A single-domain antibody targeting CD22, a chimeric antigen receptor, and their applications

Abstract

The present invention relates to the technical field of immunotherapy. The present invention provides a single-domain antibody targeting CD22, a chimeric antigen receptor (CAR) targeting CD22 constructed by using the single-domain antibody, and an engineered immune effector cell. The present invention also provides the use of the single-domain antibody targeting CD22, the CAR, and the engineered immune effector cell in the preparation of a medicament for treating CD22-related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy, and particularly relates to a single-domain antibody targeting CD22, a chimeric antigen receptor targeting CD22 constructed therefrom, and an engineered immune effector cell. The present invention also relates to a method for treating diseases or disorders in a subject, particularly to chimeric antigen receptor-based T cell immunotherapy. Background Art

[0002] CD22 is a B lymphocyte lineage differentiation antigen, also known as BL-CAM, B3, Leu-14, Lyb-8, and Siglec-2, and has been shown to be specifically expressed by B lymphocytes and is functionally important as a negative regulator of B lymphocyte activation. CD22 is an inhibitory co-receptor that can downregulate BCR signaling and block overstimulation of B cells, and plays an important role in maintaining the marginal zone B cell population, optimal B cell antigen receptor-induced proliferation, and B cell renewal. In particular, CD22 is expressed in B cell malignancies, making it a promising target for cancer treatment. In addition, some researchers have proposed treating autoimmune diseases by selectively regulating B cell activity by targeting CD22.

[0003] Chimeric antigen receptor (CAR)-modified T cells, as an immunotherapy strategy, have received extensive attention and application in tumor treatment, especially in hematological malignancies. Its principle is that through gene modification, T cells are made to express a receptor structure (single-chain antibody) that can specifically recognize tumor cell surface antigens, and after the specific binding of this receptor to tumor cell surface antigens, downstream immune co-stimulatory factors and T cells are activated, thereby activating T cells to secrete related cytokines and specifically killing tumor cells. The structure of CAR generally consists of four parts: an extracellular antigen-binding domain (usually a single-chain antibody with antigen recognition function), a hinge region, a transmembrane domain, and an intracellular signal transduction domain. Currently, according to the number of co-stimulatory molecules added to the intracellular signal transduction domain, CAR structures are usually divided into the first generation (without co-stimulatory molecules), the second generation (including one co-stimulatory molecule), and the third generation (including two co-stimulatory molecules). Currently, the second-generation CAR structure is the most widely used in marketed products and clinical research.

[0004] Although great progress has been made in the treatment of childhood and adult acute lymphoblastic leukemia (ALL), a considerable number of patients still have poor treatment outcomes, and the current standard treatment has a considerable degree of short-term and long-term toxicity. Monoclonal antibody-based therapies are expected to overcome chemotherapy resistance and potential treatment-related toxicity. Among them, the most promising is chimeric antigen receptor-modified T (CAR-T) cell therapy, which can break through MHC restriction and directly recognize tumor antigens. Currently, CAR-T cells have been widely used in hematological malignancies. In particular, CAR-T cell immunotherapy targeting CD19 as the target antigen has achieved breakthrough progress. However, CD19 CAR-T is not universally effective, and the loss of target antigen as a tumor escape mechanism after immunotherapy limits the therapeutic effect of cellular immunotherapy in hematological malignancies. The tumor cell escape mechanisms during CD19 CAR-T treatment for B-ALL mainly include alternative splicing, frameshift mutations, and missense mutations of CD19. Therefore, in the future, it is necessary to consider further enhancing the anti-tumor targeting potential of CAR-T cells, improving the therapeutic effect, and reducing the recurrence rate after tumor treatment by selecting new CAR-T treatment targets, or combining with CD19 CAR-T, or constructing multi-target CAR-T.

[0005] Similar to the CD19 antigen, CD22 is also restrictedly expressed on B cells, not expressed on other parenchymal cells, nor on hematopoietic stem cells. Therefore, it has high specificity as a B cell tumor antigen and has become an ideal treatment target in B cell malignancies. In addition, CD22 and CD19 are widely co-expressed on the surface of tumor cells, and the CD22 antigen remains after the loss of the CD19 antigen caused by CD19 CAR-T cell therapy. Therefore, CD22 CAR-T cells can be used alone to treat B cell malignancies, or for salvage treatment of patients who relapse due to antigen variation after CD19 CAR-T treatment and whose tumor cells express CD22, or in combination with CD19 CAR-T cells to avoid antigen variation, improve the effectiveness of CAR-T treatment, and reduce tumor recurrence.

[0006] Single-domain antibodies (sdAbs) differ from traditional 4-chain antibodies in that they possess the variable domain of a single monomeric antibody. For example, camelids and sharks produce antibodies that are naturally lacking in light chains, which are known as heavy-chain-only antibodies (hcAbs, or simply heavy-chain antibodies). The antigen-binding fragment in each arm of a camelid heavy-chain-only antibody has a single heavy-chain variable domain (VHH), which can have high affinity for antigens without the assistance of a light chain. Camelid VHH antibodies are referred to as the smallest functional antigen-binding fragments, with a molecular weight of only approximately 15 kD, and are thus also known as nanobodies. VHH antibodies have natural advantages such as good solubility, high stability, strong penetrability, and a wide range of binding epitopes. Since the discovery of VHH antibodies, they have gradually attracted the attention of researchers in the field, and the basic research on them has become increasingly mature. In terms of applications, they have gradually entered the clinical research stage for autoimmune diseases, blood diseases, viral infections, and orthopedic diseases, and have also shown great advantages in anti-infection, anti-inflammatory diseases, and neurodegenerative diseases.

[0007] Since CAR-T cell manufacturing technology requires the use of single-chain antibodies with good binding activity and efficient binding epitopes, one of the key technical parts of CAR-T cell therapy lies in screening high-affinity antibodies with good specificity, strong binding force, and effective binding epitopes. However, traditional CD22 antibodies are limited by disadvantages such as large molecular weight, weak binding force with antigens, low affinity, difficulty in modification, and poor stability, and further single-chain modification is required. It is difficult to effectively construct CAR-T cells using traditional CD22 antibodies (such as monoclonal antibodies, etc.).

[0008] Therefore, there is still a wide demand for the development of improved single-domain antibodies targeting CD22, chimeric antigen receptors targeting CD22 constructed therefrom, and engineered immune effector cells. For example, the development of stable and small single-domain antibodies targeting CD22 for more effective and efficient CAR-T cell therapy. Summary of the Invention

[0009] One of the objectives of the present invention is to provide a single-domain antibody targeting CD22, which has a natural single-chain structure, with advantages such as small molecular weight, high solubility, high stability, low immunogenicity, high tissue penetrability, and does not require additional folding and assembly steps or linker optimization and modification, making it a promising alternative to scFv single-chain antibodies with larger molecular weights. After constructing CAR-T cells using the single-domain antibody, it has very significant tumor cell killing ability.

[0010] The anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1-19, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20-34, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35-62.

[0011] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35.

[0012] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 36.

[0013] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 37.

[0014] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 38.

[0015] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 22, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 46.

[0016] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO:8, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO:23, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO:47.

[0017] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO:10, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO:26, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO:50.

[0018] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO:11, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO:27, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO:51.

[0019] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO:12, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO:28, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO:52.

[0020] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO:13, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO:29, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO:53.

[0021] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO:14, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO:30, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO:54.

[0022] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO: 30, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO: 56.

[0023] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 15, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO: 31, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO: 57.

[0024] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO: 31, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO: 59.

[0025] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO: 32, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO: 60.

[0026] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO: 33, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO: 61.

[0027] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO: 62.

[0028] The anti-CD22 single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein the determination of CDR1, CDR2, and CDR3 is based on the IMGT numbering scheme, Kabat numbering scheme, AbM numbering scheme, Chothia numbering scheme, or Contact numbering scheme.

[0029] The anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63-72, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 73-92, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 93-118, and wherein FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0030] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 73, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 93, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0031] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 73, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 97, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0032] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 65, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 77, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 104, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0033] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3 and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 66, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 78, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 105, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0034] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3 and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 66, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 80, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 108, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0035] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3 and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 81, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 109, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0036] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3 and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 82, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 110, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0037] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3 and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 84, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 111, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0038] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 85, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 112, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0039] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 69, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 85, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 111, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0040] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 87, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 113, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0041] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 71, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 89, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 115, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0042] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 90, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 116, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0043] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 72, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 91, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 117, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0044] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention further comprises FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO: 63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO: 92, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO: 118, and wherein said FR4 comprises the amino acid sequence shown in SEQ ID NO: 119.

[0045] Among them, the specific amino acid sequence information shown in SEQ ID NOs: 1-119 is shown in Table 1.

[0046] Table 1

[0047]

[0048]

[0049]

[0050]

[0051] The anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% similarity to the amino acid sequence shown in SEQ ID NOs: 120-136.

[0052] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% similarity to the amino acid sequence shown in SEQ ID NO: 120.

[0053] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% similarity to the amino acid sequence shown in SEQ ID NO: 121.

[0054] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 122.

[0055] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 123.

[0056] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 124.

[0057] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 125.

[0058] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 126.

[0059] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 127.

[0060] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 128.

[0061] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 129.

[0062] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 130.

[0063] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 131.

[0064] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 132.

[0065] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 133.

[0066] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 134.

[0067] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 135.

[0068] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 136.

[0069] The anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in Table 2 or SEQ ID NOs: 120-136.

[0070] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 120.

[0071] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 121.

[0072] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 122.

[0073] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 123.

[0074] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 124.

[0075] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 125.

[0076] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 126.

[0077] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 127.

[0078] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 128.

[0079] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 129.

[0080] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 130.

[0081] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 131.

[0082] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 132.

[0083] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 133.

[0084] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 134.

[0085] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 135.

[0086] In some embodiments, the anti-CD22 single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 136.

[0087] Among them, the specific amino acid sequence information shown in SEQ ID NOs: 120-136 is shown in Table 2.

[0088] Table 2

[0089]

[0090]

[0091]

[0092] One of the objectives of the present invention is to provide a chimeric antigen receptor, and after transfection to prepare CAR-T cells, the chimeric antigen receptor has a very significant ability to kill tumor cells.

[0093] The chimeric antigen receptor provided by the present invention comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signal transduction domain; wherein, the extracellular antigen-binding domain comprises the anti-CD22 single-domain antibody as described above.

[0094] In some embodiments, for the chimeric antigen receptor provided by the present invention, the transmembrane domain is derived from CD8α, CD28, CD4, CD137, CD80, CD86, CD152 or PD-1.

[0095] In some embodiments, for the chimeric antigen receptor provided by the present invention, the transmembrane domain is derived from CD8α.

[0096] In some embodiments, for the chimeric antigen receptor provided by the present invention, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 140.

[0097] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signal transduction domain is derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD66d, FcRγ, FcRβ.

[0098] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signal transduction domain is derived from CD3ζ.

[0099] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 142.

[0100] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signal transduction domain further comprises a co-stimulatory signal domain.

[0101] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the co-stimulatory signal domain is derived from CD137 (4-1BB), CD27, CD28, ICOS, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand, and combinations thereof.

[0102] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the co-stimulatory signal domain is derived from CD137 (4-1BB).

[0103] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the co-stimulatory signal domain comprises the amino acid sequence shown in SEQ ID NO: 141.

[0104] In some embodiments, the chimeric antigen receptor provided by the present invention further comprises a hinge region located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0105] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the hinge region is derived from CD8α, CD28, IgG1, or IgG4.

[0106] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the hinge region is derived from CD8α.

[0107] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO: 139.

[0108] In some embodiments, the chimeric antigen receptor provided by the present invention further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide.

[0109] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the signal peptide is derived from HLA-A, CD8α, CD33, Igκ, IL-2, GM-CSFRα.

[0110] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the signal peptide is derived from HLA-A.

[0111] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 138.

[0112] Among them, the specific amino acid sequence information shown in SEQ ID NOs: 138-142 is shown in Table 3.

[0113] Table 3

[0114]

[0115] The chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequences shown in SEQ ID NOs: 143-159.

[0116] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 143.

[0117] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 144.

[0118] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 145.

[0119] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 146.

[0120] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 147.

[0121] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 148.

[0122] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 149.

[0123] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 150.

[0124] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 151.

[0125] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 152.

[0126] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 153.

[0127] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 154.

[0128] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 155.

[0129] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 156.

[0130] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 157.

[0131] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 158.

[0132] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO: 159.

[0133] The chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in Table 4 or SEQ ID NOs: 143 - 159.

[0134] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 143.

[0135] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 144.

[0136] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 145.

[0137] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 146.

[0138] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 147.

[0139] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 148.

[0140] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 149.

[0141] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 150.

[0142] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 151.

[0143] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 152.

[0144] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 153.

[0145] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 154.

[0146] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 155.

[0147] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 156.

[0148] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 157.

[0149] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 158.

[0150] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 159.

[0151] Among them, the specific amino acid sequence information shown in SEQ ID NOs: 143-159 is shown in Table 4.

[0152] Table 4

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] One object of the present invention is to provide a nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor as described above. In some embodiments, the nucleic acid provided by the present invention comprises the nucleic acid sequences shown in SEQ ID NOs: 161-178. Among them, the specific nucleic acid sequence information shown in SEQ ID NOs: 161-178 is shown in Table 5.

[0159] Table 5

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168] One object of the present invention is to provide a vector comprising a nucleic acid encoding the nucleic acid sequence of the chimeric antigen receptor as described above.

[0169] One object of the present invention is to provide an engineered immune effector cell comprising the chimeric antigen receptor, nucleic acid, or vector as described above.

[0170] In some embodiments, the engineered immune effector cells provided by the present invention are selected from T cells, B cells, NK cells, macrophages, dendritic cells, induced pluripotent stem cells (iPSCs).

[0171] One of the objectives of the present invention is to provide a pharmaceutical composition, which comprises the aforementioned CD22-targeting single-domain antibody, engineered immune effector cells, and a pharmaceutically acceptable carrier or excipient.

[0172] One of the objectives of the present invention is further to provide a method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the aforementioned CD22-targeting single-domain antibody, engineered immune effector cells, or pharmaceutical composition.

[0173] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject, wherein the disease or disorder is a B cell-related disease or disorder and / or a CD22-related disease or disorder.

[0174] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject, wherein the disease or disorder is cancer.

[0175] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject, wherein the disease or disorder is a B cell-related malignancy. For example, the B cell-related malignancy is B cell leukemia or B cell lymphoma. More specifically, the disease or disorder is selected from marginal zone lymphoma (e.g., splenic marginal zone lymphoma), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, primary mediastinal B cell lymphoma (PMBL), small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia (B-PLL), follicular lymphoma (FL), Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia (HCL), precursor B lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), high-grade B cell lymphoma (HGBL), and multiple myeloma (MM).

[0176] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject, wherein the disease or disorder is a B cell-related autoimmune and / or inflammatory disease. More specifically, the B cell-related autoimmune and / or inflammatory disease is related to inappropriate or enhanced B cell numbers and / or activation.

[0177] One of the objectives of the present invention is further to provide the use of the aforementioned CD22-targeting single-domain antibody, engineered immune effector cells, and pharmaceutical composition in the preparation of a medicament for treating B cell-related malignancies, B cell-related autoimmune diseases, and / or inflammatory diseases.

[0178] Term Explanation

[0179] As used herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions having multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, particularly antigen-binding fragments such as Fab, F(ab')2 and Fv. In some embodiments of the invention, the terms "immunoglobulin (Ig)" and "antibody" may be used interchangeably.

[0180] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0181] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, containing ten antigen-binding sites; while IgA antibodies contain two to five basic four-chain units, which can combine with the J chain to polymerize into a multivalent assembly. In the case of IgG, the four-chain unit is typically about 150,000 daltons. Each light chain is linked to a heavy chain by a covalent disulfide bond, and the two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the heavy chain isotype. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (for each α and γ chain, CH1, CH2, and CH3) and four (for μ and ε isotypes, CH1, CH2, CH3, and CH4) constant domains (CH) and a hinge region (Hinge) located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at the N-terminus, followed by its constant domain (CL) at the other end. VL aligns with VH, and CL aligns with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form an interface between the light and heavy chain variable domains. The paired VH and VL together form an antigen-binding site. For the structure and properties of different classes of antibodies, see also Basic and Clinical Immunology. Eighth edition. Daniel P. Sties, Abba I. Terrand Tristram G. Parsolw. Appleton & Lange, Norwalk, CT. 1994, Page 71 and Chapter 6. Light chains from any vertebrate species can be grouped into one of two distinct types called κ and λ, based on the amino acid sequence of their constant domain. Immunoglobulins can be grouped into different classes or isotypes based on the amino acid sequence of their heavy chain constant domain (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains called α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes can be further divided into subclasses, for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0182] Heavy chain antibodies are antibodies derived from organisms of the Camelidae family or Chondrichthyes. Compared with the above-mentioned 4-chain antibodies, heavy chain antibodies lack the light chain and the first constant domain of the heavy chain (CH1), and only contain 2 heavy chains composed of variable regions (VHH) and other constant regions. The variable region is connected to the constant region through a structure similar to the hinge region. Each heavy chain of a Camelidae heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of a Chondrichthyes heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5). The antigen-binding fragment of a heavy chain antibody includes VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, a heavy chain antibody can have the CH2 and CH3 of human IgG Fc.

[0183] The terms "single-domain antibody", "single-domain antibody targeting CD22", "variable domain of the heavy chain of a heavy chain antibody", "VHH", and "nanobody" used in the present invention are used interchangeably, and all refer to a single-domain antibody that specifically recognizes and binds to CD22. A single-domain antibody is the variable region of a heavy chain antibody. Generally, a single-domain antibody contains three CDR regions and four FR regions. A single-domain antibody is the smallest functional antigen-binding fragment. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant domain of the heavy chain (CH1), the variable region of the antibody heavy chain is then cloned to construct a single-domain antibody composed of only one variable domain of the heavy chain.

[0184] Without substantially affecting the antibody activity, those skilled in the art can change one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody or its functional fragment sequences. These variants include, but are not limited to: deletion, insertion, and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. In the art, when conservative substitutions are made with amino acids having similar or similar properties, the function of the protein is usually not changed. For example, amino acids with similar properties are substituted in the FR and / or CDR of the variable region. Amino acid residues that can undergo conservative substitutions are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Another example is that adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. They are all considered to be included within the scope of protection of the present invention.

[0185] In some embodiments, the sequence of the variant of the present invention may have at least 95%, 96%, 97%, 98% or 99% identity with its source sequence. The sequence identity described in the present invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The present invention also includes molecules having a variable region of an antibody heavy chain with CDRs, provided that the CDRs thereof have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0186] The single-domain antibodies, nanobodies or heavy-chain antibodies of the present invention can be prepared by conventional methods in the art, such as the well-known phage display technology in the art. Alternatively, various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with the sequences encoding various antibodies of the present invention. The transformation can be carried out by any known method, for example, including packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulating the polynucleotide in liposomes and direct microinjection of DNA into the nucleus, etc. Host mammalian cell lines that can be used for expression are well known in the art, for example, various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantially CD22-binding properties.

[0187] The term "chimeric antigen receptor (CAR)" as used in the present invention comprises an extracellular antigen-binding domain, and the extracellular antigen-binding domain comprises a single-domain antibody (sdAb) that binds to CD22 disclosed in the present invention, such as VHH.

[0188] In some embodiments, the chimeric antigen receptor (CAR) disclosed in the present invention comprises a polypeptide, which comprises: (a) an extracellular antigen-binding domain, which comprises a single-domain antibody (sdAb) of CD22 disclosed in the present invention; (b) a transmembrane domain; (c) an intracellular signal transduction domain. Each domain and additional regions will be described in more detail below.

[0189] The CARs disclosed by the present invention comprise an extracellular antigen-binding domain, which comprises one or more single-domain antibodies. The sdAbs can have the same or different origins and have the same or different sizes. Exemplary sdAbs include, but are not limited to, heavy-chain variable domains (such as VHHs) from heavy-chain-only antibodies, binding molecules that naturally lack light chains, single domains (such as VH or VL) derived from conventional four-chain antibodies, humanized heavy-chain-only antibodies, human single-domain antibodies produced by transgenic mice or rats expressing human heavy-chain fragments, and engineered domains and single-domain scaffolds that are not derived from antibodies. Any sdAb known in the art or disclosed by the present invention, including the single-domain antibodies disclosed by the present invention, can be used to construct the CARs described herein. The sdAbs can be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, sharks, goats, rabbits, and cows. The single-domain antibodies contemplated by the present invention also include naturally occurring single-domain antibody molecules from species other than camelids and sharks.

[0190] In some embodiments, the extracellular antigen-binding domain disclosed by the present invention comprises at least one binding domain, and the at least one binding domain comprises a single-domain antibody that binds CD22 and is disclosed by the present invention.

[0191] In some embodiments, the anti-CD22 sdAb is camel, chimeric, human, or humanized.

[0192] In some embodiments, the CARs disclosed by the present invention that comprise a polypeptide comprise: (a) an extracellular antigen-binding domain that comprises an anti-CD22 sdAb; (b) a transmembrane domain; (c) an intracellular signaling domain; wherein the anti-CD22 sdAb comprises a polypeptide sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% similarity to the sequences SEQ ID NOs: 120-136.

[0193] In some embodiments, the CARs disclosed by the present invention that comprise a polypeptide comprise: (a) an extracellular antigen-binding domain that comprises an anti-CD22 sdAb; (b) a transmembrane domain; (c) an intracellular signaling domain; wherein the anti-CD22 sdAb comprises the amino acid sequences SEQ ID NOs: 120-136.

[0194] In addition to the antigen-binding domains disclosed by the present invention, the CARs disclosed by the present invention can also comprise one or more of the following structures: a linker (such as a peptide linker), a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory signal domain, an intracellular signaling domain, and these domains will be described in detail below.

[0195] In some embodiments, the intracellular signaling domain comprises the major intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the major intracellular signaling domain is derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD66d, FcRγ, FcRβ. In some embodiments, the major intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule that is derived from one or more of CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand. In some embodiments, the co-stimulatory signaling domain is derived from CD137 (4-1BB).

[0196] In some embodiments, the CD22 CAR further comprises a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0197] In some embodiments, the CD22 CAR further comprises a signal peptide (e.g., an HLA-A signal peptide or a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus: an HLA-A signal peptide or a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge region, a CD8α transmembrane domain, a co-stimulatory signaling domain derived from CD137 (4-1BB), and an intracellular signaling domain derived from CD3ζ.

[0198] In some embodiments, the different domains of the CAR can also be fused to each other via peptide linkers. Depending on the structure and / or functional characteristics of the single-domain antibody and / or the various domains, each peptide linker in the CAR can have the same or different length and / or sequence. Those skilled in the art can independently select and optimize each peptide linker. In some embodiments, the peptide linker comprises flexible residues (e.g., glycine and serine) such that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0199] The peptide linker can have any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids in length. In some embodiments, the peptide linker is 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 fewer amino acids in length. In some embodiments, the peptide linker has a length of from about 1 amino acid to about 10 amino acids, from about 1 amino acid to about 20 amino acids, from about 1 amino acid to about 30 amino acids, from about 5 amino acids to about 15 amino acids, from about 10 amino acids to about 25 amino acids, from about 5 amino acids to about 30 amino acids, from about 10 amino acids to about 30 amino acids in length, from about 30 amino acids to about 50 amino acids, from about 50 amino acids to about 100 amino acids, or from about 1 amino acid to about 100 amino acids.

[0200] The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a single-chain antibody can be used as a linker. See, e.g., WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSG)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0201] The present invention also discloses nucleic acids encoding the various antibodies or chimeric antigen receptors described above. The present invention provides polynucleotides encoding the heavy chain variable region, light chain variable region, heavy chain, light chain, and each CDR. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0202] As is well known to those skilled in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids can be prepared, all of which encode the antibodies or chimeric antigen receptors of the present invention. Thus, in the case where a specific amino acid sequence has been identified, those skilled in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. Therefore, the present invention also relates to polynucleotides that hybridize to the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides described in the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0203] The full-length sequences or fragments of the nucleic acids of various antibodies or chimeric antigen receptors of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis methods. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0204] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually to clone them into a vector, then transfer them into cells, and then isolate the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) involved in the present invention include biomolecules in an isolated form. At present, it is already possible to completely obtain the DNA sequence encoding the polypeptide (or its fragment, or its derivative) of the present invention by chemical synthesis. Then, the DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can be introduced into the polypeptide sequence of the present invention by chemical synthesis.

[0205] The present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above-mentioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins. Vectors generally contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The said sequences (collectively referred to as "flanking sequences" in some embodiments) generally include one or more of the following nucleotide sequences: promoters, one or more enhancer sequences, origins of replication, transcription termination sequences, complete intron sequences containing donor and acceptor splice sites, leader sequences encoding for polypeptide secretion, ribosome binding sites, polyadenylation sequences, multiple linker regions for inserting nucleic acids encoding antibodies to be expressed, and optional marker elements.

[0206] The host cells related to the present invention can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium; fungal cells of yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0207] In some embodiments, the host cells can be various functional cells well-known in the art, such as various killer cells, including but not limited to cytokine-induced killer cells (CIK), dendritic cell-stimulated cytokine-induced killer cells (DC-CIK), cytotoxic T lymphocytes (CTL), γδ T cells, natural killer cells (NK), tumor-infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), CD3AK cells (killer cells of anti-CD3 monoclonal antibody), and CAR-T / TCR-T cells. In certain embodiments, the killer cells are T cells or NK cells. Exemplary NK cells include but are not limited to primary NK cells, NK cell lines (such as NK92), and NKT cells. In certain embodiments, the NK cells are primary NK cells. Exemplary T cells include but are not limited to peripheral blood T lymphocytes, cord blood T lymphocytes, cytotoxic killer T cells (CTL), helper T cells, suppressor / regulatory T cells, γδ T cells, and T cells of mixed cell populations such as cytokine-induced killer cells (CIK), tumor-infiltrating lymphocytes (TIL), etc. In certain embodiments, the T cells are peripheral blood T lymphocytes, cord blood T lymphocytes.

[0208] Transformation of a host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps of which are well known in the art. Another method is to use MgCl2. In addition, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0209] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media, such as serum-containing medium or serum-free medium. The culture is carried out under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0210] The polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0211] The present invention also discloses a vector for cloning and expressing any one of the CARs of the present invention. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, vaccinia virus vectors, herpes simplex virus vectors, and their derivatives. Viral vector technology is well known in the art and has been described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. (2001)) and other virology and molecular biology manuals.

[0212] Many virus-based systems have been developed in the prior art for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated in vitro or ex vivo and delivered to engineered mammalian cells. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying the coding sequence of an immunomodulator (such as an immune checkpoint inhibitor) and / or self-inactivating lentiviral vectors carrying a chimeric antigen receptor can be packaged using protocols known in the art. Using methods known in the art, the resulting lentiviral vectors can be used to transduce mammalian cells (such as primary human T cells). Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow for the long-term, stable integration of the transgene and its propagation in progeny cells. Lentiviral vectors also have the advantage of low immunogenicity and can transduce non-proliferating cells.

[0213] In some embodiments, the vector comprises any nucleic acid encoding a CAR as described herein. The nucleic acid can be cloned into the vector using any known molecular cloning method in the art, including, for example, using restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in the present invention. Promoters can be further classified as constitutive promoters or regulatable promoters, such as inducible promoters.

[0214] In some embodiments, the nucleic acid encoding the CAR is operably linked to a constitutive promoter. A constitutive promoter allows for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Exemplary constitutive promoters contemplated by the present invention include, but are not limited to, the cytomegalovirus (CMV) promoter, the human elongation factor-1α (hEF1α) promoter, the ubiquitin C (UbiC) promoter, the phosphoglycerate kinase (PGK) promoter, the simian virus 40 (SV40) early promoter, and the chicken β-actin coupled with the CMV early enhancer (CAGG) promoter. The efficiency of such constitutive promoters in driving transgene expression has been widely compared in numerous studies. For example, Michael C. Milone et al. (Molecular Therapy, 17(8):1453-1464 (2009)) compared the efficiency of CMV, hEF1α, UbiC, and PGK in driving chimeric antigen receptor expression in human primary T cells and concluded that the hEF1α promoter not only induced the highest level of transgene expression but also was optimal in maintaining in CD4 and CD8 human T cells. In some embodiments, the nucleic acid encoding the CAR is operably linked to the hEF1α promoter.

[0215] In some embodiments, the nucleic acid encoding the CAR is operably linked to an inducible promoter. Inducible promoters belong to regulatable promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of engineered immune effector cells or the physiological state of engineered immune effector cells, inducers, etc.

[0216] In some embodiments, the inducing conditions do not induce the expression of endogenous genes in engineered mammalian cells and / or subjects receiving the pharmaceutical composition. In some embodiments, the inducing conditions are selected from: inducers, radiation (such as ionizing radiation, light), temperature (such as heat), redox state, tumor microenvironment, and the activation state of engineered mammalian cells.

[0217] In some embodiments, the vector further comprises a selectable marker gene or a reporter gene to select cells expressing the CAR from a population of host cells transfected with the lentiviral vector. Appropriate regulatory sequences can flank both the selectable marker and the reporter gene for expression in the host cell. For example, the vector can contain transcriptional and translational terminators, initiation sequences, and promoters for regulating the expression of nucleic acid sequences.

[0218] As used herein, the term "immune effector cell" refers to an immune cell that can perform immune effector functions. In some embodiments, the immune effector cell expresses at least FcγRIII and performs ADCC effector functions. Examples of immune effector cells mediating ADCC include T cells, B cells, NK cells, macrophages, dendritic cells, induced pluripotent stem cells (iPSCs), etc.

[0219] In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is a CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8- T cell or a combination thereof. In some embodiments, the T cell produces IL-2, TFN, and / or TNF after expressing a CAR and binding to a target cell such as a CD22+ tumor cell. In some embodiments, the CD8+ T cell lyses antigen-specific target cells after expressing a CAR and binding to the target cell.

[0220] In some embodiments, the immune effector cell is a NK cell. In other embodiments, the immune effector cell can be an established cell line, such as an NK-92 cell.

[0221] In some embodiments, the immune effector cell can be differentiated from a stem cell, such as a hematopoietic stem cell, a pluripotent stem cell, an iPSC, or an embryonic stem cell.

[0222] The engineered immune effector cell of the present invention is prepared by introducing a CAR into an immune effector cell (such as a T cell). In some embodiments, the CAR is introduced into the immune effector cell by transfecting any one of the isolated nucleic acids or any one of the above vectors.

[0223] Methods for introducing a vector or an isolated nucleic acid into a mammalian cell are known in the art. The described vectors can be transferred into the immune effector cell by physical, chemical, or biological methods.

[0224] Physical methods for introducing a vector into an immune effector cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing a vector and / or exogenous nucleic acid are well known in the art (see Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments, the vector is introduced into the cell by electroporation.

[0225] Biological methods for introducing a vector into an immune effector cell include using DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (such as human cells).

[0226] Chemical methods of introducing a vector into immune effector cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vector is a liposome.

[0227] In some embodiments, an RNA molecule encoding any CAR described herein can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into immune effector cells by known methods such as mRNA electroporation (see Peter M Rabinovich. Human Gene Therapy, 17:1027-1035 (2006)).

[0228] In some embodiments, transduced or transfected immune effector cells are proliferated ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are cultured to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days. In some embodiments, the transduced or transfected immune effector cells can be further evaluated or screened to select engineered immune effector cells.

[0229] Reporter genes can be used to identify cells that may be transfected and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide that expresses some easily detectable property, such as enzyme activity. The expression of the reporter gene is assayed at an appropriate time after introduction of the DNA into the recipient cells. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (see Kumiko Ui-Tei. FEBS Letters, 479:79-82 (2000)). Suitable expression systems are known in the art and can be prepared using known techniques or obtained commercially. Other methods of confirming the presence of a nucleic acid encoding a CAR in engineered immune effector cells include: molecular biology test methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; biochemical assay methods, such as detecting the presence or absence of a specific peptide; and immunological methods, such as ELISA.

[0230] The pharmaceutical composition disclosed by the present invention contains the CD22-targeting single-domain antibody, engineered immune effector cells, and pharmaceutically acceptable excipients or carriers described in the present invention. Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic or substantially non-toxic to the recipient at the doses and concentrations employed. Such excipients include, but are not limited to: saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition. The optimal pharmaceutical composition can be determined according to the intended route of administration, mode of delivery, and required dose.

[0231] The pharmaceutical composition for in vivo administration is generally provided in the form of a sterile preparation. Sterilization is achieved by filtration through a sterile filter membrane. This method can be used for sterilization before, during, or after lyophilization, reconstitution, and dilution of the composition. The pharmaceutical composition of the present invention can be selected for parenteral delivery. The composition for parenteral administration can be stored in lyophilized form or in solution. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The parenteral composition is usually placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of the pharmaceutically acceptable composition is within the scope of those skilled in the art. Other pharmaceutical compositions will be obvious to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Techniques for formulating various other sustained or controlled delivery modes (such as liposome carriers, bioerodible microparticles, or porous beads and depot injections) are also known to those skilled in the art.

[0232] Once formulated, the pharmaceutical composition is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal, or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form that needs to be reconstituted before administration (e.g., lyophilized). The present invention also provides a kit for generating single-dose administration units. The kit of the present invention may each contain a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, a kit containing single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyophilized syringes) is provided.

[0233] The present invention also provides a method for treating a patient (especially a patient suffering from a CD22-related disease) by administering the anti-CD22 single-domain antibody, engineered immune effector cell or pharmaceutical composition thereof according to any embodiment of the present invention. The terms "patient", "subject", "individual", "object" in the present invention are used interchangeably herein and include any living organism, preferably an animal, more preferably a mammal (such as a rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. "Treatment" refers to using the treatment method described herein for a subject to achieve at least one positive therapeutic effect (for example, a reduction in the number of cancer cells, a reduction in tumor volume, a reduction in the rate of cancer cell infiltration into surrounding organs, or a reduction in the rate of tumor metastasis or tumor growth). The treatment method for effectively treating a patient may vary according to various factors (such as the disease state, age, weight of the patient, and the ability of the therapy to stimulate the anti-cancer response of the subject).

[0234] The therapeutically effective amount of the pharmaceutical composition containing the anti-CD22 single-domain antibody or engineered immune effector cell of the present invention to be employed will depend, for example, on the degree and target of treatment. Those skilled in the art will understand that the appropriate dosage level for treatment will vary in part depending on the molecule to be delivered, the indication, the route of administration, and the patient's situation (body weight, body surface or organ size) and / or condition (age and general health status). In certain embodiments, the clinician can titrate the dose and change the route of administration to obtain the optimal therapeutic effect.

[0235] The dosing frequency will depend on the pharmacokinetic parameters of the anti-CD22 single-domain antibody or engineered immune effector cell in the formulation used. Clinicians typically administer the pharmaceutical composition until a dose that achieves the desired effect is reached. The pharmaceutical composition can thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or by continuous infusion through an implant device or catheter.

[0236] The route of administration of the pharmaceutical composition is conventional in the art, for example, oral, nasal, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection, and can also be administered by a sustained release system or through an implant device. BRIEF DESCRIPTION OF THE DRAWINGS

[0237] Figure 1A The expression rate of the CD22 CAR polypeptide molecule on CD4+ T cells in each group is shown.

[0238] Figure 1B The expression rate of the CD22 CAR polypeptide molecule on CD8+ T cells in each group is shown.

[0239] Figure 2Shows the expression of CD22 antigen on the surface of Raji cells, Namalwa cells, and K562 cells, respectively.

[0240] Figure 3A Shows the killing rate of effector cells in each group against Raji cells on the first day (D1) when the effector-to-target ratio (E:T) = 1:1.

[0241] Figure 3B Shows the killing rate of effector cells in each group against Raji cells on the third day (D3) when the effector-to-target ratio (E:T) = 1:1.

[0242] Figure 4A Shows the killing rate of effector cells in each group against Raji cells on the first day (D1) when the effector-to-target ratio (E:T) = 1:3.

[0243] Figure 4B Shows the killing rate of effector cells in each group against Raji cells on the third day (D3) when the effector-to-target ratio (E:T) = 1:3.

[0244] Figure 5A Shows the killing rate of effector cells in each group against Namalwa cells on the first day (D1) when the effector-to-target ratio (E:T) = 1:3.

[0245] Figure 5B Shows the killing rate of effector cells in each group against Namalwa cells on the third day (D3) when the effector-to-target ratio (E:T) = 1:3.

[0246] Figure 6A Shows the release of Granzyme B by effector cells in each group in the in vitro killing experiment.

[0247] Figure 6B Shows the release of TNF-α by effector cells in each group in the in vitro killing experiment.

[0248] Figure 6C Shows the release of IFN-γ by effector cells in each group in the in vitro killing experiment.

[0249] Figure 6D Shows the release of IL-2 by effector cells in each group in the in vitro killing experiment.

[0250] In the above figures: UnT is the negative control group, m971 is the positive control group, S1 is the S1-CAR-T group, S4 is the S4-CAR-T group, S9 is the S9-CAR-T group, S27 is the S27-CAR-T group, S28 is the S28-CAR-T group, S35 is the S35-CAR-T group, S36 is the S36-CAR-T group, S41 is the S41-CAR-T group, and S43 is the S43-CAR-T group.

[0251] The realization of the object of the present invention, its functional characteristics and the achievement of beneficial effects will be further described in conjunction with the following embodiments and with reference to the accompanying drawings. Detailed implementation manners

[0252] The present invention will be further illustrated by way of examples below, but the present invention is not limited to the scope of the described embodiments. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0253] Example 1. Preparation of a CD22-targeted VHH single-domain antibody

[0254] (1) Animal immunization and immune response tests

[0255] The CD22 antigen used for animal immunization is Human Siglec-2 / CD22 Protein, Fc Tag (AcroBiosystem, Cat. No. CD2-H5253).

[0256] 1) Healthy alpacas were selected as the immunization subjects.

[0257] 2) For the first immunization, complete Freund's adjuvant was mixed with the CD22 antigen (0.8 mg) at a ratio of 1:1, emulsified and then subcutaneously injected at multiple points. For subsequent booster immunizations, incomplete Freund's adjuvant was mixed with the CD22 antigen at a ratio of 1:1. The immunization interval was 2 weeks, and a total of 5 immunizations were carried out; 5 mL of peripheral blood was collected before immunization and after each immunization, the serum was separated, and the immune response was monitored by the ELISA method to confirm the serum titer.

[0258] 3) After the 5th immunization, the plasma titer reached the level of 100,000. 20 mL of blood was collected, and the lymphocytes were separated and stored in Trizol for subsequent construction of the antibody phage library.

[0259] (2) Construction of the antibody phage library

[0260] 1) After the animal immunization was completed, RNA was extracted from the separated lymphocytes and reverse transcribed using the Takara reverse transcription kit for the total RNA obtained; the above total RNA sample was divided into two parts. One part used the Oligo dT Primer in the kit as the primer, and the other part used the Random 6-mers in the kit as the primer. According to the reverse transcription kit instructions, the total RNA obtained in the previous step was reverse transcribed into cDNA and stored in 2 centrifuge tubes respectively.

[0261] 2) PCR amplification

[0262] a. Amplify specific antibody fragments from reverse-transcribed cDNA using Taq DNA Polymerase Hot Start enzyme for PCR amplification; perform 1% agarose gel electrophoresis on all PCR products, cut and recover the bands with the target fragment size around 600 - 700 bp, which are the first-round PCR amplification products, and store them at -20°C;

[0263] b. Use the first-round PCR amplification products as templates for the second-round PCR reaction. After the reaction, perform 1% agarose gel electrophoresis, finally cut and recover the bands with a single target band and a fragment size of around 400 bp, and use a universal DNA purification and recovery kit to purify the DNA in the PCR reaction solution.

[0264] 3) Digestion and Ligation

[0265] Digest the target gene fragments amplified by the second-round PCR and the pComb3XSS phage plasmid vector with the restriction endonucleases Spe I and Sac I respectively. After digestion, use ligase to ligate the VHH target gene fragments to the pComb3XSS phage plasmid vector to construct a recombinant plasmid.

[0266] 4) Bacterial Library Construction

[0267] a. Take a 50 μL aliquot of TG1 competent cells and place them on ice for 5 - 10 min to thaw;

[0268] b. Add 100 ng of the ligation product, transfer it to a pre-chilled electroporation cuvette with a 1 mm gap, set the parameters in the electroporator: 1800 V, 1 mm, and then click the button to transform;

[0269] c. Immediately add 1 mL of pre-warmed SOC culture medium at 37°C after electroporation is completed, mix well, and shake the bacteria at 37°C and 200 rpm for 1 h to recover;

[0270] d. Take more than 20 100 ng ligation systems and perform electroporation reactions with competent cells according to the above method;

[0271] e. Take 100 μL from the recovered bacterial solution, perform 10-fold serial dilutions, and spread them on plates, and culture them overnight at 37°C;

[0272] f. Collect all the remaining bacterial solution and evenly spread it on more than 20 15 cm culture plates (2×YT containing 100 μg / mL Amp, 2% agarose), and culture them inverted overnight at 37°C;

[0273] g. Calculate the number of transformed colonies that can be obtained from all reactions based on the dilution factor and the number of single colonies, which is the library capacity of the bacterial library; at the same time, randomly select several monoclonal colonies from the gradient dilution plates for colony PCR. A single band around 400 bp in the PCR product is considered a positive clone, and the cloning positive rate of the bacterial library is estimated accordingly.

[0274] h. Scrape the overnight cultured plate colonies with 2×YT liquid medium, place them in a 50 mL centrifuge tube, measure their OD600 value, and add glycerol at a final concentration of 20% and store at -80 °C.

[0275] 5) Phage library construction

[0276] a. Inoculate the bacterial library into 100 mL of 2×YT liquid medium (containing 100 μg / mL Amp) to make the initial OD600 value 0.1, and culture at 37 °C and 250 rpm until the OD600 reaches 0.5 - 0.55.

[0277] b. Add helper phage at a ratio of 1:20 (number of bacteria: number of phages), and incubate at 37 °C and 250 rpm for 30 min.

[0278] c. Add Kana at a final concentration of 50 μg / mL, culture overnight at 30 °C and 250 rpm, centrifuge, and collect the supernatant.

[0279] d. Add 1 / 4 volume of pre-cooled PEG / NaCl, mix well, incubate on ice for at least 30 min, centrifuge at 4 °C and 4000 rpm for 20 minutes to remove the supernatant, then add 1 mL of PBS buffer to dissolve the precipitate; add 1 / 4 volume of pre-cooled PEG / NaCl again and incubate on ice for 10 minutes, centrifuge at 4 °C and 12000 g for 10 minutes to remove the supernatant and dissolve the precipitate in 1 mL of PBS, and store at -80 °C to obtain the purified phage library.

[0280] (3) Phage screening

[0281] 1) First round of screening

[0282] a. Coat the screening antigen on the immunotubes (50 μg / tube, coating solution is PBS, 2 mL / tube), rotate slowly at 4 °C overnight, and at the same time coat BSA (50 μg in PBS, 2 mL / tube) in parallel as a control; discard the supernatant in the overnight-coated immunotubes, wash the immunotubes 3 times with PBS buffer at room temperature, rotate for 5 min each time, add 2 mL of blocking solution (3% skim milk powder) solution, rotate and block at room temperature for 2 h, then discard the supernatant, and add 2 mL of PBST buffer to wash the immunotubes 3 times at room temperature, rotate for 5 min each time;

[0283] b. Discard the washing solution in the immunotubes, add the prepared phage library about 1012 The pfu was used as the input phage library for the first round of screening. Add PBS buffer to 2 mL, and incubate with rotation at room temperature for 1 h. Discard the supernatant, add 2 mL of PBST (1×PBS plus 0.1% Tween 20, the same below) buffer, and wash the immunotube 20 times at room temperature, rotating for 5 min each time. Discard the liquid in the immunotube, add 1 mL of 0.25 mg / mL Trypsin solution, and elute with rotation at room temperature for 30 min. Add 10 μL of 10% AEBSF to terminate the elution, and transfer the solution in the immunotube to a new 1.5 mL centrifuge tube, which is the phage eluate for the first round of screening.

[0284] 2) Titer detection of the phage eluate in the first round

[0285] Take 10 μL of the phage eluate in the first round, perform 10-fold serial dilutions in a 1.5 mL centrifuge tube, with a total of 12 dilutions up to 10 -12 ; Add 90 μL of TG1 bacterial solution to each diluted centrifuge tube, mix well by oscillation, and incubate at 37 °C for 30 min. Take 5 μL from each diluted centrifuge tube and drop it onto a 2×YT solid medium (Amp), let it stand for a few minutes, and then place it in an inverted position at 37 °C for overnight culture. Count the number of single colonies in the dilution with clearly distinguishable single colonies on the plate, and calculate the number of phagemids in each milliliter of the phage solution, that is, the titer of the phage library, according to the following formula:

[0286] T (pfu / mL) = N × D × 400

[0287] Where T is the phage titer (pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0288] 3) Phage eluate in the third round of screening

[0289] Repeat the above experiment 3 times, using the phage in the first round as the input phage library for the second round of screening to obtain the phage eluate in the second round, and then using the phage in the second round as the input phage library for the third round of screening to obtain the phage eluate in the third round.

[0290] (4) Monoclonal ELISA detection

[0291] 1) Take the bacterial solution at an appropriate dilution after the third round of screening, evenly coat it on a solid medium plate containing 100 μg / mL Amp, and place it in an inverted position at 37 °C for overnight culture.

[0292] 2) Randomly pick 192 monoclonal colonies from the overnight-cultured medium plate into a sterile 96-well cell culture plate, add 200 μL of 2×YT medium (containing 100 μg / mL Amp) to each well, and place it in an inverted position at 37 °C for overnight static culture.

[0293] 3) Transfer 5 μL of the overnight cultured bacterial solution to a new 96-well cell culture plate containing 200 μL of 2×YT liquid medium (containing 100 μg / mL Amp) per well, and place it in a static culture at 37°C for 5 h.

[0294] 4) Add helper phage M13K07 to each well, with the ratio of the number of bacteria to the number of phages being 1:20.

[0295] 5) After incubating at 37°C for 30 min, add Kana with a final concentration of 50 μg / mL, place it in a static culture at 30°C overnight, then centrifuge the 96-well cell culture plate and store it at 4°C for later use.

[0296] 6) Coat the enzyme-linked immunosorbent assay (ELISA) plate with the screening antigen (1 ng / μL, PBS, 100 μL / well), and simultaneously coat the same concentration of BSA in parallel as a control. Place it in a coating overnight at 4°C; discard the supernatant, wash the ELISA plate 3 times with PBS buffer at room temperature for 10 min each time; add 200 μL of blocking solution (3% BSA in PBST) to each well to block the ELISA plate, and block it at room temperature for 1 h; discard the blocking solution, add 200 μL of PBST (1×PBS plus 0.1% Tween 20, the same below) buffer to each well, and wash the ELISA plate 3 times at room temperature for 10 min each time.

[0297] 7) Add 100 μL of blocking solution to each well, and then add 100 μL of the supernatant after centrifugation in step 5), and incubate at room temperature for 2 h.

[0298] 8) Discard the liquid in the ELISA plate, add 200 μL of PBST buffer to each well and wash 3 times, 10 min each time.

[0299] 9) Add M13 Bacteriophage Antibody (HRP), Mouse Mab, diluted 1:30000 in the blocking solution, 100 μL / well, and incubate at room temperature for 1 h.

[0300] 10) Discard the liquid in the ELISA plate, add 200 μL of PBST buffer to each well and wash 6 times, 5 min each time.

[0301] 11) Add 100 μL of TMB single-component chromogenic solution to each well, develop color in the dark for 1 - 3 min, then add 100 μL of 1 M HCl to each well to terminate the reaction, and read the OD450 value with an enzyme-linked immunosorbent assay reader, record and save it.

[0302] Example 2. Construction and immune cell expression of chimeric antigen receptor targeting CD22

[0303] (1) Construction of CD22 CAR

[0304] First, the CAR nucleotide sequences (SEQ ID No. 161 - 178) targeting CD22 were designed and artificially synthesized. Each group of sequences contains the HLA - A signal peptide (SEQ ID No. 138) or CD8α signal peptide, the extracellular antigen - binding domain of CD22 VHH (SEQ ID No. 120 - 136) or CD22 scFv (m971 positive control, SEQ ID No. 137), the CD8α hinge region (SEQ ID No. 139), the CD8α transmembrane domain (SEQ ID No. 140), the CD137 (4 - 1BB) co - stimulatory signal domain (SEQ ID No. 141), and the CD3ζ intracellular signal transduction domain (SEQ ID No. 142) encoding nucleotide sequences for expressing the complete CD22 CAR polypeptide molecules (SEQ ID No. 143 - 160). The CD22 CAR nucleotide sequences were inserted into the multiple cloning site of the lentiviral expression vector pK1 by homologous recombination to obtain pK1 - CD22 CAR, and the successful construction of the lentiviral expression vector sequence was verified by electrophoresis and sequencing results.

[0305] (2) Packaging of lentiviral vectors

[0306] Resuscitate 293T cells and culture them in DMEM medium containing 10% FBS; after 2 - 3 generations of cell expansion culture, inoculate them into a 2 - layer cell factory at a density of 4×10 4 cells / cm 2 . Three days after cell inoculation, perform plasmid transfection; add 40 ml of Optim - MEM to a sterile 50 - ml centrifuge tube for plasmid transfection, then add the viral packaging vector and viral envelope vector according to the ratio of pK1 - CD22 CAR:pLP1:pLP2:pLP - VSVG = 5:4:3:1, and then add 800 μL of PEI transfection reagent, mix immediately, incubate at room temperature for 15 min, and then add the plasmid / vector / transfection reagent complex drop - by - drop into the culture flask of 293T cells; collect the viral supernatant into a 50 - ml centrifuge tube 24 h later, centrifuge at 250 g for 5 min, filter the supernatant after centrifugation through a 0.45 - μm filter, and ultra - centrifuge the filtered supernatant (25000 g, 4 °C, 3 h) to obtain the concentrated CD22 CAR lentivirus; discard the supernatant after centrifugation, resuspend the lentivirus with pre - cooled PBS at 4 °C, aliquot the resuspended CD22 CAR lentivirus solution, and store it at - 80 °C for later use.

[0307] (3) Resuscitation and activation of T cells

[0308] Set the water bath temperature to 38°C and pre-warm the culture medium in advance; Take out the cryopreservation bag from the liquid nitrogen tank and immediately immerse it in the water bath. When the cryopreserved cord blood becomes transparent and completely melts, take out the cryopreservation bag; Wipe the water stains outside the cryopreservation bag with a dry cotton ball and spray and disinfect it with 75% alcohol. After the alcohol has completely evaporated, transfer it to a biosafety cabinet; Take out the cord blood and put it into a 50 mL centrifuge tube, add an appropriate amount of RPMI 1640 culture medium, mix well and take a sample for counting; Centrifuge at 300 g for 5 min, collect the cells in the lower layer after centrifugation, and resuspend them with complete medium to a T cell density of 1×10 6 cells / mL. Add the activating antibodies Anti-human CD3 antibody and Anti-human CD28 antibody according to the resuspension volume. The usage concentration of CD3 is 0.15 μg / mL and the usage concentration of CD28 is 0.625 μg / mL, and place it in an incubator at 37°C and 5% carbon dioxide for culture; After culturing for 4 hr, supplement with complete medium to adjust the T cell density to 4×10 5 cells / mL and continue culturing.

[0309] (4) Sorting and purification of T cells

[0310] After 36 hr of cell activation, mix well and take a 20 μL sample, add 10 μL of diluted antibody and stain for 10 min. After diluting 10 times with PBS, use a flow cytometer to detect and count, record the CD3+, CD4+, CD8+ T cell densities, and observe the expression of CD69 and CD25 molecules; Record the cell volume and confirm the cell quantity; Transfer the cell suspension to a centrifuge tube and centrifuge at 300 g for 5 min, discard the supernatant and collect the cells in the lower layer; Add MACS Buffer to wash, centrifuge again and collect the cells in the lower layer. The centrifugation conditions are the same as above, and resuspend the cells with an appropriate amount of MACS Buffer; Calculate the amount of magnetic beads to be added according to the cell quantity. Add 4 μL of CD4+ magnetic beads to every 1×10 6 CD4+ T cells, and add 8 μL of CD8+ magnetic beads to every 1×10 6 CD8+ T cells; After adding the magnetic beads, mix well and incubate in the dark at room temperature for 20 min. After the incubation is completed, add MACS Buffer to wash, centrifuge at 300 g for 5 min, discard the supernatant, and resuspend with an appropriate amount of MACS Buffer; Place the LS sorting column on the MACS magnetic sorting rack, rinse the column with 1 mL of Buffer, and after the rinsing is completed, pass the cell suspension through the column, and continuously add 9 mL of Buffer through the column; Take out the LS column from the MACS magnetic rack, add 5 mL of Buffer, and flush out the cells retained on the LS column; Finally, mix the cell suspension well, take a sample for staining and counting, record the CD3+, CD4+, CD8+ T cell densities, and calculate the sorting recovery rate and purity.

[0311] (5) Preparation of CD22 CAR-T cells

[0312] Lentiviral transduction of T cells: Adjust the cell density to about 400 cells / μL for plating, with a volume of 500 μL per well. According to the actual number of T cells, add the CD22 CAR lentiviral solution of each group at MOI = 25. The negative control group (UnT) is T cells without lentiviral transduction. After culturing in an incubator at 37 °C and 5% carbon dioxide for 3 days, detect the expression rate of CD22 CAR polypeptide molecules on T cells (the groups are m971, S1, S4, S9, S27, S28, S35, S36, S41, S43). The results are shown in Figure 1A and 1B .

[0313] Observe the amplification of CD22 CAR-T cells every 3 days and supplement fresh culture medium. After continuous culture for 11 days, harvest them for subsequent in vitro killing experiments.

[0314] Example 3. Verification of the tumor cell killing effect of CD22 CAR-T cells

[0315] (1) Determination of the expression of CD22 antigen on the surface of target cells

[0316] Use Raji cells (purchased from ATCC, CCL-86), Namalwa cells (purchased from ATCC, CRL-1432), and K562 cells (purchased from ATCC, CRL-3344) as target cells respectively, and detect the expression of CD22 antigen on the surface of these cells by flow cytometry. The results are shown in Figure 2 . The results show that Raji cells are CD22 high-expressing, Namalwa cells are CD22 medium-expressing, and K562 cells are CD22 negative cells. Finally, Raji cells and Namalwa cells are selected as target cells for in vitro killing experiments.

[0317] (2) Determination of the in vitro killing effect of CD22 CAR-T cells

[0318] In a 24-well plate, add the CAR-T cells of each group (the groups are m971, S1, S4, S9, S27, S28, S35, S36, S41, S43, 2×10 5cells / well, with the same ratio of CD4+ to CD8+ T cells in each group), add Raji cells with the corresponding cell amount according to the effector-to-target ratio (E:T) = 1:1 or 1:3. The negative control group (UnT) also adds the same amount of T cells and target cells according to the corresponding effector-to-target ratio, and supplement the medium to 500 μL / well, and culture in a 37°C, 5% CO2 incubator; after co-culture for 18 hr and 72 hr, detect the amount of Raji cells in each well by flow cytometry respectively. According to the killing rate = (amount of target cells reduced) / (amount of target cells plated) × 100%, calculate the D1 (18 hr) killing rate and D3 (72 hr) killing rate, and the results are shown in Figure 3A and 3B , 4A and 4B.

[0319] In a 24-well plate, add each group of CAR-T cells (the groups are m971, S1, S4, S9, S27, S28, S35, S36, S41, S43, 2×10 5 cells / well, with the same ratio of CD4+ to CD8+ T cells in each group), add Namalwa cells with the corresponding cell amount according to the effector-to-target ratio (E:T) = 1:3. The negative control group (UnT) also adds the same amount of T cells and target cells according to the corresponding effector-to-target ratio, and supplement the medium to 500 μL / well, and culture in a 37°C, 5% CO2 incubator; 18 hr and 72 hr after plating, detect the amount of Namalwa cells in each well by flow cytometry respectively. According to the killing rate = (amount of target cells reduced) / (amount of target cells plated) × 100%, calculate the D1 (18 hr) killing rate and D3 (72 hr) killing rate, and the results are shown in Figure 5A and 5B .

[0320] (3) Cytokine release assay of CD22 CAR-T cells

[0321] 18 hr after plating in the in vitro killing experiment, collect the supernatants of the co-culture media of each group of CAR-T cells (the groups are UnT, m971, S1, S4, S9, S27) and Namalwa cells, and detect by CBA (CBA detection kit: LEGENDplex TM Human CD8 / NK Panel (13-plex) with V-bottom Plate, Biolegend, Cat. No. 741065; LEGENDplex TMHuman Macrophage / Microglia Panel(13-plex) with V-bottom Plate, Biolegend, Cat.No.740503), the release of cytokines Granzyme B, TNF-α, IFN-γ, and IL-2 in each group was detected, and the results are shown in Figure 6A , 6B , 6C and 6D.

[0322] The specific operation steps are as follows:

[0323] Collection of culture medium supernatant: Collect 50 μL of the cell culture medium supernatant 18 hours after plating in the killing experiment for subsequent detection of the cytokine secretion in the supernatant.

[0324] Preparation of Beads: After restoring the required Beads to room temperature, vortex for 2 minutes to fully mix the Beads. Calculate the required amount of Beads according to the sample volume, and add 15 μL to each sample.

[0325] Preparation of Wash Buffer: Restore 20x Wash Buffer to room temperature to fully dissolve the salts in it, and dilute it with ultrapure water to 1x Wash Buffer for standby.

[0326] Preparation of standards: Dissolve the standards with 250 μL of Assay Buffer, invert multiple times to fully mix, let stand at room temperature for 10 minutes, and then transfer them to an EP tube; Take out 25 μL of the standards and put them in an EP tube, labeled as C7; Take 7 EP tubes, labeled as C6 / C5 / C4 / C3 / C2 / C1 / C0 respectively. Add 22.5 μL of Assay Buffer to each tube, and take out 7.5 μL of the standards from C7 and perform serial dilutions at a 4-fold ratio until dilution to C1. C0 is Assay Buffer (0 pg / ml).

[0327] Preparation of standard wells and sample wells: Prepare the cell supernatant standard curve wells by adding 15 μL of Assay Buffer, standards, and Beads to an EP tube and mixing; Prepare the sample wells: Add 15 μL of Assay Buffer, samples, and Beads to an EP tube and mix.

[0328] Binding of capture Beads to the analyte of interest: Shake the standard wells and sample wells at 500 rpm and incubate for 2 hours under light protection.

[0329] Washing: Centrifuge the sample at 2000g for 5 min, discard the supernatant, and the Beads can be seen at the bottom; Add 200 μL of 1x Wash buffer to each EP tube, vortex briefly, then centrifuge at 2000g for 5 min, and discard the supernatant.

[0330] Capture of the binding of Beads, the analyte of interest, biotinylated detection antibody, and SA-PE: Add 15 μL of the detection antibody to each EP tube, pipette to mix well, shake at 500 rpm, and incubate for 1 hr in the dark; Add 15 μL of streptavidin-phycoerythrin (SA-PE) to each tube, shake at 500 rpm, and incubate for 0.5 hr in the dark.

[0331] Secondary washing: Add 200 μL of 1x Wash buffer to each EP tube, vortex briefly, then centrifuge at 2000g for 5 min, and discard the supernatant.

[0332] Detection: Add 200 μL of 1x Wash buffer to each tube, vortex, and then perform flow cytometry detection. Check the FSC, SSC, APC, and PE channels on the flow cytometer.

[0333] Detection result: Export the file detected by the flow cytometer as an FSC format for analysis, and use LEGENDplex TM The data analysis software determines the concentration of the target cytokine according to the known standard curve.

Claims

1. A single-domain antibody targeting CD22, characterized in that, The antibody comprises CDR1, CDR2 and CDR3; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:1, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:20, wherein CDR3 is the amino acid sequence shown in SEQ ID NO:35, and wherein the determination of CDR1, CDR2 and CDR3 is according to the IMGT numbering scheme.

2. The single-domain antibody according to claim 1, wherein The antibody further comprises FR1, FR2, FR3 and FR4; wherein FR1 comprises the amino acid sequence shown in SEQ ID NO:63, wherein FR2 comprises the amino acid sequence shown in SEQ ID NO:73, wherein FR3 comprises the amino acid sequence shown in SEQ ID NO:93, and wherein FR4 comprises the amino acid sequence shown in SEQ ID NO:

119.

3. The single-domain antibody according to any one of claims 1 or 2, characterized in that The antibody comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO:

120.

4. A chimeric antigen receptor, characterized in that, Comprising: (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signal transduction domain; wherein the extracellular antigen-binding domain comprises the anti-CD22 single-domain antibody according to any one of claims 1-3.

5. The chimeric antigen receptor according to claim 4, wherein Wherein the transmembrane domain is derived from CD8α, CD4, CD28, CD137, CD80, CD86, CD152 or PD-1.

6. The chimeric antigen receptor according to claim 5, wherein Wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

140.

7. The chimeric antigen receptor according to claim 4, wherein Wherein the intracellular signal transduction domain is derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD66d, FcRγ or FcRβ.

8. The chimeric antigen receptor according to claim 7, wherein Wherein the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:

142.

9. The chimeric antigen receptor according to claim 7, wherein Wherein the intracellular signal transduction domain further comprises a co-stimulatory signal domain, and wherein the co-stimulatory signal domain is derived from CD137, CD27, CD28, ICOS, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand and combinations thereof.

10. The chimeric antigen receptor according to claim 9, wherein, Wherein the co-stimulatory signal domain comprises the amino acid sequence shown in SEQ ID NO:

141.

11. The chimeric antigen receptor according to claim 4, wherein Further comprising a hinge region located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, and wherein the hinge region is derived from CD8α, CD28, IgG1 or IgG4.

12. The chimeric antigen receptor according to claim 11, wherein Wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:

139.

13. The chimeric antigen receptor according to claim 4, wherein Further comprising a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide, and wherein the signal peptide is derived from HLA-A, CD8α, CD33, Igκ, IL-2 or GM-CSFRα.

14. The chimeric antigen receptor according to claim 13, wherein The signal peptide comprises the amino acid sequence shown in SEQ ID NO:

138.

15. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor is the amino acid sequence shown in SEQ ID NO:

143.

16. A nucleic acid, characterized in that, Comprises a nucleic acid sequence encoding the chimeric antigen receptor according to any one of claims 4-15.

17. A carrier, characterized in that, Comprises the nucleic acid according to claim 16.

18. An engineered immune effector cell, characterized in that, Comprises the chimeric antigen receptor according to any one of claims 4-15, the nucleic acid according to claim 16, or the vector according to claim 17.

19. The engineered immune effector cell according to claim 18, wherein, The immune effector cells are selected from T cells, B cells, NK cells, macrophages or dendritic cells.

20. A pharmaceutical composition, characterized in that, Comprises the anti-CD22 single-domain antibody according to any one of claims 1-3, the engineered immune effector cells according to claim 18 or 19, and a pharmaceutically acceptable carrier or excipient.

21. Use of the anti-CD22 single-domain antibody according to any one of claims 1-3, the engineered immune effector cells according to claim 18 or 19, or the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating leukemia or lymphoma.

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