Anti-fcrh5 nanobodies and uses thereof

Abstract

The present application relates to the technical field of nanobody, in particular to anti-FcRH5 nanobody and its application. The present application provides a kind of anti-FcRH5 nanobody with high affinity and specificity, which is based on the single domain heavy chain variable region structure of camelid origin, with the advantages of small molecular weight, high stability, strong tissue penetration, easy to be engineered and the like. The nanobody specifically recognizes and binds to the target point highly expressed and stably expressed on the surface of multiple myeloma cells through its complementarity determining region. The nanobody of the present application can be used as a core recognition element to construct a variety of therapeutic or diagnostic tools such as chimeric antigen receptor T cells, bispecific antibodies, antibody drug conjugates or immunodetection probes. The technical scheme can solve the technical problem that there is a lack of high-quality, functionally clear anti-FcRH5 antibody in the prior art. The nanobody of the present application has significant clinical transformation and industrialization advantages, and promotes the treatment of multiple myeloma to multiple target points.

Description

Technical Field

[0001] This case is a divisional application of the invention patent with application number CN202511982813.X. The specific information of the parent case is as follows: application date: December 26, 2025; title: Anti-FcRH5 nanobody and its application; applicant: Chongqing Tianyimei Life Science Co., Ltd.; application type: invention patent.

[0002] This invention relates to the field of nanobody technology, specifically to anti-FcRH5 nanobodies and their applications. Background Technology

[0003] Multiple myeloma (MM) is the second most common hematologic malignancy, accounting for 10-15% of all hematologic malignancies. It primarily affects the elderly and is an incurable plasma cell malignancy originating in the bone marrow, characterized by the malignant proliferation of monoclonal terminally differentiated plasma cells in the bone marrow or extramedullary space. It leads to end-organ damage and corresponding clinical features including hypercalcemia, renal failure, anemia, and osteolytic bone destruction. In recent years, although the use of immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs), histone deacetylase inhibitors, and monoclonal antibodies has significantly improved the prognosis of MM patients, the relapse / refractory (R / R) rate remains as high as 70-80%, and the 5-year survival rate is less than 50%. Patients ultimately face treatment difficulties due to drug resistance caused by altered drug targets, overexpression of drug efflux pumps, the tumor immunosuppressive microenvironment, immune cell dysfunction, and antigen loss.

[0004] FcRH5 (Fc receptor homolog 5, also known as IRTA2) is an Fc receptor homolog belonging to the immunoglobulin superfamily. It begins to be expressed in early B cells and is fully expressed only in mature B cells. It can also promote B cell proliferation and the expression of different B cell antibody types after antigen exposure. It is highly expressed in malignant plasma cells; for example, in patients with multiple myeloma, more than 78.57% of patients have more than 50% of their bone marrow plasma cells expressing FcRH5. Compared to B cell maturation antigen (BCMA), FcRH5 expression is more stable in multiple myeloma (MM) cells. Preclinical studies have shown that its antigen loss rate is significantly lower than BCMA, and it is associated with disease progression and poor prognosis. Its expression is extremely low in normal tissues, making it an ideal target for MM treatment.

[0005] However, the lack of effective antibody products targeting FcRH5 in current technologies results in significant unmet clinical needs in the treatment of multiple myeloma (MM). For patients with relapsed or refractory MM who have failed BCMA-targeted therapy, alternative treatments could have been developed based on the advantages of stable FcRH5 expression and low antigen loss rate. However, the absence of effective antibody products leaves them in a drug-free predicament, forcing them to rely on traditional chemotherapy with limited efficacy, significantly shortening their survival. Novel targeted therapies based on FcRH5, such as bispecific antibodies and antibody-drug conjugates (ADCs), all require high-quality antibodies as a foundation. The lack of current technology directly hinders the development of these therapies, delaying the technological iteration of MM treatment from BCMA single-target dependence to multi-target synergy. Therefore, a breakthrough is urgently needed across the entire chain from basic research to clinical translation to develop high-quality, functionally defined anti-FcRH5 antibodies, and to build a diversified targeted therapy system based on these antibodies. Summary of the Invention

[0006] The present invention aims to provide anti-FcRH5 nanobodies to solve the technical problem of the lack of high-quality, functionally defined anti-FcRH5 antibodies in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The anti-FcRH5 nanobody has an amino acid sequence of CDR1 that is any one of SEQ ID NO.1, 10, and 19; or, any one of amino acid sequences that differs from the sequence of SEQ ID NO.1, 10, or 19 by 4, 3, 2, or 1 amino acid. The amino acid sequence of its CDR2 is any one of SEQ ID NO.2, 11 and 20; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO.2, 11 or 20 by 4, 3, 2 or 1 amino acid; The amino acid sequence of its CDR3 is any one of SEQ ID NO.3, 12 and 21; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO.3, 12 or 21 by 4, 3, 2 or 1 amino acids.

[0008] Furthermore, the amino acid sequence of its CDR1 is SEQ ID NO.1; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO.1 by 4, 3, 2 or 1 amino acids; the amino acid sequence of its CDR2 is SEQ ID NO.2; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO.2 by 4, 3, 2 or 1 amino acids; the amino acid sequence of its CDR3 is SEQ ID NO.3; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO.3 by 4, 3, 2 or 1 amino acids.

[0009] Furthermore, the amino acid sequence of its CDR1 is SEQ ID NO.10; or, any one of the amino acid sequences that differ from the sequence of SEQ ID NO.10 by 4, 3, 2 or 1 amino acids; the amino acid sequence of its CDR2 is SEQ ID NO.11; or, any one of the amino acid sequences that differ from the sequence of SEQ ID NO.11 by 4, 3, 2 or 1 amino acids; the amino acid sequence of its CDR3 is SEQ ID NO.12; or, any one of the amino acid sequences that differ from the sequence of SEQ ID NO.12 by 4, 3, 2 or 1 amino acids.

[0010] Furthermore, the amino acid sequence of its CDR1 is SEQ ID NO.19; or, any one of the amino acid sequences that differ from the sequence of SEQ ID NO.19 by 4, 3, 2 or 1 amino acids; the amino acid sequence of its CDR2 is SEQ ID NO.20; or, any one of the amino acid sequences that differ from the sequence of SEQ ID NO.20 by 4, 3, 2 or 1 amino acids; the amino acid sequence of its CDR3 is SEQ ID NO.21; or, any one of the amino acid sequences that differ from the sequence of SEQ ID NO.21 by 4, 3, 2 or 1 amino acids.

[0011] Furthermore, the amino acid sequence of its FR1 is any one of SEQ ID NO.4, 13 and 22; or, any one of the amino acid sequences that differ from the sequence of SEQ ID NO.4, 13 or 22 by 4, 3, 2 or 1 amino acid; The amino acid sequence of its FR2 is any one of SEQ ID NO.5, 14 and 23; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO.5, 14 or 23 by 4, 3, 2 or 1 amino acid; The amino acid sequence of its FR3 is any one of SEQ ID NO. 6, 15 and 24; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO. 6, 15 or 24 by 4, 3, 2 or 1 amino acids. The amino acid sequence of its FR4 is any one of SEQ ID NO.7, 16 and 25; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO.7, 16 or 25 by 4, 3, 2 or 1 amino acid.

[0012] Furthermore, its amino acid sequence is any one of SEQ ID NO. 8, 17 and 26; or, any one of the amino acid sequences that differs from the sequence of SEQ ID NO. 8, 17 or 26 by 4, 3, 2 or 1 amino acid.

[0013] This technical solution also provides the application of anti-FcRH5 nanobody in the preparation of products for the treatment of multiple myeloma.

[0014] This technical solution provides an anti-FcRH5 antibody, which contains at least one anti-FcRH5 nanobody.

[0015] Traditional antibodies, represented by IgG, consist of heavy and light chains, and their antigen recognition relies on the synergistic action of six complementarity-determining regions (CDRs) on the variable region (VH) of the heavy chain and the variable region (VL) of the light chain. Nanobodies, on the other hand, contain only the heavy chain variable region (VHH) and are the smallest naturally occurring functional antigen-binding fragments. The nanobodies in this scheme can serve as heavy chain variable region components, assembling with other fragments to form traditional antibodies. The term "antibody" as used herein refers to traditional antibodies containing the nanobody chains described in this scheme.

[0016] This technical solution also provides a CAR-engineered immune cell, wherein the immune cell expresses a chimeric antigen receptor; the antigen-binding domain of the chimeric antigen receptor includes an anti-FcRH5 nanobody; and the immune cell is a T cell or an NK cell. Preferably, the chimeric antigen receptor comprises the following sequentially connected parts: a signal peptide, a single-domain antibody, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3zeta intracellular region. The nucleotide sequence of the signal peptide is SEQ ID NO.28; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.28, and whose translated protein sequence differs from the translated protein sequence of SEQ ID NO.28 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the CD8 hinge region is SEQ ID NO.29; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.29, and the translated protein sequence differs from the translated protein sequence of SEQ ID NO.29 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.30; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.30, and the translated protein sequence differs from the translated protein sequence of SEQ ID NO.30 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the CD28 intracellular region is SEQ ID NO.31; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.31, and the translated protein sequence differs from the translated protein sequence of SEQ ID NO.31 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the CD3zeta intracellular region is SEQ ID NO.32; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.32, and whose translated protein sequence differs from the protein sequence translated from SEQ ID NO.32 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the single-domain antibody is any one of SEQ ID NO. 9, 18 and 27; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO. 9, 18 or 27, and the translated protein sequence differs from SEQ ID NO. 8, 17 or 26 by only 4, 3, 2 or 1 amino acids.

[0017] This technical solution also provides a chimeric antigen receptor fusion protein, comprising the following sequentially connected parts: a signal peptide, a single-domain antibody, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3zeta intracellular region. The nucleotide sequence of the signal peptide is SEQ ID NO.28; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.28, and whose translated protein sequence differs from the translated protein sequence of SEQ ID NO.28 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the CD8 hinge region is SEQ ID NO.29; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.29, and the translated protein sequence differs from the translated protein sequence of SEQ ID NO.29 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.30; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.30, and the translated protein sequence differs from the translated protein sequence of SEQ ID NO.30 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the CD28 intracellular region is SEQ ID NO.31; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.31, and the translated protein sequence differs from the translated protein sequence of SEQ ID NO.31 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the CD3zeta intracellular region is SEQ ID NO.32; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO.32, and whose translated protein sequence differs from the protein sequence translated from SEQ ID NO.32 by only 4, 3, 2 or 1 amino acids. The nucleotide sequence of the single-domain antibody is any one of SEQ ID NO. 9, 18 and 27; or, it is a nucleotide sequence that has ≥90% homology with the nucleotide sequence shown in SEQ ID NO. 9, 18 or 27, and the translated protein sequence differs from SEQ ID NO. 8, 17 or 26 by only 4, 3, 2 or 1 amino acids.

[0018] In summary, the technical principle of this solution is as follows: This invention provides a class of anti-FcRH5 nanobodies with high affinity and specificity. Based on a single-domain heavy chain variable region (VHH) structure derived from camelids, these nanobodies consist of only one variable domain and possess advantages such as small molecular weight, high stability, strong tissue penetration, and ease of engineering modification. The nanobodies specifically recognize and bind to FcRH5 (Fc receptorhomolog 5), a target highly expressed and stably expressed on the surface of multiple myeloma (MM) cells, through their complementarity-determining regions (CDR1, CDR2, and CDR3), thereby mediating downstream immune effects or serving as functional modules embedded in various therapeutic platforms.

[0019] In terms of technical principle, the anti-FcRH5 nanobody disclosed in this invention can precisely target the FcRH5 antigen on the surface of tumor cells, while this antigen is almost not expressed in normal tissues, significantly reducing the risk of off-target toxicity. More importantly, compared with the current mainstream target B cell maturation antigen (BCMA), FcRH5 exhibits higher expression stability and a lower antigen loss rate in relapsed / refractory MM patients, effectively avoiding the problem of treatment failure caused by target escape. Based on this, the nanobody of this invention can be used as a core recognition element to construct various therapeutic or diagnostic tools such as chimeric antigen receptor T cells (CAR-T), chimeric antigen receptor NK cells (CAR-NK), bispecific antibodies, antibody-drug conjugates (ADCs), or immunodetection probes.

[0020] The beneficial effects of this technical solution are as follows: (1) Filling the technological gap of high-quality anti-FcRH5 antibodies: In view of the lack of effective anti-FcRH5 antibodies in the existing technology, the present invention provides anti-FcRH5 nanobodies with well-defined sequences and stable functions. The optimized combination of its CDR and FR regions ensures high specificity binding to the FcRH5 target, solving the dilemma of no available drugs for relapsed and refractory multiple myeloma after failure of BCMA targeted therapy, and providing a core foundation for multi-target synergistic therapy.

[0021] (2) Improve the efficacy and stability of targeted therapy: FcRH5 is stably expressed in multiple myeloma cells and its antigen loss rate is significantly lower than that of BCMA. This invention uses it as a target and combines the advantages of nanobodies in recognizing cryptic epitopes to effectively avoid the relapse problem caused by antigen escape in existing CAR-T therapies. At the same time, nanobodies have structural stability that is resistant to high temperature, acid and alkali and resistant to protease degradation, ensuring that antibodies, CAR-T cells, antibodies and immune conjugates maintain their activity in the complex environment in vivo and prolong the duration of action.

[0022] (3) Optimize treatment safety and reduce the risk of side effects: FcRH5 is expressed at low levels in normal tissues. The high specificity of the nanobody of the present invention to FcRH5 can reduce cross-reactivity with normal cells. The nanobody has a small molecular weight and low immunogenicity (high homology with human VH region), which can reduce the body's immune rejection response and improve treatment tolerance.

[0023] (4) Nanobodies can construct diversified treatment systems: The anti-FcRH5 nanobody of the present invention can be used as a single functional unit, or assembled into traditional antibodies, CAR-T cells, immunoconjugates and other product forms to meet different needs such as targeted therapy and diagnostic testing for multiple myeloma. Among them, CAR-T cells can be used for salvage therapy for relapsed and refractory patients, and immunoconjugates can be used for precision chemotherapy. The synergy of multiple products can achieve full-cycle coverage from first-line treatment to salvage therapy after relapse, and promote the technological iteration of multiple myeloma treatment from single-target dependence to multi-target synergy.

[0024] (5) Advantages in industrialization and clinical translation: Nanobodies can be efficiently expressed through bioengineering technology, with production costs significantly lower than traditional four-chain antibodies. Furthermore, they offer high flexibility in genetic engineering modification, allowing for rapid optimization of properties such as affinity and half-life. The lentiviral packaging system and T-cell transfection protocol used in CAR-T cell construction are mature and controllable, facilitating large-scale production. All core sequences of the nanobodies in this protocol are well-defined and reproducible, providing a solid foundation for subsequent clinical translation and industrial applications.

[0025] In summary, this invention provides a class of anti-FcRH5 nanobodies based on the VHH structure of the Camelidae family, exhibiting high affinity and specificity. Their single-domain structure provides advantages such as small molecular weight, high stability, excellent tissue penetration, and ease of engineering modification. The CDR region precisely recognizes and binds to the highly expressed and stable FcRH5 target on MM cells. Simultaneously, their low immunogenicity and low off-target toxicity enhance therapeutic safety, making them suitable as core components for constructing diverse therapeutic and diagnostic tools such as CAR-T cells, CAR-NK cells, ADCs, and bispecific antibodies. This nanobody approach, relying on efficient expression, low cost, and mature large-scale production technology, possesses significant advantages in clinical translation and industrialization, driving the iterative development of MM treatment towards multi-target synergistic approaches. Attached Figure Description

[0026] Figure 1 The titer of the antiserum obtained from immunizing alpacas with the FcRH5 antigen protein in Example 1 is shown.

[0027] Figure 2 The results of bacterial PCR (polymerase chain reaction) identification of the alpaca nanobody library in Example 2 are shown (1-24 represent the 24 nanobody sequences H1-H24 obtained in this study).

[0028] Figure 3 The affinity test results for the eukaryotically expressed nanobody in Example 3 are shown in A: H3-VHH-Fc.

[0029] Figure 4 The affinity test results for the eukaryotically expressed nanobody in Example 3 are shown in B: H4-VHH-Fc.

[0030] Figure 5 The affinity test results for the eukaryotically expressed nanobody in Example 3 are shown (C: H10-VHH-Fc).

[0031] Figure 6 The statistical results of the 24-hour killing rate of three types of CAR-T cells targeting FcRH5 against target cells H929 in Example 4 are as follows (H3: CAR-T cells expressing H3 nanobodies; H4: CAR-T cells expressing H4 nanobodies; H10: CAR-T cells expressing H10 nanobodies; PC: positive control; MOCK: negative control; mean ± standard deviation, n=3).

[0032] Figure 7 The statistical results of the killing rate of three types of CAR-NK cells targeting FcRH5 against target cells H929 over 4 hours in Example 5 are as follows (H3: CAR-NK cells expressing H3 nanobodies; H4: CAR-NK cells expressing H4 nanobodies; H10: CAR-NK cells expressing H10 nanobodies; PC: positive control; MOCK: negative control; mean ± standard deviation, n=3). Detailed Implementation

[0033] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0034] To facilitate understanding of this technical solution, the relevant technical terms and concepts are explained in detail below: Nanobodies (Nb) are a class of single-domain antibodies derived from camel species (such as alpacas and camels), consisting solely of the variable heavy chain region (VHH). The VHH structure, cloned and expressed independently, possesses structural stability and antigen-binding activity comparable to the original heavy chain antibody. It is the smallest known unit capable of binding to target antigens, with a molecular weight of 15 kDa and a volume only one-tenth that of a traditional antibody; hence, it is also known as a nanobody.

[0035] Antibodies (also known as immunoglobulins, Ig) are proteins produced by the proliferation and differentiation of B lymphocytes after stimulation by antigens. They specifically recognize and bind to antigens and are core molecules in the humoral immune function of the immune system. Antibodies are generally composed of two heavy chains and two light chains linked by disulfide bonds, with a typical Y-shaped four-chain structure (represented by IgG), containing an antigen-binding domain (Fab region) and an effector domain (Fc region). Through the antigen-binding domain (CDR region of VH and VL), they specifically recognize antigens such as bacteria and viruses, subsequently mediating effects such as toxin neutralization, complement activation, and binding to immune cells to eliminate pathogens or abnormal cells. Based on the amino acid composition of the heavy chain constant region, they can be divided into five classes: IgG, IgA, IgM, IgD, and IgE.

[0036] Heavy chain (H chain): Two identical polypeptide chains in an antibody molecule, with a molecular weight of approximately 50 kDa (twice that of the light chain). The type determines the antibody class, which is divided into five classes: γ, α, μ, δ, and ε, corresponding to IgG, IgA, IgM, IgD, and IgE, respectively. Each heavy chain contains an N-terminal variable region (VH) and a C-terminal constant region (CH). The constant region mediates antibody effector functions (such as activating complement and binding to immune cells).

[0037] Light chain (L chain): Two identical small polypeptide chains in the antibody molecule, with a molecular weight of approximately 25 kDa. Each light chain contains an N-terminal variable region (VL) and a C-terminal constant region (CH). The VL and VH work together to form the antigen binding site.

[0038] VH (Variable Region of Heavy Chain): A variable structural domain at the N-terminus of the heavy chain. It is one of the core units of antigen binding and must bind to VL to form a functional antigen-binding pocket. It contains 3 complementarity-determining regions (CDR1-CDR3) and 4 framework regions (FR1-FR4). The CDR regions directly participate in antigen recognition.

[0039] VL (Variable Region of Light Chain): A variable domain at the N-terminus of the light chain. Together with VH, it forms the antigen-binding region of the antibody, enhancing the specificity and affinity of antigen binding. Its structure is similar to VH, containing 3 CDR regions and 4 FR regions, assisting VH in recognizing antigenic epitopes.

[0040] CDR (Complementarity Determining Region): Highly variable regions in the VH and VL sequences, with three CDRs in each class (CDR1, CDR2, and CDR3). These six CDRs together form an antigen-binding pocket, directly binding complementary to the antigen epitope and determining antibody specificity.

[0041] FR (Framework Region): A conserved sequence between the CDR regions in VH and VL, with four FRs (FR1-FR4) in each type of variable region. It provides structural support for the CDR region, ensuring it can be properly exposed and bind to the antigen.

[0042] Fab (Antigen-binding Fragment): A fragment produced after an antibody is digested with papain. Each antibody molecule can produce two identical Fab fragments, containing complete VH, VL, CL, and CH1 domains. It is solely responsible for antigen binding (preserving antibody specificity) and has no Fc-related effector function.

[0043] Fc (crystallizable fragment): A fragment produced after antibody digestion with papain. It consists of CH2 and CH3 domains of two heavy chains, is crystallizable, and has no antigen-binding activity. Its core function is to mediate antibody effects, such as binding to Fc receptors on immune cells, activating complement, and determining antibody half-life.

[0044] VHH (Variable Domain of Heavy Chain-only Antibody) is the only antigen-binding domain in heavy chain antibodies (HCAbs) from camels (alpacas, camels, etc.), and is the smallest naturally occurring functional antigen-binding fragment. It consists of a framework region (FR1-FR4) and a complementarity-determining region (CDR1-CDR3).

[0045] Immunoconjugates are complexes formed by chemically linking an antibody to another "effect molecule" with a specific function.

[0046] M13KO7 helper phage is a specialized helper phage modified from wild-type M13 filamentous phage. It provides structural proteins and packaging signals required for phage particle assembly to support the formation of recombinant phages.

[0047] CAR-T cells (Chimeric Antigen Receptor T-Cells) are a novel immunocellular therapy that uses genetic engineering to modify T lymphocytes to express chimeric antigen receptors (CARs). In other words, they are T cells that express chimeric antigen receptors. CARs consist of an antigen-binding domain (e.g., monoclonal antibody scFv, nanobody VHH), a transmembrane domain, and an intracellular signaling domain. CARs can specifically recognize tumor cell surface antigens, activate T cell killing activity, and achieve precise targeted elimination of tumor cells.

[0048] CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cells) are novel engineered immune cells obtained by modifying natural killer cells (NK cells) using genetic engineering technology. The core feature is the introduction of chimeric antigen receptors (CARs) into the NK cell genome, enabling them to specifically recognize target cells (such as tumor cells) and kill them efficiently, while retaining the natural immune advantages of NK cells.

[0049] PBMCs (Peripheral Blood Mononuclear Cells) are a population of cells with single nuclei isolated from peripheral venous blood. They mainly consist of lymphocytes (T cells, B cells, NK cells), monocytes, a small number of dendritic cells, and do not contain erythrocytes or multinucleated granulocytes. They can be used for immune cell research (e.g., T cell isolation, starting material for CAR-T cell preparation), cytokine detection, and immune function assessment.

[0050] 293T cells (Human Embryonic Kidney 293T Cell): An adherent cell line derived from human embryonic kidney 293 cells, obtained through transfection with the SV40 large T antigen gene. They exhibit adherent growth, rapid proliferation, and extremely high transfection efficiency (commonly transfected via liposomes and viruses). They stably express the SV40 large T antigen, promoting the amplification of plasmids containing the SV40 origin of replication, facilitating the production of recombinant proteins or viral vectors (such as lentiviruses). Their main applications include recombinant protein expression, viral vector packaging (commonly used in CAR-T cell preparation), and gene function validation.

[0051] 293F cells (Human Embryonic Kidney 293F Cell): A suspension-derived cell line of 293 cells. It exhibits high growth density, good compatibility with serum-free culture, and is suitable for large-scale production of recombinant proteins (such as antibodies and cytokines). Its main applications are industrial-grade recombinant protein expression and protein raw material production in biopharmaceuticals.

[0052] H929 cells are a human multiple myeloma cell line widely used in research on hematologic malignancies, particularly in the pathogenesis, drug screening, and development of treatment strategies for multiple myeloma.

[0053] Example 1: Preparation of FcRH5 antigen and alpaca anti-FcRH5 serum (1) Preparation of FcRH5 antigen Based on the FcRH5 protein and gene sequence information on the NCBI website (NCBI ID: 83416), we analyzed and designed a method to effectively induce alpacas to produce a specific antigen against the extracellular fragment of FcRH5. A His-tag (histidine tag, forming a fusion protein abbreviated as hFcRH5-his) was linked to the C-terminus for subsequent purification and detection. Following standard molecular biology procedures, an expression vector integrating the corresponding gene fragment of hFcRH5-his was prepared for 293F cell transfection and antigen protein expression. The recombinant protein was purified using conventional nickel affinity chromatography and validated by SDS-PAGE gel chromatography to obtain the FcRH5 antigen protein, which was used for serum titer assays. Simultaneously, we constructed an AAV9 expression plasmid expressing the specific extracellular fragment of human FcRH5 protein, transiently transfected into 293F cells for expression validation. After successful validation, the FcRH5-AAV9 virus was packaged, purified, and concentrated.

[0054] (2) Alpaca immunity and the acquisition of antiserum Immunization dosage: 1 × 10⁹ FcRH5 AAV9 virus per injection 12 vg.

[0055] First immunization: The sample was emulsified by mixing Freund's complete adjuvant with the sample at a volume ratio of 1:1 and then injected subcutaneously at multiple points.

[0056] The 2nd, 3rd, 4th, and 5th immunizations were performed by subcutaneous injection of a mixture of Freund's incomplete adjuvant and the sample at a 1:1 volume ratio. On days 21, 35, 49, and 63, booster immunizations were administered using FcRH5 AAV9 (adeno-associated virus type 9) virus and Freund's incomplete adjuvant at a 1:1 volume ratio. After the 4th and 5th immunizations, blood samples were collected to measure antiserum titers. One week after the 5th immunization, 100 mL of blood was collected for the construction of a phage antibody library.

[0057] Serum titers were determined using ELISA (Enzyme-Linked Immunosorbent Assay): On day 1, the test antigen was diluted to 1.0 μg / mL with antigen dilution buffer, and then 100 μL of antigen was added to each well. After sealing, the wells were incubated overnight at 4°C. On day 2, the supernatant was discarded; 200 μL of blocking buffer was added to each well and incubated at 37°C for 30 min, ready for use (the supernatant was discarded before use). Serum and negative control samples were serially diluted 10-fold using a 10-fold serial dilution method. 6 Prepare 10 times. 2 Up to 10 6Five dilutions of samples were prepared. 100 μL of sample and negative control were added to appropriate wells, sealed, and incubated at 37°C for 1 h. The supernatant was discarded, and the sample was washed three times with washing buffer. 100 μL of HRP (horseradish peroxidase)-labeled goat anti-alpaca antibody IgG H&L (H&L refers to heavy and light chains, product number: SPAB02) was added to each well. This antibody was diluted 1:15000 before use. The sample was incubated at 37°C for 40 min, the supernatant was discarded, and the sample was washed five times with washing buffer. 100 μL of TMB (tetramethylbenzidine) one-step substrate reagent (mixed solutions A and B) was added to each well. The sample was incubated at room temperature for 15 min with gentle shaking. 50 μL of stop solution was added to each well, and the reading was taken quickly at 450 nm. The antigen dilution buffer and blocking buffer were standard reagents for serum titer ELISA testing and were commercially available; details are omitted here.

[0058] Principles for calculating serum positive titers: At the same dilution factor, the sample OD... 450 A value (optical density at 450 nm) ≥ 2.1 times that of the negative control is considered positive. If the absorbance value of the negative control is < 0.05, it is calculated as 0.05. Results are as follows... Figure 1 As shown, the effective titer of the antiserum after 5 immunizations was 10. 5 Therefore, this antigen can induce alpacas to produce high-titer antiserum specifically targeting the human FcRH5 antigen protein.

[0059] Example 2: Alpaca phage library and screening 100 mL of peripheral blood was collected from immunized alpacas. Peripheral blood mononuclear cells (PBMCs) were obtained by separating the blood using lymphocyte separation medium. RNA (ribonucleic acid) was extracted from the PBMCs according to the instructions of the QIAGEN Plus MiniRNA Extraction Kit. cDNA (complementary deoxyribonucleic acid) reverse transcription was performed on the extracted RNA according to the instructions of the Invitrogen Super III First-Strand Synthesis System Extraction Kit.

[0060] Next, FcRH5 nanobody amplification was performed. The reverse cDNA library was amplified using a gene template (two rounds of amplification), followed by the construction of the FcRH5 nanobody display vector. The PCR product was double-digested with Pst-I-HF and Not-I-HF enzymes, then cloned into the pMECS phage display vector. The digested products were recovered using a PCR product recovery kit according to the manufacturer's instructions, and the concentration of the recovered product was measured for subsequent experiments.

[0061] Next, the enzyme digestion and ligation products were transformed into TG1 competent cells, and a phage antibody library was obtained. Then, VHH (nanobody) phage antibody library rescue was performed. After determining the recombinant phage titer, the recombinant phages were screened. The screening process consisted of three rounds, as detailed below: Antigen coating: Dilute FcRH5 antigen protein to 4 μg / mL with PBS buffer. Take a 96-well microplate, select 3 replicates, add 100 μL (400 ng / well) to each well, and coat overnight at 4°C. PBS is used as a negative control.

[0062] Blocking: Discard the coating solution, add 150 μL of 2% skim milk powder to each well, and block at room temperature for 1 hour.

[0063] Incubate bacteriophages: Wash four times with PBST, take the bacteriophage solution, and dilute to 5 × 10⁻⁶ with 2% milk powder. 11 Add pfu / mL to the microplate, 100 μL / well, and incubate at room temperature for 2 h.

[0064] Elution: Discard the phage sample, wash 10 times with PBST (phosphate buffer containing Tween), then wash 5 times with PBS (phosphate buffer). Add 100 μL of freshly prepared 0.1 M triethylamine to each well, let stand at room temperature for 10 min, aspirate the eluent and quickly neutralize with an equal volume of 1 M Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride buffer, pH 7.4.

[0065] Eluent phage titer determination: The enrichment effect of specific VHH recombinant phages was evaluated by detecting the titer of recombinant phages in each round of elution. The enrichment effect was assessed by counting enriched clones (Table 1).

[0066] Infection: Take 400 μL of eluent and infect 4 mL of logarithmic TG1 cells. Shake well and incubate at 37°C for 30 min. Add 16 mL of 2×YT medium (containing ampicillin and glucose) and culture at 37°C and 200 rpm until OD500. 600 It reaches 0.6-0.8.

[0067] Rescue: Add 20 μL of M13KO7 helper phage to the culture medium, shake well, let stand at room temperature for 1 h, centrifuge at 2800g for 10 min, discard the supernatant, resuspend the cells in 100 mL of 2×YT medium (containing ampicillin and kanamycin), and incubate at 37℃ and 225 rpm for 14 h.

[0068] Next, the phage particles were concentrated and purified for the next round of screening. This process was repeated twice to complete the second and third rounds of screening. The results of the nanobody library screening are detailed in Table 1. The O / I ratio represents the ratio of the number of output phages to the number of input phages, used to quantify the enrichment efficiency of specifically binding phages in each round of screening.

[0069] Table 1: Screening Results of Nanobody (VHH) Library

[0070] To further verify the positive phage rate binding to human FcRH5-VHH (FcRH5 nanobody) protein in the enriched library, 192 clones were selected from the third-round enriched library for single phage ELISA detection. The results showed that most of the phage clones in the third-round library were positive, and the OD450 value / PBS control (P / N) was greater than 3.0 (Tables 2-1 and 2-2, underlined to indicate positive). A high-binding FcRH5-VHH phage library was successfully enriched through human FcRH5 protein screening.

[0071] Table 2-1: ELISA detection effect on phage library enrichment

[0072] Table 2-2: ELISA detection effect on phage library enrichment

[0073] Next, prokaryotic expression of the nanobodies was performed: 100 μL of LB medium (containing ampicillin and glucose) was added to each well of a 96-well cell culture plate. Simultaneously, 96 single colonies were randomly picked from the plates used in the third round of titer determination and added to the medium. The plates were incubated at 37°C and 200 rpm for 6 hours. 1 mL of TB medium was added to each well of four 24-well cell culture plates, and single-clone bacterial cultures were transferred to the plates at a 1:100 ratio. The plates were incubated at 37°C and 200 rpm until the logarithmic growth phase. IPTG was added to each well to a final concentration of 1 mM, and the plates were induced overnight at 37°C and 200 rpm. The cell culture plates were centrifuged at 4°C and 12,000 rpm for 2 minutes, the supernatant was discarded, and the cells were frozen at -80°C for 30 minutes. After the cells returned to room temperature, 500 μL of PBS was added to each well to resuspend the cells. The plates were then centrifuged at 4°C and 12,000 rpm for 2 minutes, and the supernatant was collected. The supernatant was the crude nanobodies.

[0074] Sequencing of nanobodies: Fractional bacterial cultures from each of the above-mentioned monoclonal strains were sequenced. Twenty-four clones were randomly selected for sequencing, successfully yielding 24 nanobodily sequences, of which 23 were non-repetitive antibody sequences. These 24 sequences were named H1-H24. Alignment results showed that most of the differential sequences were located in the CDR (complementarity-determining region) binding region. The constructed FcRH5-VHH phage antibody library had a volume of 5.7 × 10⁻⁶. 10 The library exhibited a diversity of 95.8% and an insertion rate of 100%. For PCR identification of the alpaca nanobody library using bacterial culture, please refer to [link to relevant documentation]. Figure 2 (Where lanes 1-24 represent H1-H24 respectively, targeting the amplification of the VHH fragment in the heavy chain variable region, with a positive insertion rate of 100%).

[0075] Of these sequences, H3, H4, and H10 (which exhibit stronger in vitro killing effects than other candidate sequences) Figure 2 The nanobodies located in lanes 3, 4, and 10 are the final selected nanobodies. For specific sequence information, please refer to Tables 3, 4, and 5.

[0076] Table 3: Sequence information of nanobody H3

[0077] Table 4: Sequence information of nanobody H4

[0078] Table 5: Sequence information of nanobody H10

[0079] Example 3: Construction of a eukaryotic expression library of nanobodies and expression of nanobodies To construct the recombinant expression plasmid, the nanobody sequence determined in Example 2 was recombined with the eukaryotic expression plasmid pcDNA3.4 using conventional molecular cloning techniques to form a eukaryotic protein expression plasmid, which was then transfected into 293F cells.

[0080] One day before transfection, count the normally cultured 293F cells, aspirate an appropriate amount of cell suspension and centrifuge (1000 rpm, 5 min), discard the supernatant, and resuspend in fresh culture medium (specifically Gibco). TM Expi293 TM Expression medium (containing 6 mM L-glutamine) resuspended to 1 × 10⁻⁶ 6Cells / mL (total volume 50mL), continue incubation at 37℃, 5% CO2, 125rpm overnight. The next day, prepare the transfection complex: add 2.5mL of culture medium and 50μg of plasmid DNA (eukaryotic protein expression plasmid) to a 15mL sterile centrifuge tube and vortex to mix. Add 2.5mL of culture medium and 150μL of liposome transfection reagent (Lipofectin) to the diluted DNA solution in another 15mL sterile centrifuge tube and vortex again to mix. After incubation at room temperature for 5 min, add the transfection complex to 45mL of 293F culture medium and continue incubation at 37℃, 5% CO2, 125rpm. After 4-5 days, centrifuge to collect the expressing cells and supernatant (1000rpm, 5min) for protein purification.

[0081] Purification was performed using nickel column affinity chromatography. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) showed successful purification of the FcRH5 nanobody, yielding three eukaryotic expression antibodies for VHH-Fc (FcRH5 Nb, FcRH5 nanobody): H3-VHH-Fc, H4-VHH-Fc, and H10-VHH-Fc (formed from H3, H4, or H10 nanobody + Fc sequence + His tag). The Fc sequence + His tag sequence is (SEQ ID NO. 33; the sequence of Fc sequence + His tag directly linked to the C-terminus of VHH of H3, H4, or H10 nanobody): EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH.

[0082] To detect the affinity of VHH-Fc for human FcRH5 protein, the detection antigen used is a specific functional region of the FCHR5 antigen protein, which carries a histidine (His) and avidin (Avi) tag. The sequence of the detection antigen is (SEQ ID NO.34, bold text indicates the histidine (His) and avidin (Avi) tag): VAVPVSRPVLTLRAPGTHAAVGDLLELHCEALRGSPLILYRFFHEDVTLGNRSSPSGGASLNLSLTAEHSGNYSCEADNGLGAQRSETVTLYITGLTANRSGPFATHHHHHHGLNDIFEAQKIEWHE.

[0083] The specific operation process of the detection method is as follows: On day 1, the detection antigen (a specific functional region of the FCHR5 antigen protein, tagged with histidine His and avidin Avi) was diluted to 2.0 μg / mL with antigen dilution buffer, and then 100 μL of antigen was added to each well. After sealing, the wells were incubated overnight at 4°C. On day 2, the supernatant was discarded; 200 μL of blocking buffer was added to each well and incubated at 37°C for 30 min, ready for use (the supernatant was discarded before use). The VHH-Fc antibody was uniformly diluted to 20 μg / mL according to the layout, starting with a 5-fold serial dilution to form one gradient, followed by 3-fold dilutions to form 14 gradients, resulting in 16 gradients of antibody protein dilution. More specifically, the 16 gradients, from highest to lowest concentration, were: initial concentration, 1 / 5 of the initial concentration, 1 / (5×3) of the initial concentration, 1 / (5×3) of the initial concentration, and so on. 2 Initial concentration, 1 / (5×3) 3 Initial concentration, 1 / (5×3) 4 Initial concentration, 1 / (5×3) 5 Initial concentration, 1 / (5×3) 6 Initial concentration, 1 / (5×3) 7 Initial concentration, 1 / (5×3) 8 Initial concentration, 1 / (5×3) 9 Initial concentration, 1 / (5×3) 10 Initial concentration, 1 / (5×3) 11 Initial concentration, 1 / (5×31) 12 Initial concentration, 1 / (5×3) 13 Initial concentration, 1 / (5×3) 14 Initial concentration.

[0084] Add 100 μL of sample to the appropriate wells, seal, and incubate at 37°C for 1 h. Discard the supernatant and wash twice with washing buffer. Add 100 μL of HRP-labeled secondary antibody (diluted with blocking buffer to 0.2 μg / mL) to each well and incubate at 37°C for 1 h. Discard the supernatant and wash five times with washing buffer. Add 100 μL of TMB (a mixture of solutions A and B) one-step substrate reagent to each well, incubate at room temperature in the dark for 15 min, and gently shake. Add 50 μL of stop solution to each well and quickly read the value at 450 nm. The antigen dilution buffer and blocking buffer are standard reagents for serum titer ELISA assays and are commercially available; details are omitted here. Calculation: EC (effective concentration, nM) = antibody concentration (g / L) / dilution factor / antibody molecular weight × 10 9 .

[0085] For detailed experimental results, please refer to Figure 3 , Figure 4 and Figure 5 The initial concentrations of H3-VHH-Fc, H4-VHH-Fc, and H10-VHH-Fc were all 0.02 mg / mL, and affinity tests were performed on all three for the same antigen protein. The EC50 of H3-VHH-Fc... 50 The half-maximal effective concentration (MCD) was 30.74 nM, and the affinity was 16.0373 nM; the EC50 of H4-VHH-Fc was... 50 The α-hydroxyl content is 12.98 nM, and the affinity is 38.896 nM; the EC50 of H10-VHH-Fc is... 50 The molecular weight is 30.72 nM, and the affinity is 16.3559 nM. Experimental data show that the EC50 of H4-VHH-Fc is... 50 The lowest concentration is required to achieve half the efficacy, while H3-VHH-Fc and H10-VHH-Fc have higher affinity for the detected antigen.

[0086] Example 4: Preparation and efficacy study of CAR-T The original lentiviral expression vector (empty vector) used was pALD (a product of ALDEVRON). GenScript was commissioned to synthesize the entire genome, including the chimeric antigen receptor CAR sequence and downstream co-stimulatory signal domain, which were then inserted into the pALD plasmid to obtain the pALD-EF1a-anti-FcRH5-28z expression vector. Sequencing confirmed its correctness.

[0087] The chimeric antigen receptor CAR (FcRH5 CAR) is constructed based on the FcRH5 antibody sequence. In addition to the FcRH5 nanobody sequence proposed in this protocol, the remaining sequences are conventional chimeric antigen receptor-related sequences in the prior art, including the following parts, in order from extracellular to intracellular: signal peptide (SEQ ID NO.28), single-domain antibody (one of the three nanobodies provided in this protocol, namely: SEQ ID NO.9, SEQ ID NO.18, or SEQ ID NO.27), CD8 hinge region (SEQ ID NO.29), CD28 transmembrane region (CD28 TM, SEQ ID NO.30), CD28 intracellular region (CD28 cyto, SEQ ID NO.31), and CD3zeta intracellular region (CD3zeta cyto, CD3ζ, SEQ ID NO.32). The above sequences are linked together from the 5' end to the 3' end (sequence information is detailed in Table 6), forming the CAR sequence (nucleotide sequence corresponding to the CAR fusion protein) of this embodiment. This sequence is integrated into the pALD plasmid using conventional molecular cloning methods to obtain the expression vector. The virus prepared from this expression vector expresses the CAR fusion protein after infecting T cells. The CAR fusion protein is a transmembrane protein that guides T cells to kill target cells. This protocol tested the efficacy of three CAR fusion proteins: a CAR fusion protein with anti-FcRH5 antibody H3, a CAR fusion protein with anti-FcRH5 antibody H4, and a CAR fusion protein with anti-FcRH5 antibody H10.

[0088] Table 6: Chimeric antigen receptor CAR sequence information (see Tables 3-5 for single-domain antibody information).

[0089] First, lentiviruses were prepared and seeded into 293T cells (day 0) in T25 culture flasks. On day 1, virus packaging was performed. In a DNA tube, 4.8 µg of transfer plasmid (pALD-EF1a-anti-FcRH5-28z expression vector), 2.7 µg of helper plasmid pLP1, 1.8 µg of helper plasmid pLP2, and 2.7 µg of helper plasmid pLP-VSVG were added, totaling 12 µg, and Opti-MEM medium was added to bring the volume to 100 µL. In a PEI (polyethyleneimine) tube, 36 µg of PEI was added, and Opti-MEM was added to bring the volume to 100 µL, mixing well with the DNA. The supernatant in the T25 culture flask was discarded, and 4.8 mL of fresh culture medium and the DNA-PEI complex (obtained by mixing the contents of the DNA and PEI tubes) were added to the flask. The flask was gently shaken back and forth and side to side to mix. The flask was then incubated at 37°C in a 5% CO2 incubator for 6 hours, after which the medium was changed. On day 3, after 48 hours of culture, the virus was harvested. The viral supernatant was collected (temporarily stored at 4°C), and cultured further with added culture medium. On day 4, the viral supernatant was collected again after 72 hours and combined with the 48-hour viral supernatant. After centrifugation at 1300g for 15 minutes (4°C), the mixture was filtered through a 0.45μm filter. 3 mL of Takara Lenti-X lentiviral concentrate was added to the supernatant and incubated overnight at 4°C. Centrifugation was then performed again at 1300g for 45 minutes (4°C). The supernatant was discarded, the precipitate was resuspended in PBS, and aliquoted to obtain lentivirus containing the expression vector (stored at -80°C).

[0090] Next, CAR-T cell preparation was performed. On day 0, PBMCs were resuscitated and initially activated. PBMCs were removed from liquid nitrogen and rapidly resuscitated in a 37°C water bath, centrifuged at 350g for 5 min, washed once with PBS, and incubated with anti-CD3 magnetic beads for 20 min. CD3 cells were then separated using a magnetic separation column. + T cells were resuspended and counted using CAR-T medium (Lonza X-vivo 15). Cells were seeded in T75 flasks, topped with 17 mL of CAR-T medium, and T cell activation and expansion reagent (T Cell TransAct) was added. TM170 μL of CAR-T cells were collected from T75 culture flasks and transferred to 50 mL centrifuge tubes. The cells were centrifuged at 350 g for 5 min, the supernatant was discarded, and the cells were resuspended in 6 mL of CAR-T medium. 1 mL of CAR-T medium was added to each well of a 24-well plate (6 wells total), with 200 μL of lentivirus and 8 μg / mL polybrene added to each well. The cells were centrifuged at 1500 g for 1.5 h at 32 °C. The cells were then transferred to T25 culture flasks, the medium was brought to 5 mL, and interleukin-2 (IL-2, 100 U / mL) was added. The transfected cells were then expanded and cultured, and the cells were used for cell killing experiments. The above procedure for preparing CAR-T cells is a standard method in existing technology. The difference between this protocol and existing technologies lies only in the sequence of the CAR fusion protein, resulting in T cells expressing a specific CAR fusion protein, which can effectively recognize and kill MM cells. This step yields CAR-T cells, including three types: CAR-T cells expressing H3 nanobodies, CAR-T cells expressing H4 nanobodies, and CAR-T cells expressing H10 nanobodies.

[0091] A co-incubation system was established using an E:T ratio of 2 (effect-to-target ratio). H929 cells (expressing luciferase) were used as target cells, and CAR-T cells were used as effector cells. Positive controls (PC: CAR-T cells with a positive control sequence), negative controls (MOCK: T cells without CAR gene modification), and a single-target cell group (H929 cells only) were also included. After 48 hours of co-culture, the cell culture plates were removed from the incubator and incubated at room temperature for 30 minutes to allow the temperature to equilibrate. An equal volume of Bio-Lite assay reagent, also equilibrated to room temperature, was added. Cells were incubated at room temperature for at least 3 minutes to allow for complete lysis before detection. Detection was performed using a microplate reader in chemiluminescence mode. The cell kill rate was calculated as: Kill rate % = (1 - RLU of the co-incubation group / RLU of the single-target cell group) × 100%, where RLU is the relative intensity of the fluorescence signal output by the detection device.

[0092] For detailed experimental results, please refer to Figure 6 The killing rate of CAR-T cells expressing H3 nanobodies was 90.65%, that of CAR-T cells expressing H4 nanobodies was 72.67%, and that of CAR-T cells expressing H10 nanobodies was 66.30%. The killing rate of the positive control was 99.85%, and that of the negative control was 7.26%. Therefore, when H3 nanobodies are used in the preparation of CAR-T cells, the in vitro tumor-killing effect of CAR-T cells is the most ideal, followed by H4 and H10 nanobodies.

[0093] Example 5: Preparation and efficacy study of CAR-NK The original retroviral expression vector (empty vector) used was pMSCV. GenScript was commissioned to synthesize the entire genome, including a chimeric antigen receptor CAR sequence and a downstream co-stimulatory signal domain, which were then inserted into the pMSCV plasmid to obtain the pMSCV-EF1a-anti-FcRH5-28z expression vector. Sequencing confirmed its correctness.

[0094] The chimeric antigen receptor CAR (FcRH5 CAR) is constructed based on the FcRH5 antibody sequence, which is identical to the components of CAR-T, including a signal peptide (SEQ ID NO. 28), a single-domain antibody (one of the three nanobodies provided in this protocol, namely: SEQ ID NO. 9, SEQ ID NO. 18, or SEQ ID NO. 27), a CD8 hinge region (SEQ ID NO. 29), a CD28 transmembrane region (CD28™, SEQ ID NO. 30), a CD28 intracellular region (CD28cyto, SEQ ID NO. 31), and a CD3zeta intracellular region (CD3zeta cyto, CD3ζ, SEQ ID NO. 32). These sequences are linked sequentially from the 5' end to the 3' end (sequence information is detailed in Table 6). They are then integrated into the pMSCV plasmid using conventional molecular cloning methods to obtain the expression vector. The virus prepared by this expression vector expresses a CAR fusion protein after infecting NK cells (natural killer cells). The CAR fusion protein is a transmembrane protein that can guide NK cells to kill target cells. This protocol tested the efficacy of three CAR fusion proteins: a CAR fusion protein with anti-FcRH5 antibody H3, a CAR fusion protein with anti-FcRH5 antibody H4, and a CAR fusion protein with anti-FcRH5 antibody H10.

[0095] First, retroviruses were prepared and 293T cells were seeded in T75 culture flasks (day 0). On day 1, virus packaging was performed. In a DNA tube, 10.6 µg of transfer plasmid (pMSCV-EF1a-anti-FcRH5-28z expression vector), 6.4 µg of helper plasmid pUMVC, and 10.6 µg of envelope plasmid pMD2G-BaEV (using the envelope of baboon endogenous retrovirus BaEV) were added, totaling 27.6 µg, and Opti-MEM medium was added to bring the volume to 300 µL. In a PEI (polyethyleneimine) tube, 108 µg of PEI was added, and Opti-MEM was added to bring the volume to 300 µL, mixing well with the DNA. The supernatant in the T75 culture flask was discarded, and 15 mL of fresh culture medium and the DNA-PEI complex (obtained by mixing the contents of the DNA and PEI tubes) were added to the flask. Gently mix the contents by shaking the bottle back and forth and side to side; incubate at 37℃ in a 5% CO2 incubator for 6 hours, then change the medium. On day 3, after 48 hours of incubation, harvest the virus. Collect the viral supernatant (temporarily store the viral supernatant at 4℃), add culture medium, and continue culturing the cells. On day 4, collect the viral supernatant again after 72 hours and combine it with the viral supernatant after 48 hours. Centrifuge at 1300g for 15 minutes (4℃), filter through a 0.45μm filter membrane, add 10mL of Takara Retro-X retrovirus concentrate to the supernatant, and incubate overnight at 4℃. Centrifuge again at 1300g for 45 minutes (4℃). Discard the supernatant, resuspend the precipitate in PBS, and aliquot to obtain the retrovirus containing the expression vector (store at -80℃).

[0096] Next, CAR-NK cell preparation was performed. On day 0, PBMCs were resuscitated and activated. PBMCs were removed from liquid nitrogen and rapidly resuscitated in a 37°C water bath, centrifuged at 350g for 5 min, washed once with PBS, and NK cells were sorted using the Miltenyi NK sorting kit. The cells were resuspended in 6 mL of NK cell culture medium (Yokang Biotechnology) and a T25 culture flask pre-coated with 5 μg / mL CD16 antibody at 4°C overnight was added. Cytokines were supplemented at 1000 U / mL IL-2, 10 ng / mL IL-15, and 45 ng / mL IL-18. On day 5, NK cells were added at 1 mL / well to 6 wells of a 24-well plate, each well pre-coated with 5 μg / mL CD16 antibody at 4°C overnight. 2Retronectin (Takara) was added, followed by retrovirus. The cells were centrifuged at 1500g for 1.5 hours at 32°C, and supplemented with interleukin-2 and IL-15 (1000 U / mL IL-2, 10 ng / mL IL-15). The transfected cells were then expanded and cultured, and cytotoxicity experiments were performed using these cells. The above procedure for preparing CAR-NK cells is a standard method in existing technology. The only difference between this method and existing technologies is the sequence of the CAR fusion protein, resulting in NK cells expressing a specific CAR fusion protein, which can effectively recognize and kill target cells. This step yields three types of CAR-NK cells: CAR-NK cells expressing H3 nanobodies, CAR-NK cells expressing H4 nanobodies, and CAR-NK cells expressing H10 nanobodies.

[0097] A co-incubation system was established using an E:T ratio (effectant-to-target ratio). H929 cells (expressing luciferase) were used as target cells, and CAR-NK cells were used as effector cells. Positive controls (PC: CAR-NK cells with a positive control sequence), negative controls (MOCK: NK cells without CAR gene modification), and a single-target cell group (H929 cells only) were also included. After 4 hours of co-culture, the cell culture plates were removed from the incubator and incubated at room temperature for 30 minutes to allow the temperature to equilibrate. An equal volume of Bio-Lite assay reagent, also equilibrated to room temperature, was added. Cells were incubated at room temperature for at least 3 minutes to allow for complete lysis before detection. Detection was performed using a microplate reader in chemiluminescence mode. The cell kill rate was calculated as: Kill rate % = (1 - RLU of the co-incubation group / RLU of the single-target cell group) × 100%, where RLU is the relative intensity of the fluorescence signal output by the detection device.

[0098] For detailed experimental results, please refer to Figure 7 The killing rate of CAR-NK cells expressing H3 nanobodies was 42.25%, that of CAR-NK cells expressing H4 nanobodies was 36.48%, and that of CAR-NK cells expressing H10 nanobodies was 42.43%. The killing rate of the positive control was 40.59%, and that of the negative control was 14.12%. Therefore, when H3 and H10 nanobodies are used in the preparation of CAR-NK cells, the in vitro tumor-killing effect of CAR-NK cells is the most ideal, followed by H4 nanobodies.

[0099] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An anti-FcRH5 nanobody, characterized in that, The amino acid sequence of its CDR1 is SEQ ID NO.19, the amino acid sequence of its CDR2 is SEQ ID NO.20, and the amino acid sequence of its CDR3 is SEQ ID NO.

21.

2. The anti-FcRH5 nanobody according to claim 1, characterized in that, The amino acid sequence of its FR1 is SEQ ID NO.22; or, it is any one of the amino acid sequences that differs from the sequence of SEQ ID NO.22 by 4, 3, 2 or 1 amino acids. The amino acid sequence of its FR2 is any one of those in SEQ ID NO.23; or, any one of those amino acid sequences that differs from the sequence of SEQ ID NO.23 by 4, 3, 2 or 1 amino acid. The amino acid sequence of its FR3 is any one of those in SEQ ID NO.24; or, any one of those amino acid sequences that differs from the sequence of SEQ ID NO.24 by 4, 3, 2 or 1 amino acid; The amino acid sequence of its FR4 is any one of those in SEQ ID NO.25; or, any one of those amino acid sequences that differs from the sequence of SEQ ID NO.25 by 4, 3, 2 or 1 amino acid.

3. The anti-FcRH5 nanobody according to claim 1, characterized in that, Its amino acid sequence is SEQ ID NO.

26.

4. The use of the anti-FcRH5 nanobody according to any one of claims 1-3 in the preparation of products for the treatment of multiple myeloma.

5. An antibody against FcRH5, characterized in that, It contains at least one anti-FcRH5 nanobody as described in any one of claims 1-3.

6. A CAR-engineered immune cell, characterized in that, The immune cells express chimeric antigen receptors; the immune cells are T cells or NK cells. The chimeric antigen receptor comprises the following sequentially connected parts: a signal peptide, a single-domain antibody, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3zeta intracellular region. The nucleotide sequence of the signal peptide is SEQ ID NO.28; the nucleotide sequence of the CD8 hinge region is SEQ ID NO.29; the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.30; the nucleotide sequence of the CD28 intracellular region is SEQ ID NO.31; the nucleotide sequence of the CD3zeta intracellular region is SEQ ID NO.32; and the nucleotide sequence of the single-domain antibody is SEQ ID NO.

27.

7. A chimeric antigen receptor fusion protein, characterized in that, It includes the following sequentially connected parts: signal peptide, single-domain antibody, CD8 hinge region, CD28 transmembrane region, CD28 intracellular region and CD3zeta intracellular region; The nucleotide sequence of the signal peptide is SEQ ID NO.28; the nucleotide sequence of the CD8 hinge region is SEQ ID NO.29; the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.30; the nucleotide sequence of the CD28 intracellular region is SEQ ID NO.31; the nucleotide sequence of the CD3zeta intracellular region is SEQ ID NO.32; and the nucleotide sequence of the single-domain antibody is SEQ ID NO.27.

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