Single-domain antibody targeting CD79b, chimeric antigen receptor, and use thereof

Abstract

A single-domain antibody targeting CD79B, a chimeric antigen receptor, and a use thereof. Provided are an anti-CD79B single-domain antibody, a chimeric antigen receptor constructed using the single-domain antibody, and an engineered immune effector cell. Also provided is a use of the single-domain antibody, the chimeric antigen receptor, and the engineered immune effector cell in the preparation of a drug for the treatment of CD79B and / or B cell-related diseases.

Description

A single-domain antibody and chimeric antigen receptor targeting CD79B and their applications Technical Field

[0001] The present invention relates to the field of immunotherapy technology, and particularly to a single-domain antibody and chimeric antigen receptor targeting CD79B and applications thereof. Background Art

[0002] CD79B is part of the B cell receptor (BCR) signaling complex and is a key cell surface receptor for the successful development and maintenance of mature B cells. CD79B expression is generally restricted to B cell lineages and remains highly expressed in most non-Hodgkin lymphoma subtypes, making it one of the ideal tumor antigens for immunotherapy targeting B cell-related tumors.

[0003] Unlike traditional four-chain antibodies, single-domain antibodies (sdAbs) possess a single single-chain antibody variable domain. For example, camelids and sharks naturally produce antibodies lacking light chains, known as heavy-chain-only antibodies (hcAbs, or simply heavy-chain antibodies). The antigen-binding fragment in each arm of a camelid heavy-chain-only antibody contains a single heavy-chain variable domain (VHH), which possesses a high affinity for antigen without the aid of light chains. Camelid VHH antibodies are considered the smallest functional antigen-binding fragment, with a molecular weight of only approximately 15 kDa, hence the term nanobody. VHH antibodies possess the inherent advantages of good solubility, high stability, strong penetrating power, and broad epitope binding. Since their discovery, VHH antibodies have gradually attracted the attention of researchers in the field, and basic research on them has matured. In terms of application, VHH antibodies have gradually entered the clinical research stage for targets such as autoimmune diseases, blood disorders, viral infections, and orthopedic diseases. They have also demonstrated significant advantages in combating infections, inflammatory diseases, and neurodegenerative diseases.

[0004] Chimeric Antigen Receptor (CAR)-modified T cells (CAR-T cells) have gained widespread attention and application as an immunotherapy strategy in cancer treatment, particularly for hematologic malignancies. The principle behind the development of CAR-T cells is to genetically modify T cells to express a CAR structure that specifically recognizes tumor cell surface antigens. After the CAR specifically binds to the tumor cell surface antigen, it activates downstream immune co-stimulatory factors and T cells, thereby activating the T cells to secrete relevant cytokines, which specifically kill tumor cells. The CAR structure generally consists of four components: an extracellular antigen-binding region (often an antibody with antigen recognition function, such as a single-chain antibody), a hinge region, a transmembrane region, and an intracellular signaling domain. Currently, CAR structures are generally classified into first-generation (no co-stimulatory molecules), second-generation (containing one co-stimulatory molecule), and third-generation (containing two co-stimulatory molecules), based on the number of co-stimulatory molecules incorporated into the intracellular signaling domain. Second-generation CAR structures are currently the most widely used in marketed products and clinical trials.

[0005] Since CAR-T cell manufacturing technology requires the use of single-chain antibodies with good binding activity and efficient epitope binding, one of the key technical components of CAR-T cell therapy is the screening of high-affinity antibodies with good specificity, strong binding force, and efficient epitope binding. However, traditional anti-CD79B antibodies, such as monoclonal anti-CD79B antibodies or anti-CD79B single-chain variable region fragments (scFv), are limited by disadvantages such as large molecular weight, weak binding force with antigens, low affinity, high immunogenicity, difficulty in modification, and poor stability, and further modification is needed.

[0006] Therefore, there is still a wide demand for the development of improved anti-CD79B single-domain antibodies, CD79B-targeting CAR structures constructed based thereon, and engineered immune effector cells expressing the CAR structures. For example, the development of stable, high-affinity, small, and less immunogenic anti-CD79B single-domain antibodies for more effective and efficient CAR-T cell therapy.

[0007] Summary of the Invention

[0008] In view of this, in order to at least solve one of the above technical problems, the present invention provides an anti-CD79B single-domain antibody in one aspect, wherein the anti-CD79B single-domain antibody comprises a CDR-H1 region, a CDR-H2 region and a CDR-H3 region; the CDR-H1 region comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to the amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 24 or SEQ ID NO: 29; the CDR-H2 region comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to the amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 13 or SEQ ID NO:20; and the CDR-H3 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:26 or SEQ ID NO:31.

[0009] In some embodiments of the present invention, the anti-CD79B single-domain antibody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region;

[0010] wherein the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO: 3, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO: 5, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO: 7; or

[0011] wherein the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO: 11, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO: 13, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO: 15; or

[0012] wherein the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO: 19, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO: 20, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO: 22; or

[0013] wherein the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO: 24, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO: 20, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO: 26; or

[0014] Wherein, the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO: 29, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO: 20, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO: 31.

[0015] In some embodiments of the present invention, the anti-CD79B single domain antibody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the CDR-H1 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 3; the CDR-H2 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 5; the CDR-H3 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: The amino acid sequence shown in NO:7 has an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical.

[0016] In some embodiments of the present invention, the anti-CD79B single domain antibody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the CDR-H1 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 11; the CDR-H2 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; the CDR-H3 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: The amino acid sequence shown in NO:15 has an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical.

[0017] In some embodiments of the present invention, the anti-CD79B single domain antibody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the CDR-H1 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 19; the CDR-H2 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 20; the CDR-H3 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: The amino acid sequence shown in NO:22 has an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical.

[0018] In some embodiments of the present invention, the anti-CD79B single domain antibody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the CDR-H1 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 24; the CDR-H2 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 20; the CDR-H3 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: The amino acid sequence shown in NO:26 has an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical.

[0019] In some embodiments of the present invention, the anti-CD79B single domain antibody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the CDR-H1 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 29, the CDR-H2 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: 20, and the CDR-H3 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: The amino acid sequence shown in NO:31 has an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical.

[0020] In some embodiments of the present invention, the anti-CD79B single-domain antibody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; or

[0021] wherein the amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 3, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 5, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 7; or

[0022] wherein the amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 11, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 13, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 15; or

[0023] wherein the amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 19, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 20, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 22; or

[0024] wherein the amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 24, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 20, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 26; or

[0025] The amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 29, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 20, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 31.

[0026] In some embodiments of the present invention, the anti-CD79B single-domain antibody comprises CDR1, CDR2 and CDR3 regions; wherein the CDR-H1 region, CDR-H2 region and CDR-H3 region are determined according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme or the Contact numbering scheme.

[0027] In some embodiments of the present invention, the CDR-H1 region, CDR-H2 region and CDR-H3 region are determined according to the Martin numbering scheme.

[0028] In some embodiments of the present invention, the anti-CD79B single-domain antibody further comprises a FR1 region, a FR2 region, a FR3 region and a FR4 region; the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18 or SEQ ID NO: 28; the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 30; the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 16.

[0029] In some embodiments of the present invention, the anti-CD79B single-domain antibody further comprises a FR1 region, a FR2 region, a FR3 region and a FR4 region; the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 2, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 4, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 6, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 8.

[0030] In some embodiments of the present invention, the anti-CD79B single-domain antibody further comprises a FR1 region, a FR2 region, a FR3 region and a FR4 region; the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 10, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 14, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16.

[0031] In some embodiments of the present invention, the anti-CD79B single-domain antibody further comprises a FR1 region, a FR2 region, a FR3 region, and a FR4 region;

[0032] wherein the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 2, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 4, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 6, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 8; or

[0033] wherein the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 10, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 14, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16; or

[0034] wherein the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 18, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 21, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16; or

[0035] wherein the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 10, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 25, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16; or

[0036] Wherein, the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 28, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 30, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16.

[0037] In some embodiments of the present invention, the anti-CD79B single-domain antibody further comprises a FR1 region, a FR2 region, a FR3 region and a FR4 region; the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 18, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 21, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16.

[0038] In some embodiments of the present invention, the anti-CD79B single-domain antibody further comprises a FR1 region, a FR2 region, a FR3 region and a FR4 region; the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 10, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 25, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16.

[0039] In some embodiments of the present invention, the anti-CD79B single-domain antibody further comprises a FR1 region, a FR2 region, a FR3 region and a FR4 region; the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 28, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 30, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16.

[0040] In some embodiments of the present invention, the anti-CD79B single domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO: 27, or an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO: 27.

[0041] In some embodiments of the present invention, the amino acid sequence of the anti-CD79B single-domain antibody is shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO: 27.

[0042] In some embodiments of the present invention, the anti-CD79B single-domain antibody can specifically bind to human CD79B protein.

[0043] In some embodiments of the present invention, the amino acid sequence of the anti-CD79B single domain antibody and the amino acid sequences of its CDR-H1 region, CDR-H2 region, and CDR-H3 region are shown in Table 1 below.

[0044] Table 1

[0045] In another aspect, the present invention discloses an isolated nucleic acid encoding any of the aforementioned anti-CD79B single domain antibodies.

[0046] In another aspect, the present invention discloses a chimeric antigen receptor (CAR), comprising:

[0047] (a) Extracellular antigen-binding region;

[0048] (b) a transmembrane region; and

[0049] (c) intracellular signal transduction domain;

[0050] Wherein, the extracellular antigen binding region comprises any one of the aforementioned anti-CD79B single-domain antibodies.

[0051] In some embodiments of the present invention, the transmembrane region of the CAR is derived from the transmembrane region of at least one of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcεRIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D and CS1;

[0052] Preferably, the transmembrane region of the CAR is derived from the transmembrane region of CD8α.

[0053] In some embodiments of the present invention, the transmembrane region of the CAR is derived from the transmembrane region of CD8α.

[0054] In some embodiments of the present invention, the amino acid sequence of the transmembrane region of CD8α is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:34.

[0055] In some embodiments of the present invention, the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of at least one of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79B, FcεRIγ, FcεRβ, FcγRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP12 and other intracellular signaling domains containing at least one ITAM protein intracellular signaling domain;

[0056] Preferably, the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of CD3ζ.

[0057] In some embodiments of the present invention, the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of CD3ζ.

[0058] In some embodiments of the present invention, the amino acid sequence of the intracellular signaling domain of CD3ζ is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:36.

[0059] In some embodiments of the present invention, the chimeric antigen receptor further comprises a hinge region, wherein the hinge region connects the extracellular antigen binding region and the transmembrane region;

[0060] Preferably, the hinge region is derived from the hinge region of at least one of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154;

[0061] More preferably, the hinge region is derived from the hinge region of CD8α.

[0062] In some embodiments of the present invention, the hinge region of the CAR connects the extracellular antigen binding region and the transmembrane region, and the hinge region is located between the C-terminus of the extracellular antigen binding region and the N-terminus of the transmembrane region.

[0063] In some embodiments of the present invention, the hinge region of the CAR is derived from the hinge region of CD8α.

[0064] In some embodiments of the present invention, the amino acid sequence of the hinge region of CD8α is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:33.

[0065] In some embodiments of the present invention, the chimeric antigen receptor further comprises a co-stimulatory signaling domain;

[0066] Preferably, the costimulatory signaling domain comprises a costimulatory signaling domain of at least one of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, and MyD88;

[0067] More preferably, the costimulatory signaling domain comprises the costimulatory signaling domain of 4-1BB.

[0068] In some embodiments of the present invention, the costimulatory signaling domain of the CAR is the costimulatory signaling domain of 4-1BB.

[0069] In some embodiments of the present invention, the amino acid sequence of the costimulatory signaling domain of 4-1BB is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.5% or 100% identical to SEQ ID NO: 35.

[0070] In some embodiments of the present invention, the chimeric antigen receptor further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor;

[0071] Preferably, the signal peptide is derived from the signal peptide of at least one of the following proteins: HLA-A, CD8α, CD33, Igκ, IL-2 and GM-CSFRα;

[0072] More preferably, the signal peptide is derived from the signal peptide of CD8α.

[0073] In some embodiments of the present invention, the signal peptide is derived from the signal peptide of CD8α.

[0074] In some embodiments of the present invention, the amino acid sequence of the CD8α signal peptide is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:32.

[0075] In some embodiments of the present invention, the CAR can specifically bind to human CD79B protein.

[0076] In another aspect, the present invention discloses an isolated nucleic acid encoding any of the aforementioned chimeric antigen receptors.

[0077] In some embodiments of the present invention, the nucleic acid further comprises a nucleic acid encoding a signal peptide, and the signal peptide is located at the N-terminus of the chimeric antigen receptor.

[0078] In some embodiments of the present invention, the signal peptide is derived from the signal peptide of CD8α.

[0079] In another aspect, the present invention discloses a vector comprising an isolated nucleic acid encoding any one of the aforementioned chimeric antigen receptors.

[0080] In some embodiments of the present invention, the vector is selected from at least one of lipid nanoparticles, virus-like particles, adenovirus, adeno-associated virus, lentiviral vectors, and retroviral vectors;

[0081] Preferably, the vector is a lentiviral vector or a retroviral vector.

[0082] In some embodiments of the present invention, the vector is a lentiviral vector or a retroviral vector.

[0083] In another aspect, the present invention discloses a targeting vector, the surface of which comprises a targeting molecule, wherein the targeting molecule comprises any one of the aforementioned anti-CD79B single-domain antibodies disclosed in the present invention.

[0084] In some embodiments of the present invention, the targeting vector is a lentiviral vector or a retroviral vector, the targeting molecule further comprises a transmembrane region, and the anti-CD79B single-domain antibody is directly or indirectly connected to the transmembrane region and exposed on the surface of the lentiviral vector or retroviral vector;

[0085] Preferably, the transmembrane region is derived from the transmembrane region of any of the following proteins:

[0086] CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcεRIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1;

[0087] More preferably, the transmembrane region is derived from the transmembrane region of CD8α.

[0088] In some embodiments of the present invention, the transmembrane region of the targeting molecule is the transmembrane region of CD8α; preferably, the amino acid sequence of the transmembrane region of CD8α is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:34.

[0089] In some embodiments of the present invention, the targeting molecule further comprises a linker domain, and the anti-CD79B single domain antibody is indirectly connected to the transmembrane region of the targeting molecule via the linker domain;

[0090] Preferably, the linker domain is selected from:

[0091] (i) an immunoglobulin hinge region selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0092] (ii) a hinge region selected from the wild-type or modified hinge region of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0093] (iii) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and

[0094] (iv) a stem region of a type II C-lectin, wherein the type II C-lectin is selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0095] More preferably, the connecting domain is the hinge region of CD8α.

[0096] In some embodiments of the present invention, the connecting domain of the targeting molecule is the hinge region of CD8α;

[0097] Preferably, the amino acid sequence of the hinge region of CD8α is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:33.

[0098] In some embodiments of the present invention, the viral envelope of the lentiviral vector or retroviral vector comprises the targeting molecule.

[0099] In another aspect, the present invention discloses an engineered effector cell, comprising any one of the aforementioned chimeric antigen receptors or a nucleic acid encoding any one of the aforementioned chimeric antigen receptors;

[0100] Preferably, the engineered immune effector cells are selected from at least one of T cells, B cells, NK cells, macrophages, dendritic cells and induced pluripotent stem cells;

[0101] More preferably, the engineered immune effector cells are engineered T cells.

[0102] In some embodiments of the present invention, the engineered immune effector cells are engineered T cells that express the chimeric antigen receptor (CAR-T cells).

[0103] In some embodiments of the present invention, the CAR-T cells are prepared in vivo or in vitro in a subject;

[0104] When the CAR-T cells are prepared outside the body of a subject, the subject is an individual to whom the CAR-T cells are administered;

[0105] When the CAR-T cells are prepared in a subject, the subject is an individual who is administered a vector comprising a nucleic acid encoding the CAR.

[0106] In another aspect, the present invention discloses a composition comprising a pharmaceutically acceptable excipient or carrier and any one of the following components: any one of the aforementioned (a) anti-CD79B single domain antibodies, (b) chimeric antigen receptors, (c) engineered immune effector cells, (d) nucleic acids encoding any one of the aforementioned anti-CD79B single domain antibodies, or (e) nucleic acids encoding any one of the aforementioned CARs.

[0107] In some embodiments of the present invention, the composition comprises a pharmaceutically acceptable excipient or carrier and any one of the following ingredients: any one of the aforementioned anti-CD79B single-domain antibodies, a chimeric antigen receptor, a vector comprising a nucleic acid encoding CAR, and an engineered immune effector cell.

[0108] In another aspect, the present invention discloses a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of any one of the aforementioned anti-CD79B single-domain antibodies, vectors comprising a nucleic acid encoding a CAR, engineered immune effector cells, or compositions;

[0109] Preferably, the disease is a B cell-related disease and / or a CD79B-related disease;

[0110] More preferably, the disease is a B cell-related and / or CD79B-related malignancy;

[0111] Still further preferably, the B cell-related malignancy is a B cell leukemia or a B cell lymphoma, and the B cell leukemia or the B cell lymphoma is selected from marginal zone lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B cell lymphoma, small lymphocytic lymphoma, B cell prolymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B lymphocytic leukemia, non-Hodgkin's lymphoma, high grade B cell lymphoma and multiple myeloma.

[0112] In some embodiments of the present invention, the disease is a B cell-related and / or CD79B-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 number and / or activation.

[0113] In another aspect, the present invention discloses the use of any of the aforementioned anti-CD79B single-domain antibodies, isolated nucleic acids encoding the anti-CD79B single-domain antibodies, chimeric antigen receptors, isolated nucleic acids encoding the CARs, vectors, targeting vectors, engineered immune effector cells or compositions in the preparation of drugs for treating B cell-related and / or CD79B-related malignancies, B cell-related and / or CD79B-related autoimmune diseases and / or inflammatory diseases;

[0114] Preferably, the B cell-related malignancy is a B cell leukemia or a B cell lymphoma, and the B cell leukemia or B cell lymphoma is selected from marginal zone lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B cell lymphoma, small lymphocytic lymphoma, B cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B lymphocytic leukemia, non-Hodgkin's lymphoma, high grade B cell lymphoma and multiple myeloma.

[0115] In another aspect, the present invention discloses a reagent for detecting CD79B protein or a functional domain comprising its antigenic determinants (Epitopes), wherein the reagent comprises any of the aforementioned anti-CD79B single domain antibodies;

[0116] Preferably, the CD79B protein is human CD79B protein;

[0117] More preferably, the reagent further comprises a modification portion linked to the anti-CD79B single domain antibody, wherein the modification portion comprises a detectable label or a therapeutic agent;

[0118] More preferably, the detectable label comprises at least one of an enzyme, a tag protein, a radionuclide, a fluorescent dye, a luminescent substance and biotin.

[0119] In another aspect, the present invention discloses a method for detecting CD79B protein or a functional domain comprising its antigenic determinant for non-diagnostic purposes, the method comprising obtaining a sample suspected of containing CD79B protein or a functional domain comprising its antigenic determinant, contacting the sample with any of the aforementioned anti-CD79B single domain antibodies or reagents to form an antibody-antigen complex, and detecting the presence of the antibody-antigen complex;

[0120] Preferably, the CD79B protein is human CD79B protein.

[0121] The beneficial effects of the present invention include:

[0122] The anti-CD79B single-domain antibody provided by the present invention has the advantages of small molecular weight, high solubility, high stability, low immunogenicity, high tissue permeability, and no need for additional folding and assembly steps or linker optimization and modification, making it a promising alternative to the larger molecular weight scFv single-chain antibody; and the chimeric antigen receptor constructed using the anti-CD79B single-domain antibody and the CAR-T cell prepared using the chimeric antigen receptor have significant and efficient CD79B +Tumor cell killing ability.

[0123] Explanation of terms:

[0124] The term "antibody" as used herein includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies, single-chain variable region fragments (Single-Chain Fragment Variable, "scFv", also known as single-chain antibodies), single-domain antibodies (VHH or nanobodies) and antibody fragments, in particular antigen-binding fragments, such as Fab', F(ab') and Fv2. In some embodiments of the present invention, the terms "immunoglobulin (Ig)" and "antibody" are used interchangeably.

[0125] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0126] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites; whereas IgA antibodies contain 2-5 basic 4-chain units, which can be polymerized in combination with the J chain to form multivalent assemblies. In the case of IgG, the 4-chain unit is typically approximately 150,000 daltons. Each light chain is linked to a heavy chain by a single covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the heavy chain isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH1, CH2 and CH3 for each α and γ chain) and four (CH1, CH2, CH3 and CH4 for μ and ε isotypes) constant domains (CH) and a hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. VL is arranged together with VH, while CL is arranged together with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. Pairs of VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies, see also Basic and Clinical Immunology. Eighth edition. Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw. Appleton & Lange, Norwalk, CT. 1994, P71 and Chapter 6. Light chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes, or isotypes, based on the amino acid sequence of their heavy chain constant domains (CHs). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes can be further divided into subclasses based on relatively minor differences in CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0127] Heavy chain antibodies are antibodies derived from camelids or cartilaginous fish. Compared to the above-mentioned four-chain antibodies, heavy chain antibodies lack light chains and heavy chain constant region 1 (CH1), and only contain two heavy chains composed of variable regions (VHH) and other constant regions. The variable regions are connected to the constant regions through a hinge-like structure. Each heavy chain of camelid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3), and each heavy chain of cartilaginous fish heavy chain antibodies contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. By fusing with the Fc constant region of human IgG, heavy chain antibodies can have the CH2 and CH3 of the Fc region of human IgG.

[0128] The terms "anti-CD79B single-domain antibody", "heavy chain variable region domain of an anti-CD79B heavy chain antibody", "anti-CD79B VHH" and "anti-CD79B nanobody" used in the present invention are used interchangeably and all refer to single-domain antibodies that specifically recognize and bind to CD79B. A single-domain antibody is the variable region of a heavy chain antibody. Typically, a single-domain antibody contains three CDR regions and four FR regions. A single-domain antibody is the smallest functional antigen-binding fragment. Usually, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0129] Without substantially affecting the activity of the antibody, those skilled in the art may modify one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acids in the sequence of the present invention to obtain variants of the antibody or its functional fragment sequence. These variants include, but are not limited to, deletions, insertions, and / or substitutions of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and additions of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids having similar or similar properties generally do not alter protein function. For example, amino acids with similar properties may be substituted in FR and / or CDR regions. Amino acid residues that can be conservatively substituted are well known in the art, and the amino acid residues substituted by the aforementioned methods may or may not be encoded by the genetic code. For example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein and is considered to be included in the scope of protection of the present invention.

[0130] "Sequence identity": Generally speaking, "sequence identity" or "sequence homology" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity." Whether it is a nucleic acid or amino acid sequence, the percent identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. For example, the advanced BLAST computer program available from the National Institutes of Health can also be used to compare sequence information to determine the percent identity. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). Briefly, the BLAST program defines identity as the number of aligned symbols (usually nucleotides or amino acids) that are identical divided by the total number of shorter symbols in the two sequences. The program can be used to determine percent identity over the entire length of the compared proteins.

[0131] In some embodiments of the present invention, the sequence of the variant of the present invention can be at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to its source sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters is used. The present invention also includes molecules having antibody heavy chain variable regions with CDR regions, as long as their CDR regions have more than 90% (preferably more than 95%, and most preferably more than 98%) homology to the CDR regions identified herein.

[0132] The single domain antibodies, nano antibodies or heavy chain antibodies of the present invention can be prepared by conventional methods in the art, such as phage display technology well known in the art; alternatively, the various antibodies of the present invention can be expressed in other cell lines. Sequences encoding the various antibodies of the present invention can be used to transform suitable mammalian host cells. Transformation can be carried out by any known method, for example, including packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Host mammalian cell lines that can be used for expression are well known in the art, such as the various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with essential CD79B binding properties.

[0133] "Specific binding": As used herein, the term "specific binding" refers to the binding that occurs between paired molecular species (e.g., a receptor and a ligand). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, the specific binding between one or more species is direct. In some embodiments of the invention, the affinity of the specific binding is about 2 times greater than nonspecific binding, about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.

[0134] The term "chimeric antigen receptor (CAR)" used in the present invention comprises an extracellular antigen binding region, which comprises any anti-CD79B single domain antibody (sdAb) disclosed in the present invention, such as VHH.

[0135] In some embodiments of the present invention, the CAR disclosed in the present invention comprises: (a) an extracellular antigen binding region; (b) a transmembrane region; and (c) an intracellular signal transduction domain; the extracellular antigen binding region comprises the anti-CD79B single domain antibody (sdAb) disclosed in the present invention.

[0136] The present invention discloses a CAR comprising an extracellular antigen binding region comprising one or more sdAbs. The sdAbs may be of the same or different origins and of the same or different sizes. Any sdAb known in the art or disclosed by the present invention, including the single domain antibodies disclosed by the present invention, may be used to construct the CAR described herein. The sdAbs may be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, sharks, goats, rabbits, and cattle. The single domain antibodies contemplated by the present invention also include naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0137] In some embodiments of the present invention, the extracellular antigen binding region disclosed in the present invention comprises at least one binding domain, and the at least one binding domain comprises the anti-CD79B single domain antibody disclosed in the present invention.

[0138] In some embodiments of the invention, the anti-CD79B sdAb is camelid, chimeric, human, or humanized.

[0139] In some embodiments of the present invention, the chimeric antigen receptor disclosed in the present invention comprises: (a) an extracellular antigen binding region, wherein the extracellular antigen binding region comprises any one of the anti-CD79B single domain antibodies described above; (b) a transmembrane region; and (c) an intracellular signaling domain; wherein the anti-CD79B single domain antibody comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO: 27.

[0140] In some embodiments of the present invention, the chimeric antigen receptor disclosed in the present invention comprises any of the aforementioned anti-CD79B single-domain antibodies, wherein the anti-CD79B single-domain antibody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the CDR-H1 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 24 or SEQ ID NO: 29; the CDR-H2 region comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 5, SEQ ID NO: 13 or SEQ ID NO:20 has an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical; and the CDR-H3 region has an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:26 or SEQ ID NO:31.

[0141] In some embodiments of the present invention, the chimeric antigen receptor disclosed in the present invention comprises: (a) an extracellular antigen binding region, wherein the extracellular antigen binding region comprises any one of the aforementioned anti-CD79B single-domain antibodies; (b) a transmembrane region; and (c) an intracellular signaling domain; wherein the anti-CD79B single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO: 27.

[0142] In addition to the extracellular antigen-binding region disclosed in the present invention, the chimeric antigen receptor disclosed in the present invention may further comprise one or more of the following structures: a linker (e.g., a peptide linker, including a flexible linker and a rigid linker), a signal peptide, a hinge region, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0143] In some embodiments of the present invention, different domains of chimeric antigen receptors can also be fused to each other through peptide linkers. This depends on the structure and / or functional characteristics of single domain antibodies and / or various domains, and each peptide linker in a chimeric antigen receptor can have the same or different lengths and / or sequences. Those skilled in the art can independently select and optimize each peptide linker. In some embodiments of the present invention, the peptide linker comprises flexible residues (e.g., glycine and serine) so that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0144] The peptide linker can have any suitable length. In some embodiments of the present invention, 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, 60, 70, 80, 90, 100 or more amino acids in length.

[0145] In some embodiments of the invention, the peptide linker is no more than about 100, 90, 80, 75, 70, 60, 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 of the invention, 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, 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.

[0146] The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain antibody alone can be used as a linker. See, for example, WO 1996 / 34103.

[0147] In some embodiments of the present invention, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSG)n, (GGGS)n, and (GGGGS)n, wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0148] Flexible linkers are usually used when the connected domains need to move or interact to a certain extent (Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69.). Flexible linkers are usually composed of small, non-polar (such as Gly) or polar (such as Ser or Thr) amino acids (Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990; 211: 943–958.). These small amino acids provide flexibility while also allowing the movement of the connected functional domains. For commonly used flexible linkers, see Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69, which is incorporated herein by reference in its entirety.

[0149] The present invention also discloses nucleic acids encoding any of the aforementioned anti-CD79B single-domain antibodies or chimeric antigen receptors. The nucleic acids / polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0150] "Nucleic acid" refers to any compound and / or substance including a polymer containing nucleotides, such as a polynucleotide. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed from 5' to 3'. As used herein, the term "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. "Nucleic acid" can be linear or circular. In addition, "nucleic acid" includes both a sense strand (coding strand) and an antisense strand (template strand), as well as single-stranded and double-stranded forms. Moreover, the "nucleic acids" described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues.

[0151] As is well known to those skilled in the art, due to the degeneracy of the genetic code, a vast number of nucleic acids can be produced, all of which encode the antibodies or chimeric antigen receptors of the present invention. Therefore, once a specific amino acid sequence has been identified, one skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons without changing the amino acid sequence of the encoded protein. Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0152] The full-length nucleic acid sequences of the various antibodies or chimeric antigen receptors of the present invention, or fragments thereof, can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. One feasible method is to synthesize the relevant sequences using synthetic methods, especially when the fragment length is relatively short. Generally, very long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0153] Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) involved in the present invention include biomolecules in isolated form. Currently, DNA sequences encoding the polypeptides of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the polypeptide sequences of the present invention through chemical synthesis.

[0154] The present invention further relates to nucleic acid constructs comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence, such as expression vectors and recombinant vectors. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors generally contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (generally referred to as "flanking sequences" in certain embodiments) generally include one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, origin of replication, transcription termination sequence, complete intron sequence containing donor and acceptor splice sites, leader sequence encoding for polypeptide secretion, ribosome binding site, polyadenylation sequence, multiple linker region and selectable marker element for inserting nucleic acid encoding the antibody to be expressed.

[0155] The host cells of the present invention can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include bacterial cells of Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal yeast cells; insect cells of Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0156] In some embodiments of the present invention, the host cell can be various functional cells well known in the art, such as various killer cells, including but not limited to cytokine-induced killer cells (CIK), cytokine-induced killer cells stimulated by dendritic cells (DC-CIK), cytotoxic T lymphocytes (CTL), γδT cells, natural killer cells (NK), tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), CD3AK cells (killer cells of anti-CD3 monoclonal antibodies) and CAR-T / TCR-T cells. In some embodiments of the present invention, the killer cell is a T cell or a NK cell. Exemplary NK cells include but are not limited to primary NK cells, NK cell lines (such as NK92) and NKT cells. In some embodiments of the present invention, the NK cell is a primary NK cell. Exemplary T cells include, but are not limited to, peripheral blood T lymphocytes, umbilical cord blood T lymphocytes, cytotoxic killer T cells (CTLs), helper T cells, suppressor / regulatory T cells, γδ T cells, cytokine-induced killer cells (CIKs), tumor-infiltrating lymphocytes (TILs), and other mixed cell populations. In certain embodiments, the T cells are peripheral blood T lymphocytes and umbilical cord blood T lymphocytes.

[0157] 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 prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method, using procedures well known in the art. Another method uses MgCl2. In addition, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0158] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media, such as serum-containing medium or serum-free medium. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0159] The polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0160] The present invention also discloses vectors for cloning and expressing any chimeric antigen receptor of the present invention. In some embodiments of the present invention, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments of the present invention, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and has been described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. (2001)) and other virology and molecular biology manuals.

[0161] Numerous viral-based systems have been developed in the prior art for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art. 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 of the present invention, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments of the present invention, lentiviral vectors are used. In some embodiments of the present invention, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying coding sequences for immunomodulators (e.g., immune checkpoint inhibitors) and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged using protocols known in the art. Using methods known in the art, the resulting lentiviral vectors can be used to transduce mammalian cells (e.g., primary human T cells). Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their propagation in progeny cells. Lentiviral vectors also have the advantages of low immunogenicity and the ability to transduce non-proliferating cells.

[0162] In some embodiments of the present invention, the vector comprises any nucleic acid encoding the CAR of the present invention. Any molecular cloning method known in the art can be used to clone the nucleic acid into the vector, including, for example, the use of restriction endonuclease sites and one or more selection markers. In some embodiments of the present invention, the nucleic acid is operably connected to the promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in the present invention. Promoters can be further divided into constitutive promoters or regulated promoters, such as inducible promoters.

[0163] In some embodiments of the present invention, the nucleic acid encoding CAR is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters contemplated by the present invention include, but are not limited to, cytomegalovirus (CMV) promoter, human elongation factor-1α (hEF1α) promoter, ubiquitin C (UbiC) promoter, phosphoglycerol kinase (PGK) promoter, simian virus 40 (SV40) early promoter, and chicken β-actin coupled to CMV early enhancer (CAGG) promoter. The efficiency of this constitutive promoter in driving transgene expression has been extensively compared in a large number of studies. For example, Michael C. Milone et al. (Molecular Therapy, 17 (8): 1453-1464 (2009)) compared the efficiency of CMV, hEF1α, UbiC, and PGK in driving chimeric antigen receptor expression in human primary T cells and concluded that the hEF1α promoter not only induces the highest level of transgene expression, but also maintains the expression of CD4 + and CD8 + In some embodiments of the present invention, the nucleic acid encoding CAR is operably linked to the hEF1α promoter.

[0164] In some embodiments of the present invention, the nucleic acid encoding the CAR is operably linked to an inducible promoter. An inducible promoter is a regulated promoter. An inducible promoter can be induced by one or more conditions, such as physical conditions, the microenvironment of the engineered immune effector cell, or the physiological state of the engineered immune effector cell, an inducer, and the like.

[0165] In some embodiments of the present invention, the inducing conditions do not induce expression of endogenous genes in the engineered mammalian cells and / or in the subject receiving the pharmaceutical composition. In some embodiments of the present invention, the inducing conditions are selected from the group consisting of: an inducing agent, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and activation state of the engineered mammalian cells.

[0166] In some embodiments of the present invention, the vector further comprises a selection marker gene or a reporter gene to select cells expressing CAR from a population of host cells transfected by a lentiviral vector. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences for expression in the host cell. For example, the vector may contain transcriptional and translational terminators, initiation sequences, and promoters for regulating expression of nucleic acid sequences.

[0167] "Signal peptide": Signal peptide, sometimes also called signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), present at the N-terminus of most newly synthesized proteins that enter the secretory pathway (occasionally non-classically present at the C-terminus or internally) (Owji, et al., A comprehensive review of signal peptides: Structure, roles, and applications, European Journal of Cell Biology. 97(6): 422-441. (2018)) (Blobel G, Dobberstein B, et al., Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma, The Journal of Cell Biology, 67(3):835-51.(1975)).

[0168] The effect of signal peptide is to promote cell transfer protein, usually transferred to the cell membrane. In prokaryotes, the newly synthesized protein is guided to the SecYEG protein conduction channel present in the plasma membrane by signal peptide. There is a homologous system in eukaryotes, in which the newly synthesized protein is guided to the Sec6L channel by signal peptide, which has structural and sequence homology with SecYEG, but is present in the endoplasmic reticulum (Rapoport TA, Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes, Nature.450(7170):663-9(2007).). SecYEG and Sec6L channels are commonly referred to as transporters, and the transport by the channel is called translocation. When secreted protein passes through the channel, the transmembrane region may diffuse through the side gate in the translocon to be distributed in the surrounding membrane.

[0169] "Lentiviral vector": A lentiviral vector is a vector derived from a lentivirus and contains one or more lentiviral packaging proteins and the lentiviral proteins necessary for expressing one or more genes carried by the vector. Lentiviral vectors are produced by multiple attenuation of the virulence genes of lentiviruses such as HIV through gene editing and genetic engineering techniques. For example, deletion of the env, vif, vpr, vpu, and nef genes makes lentiviral vectors biosafe.

[0170] Lentiviral vectors or retroviral vectors are generally packaged in packaging cells using a lentiviral vector packaging system or a retroviral vector packaging system. For example, the process and method of packaging lentiviral vectors are described in Merten OW, et al., Production of lentiviral vectors. Mol Ther Methods Clin Dev. (2016), 3, 16017, which is incorporated herein by reference in its entirety.

[0171] Commonly used pseudotyped lentiviral vectors include so-called third-generation lentiviral vector packaging systems. Third-generation lentiviral vector packaging systems typically consist of four plasmids: a transfer plasmid encoding a gene of interest ("GOI"), such as a transgene; a GagPol plasmid; a Rev plasmid; and an envelope plasmid (containing viral glycoprotein genes, such as VSV-G or its variants or Cocal-G or its variants).

[0172] The "transfer plasmid" contains the lentiviral vector backbone genome and transgene. The transfer plasmid usually has one or more transgenes flanked by long terminal repeat (LTRs) sequences, which facilitate the integration of the transgene contained in the transfer plasmid into the host genome. LTRs are responsible for the reverse transcription and integration process of the viral genome. Through these sequences, the lentivirus can integrate the transgene into the genome of the host cell. For safety reasons, the transfer plasmid is usually designed so that the resulting viral vector cannot replicate itself. For example, the transfer plasmid lacks the genetic elements necessary to produce infectious lentiviral particles in host cells. In addition, the transfer plasmid can be designed to lack the 3'LTR, thereby making the virus "self-inactivating". Compared with the traditional second-generation pseudotype lentiviral vector packaging system (usually a single packaging plasmid containing nucleic acids encoding Gag, Pol, Rev and Tat and a separate envelope plasmid), the TAT gene is eliminated from the third-generation pseudotype lentiviral vector packaging system by adding a chimeric 5'LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) to the transfer plasmid. The transfer plasmid usually contains a Ψ sequence (Psi sequence, also known as Ψ packaging signal) located downstream of the 5'LTR. The Ψ sequence is responsible for packaging the transgenic RNA into the viral particles. The Ψ sequence ensures that only RNA containing the transgene is packaged into the viral particles. The transfer plasmid may also optionally contain an internal ribosome entry site (IRENS). In some embodiments, the transfection site (IRES) can be used to allow simultaneous translation of two or more open reading frames (ORFs) on a single mRNA, thereby achieving multi-gene expression. Some transfer plasmids, such as the lentiviral-GFP plasmid used in some embodiments of the present invention, may also contain a selectable marker gene, such as an antibiotic resistance gene (e.g., PuroR, encoding puromycin resistance) or a fluorescent protein gene (e.g., GFP), for screening or tracking transduced cells.

[0173] For details on transfer plasmids in lentiviral vector packaging systems, see Dull, et al., J. Virol. 72: 8463-71 (1998); Miyoshi, et al., J. Virol. 72: 8150-57 (1998).

[0174] Exemplarily, the packaging plasmid includes but is not limited to pMD2.G, pRSV-rev, pMDLG-pRRE and pRRL-GOI.

[0175] Lentiviral vectors and lentiviral vector backbone genomes are known in the art, see Naldini, et al., (1996) Science 272: 263-7; Zufferey, et al., (1998) J. Virol. 72: 9873-9880; Dull, et al., (1998) J. Virol. 72: 8463-8471, U.S. Pat. No. 6,013,516, and U.S. Pat. No. 5,994,136, each of which is herein incorporated by reference in its entirety.

[0176] "MOI": Multiplicity of Infection (MOI) refers to the number of viral particles added to each cell during infection. For example, when one million viral particles are added to one million cells, MOI = 1.

[0177] As used herein, the term "immune effector cell" refers to an immune cell that can perform immune effector functions. In some embodiments of the present invention, the immune effector cell expresses at least FcγRIII and performs ADCC effector functions. Examples of immune effector cells that mediate ADCC include T cells, B cells, NK cells, macrophages, dendritic cells, and induced pluripotent stem cells (iPSCs).

[0178] In some embodiments of the present invention, the immune effector cells are T cells. In some embodiments of the present invention, the T cells are CD4 + / CD8 - 、CD4 - / CD8 + 、CD4 + / CD8 + 、CD4 - / CD8 - In some embodiments of the present invention, T cells express CAR and bind to target cells such as CD79B + In some embodiments of the present invention, CD8 + T cells lyse antigen-specific target cells after expressing CAR and binding to target cells.

[0179] In some embodiments of the present invention, the immune effector cells are NK cells. In other embodiments, the immune effector cells can be established cell lines, such as NK-92 cells.

[0180] In some embodiments of the present invention, immune effector cells can be differentiated from stem cells, such as hematopoietic stem cells, pluripotent stem cells, iPSCs, or embryonic stem cells.

[0181] The engineered immune effector cells of the present invention are prepared by introducing CAR into immune effector cells (eg, T cells). In some embodiments of the present invention, CAR is introduced into immune effector cells by transfection of any isolated nucleic acid encoding CAR or any of the above-mentioned vectors comprising nucleic acid encoding CAR.

[0182] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into immune effector cells by physical, chemical or biological methods.

[0183] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, EF and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments of the present invention, vectors are introduced into cells by electroporation. In some embodiments of the present invention, vectors are introduced into cells by PEI (Polyethylenimine, polyethyleneimine transfection reagent) transfection reagent.

[0184] Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.

[0185] Chemical methods for introducing vectors into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome.

[0186] In some embodiments of the present invention, RNA molecules encoding any CAR described herein can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into immune effector cells by known methods such as mRNA electroporation (see Peter M Rabinovich. Human Gene Therapy, 17: 1027-1035 (2006)).

[0187] In some embodiments of the present invention, the transduced or transfected immune effector cells are proliferated in vitro after the introduction of a vector or isolated nucleic acid or are proliferated in a subject to which the transfected / transduced immune effector cells are administered. In some embodiments of the present invention, 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, 11, 12, 13 or 14 days. In some embodiments of the present invention, the transduced or transfected immune effector cells can be further evaluated or screened to select engineered immune effector cells.

[0188] Reporter gene can be used for identifying the cell that may be transfected and the function of assessment regulating sequence. Generally speaking, reporter gene is not present in recipient organism or tissue or is not expressed by recipient organism or tissue, and its coded polypeptide, some easily detectable characteristics of this polypeptide expression, such as enzymatic activity. The expression of reporter gene is measured at the appropriate time after DNA is introduced into recipient cell. Suitable reporter gene can include the gene of coding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (referring to Kumiko Ui-Tei.FEBS Letters, 479:79-82 (2000)). Suitable expression system is known in the art and can use known technology to prepare or commercially obtain.

[0189] Other methods for confirming the presence of CAR-encoding nucleic acids in engineered immune effector cells include: molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RTPCR and PCR; biochemical assays, such as detecting the presence or absence of specific peptides; and immunological methods, such as ELISA.

[0190] The composition disclosed in the present invention comprises a pharmaceutically acceptable excipient or carrier and any of the following ingredients: any of the aforementioned anti-CD79B single domain antibodies, CARs and engineered immune effector cells disclosed in the present invention. Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives and / or adjuvants. The excipients are preferably non-toxic or substantially non-toxic to the recipient at the dose 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, the pH, osmotic properties, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. The optimal pharmaceutical composition can be determined based on the intended route of administration, mode of delivery and desired dosage.

[0191] Pharmaceutical compositions for in vivo administration are typically provided as sterile formulations. Sterilization is achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization, reconstitution, or dilution. The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, they can be prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are typically placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers pierceable by hypodermic needles. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposomal carriers, bioerodible microparticles or porous beads, and depot injections) are also known to those skilled in the art.

[0192] Once the pharmaceutical composition is formulated, it is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form (e.g., lyophilized) that is redissolved before administration. The present invention also provides a test kit for producing a single-dose administration unit. The test kit of the present invention can each contain a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, a test kit containing a single-lumen and multi-lumen prefilled syringe (e.g., a liquid syringe and a lyophilizing syringe) is provided.

[0193] The present invention also provides a method for treating a patient (especially a patient with a CD79B-related disease) by administering an anti-CD79B single domain antibody, engineered immune effector cell, or pharmaceutical composition thereof as described in any embodiment of the present invention. The terms "patient," "subject," "individual," or "object" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human.

[0194] "Treatment" refers to treating a subject with the treatments described herein to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor size, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth). The treatment methods that effectively treat a patient can vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the treatment to elicit an anti-cancer response in the subject.

[0195] "Therapeutically effective amount": i.e., Therapeutically Effective Amount, as used herein, "therapeutically effective amount" is a composition or its active substance administered to an individual sufficient to provide a beneficial effect or otherwise reduce harmful, non-beneficial events, such as the amount of anti-CD79B single domain antibodies and CAR-T cells provided herein. Exemplarily, "therapeutically effective dose" means herein a dosage that produces one or more desired or expected (e.g., beneficial) effects due to its administration, which is administered once or multiple times within a specified time period. The exact dosage will depend on the purpose of treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0196] The therapeutically effective amount of the pharmaceutical composition containing the anti-CD79B single domain antibody disclosed in the present invention or the engineered immune effector cells such as CAR-T cells to be used will depend on, for example, the degree of treatment and the target. It will be understood by those skilled in the art that the appropriate dosage level for treatment will vary depending in part on the delivered molecule, indication, route of administration, and patient condition (body weight, body surface or organ size) and / or condition (age and general health). In certain embodiments, the clinician can titrate the dosage and change the route of administration to obtain the best therapeutic effect.

[0197] The frequency of administration will depend on the pharmacokinetic parameters of the anti-CD79B single domain antibody or engineered immune effector cell in the formulation used. Clinicians typically administer the pharmaceutical composition until the dose that achieves the desired effect is reached. The pharmaceutical composition can therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or administered as a continuous infusion via an implantable device or catheter.

[0198] The routes of administration / administration of the pharmaceutical composition are conventional in the art, such as oral, nasal, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection, and can also be administered by sustained release system or by implantation device.

[0199] “And / or”: should be understood to mean one or two alternatives.

[0200] "About" / "approximately": As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. It should be understood that reference herein to "about" a value, range, or parameter includes (and describes) embodiments for the value, range, or parameter itself. For example, description referring to "about X" includes description of "X."

[0201] "Comprising": As used herein, unless the context requires otherwise, the word "comprising" will be understood to mean the inclusion of the specified steps, elements, or groups of steps or elements, but not the exclusion of any other steps, elements, or groups of steps or elements. In some embodiments of the present invention, the terms "including," "having," "containing," and "comprising" are used synonymously.

[0202] "Embodiments": Reference throughout this specification to "some embodiments," "some implementations," "some embodiments," or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, various appearances of the foregoing phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0203] "Operably": A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned so as to promote translation, the ribosome binding site is operably linked to a coding sequence. Generally speaking, "operably linked" means that the nucleic acid sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0204] All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety, just as if each individual publication, document, or patent not specifically and individually indicated as incorporated by reference in its entirety were incorporated by reference in its entirety. In the event of a conflict, the present application (including any definitions herein) will control. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be construed as an admission or any form of recommendation.

[0205] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. BRIEF DESCRIPTION OF THE DRAWINGS

[0206] Figure 1: Figure 1 is a flow cytometry graph showing that the anti-CD79B single domain antibodies JYB4, JYB10, JYB8, JYB11 and JYB14 specifically bind to the CD79B antigen;

[0207] Figure 2: Figure 2 is a graph showing the killing efficiency of the CAR-T-JYB4 cells, CAR-T-JYB10 cells, CAR-T-JYB8 cells, CAR-T-JYB11 cells and CAR-T-JYB14 cells in killing target cells Ramos cells. DETAILED DESCRIPTION

[0208] The following is a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments, so that the purpose, features and effects of the present invention are fully understood. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

[0209] In the following examples, the experimental methods without specific conditions are based on conventional methods and conditions known in the art, or are selected according to the product specifications. Reagents and raw materials not specified in the present invention are all commercially available.

[0210] Example 1

[0211] Immunize animals to establish a phage library to prepare anti-CD79B single domain antibodies.

[0212] A. Animal Immunization and Immune Response Tests

[0213] (1) Select healthy alpacas as immunization subjects.

[0214] (2) For the first immunization, complete Freund's adjuvant was mixed with CD79B antigen (0.4 mg) in a 1:1 ratio, emulsified and injected subcutaneously at multiple points. For subsequent booster immunizations, incomplete Freund's adjuvant was mixed with CD79B antigen in a 1:1 ratio. The immunization interval was 2 weeks, and a total of 4 immunizations were performed. 5 mL of peripheral blood was collected before and after each immunization, and serum was separated. The immune response was monitored by ELISA and the serum titer was confirmed.

[0215] CD79B antigen used for immunization of animals:

[0216] Product Name: Human CD79A & CD79B Heterodimer Protein, Mouse IgG2a Fc, Flag Tag & Mouse IgG2a Fc, His Tag (MALS verified); Catalog Number: #CDB-H52W3-1mg; Brand: ACRO;

[0217] Antigen used in ELISA: Human CD79B Protein, His Tag (MALS verified), Catalog Number: #CDB-H52H3-1mg; Brand: ACRO.

[0218] (3) After the fourth immunization, the plasma titer reached 10,000, and 20 mL of blood was collected. The lymphocytes were separated and stored in TRIzol reagent for subsequent antibody phage library construction.

[0219] B. Construction of Antibody Phage Library

[0220] (1) Reverse transcription

[0221] After animal immunization, total RNA was extracted from the separated peripheral blood lymphocytes and reverse transcribed using the Takara reverse transcription kit. The total RNA sample was divided into two parts, one using the Oligo dT Primer in the kit as a primer, and the other using the Random 6-mers in the kit as a primer. The total RNA obtained in the previous step was reverse transcribed into cDNA according to the instructions of the reverse transcription kit and stored in two centrifuge tubes respectively.

[0222] (2) PCR amplification

[0223] a. PCR amplification: Amplify specific antibody fragments from reverse-transcribed cDNA using Taq DNA Polymerase Hot Start enzyme. Run all PCR products on a 1% agarose gel, excise the gel, and recover the target fragment (600-700 bp in size) as the first-round PCR amplification product. Store at -20°C.

[0224] b. The first-round PCR amplification product was used as a template for the second-round PCR reaction. After the reaction, 1% agarose gel electrophoresis was performed. The gel was finally cut to recover a single target band with a fragment size of approximately 400 bp. The DNA was purified from the PCR reaction solution using a universal DNA purification kit.

[0225] (3) Enzyme digestion and ligation

[0226] The target gene fragment amplified by the second round of PCR and the pComb3XSS phage plasmid vector were digested with restriction endonucleases SpeⅠ and SacⅠ, respectively. After the digestion, the VHH target gene fragment was ligated to the pComb3XSS phage plasmid vector using ligase to construct a recombinant plasmid.

[0227] (4) Bacterial library construction

[0228] a. Take a 50 μL aliquot of TG1 competent cells and place on ice for 5-10 minutes to thaw;

[0229] b. Add 100 ng of ligation product and transfer to a pre-cooled electroporation cuvette with a 1 mm gap. Set the electroporation parameters to 1800 V and 1 mm, and click the "Transform" button.

[0230] c. After electroporation is complete, add 1 mL of 37°C preheated SOC culture medium, mix thoroughly, and shake at 37°C, 200 rpm, to resuscitate the bacteria for 1 hour.

[0231] d. Take 21 100ng ligation systems and perform electroporation reaction using competent cells as described above;

[0232] e. Take 100 μL of the recovered bacterial solution, perform a 10-10 serial dilution, plate the solution, and culture overnight at 37°C.

[0233] f. Collect all remaining bacterial suspension and evenly spread it into five 245 mm square culture plates (2×YT containing 100 μg / mL Amp, 2% agarose), and culture inverted at 37°C overnight;

[0234] g. Calculate the number of transformed colonies obtained from all reactions based on the dilution factor and the number of single colonies, which is the library capacity of the bacterial library. Simultaneously, randomly select several single colonies from the gradient dilution plate for colony PCR. A single PCR product with a band of approximately 400 bp is considered a positive clone, which is used to estimate the positive clone rate of the bacterial library.

[0235] h. Scrape the overnight cultured colonies from the plate using 2×YT liquid medium, place them in a 50 mL centrifuge tube, measure their OD600 value, add glycerol to a final concentration of 20%, and store at -80°C.

[0236] (5) Phage library construction

[0237] a. Inoculate the bacterial library into 100 mL of 2×YT liquid medium (containing 100 μg / mL Amp) to an initial OD600 value of 0.1 and culture at 37°C and 250 rpm until the OD600 reaches 0.5-0.55;

[0238] b. Add helper phage at a ratio of 1:20 (number of bacteria: number of phages) and incubate at 37°C and 250 rpm for 30 min;

[0239] c. Add Kana to a final concentration of 50 μg / mL, incubate overnight at 30°C and 250 rpm, centrifuge, and collect the supernatant;

[0240] d. Add 1 / 4 volume of pre-cooled PEG / 2.5M NaCl, mix well, incubate on ice for at least 30 minutes, centrifuge at 4000 rpm at 4°C for 20 minutes, remove the supernatant, and add 1 mL of PBS buffer to dissolve the precipitate. Add 1 / 4 volume of pre-cooled PEG / NaCl again and incubate on ice for 10 minutes. Centrifuge at 12000 g at 4°C for 10 minutes, remove the supernatant, dissolve the precipitate in 1 mL of PBS, and store at -80°C to obtain the purified phage library.

[0241] C. Phage Screening

[0242] (1) First round of screening

[0243] a. Remove the screening antigen (Human CD79B Protein, His Tag) from the -80°C freezer and place on ice to thaw;

[0244] b. Coat the screening antigen onto the immunotube (40 μg / tube, coating solution: CBS, pH 9.6, 2 mL / tube) and rotate slowly at 4°C overnight. Simultaneously, coat the tube with BSA as a control.

[0245] c. Discard the liquid in the overnight coated immunotubes and wash the immunotubes three times with 2 mL of PBS buffer at room temperature, rotating for 5 minutes each time.

[0246] d. Add 2 mL of blocking solution (3% BSA) and rotate at room temperature for 2 h;

[0247] e. Discard the liquid in the blocked immunotube and wash the immunotube three times with 2 mL of PBS buffer at room temperature, rotating for 5 minutes each time.

[0248] f. Discard the washing solution in the immune tube, add 2 mL of PBS, calculate according to the following formula and add the prepared phage library, 28 μL (about 10 12pfu) as the input phage library for the first round of screening and incubate with rotation at room temperature for 1 hour:

[0249] Where, V is the volume of phage added (unit: μL), T library is the phage titer;

[0250] Discard the liquid in the immunotube and add 2 mL of PBST (1× PBS plus 0.1% Tween 20, the same below) buffer to wash the immunotube 20 times at room temperature, rotating for 5 minutes each time;

[0251] h. Discard the liquid in the immunotube and remove as much residual liquid as possible. Add 1 mL of 0.25 mg / mL Trypsin solution and elute with rotation at room temperature for 30 minutes.

[0252] i. Add 10 μL of 10% AEBSF to terminate elution and transfer the solution in the immunotube to a new 1.5 mL centrifuge tube, which is the phage eluate for the first round of screening.

[0253] (2) First round of phage eluate titer detection

[0254] a. Streak a single colony of the TG1 strain stored at -80°C onto 2×YT solid medium and culture overnight at 37°C (store at 4°C for one week). Pick a single colony from the single colony plate and transfer it to 5 mL of 2×YT medium and culture overnight at 37°C.

[0255] b. Transfer 500 μL of overnight culture to 5 mL of 2×YT liquid medium and incubate at 37°C, 250 rpm for approximately 45–60 min until the OD600 reaches 0.5–0.55.

[0256] c. Take 10 μL of the first round of phage eluate and dilute it 10-fold in a 1.5 mL centrifuge tube. Dilute it 12 times in total. That is, dilute 10 μL of the first round of phage eluate to 100 μL, then take 10 μL and dilute it to 100 μL. Repeat this process for a total of 12 dilutions to 10. -12 , oscillate to mix;

[0257] d. Add 90 μL of TG1 bacterial solution to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 minutes;

[0258] e. Take 5 μL from each dilution centrifuge tube and add it dropwise to 2×YT solid medium (Amp) and culture it upside down at 37°C overnight;

[0259] f. Count the number of single colonies on the plate at the dilution that can clearly distinguish single colonies, and calculate the number of phage particles per milliliter of phage solution, i.e., the phage library titer, according to the following formula: T (pfu / ml) = N × D × 400

[0260] Wherein, T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0261] (3) Amplification of the first round of phage eluate

[0262] a. Streak a single colony of the TG1 strain, stored at -80°C, onto 2×YT solid medium and culture overnight at 37°C (store at 4°C for one week). Transfer a single colony from the single colony plate to 5 mL of 2×YT medium and culture overnight at 37°C.

[0263] b. Transfer 500 μL of overnight culture to 5 mL of 2×YT liquid medium and incubate at 37°C, 250 rpm for approximately 45–60 min until the OD600 value reaches 0.5–0.55.

[0264] c. Add 500 μL of the phage eluate obtained after the first round of screening to the bacterial solution with an OD600 of 0.5-0.55 (store the remaining eluate at 4°C);

[0265] d. Continue incubation at 37°C, 250 rpm for 30 min;

[0266] e. Spread the entire bacterial suspension evenly onto a 245 mm square plate containing 2% agarose containing 100 μg / mL Amp and culture overnight at 37°C.

[0267] f. Take the square plate from the overnight culture and add 6 mL of 2×YT liquid medium (containing 100 μg / mL Amp) to the surface of the culture plate. Use a spreading stick to gently scrape the colonies off the square plate and collect the bacterial liquid into a 15 mL centrifuge tube. This is the amplified bacterial sub-library. At the same time, use a spectrophotometer to measure the OD600 value of the bacterial liquid, which is the OD600 value of the eluate bacterial library. Add glycerol to a final concentration of 20%, which is the first round bacterial library.

[0268] g. Calculate the amount of bacterial solution from the eluate bacterial library according to the following formula and transfer it to 100 mL of 2×YT liquid medium (containing 100 μg / mL Amp) to an initial OD600 of 0.1:

[0269] Wherein, V is the volume of the transferred bacterial solution (unit: μL), and OD600 is the OD600 of the constructed eluate bacterial library;

[0270] h. Incubate at 37°C, 250 rpm until the OD600 of the bacterial solution reaches 0.5-0.55;

[0271] i. Calculate and add helper phage M13K07 according to the following formula to make the number of bacteria: number of phage = 1:20:

[0272] Where V is the volume of helper phage added (unit: mL), T helper-phage is the titer of the helper phage used, OD600 is the OD600 value of the bacterial solution;

[0273] j. Continue incubation at 37°C, 250 rpm for 30 min;

[0274] k. Add Kana to a final concentration of 50 μg / mL and culture overnight at 30°C and 250 rpm.

[0275] (4) First round of phage purification

[0276] a. Transfer the overnight culture to a new 50 mL centrifuge tube and centrifuge at 4000 rpm at 4°C for 10 min.

[0277] b. Transfer the supernatant after centrifugation to a new 50 mL centrifuge tube, add 1 / 4 volume of 4°C pre-cooled 20% PEG / 2.5 M NaCl, mix thoroughly, and place on ice for 30 minutes;

[0278] c. Centrifuge at 4000 rpm, 4°C for 20 min, discard the supernatant, and invert on paper for 2 min;

[0279] d. Add 1 mL of PBS to resuspend the pellet, transfer the resuspension to a new 1.5 mL centrifuge tube, and centrifuge at 12,000 rpm at 4°C for 20 min.

[0280] e. Transfer the supernatant after centrifugation to a new 1.5 mL centrifuge tube, add 1 / 4 volume of pre-chilled 20% PEG / 2.5 M NaCl solution, mix well, and place on ice for 10 minutes;

[0281] f. Centrifuge at 13,000 rpm at 4°C for 10 min, discard the supernatant, and resuspend the pellet in 1 mL of PBS.

[0282] Centrifuge at 13000 rpm and 4°C for 2 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube, which is the first round of screening of the phage sub-library. Store at -80°C for a long term and at -20°C for a short term (1-2 weeks).

[0283] h. The titer of the phage sub-library was tested in the first round of screening using the same method as described in C(2).

[0284] (5) Second round of screening

[0285] The screening method was the same as described in C(1). The input phage was 0.25 mL of the phage sublibrary obtained in the first round of screening, and the input phage library was used as the second round of screening to obtain the second round of screening phage eluate.

[0286] (6) Second round of phage eluate titer detection

[0287] The method is the same as that of C(2) above.

[0288] (7) Monoclonal ELISA test (Phage-ELISA)

[0289] After immunotube screening, 192 monoclonal clones were randomly selected for ELISA testing and secondary verification of positive clones.

[0290] a. Streak a single colony of the TG1 strain, stored at -80°C, onto 2×YT solid medium and culture overnight at 37°C (store at 4°C for one week). Transfer a single colony from the single colony plate to 5 mL of 2×YT medium and culture overnight at 37°C.

[0291] b. Transfer 500 μL of overnight culture to 5 mL of 2×YT liquid medium and incubate at 37°C, 250 rpm for approximately 45–60 min until the OD600 value reaches 0.5–0.55.

[0292] c. Take 10 μL of the phage eluate after the second round of screening and dilute it 10-fold in a 1.5 mL centrifuge tube. Dilute it 12 times in a 10-fold gradient. That is, dilute 10 μL of the phage library to 100 μL, then dilute 10 μL to 100 μL, and so on. Dilute it 12 times in a 10-fold gradient. Mix thoroughly by shaking.

[0293] d. Add 90 μL of bacterial solution with an OD600 value of 0.5-0.55 to each dilution centrifuge tube and mix well;

[0294] e. Continue incubation at 37°C, 250 rpm for 30 min;

[0295] f. Spread the bacterial solution evenly onto a solid culture medium plate containing 100 μg / mL Amp and culture overnight at 37°C;

[0296] g. Randomly pick single colonies from the culture medium plate after overnight culture and place them in sterile 96-well cell culture plates (P1-P3). Add 200 μL of 2×YT medium (containing 100 μg / mL Amp) to each well and culture at 37°C overnight.

[0297] h. Transfer 2 μL of the overnight culture to 200 μL of 2×YT liquid medium (containing 100 μg / mL Amp) per well of a new 96-well cell culture plate. Incubate at 37°C for 3-5 hours. Store the overnight culture at 4°C before transfer.

[0298] i. Calculate according to the following formula and add helper phage M13K07 to each well so that the number of bacteria: number of phage = 1:20:

[0299] Where V is the volume of helper phage added (unit: mL), T helper-phage is the helper phage titer used;

[0300] j. Incubate at 37°C for 30 min, add Kana to a final concentration of 50 μg / mL, and incubate at 30°C overnight;

[0301] k. Centrifuge the 96-well culture plate after overnight culture at 4°C, 4000 rpm for 10 min and store at 4°C until use;

[0302] 1. Coat the screening antigen onto an ELISA plate (1 ng / μL, coating solution: CBS, pH 9.6, 100 μL / well). Coat the plate with BSA as a control in parallel at 4°C overnight.

[0303] m. Discard the liquid in the overnight coated ELISA plate, add 200 μL PBS buffer to each well, and wash the ELISA plate three times at room temperature, each time for 10 minutes;

[0304] n. Add 200 μL of blocking solution (3% BSA) to each well to block the ELISA plate and block at room temperature for 1 hour;

[0305] o. Discard the blocking solution and add 200 μL of PBST (1× PBS plus 0.1% Tween 20, the same below) buffer to each well. Wash the plate three times at room temperature for 10 minutes each time.

[0306] p. Add 120 μL of 3% BSA to each well and then add 80 μL of the supernatant after centrifugation in step (7) (k) and incubate at room temperature for 2 h;

[0307] q. Discard the liquid in the ELISA plate and add 200 μL of PBST buffer to each well and wash three times for 10 minutes each time;

[0308] r. Add M13 Bacteriophage Antibody (HRP) and Mouse Mab to each well, diluted 1:55000 in PBS, 100 μL / well, and incubate at room temperature for 1 hour;

[0309] s. Discard the liquid in the ELISA plate and add 200 μL of PBST buffer to each well and wash three times for 10 minutes each time;

[0310] t. Add 100 μL of TMB single-component colorimetric solution to each well and develop for 2-3 min in the dark. Add 100 μL of 1 M HCl to each well to terminate the reaction. Read the OD450 nm value using a microplate reader, record, and save.

[0311] (8) Secondary verification of positive clones by ELISA

[0312] To exclude false positive results, a secondary ELISA validation was performed using the same method as described in C (7).

[0313] (9) Sequencing of positive clones

[0314] Positive monoclones were selected based on the ELISA test data and secondary verification data: 5 μL of the positive clone bacterial solution from the monoclonal ELISA test (7)(g) plate was inoculated into 1 mL of 2×YT medium (containing 100 μg / mL of Amp), cultured at 37°C, 250 rpm until the OD600 reached 0.8-1.0 (approximately 6-8 h), 0.5 mL of the bacterial solution was taken for sequencing, and the remaining bacterial solution was added with a final concentration of 20% glycerol and stored at -20°C.

[0315] (10) Results of two rounds of screening

[0316] The results of the two rounds of screening are shown in Table 2 below:

[0317] Table 2

[0318] D. ELISA detection of purified single domain / VHH antibodies binding to various antigens

[0319] The multiple positive monoclonal clones after verification were sequenced to obtain multiple different anti-CD79B single-domain antibody sequences. After preparing and purifying multiple antibodies, ELISA tests were performed with human CD79B antigen, mouse CD79B antigen, human CD79A+CD79B antigen, and mouse CD79A+CD79B antigen, respectively. The anti-CD79B single-domain antibodies JYB4, JYB10, JYB8, JYB11, and JYB14 that can specifically bind to the human CD79B antigen were screened. The specific detection method is as follows:

[0320] (1) Antigen was coated onto an ELISA plate (1 ng / μL, coating solution was CBS, pH 9.6, 100 μL / well) and BSA was coated in parallel as a control at 4°C overnight.

[0321] (2) Discard the liquid in the overnight coated ELISA plate, add 200 μL PBS buffer to each well, and wash the ELISA plate three times at room temperature, each time for 10 minutes;

[0322] (3) Add 200 μL of blocking solution (3% BSA) to each well to block the ELISA plate and block at room temperature for 1 h;

[0323] (4) Discard the blocking solution and add 200 μL of PBST (1× PBS plus 0.1% Tween 20, the same below) buffer to each well. Wash the ELISA plate three times at room temperature for 10 min each time.

[0324] (5) The purified nano / single domain antibody was diluted to 0.1 μg / μL with PBS, and then 500 μL was aspirated for 10-fold gradient dilution, for a total of 6 dilution gradients, that is, 50 μg of nano antibody was aspirated and diluted to 500 μL, and then 50 μL was aspirated and diluted to 500 μL, and so on, for a total of 6 dilution gradients;

[0325] (6) Add 200 μL of each dilution of nanoantibodies to each well and incubate at room temperature for 2 h;

[0326] (7) Discard the liquid in the ELISA plate and add 200 μL PBST buffer to each well and wash three times for 10 min each time;

[0327] (8) Anti-myc was added to each well, diluted 1:10000 in PBS, 100 μL / well, and incubated at room temperature for 1 h;

[0328] (9) Discard the liquid in the ELISA plate and add 200 μL PBST buffer to each well and wash six times, each time for 5 minutes;

[0329] (10) Add 100 μL of TMB single-component colorimetric solution to each well and develop in the dark for 2–3 min. Add 100 μL of 1 M HCl to each well to terminate the reaction. Read the OD450 nm value using a microplate reader, record, and save.

[0330] The test results are shown in Tables 3-7 below.

[0331] Table 3

[0332] Table 4

[0333] Table 5

[0334] Table 6

[0335] Table 7

[0336] As can be seen from Table 3-7, the anti-CD79B single-domain antibodies JYB4, JYB10, JYB8, JYB11 and JYB14 can all effectively and specifically bind to human anti-CD79B antigens, but cannot effectively bind to mouse CD79B antigens.

[0337] The amino acid sequence of the anti-CD79B single-domain antibody JYB4 is shown in SEQ ID NO: 1; the anti-CD79B single-domain antibody JYB4 comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 3, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 5, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 7;

[0338] The amino acid sequence of the anti-CD79B single-domain antibody JYB10 is shown in SEQ ID NO: 9; the anti-CD79B single-domain antibody JYB10 comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 11, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 13, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 15;

[0339] The amino acid sequence of the anti-CD79B single-domain antibody JYB8 is shown in SEQ ID NO: 17; the anti-CD79B single-domain antibody JYB8 comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 19, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 20, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 22;

[0340] The amino acid sequence of the anti-CD79B single-domain antibody JYB11 is shown in SEQ ID NO: 23; the anti-CD79B single-domain antibody JYB11 comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; the amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 24, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 20, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 26;

[0341] The amino acid sequence of the anti-CD79B single-domain antibody JYB14 is shown in SEQ ID NO: 27; the anti-CD79B single-domain antibody JYB14 comprises a CDR-H1 region, a CDR-H2 region and a CDR-H3 region; the amino acid sequence of the CDR-H1 region is shown in SEQ ID NO: 29, the amino acid sequence of the CDR-H2 region is shown in SEQ ID NO: 20, and the amino acid sequence of the CDR-H3 region is shown in SEQ ID NO: 31.

[0342] E. Flow cytometry

[0343] A CD79B-GFP molecule was designed, wherein the nucleic acid sequence encoding the CD79B-GFP molecule included, from the 5' end to the 3' end, a nucleic acid sequence encoding a human CD79B antigen (a full-length protein including its signal peptide, UniProt ID: P40259), a nucleic acid sequence encoding an F2A peptide (a self-cleaving peptide), and a nucleic acid sequence encoding a GFP molecule (Green Fluorescent Protein); a plasmid (CD79B-GFP plasmid) containing the nucleic acid sequence encoding the CD79B-GFP molecule was constructed; the CD79B-GFP plasmid was transfected into HEK-293T cells using a PEI reagent to overexpress the CD79B-GFP molecule; the method for constructing the CD79B-GFP plasmid is well known to those skilled in the art.

[0344] HEK-293T cells overexpressing the CD79B-GFP molecule were digested and divided into five groups;

[0345] In this example, when synthesizing the anti-CD79B single-domain antibodies JYB4, JYB10, JYB8, JYB11, and JYB14, a nucleic acid sequence encoding a His-tag protein was added to the 3' end of the nucleic acid sequence encoding each of the anti-CD79B single-domain antibodies, so that the His-tag protein could be expressed together with each of the anti-CD79B single-domain antibodies;

[0346] The digested HEK-293T cells in each group were incubated with the anti-CD79B single-domain antibodies JYB4, JYB10, JYB8, JYB11 and JYB14 (antibody concentration was 0.1 mg / mL) carrying the His-tag protein at the tail end at 4°C for 30 minutes, and then incubated with APC-His tag antibody (brand: BIOLEGEND, catalog number: #362605) at 4°C for 30 minutes; the control group was incubated with digested HEK-293T cells and commercial anti-CD79B monoclonal antibody (brand: BIOLEGEND, catalog number: #341404, with fluorescent labeling); on Day 2, the cells in each group were detected by flow cytometry, and the results are shown in Figure 1.

[0347] As shown in Figure 1, the ability of the anti-CD79B single-domain antibodies JYB4, JYB10, JYB8, JYB11 and JYB14 to specifically bind to the human CD79B antigen is comparable to that of the commercial anti-CD79B antibodies (monoclonal antibodies) with high specificity and strong affinity.

[0348] Example 2

[0349] CAR-T cell killing ability detection

[0350] A. Construction of CD79B-CAR

[0351] Designing a chimeric antigen receptor targeting CD79B (CD79B-CAR), wherein the structure of the CD79B-CAR from the N-terminus to the C-terminus is an extracellular antigen binding region, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB costimulatory signaling domain, and a CD3ζ intracellular signaling domain;

[0352] The extracellular antigen binding region is the anti-CD79B single domain antibody JYB4, JYB10, JYB8, JYB11 or JYB14;

[0353] The 5' end of the nucleic acid sequence encoding the CD79B-CAR is operably linked to the 3' end of the nucleic acid sequence encoding the CD8α signal peptide, so that the CD79B-CAR can be expressed on the cell surface under the action of the CD8α signal peptide;

[0354] To efficiently detect the expression of each CD79B-CAR, a nucleic acid sequence encoding the F2A peptide was connected to the 3' end of the nucleic acid sequence encoding each CD79B-CAR, and a nucleic acid sequence encoding the GFP molecule was connected to the 3' end of the nucleic acid sequence encoding the F2A peptide to construct a CD79B-CAR-GFP molecule;

[0355] (1) The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 32;

[0356] (2) The extracellular antigen binding region is the anti-CD79B single domain antibody JYB4, JYB10, JYB8, JYB11 or JYB14; the amino acid sequences of the anti-CD79B single domain antibodies JYB4, JYB10, JYB8, JYB11 and JYB14 are shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID NO: 27, respectively;

[0357] (3) the amino acid sequence of the hinge region of CD8α is shown in SEQ ID NO: 33;

[0358] (4) The amino acid sequence of the transmembrane region of CD8α is shown in SEQ ID NO: 34;

[0359] (5) the amino acid sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO: 35;

[0360] (6) the amino acid sequence of the intracellular signaling domain of CD3ζ is shown in SEQ ID NO: 36;

[0361] (7) The amino acid sequence of the F2A peptide is shown in SEQ ID NO: 37;

[0362] (8) The amino acid sequence of the GFP is shown in SEQ ID NO: 38.

[0363] The amino acid sequences of each domain of CD79B-CAR are shown in Table 8 below.

[0364] Table 8

[0365] B. Lentiviral Vector Packaging

[0366] Prepare the following four plasmids: pMD2.G envelope plasmid (containing a gene encoding wild-type VSV-G), pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and a main plasmid carrying the nucleic acid encoding the CD79B-CAR-GFP and the lentiviral backbone genome (the main plasmid is synthesized by conventional molecular cloning methods); the four plasmids are transfected into HEK-293T cells using PEI reagent to package lentiviral vectors CD79B-JYB4, CD79B-JYB10, CD79B-JYB8, CD79B-JYB11, and CD79B-JYB14 carrying the nucleic acid sequence of CAR containing different anti-CD79B single-domain antibodies; the specific packaging steps are as follows:

[0367] Add 9ug of the main plasmid, 4ug of the pMDLg / pRRE packaging plasmid, 2ug of the pRSV-REV packaging plasmid, and 2ug of the pMD2.G envelope plasmid to 1mL of Opti-MEM alpha reduced serum medium, shake well, add 64uL of PEI reagent, pipette evenly, and let stand for 10 minutes. Then add it to the culture medium of HEK-293T cells. Renew the culture medium after 6 hours. Collect the culture supernatant 48 hours after transfection, filter with a 0.45um filter membrane, centrifuge at 50000g for 2.5h, discard the supernatant, resuspend the lentiviral vector in 200uL of F12 medium, and freeze at -80℃.

[0368] Opti-MEM alpha Reduced Serum Medium, Brand: GIBCO, Catalog Number: #SP0272;

[0369] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog Number: #C12430500BT; FBS: Brand: EXCELL, Catalog Number: #FSP500;

[0370] F12 culture medium: Brand: GIBCO, catalog number: #C11330500BT;

[0371] Syringe filter: Brand: SORFA, item number: #622120.

[0372] C. Infection of activated T cells (MOI=5)

[0373] Day 0, take 5 groups of 1×10 7 Individual resting PBMCs were adjusted to a cell concentration of 3×10 using XVT medium + 1000 IU / mL IL-2. 7 cells / mL, anti-CD3 antibody at a final concentration of 1 μg / mL and anti-CD28 antibody at a final concentration of 2 μg / mL were added to stimulate and activate T cells (Mock T cells);

[0374] On Day 1, the original culture medium of each T cell culture system was washed away, and XVT medium + 1000 IU / mL IL-2 was added to adjust the cell concentration to 4×10 6cells / mL; at an MOI of 5, the lentiviral vectors CD79B-JYB4, CD79B-JYB10, CD79B-JYB8, CD79B-JYB11, and CD79B-JYB14 were added to the T cell culture systems of each group, respectively, to prepare CAR-T-JYB4 cells, CAR-T-JYB10 cells, CAR-T-JYB8 cells, CAR-T-JYB11 cells, and CAR-T-JYB14 cells, respectively;

[0375] On Day 2, the original culture medium of each T cell culture system was washed away, and the cell concentration of each group was adjusted to 1×10 6 -2×10 6 cells / mL;

[0376] Day 8, take 3×10 5 The CAR-T-JYB4 cells, CAR-T-JYB10 cells, CAR-T-JYB8 cells, CAR-T-JYB11 cells and CAR-T-JYB14 cells prepared by the T cell culture system of each group were added with 1×10 5 CD79B + Killing tests were performed on target Ramos cells (human B lymphoma cells); the control group was a mock T cell group (activated T cells). On Day 10, flow cytometry was performed on each group, and the results are shown in Figure 2.

[0377] XVT medium: Brand: IRVINE (Fujifilm), catalog number: #91154;

[0378] IL-2: Brand: Beijing Sihuan, Product No.: National Medicine Standard S20040008;

[0379] Anti-CD3 antibody: Brand: BIOLEGEND, Product Number: #317354

[0380] Anti-CD28 antibody: Brand: INVITROGEN, Catalog Number: #16-0289-81.

[0381] As can be seen from Figure 2, the CAR-T-JYB4 cells, CAR-T-JYB10 cells, CAR-T-JYB8 cells, CAR-T-JYB11 cells and CAR-T-JYB14 cells can all effectively kill the target cells Ramos cells.

[0382] Antibodies used in flow cytometry: Brand: BD, Catalog Number: #550955.

Claims

1. An anti-CD79B single domain antibody, characterized in that: The anti-CD79B single-domain antibody comprises a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region; wherein the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO:3, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO:5, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO:7; or wherein the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO: 11, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO: 13, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO: 15; or wherein the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO: 19, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO: 20, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO: 22; or wherein the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO: 24, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO: 20, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO: 26; or Wherein, the CDR-H1 region comprises the amino acid sequence shown in SEQ ID NO:29, the CDR-H2 region comprises the amino acid sequence shown in SEQ ID NO:20, and the CDR-H3 region comprises the amino acid sequence shown in SEQ ID NO:

31.

2. The anti-CD79B single domain antibody according to claim 1, characterized in that The anti-CD79B single-domain antibody further comprises a FR1 region, a FR2 region, a FR3 region and a FR4 region; wherein the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 2, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 4, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 6, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 8; or wherein the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 10, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 14, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16; or wherein the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 18, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 21, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16; or wherein the FR1 region comprises the amino acid sequence shown in SEQ ID NO: 10, the FR2 region comprises the amino acid sequence shown in SEQ ID NO: 12, the FR3 region comprises the amino acid sequence shown in SEQ ID NO: 25, and the FR4 region comprises the amino acid sequence shown in SEQ ID NO: 16; or Wherein, the FR1 region comprises the amino acid sequence shown as SEQ ID NO:28, the FR2 region comprises the amino acid sequence shown as SEQ ID NO:12, the FR3 region comprises the amino acid sequence shown as SEQ ID NO:30, and the FR4 region comprises the amino acid sequence shown as SEQ ID NO:

16.

3. The anti-CD79B single domain antibody according to any one of claims 1 or 2, characterized in that The anti-CD79B single-domain antibody comprises an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO: 27, or an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO:

27.

4. An isolated nucleic acid, characterized in that The nucleic acid encodes the anti-CD79B single-domain antibody according to any one of claims 1-3.

5. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises: (a) Extracellular antigen binding region; (b) a transmembrane region; and (c) intracellular signal transduction domain; Wherein, the extracellular antigen binding region comprises the anti-CD79B single domain antibody according to any one of claims 1-3.

6. The chimeric antigen receptor according to claim 5, characterized in that The transmembrane region is derived from the transmembrane region of at least one of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX4 0. ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, B TLA, DNAM-1, DR3, FcεRIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; Preferably, the transmembrane region is derived from the transmembrane region of CD8α.

7. The chimeric antigen receptor according to claim 5 or 6, characterized in that The intracellular signaling domain is derived from the intracellular signaling domain of at least one of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FcεRIγ, FcεRβ, FcγRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP12 and other intracellular signaling domains containing at least one ITAM protein intracellular signaling domain; Preferably, the intracellular signaling domain is derived from the intracellular signaling domain of CD3ζ.

8. The chimeric antigen receptor according to any one of claims 5 to 7, characterized in that The chimeric antigen receptor further comprises a hinge region, which connects the extracellular antigen binding region and the transmembrane region; Preferably, the hinge region is derived from the hinge region of at least one of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; More preferably, the hinge region is derived from the hinge region of CD8α.

9. The chimeric antigen receptor according to any one of claims 5 to 8, characterized in that The chimeric antigen receptor further comprises a co-stimulatory signaling domain; Preferably, the costimulatory signaling domain comprises a costimulatory signaling domain of at least one of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88; More preferably, the costimulatory signaling domain comprises the costimulatory signaling domain of 4-1BB.

10. The chimeric antigen receptor according to any one of claims 5 to 9, characterized in that The chimeric antigen receptor further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor; Preferably, the signal peptide is derived from the signal peptide of at least one of the following proteins: HLA-A, CD8α, CD33, Igκ, IL-2 and GM-CSFRα; More preferably, the signal peptide is derived from the signal peptide of CD8α.

11. An isolated nucleic acid, characterized in that The nucleic acid encodes the chimeric antigen receptor according to any one of claims 5 to 10.

12. A carrier, characterized in that The vector comprises the isolated nucleic acid of claim 11; Preferably, the vector is selected from at least one of lipid nanoparticles, virus-like particles, adenovirus, adeno-associated virus, lentiviral vector and retroviral vector; More preferably, the vector is a lentiviral vector or a retroviral vector.

13. A targeting vector, characterized in that: The surface of the targeting carrier comprises a targeting molecule, and the targeting molecule comprises the anti-CD79B single-domain antibody according to any one of claims 1-3.

14. The targeting vector according to claim 13, characterized in that The targeting vector is a lentiviral vector or a retroviral vector, the targeting molecule further comprises a transmembrane region, the anti-CD79B single-domain antibody is directly or indirectly connected to the transmembrane region and exposed on the surface of the lentiviral vector or the retroviral vector; Preferably, the transmembrane region is derived from the transmembrane region of any of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcεRIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; More preferably, the transmembrane region is derived from the transmembrane region of CD8α.

15. The targeting vector according to claim 14, characterized in that The targeting molecule further comprises a connecting domain, and the anti-CD79B single-domain antibody is indirectly connected to the transmembrane region via the connecting domain; Preferably, the linking domain is selected from: (i) an immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge region; (ii) a hinge region selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (iii) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and (iv) a stem region of a type II C-lectin, wherein the type II C-lectin is selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; More preferably, the connecting domain is the hinge region of CD8α.

16. An engineered immune effector cell, characterized in that: The engineered immune effector cell comprises the chimeric antigen receptor of any one of claims 5-10 or the nucleic acid of claim 11; Preferably, the engineered immune effector cells are selected from at least one of T cells, B cells, NK cells, macrophages, dendritic cells and induced pluripotent stem cells; More preferably, the engineered immune effector cells are engineered T cells.

17. The engineered immune effector cell according to claim 16, characterized in that The engineered immune effector cells are engineered T cells that express the chimeric antigen receptor.

18. A composition, characterized in that The composition comprises a pharmaceutically acceptable excipient or carrier and any one of the following ingredients: (a) an anti-CD79B single domain antibody as described in any one of claims 1-3, (b) a chimeric antigen receptor as described in any one of claims 5-10, or (c) an engineered immune effector cell as described in claim 16 or 17.

19. A method for treating a disease in a subject, characterized in that: Administering to a subject a therapeutically effective amount of the anti-CD79B single domain antibody of any one of claims 1 to 3, the engineered immune effector cell of claim 16 or 17, or the composition of claim 18; Preferably, the disease is a B cell-related disease and / or a CD79B-related disease; More preferably, the disease is a B cell-related and / or CD79B-related malignancy; Still further preferably, the B cell-related malignancy is B cell leukemia or B cell lymphoma The B cell leukemia or B cell lymphoma is selected from the group consisting of marginal zone lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B cell lymphoma, small lymphocytic lymphoma, B cell prolymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B lymphocytic leukemia, non-Hodgkin's lymphoma, high grade B cell lymphoma and multiple myeloma.

20. Use of the anti-CD79B single domain antibody according to any one of claims 1 to 3, the chimeric antigen receptor according to any one of claims 5 to 10, the vector according to claim 12, the engineered immune effector cell according to claim 16 or 17, or the composition according to claim 18 in the preparation of a drug for treating B cell-related and / or CD79B-related malignant tumors, B cell-related and / or CD79B-related autoimmune diseases and / or inflammatory diseases; Preferably, the B cell-related malignancy is a B cell leukemia or a B cell lymphoma, and the B cell leukemia or the B cell lymphoma is selected from the group consisting of marginal zone lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B cell lymphoma, small lymphocytic lymphoma, B cell prolymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B lymphocytic leukemia, non-Hodgkin's lymphoma, high grade B cell lymphoma and multiple myeloma.

21. A reagent for detecting CD79B protein or a functional domain containing its antigenic determinants (Epitopes), characterized in that: The reagent comprises the anti-CD79B single domain antibody according to any one of claims 1 to 3; Preferably, the CD79B protein is human CD79B protein; More preferably, the reagent further comprises a modified portion connected to the anti-CD79B single domain antibody, The modified moiety includes a detectable label or a therapeutic agent; More preferably, the detectable label comprises at least one of an enzyme, a label protein, a radionuclide, a fluorescent dye, a luminescent substance and biotin.

22. A method for detecting CD79B protein or a functional domain containing its antigenic determinant for non-diagnostic purposes, characterized in that: The method comprises obtaining a sample suspected of containing CD79B protein or a functional domain containing an antigenic determinant thereof, contacting the sample with the anti-CD79B single domain antibody of any one of claims 1 to 3 or the reagent of claim 21 to form an antibody-antigen complex, and detecting the presence of the antibody-antigen complex; Preferably, the CD79B protein is human CD79B protein.

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