method for treating an autoimmune disease (AID) in a subject in need thereof, the method comprising administering to the subject an effective amount of CD20, CD19, and CD22 antagonists.

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

3. The method according to claim 1, wherein the autoimmune disease is systemic sclerosis (SSc), ANCA-associated vasculitis (AAV), idiopathic inflammatory myopathies (IIM), or a neurological autoimmune disease.

4. The method according to claim 3, wherein the neurological autoimmune disease is multiple sclerosis (MS), neuromyelitis optica spectrum disease (NMOSD) / anti-myelin oligodendrocyte glycoprotein-IgG associated disorders (MOGAD), or myasthenia gravis (MG).

5. The method according to any one of claims 1-4, wherein the autoimmune disease is a relapsed / refractory autoimmune disease.

6. The method according to claim 2, wherein the subject has an insufficient response to at least one drug for treating systemic lupus erythematosus, the drugs selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid esters or their derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, taitanicimab, rituximab, anifrolumab, chloroquine, hydroxychloroquine, stem cell therapy, and immunocyte therapy.

7. The method according to claim 2 or 6, wherein the subject has an insufficient response to at least two drugs for treating systemic lupus erythematosus, the drugs selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid esters or their derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, taitanicimab, rituximab, anifrolumab, chloroquine, hydroxychloroquine, stem cell therapy, and immunocyte therapy.

8. The method according to any one of claims 2 and 6 - 7, wherein the subject has an insufficient response to glucocorticoids in combination with at least one drug for treating systemic lupus erythematosus, and the drug is selected from mycophenolate mofetil, cyclophosphamide, mycophenolic acid ester or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, taitanicimab, rituximab, anifrolumab, chloroquine, hydroxychloroquine, stem cell therapy, and immune cell therapy.

9. The method according to any one of claims 2 and 6 - 8, wherein the subject has an insufficient response to glucocorticoids in combination with at least two drugs for treating systemic lupus erythematosus, and the drug is selected from mycophenolate mofetil, cyclophosphamide, mycophenolic acid ester or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, taitanicimab, rituximab, anifrolumab, chloroquine, hydroxychloroquine, stem cell therapy, and immune cell therapy.

10. The method according to any one of claims 6 - 9, wherein the immune cell therapy is CAR - T or CAR - NK targeting at least one of CD19, CD20, and BCMA.

11. The method according to any one of claims 2 and 6 - 10, wherein the subject has concurrent lupus nephritis (LN).

12. The method according to claim 3, wherein the autoimmune disease is systemic sclerosis.

13. The method according to claim 12, wherein the subject has an insufficient response to at least one drug for treating systemic sclerosis, and the drug is selected from anti - fibrotic drugs (such as nintedanib, pirfenidone), vasoprotective agents (such as PDE5 inhibitors, endothelin receptor inhibitors, calcium channel blockers), glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid ester or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, belimumab, stem cell therapy, and immune cell therapy.

14. The method according to claim 12 or 13, wherein the subject has an insufficient response to anti - fibrotic drugs (such as nintedanib, pirfenidone) and / or vasoprotective agents (such as PDE5 inhibitors, endothelin receptor inhibitors, calcium channel blockers) in combination with at least one drug for treating systemic sclerosis, and the drug is selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid ester or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, belimumab, stem cell therapy, and immune cell therapy.

15. The method according to claim 13 or 14, wherein the immune cell therapy is CAR - T or CAR - NK targeting at least one of CD19, CD20, and BCMA.

16. The method according to claim 3, wherein the autoimmune disease is ANCA - associated vasculitis.

17. The method according to claim 16, wherein the subject has an insufficient response to at least one drug for treating ANCA-associated vasculitis, and the drug is selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid esters or their derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tacirolimus, rituximab, mepolizumab, tocilizumab, avacopan, and JAK inhibitors.

18. The method according to claim 16 or 17, wherein the subject has an insufficient response to a combination of glucocorticoids and at least one drug for treating ANCA-associated vasculitis, and the drug is selected from mycophenolate mofetil, cyclophosphamide, mycophenolic acid esters or their derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, belimumab, tacirolimus, rituximab, mepolizumab, tocilizumab, avacopan, and JAK inhibitors.

19. The method according to claim 3, wherein the autoimmune disease is idiopathic inflammatory myopathy.

20. The method according to claim 19, wherein the subject has an insufficient response to at least one drug for treating idiopathic inflammatory myopathy, and the drug is selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid esters or their derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, and JAK inhibitors.

21. The method according to claim 19 or 20, wherein the subject has an insufficient response to a combination of glucocorticoids and at least one drug for treating idiopathic inflammatory myopathy, and the drug is selected from mycophenolate mofetil, cyclophosphamide, mycophenolic acid esters or their derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, tocilizumab, and JAK inhibitors.

22. The method according to claim 4, wherein the neurological autoimmune disease is multiple sclerosis.

23. The method according to claim 22, wherein the subject has an insufficient response to at least one drug for treating multiple sclerosis, and the drug is selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid esters or their derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, alemtuzumab, cladribine, natalizumab, ocrelizumab, rituximab, ofatumumab, and sphingosine 1-phoshate receptor (S1PR) modulators.

24. The method according to claim 4, wherein the neurological autoimmune disease is neuromyelitis optica spectrum disorder / antimyelin oligodendrocyte glycoprotein immunoglobulin G antibody-related disease.

25. The method according to claim 24, wherein the subject has an insufficient response to at least one drug for treating neuromyelitis optica spectrum disorder / anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-related disease, and the drug is selected from glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid ester or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, satralizumab, rituximab, inebilizumab, ravulizumab, eculizumab, tocilizumab.

26. The method according to claim 4, wherein the neurological autoimmune disease is myasthenia gravis.

27. The method according to claim 26, wherein the subject has an insufficient response to at least one drug for treating myasthenia gravis, and the drug is selected from acetylcholinesterase inhibitors, glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid ester or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, eculizumab, ravulizumab, belimumab, efgartigimod, rozanolixizumab, or requires repeated administration of intravenous immunoglobulin (IVIG) or plasma exchange to relieve the condition.

28. The method according to claim 26 or 27, wherein the subject has an insufficient response to at least two drugs for treating myasthenia gravis, and the drugs are selected from acetylcholinesterase inhibitors, glucocorticoids, mycophenolate mofetil, cyclophosphamide, mycophenolic acid ester or its derivatives, azathioprine, methotrexate, leflunomide, tacrolimus, cyclosporine, rituximab, eculizumab, ravulizumab, belimumab, efgartigimod, rozanolixizumab.

29. The method according to any one of claims 1-28, wherein the CD20, CD19 and CD22 antagonists are selected from engineered immune effector cells, engineered receptors, antibodies, antibody-drug conjugates (ADCs), aptamers and small RNAs.

30. The method according to any one of claims 1-29, wherein the CD20, CD19 and CD22 antagonists are a single antagonist, a combination of two antagonists, or a combination of three antagonists.

31. The method according to any one of claims 1-30, wherein the CD20, CD19 and CD22 antagonists are a combination of three antagonists including a CD20 antagonist, a CD19 antagonist and a CD22 antagonist, wherein: (1) The CD20 antagonist is a first group of engineered immune effector cells comprising an engineered receptor that specifically targets CD20, and the engineered immune effector cells comprise: an extracellular antigen-binding domain comprising at least one anti-CD20 binding moiety, a transmembrane domain and an intracellular signaling domain; (2) CD19 antagonists are a second group of engineered immune effector cells comprising engineered receptors that specifically target CD19, which comprise: an extracellular antigen-binding domain comprising at least one anti-CD19 binding moiety, a transmembrane domain, and an intracellular signaling domain; and (3) CD22 antagonists are a third group of engineered immune effector cells comprising engineered receptors that specifically target CD22, which comprise: an extracellular antigen-binding domain comprising at least one anti-CD22 binding moiety, a transmembrane domain, and an intracellular signaling domain.

32. The method according to any one of claims 1-30, wherein the CD20, CD19, and CD22 antagonists are engineered immune effector cells comprising a first engineered receptor that specifically targets CD20, a second engineered receptor that specifically targets CD19, and a third engineered receptor that specifically targets CD22, wherein (1) the first engineered receptor that specifically targets CD20 comprises: a first extracellular antigen-binding domain comprising at least one anti-CD20 binding moiety, a first transmembrane domain, and a first intracellular signaling domain; (2) the second engineered receptor that specifically targets CD19 comprises: a second extracellular antigen-binding domain comprising at least one anti-CD19 binding moiety, a second transmembrane domain, and a second intracellular signaling domain; and (3) the third engineered receptor that specifically targets CD22 comprises: a third extracellular antigen-binding domain comprising at least one anti-CD22 binding moiety, a third transmembrane domain, and a third intracellular signaling domain.

33. The method according to any one of claims 1-30, wherein the CD20, CD19, and CD22 antagonists are engineered immune effector cells comprising an engineered receptor that simultaneously targets CD20, CD19, and CD22, the receptor comprising: (a) an extracellular antigen-binding domain comprising at least one anti-CD20 binding moiety, at least one anti-CD19 binding moiety, and at least one anti-CD22 binding moiety; (b) a transmembrane domain; and (c) an intracellular signaling domain.

34. The method according to any one of claims 31-33, wherein the engineered receptor is selected from the group consisting of engineered T cell receptors (TCRs), chimeric antigen receptors (CARs), T cell antigen conjugates (TACs), or portions thereof.

35. The method according to claim 33 or 34, wherein the CD20, CD19, and CD22 antagonists are engineered immune effector cells comprising a chimeric antigen receptor (CAR) that simultaneously targets CD20, CD19, and CD22, the CAR comprising: (a) an extracellular antigen-binding domain comprising at least one anti-CD20 binding moiety, at least one anti-CD19 binding moiety, and at least one anti-CD22 binding moiety; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD20 binding moiety is closer to the transmembrane domain than the anti-CD19 binding moiety or the anti-CD22 binding moiety.

36. The method according to claim 35, wherein the anti-CD19 binding portion is at the N-terminus or C-terminus of the anti-CD22 binding portion.

37. The method according to any one of claims 33-36, wherein the CD20, CD19 and CD22 antagonists are engineered immune effector cells comprising a chimeric antigen receptor that simultaneously targets CD20, CD19 and CD22, the CAR comprising: (a) an extracellular antigen-binding domain comprising at least one anti-CD20 binding portion, at least one anti-CD19 binding portion and at least one anti-CD22 binding portion; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD20 binding portion, the anti-CD19 binding portion and the anti-CD22 binding portion are fused to each other directly or via one or more peptide linkers; and wherein the one or more peptide linkers comprise no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids.

38. The method according to claim 37, wherein the one or more peptide linkers are (GGGGS)n, where n is 1, 2, 3 or 4.

39. The method according to any one of claims 31-38, wherein the anti-CD20 binding portion, the anti-CD19 binding portion and the anti-CD22 binding portion are selected from Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), minibody, diabody, single-domain antibody (sdAb) or VHH domain.

40. The method according to any one of claims 31-39, wherein the anti-CD20 binding portion is an anti-CD20 single-domain antibody (sdAb) comprising: (i) a CDR1 comprising the amino acid sequence of SEQ ID NO:1 or 7; a CDR2 comprising the amino acid sequence of SEQ ID NO:2; and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:5; and a CDR3 comprising the amino acid sequence of SEQ ID NO:6; (iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:1 or 7; a CDR2 comprising the amino acid sequence of SEQ ID NO:8; and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; (iv) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:9; and a CDR3 comprising the amino acid sequence of SEQ ID NO:6; (v) a CDR1 comprising the amino acid sequence of SEQ ID NO:12 or 18; a CDR2 comprising the amino acid sequence of SEQ ID NO:13; and a CDR3 comprising the amino acid sequence of SEQ ID NO:14; or (vi) CDR1 comprising the amino acid sequence of SEQ ID NO:15; CDR2 comprising the amino acid sequence of SEQ ID NO:16; and CDR3 comprising the amino acid sequence of SEQ ID NO:

17.

41. The method according to any one of claims 31-39, wherein the anti-CD19 binding portion is an anti-CD19 single domain antibody (sdAb) comprising: (i) CDR1 comprising the amino acid sequence of SEQ ID NO:21 or 27; CDR2 comprising the amino acid sequence of SEQ ID NO:22; and CDR3 comprising the amino acid sequence of SEQ ID NO:23; or (ii) CDR1 comprising the amino acid sequence of SEQ ID NO:24; CDR2 comprising the amino acid sequence of SEQ ID NO:25; and CDR3 comprising the amino acid sequence of SEQ ID NO:

26.

42. The method according to any one of claims 31-39, wherein the anti-CD22 binding portion is an anti-CD22 single domain antibody (sdAb) comprising: (i) CDR1 comprising the amino acid sequence of SEQ ID NO:30 or 36; CDR2 comprising the amino acid sequence of SEQ ID NO:31; and CDR3 comprising the amino acid sequence of SEQ ID NO:32; or (ii) CDR1 comprising the amino acid sequence of SEQ ID NO:33; CDR2 comprising the amino acid sequence of SEQ ID NO:34; and CDR3 comprising the amino acid sequence of SEQ ID NO:

35.

43. The method according to any one of claims 35-42, wherein the CAR comprises: (a) an extracellular antigen-binding domain comprising an anti-CD20 sdAb, an anti-CD19 sdAb, and an anti-CD22 sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein: (I) the anti-CD20 sdAb comprises: (i) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO:10, respectively; (ii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO:11, respectively; (iii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO:19, respectively; or (iv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO:20, respectively; (II) the anti-CD19 sdAb comprises: (i) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO:28, respectively; or (ii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 shown in SEQ ID NO: 29, respectively; (III) The anti-CD22 sdAb comprises: (i) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 shown in SEQ ID NO: 37, respectively; or (ii) CDR1, CDR2 and CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 shown in SEQ ID NO: 38, respectively.

44. The method according to any one of claims 40 - 43, wherein the anti-CD20 sdAb, the anti-CD19 sdAb and the anti-CD22 sdAb are each independently a camelid sdAb or a humanized sdAb.

45. The method according to any one of claims 40 - 44, wherein the CAR comprises: (I) The anti-CD20 sdAb comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: 20; (II) The anti-CD19 sdAb comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29; and (III) The anti-CD22 sdAb comprises the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO:

38.

46. The method according to any one of claims 35 - 45, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

47. The method according to claim 46, wherein the transmembrane domain is derived from CD8α.

48. The method according to any one of claims 35 - 47, wherein the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell.

49. The method according to claim 48, wherein the primary intracellular signaling domain is derived from CD3ζ.

50. The method according to claim 48 or 49, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain.

51. The method according to claim 50, wherein the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, the ligand of B7-H3, CD83 and combinations thereof.

52. The method according to claim 51, wherein the co-stimulatory signaling domain is derived from CD137.

53. The method according to any one of claims 35 - 52, wherein the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

54. The method according to claim 53, wherein the hinge domain is derived from CD8α.

55. The method according to any one of claims 35 - 54, wherein the CAR comprises (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 81 - 85; or (ii) an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 81 - 85.

56. The method according to any one of claims 35 - 55, wherein the CAR further comprises a signal peptide located at the N-terminus of the polypeptide.

57. The method according to claim 56, wherein the signal peptide is derived from CD8α.

58. The method according to any one of claims 35 - 57, wherein the CAR comprises (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 67 - 71; or (ii) an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 67 - 71.

59. The method according to any one of claims 31 - 58, wherein the engineered immune effector cells are selected from the group consisting of T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

60. The method according to any one of claims 31-59, wherein the amount of the engineered immune effector cells administered to the subject is 0.1 to 1×10 6 cells / kg.

61. The method according to any one of claims 1 - 60, wherein the subject is lymphodepleted before or at the time of administration of the antagonist.

62. The method according to claim 61, wherein lymphodepletion comprises administration of cyclophosphamide and fludarabine.

63. The method according to claim 61 or 62, wherein lymphodepletion comprises: (1) Administer cyclophosphamide at 500 - 1500 mg / m 2 once only and fludarabine at 15 - 35 mg / m 2 once daily for up to 3 days; or (2) Administer cyclophosphamide at 200 - 400 mg / m 2 once a day for up to 3 days, and fludarabine at 15 - 35 mg / m 2 once a day for up to 3 days.

64. The method according to any one of claims 61-63, wherein lymphocyte depletion comprises intravenous infusion of cyclophosphamide at 1000 mg / m 2 for 1 time and fludarabine at 25 mg / m 2 once daily for up to 3 days.

65. The method according to any one of claims 61-63, wherein the lymphocyte depletion comprises intravenous infusion of cyclophosphamide at 300 mg / m 2 once daily for up to 3 days, and fludarabine at 25 mg / m 2 once daily for up to 3 days.

66. The method according to any one of claims 61 - 65, wherein lymphodepletion is initiated at least 5 days before administration of the antagonist.

67. The method according to any one of claims 1 - 60, wherein the subject is not lymphodepleted before administration of the antagonist.

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