CN117164712A

CN117164712A - CD 22-targeted single domain antibody, chimeric antigen receptor and application thereof

Info

Publication number: CN117164712A
Application number: CN202311189542.3A
Authority: CN
Inventors: 邹强; 孙海; 周丹; 袁肖; 何雨辰
Original assignee: Chengdu Yousainuo Biotechnology Co ltd
Current assignee: Chengdu Yousainuo Biotechnology Co ltd
Priority date: 2023-02-22
Filing date: 2023-02-22
Publication date: 2023-12-05
Also published as: CN116199781A

Abstract

The invention relates to the technical field of immunotherapy, and provides a single domain antibody targeting CD22, a Chimeric Antigen Receptor (CAR) targeting CD22 constructed by using the single domain antibody and an engineered immune effector cell. The invention also provides application of the single domain antibody and CAR of the targeting CD22 and the engineered immune effector cells in preparing medicines for treating CD22 related diseases.

Description

CD 22-targeted single domain antibody, chimeric antigen receptor and application thereof

Technical Field

The invention belongs to the technical field of immunotherapy, and particularly relates to a single-domain antibody targeting CD22, a chimeric antigen receptor which is constructed by the single-domain antibody and targets CD22, and an engineered immune effector cell. The invention also relates to a method of treating a disease or disorder in a subject, in particular to T cell immunotherapy based on chimeric antigen receptors.

Background

CD22 is a B lymphocyte lineage differentiation antigen, also known as BL-CAM, B3, leu-14, lyb-8, and Siglec-2, has been shown to be specifically expressed by B lymphocytes and is functionally important as a negative regulator of B lymphocyte activation. CD22 is an inhibitory co-receptor, can down regulate BCR signals, block B cell over-stimulation, plays an important role in maintaining marginal zone B cell population, optimal B cell antigen receptor induced proliferation, B cell renewal and the like, and particularly, CD22 is expressed in B cell malignant tumors, so that the CD22 becomes a promising target point for cancer treatment. In addition, there have been researchers proposed to treat autoimmune diseases by selectively modulating B cell activity by targeting CD 22.

Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) modified T cells are widely appreciated and used as an immunotherapeutic strategy in tumor therapy, particularly in hematological malignancies. The principle is that through gene modification, T cell expresses receptor structure capable of specifically recognizing tumor cell surface antigen (single chain antibody), and after the receptor is specifically combined with tumor cell surface antigen, the downstream immune co-stimulatory factor and T cell are activated, so that T cell is activated to secrete relevant cytokine, and tumor cell is specifically killed. The structure of a CAR generally consists of four parts, an extracellular antigen binding domain (often a single chain antibody with antigen recognition), a hinge region, a transmembrane domain, and an intracellular signaling domain. Currently, CAR structures are generally classified into first generation (without costimulatory molecules), second generation (comprising one costimulatory molecule) and third generation (comprising two costimulatory molecules) according to the number of costimulatory molecules added to intracellular signaling domains, with the second generation CAR structure being the most used in the current market and clinical research stages.

Although humans have made great progress in the treatment of Acute Lymphoblastic Leukemia (ALL) in children and adults, there are still a significant number of patients who are poorly treated and current standard therapies have a significant degree of short-term and long-term toxicity. Monoclonal antibody-based therapies are expected to overcome chemotherapy resistance and treatment-related potential toxicities. Among the most promising are chimeric antigen receptor-modified T (CAR-T) cell therapies, which can break through MHC restriction, recognizing tumor antigens directly. Currently CAR-T cells have been widely used in hematologic malignancies, and in particular CAR-T cell immunotherapy with CD19 as target antigen has made breakthrough progress. Targeting CD19CAR-T is not universally effective, however, and loss of target antigen serves as a tumor escape mechanism after immunotherapy, limiting the therapeutic efficacy of cellular immunotherapy in hematological malignancies. Tumor cell evasion mechanisms of CD19CAR-T during treatment of B-ALL mainly include alternative splicing, frameshift mutations, and missense mutations of CD 19. Therefore, in the future, it is also considered to select a new CAR-T therapeutic target or apply the novel CAR-T therapeutic target in combination with CD19CAR-T or construct a multi-target CAR-T, so that the anti-tumor targeting potential of CAR-T cells is further enhanced, the therapeutic effect is improved, and the recurrence rate after tumor treatment is reduced.

Similarly to the CD19 antigen, CD22 is also B cell restricted expressed, not expressed in other parenchymal cells, nor expressed in hematopoietic stem cells, and thus has been an ideal therapeutic target in B cell malignancies because of its high specificity as a B cell tumor antigen. In addition, CD22 has extensive co-expression with CD19 on the surface of tumor cells, and CD22 antigen remains after CD19 CAR-T cell therapy causes loss of CD19 antigen. Therefore, the CD22 CAR-T cells can be used for treating B cell malignant tumors independently, can also be used for salvage treatment of patients with the tumor cells expressing CD22, which relapse due to antigen variation after the CD19 CAR-T treatment, or can be used for combined treatment with the CD19 CAR-T cells, so that the antigen variation is avoided, the effectiveness of CAR-T treatment is improved, and the relapse of tumors is reduced.

Single domain antibodies (sdabs) differ from traditional 4-chain antibodies by having a single, monomeric antibody variable domain. For example, camelids and sharks produce antibodies that naturally lack light chains, which are referred to as heavy chain-only antibodies (hcabs, or simply heavy chain antibodies). The antigen binding fragment in each arm of a camelid heavy chain antibody has a single heavy chain variable domain (VHH) which can have a high affinity for the antigen without the aid of a light chain. Camelid VHH antibodies are referred to as the smallest functional antigen binding fragments, have a molecular weight of only about 15kD and are thus also referred to as nanobodies. The VHH antibody has the natural advantages of good solubility, high stability, strong penetrating power and wide binding epitope. VHH antibodies have been increasingly focused by researchers in the field since their discovery, and their basic research has grown. In application, the medicine has gradually entered clinical research stage aiming at autoimmune diseases, blood diseases, virus infection, orthopedic diseases and the like, and has great advantages in the aspects of anti-infection, anti-inflammatory diseases, neurodegenerative diseases and the like.

Because CAR-T cell manufacturing techniques require the use of single chain antibodies that have good binding activity and high binding epitope efficiency, one of the key technical parts of CAR-T cell therapies is the screening of high affinity antibodies that have good specificity, strong binding capacity, and efficient binding epitope. However, conventional CD22 antibodies are limited by disadvantages of large molecular weight, weak binding force with antigen, low affinity, difficult transformation, poor stability, etc., and further single-chain transformation is required, and effective construction of CAR-T cells is difficult to achieve using conventional CD22 antibodies (e.g., monoclonal antibodies, etc.).

Thus, there remains a broad need to develop improved CD 22-targeting single domain antibodies, CD 22-targeting chimeric antigen receptors constructed thereof, and engineered immune effector cells. For example, stable and small CD 22-targeting single domain antibodies were developed for more effective and more efficient CAR-T cell therapies.

Disclosure of Invention

One of the purposes of the present invention is to provide a CD 22-targeting single-domain antibody, which has a natural single-chain structure, has the advantages of small molecular weight, high solubility, high stability, low immunogenicity, high tissue permeability, and no need of additional folding and assembly steps or linker optimization modification, and is a promising alternative to scFv single-chain antibodies with larger molecular weight, and has very remarkable tumor cell killing ability after being constructed into CAR-T cells.

The single domain antibody for targeting CD22 provided by the invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequences shown in SEQ ID NOS.1-19, wherein CDR2 comprises the amino acid sequences shown in SEQ ID NOS.20-34, and wherein CDR3 comprises the amino acid sequences shown in SEQ ID NOS.35-62.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 20, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 35.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 20, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 36.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 3, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 20, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 37.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 20, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 38.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 7, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 22, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 46.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 8, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 23, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 47.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 10, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 26, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 50.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 11, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 27, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 51.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising 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, wherein CDR3 comprises the amino acid sequence shown in SEQ ID NO. 52.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 13, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 29, and wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 53.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 14, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 30, and wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 54.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 14, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 30, and wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 56.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 15, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 31, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 57.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 16, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 31, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 59.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 17, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 32, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 60.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 18, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 33, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 61.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 19, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 34, and wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 62.

The single domain antibody for targeting CD22 provided by the invention comprises CDR1, CDR2 and CDR3 regions; wherein the determination of CDR1, CDR2 and CDR3 is according to the IMGT numbering scheme, the Kabat numbering scheme, the AbM numbering scheme, the Chothia numbering scheme or the Contact numbering scheme.

The single domain antibody targeting CD22 provided by the 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.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 63, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 73, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 93, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 63, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 73, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 97, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 65, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 77, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 104, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 66, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 78, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 105, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 66, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 80, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 108, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 63, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 81, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 109, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 63, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 82, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 110, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 63, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 84, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 111, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 63, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 85, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 112, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO:69, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO:85, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO:111, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO: 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 63, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 87, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 113, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO:71, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO:89, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO:115, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO: 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 63, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 90, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 116, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 72, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 91, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 117, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

In some embodiments, the present invention provides a CD 22-targeting single domain antibody further comprising FR1, FR2, FR3, and FR4 regions; wherein FR1 comprises the amino acid sequence shown as SEQ ID NO. 63, wherein FR2 comprises the amino acid sequence shown as SEQ ID NO. 92, wherein FR3 comprises the amino acid sequence shown as SEQ ID NO. 118, wherein said FR4 comprises the amino acid sequence shown as SEQ ID NO. 119.

Wherein, the specific amino acid sequence information shown in SEQ ID NOS: 1-119 is shown in Table 1.

TABLE 1

The single domain antibody for targeting CD22 provided by the invention comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity with the amino acid sequence shown in SEQ ID NO. 120-136.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 120.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 121.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 122.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO. 123.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 124.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 125.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 126.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO 127.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 128.

In some embodiments, a single domain antibody targeting CD22 provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence shown in SEQ ID NO. 129.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 130.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 131.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 132.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO 133.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO 134.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 135.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 136.

The single domain antibody for targeting CD22 provided by the invention comprises an amino acid sequence shown in a table 2 or shown in SEQ ID NO. 120-136.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 120.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 121.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 122.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise an amino acid sequence as shown in SEQ ID NO. 123.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising the amino acid sequence shown as SEQ ID NO. 124.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 125.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising the amino acid sequence shown as SEQ ID NO. 126.

In some embodiments, the single domain antibodies targeting CD22 provided herein comprise the amino acid sequence shown as SEQ ID NO. 127.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise an amino acid sequence as set forth in SEQ ID NO. 128.

In some embodiments, the present invention provides a single domain antibody targeting CD22 comprising the amino acid sequence shown as SEQ ID NO. 129.

In some embodiments, the single domain antibodies targeting CD22 provided herein comprise the amino acid sequence shown as SEQ ID NO. 130.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 131.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 132.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 133.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 134.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 135.

In some embodiments, the CD 22-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 136.

Wherein, the specific amino acid sequence information shown in SEQ ID NOS.120-136 is shown in Table 2.

TABLE 2

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One of the purposes of the present invention is to provide a chimeric antigen receptor, which has a very remarkable capability of killing tumor cells after transfection to prepare CAR-T cells.

The present invention provides chimeric antigen receptors comprising (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain; wherein the extracellular antigen-binding domain comprises a single domain antibody that targets CD22 as described previously.

In some embodiments, the present invention provides chimeric antigen receptors wherein the transmembrane domain is derived from CD8 a, CD28, CD4, CD137, CD80, CD86, CD152 or PD-1.

In some embodiments, the present invention provides chimeric antigen receptors wherein the transmembrane domain is derived from CD8 a.

In some embodiments, the present invention provides chimeric antigen receptors in which the transmembrane domain comprises the amino acid sequence shown as SEQ ID NO. 140.

In some embodiments, the present invention provides chimeric antigen receptors wherein the intracellular signaling domain is derived from cd3ζ, cd3γ, cd3δ, cd3ε, CD22, CD79a, CD79b, CD66d, fcrγ, fcrβ.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain is derived from cd3ζ.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signal transduction domain comprises the amino acid sequence shown as SEQ ID NO. 142.

In some embodiments, the invention provides chimeric antigen receptors in which the intracellular signaling domain further comprises a costimulatory signaling domain.

In some embodiments, the present invention provides chimeric antigen receptors in which the costimulatory signaling domain is derived from CD137 (4-1 BB), CD27, CD28, ICOS, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B-H3, CD83 ligand, and combinations thereof.

In some embodiments, the present invention provides chimeric antigen receptors in which the costimulatory signaling domain is derived from CD137 (4-1 BB).

In some embodiments, the present invention provides chimeric antigen receptors in which the costimulatory signaling domain comprises the amino acid sequence depicted as SEQ ID NO. 141.

In some embodiments, the chimeric antigen receptor provided herein further comprises a hinge region located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

In some embodiments, the invention provides chimeric antigen receptors wherein the hinge region is derived from CD8 a, CD28, igG1 or IgG4.

In some embodiments, the invention provides chimeric antigen receptors in which the hinge region is derived from CD8 a.

In some embodiments, the present invention provides chimeric antigen receptors in which the hinge region comprises the amino acid sequence shown as SEQ ID NO. 139.

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

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from HLA-A, CD8 a, CD33, igkappa, IL-2, GM-CSFR alpha.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from HLA-A.

In some embodiments, the present invention provides chimeric antigen receptors in which the signal peptide comprises the amino acid sequence shown as SEQ ID NO. 138.

Wherein, the specific amino acid sequence information shown in SEQ ID NOS: 138-142 is shown in Table 3.

TABLE 3 Table 3

The chimeric antigen receptor provided by the invention comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity with the amino acid sequence shown in SEQ ID NO 143-159.

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

In some embodiments, the chimeric antigen receptor provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 144.

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

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

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

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

In some embodiments, the chimeric antigen receptor provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in SEQ ID NO. 149.

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

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

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

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

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

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

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

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

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

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

The chimeric antigen receptor provided by the invention comprises an amino acid sequence shown in table 4 or SEQ ID NO 143-159.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO 143.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 144.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 145.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 146.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO: 147.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 148.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO: 149.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 150.

In some embodiments, the chimeric antigen receptor provided by the invention comprises the amino acid sequence as set forth in SEQ ID NO. 151.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 152.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 153.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 154.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO: 155.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 156.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 157.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 158.

In some embodiments, the chimeric antigen receptor provided by the invention comprises the amino acid sequence shown as SEQ ID NO. 159.

Wherein, the specific amino acid sequence information shown in SEQ ID NOS: 143-159 is shown in Table 4.

TABLE 4 Table 4

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It is an object of the present invention to provide a nucleic acid comprising a nucleic acid sequence encoding a chimeric antigen receptor as described above. In some embodiments, the nucleic acids provided herein comprise the nucleic acid sequences set forth in SEQ ID NOS.161-178. Wherein, the specific nucleic acid sequence information shown in SEQ ID NOS.161-178 is shown in Table 5.

TABLE 5

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It is an object of the present invention to provide a vector comprising a nucleic acid encoding a nucleic acid sequence of a chimeric antigen receptor as described above.

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

In some embodiments, the engineered immune effector cells provided herein are selected from T cells, B cells, NK cells, macrophages, dendritic cells, induced pluripotent stem cells (ipscs).

It is an object of the present invention to provide a pharmaceutical composition comprising a single domain antibody targeting CD22 as described above, an engineered immune effector cell, and a pharmaceutically acceptable carrier or excipient.

It is an object of the present invention to also provide a method of treating a disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of a CD 22-targeting single domain antibody, engineered immune effector cell, or pharmaceutical composition as described previously.

In some embodiments, the 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.

In some embodiments, the invention provides a method of treating a disease or disorder in a subject, wherein the disease or disorder is cancer.

In some embodiments, the invention provides a method of treating a disease or disorder in a subject, wherein the disease or disorder is a B cell-related malignancy. For example, the B cell-related malignancy is B cell leukemia or B cell lymphoma. More specifically, 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 mediastinum lymphoma B-cell lymphoma (PMBL), small Lymphocytic Lymphoma (SLL), B-cell prolymphocytic leukemia (B-PLL), follicular Lymphoma (FL), burkitt lymphoma, primary intraocular lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy Cell Leukemia (HCL), precursor B-cell leukemia, non-hodgkin lymphoma (NHL), high grade B-cell lymphoma (HGBL), and Multiple Myeloma (MM).

In some embodiments, the invention provides a method of treating a disease or disorder in a subject, wherein the disease or disorder is a B-cell related autoimmune and/or inflammatory disease. More specifically, wherein the B cell-related autoimmune and/or inflammatory disease is associated with inappropriate or enhanced B cell numbers and/or activation.

It is an object of the present invention to provide a CD 22-targeting single domain antibody, an engineered immune effector cell, and a use of the pharmaceutical composition in the preparation of a medicament for treating a B cell-related malignancy, a B cell-related autoimmune disease, and/or an inflammatory disease.

Interpretation of the terms

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

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

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 heterotetramer units and a further polypeptide called a J chain, comprising 10 antigen binding sites; whereas IgA antibodies comprise 2-5 basic 4-chain units, which can polymerize with J-chains to form multivalent assemblies. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds being dependent 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 (CH 1, CH2 and CH3 for each alpha and gamma chain) and four (CH 1, CH2, CH3 and CH 4) constant domains (CH) for the mu and epsilon isoforms 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 and VH are aligned together, while CL and the first constant domain of the heavy chain (CH 1) are aligned together. 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, weight edition, daniel p. Sties, abba i.terr and Tristram g. Parsol w. Appleton & Lange, norwalk, ct.1994, page 71and Chapter 6. Light chains from any vertebrate species can be classified, based on their constant domain amino acid sequences, into one of two distinct types called kappa and lambda. Immunoglobulins may be assigned to different classes or isotypes depending on their heavy chain constant domain (CH) amino acid sequence. There are five classes of immunoglobulins: igA, igD, igE, igG and IgM have heavy chains called α, δ, ε, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the gamma and alpha classes can be further divided into subclasses, e.g., humans express the following subclasses: igG1, igG2A, igG2B, igG3, igG4, igA 1and IgA2.

Heavy chain antibodies are antibodies derived from camelidae or cartilaginous fish organisms. In contrast to the 4-chain antibodies described above, the heavy chain antibody lacks the light and heavy chain constant regions 1 (CH 1), comprising only 2 heavy chains consisting of variable regions (VHH) and other constant regions, which are linked to the constant regions by hinge-like structures. Each heavy chain of a camelidae heavy chain antibody comprises 1 variable region (VHH) and 2 constant regions (CH 2 and CH 3), and each heavy chain of a cartilaginous fish heavy chain antibody comprises 1 variable region and 5 constant regions (CH 1-CH 5). Antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion to the constant region of human IgG Fc.

The terms "single domain antibody", "single domain antibody targeting CD 22", "heavy chain variable region domain of heavy chain antibody", "VHH", "nanobody" as used herein are used interchangeably and refer to a single domain antibody that specifically recognizes and binds to CD 22. Single domain antibodies are the variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDR regions and four FR regions. Single domain antibodies are the smallest functional antigen binding fragments. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed.

One skilled in the art can alter the sequences of the invention by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain variants of the antibody or functional fragment sequences thereof without substantially affecting the activity of the antibody. These variants include, but are not limited to: deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. Conservative substitutions with amino acids of similar or similar properties generally do not alter the function of the protein in the art. Amino acids having similar properties are substituted, for example, in the FR and/or CDR of the variable region. Amino acid residues that can be conservatively substituted are known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Also for example, the addition of one or several amino acids at the C-terminal and/or N-terminal end will not generally alter the function of the protein. They are all considered to be included within the scope of the present invention.

In some embodiments, the sequences of the variants of the invention may have at least 95%, 96%, 97%, 98% or 99% identity to the sequence from which they were derived. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The invention also includes those molecules having antibody heavy chain variable regions with CDRs, provided that the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.

The single domain antibodies, nanobodies or heavy chain antibodies of the invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the various antibodies of the invention may be expressed in other cell lines. Suitable mammalian host cells may be transformed with sequences encoding the various antibodies of the invention. Transformation may be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus, etc. Host mammalian cell lines useful for expression are well known in the art, for example, 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., hepG 2), and the like. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and producing antibodies with substantial CD 22 binding properties.

The term "Chimeric Antigen Receptor (CAR)" as used herein, comprises an extracellular antigen-binding domain comprising a single domain antibody (sdAb), e.g., VHH, that binds CD22 as disclosed herein.

In some embodiments, a Chimeric Antigen Receptor (CAR) disclosed herein comprises a polypeptide comprising (a) an extracellular antigen binding domain comprising a single domain antibody (sdAb) of CD22 disclosed herein; (b) a transmembrane domain; (c) an intracellular signaling domain. Each domain and additional regions will be described in more detail below.

The invention discloses CARs comprising an extracellular antigen-binding domain comprising one or more single domain antibodies. The sdabs 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-only antibodies, binding molecules that are naturally devoid of light chains, single domains (e.g., VH or VL) derived from conventional 4-chain antibodies, humanized-only heavy chain antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain fragments, and engineering domains and single domain scaffolds that are not derived from antibodies. Any sdAb known in the art or disclosed by the invention, including the single domain antibodies disclosed herein, can be used to construct a CAR described herein. The sdAb may be derived from any species, including but not limited to mouse, rat, human, camel, llama, lamprey, shark, goat, rabbit, and cow. The single domain antibodies contemplated by the present invention also include naturally occurring single domain antibody molecules from species other than camelidae and shark.

In some embodiments, the extracellular antigen-binding domains disclosed herein comprise at least one binding domain, and the at least one binding domain comprises a single domain antibody disclosed herein that binds CD 22.

In some embodiments, the anti-CD 22 sdAb is camelid, chimeric, human, or humanized.

In some embodiments, a CAR of the present disclosure comprises a polypeptide comprising (a) an extracellular antigen-binding domain comprising an anti-CD 22 sdAb; (b) a transmembrane domain; (c) an intracellular signaling domain; wherein the anti-CD 22 sdAb comprises a polypeptide sequence that has at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the sequences SEQ ID NO 120-136.

In some embodiments, a CAR of the present disclosure comprises a polypeptide comprising (a) an extracellular antigen-binding domain comprising an anti-CD 22 sdAb; (b) a transmembrane domain; (c) an intracellular signaling domain; wherein the anti-CD 22 sdAb comprises the amino acid sequences SEQ ID NOS 120-136.

In addition to the antigen binding domains disclosed herein, the CARs disclosed herein may further comprise one or more of the following structures: a linker (e.g., a peptide linker), a signal peptide, a hinge region, a transmembrane domain, a costimulatory signaling domain, an intracellular signaling domain, which are described in detail below.

In some embodiments, the intracellular signal transduction domain comprises a primary intracellular signal transduction domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from cd3ζ, cd3γ, cd3δ, cd3ε, CD22, CD79a, CD79b, CD66d, fcrγ, fcrβ. In some embodiments, the primary intracellular signaling domain is derived from cd3ζ. In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signal domain is derived from a costimulatory molecule derived from one or more of CD27, CD28, CD137 (4-1 BB), OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand. In some embodiments, the costimulatory signaling domain is derived from CD137 (4-1 BB).

In some embodiments, the CD22 CAR further comprises a hinge domain (e.g., a CD8 a hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

In some embodiments, the CD22 CAR further comprises a signal peptide (e.g., HLA-A signal peptide or CD8 a signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus: HLA-A signal peptide or CD8 a signal peptide, extracellular antigen binding domain, CD8 a hinge region, CD8 a transmembrane domain, costimulatory signal domain derived from CD137 (4-1 BB), and intracellular signal transduction domain derived from cd3ζ.

In some embodiments, the different domains of the CAR can also be fused to each other by a peptide linker. Depending on the structural and/or functional characteristics of the single domain antibody and/or the various domains, each peptide linker in the CAR may have the same or different length and/or sequence. Each peptide linker can be independently selected and optimized by one skilled in the art. In some embodiments, the peptide linker comprises flexible residues (e.g., glycine and serine) such that adjacent domains can move freely relative to each other. For example, glycine-serine duplex may be a suitable peptide linker.

The peptide linker may have any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more amino acids in length. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or less amino acids in length. In some embodiments, the peptide linker is about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids in length.

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

The invention also discloses nucleic acids encoding the above antibodies or chimeric antigen receptors. The present invention provides polynucleotides encoding the heavy chain variable region, the light chain variable region, the heavy chain, the light chain, and the CDRs. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

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 made, all of which encode an antibody or chimeric antigen receptor of the invention. Thus, where a particular amino acid sequence has been identified, one of skill in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence encoding the protein. Thus, the present invention also relates to polynucleotides which hybridize to the above polynucleotide sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleic acid sequences of the various antibodies or chimeric antigen receptors of the invention, or fragments thereof, can be obtained generally by PCR amplification, recombinant methods, or synthetic methods. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) to which the present invention relates include biomolecules that exist in isolated form. At present, it is entirely possible to obtain DNA sequences encoding the polypeptides of the invention (or fragments or derivatives thereof) by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can be introduced into the polypeptide sequences of the invention by chemical synthesis.

The invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a leader sequence encoding for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a multiple linker region for inserting a nucleic acid encoding an antibody to be expressed, and selectable marker elements.

The host cell to which the present invention relates may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are bacterial cells of E.coli, streptomyces, salmonella typhimurium; a fungal cell of a yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.

In some embodiments, the host cell may be a variety of functional cells well known in the art, such as a variety of killer cells, including, but not limited to, cytokine-induced killer Cells (CIK), dendritic cell-stimulated cytokine-induced killer cells (DC-CIK), cytotoxic T Lymphocytes (CTLs), γδ T cells, natural killer cells (NK), tumor Infiltrating Lymphocytes (TIL), lymphokine activated killer cells (LAK), CD3AK cells (anti-CD 3 mab killer cells), and CAR-T/TCR-T cells. In certain embodiments, the killer cells are T cells or NK cells. Exemplary NK cells include, but are not limited to, primary NK cells, NK cell lines (e.g., NK 92), and NKT cells. In certain embodiments, the NK cells are primary NK cells. Exemplary T cells include, but are not limited to, T cells of mixed cell populations such as peripheral blood T lymphocytes, umbilical cord blood T lymphocytes, cytotoxic killer T Cells (CTLs), helper T cells, suppressor/regulatory T cells, γδ T cells, and cytokine-induced killer Cells (CIKs), tumor Infiltrating Lymphocytes (TILs), and the like. In certain embodiments, the T cells are peripheral blood T lymphocytes, umbilical cord blood T lymphocytes.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Alternatively, transformation may be performed by electroporation. When the host isIs eukaryotic, and the following DNA transfection method can be selected: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media, such as a serum-containing medium or a serum-free medium, depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

The invention also discloses vectors for cloning and expressing any of the CARs of the invention. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and is 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.

Many virus-based systems have been developed in the art for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated in vitro or ex vivo and delivered to engineered mammalian cells. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is 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 an immunomodulatory agent (e.g., immune checkpoint inhibitor) coding sequence and/or self-inactivating lentiviral vectors carrying a chimeric antigen receptor may be packaged using protocols known in the art. The resulting lentiviral vector may be used to transduce mammalian cells (e.g., primary human T cells) using methods known in the art. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their propagation in offspring cells. Lentiviral vectors also have the advantage of low immunogenicity and can transduce non-proliferating cells.

In some embodiments, the vector comprises any nucleic acid encoding a CAR of the invention. The nucleic acid may 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 linked to a promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art may be used in the present invention. Promoters can be further classified as constitutive or regulated, e.g., inducible.

In some embodiments, the nucleic acid encoding the CAR is operably linked to a constitutive promoter. Constitutive promoters allow for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Exemplary constitutive promoters contemplated by the present invention include, but are not limited to, the Cytomegalovirus (CMV) promoter, the human elongation factor-1 alpha (hEF 1 alpha) promoter, the ubiquitin C (Ubic) promoter, the phosphoglycerate kinase (PGK) promoter, the Simian Virus 40 (SV 40) early promoter, and the chicken beta-actin coupled to CMV early enhancer (CAGG) promoter. The efficiency of such constitutive promoters in driving transgene expression has been widely compared in a number of studies. For example Michael C.Milone et al (Molecular Therapy,17 (8): 1453-1464 (2009)) compared the efficiencies of CMV, hEF 1. Alpha., ubic and PGK in driving chimeric antigen receptor expression in human primary T cells and concluded that the hEF 1. Alpha. Promoter not only induced the highest levels of transgene expression, but was also optimally maintained in CD4 and CD8 human T cells. In some embodiments, the nucleic acid encoding the CAR is operably linked to the hef1α promoter.

In some embodiments, the nucleic acid encoding the CAR is operably linked to an inducible promoter. Inducible promoters are among the regulatory promoters. The inducible promoter may be induced by one or more conditions, such as physical conditions, microenvironment of the engineered immune effector cell or physiological state of the engineered immune effector cell, an inducer, etc.

In some embodiments, the induction conditions do not induce expression of an endogenous gene in the engineered mammalian cell and/or in the subject receiving the pharmaceutical composition. In some embodiments, the induction conditions are selected from: inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox status, tumor environment, and activation status of the engineered mammalian cells.

In some embodiments, the vector further comprises a selectable marker gene or reporter gene to select a CAR expressing cell from a population of host cells transfected with the lentiviral vector. The selectable marker and the reporter gene may be flanked by appropriate regulatory sequences for expression in the host cell. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the nucleic acid sequences.

The term "immune effector cell" as used herein is an immune cell that can perform an immune effector function. In some embodiments, the immune effector cells express at least fcyriii and perform 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 (ipscs), and the like.

In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is a CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-T cell or a combination thereof. In some embodiments, the T cells produce IL-2, TFN, and/or TNF after expressing the CAR and binding to a target cell, such as a cd22+ tumor cell. In some embodiments, the cd8+ T cells lyse antigen-specific target cells after expression of the CAR and binding to the target cells.

In some embodiments, the immune effector cell is an NK cell. In other embodiments, the immune effector cell may be an established cell line, such as NK-92 cells.

In some embodiments, immune effector cells may be differentiated from stem cells, such as hematopoietic stem cells, pluripotent stem cells, ipscs, or embryonic stem cells.

The engineered immune effector cells of the invention are prepared by introducing a CAR into an immune effector cell (e.g., T cell). In some embodiments, the CAR is introduced into the immune effector cell by transfection of any one of the isolated nucleic acids or any one of the vectors described above.

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

Physical methods for introducing the vector into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art (see Sambrook, j., fritsch, e.f. and manitis, 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.

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 mammals (e.g., human cells).

Chemical methods for introducing the carrier into immune effector cells include colloidal dispersion systems, including, for example, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including, for example, oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome.

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

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

Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue and which encodes a polypeptide that expresses some readily detectable property, such as enzymatic activity. The expression of the reporter gene is determined at a suitable time after introduction of the DNA into the recipient cell. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (see Kumiko Ui-tei.febs Letters,479:79-82 (2000)). Suitable expression systems are known in the art and may be prepared or commercially available using known techniques. Other methods of confirming the presence of a nucleic acid encoding a CAR in an engineered immune effector cell include: molecular biological testing methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assay methods, such as detecting the presence or absence of a particular peptide; immunological methods such as ELISA.

The pharmaceutical composition disclosed by the invention contains the CD 22-targeted single domain antibody, the engineered immune effector cells and pharmaceutically acceptable excipients or carriers. Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives and/or adjuvants. The adjuvant is preferably non-toxic or substantially non-toxic to the recipient at the dosage and concentration employed. 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, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required.

Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. In lyophilizing a composition, the method may be used to sterilize prior to or after lyophilization, reconstitution, dilution. The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Compositions for parenteral administration may be stored in lyophilized form or in solution. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of the pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles or porous beads, and depot injections, are also known to those skilled in the art.

Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, or as lyophilized powders. The formulation may be stored in a ready-to-use form or in a form that is reconstituted prior to administration (e.g., lyophilized). The invention also provides kits for producing single dose administration units. Kits of the invention may each contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the invention, kits are provided that contain single and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes).

The invention also provides methods of treating a patient, particularly a patient suffering from a CD 22-associated disease, by administering a CD 22-targeted single domain antibody, an engineered immune effector cell, or a pharmaceutical composition thereof according to any of the embodiments of the invention. The terms "patient", "subject", "individual", "subject" are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. "treating" refers to a subject employing the methods of treatment described herein to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, or reduced rate of tumor metastasis or tumor growth). The therapeutic methods effective to treat a patient can vary depending on a variety of factors, such as the disease state, age, weight, and ability of the therapy to elicit an anti-cancer response in the subject.

The therapeutically effective amount of the pharmaceutical composition comprising the CD 22-targeting single domain antibodies or engineered immune effector cells of the invention to be employed will depend, for example, on the extent of treatment and the goal. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the molecule delivered, the indication, the route of administration, and the patient's condition (body weight, body surface or organ size) and/or condition (age and general health). In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect.

The frequency of administration will depend on the pharmacokinetic parameters of the CD 22-targeting single domain antibodies or engineered immune effector cells in the formulation used. The clinician typically administers the pharmaceutical composition until a dose is reached that achieves the desired effect. The pharmaceutical composition may thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.

The route of administration of the pharmaceutical composition is conventional in the art, e.g. oral, nasal, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional route injection, and may also be administered by a sustained release system or by an implanted device.

Drawings

Figure 1A shows the expression rate of CD22 CAR polypeptide molecules on each set of cd4+ T cells.

Figure 1B shows the expression rate of CD22 CAR polypeptide molecules on each set of cd8+ T cells.

FIG. 2 shows the expression of CD22 antigen on the surfaces of Raji cells, namalwa cells and K562 cells, respectively.

Fig. 3A shows the killing rate of Raji cells by each group of effector cells on the first day (D1) when the effective target ratio (E: T) =1:1.

Fig. 3B shows the killing rate of Raji cells by each group of effector cells on the third day (D3) when the effective target ratio (E: T) =1:1.

Fig. 4A shows the killing rate of Raji cells by each group of effector cells on the first day (D1) when the effective target ratio (E: T) =1:3.

Fig. 4B shows the killing rate of Raji cells by each group of effector cells on the third day (D3) when the effective target ratio (E: T) =1:3.

Fig. 5A shows the killing rate of Namalwa cells by each group of effector cells on the first day (D1) when the effective target ratio (E: T) =1:3.

Fig. 5B shows the killing rate of Namalwa cells by each group of effector cells on the third day (D3) at an effective target ratio (E: T) =1:3.

FIG. 6A shows the release of Granzyme B in an in vitro killing experiment for each group of effector cells.

FIG. 6B shows TNF- α release from various groups of effector cells in an in vitro killing assay.

FIG. 6C shows IFN-gamma release from various groups of effector cells in an in vitro killing assay.

FIG. 6D shows IL-2 release from various groups of effector cells in an in vitro killing assay.

In the above figures: unT A negative control group, m 971A positive control group, S1-CAR-T group, S4-CAR-T group, S9-CAR-T group, S27-CAR-T group S28 is S28-CAR-T group, S35 is S35-CAR-T group, S36 is S36-CAR-T group, S41 is S41-CAR-T group, and S43 is S43-CAR-T group.

The achievement of the objects, functional features and advantageous effects of the present invention will be further described with reference to the following embodiments and with reference to the accompanying drawings.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the invention thereto. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 preparation of CD22 VHH-targeting Single-Domain antibodies

(1) Animal immunity and immune response test

The CD22 antigen used for immunization of animals was Hμman Siglec-2/CD22 Protein, fc Tag (Acro Biosystem, cat. No. CD2-H5253).

1) Healthy alpaca was selected as the immunization subject.

2) Immunization for the 1 st time, mixing complete Freund's adjuvant with CD22 antigen (0.8 mg) 1:1, emulsifying, performing subcutaneous multipoint injection, mixing incomplete Freund's adjuvant with CD22 antigen 1:1 for subsequent booster immunization, and immunizing for 5 times at intervals of 2 weeks; serum was collected at 5mL of peripheral blood before and after each immunization, and the immune response was monitored by ELISA to confirm serum titer.

3) After the 5 th immunization, the plasma titer reached 100000 level, 20mL was collected, lymphocytes were isolated and stored in Trizol for subsequent antibody phage library construction.

(2) Construction of antibody phage libraries

1) After animal immunization is finished, RNA is extracted by using the separated lymphocytes and the obtained total RNA is subjected to reverse transcription by using a Takara reverse transcription kit; dividing the total RNA sample into two parts, wherein one part uses Oligo dT Primer in the kit as a Primer, the other part uses Random 6-mers in the kit as a Primer, and the total RNA obtained in the last step is reversely transcribed into cDNA according to the instruction of a reverse transcription kit and is respectively stored in 2 centrifuge tubes.

2) PCR amplification

a. Amplifying a specific antibody fragment from the reverse transcribed cDNA, and performing PCR amplification using Taq DNAPolymerase Hot Start enzyme; carrying out 1% agarose gel electrophoresis on all PCR products, cutting gel, recovering a band with the size of a target fragment of about 600-700bp, namely the PCR amplification product of the first round, and storing at the temperature of minus 20 ℃;

b. And (3) taking the PCR amplification product of the first round as a template to perform a second round of PCR reaction, performing 1% agarose gel electrophoresis after the reaction is finished, finally cutting gel to recover the single target band with the fragment size of about 400bp, and performing DNA purification on the PCR reaction liquid by using a universal DNA purification recovery kit.

3) Enzyme digestion and ligation

The target gene fragment amplified by the second round of PCR and the pComb3XSS phage plasmid vector are respectively digested by restriction enzymes SpeI and SacI, and the VHH target gene fragment is connected to the pComb3XSS phage plasmid vector by ligase after the digestion is completed, so as to construct a recombinant plasmid.

4) Bacterial library construction

a. Taking a 50 mu L TG1 competent cell, placing on ice for 5-10min to melt;

b. 100ng of the ligation product was added and transferred to pre-chilled 1mm apart electrotransfer cups, and the parameters were set in the electrotransfer apparatus: after 1800V and 1mm, clicking a button for conversion;

c. immediately adding 1mL of preheated SOC culture solution at 37 ℃ after the completion of electrotransformation, shaking bacteria at 200rpm at 37 ℃ for recovery for 1h after uniform mixing;

d. taking more than 20 100ng of connection systems, and performing electrotransformation reaction by using competence according to the method;

e. taking 100 mu L of the recovered bacterial liquid, carrying out 10-gradient dilution, then coating the bacterial liquid on a plate, and culturing at 37 ℃ overnight;

f. All the remaining bacterial liquid was collected and spread evenly over 20 more 15cm plates (2 XYT containing 100. Mu.g/mLAmp, 2% agarose) and incubated overnight at 37℃with inversion;

g. calculating the number of transformed colonies obtained by all reactions according to the dilution times and the number of single colonies, namely the library capacity of the bacterial library; meanwhile, randomly selecting a plurality of monoclone from the gradient dilution plate to perform colony PCR, wherein a single band with about 400bp of PCR product is considered as positive clone, so that the cloning positive rate of the bacterial library is estimated;

h. the overnight cultured plate colonies were scraped off using 2 XYT liquid medium, placed in 50mL centrifuge tubes, OD600 values were measured, and glycerol at a final concentration of 20% was added for preservation at-80 ℃.

5) Phage library construction

a. Inoculating the bacterial library into 100mL 2 XYT liquid culture medium (containing 100. Mu.g/mLAMP) to make initial OD600 value 0.1, 37 deg.C, and culturing at 250rpm until OD600 is 0.5-0.55;

b. according to 1:20 (bacterial count: phage count) helper phage was added and incubated at 37℃and 250rpm for 30min;

c. adding 50 mug/ml Kana, culturing at 30deg.C and 250rpm overnight, centrifuging, and collecting supernatant;

d. adding 1/4 volume of precooled PEG/NaCl, uniformly mixing, standing on ice for incubation for at least 30min, centrifuging at 4000rpm for 20 min at 4 ℃, removing the supernatant, and adding 1mLPBS buffer to dissolve precipitate; after adding again 1/4 volume of pre-chilled PEG/NaCl and incubating on ice for 10 minutes, centrifuging at 4℃and 12000g for 10 minutes, removing the supernatant and dissolving the precipitate in 1mLPBS, and preserving at-80℃to obtain a purified phage library.

(3) Phage selection

1) First round screening

a. Screening antigen coated immune tube (50. Mu.g/tube, coating solution PBS,2 mL/tube), slowly rotating at 4deg.C overnight, while parallel coating BSA (50. Mu.g in PBS,2 mL/tube) was used as control; removing the supernatant from the immune tube coated overnight, washing the immune tube with PBS buffer solution at room temperature for 3 times, rotating for 5 min/time, adding 2mL of sealing solution (3% skimmed milk powder) solution, rotating at room temperature for 2h, removing the supernatant, adding 2mL of BST buffer solution, washing the immune tube at room temperature for 3 times, and rotating for 5 min/time;

b. removing the wash solution from the immune tube and adding about 10% of the phage library prepared ¹² pfu is used as a first round of screening an input phage library, PBS buffer is added to 2mL, and the phage library is rotated and incubated for 1h at room temperature; the supernatant was discarded, and the immune tube was washed 20 times with 2ml of buffer solution (1 XPBS plus 0.1% Tween20, the same applies below) at room temperature, each time rotated for 5min; the liquid in the immune tube is discarded, 1mL of 0.25mg/mL Trypsin solution is added, and the solution is subjected to rotary elution at room temperature for 30min; the elution was stopped by adding 10. Mu.L of 10% AEBSF and the solution in the immune tube was transferred to a new 1.5mL centrifuge tube, the phage eluate from the first round of screening.

2) First round phage eluate titer detection

Taking 10 mu L of phage eluate of the first round, carrying out 10-time gradient dilution in a 1.5mL centrifuge tube, and diluting for 12 gradients to 10 ^-12 The method comprises the steps of carrying out a first treatment on the surface of the Adding 90 mu L of TG1 bacterial liquid into each dilution centrifuge tube, shaking and uniformly mixing, and then incubating for 30min at 37 ℃; from each dilution centrifuge tube, 5. Mu.L was added dropwise to 2 XYT solid medium (Amp), and after standing for several minutes, the mixture was placed in 37℃and incubated overnight upside down; the number of single colonies at the dilution of single colonies can be clearly distinguished on the statistical plate and the number of phagemids per ml of phage solution, i.e. phage library titers, is calculated according to the following formula:

T(pfu/mL)＝N×D×400

wherein T is phage titer (pfu/mL), D is dilution factor, and N is single colony number of corresponding dilution factor.

3) Third round of screening phage eluate

Repeating the above experiment for 3 times, and taking the phage of the first round as the input phage library for the second round to obtain a phage eluent for the second round, and taking the phage of the second round as the phage library for the third round to obtain a phage eluent for the third round.

(4) Monoclonal ELISA detection

1) The bacterial liquid with proper dilution after the third round of screening is evenly coated on a solid culture medium plate containing 100 mug/mLAMP, and the solid culture medium plate is placed at 37 ℃ for overnight culture.

2) 192 monoclonal colonies were randomly picked from the overnight culture medium plates and placed in sterile 96 well cell culture plates, 200. Mu.L of 2 XYT medium (containing 100. Mu.g/mLAMP) was added to each well, and the plates were allowed to stand overnight at 37 ℃.

3) mu.L of the overnight cultured bacterial liquid was transferred to a new 96-well cell culture plate containing 200. Mu.L of 2 XYT liquid medium (containing 100. Mu.g/mLAmp) per well, and the cell culture plate was allowed to stand at 37℃for 5 hours.

4) Helper phage M13K07 was added per well, where bacterial count: phage number 1:20.

5) After incubation at 37℃for 30min, kana was added at a final concentration of 50. Mu.g/mL, and the mixture was allowed to stand at 30℃overnight for incubation, and then 96-well cell culture plates were centrifuged and stored at 4℃for further use.

6) Screening antigen coated ELISA plates (1 ng/. Mu.L, PBS, 100. Mu.L/well) were coated in parallel with BSA of the same concentration as a control, and placed at 4deg.C overnight; discarding the supernatant, and washing the ELISA plate with PBS buffer solution for 3 times at room temperature for 10min each time; adding 200 mu L of blocking solution (3% BSA in PBST) to each well to block the ELISA plate, and blocking for 1h at room temperature; the blocking solution was discarded, 200. Mu.LPBST (1 XPBS plus 0.1% Tween20, the same applies below) buffer was added to each well, and the ELISA plates were washed 3 times at room temperature for 10min each time.

7) After adding 100. Mu.L of blocking solution per well, 100. Mu.L of the supernatant after centrifugation in step 5) was added and incubated at room temperature for 2 hours.

8) The liquid in the ELISA plate was discarded, and 200. Mu.L of PBST buffer was added to each well and washed 3 times for 10min each.

9) M13 BacteriophageAntibody (HRP), mouse Mab, was added to each well, diluted 1:30000 in blocking solution, 100. Mu.L/well, and incubated for 1h at room temperature.

10 Liquid in ELISA plate was discarded, and 200. Mu.LPBST buffer was added to wash 6 times per well for 5min.

11 100 mu LTMB single-component color development liquid is added into each hole, color development is carried out for 1-3min in a dark place, then 100 mu L of 1M HCl is added into each hole to terminate the reaction, and an OD450 value is read by an enzyme-labeling instrument, recorded and stored.

Example 2 construction of CD 22-targeting chimeric antigen receptor and immune cell expression

(1) Construction of CD22 CAR

First, each set of CD 22-targeting CAR nucleotide sequences (SEQ ID Nos. 161-178) was designed and artificially synthesized, each set comprising the coding nucleotide sequence of the HLA-A signal peptide (SEQ ID No. 138) or CD8 a signal peptide, the extracellular antigen binding domain of CD22 VHH (SEQ ID Nos. 120-136) or CD22 scFv (m 971 positive control, SEQ ID No. 137), the CD8 a hinge region (SEQ ID No. 139), the CD8 a transmembrane domain (SEQ ID No. 140), the CD137 (4-1 BB) co-stimulatory signal domain (SEQ ID No. 141) and the CD3 zeta intracellular signal transduction domain (SEQ ID No. 142) for expressing the complete CD22 CAR polypeptide molecule (SEQ ID Nos. 143-160). The nucleotide sequence of the CD22 CAR is inserted into the multiple cloning site of the lentiviral expression vector pK1 through homologous recombination to obtain the pK1-CD22 CAR, and the successful construction of the lentiviral expression vector sequence is verified through electrophoresis and sequencing results.

(2) Packaging of lentiviral vectors

Resuscitate 293T cells and culture in DMEM medium containing 10% FBS; after 2-3 generation cell expansion culture, the method is carried out according to 4 multiplied by 10 ⁴ Individual/cm ² Is seeded into a 2-layer cell factory; plasmid transfection was performed 3 days after cell inoculation; plasmid transfection 40ml Optim-MEM was added to a sterile 50ml centrifuge tube and the tube was sterilized according to pK1-CD22 CAR: pLP1: pLP2: pLP-vsvg=5: 4:3:1, adding a virus packaging vector and a virus envelope vector in proportion, then adding 800 mu LPEI transfection reagent, immediately mixing, incubating for 15min at room temperature, and then adding the plasmid/vector/transfection reagent compound dropwise into a culture flask of 293T cells; collecting the virus supernatant after 24 hours into a 50ml centrifuge tube, centrifuging 250g for 5 minutes, filtering the supernatant after centrifugation by a 0.45 mu m filter, and ultracentrifugating the filtered supernatant (25000 g,4 ℃ C., 3 hours) to obtain concentrated CD22 CAR lentivirus; centrifuging, discarding supernatant, re-suspending lentivirus with PBS pre-cooled at 4deg.C, packaging the re-suspended CD22 CAR lentivirus liquid, and storing at-80deg.C.

(3) Resuscitation and activation of T cells

Setting the temperature of the water bath kettle to 38 ℃, and preheating the culture medium in advance; taking out the freezing bag from the liquid nitrogen tank, immediately immersing the bag in a water bath, and taking out the freezing bag when the navel blood to be frozen is completely melted in a transparent state; wiping water stains outside the freezing bag by using a dry cotton ball, spraying and sterilizing by using 75% alcohol, and transferring to a biosafety cabinet after the alcohol is volatilized completely; taking out umbilical blood, placing the umbilical blood into a 50mL centrifuge tube, adding a proper amount of RPMI 1640 culture medium, uniformly mixing, and sampling and counting; centrifuging at 300g for 5min, collecting lower layer cells after centrifuging, and re-suspending with complete medium to T cell density of 1×10 ⁶ Adding activated antibodies Anti-h [ mu ] man CD3 Anti-ibody and Anti-h [ mu ] man CD28 Anti-ibody according to the re-suspension volume, wherein the concentration of CD3 is 0.15 [ mu ] g/mL, the concentration of CD28 is 0.625 [ mu ] g/mL, and culturing in a 5% carbon dioxide incubator at 37 ℃; culturing for 4hr, adding complete culture medium, and adjusting to T cell density of 4×10 ⁵ Culture was continued at a concentration of each mL.

(4) Sorting and purification of T cells

After 36hr of cell activation, mixing and sampling 20 μl, adding diluted antibody 10 μl, staining for 10min, diluting 10 times with PBS, detecting and counting by flow cytometry, recording cell density of CD3+, CD4+, CD8+ T, and observing CD69 and CD25 molecule expression; recording the cell volume and confirming the cell quantity; transferring the cell suspension to a centrifuge tube for centrifugation at 300g for 5min, and discarding the supernatant to collect the lower cells; adding MACS Buffer for washing, centrifuging again, collecting lower cells, centrifuging under the same conditions, and re-suspending cells with appropriate amount of MACS Buffer; the amount of the magnetic beads was calculated from the amount of cells per 1X 10 ⁶ mu.L of CD4+ magnetic beads were added to each 1X 10 CD4+ T cell ⁶ mu.L of CD8+ magnetic beads were added to the CD8+ T cells; adding magnetic beads, uniformly mixing, incubating for 20min at room temperature in a dark place, adding MACS Buffer for washing after incubation is completed, centrifuging 300g for 5min, discarding supernatant, and re-suspending by using a proper amount of MACS Buffer; placing the LS sorting column on a MACS magnetic sorting frame, washing the column with 1mLBuffer, passing the cell suspension through the column after washing, and continuously adding Buffer 9mL through the column; the LS column was removed from the MACS magnet and 5mL of buffer was added to wash the cells trapped on the LS column Discharging; and finally, uniformly mixing the cell suspension, sampling, dyeing and counting, recording the densities of CD3+, CD4+ and CD8+ T cells, and calculating the sorting recovery rate and purity.

(5) Preparation of CD22 CAR-T cells

Lentivirus transduced T cells: cell density was adjusted to about 400 cells/μl, plated at 500 μl per well volume, and each group of CD22 CAR slow virus liquid was added at moi=25 according to the number of actual T cells, negative control group (UnT) was T cells without lentivirus transduction, and the expression rate of CD22 CAR polypeptide molecules on T cells (group m971, S1, S4, S9, S27, S28, S35, S36, S41, S43) was detected after 3 days of culture in a 5% carbon dioxide incubator at 37 ℃, and the results are shown in fig. 1A and 1B.

CD22 CAR-T cell expansion was observed every 3 days and fresh culture medium was supplemented, and harvested after 11 days of continuous culture for subsequent in vitro killing experiments.

Example 3 tumor cell killing Effect validation of CD22 CAR-T cells

(1) Determination of expression of CD22 antigen on target cell surface

The expression of these cell surface CD22 antigens was examined by flow cytometry using Raji cells (from ATCC, CCL-86), namalwa cells (from ATCC, CRL-1432) and K562 cells (from ATCC, CRL-3344) as target cells, respectively, as shown in FIG. 2. The result shows that Raji cells are highly expressed in CD22, namalwa cells are moderately expressed in CD22, K562 cells are CD22 negative cells, and Raji cells and Namalwa cells are finally selected as target cells for in vitro killing experiments.

(2) Determination of in vitro killing effect of CD22 CAR-T cells

Each set of CAR-T cells (m 971, S1, S4, S9, S27, S28, S35, S36, S41, S43, 2X 10) was added to a 24-well plate ⁵ Each group of CD4+ and CD8+ T cells in the same ratio), raji cells with corresponding cell quantity are added according to the effective target ratio (E: T) =1:1 or 1:3, the negative control group (UnT) is also added with the same quantity of T cells and target cells according to the corresponding effective target ratio, and the culture medium is added to 500 mu L/hole, and the mixture is placed in a 5% CO2 incubator at 37 ℃ for culture; after 18hr and 72hr of co-culture, the Raji cell amount in each well was measured by flow cytometryThe killing rate of D1 (18 hr) and the killing rate of D3 (72 hr) were calculated as killing rate=target cell reduction/target cell plating cell amount×100%, and the results are shown in fig. 3A, 3B, 4A and 4B.

Each set of CAR-T cells (m 971, S1, S4, S9, S27, S28, S35, S36, S41, S43, 2X 10) was added to a 24-well plate ⁵ Each group of CD4+ and CD8+ T cells in the same proportion), namalwa cells with corresponding cell quantity are added according to the effective target ratio (E: T) =1:3, the negative control group (UnT) is also added with the same quantity of T cells and target cells according to the corresponding effective target ratio, and the culture medium is added to 500 mu L/hole, and the mixture is placed in a 5% CO2 incubator at 37 ℃ for culture; the Namalwa cell amount in each well was measured by flow cytometry at 18hr and 72hr after plating, and D1 (18 hr) killing rate and D3 (72 hr) killing rate were calculated as killing rate=target cell reduction/target cell plated cell amount×100%, and the results are shown in FIGS. 5A and 5B.

(3) Cytokine release assay for CD22 CAR-T cells

The supernatants of each group of CAR-T cells (group UnT, m971, S1, S4, S9, S27) and Namalwa cell co-culture broth were collected 18hr after plating of the in vitro killing experiments, and were assayed by CBA assay (CBA assay kit: LEGENDplex) ^TM Human CD8/NK Panel(13-plex)with V-bottom Plate,Biolegend,Cat.No.741065；LEGENDplex ^TM The release of each group of cytokines Granzyme B, TNF- α, IFN- γ, IL-2 was examined by Human macrogel/Microglia Panel (13-plex) with V-bottom Plate, biolegend, cat. No.740503, and the results are shown in FIGS. 6A, 6B, 6C and 6D.

The specific operation steps are as follows:

collecting culture solution supernatant: cell culture supernatant, 50. Mu.L, 18hr after plating of the killing experiment was collected and used for subsequent detection of cytokine secretion in the supernatant.

Preparing loads: after the desired Beads were returned to room temperature, the Beads were vortexed for 2min to thoroughly mix the Beads, and the amount of Beads required was calculated based on the sample volumes, each sample added at a volume of 15 μl.

Wash Buffer preparation: the 20 xWash Buffer is restored to room temperature, so that the salt in the Buffer is fully dissolved, and the Buffer is prepared into 1xWash Buffer for standby by using up water.

Standard preparation: dissolving the standard substance with 250 mu LAssay Buffer, reversing for multiple times to fully and uniformly mix the standard substance, standing for 10min at room temperature, and then transferring the standard substance into an EP tube; 25 μl of standard was removed from the EP tube and labeled C7; 7 EP tubes, labeled C6/C5/C4/C3/C2/C1/C0, were taken, 22.5. Mu.LAssay Buffer was added to each tube, 7.5. Mu.L of standard was removed from C7 and serially diluted in a 4-fold ratio, and C0 was Assay Buffer (0 pg/ml) until dilution was reached C1.

Standard well and sample well preparation: preparing a standard curve hole of cell supernatant, adding 15 mu L of each of an assay buffer, a standard substance and a Beads into an EP tube, and uniformly mixing; sample wells were prepared by adding 15. Mu.L each of Assay Buffer, sample and Beads to the EP tube and mixing well.

Capture binding of the Beads to the analyte of interest: the standard wells and sample wells were incubated at 500rpm with shaking for 2hr in the dark.

Washing: centrifuging the sample at 2000g for 5min, discarding the supernatant, and allowing the Beads to be visible at the bottom; 200. Mu.L of 1xWash buffer was added to each EP tube, after brief vortexing, 2000g was centrifuged for 5min and the supernatant discarded.

Capture of the binding of the Beads, analyte of interest to biotinylated detection antibody, SA-PE: adding 15 μl of detection antibody into each EP tube, blowing, mixing, shaking at 500rpm, and incubating for 1hr under dark condition; add 15. Mu.L of streptavidin-phycoerythrin (SA-PE) to each tube, shake at 500rpm, incubate for 0.5hr in the dark.

And (3) secondary washing: 200. Mu.L of 1xWash buffer was added to each EP tube, after brief vortexing, 2000g was centrifuged for 5min and the supernatant discarded.

And (3) detection: 200 μL of 1xWash buffer was added to each tube, and after vortexing, flow cytometry was performed to check for FSC, SSC, APC, PE channels.

Detection result: the files detected by the flow cytometry are exported to FSC format for analysis, and LEGENDplex is used ^TM The data analysis software determines the concentration of the target cytokine according to a known standard curve.

Claims

1. A single domain antibody targeting CD22, wherein the single domain antibody comprises CDR1, CDR2, and CDR3;

wherein said CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein said CDR2 comprises the amino acid sequence shown as SEQ ID NO. 20, wherein said CDR3 comprises the amino acid sequence shown as SEQ ID NO. 35;

wherein said CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein said CDR2 comprises the amino acid sequence shown as SEQ ID NO. 20, wherein said CDR3 comprises the amino acid sequence shown as SEQ ID NO. 36;

wherein said CDR1 comprises an amino acid sequence as shown in SEQ ID NO. 3, wherein said CDR2 comprises an amino acid sequence as shown in SEQ ID NO. 20, wherein said CDR3 comprises an amino acid sequence as shown in SEQ ID NO. 37;

wherein said CDR1 comprises the amino acid sequence shown as SEQ ID NO. 7, wherein said CDR2 comprises the amino acid sequence shown as SEQ ID NO. 22, wherein said CDR3 comprises the amino acid sequence shown as SEQ ID NO. 46;

wherein said CDR1 comprises the amino acid sequence shown as SEQ ID NO. 8, wherein said CDR2 comprises the amino acid sequence shown as SEQ ID NO. 23, wherein said CDR3 comprises the amino acid sequence shown as SEQ ID NO. 47;

Wherein said CDR1 comprises the amino acid sequence shown as SEQ ID NO. 13, wherein said CDR2 comprises the amino acid sequence shown as SEQ ID NO. 29, wherein said CDR3 comprises the amino acid sequence shown as SEQ ID NO. 53;

wherein said CDR1 comprises the amino acid sequence shown as SEQ ID NO. 14, wherein said CDR2 comprises the amino acid sequence shown as SEQ ID NO. 30, wherein said CDR3 comprises the amino acid sequence shown as SEQ ID NO. 54;

wherein said CDR1 comprises the amino acid sequence shown as SEQ ID NO. 15, wherein said CDR2 comprises the amino acid sequence shown as SEQ ID NO. 31, wherein said CDR3 comprises the amino acid sequence shown as SEQ ID NO. 57; or (b)

Wherein said CDR1 comprises the amino acid sequence shown as SEQ ID NO. 16, wherein said CDR2 comprises the amino acid sequence shown as SEQ ID NO. 31, wherein said CDR3 comprises the amino acid sequence shown as SEQ ID NO. 59.

2. The single domain antibody of claim 1, wherein the single domain antibody comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth 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. The single domain antibody of claim 1, wherein the single domain antibody comprises an amino acid sequence as set forth 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.

4. A chimeric antigen receptor comprising:

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen binding domain comprises the CD 22-targeting single domain antibody of any one of claims 1-3.

5. The chimeric antigen receptor according to claim 4, wherein the transmembrane domain is derived from CD8 a, CD4, CD28, CD137, CD80, CD86, CD152 or PD-1.

6. The chimeric antigen receptor according to claim 5, wherein the transmembrane domain is derived from CD8 a comprising the amino acid sequence shown in SEQ ID No. 140.

7. The chimeric antigen receptor according to claim 4, wherein the intracellular signaling domain is derived from cd3ζ, cd3γ, cd3δ, cd3ε, CD22, CD79a, CD79b, CD66d, fcrγ, fcrβ.

8. The chimeric antigen receptor according to claim 7, wherein the intracellular signaling domain is derived from cd3ζ comprising the amino acid sequence shown in SEQ ID No. 142.

9. The chimeric antigen receptor according to claim 4, wherein the intracellular signaling domain further comprises a costimulatory signaling domain, wherein the costimulatory signaling domain is derived from CD137 (4-1 BB), CD27, CD28, ICOS, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand, and combinations thereof.

10. The chimeric antigen receptor according to claim 9, wherein the costimulatory signaling domain is derived from CD137 (4-1 BB), which comprises the amino acid sequence depicted as SEQ ID No. 141.

11. The chimeric antigen receptor according to claim 4, further comprising a hinge region 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 CD8 a, CD28, igG1 or IgG4.

12. The chimeric antigen receptor according to claim 11, wherein the hinge region is derived from CD8 a comprising the amino acid sequence shown in SEQ ID No. 139.

13. The chimeric antigen receptor according to claim 4, further comprising a signal peptide at the N-terminus of the chimeric antigen receptor polypeptide, wherein the signal peptide is derived from HLA-A, CD8 a, CD33, igκ, IL-2, GM-csfra.

14. The chimeric antigen receptor according to claim 13, wherein the signal peptide is derived from HLA-A comprising the amino acid sequence shown in SEQ ID No. 138.

15. A chimeric antigen receptor comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% similarity to the amino acid sequence set forth in any one of SEQ ID No. 143, 144, 145, 147, 148, 152, 153, 155, 156.

16. A chimeric antigen receptor, characterized by comprising 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, and SEQ ID NO: 156.

17. An isolated nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor of any one of claims 4-16.

18. A vector comprising the nucleic acid of claim 17.

19. An engineered immune effector cell comprising the chimeric antigen receptor of any one of claims 4-16, the isolated nucleic acid of claim 17, or the vector of claim 18.

20. The engineered immune effector cell of claim 19, wherein the immune effector cell is selected from the group consisting of T cells, B cells, NK cells, macrophages, dendritic cells, induced pluripotent stem cells.

21. A pharmaceutical composition comprising the CD 22-targeting single domain antibody of any one of claims 1-3, the engineered immune effector cell of claim 19 or 20, and a pharmaceutically acceptable carrier or excipient.

22. Use of a CD 22-targeting single domain antibody according to any one of claims 1-3, an engineered immune effector cell according to claim 19 or 20, or a pharmaceutical composition according to claim 21 for the preparation of a medicament for the treatment of a B-cell related malignancy, a B-cell related autoimmune disease and/or an inflammatory disease.