CD22-targeted single-domain antibody, chimeric antigen receptor and application of CD22-targeted single-domain antibody and chimeric antigen receptor

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy technology, and specifically 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 a disease or disorder in a subject, and in particular to T cell immunotherapy based on a chimeric antigen receptor. Background Art

[0002] CD22 is a B lymphocyte lineage differentiation antigen, also known as BL-CAM, B3, Leu-14, Lyb-8 and Siglec-2. It has been shown to be specifically expressed by B lymphocytes and is functionally very important as a negative regulator of B lymphocyte activation. CD22 is an inhibitory co-receptor that can downregulate BCR signals and block excessive stimulation of B cells. It plays an important role in maintaining marginal zone B cell populations, 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 to treat autoimmune diseases by selectively regulating B cell activity by targeting CD22.

[0003] As an immunotherapy strategy, T cells modified with chimeric antigen receptors (CARs) have received extensive attention and application in tumor treatment, especially in hematological malignancies. The principle is to enable T cells to express receptor structures (single-chain antibodies) that can specifically recognize tumor cell surface antigens through gene modification, and after the receptor specifically binds to tumor cell surface antigens, activate its downstream immune co-stimulatory factors and T cells, thereby activating T cells to secrete related cytokines and specifically kill tumor cells. The structure of CAR generally consists of four parts: an extracellular antigen binding domain (often a single-chain antibody with antigen recognition function), a hinge region, a transmembrane domain, and an intracellular signal transduction domain. At present, according to the number of co-stimulatory molecules added to the intracellular signal transduction domain, the CAR structure is usually divided into the first generation (no co-stimulatory molecules), the second generation (containing one co-stimulatory molecule), and the third generation (containing two co-stimulatory molecules). The second generation CAR structure is currently the most widely used in marketed products and clinical research stages.

[0004] Although great progress has been made in the treatment of acute lymphoblastic leukemia (ALL) in children and adults, a considerable number of patients still do not respond well to treatment, and the current standard of care has considerable short-term and long-term toxicity. Monoclonal antibody-based therapy is expected to overcome chemotherapy resistance and potential treatment-related toxicity. The most promising of these 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, especially CAR-T cell immunotherapy with CD19 as the target antigen has made breakthrough progress. However, CD19-targeted 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 of CD19CAR-T during the treatment of 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 therapeutic targets or combining them with CD19 CAR-T, or constructing multi-target CAR-T.

[0005] Similar to the CD19 antigen, CD22 is also restricted to B cells and is not expressed in other parenchymal cells or hematopoietic stem cells. Therefore, it has high specificity as a B cell tumor antigen and has become an ideal therapeutic target for B cell malignancies. In addition, CD22 and CD19 are widely co-expressed on the surface of tumor cells, and after CD19 CAR-T cell therapy causes the loss of CD19 antigen, the CD22 antigen is still retained. 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 mutation after CD19 CAR-T treatment and whose tumor cells express CD22, or in combination with CD19 CAR-T cells to avoid antigen mutation, improve the effectiveness of CAR-T treatment, and reduce tumor recurrence.

[0006] Single domain antibodies (sdAb) are different from traditional 4-chain antibodies because they have variable domains of a single monomer antibody. For example, camelids and sharks produce antibodies that naturally lack light chains, which are called heavy chain antibodies only (hcAb, or simply heavy chain antibodies). The antigen binding fragment in each arm of the camelid heavy chain antibody only has a single heavy chain variable domain (VHH), which can have a high affinity for antigens without the help of light chains. Camelid VHH antibodies are known as the smallest functional antigen binding fragments, with a molecular weight of only about 15kD, and are therefore also known as nanobodies. VHH antibodies have the natural advantages of good solubility, high stability, strong penetration, and wide binding epitopes. Since their discovery, VHH antibodies have gradually attracted the attention of researchers in the field, and basic research on them has become increasingly mature. In terms of application, 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 is to screen high-affinity antibodies with good specificity, strong binding force, and effective binding epitopes. However, traditional CD22 antibodies are limited by their large molecular weight, weak binding force with antigens, low affinity, difficulty in transformation, and poor stability. Further single-chain transformation is required, and it is difficult to achieve effective construction of 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 purposes of the present invention is to provide a single-domain antibody targeting CD22, which has a natural single-chain structure, has the advantages of small molecular weight, high solubility, high stability, low immunogenicity, high tissue permeability, 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 weight. After the single-domain antibody is used to construct CAR-T cells, it has a very significant tumor cell killing ability.

[0010] The single-domain antibody targeting CD22 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, wherein CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20-34, and wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35-62.

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

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

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

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

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

[0016] In some embodiments, the single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1, CDR2 and CDR3 are determined according to the IMGT numbering scheme, the Kabat numbering scheme, the AbM numbering scheme, the Chothia numbering scheme or the Contact numbering scheme.

[0029] The single-domain antibody targeting CD22 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 single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0031] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0032] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0033] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0034] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0035] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0036] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0037] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0038] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0039] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0040] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0041] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0042] In some embodiments, the single-domain antibody targeting CD22 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 the FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0043] In some embodiments, the single-domain antibody targeting CD22 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 FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0044] In some embodiments, the single-domain antibody targeting CD22 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 FR4 comprises the amino acid sequence shown in SEQ ID NO:119.

[0045] Among them, the specific amino acid sequence information shown by SEQ ID NO:1-119 is shown in Table 1.

[0046] Table 1

[0047]

[0048]

[0049]

[0050]

[0051] The single-domain antibody targeting CD22 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: 120-136.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0069] The single-domain antibody targeting CD22 provided by the present invention comprises an amino acid sequence as shown in Table 2 or as shown in SEQ ID NO: 120-136.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0086] In some embodiments, the single-domain antibody targeting CD22 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 by SEQ ID NO:120-136 is shown in Table 2.

[0088] Table 2

[0089]

[0090]

[0091]

[0092] One of the purposes of the present invention is to provide a chimeric antigen receptor, and the chimeric antigen receptor has a very significant ability to kill tumor cells after transfection to prepare CAR-T 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 a single domain antibody targeting CD22 as described above.

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

[0095] In some embodiments, the present invention provides a chimeric antigen receptor wherein the transmembrane domain is derived from CD8α.

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

[0097] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the intracellular signaling domain is derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD66d, FcRγ, or FcRβ.

[0098] In some embodiments, the present invention provides a chimeric antigen receptor wherein the intracellular signaling domain is derived from CD3ζ.

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

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

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

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

[0103] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the co-stimulatory signaling 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 present invention provides a chimeric antigen receptor, wherein the hinge region is derived from CD8α, CD28, IgG1 or IgG4.

[0106] In some embodiments, the present invention provides a chimeric antigen receptor 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 present invention provides a chimeric antigen receptor 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 by SEQ ID NO: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 sequence shown in SEQ ID NO:143-159.

[0116] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar 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 that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar to the amino acid sequence shown in SEQ ID NO:159.

[0133] The chimeric antigen receptor provided by the present invention comprises an amino acid sequence as shown in Table 4 or as shown in SEQ ID NO: 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 by SEQ ID NO:143-159 is shown in Table 4.

[0152] Table 4

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] One of the purposes of the present invention is to provide a nucleic acid comprising a nucleic acid sequence encoding a chimeric antigen receptor as described above. In some embodiments, the nucleic acid provided by the present invention comprises a nucleic acid sequence as shown in SEQ ID NO: 161-178. The specific nucleic acid sequence information shown in SEQ ID NO: 161-178 is shown in Table 5.

[0159] Table 5

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168] One of the objectives 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 of the objects of the present invention is to provide an engineered immune effector cell, which comprises 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, and induced pluripotent stem cells (iPSCs).

[0171] One of the objects of the present invention is to provide a pharmaceutical composition comprising the single domain antibody targeting CD22 as described above, engineered immune effector cells and a pharmaceutically acceptable carrier or excipient.

[0172] One of the objects of the present invention is to provide a method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a single domain antibody, engineered immune effector cell or pharmaceutical composition targeting CD22 as described above.

[0173] In some embodiments, the present invention provides a method of 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 of 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 a B cell leukemia or a B cell lymphoma. More specifically, wherein 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 lymphoma B-cell lymphoma (PMBL), small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia (B-PLL), follicular lymphoma (FL), Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia (HCL), precursor B-lymphocytic leukemia, non-Hodgkin's lymphoma (NHL), high-grade B-cell lymphoma (HGBL) and multiple myeloma (MM).

[0176] In some embodiments, the invention provides a method for treating a subject's disease or disorder, wherein the disease or disorder is a B cell-related autoimmune and / or inflammatory disease. More specifically, wherein the B cell-related autoimmune and / or inflammatory disease is associated with inappropriate or enhanced B cell quantity and / or activation.

[0177] One of the purposes of the present invention is to provide a use of a single domain antibody targeting CD22, an engineered immune effector cell, and a pharmaceutical composition as described above in the preparation of a drug for treating B cell-related malignancies, B cell-related autoimmune diseases and / or inflammatory diseases.

[0178] Explanation of terms

[0179] The term "antibody" used in the present invention includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2 and Fv. In some embodiments of the present invention, the terms "immunoglobulin (Ig)" and "antibody" are 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 an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.

[0181] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of 5 basic heterotetrameric units and an additional polypeptide called J chain, containing 10 antigen binding sites; while IgA antibodies contain 2-5 basic 4-chain units, which can be combined with J chains to form multivalent assemblies. In the case of IgG, the 4-chain unit is usually about 150,000 Daltons. Each light chain is connected to the heavy chain by one covalent disulfide bond, while the two heavy chains are connected to each other by one or more disulfide bonds, the number of which depends on the isotype of the heavy chain. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH1, CH2 and CH3 for each α and γ chain) and four (CH1, CH2, CH3 and CH4 for μ and ε isotypes) constant domains (CH) and a hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. VL is arranged together with VH, while CL is arranged together with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain 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. The light chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on their constant domain amino acid sequences. Immunoglobulins can be assigned to different classes or isotypes based on their heavy chain constant domain (CH) amino acid sequences. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequences and functions, 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 camelids or cartilaginous fish. Compared with the above-mentioned 4-chain antibodies, heavy chain antibodies lack light chains and heavy chain constant region 1 (CH1), and only contain 2 heavy chains composed of variable regions (VHH) and other constant regions, and the variable regions are connected to the constant regions through a hinge region-like structure. Each heavy chain of camelid heavy chain antibodies contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of cartilaginous fish heavy chain antibodies contains 1 variable region and 5 constant regions (CH1-CH5). The antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can have CH2 and CH3 of human IgG Fc.

[0183] The terms "single domain antibody", "single domain antibody targeting CD22", "heavy chain variable region domain of heavy chain antibody", "VHH", and "nanoantibody" used in the present invention are used interchangeably, all referring to single domain antibodies that specifically recognize and bind to CD22. Single domain antibodies are the variable regions of heavy chain antibodies. Generally, single domain antibodies contain three CDR regions and four FR regions. Single domain antibodies are the smallest functional antigen-binding fragments. Usually, antibodies that naturally lack light chains and heavy chain constant regions 1 (CH1) are first obtained, and then the variable regions of the antibody heavy chains are cloned to construct single domain antibodies consisting of only one heavy chain variable region.

[0184] Under the premise of not substantially affecting the activity of the antibody, those skilled in the art can change one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the antibody or its functional fragment sequence. These variants include but are not limited to: one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein. For example, amino acids with similar properties are substituted in the FR and / or CDR of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.

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

[0186] The single domain antibody, nanobody or heavy chain antibody of the present invention can be prepared by conventional methods in the art, such as phage display technology well known 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 sequences encoding various antibodies of the present invention. Transformation can be carried out by any known method, for example, including packaging the polynucleotide in a virus (or a 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, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Host mammalian cell lines that can be used for expression are well known in the art, such as a variety of 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 substantial CD22 binding properties.

[0187] The term "chimeric antigen receptor (CAR)" used in the present invention comprises an extracellular antigen binding domain, wherein the extracellular antigen binding domain comprises a single domain antibody (sdAb) binding 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 comprising: (a) an extracellular antigen binding domain comprising 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 region will be described in more detail below.

[0189] The present invention discloses that CAR comprises an extracellular antigen binding domain, which comprises one or more single domain antibodies. sdAb may have the same or different sources and have the same or different sizes. Exemplary sdAbs include, but are not limited to, heavy chain variable domains (e.g., VHH) from heavy chain antibodies only, binding molecules without light chains naturally, single domains (e.g., VH or VL) derived from conventional 4-chain antibodies, humanized heavy chain antibodies only, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain fragments, and non-derived from antibody engineering domains and single domain scaffolds. Any sdAb known in the art or disclosed by the present invention, including single domain antibodies disclosed by the present invention, can be used to construct CAR described herein. sdAbs may be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, sharks, goats, rabbits, and cattle. 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 herein comprises at least one binding domain, and the at least one binding domain comprises a single domain antibody that binds CD22 disclosed herein.

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

[0192] In some embodiments, the present invention discloses a CAR comprising a polypeptide, wherein the polypeptide comprises: (a) an extracellular antigen binding domain comprising an anti-CD22sdAb; (b) a transmembrane domain; (c) an intracellular signaling domain; wherein the anti-CD22sdAb comprises a polypeptide sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the sequence SEQ ID NO: 120-136.

[0193] In some embodiments, the present invention discloses a CAR comprising a polypeptide, wherein the polypeptide comprises: (a) an extracellular antigen binding domain comprising an anti-CD22sdAb; (b) a transmembrane domain; (c) an intracellular signaling domain; wherein the anti-CD22sdAb comprises the amino acid sequence of SEQ ID NO: 120-136.

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

[0195] In some embodiments, the intracellular signal transduction domain includes the main intracellular signal transduction domain of an immune effector cell (e.g., T cell). In some embodiments, the main intracellular signal transduction domain is derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD66d, FcRγ, FcRβ. In some embodiments, the main intracellular signal transduction domain is derived from CD3ζ. In some embodiments, the intracellular signal transduction domain further includes a costimulatory signal domain. In some embodiments, the costimulatory signal domain is derived from a costimulatory molecule, which is derived from one or more of CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83 ligands. In some embodiments, the costimulatory signal 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, CD22 CAR also includes a signal peptide (e.g., HLA-A signal peptide or CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide includes from N-terminus to C-terminus: HLA-A signal peptide or CD8α signal peptide, extracellular antigen binding domain, CD8α hinge region, CD8α transmembrane domain, costimulatory signal domain derived from CD137 (4-1BB), and intracellular signal transduction domain derived from CD3ζ.

[0198] In some embodiments, different domains of CAR can also be fused to each other through peptide linkers. This depends on the structure and / or functional characteristics of single domain antibodies and / or various domains, and each peptide linker in CAR can have the same or different lengths and / or sequences. Those skilled in the art can independently select and optimize each peptide linker. In some embodiments, the peptide linker includes flexible residues (e.g., glycine and serine) so that adjacent domains can move freely relative to each other. For example, glycine-serine doublets can be suitable peptide linkers.

[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 acid lengths. 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 less amino acid lengths. In some embodiments, the peptide linker is 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, 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 in length.

[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 only a heavy chain antibody can be used as a linker. See, for example, 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, wherein 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 above-mentioned various antibodies or chimeric antigen receptors. The present invention provides polynucleotides encoding heavy chain variable regions, light chain variable regions, heavy chains, light chains 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 artificially synthesized 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, a very large number of nucleic acids can be produced, all of which encode the antibodies or chimeric antigen receptors of the present invention. Therefore, in the case where a specific amino acid sequence has been identified, those skilled in the art can produce 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 with the above-mentioned polynucleotide sequence and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturant 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%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

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

[0204] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the host cells after proliferation by conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) involved in the present invention include biomolecules in isolated form. At present, the DNA sequence encoding the polypeptide of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also 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 appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vector usually contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) usually include one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, replication origin, transcription termination sequence, complete intron sequence containing donor and acceptor splice sites, leader sequence encoding for polypeptide secretion, ribosome binding site, polyadenylation sequence, multiple linker region and selectable marker element for inserting nucleic acid encoding the antibody to be expressed.

[0206] The host cells involved in 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, and Salmonella typhimurium; fungal cells of yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells, etc.

[0207] In some embodiments, the host cell 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), cytokine-induced killer cells (DC-CIK) stimulated by dendritic cells, cytotoxic T lymphocytes (CTL), γδT cells, natural killer cells (NK), tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), CD3AK cells (killer cells of anti-CD3 monoclonal antibodies) and CAR-T / TCR-T cells. In certain embodiments, the killer cell is a T cell or a NK cell. Exemplary NK cells include but are not limited to primary NK cells, NK cell strains (such as NK92) and NKT cells. In certain embodiments, the NK cell is a primary NK cell. 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, inhibitory / regulatory T cells, γδT cells, cytokine-induced killer cells (CIK), tumor infiltrating lymphocytes (TIL), and other mixed cell populations of T cells. In certain embodiments, the T cells are peripheral blood T lymphocytes and cord blood T lymphocytes.

[0208] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2Another method is to use MgCl 2 In addition, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0209] The obtained transformant 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 culture medium or serum-free culture medium. Culture is carried out under conditions suitable for the growth of the host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0210] The polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using 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 a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0211] The present invention also discloses a vector for cloning and expressing any CAR 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, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, vaccinia vectors, herpes simplex virus vectors and derivatives thereof. 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 in retroviral particles using techniques known in the art. Recombinant viruses 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 immunomodulatory agents (e.g., immune checkpoint inhibitors) coding sequences and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged with protocols known in the art. Using methods known in the art, the resulting lentiviral vectors can be used to transduce mammalian cells (e.g., primary human T cells). Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their propagation in progeny cells. Lentiviral vectors also have the advantages of low immunogenicity and can transduce non-proliferating cells.

[0213] In some embodiments, the vector comprises any nucleic acid encoding the CAR of the present invention. The nucleic acid can be cloned into a vector using any molecular cloning method known in the art, including, for example, using restriction endonuclease sites and one or more selection markers. In some embodiments, the nucleic acid is operably connected 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 divided into constitutive promoters or regulated promoters, such as inducible promoters.

[0214] In some embodiments, the nucleic acid encoding CAR is operably connected to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. The exemplary constitutive promoters considered by the present invention include, but are not limited to, cytomegalovirus (CMV) promoters, human elongation factor-1α (hEF1α) promoters, ubiquitin C (UbiC) promoters, phosphoglycerol kinase (PGK) promoters, simian virus 40 (SV40) early promoters, and chicken β-actin coupled to CMV early enhancer (CAGG) promoters. The efficiency of this constitutive promoter in driving transgenic expression has been extensively compared in a large number of 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 induces the highest level of transgenic expression, but also maintains optimal expression in CD4 and CD8 human T cells. In some embodiments, the nucleic acid encoding the CAR is operably linked to a hEF1α promoter.

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

[0216] In some embodiments, the inducing conditions do not induce expression of endogenous genes in the engineered mammalian cells and / or the subject receiving the pharmaceutical composition. In some embodiments, the inducing conditions are selected from: an inducing agent, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and the activation state of the engineered mammalian cells.

[0217] In some embodiments, the vector also includes a selection marker gene or a reporter gene to select cells expressing CAR from a host cell population transfected by a lentiviral vector. Both the selection marker and the reporter gene can have appropriate regulatory sequences on both sides to express in the host cell. For example, the vector can contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of nucleic acid sequences.

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

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

[0220] In some embodiments, the immune effector cells are NK cells. In other embodiments, the immune effector cells can be established cell lines, such as NK-92 cells.

[0221] In some embodiments, immune effector cells can be differentiated from stem cells, such as hematopoietic stem cells, pluripotent stem cells, iPSCs, or embryonic stem cells.

[0222] The engineered immune effector cells of the present invention are prepared by introducing CAR into immune effector cells (e.g., T cells). In some embodiments, CAR is introduced into immune effector cells by transfecting any isolated nucleic acid or any of the above-mentioned vectors.

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

[0224] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, EF 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 vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g. human) cells.

[0226] Chemical methods for introducing vectors 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 vehicle is a liposome.

[0227] In some embodiments, RNA molecules 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, the transduced or transfected immune effector cells are propagated in vitro after the introduction of a 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 gene can be used to identify cells that may be transfected and to evaluate the function of regulatory sequences. In general, reporter gene is a gene that is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide that expresses some easily detectable properties, such as enzyme activity. The expression of reporter gene is measured at the appropriate time after DNA is introduced into the receptor cell. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory 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 or commercially obtained using known techniques. Other methods for confirming the presence of nucleic acids encoding CAR in engineered immune effector cells include: molecular biological test methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of specific peptides; immunological methods, such as ELISA.

[0230] The pharmaceutical composition disclosed in the present invention contains a single domain antibody targeting CD22 according to the present invention, an engineered immune effector cell, and a pharmaceutically acceptable excipient or carrier. 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 dose and concentration used. Such excipients include, but are not limited to, saline, buffer, 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, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition. The optimal pharmaceutical composition can be determined based on the intended route of administration, delivery method, and desired dosage.

[0231] Pharmaceutical compositions for in vivo administration are usually provided in the form of sterile preparations. Sterilization is achieved by filtering through a sterile filtration membrane. When the composition is freeze-dried, this method can be used for sterilization before or after freeze-drying, reconstitution, dilution. The pharmaceutical composition of the present invention can be selected for parenteral delivery. The composition for parenteral administration can be stored in a freeze-dried form or in a solution. For example, it is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually placed in a container with a sterile access hole, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as oral). The preparation of the pharmaceutically acceptable composition is within the technical scope of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Technologies for preparing a variety of other sustained or controlled delivery methods (such as liposome carriers, bioerodible microparticles or porous beads and accumulated injections) are also known to those skilled in the art.

[0232] Once the pharmaceutical composition is formulated, it 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 (e.g., lyophilized) that is reconstituted before administration. The present invention also provides a kit for producing a single-dose administration unit. The kit of the present invention can 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 a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[0233] The present invention also provides a method for treating a patient (especially a patient with a CD22-related disease) by administering a single domain antibody targeting CD22, an engineered immune effector cell, or a pharmaceutical composition thereof as described in any embodiment of the present invention. The terms "patient", "subject", "individual", and "object" in the present invention are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., rats, mice, dogs, cats, rabbits, etc.), and most preferably a human. "Treatment" refers to the subject using the treatment methods described herein to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a reduction in the rate of cancer cell infiltration to peripheral 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 a variety of factors (such as the patient's disease state, age, weight, and the ability of the therapy to stimulate an anti-cancer response in the subject).

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

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

[0236] The administration routes of the pharmaceutical composition are conventional in the art, such as oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection, and can also be administered by sustained release system or by implantation device. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0240] Figure 3A The graph shows the killing rate of Raji cells by effector cells in each group on the first day (D1) when the effector-target ratio (E:T) is 1:1.

[0241] Figure 3B The graph shows the killing rate of Raji cells by effector cells in each group on the third day (D3) when the effector-target ratio (E:T) is 1:1.

[0242] Figure 4A The graph shows the killing rate of Raji cells by effector cells in each group on the first day (D1) when the effector-target ratio (E:T) is 1:3.

[0243] Figure 4B The graph shows the killing rate of Raji cells by effector cells in each group on the third day (D3) when the effector-target ratio (E:T) is 1:3.

[0244] Figure 5A The graph shows the killing rate of Namalwa cells by effector cells in each group on the first day (D1) when the effector-target ratio (E:T) is 1:3.

[0245] Figure 5B The graph shows the killing rate of Namalwa cells by effector cells in each group on the third day (D3) when the effector-target ratio (E:T) is 1:3.

[0246] Fig. 6A The release of Granzyme B by each group of effector cells in the in vitro killing experiment is shown.

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

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

[0249] Fig.6D The release of IL-2 by effector cells of each group in the in vitro killing experiment is shown.

[0250] In the above figure: 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 purpose of the present invention, its features and beneficial effects will be further described with reference to the following embodiments and accompanying drawings. DETAILED DESCRIPTION

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

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

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

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

[0256] 1) Select healthy alpacas as immunization subjects.

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

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

[0259] (2) Construction of antibody phage library

[0260] 1) After the animal immunization is completed, RNA is extracted from the separated lymphocytes and the total RNA obtained is reverse transcribed using the Takara reverse transcription kit; the total RNA sample is divided into two parts, one using the Oligo dTPrimer in the kit as a primer, and the other using the Random 6-mers in the kit as a primer. According to the instructions of the reverse transcription kit, the total RNA obtained in the previous step is reverse transcribed into cDNA and stored in two centrifuge tubes respectively.

[0261] 2) PCR amplification

[0262] a. Amplify specific antibody fragments from reverse transcribed cDNA and perform PCR amplification using Taq DNA Polymerase Hot Start enzyme; perform 1% agarose gel electrophoresis on all PCR products, and cut the gel to recover the target fragment with a size of about 600-700 bp, which is the first round of PCR amplification product and store it at -20℃;

[0263] b. The first round of PCR amplification products were used as templates for the second round of PCR reaction. After the reaction was completed, 1% agarose gel electrophoresis was performed, and the gel was finally cut to recover a single target band with a fragment size of about 400 bp. The PCR reaction solution was purified using a universal DNA purification and recovery kit.

[0264] 3) Enzyme digestion and ligation

[0265] The target gene fragment amplified by the second round of PCR and the pComb3XSS phage plasmid vector were digested with restriction endonucleases Spe I and Sac I, respectively. After the digestion was completed, the VHH target gene fragment was ligated to the pComb3XSS phage plasmid vector with a ligase to construct a recombinant plasmid.

[0266] 4) Bacterial library construction

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

[0268] b. Add 100 ng of ligation product and transfer to a pre-cooled electroporation cup with a spacing of 1 mm. Set the parameters in the electroporation instrument: 1800 V, 1 mm, and click the button to transform;

[0269] c. After the electroporation is completed, add 1 mL of SOC culture medium preheated at 37°C, mix well, and shake at 37°C, 200 rpm for 1 hour to revive the bacteria;

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

[0271] e. Take 100 μL of the recovered bacterial solution, dilute it in 10 steps, and then plate it and culture it at 37°C overnight;

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

[0273] g. Calculate the number of transformed colonies that can be obtained from all reactions according to the dilution multiple and the number of single colonies, which is the library capacity of the bacterial library; at the same time, randomly select several single clones from the gradient dilution plate for colony PCR. The PCR product with a single band of about 400 bp is considered to be a positive clone, so as to estimate the clone positive rate of the bacterial library;

[0274] h. Scrape the overnight cultured plate colonies using 2×YT liquid medium, place them in a 50 mL centrifuge tube, measure their OD600 value, 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 2×YT liquid medium (containing 100 μg / mL Amp) to an initial OD600 value of 0.1, and culture at 37°C and 250 rpm until the OD600 is 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, 4000 rpm for 20 min, remove the supernatant, and 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 min, centrifuge at 4°C, 12000 g for 10 min, 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 immunotube (50 μg / tube, coating solution is PBS, 2 mL / tube), rotate slowly overnight at 4°C, and coat BSA (50 μg in PBS, 2 mL / tube) in parallel as a control; discard the supernatant in the overnight coated immunotube, wash the immunotube 3 times at room temperature with PBS buffer, rotate for 5 min / time, add 2 mL of blocking solution (3% skim milk powder), rotate and block at room temperature for 2 hours, discard the supernatant, and add 2 mL of PBST buffer to wash the immunotube 3 times at room temperature, rotate for 5 min / time;

[0283] b. Discard the washing solution in the immunotube and add about 1012 pfu was used as the input phage library for the first round of screening, PBS buffer was added to 2 mL, and incubated at room temperature for 1 h; the supernatant was discarded, and 2 mL PBST (1×PBS plus 0.1% Tween20, the same below) buffer was added to wash the immunotube 20 times at room temperature, and each time was rotated for 5 min; the liquid in the immunotube was discarded, 1 mL 0.25 mg / mL Trypsin solution was added, and the elution was rotated at room temperature for 30 min; 10 μL 10% AEBSF was added to terminate the elution, and the solution in the immunotube was transferred to a new 1.5 mL centrifuge tube, which was the phage eluate for the first round of screening.

[0284] 2) First round of phage eluate titer detection

[0285] Take 10 μL of the first round of phage eluate and dilute it 10 times in a 1.5 mL centrifuge tube. Dilute 12 times in total to 10. -12 ; Add 90 μL of TG1 bacterial solution to each dilution centrifuge tube, shake and mix, and incubate at 37°C for 30 minutes; Take 5 μL from each dilution centrifuge tube and add it dropwise to 2×YT solid culture medium (Amp), let it stand for a few minutes, and then invert and culture it at 37°C overnight; Count the number of single colonies on the plate at the dilution that can clearly distinguish single colonies, and calculate the number of phage particles in each milliliter of phage solution, that is, the phage library titer, according to the following formula:

[0286] T (pfu / mL) = N × D × 400

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

[0288] 3) The third round of screening of phage eluate

[0289] The above experiment was repeated three times, and the phages from the first round were used as the input phage library for the second round of screening to obtain the second round of screening phage eluate, and the phages from the second round were used as the input phage library for the third round of screening to obtain the third round of screening phage eluate.

[0290] (4) Monoclonal ELISA test

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

[0292] 2) Randomly pick 192 monoclonal colonies from the culture medium plate after overnight culture and place them in a sterile 96-well cell culture plate. Add 200 μL of 2×YT culture medium (containing 100 μg / mL Amp) to each well and place them in a static culture at 37°C overnight.

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

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

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

[0296] 6) Coat the screening antigen on the ELISA plate (1 ng / μL, PBS, 100 μL / well) and coat the same concentration of BSA in parallel as a control, and place it at 4°C for overnight coating; discard the supernatant, wash the ELISA plate 3 times with PBS buffer at room temperature, 10 min each time; add 200 μL of blocking solution (3% BSA in PBST) to each well to block the ELISA plate, and block at room temperature for 1 h; discard the blocking solution, add 200 μL of PBST (1×PBS plus 0.1% Tween20, the same below) buffer to each well, and wash the ELISA plate 3 times at room temperature, 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 hours.

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

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

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

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

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

[0303] (1) Construction of CD22 CAR

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

[0305] (2) Packaging of lentiviral vectors

[0306] Resuscitated 293T cells were cultured in DMEM medium containing 10% FBS. After 2-3 generations of cell expansion, 4×10 4 Pieces / cm 2 The density was inoculated into the 2-layer cell factory; plasmid transfection was performed 3 days after cell inoculation; 40 ml of Optim-MEM was added to a sterile 50 ml centrifuge tube for plasmid transfection, and then the viral packaging vector and the viral envelope vector were added according to the ratio of pK1-CD22 CAR: pLP1: pLP2: pLP-VSVG = 5:4:3:1, and then 800 μL of PEI transfection reagent was added, mixed immediately, incubated at room temperature for 15 minutes, and then the plasmid / vector / transfection reagent complex was added dropwise to the culture bottle of 293T cells; after 24 hours, the viral supernatant was collected into a 50 ml centrifuge tube, centrifuged at 250g for 5 minutes, and the supernatant after centrifugation was filtered with a 0.45 μm filter, and the filtered supernatant was ultracentrifuged (25000g, 4°C, 3h) to obtain concentrated CD22 CAR lentivirus; after centrifugation, the supernatant was discarded, and the lentivirus was resuspended with PBS precooled at 4°C, and the resuspended CD22 The CAR lentivirus solution was packaged and stored at -80°C for later use.

[0307] (3) T cell recovery and activation

[0308] Set the water bath temperature to 38°C and preheat 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 frozen umbilical cord blood becomes transparent and completely melts, take out the cryopreservation bag; wipe the water stains on the outside of the cryopreservation bag with a dry cotton ball and spray it with 75% alcohol for disinfection. After the alcohol evaporates completely, transfer it to the biosafety cabinet; take out the umbilical cord blood and put it in a 50mL centrifuge tube, add an appropriate amount of RPMI 1640 culture medium, mix well, and take samples for counting; centrifuge at 300g for 5min, collect the lower layer cells after centrifugation, and resuspend them with complete culture medium to a T cell density of 1×10 6 / mL, add activation antibodies Anti-hμman CD3 antibody and Anti-hμman CD28 antibody according to the resuspension volume, where CD3 is used at a concentration of 0.15μg / mL and CD28 is used at a concentration of 0.625μg / mL, and culture in a 37℃, 5% carbon dioxide incubator; after culturing for 4 hours, add complete culture medium to adjust the T cell density to 4×10 5 / mL, and continue culturing.

[0309] (4) T cell sorting and purification

[0310] After the cells were activated for 36 hours, 20 μL of the sample was mixed and added with 10 μL of the diluted antibody for staining for 10 minutes. After adding PBS to dilute 10 times, the cells were counted using a flow cytometer, and the density of CD3+, CD4+, and CD8+ T cells was recorded. The expression of CD69 and CD25 molecules was observed. The cell volume was recorded to confirm the cell amount. The cell suspension was transferred to a centrifuge tube and centrifuged at 300 g for 5 minutes, and the supernatant was discarded to collect the lower layer of cells. MACS Buffer was added for washing, and the lower layer of cells was collected after centrifugation again. The centrifugation conditions were the same as above, and the cells were resuspended with an appropriate amount of MACS Buffer. The amount of magnetic beads added was calculated according to the cell amount, and each 1×10 6 Add 4 μL CD4+ magnetic beads to each 1×10 CD4+ T cells. 6 Add 8μL CD8+ magnetic beads to each CD8+T cell; after adding the magnetic beads, mix well and incubate in the dark for 20min at room temperature. After incubation, add MACS Buffer to wash, centrifuge at 300g for 5min, 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 1mL Buffer, after rinsing, pass the cell suspension through the column, and continue to add 9mL Buffer to pass the column; remove the LS column from the MACS magnetic rack, and add 5mL Buffer to flush out the cells trapped on the LS column; finally, mix the cell suspension, take samples for staining and counting, record the density of CD3+, CD4+, and CD8+T cells, and calculate the sorting recovery rate and purity.

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

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

[0313] The proliferation of CD22 CAR-T cells was observed every 3 days and fresh culture medium was added. After 11 days of continuous culture, the cells were harvested for subsequent in vitro killing experiments.

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

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

[0316] Raji cells (purchased from ATCC, CCL-86), Namalwa cells (purchased from ATCC, CRL-1432) and K562 cells (purchased from ATCC, CRL-3344) were used as target cells, and the expression of CD22 antigen on the surface of these cells was detected by flow cytometry. The results are shown in Figure 2 The results showed that Raji cells highly expressed CD22, Namalwa cells moderately expressed CD22, and K562 cells were CD22 negative cells. Finally, Raji cells and Namalwa cells were selected as target cells for in vitro killing experiments.

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

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

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

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

[0321] 18 hours after plating in vitro killing experiment, the supernatant of co-culture fluid of CAR-T cells (UnT, m971, S1, S4, S9, S27) and Namalwa cells was collected and detected 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) was used to detect the release of cytokines Granzyme B, TNF-α, IFN-γ, and IL-2 in each group. The results are shown in Fig. 6A , 6B , 6C and 6D.

[0322] The specific steps are as follows:

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

[0324] Beads preparation: After the required beads are restored to room temperature, vortex for 2 minutes to fully mix the beads. Calculate the required amount of beads based on the sample volume, with the volume added to each sample being 15 μL.

[0325] Preparation of Wash Buffer: Return 20x Wash Buffer to room temperature to fully dissolve the salt, and prepare 1x Wash Buffer with up-water for later use.

[0326] Preparation of standard: dissolve the standard in 250μL Assay Buffer, invert several times to mix thoroughly, let stand at room temperature for 10 min, and then transfer to an EP tube; take out 25μL of the standard and put it in an EP tube, marked as C7; take 7 EP tubes, marked as C6 / C5 / C4 / C3 / C2 / C1 / C0, add 22.5μL Assay Buffer to each tube, take out 7.5μL of the standard from C7 and dilute it in a 4-fold gradient until it is diluted to C1. C0 is Assay Buffer (0pg / ml).

[0327] Preparation of standard wells and sample wells: prepare cell supernatant standard curve wells, add 15 μL each of AssaBuffer, standard, and Beads into the EP tube and mix well; prepare sample wells: add 15 μL each of Assay Buffer, sample, and Beads into the EP tube and mix well.

[0328] Capture the binding of beads to the target analyte: shake the standard wells and sample wells at 500 rpm and incubate for 2 hours in the dark.

[0329] Washing: Centrifuge the sample at 2000g for 5 min, discard the supernatant, and beads can be seen at the bottom; add 200μL 1xWashbuffer to each EP tube, vortex briefly, centrifuge at 2000g for 5 min, and discard the supernatant.

[0330] Binding of capture beads, target analytes, biotinylated detection antibodies, and SA-PE: add 15 μL of detection antibody to each EP tube, pipette to mix, shake at 500 rpm, and incubate for 1 hour 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 1xWash buffer to each EP tube, vortex briefly, centrifuge at 2000 g for 5 min, and discard the supernatant.

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

[0333] Test results: The flow cytometer test files were exported to FSC format for analysis using LEGENDplex TM The data analysis software determines the concentration of the target cytokine based on a known standard curve.

Claims

1. A single domain antibody targeting CD22, It is characterized in that The single domain antibody comprises CDR1, CDR2 and CDR3; wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, wherein the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35; wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, wherein the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 36; wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO:3, wherein the CDR2 comprises the amino acid sequence shown in SEQ ID NO:20, wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO:37; wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO:7, wherein the CDR2 comprises the amino acid sequence shown in SEQ ID NO:22, wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO:46; wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO:8, wherein the CDR2 comprises the amino acid sequence shown in SEQ ID NO:23, wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO:47; wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 13, wherein the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 29, and wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 53; wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, wherein the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 30, wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 54; wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 15, wherein the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 31, wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 57; or wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO:16, wherein the CDR2 comprises the amino acid sequence shown in SEQ ID NO:31, wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO:

59.

2. The single domain antibody according to claim 1, It is characterized in that The single-domain antibody comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar to the amino acid sequence shown in any one of SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:132 or SEQ ID NO:133; or the single-domain antibody comprises an amino acid sequence as shown in any one of SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:132 or SEQ ID NO:

133.

3. A chimeric antigen receptor, It is characterized in that Include: (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) intracellular signal transduction domain; Wherein, the extracellular antigen binding domain comprises a single domain antibody targeting CD22 as described in any one of claims 1-2.

4. The chimeric antigen receptor according to claim 3, It is characterized in that wherein the transmembrane domain is derived from CD8α, CD4, CD28, CD137, CD80, CD86, CD152 or PD-1; preferably, wherein the transmembrane domain is derived from CD8α or CD28; or wherein the intracellular signal transduction domain is derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD66d, FcRγ or FcRβ; preferably, wherein the intracellular signal transduction domain is derived from CD3ζ ; or wherein the intracellular signal transduction domain further comprises a costimulatory signaling domain, wherein the costimulatory signaling 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 a combination thereof; preferably, the costimulatory signaling domain is derived from CD137 (4-1BB) or CD28.

5. The chimeric antigen receptor according to claim 3, It is characterized in that Further comprises a hinge region located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain, wherein the hinge region is derived from CD8α, CD28, IgG1 or IgG4; preferably, wherein the hinge region is derived from CD8α or CD28; or further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide, wherein the signal peptide is derived from HLA-A, CD8α, CD33, Igκ, IL-2 or GM-CSFRα; preferably, wherein the signal peptide is derived from HLA-A or CD8α.

6. A chimeric antigen receptor, It is characterized in that The chimeric antigen receptor comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similar to the amino acid sequence shown in any one of SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155 or SEQ ID NO: 156; or the chimeric antigen receptor comprises an amino acid sequence as shown in any one of SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155 or SEQ ID NO:

156.

7. An isolated nucleic acid, It is characterized in that The method comprises a nucleic acid sequence encoding a chimeric antigen receptor according to any one of claims 3 to 6.

8. An engineered immune effector cell, It is characterized in that Comprising the chimeric antigen receptor according to any one of claims 3 to 6 or the isolated nucleic acid according to claim 7; wherein the immune effector cells are selected from T cells, B cells, NK cells, macrophages, dendritic cells or induced pluripotent stem cells; preferably, wherein the immune effector cells are selected from T cells.

9. A pharmaceutical composition, It is characterized in that It comprises the single domain antibody targeting CD22 as described in any one of claims 1-2, the engineered immune effector cell as described in claim 8, and a pharmaceutically acceptable carrier or excipient.

10. Use of the single domain antibody targeting CD22 as described in any one of claims 1-2, the engineered immune effector cell as described in claim 8, or the pharmaceutical composition as described in claim 9 in the preparation of a drug for treating B cell-related malignancies, B cell-related autoimmune diseases and / or inflammatory diseases.

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