A multifunctional antibody binding human CD19, CD3 and Fc gamma R

Abstract

The present application relates to the technical field of biological medicine, and more particularly to a multifunctional antibody K1932 binding human CD19, CD3 and Fc gamma R, wherein the multifunctional antibody is composed of a bivalent Fab fragment specifically recognizing CD19 on the surface of B lymphocyte, a bivalent single-chain antibody recognizing CD3 molecule on the surface of T lymphocyte, and a human immunoglobulin Fc domain binding with Fc receptor, wherein the single-chain antibody recognizing CD3 molecule is connected with the C-terminal of Fab light chain through a connecting peptide Linker1, and the heavy chain and the light chain of the CD3 single-chain antibody are connected by a flexible Linker2; the biological macromolecule with the three functional domains is a highly directional immunotherapy drug, which is suitable for injection administration, can guide T cells to attack CD19-positive B lymphocytes, and the Fc domain is combined with Fc gamma R, so that the in-vivo half-life of the biological macromolecule is adjusted and the unnecessary influence on normal cells is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a multifunctional antibody K1932 that binds to human CD19, CD3 and FcγR. Background Technology

[0002] Pre-B cells are a histological and embryological term introduced in 2014. They are defined as cells derived from progenitor B cells and comprise approximately 5% of nucleated cells in adult bone marrow. They have completed heavy chain gene rearrangement, but light chain gene rearrangement has not yet begun. They are relatively large, do not express membrane immunoglobulins, but can express CD19. B-cell lymphoma is a solid tumor originating from B cells in the bloodstream. It includes Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL), with numerous subtypes. Classical Hodgkin's lymphoma and nodular lymphocyte-predominant Hodgkin's lymphoma are now considered to originate from B cells. Diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma / chronic lymphocytic leukemia, and mantle cell lymphoma (MCL) are the five most common types of B-cell non-Hodgkin lymphoma, accounting for approximately three-quarters of all non-Hodgkin lymphomas. Non-Hodgkin lymphoma is a group of malignant tumors originating from lymphoid tissue and spreading throughout the body. Its incidence and mortality rates rank fifth among malignant tumors, and most NHLs originate from B lymphocytes (B-NHL).

[0003] Lymphoma is one of the most common malignant tumors in China. According to the WHO GLOBOCAN 2020 report, there were 6,829 new cases of Hodgkin's lymphoma in China in 2020, including 4,506 males and 2,323 females; 2,807 deaths were reported, including 1,865 males and 942 females. In 2020, there were 92,834 new cases of non-Hodgkin's lymphoma in China, including 50,125 males and 42,709 females; 54,351 deaths were reported, including 29,721 males and 24,630 females. In men, NHL ranks 10th in both incidence and mortality among all malignant tumors; in women, NHL does not rank among the top 10 in either incidence or mortality. Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy originating from B-cell or T-cell lymphoprogenitor cells, resulting from abnormal differentiation of hematopoietic stem cells. ALL is more common in children and adolescents, and relatively rare in adults. In the United States, approximately 6,020 new cases are diagnosed annually, with about 2,400 occurring in adults. In the European Union, over 7,200 new cases are diagnosed annually, with approximately 40% (about 3,000) occurring in adults. In Europe and the United States, the prevalence of ALL is approximately 25 per 100,000 people. In China, there is no national-level epidemiological data on the incidence of ALL. From 2002 to 2006, the average annual incidence of ALL in Shanghai was 0.81 per 100,000 people. Most ALL cases are B-cell and Philadelphia chromosome (Ph) negative. First-line treatment for ALL is combination chemotherapy, including induction remission and post-remission therapy (consolidation and maintenance). Induction remission drugs include vincristine, glucocorticoids, L-asparaginase, and anthracyclines. Commonly used consolidation and intensive treatment regimens include high-dose cytarabine and high-dose methotrexate. Standard maintenance therapy is based on 6-mercaptopurine and methotrexate. Intrathecal administration of methotrexate, cytarabine, and dexamethasone is required during treatment. Patients with poor prognostic factors should be actively considered for allogeneic hematopoietic stem cell transplantation (allo-HSCT) after initial complete remission. Generally, children and adolescents have higher complete remission rates after treatment than adults; however, complete remission (CR) rates in adult studies can reach 85% or higher, and ALL is generally considered a curable malignancy. Nevertheless, nearly 50% of adult pre-B-cell ALL patients and up to 20% of pediatric pre-B-cell ALL patients eventually relapse or develop primary resistance. Ph-positive ALL is a rare subtype of ALL, accounting for approximately 3% of pediatric patients and 25% of adult patients. It is a poor prognostic factor. Since the introduction of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion gene, the objective response rate of Ph-positive ALL has improved, becoming similar to that of Ph-negative ALL patients, but the duration of remission and relapse-free survival remain unsatisfactory.For relapsed / refractory adult B-cell ALL, the NCCN guidelines recommend regimens including belintolimab, innotuzumab, oxogamicin, CAR-T products, and combination chemotherapy regimens based on cytarabine, alkylating agents, or fludarabine / clofarabine. In China, there is no unified opinion on the treatment of relapsed / refractory Ph-negative ALL, but combination chemotherapy remains the primary recommendation. Relapsed / refractory Ph-positive patients should be tested for BCR-ABL mutations, and TKI substitution may be considered in salvage therapy; other treatment principles are consistent with Ph-negative patients. Once a patient relapses, the prognosis is extremely poor. Before the availability of belintolimab, the median survival for adult patients with relapsed / refractory ALL was approximately 3-6 months, with a complete response (CR) rate of only 8% (95% CI: 4%-14%) for single-agent chemotherapy and approximately 25%-46% for combination chemotherapy. Advanced age, first remission time less than 12 months, multiple relapses, and relapse after HSCT are considered to be associated with lower CR rates. In another recent retrospective observational study, 270 patients with relapsed / refractory Ph-negative ALL treated at 14 centers in China between 2005 and 2014 were observed. The complete remission (CR) + CRh rate was approximately 31%, and the median duration of CR / CRh was 2.7 months. The CR + CRh rate was 41% with initial salvage therapy, 24% with secondary salvage therapy, and only 17% with subsequent salvage therapy. In China, the CAG regimen containing aclarubicin differs from the treatment regimens used in Western countries. While it can achieve a CR rate of around 50% in relapsed / refractory ALL patients, the median overall survival (OS) is only about 3 months. Therefore, there is a significant clinical need for new treatments and therapies that can effectively improve remission rates and prolong survival in patients with relapsed / refractory ALL.

[0004] The development of genetic engineering technology has made it possible to research and develop bispecific and multispecific antibodies. More than ten types of bispecific genetically engineered antibodies for tumor treatment have been developed, mainly including antibody fragments, tandem single-chain antibodies, miniature bifunctional antibodies, single-chain miniature bifunctional antibodies, bispecific trivalent molecules, bispecific tetravalent molecules, and trifunctional antibodies. Although numerous molecular forms of bifunctional antibodies have been reported in basic research, very few have entered clinical trials.

[0005] Currently, there are two internationally approved bispecific antibody products: one is Catumaxomab antibody developed by Trion Pharma, which targets the tumor surface antigen EpCAM and the T cell surface receptor CD3; the other is Blinatumomab antibody jointly developed by Micromet and Amgen, which targets CD19 and CD3. Both achieve the goal of treating tumors by activating and recruiting cytotoxic T cells.

[0006] Catumaxomab, based on the Triomab technology platform, consists of mouse IgG2a targeting tumors and rat IgG2b targeting human CD3ε. It simultaneously activates monocytes, macrophages, stellate cells, and NK cells via the Fcγ receptor, achieving trifunctional antibody activity. Catumaxomab was the first approved bispecific antibody, but it has significant drawbacks. These include a complex and difficult-to-control manufacturing process on the Triomab platform, resulting in low yields, difficult purification, and poor stability; additionally, heterologous antibodies present immunogenicity issues.

[0007] Blinatumomab is a bispecific antibody based on the BiTE technology platform, consisting of two single-chain antibodies containing variable regions linked by a peptide. Unlike Triomab platform antibodies, BiTE platform antibodies are mass-produced using Chinese hamster ovary (CHO) cells, and contain only two binding domains: one with high affinity targeting cancer cell surface antigens (e.g., CD19), and the other with lower affinity targeting CD3. Clinical trials have demonstrated that Blinatumomab can effectively activate T cells and eliminate tumor cells even at very low doses. In July 2017, the US FDA approved Blinatumomab for the treatment of B-cell lymphoma, marking a breakthrough in the use of genetically engineered bispecific antibodies for immunotherapy of malignant tumors.

[0008] Currently, bispecific antibody molecules targeting human CD19 and CD3 have shown significant efficacy in animal models and some limited clinical trials. However, their effectiveness varies greatly depending on the platform technology used. Prokaryotic expression systems are frequently reported in the literature; these systems are rapid and simple to operate, but the bispecific antibody molecules obtained often exhibit unsatisfactory effects and poor stability, easily forming polymers and losing activity completely, requiring cryopreservation or lyophilization. Another method for producing bispecific antibodies involves hybridoma technology and chemical reactions to covalently couple monoclonal antibodies. This method generally has lower biological activity, requiring higher doses to achieve any noticeable effect.

[0009] Markers on the surface of lymphocytes are widely recognized as targets for autoimmune diseases such as B-cell lymphoma and B-cell disorders. Markers present on the surface of B lymphocytes include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, and CD81–CD86. Currently, monoclonal antibody drugs have been developed targeting molecules highly expressed on the surface of B lymphocytes, such as CD20 and CD19, for the treatment of autoimmune diseases such as B-cell lymphoma, rheumatoid arthritis, and systemic lupus erythematosus. In particular, anti-human CD20 monoclonal antibodies (such as rituximab) have become the first-line drugs for the treatment of non-Hodgkin's lymphoma and are the most widely used drugs worldwide. It is a well-known fact that acute lymphoblastic leukemia (ALL) and many other B-cell malignancies do not express CD20, or express it at low levels. Approximately half of non-Hodgkin's lymphoma patients respond to CD20-controlled immunotherapy. CD19 is an important membrane antigen associated with B lymphocyte differentiation, activation, proliferation, and antibody production, and is the best marker for diagnosing B-cell lineage tumors (leukemia, lymphoma) and identifying B lymphocytes. CD19 is a specific marker on the surface of B lymphocytes, belonging to the immunoglobulin superfamily. It is involved in B cell activation and signal transduction, and is expressed in pre-B lymphocytes, immature B lymphocytes, mature B lymphocytes, and activated B lymphocytes, but not in lymphoplastic stem cells or other tissues. Most non-Hodgkin's lymphomas originate from B lymphocytes, and more than 95% of B-cell NHLs express CD19 antigen. Furthermore, CD19 antigen is relatively exposed, and free CD19 is not present in human serum. Therefore, CD19 can serve as a target for the treatment of B-cell lymphomas. CD19 is a more widely distributed B-cell surface marker than CD20. It is a receptor expressed on the B-cell surface and belongs to the immunoglobulin superfamily. Its ligands and related molecules include CR2 (CD21), TAPA-1 (CD81), Leu-13, PI-3K, Vav, lyn, and fyn. CD19 is an important signaling molecule that regulates the growth, activation, and differentiation of B lymphocytes. CD19 modulates signaling responses and plays a crucial role in regulating the signal thresholds of B lymphocyte antigen receptors or other surface receptors. CD19 is a pan-B-cell membrane glycoprotein expressed from early pre-B cell development to terminal differentiation, regulating B lymphocyte development and function. CD19 expression has been identified in most lymphoid tumors, most non-Hodgkin's lymphomas (NHL), and leukemias including chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and Waldenström's macroglobulinemia (WM).CD19 expression occurs throughout the entire lifecycle of B lymphocytes, from primitive B cells, pre-B cells, early-developing B cells, mature B cells, plasma cells derived from mature B cells, and malignant B lymphoma cells. The vast majority of tumor cells derived from B lymphocytes, such as pre-B acute lymphoblastic leukemia, chronic B lymphocytic leukemia, pre-lymphocytic leukemia, non-Hodgkin's lymphoma, hairy cell leukemia, ordinary acute lymphoblastic leukemia, and some non-acute lymphoblastic leukemias, multiple myeloma, and plasmacytoma, express CD19 molecules.

[0010] CD3 is a marker present on the surface of all T lymphocytes. CD3 is also known as T3 or Leu-4. There are three isoforms: CD3δ, CD3ε, and CD3γ. CD3δ and CD3ε each have a molecular weight of 20 kDa, while CD3γ has a molecular weight of 26 kDa. It is expressed on the surface of T lymphocytes, thymocytes, and NK cells. It is expressed in 61%–85% of normal peripheral blood lymphocytes and 60%–85% of thymocytes. It belongs to the immunoglobulin superfamily. CD3 is a component of the T lymphocyte receptor (TCR) complex, forming a complex with α / β and γ / δ T lymphocyte receptors (TCRs), and is the main membrane antigen transmitting TCR signals bound to peptide / MHC. TCRs are essential for cell surface expression, antigen recognition, and signal transduction. CD3 is a T lymphocyte-specific molecule, through which cytotoxic T lymphocytes can be recruited. Monoclonal antibodies against CD3 can induce or inhibit T lymphocyte activation. In the presence of anti-CD28 antibodies or IL-2, anti-CD3 antibodies can induce apoptosis of T lymphocytes. CD3 is one of the best markers of mature T lymphocytes in peripheral blood, and the determination of CD3+ T lymphocytes is of great significance for the subtyping and diagnosis of immunodeficiency diseases (T lymphocytopenia), leukemia, and lymphoma (T lymphocytic type). Anti-CD3 monoclonal antibodies can be used for immunosuppressive therapy during organ transplantation or bone marrow transplantation, and can also be used for immunomodulatory therapy in severe autoimmune diseases to eliminate T lymphocytes. US Patent 4,361,549 describes a murine hybrid cell line for producing the monoclonal antibody OKT3 against antigens found on normal human T cells and cutaneous T lymphoma cells. US Patent 5,885,573 describes the construction of a humanized monoclonal antibody by transferring murine OKT3 into a human antibody framework, aiming to reduce its immunogenicity in human application and decrease the probability of human anti-mouse antibody (HAMA) reactions. OKT3 was the first murine monoclonal antibody approved by the US FDA in 1986 for the treatment of acute rejection in organ transplant recipients, and it was also the world's first monoclonal antibody drug approved by a government drug regulatory authority. The main drawback of murine OKT3 monoclonal antibody therapy was the T cell activation and HAMA response caused by cytokine release resulting from cross-linking between T cells and FcγR carrier cells. After more than a decade on the market, OKT3 was eventually replaced by humanized antibodies and new small-molecule immunosuppressants. On the other hand, OKT3 or other anti-CD3 antibodies can be used as immune enhancers to stimulate T cell activation and proliferation. In in vitro cell culture, anti-CD3 monoclonal antibodies, in combination with anti-CD28 antibodies or interleukin-2, can induce T cell proliferation. OKT3 has also been used alone or as a component of bispecific antibodies to target cytotoxic T cells to tumor cells and virus-infected cells.To date, the use of antibodies as agents for recruiting T cells has been hampered by several findings. First, natural or modified antibodies with high affinity for T cells often fail to activate the T cells they bind to; second, natural or modified antibodies with low affinity for T cells are generally ineffective or inefficient in inducing T cell-mediated cell lysis. Therefore, selecting an anti-CD3 monoclonal antibody with appropriate affinity is crucial.

[0011] FcγR plays a crucial role in the uptake, processing, and presentation of antigens, which in turn activates T cells, and is a key process in adaptive immunity. Effective antigen recognition by T cells requires first uptake and processing of the antigen, followed by presentation on MHC molecules on the cell surface. A key step in antigen presentation is the transport of foreign antigens to intracellular compartments, where they can be processed and loaded onto MHC molecules. FcγR plays an important role in antigen uptake, processing, and presentation.

[0012] Based on receptor affinity, Fcγ receptors are classified into three main classes: high-affinity receptors FcγRI (CD64), low-affinity receptors FcγRII (CD32), and FcγRIII (CD16). All three classes of Fcγ receptors contain highly conserved extracellular Ig domains. Fcγ receptor I (CD64) is primarily expressed on monocytes and neutrophils. FcγRI binds to ligands with high affinity. Fcγ receptor I binds to monomeric Ig and immune complexes. FcγRI is the only IgG Fc receptor whose monomeric ligand binding level can be directly measured. Interferon-gamma (IFN-γ) can enhance FcγRI expression by 20-fold. FcγRII (CD32) is expressed on every cell carrying FcγR (except NK cells). FcγRII binds to ligands with low affinity and cannot be directly measured by binding to monomeric ligands. FcγRII promotes phagocytosis / endocytosis of immune complexes and B cell activation. FcγRIII (CD16) is expressed on macrophages and macrophage lines, NK cells, bone marrow progenitor cells, and neutrophil lines. FcγRIII expression on macrophages is regulated by IFN-γ. FcγRIII can mediate antibody-dependent cytotoxicity (ADCC).

[0013] Traditional bispecific antibodies lack specificity, and while attacking leukemia cells, they can also unnecessarily affect normal cells. During treatment, they have a certain probability of triggering nonspecific immune responses. Due to their lack of high specificity, these antibodies may attack normal cells, not just leukemia cells, leading to so-called "nonspecific toxicity"—damage to normal tissues, resulting in poor treatment efficacy and requiring higher doses to achieve therapeutic effects. This increases patient discomfort and risk. Furthermore, traditional bispecific antibodies have limitations in their selection of specific affinity or effector linkages, potentially leading to overactivation of T cells or other adverse reactions. If the selected affinity is too high or the effector linkage is too strong, they may cause excessive T cell activation, potentially triggering strong immune responses, including cytokine release syndrome, reducing treatment safety. Therefore, there is an urgent need for a highly targeted, multifunctional antibody drug that binds with low affinity during treatment. Summary of the Invention

[0014] Therefore, the present invention provides a multifunctional antibody that combines human CD19, CD3 and FcγR to overcome the problems of low specificity of existing bispecific antibodies, which may have unnecessary effects on normal cells and have defects in the selection of specific affinity or linker.

[0015] To achieve the above objectives, in one aspect, the present invention provides a multifunctional antibody that combines human CD19, CD3, and FcγR, comprising:

[0016] 1) It specifically binds to the domain of B lymphocyte membrane antigen CD19, wherein the Fab fragment that binds CD19 contains a humanized light chain with the amino acid sequence SEQ No. 3, and a heavy chain variable region VH with the amino acid sequence SEQ No. 2;

[0017] 2) A single-chain antibody domain that specifically binds to the T cell surface antigen CD3 molecule, wherein the single-chain antibody recognizing the CD3 molecule includes a light chain VL and a heavy chain VH, which are linked by Linker1 SEQ No.6 and Linker2 SEQ No.7, respectively;

[0018] 3) A domain that binds monovalently to the NK cell FcγR receptor, wherein the structure of the Fc fragment includes CH1, a hinge region, CH2 and CH3, and the amino acid sequence is SEQ No. 1;

[0019] The single-chain antibody that recognizes CD3 molecules in the bivalent single-chain antibody domain that specifically binds to the CD3 molecule on the surface of T cells is linked to the C-terminus of the CD19 antibody light chain via a hydrophilic linker peptide.

[0020] The structure of the multifunctional antibody is as follows:

[0021] ,

[0022] Furthermore, the FcγR binding sequence is CH1-Hinge Region-CH2-CH3, and the amino acid sequence is SEQ No. 1;

[0023] The humanized CD19 binding domain sequences are SEQ No. 2 and SEQ No. 3, which constitute the Fab domain.

[0024] The amino acid sequences of the CD3 single-chain antibody are SEQ No. 4 and SEQ No. 5.

[0025] Furthermore, the single-chain antibody structure that recognizes the CD3 molecule adopts the form of ScFv and is targeted at human CD3ε, including but not limited to humanized CD3-specific antibodies such as OKT3, X35-3, WT31, WT32, SPv-T3b, TR-66, 11D8, 12F6, M-T301, SMC2 and F101.01.

[0026] Furthermore, the preferred sequence for FcR is the γ chain.

[0027] Furthermore, CD3 is a bivalent humanized antibody, and CD19 is a bivalent humanized antibody.

[0028] The present invention also provides a method for preparing the above-mentioned multifunctional antibody, which is prepared by gene recombination technology and uses different types of mammalian cell expression vectors. Preferably, the expression is carried out in CHO cells. The CHO cells are cultured using a chemically defined culture medium, and no hormones or various animal-derived proteins or their hydrolysates are added during the culture process.

[0029] Furthermore, the preparation steps include:

[0030] The plasmid containing the multifunctional antibody gene was tandemly linearized by digestion with an endonuclease.

[0031] Positive clones were obtained after transfecting CHO cells, and then cultured in a bioreactor, with the products secreted into the culture supernatant.

[0032] Multifunctional antibodies can be obtained by purification using ion exchange chromatography media or a combination of affinity chromatography and ion exchange chromatography.

[0033] The application of the multifunctional antibody provided by this invention in the preparation of drugs for treating relapsed and refractory B-cell lymphoma. The multifunctional antibody is a T-cell-dependent immunotherapeutic drug that kills tumor cells.

[0034] The present invention also discloses a pharmaceutical composition containing the above-described multifunctional antibody.

[0035] Furthermore, the method of using the pharmaceutical composition includes: preparing it as a liquid formulation, or administering it via subcutaneous or intramuscular injection.

[0036] Furthermore, the pharmaceutical composition formulation includes: acetic acid, histidine, methionine, sucrose, and Tween 20.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The K1932 antibody of the present invention is a novel multifunctional antibody with a novel antibody structure that simultaneously targets CD3, CD19 and FcγR.

[0039] The innovative mechanism of action of this antibody is to utilize the patient's own cytotoxic T cells to attack malignant B cells. B lymphocytes bind to the anti-CD19 portion of K1932, T cells bind to the anti-CD3 portion, and the Fc portion binds to monocytes, macrophages, stellate cells, and NK cells, regulating the in vivo half-life of biomolecules while reducing unnecessary impacts on normal cells. This characteristic of K1932 allows it to temporarily link malignant cells with T cells, thereby inducing the killing effect of T cells on the bound malignant cells. K1932-mediated T cell activation involves the temporary release of inflammatory cytokines and T cell proliferation. The subsequent series of malignant cell lysis reactions generated by K1932-activated T cells are very similar to the natural cytotoxic T cell response.

[0040] This invention is a multifunctional antibody targeting CD19, CD3, and FcγR. It can utilize the body's own T cells to kill CD19-positive tumor cells, thereby achieving the purpose of treating lymphoma and leukemia.

[0041] The molecular structure of K1932 differs fundamentally from other specific antibodies currently in clinical trials, offering superior targeting capabilities. It utilizes low-affinity CD3ε-ScFv as the effector-T cell binding molecule. Even though each K1932 molecule contains two CD3 binding sites, its lower concentration prevents it from activating T cells like OKT3. Structurally, K1932's use of low-affinity CD3ε-ScFv as the effector linker results in weaker affinity for T cells. This helps prevent over-activation of T cells, reducing adverse reactions, while ensuring preferential binding to the CD19 target. It also attracts effector cells by binding to FcγR.

[0042] This solves the problems of low specificity of existing bispecific antibodies, which can have unnecessary effects on normal cells, and defects in the selection of specific affinity or linker effectors. Attached Figure Description

[0043] Figure 1 A map showing the construction of the gene co-expression vector of the present invention;

[0044] Figure 2 The electrophoretic detection results of plasmid linearization according to the present invention (1% agarose gel).

[0045] Figure 3 The purity spectrum (SEC-HPLC) of the K1932 antibody of this invention;

[0046] Figure 4 The flow cytometry and dose-response curves of the specific binding reaction between the K1932 antibody of the present invention and the CD19 site of Raji, Daudi, IM-9, and K562 cells are shown.

[0047] Figure 5 The flow cytometry and dose-response curves of the specific binding reactions of the K1932 antibody, K19 monoclonal antibody, and OKT3 monoclonal antibody to the CD19 site in Raji cells are shown below.

[0048] Figure 6 The flow cytometry and dose-response curves of the K1932 antibody, OKT3 and CD3 binding reaction of the present invention are shown.

[0049] Figure 7 The flow cytometry pattern and dose-response curve of CD69 expression in T lymphocytes activated by the K1932 antibody of this invention are shown.

[0050] Figure 8 The reaction curves of the K1932 antibody of the present invention with T+B cells, T cells, and B cells show the CD69 expression level.

[0051] Figure 9 The relative binding activity of the K1932 antibody stock solution of the present invention;

[0052] Figure 10 CD8 mediated by the K1932 antibody of this invention + T cells kill B cells;

[0053] Figure 11 The results of the K1932 antibody-mediated T cell activation assay (PBMC) of this invention;

[0054] Figure 12 The curve of T lymphocyte activation and B lymphocyte killing mediated by the K1932 antibody of the present invention is shown. Detailed Implementation

[0055] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0056] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0057] The present invention will now be described in further detail with reference to the accompanying drawings:

[0058] Supplementing some of the illustrations in the description of the accompanying drawings:

[0059] Figure 4 :

[0060] Mean PE-H: Average fluorescence intensity of the PE channel

[0061] Antibody content (ng / ml): Antibody concentration (ng / ml)

[0062] K1932+Raji: K1932 antibody and Raji cells

[0063] K1932+Daudi: K1932 antibody and Daudi cells

[0064] K1932+IM-9: K1932 antibody and IM-9 cells

[0065] K1932+K562: K1932 antibody and K562 cells

[0066] Figure 5 :

[0067] Raji+K1932: Raji cells + K1932 antibody

[0068] Raji+K19: Raji cells + K19 monoclonal antibody

[0069] Raji+OKT3: Raji cells + OKT3 monoclonal antibody

[0070] PE-H: Fluorescence intensity of the PE channel

[0071] Figure 6 :

[0072] Jurkat+K1932: Jurkat cells + K1932 antibody

[0073] Jurkat+OKT3: Jurkat cells + OKT3 antibody

[0074] Mean FITC-H: Average fluorescence intensity of the FITC channel

[0075] Antibody content (ng / ml): Antibody concentration (ng / ml)

[0076] Figure 7 :

[0077] Mean PE-H (CD69): Average fluorescence intensity of the PE channel (CD69)

[0078] Antibody content (pg / ml): Antibody concentration (pg / ml)

[0079] K1932 (20240601): K1932 antibody batch (20240601)

[0080] K1932 (20240607): K1932 antibody batch (20240607)

[0081] Figure 8 :

[0082] Mean PE-H (CD69): Average fluorescence intensity of the PE channel (CD69)

[0083] Antibody content (pg / ml): Antibody concentration (pg / ml)

[0084] T+B: T+B cell co-culture

[0085] T: T cells cultured alone

[0086] B: B cells cultured alone

[0087] Figure 10 : Activated CD8⁺T + Raji-Luc2 killing curve

[0088] Killing percentage (%)

[0089] Log10[Test Articles](ng / ml): Logarithmic value of the concentration of the tested substance (ng / ml)

[0090] K1932 Stock Solution: K1932 stock solution

[0091] k193 Reference Solution: K193 reference solution

[0092] Blinatumomab: Blinatumomab antibody

[0093] Figure 11 :

[0094] CD69 expression curve

[0095] CD4⁺CD69: CD4⁺ T cell CD69 expression

[0096] CD8⁺CD69: CD8⁺ T cell CD69 expression

[0097] Perforin expression curve

[0098] CD4⁺Perforin: CD4⁺ T cell perforin expression

[0099] CD8⁺Perforin: CD8⁺ T cell perforin expression

[0100] Granzyme B expression curve

[0101] CD4⁺ Granzyme B: CD4⁺ T cell granzyme B expression

[0102] CD8⁺ Granzyme B: CD8⁺ T cell granzyme B expression

[0103] Figure 12 :

[0104] Killing percentage (%)

[0105] Antibody content (pg / ml): Antibody concentration (pg / ml)

[0106] 18h, 24h, 42h, 72h: 18 hours, 24 hours, 42 hours, 72 hours.

[0107] Example 1

[0108] Sequence Design: The Fcγ amino acid sequence is SEQ No. 1. The constituent components of the sequence are CH1-Hinge Region-CH2-CH3. This sequence can form an interchain disulfide bond at the cysteine-proline-proline-cysteine ​​sequence. The resulting structure can bind to the FcγR receptors of monocytes, macrophages, stellate cells, and NK cells, exerting effector functions of cell killing and antigen presentation. The preferred sequence is derived from human IgG1, but IgG2, IgG3, IgG4, or other appropriate artificially mutated sequences can also be used. The core function of this design is to form an interchain disulfide bond and bind to the FcγR receptor. Different sequences can have different affinities and target different dominant cells, thereby exerting effector functions.

[0109] The CD19-binding domains are SEQ No. 2 and SEQ No. 3. SEQ No. 2 is VH, and SEQ No. 3 is VL-Cκ. SEQ No. 2 and SEQ No. 3 constitute the CD19-binding region, i.e., the Fab domain. This invention can be applied to the variable regions of other natural or artificial antibodies that bind to CD19. Different artificial mutations can adjust the binding affinity to CD19, and different binding energies can adjust the molecular targeting ability. Stronger binding affinity to CD19 results in stronger targeting ability, and vice versa. The sequences used in this invention have been tested and show relatively strong binding affinity; specific data can be found in the subsequent test results of this invention. It should be noted that there is no disulfide bond between SEQ No. 2 and SEQ No. 3. The C-terminal amino acid of SEQ No. 2 is connected to the N-terminal amino acid of SEQ No. 1 via a peptide bond; the Cκ portion of SEQ No. 3 forms a disulfide bond with the CH1 portion of SEQ No. 1. The VL portion of SEQ No. 3 is not covalently connected to SEQ No. 2.

[0110] The CD3-binding domains are SEQ No. 4 and SEQ No. 5. SEQ No. 4 and SEQ No. 5 are linked by a linker peptide chain. The domains of SEQ No. 4 and SEQ No. 5 are combined through binding forces such as salt bridges and hydrogen bonds to form a single-chain antibody domain. After pairing, SEQ No. 4 and SEQ No. 5 can bind to CD3, enabling the molecule of this invention to target T cells. Binding to CD3 activates T cells. The design of this invention weakens the binding to CD3, thus reducing the occurrence of accidental T cell activation, i.e., reducing off-target T cell activation. Of course, the affinity of SEQ No. 4 and SEQ No. 5 to CD3 can also be adjusted through artificial mutation. This invention incorporates two designs: one where SEQ No. 4 and SEQ No. 5 are located downstream of the Cκ sequence of SEQ No. 3, effectively forming steric hindrance; and another where the CDR sequence of SEQ No. 4 and SEQ No. 5 is optimized to ensure appropriate CD3 binding affinity, reducing off-target (CD19) T cell activation. This invention combines two technological considerations in its design, achieving excellent efficacy. Subsequent testing of its CD3 binding activity was conducted, as detailed in the examples below.

[0111] SEQ No. 4 and SEQ No. 5 are SvFc single-chain antibodies. The combination of the two sequence fragments forms a CD3-binding domain. To provide flexible adjustment space for SEQ No. 4 and SEQ No. 5, this invention optimizes the design of two linkers (Linker1 and Linker2). The Linker1 sequence is SEQ No. 6: Gly Ser Thr Ser Gly Ser Gly Lys ProGly Ser Gly Glu Gly Ser Thr Lys Gly Gly Ser. The Linker2 sequence is SEQ No. 7: Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser. The Linker1 sequence design focuses on factors such as sequence length, hydrophilicity / hydrophobicity, peptide secondary structure, and steric hindrance to neighboring peptide chains. The Linker2 sequence design focuses on providing flexibility for SEQ No.5, enabling SEQ No.5 to functionally combine with SEQ No.4. The glycine and serine combination is a classic flexible linker. Based on experiments, this invention selected the (GlyGlyGlyGlySer)3 combination, which does not affect the activity of SEQ No.4 and SEQ No.5, does not affect the binding to CD3, and the resulting binding force meets the design criteria.

[0112] As described above, the overall molecular design consists of three functional domains: an Fc peptide chain that binds to the Fc receptor, a CD19-binding domain, and a CD3-binding domain. To facilitate the connection of these three polypeptide chains and subsequent protein expression and purification, two polypeptide chains are designed in combination: SEQ No. 2 and SEQ No. 1 are fused in tandem, resulting in SEQ No. 8; SEQ No. 3, SEQ No. 4, and SEQ No. 5 are fused in tandem, with SEQ No. 3 and SEQ No. 4 linked by Linker1 and SEQ No. 4 and SEQ No. 5 linked by Linker2, resulting in SEQ No. 9.

[0113] Example 2: Optimizing the codon

[0114] The two polypeptide chains designed in Example 1 were codon-optimized to suit expression in CHO cells. The optimized nucleotide sequence of the target gene amino acid sequence SEQ No. 8 is SEQ No. 10. The optimized nucleotide sequence of the target gene amino acid sequence SEQ No. 9 is SEQ No. 11.

[0115] Example 3: Construction of cloning plasmids

[0116] The expression vector was double-digested with HindIII / EcoRI restriction endonucleases, followed by alkaline phosphatase dephosphorylation to obtain a nucleotide sequence fragment with sticky ends. The gene sequence SEQ No. 10 was ligated into a cloning plasmid. The plasmid was amplified and extracted, and the target gene sequence was obtained by double digestion with HindIII / EcoRI. The two digestion products were ligated using the TaKaRa DNA Ligation Kit LONG, transformed into DH5α, and positive clones were screened. After transformation of *E. coli* DH5α, three single colonies were randomly selected for expansion culture. The plasmid was extracted using a commercial kit, and double digestion with HindIII / EcoRI confirmed correct ligation. The first peptide chain was constructed and named plasmid p21. Figure 1 The diagram shows the construction of the gene co-expression vector, where EQ No.10 and SEQ No.11 are nucleotide sequences encoding multifunctional antibodies, corresponding to the heavy chain and light chain DNA sequences, respectively; SEQ No.8 and SEQ No.9 are the amino acid sequences translated from SEQ No.10 and SEQ No.11, representing the heavy chain protein sequence and light chain protein sequence of the multifunctional antibody, respectively.

[0117] The nucleotide sequence of SEQ No. 10 is a DNA fragment encoding the heavy chain variable region (VH) and constant region (CH1-HingeRegion-CH2-CH3); the nucleotide sequence of SEQ No. 11 is a DNA fragment encoding the light chain variable region (VL) and constant region (Cκ).

[0118] SEQ No. 8 and SEQ No. 9 were obtained by translating nucleotide sequences (SEQ No. 10 and SEQ No. 11), in which the VH and VL sequences bind to CD19 and CD3 targets, respectively; the heavy chain constant region (CH1-Hinge Region-CH2-CH3) binds to FcγR on NK cells through its Fc segment, and the light chain constant region (Cκ) ensures the overall conformation and functionality of the antibody through its stabilizing effect.

[0119] Using the same method, a sequence containing SEQ No. 11 was constructed into another vector plasmid. Double enzyme digestion confirmed that the ligation was correct, and the construction of the second peptide chain was completed. The plasmid was named p345.

[0120] Example 4: Constructing a tandem expression plasmid by cascading plasmids p21 and p345.

[0121] The target fragments were prepared by double digestion of plasmids p21 and p345 with restriction endonucleases PvuI and NotI using a commercial gel extraction kit. The plasmids were then linearized to approximately 4700 bp and 6500 bp in size, respectively. See details... Figure 2 Using a commercial DNA ligation kit, the two fragments were ligated overnight at 16°C using sticky end ligation technology, and then transformed into DH5α. The plasmid was named p1932. Clones were randomly selected, and the target gene was amplified from the clones using colony PCR to screen for positive clones.

[0122] Example 5: Preparation of plasmids for transfecting CHO cells

[0123] The positive clones from Example 4 were inoculated into 300 ml of LB (Amp+) medium and cultured at 37°C and 180 rpm for 16 h. Plasmid p1932 was extracted using a large / large plasmid extraction kit (Beijing Bomed Gene Technology Co., Ltd.). After linearization with the restriction enzyme PvuI (TaKaRa), the plasmid was purified by phenol / chloroform / isoamyl alcohol extraction, precipitated with ethanol, and then reconstituted in sterile TE buffer (TaKaRa). The agarose gel electrophoresis results for plasmid p1932 are shown below. Figure 2 As shown, Figure 2The plasmid contains lane M: DNA Marker (10000bp, 7000bp, 4000bp, 2000bp, 1000bp, 500bp and 250bp), where lane 1: intact circular plasmid, lane 2: product of plasmid digestion with PvuI, and lane 3: product of plasmid digestion, extraction and purification with PvuI. Figure 2 The sample contained a complete circular plasmid (lane 1), the product digested with PvuI (lane 2), and the purified linear plasmid product (lane 3). Electrophoresis showed that the enzyme digestion reaction was complete and the target band was consistent with the expected result.

[0124] Lane 1: Intact circular plasmid;

[0125] Lane 2: The product of plasmid digestion with PvuI;

[0126] Lane 3: The product of plasmid digestion, extraction and purification by PvuI enzyme.

[0127] After complete enzyme digestion, a DNA fragment of approximately 11200 bp was obtained. This fragment migrated more slowly than the plasmid in agarose gel electrophoresis. The single enzyme digestion reaction was complete, and the target band matched the expected result.

[0128] Example 6 Construction of engineered cell lines

[0129] Establishment and screening of stable clones: In a sterile laminar flow hood, the Xcell (Bio-Rad) gene pulse generator was set to a perforation voltage of 300V, a single pulse of 900μF, and infinite resistance. A disposable electroporation cup (Bio-Rad) with a 4mm gap was removed, and 40μg of linearized plasmid DNA (100μl) and 0.7ml of CHO-K1 cell suspension (1.43×10⁻⁶) were added. 7The linearized plasmid p1932 was directly transfected into CHO-K1 cells using electroporation. Cells from the electroporation cuvettes were then transferred to Erlenmeyer flasks, and 30 ml of CD-CHO culture medium was added. Cells were incubated at 36–37°C on a 5% CO2 shaker at 135 rpm for 24 hours. Cells were then collected by low-speed centrifugation and replaced with CD-CHO culture medium (glutamine-free) containing 50 μM MSX. Cells were then transferred to 96-well flat-bottomed culture plates using limiting dilution. The plates were incubated at 37°C on a 10% CO2 incubator. Observation was performed under an inverted microscope, and wells containing monoclonal cells were labeled. Positive cells in 96-well, 24-well, and 6-well cell culture plates were then screened using ELISA (Recombinant Human CD3E protein + expression product double antibody Fc + goat anti-human IgG-HRP). Monoclonal lines with high expression levels were selected, passaged, and tested repeatedly to obtain cell clones with high expression of the target gene. The protein loading was then adjusted accordingly. The expression level of recombinant protein in the culture supernatant was detected by HPLC column chromatography using gel A, and the 7D11 clone was selected for amplification. An engineered cell line was established and named K1932 cells.

[0130] Example 7: Fermentation and purification of engineered cells K1932

[0131] K1932 cells were seeded into 2L or 3L Erlenmeyer flasks containing 500ml of CD-CHO culture medium, the caps were tightened, and the flasks were cultured on a shaker at 36-37℃ and 5% CO2 at 130-140 rpm for 3-6 days. Afterward, the cells were transferred to a bioreactor. Bioreactor culture conditions: initial seeding density: 500,000-2,000,000 cells per milliliter; culture temperature: 32-37℃; pH: 6.5-7.5; stirring speed: 50-120 rpm; dissolved oxygen: 50% ± 20%; intermittent feeding (feeds such as sugar, culture medium, or alkali solution) during culture; culture time: 12-18 days; harvested when the viable cell density drops to 60%-70%.

[0132] The harvested culture medium is centrifuged at 10,000 rpm for 30 minutes to remove insoluble particles such as cells, or a deep filter is used to remove cells and cell debris, and the cell culture supernatant is collected. To ensure aseptic operation during the process, a 0.45 μm clarifying filter is typically used to filter the culture supernatant. The filtered intermediate can be stored at 2-8°C or directly proceeded to the next purification step. Protein A affinity chromatography is a common method for antibody purification, utilizing the presence of the Fc domain in antibodies. The K1932 peptide chain contains an Fc domain, and experimental testing has shown that the Fc portion of the K1932 structure can effectively bind to affinity gels containing protein A. This invention can use protein A or protein G affinity gels for affinity purification. We will use protein A as an example below. The above-mentioned filtered harvested liquid is purified by passing it through a protein A affinity chromatography gel (such as MaXtar ARPA, Protein At Bead LX, Mabselect Sure LX). The affinity chromatography column was equilibrated with 20 mM PBS equilibration buffer, and then washed with 20 mM equilibration buffer until the UV detector reading was A. 280 Return to near the baseline; then replace the mobile phase with a dissociation buffer (0.1M citrate buffer, acetate buffer, or glycine buffer, pH 3.0–4.5) for elution and dissociation, according to A. 280 Collect the elution peak using absorbance values.

[0133] Incubate the affinity chromatography buffer at room temperature for 60 minutes to inactivate the virus, then add 1M Tris neutralization buffer and neutralize to pH 7.2. Dilute with purified water to adjust the ionic strength of the solution to meet the requirements of the next step, anion exchange chromatography.

[0134] The packing material used in the anion exchange chromatography was DEAE Sepharose 6FF. The pH of the intermediate product for virus removal, incubated at low pH, was adjusted to 7.2 and appropriately diluted with pure water before being directly passed through the anion exchange chromatography column. According to A... 280 Collect the flow-through liquid based on the absorbance value until the absorbance value drops to near the baseline level, at which point the sample collection is stopped; this is the anion exchange chromatography liquid.

[0135] The liquid obtained after anion exchange chromatography was loaded onto a ChromaX-filled container. TM A Baron CHT II hydroxyapatite chromatography column was used. After equilibration, 70 mM phosphate solution (pH 6.8) was used as the dissociation buffer, and UV detector A was used. 280 When the absorbance increases, start collecting the column eluent and stop collecting when the absorbance drops to near the baseline level. The purity of the target protein can reach over 99%. Figure 3The SEC-HPLC chromatogram of K1932 antibody purity detection is shown. The single peak in the figure indicates that the purity meets the design standard.

[0136] The purified liquid was concentrated by ultrafiltration, and after adding a protective agent, it was filtered through a 0.2-micron sterile filter membrane to prepare the K1932 antibody stock solution.

[0137] The K1932 antibody purification process described in this embodiment is a combination of techniques, intended to illustrate the process of obtaining K1932 with biological function. Of course, other suitable purification routes can also be selected or combined to obtain high-purity K1932 antibodies. All combinations aim to obtain K1932 antibodies and are within the scope of protection of this invention.

[0138] Example 8: K1932 stock solution stabilizer formulation

[0139] K1932 is an innovative molecular design comprising three functional domains. Structurally, it includes an antibody Fab segment, an Fc segment, and a ScFv single-chain antibody. To maintain molecular stability, it was designed following general principles for antibody stabilizers. Various buffer systems are available, such as phosphate buffers, citrate buffers, and acetate-sodium acetate buffers. This invention compared several options and preferably selected an acetate-histidine buffer system with a pH of 5.2 ± 0.2. For nitrogen-hydrogen bond and oxygen-hydrogen bond stabilization, lysine, arginine, glycine, glutamine, etc., can be selected; preferably, L-methionine is added. Carbohydrates are polyhydroxy protective agents, and various combinations of monosaccharides, disaccharides, or other polyhydroxy alcohols can be used, such as glucose, trehalose, sucrose, lactose, maltose, sorbitol, etc. This invention preferably selects sucrose as the protective agent component. Surfactants reduce protein adsorption to the container and have excellent dispersing ability, providing protection for the target protein. Suitable surfactants include Tween-20, Tween-80, and poloxamer 188. This invention, through testing, preferentially uses Tween-20. The resulting protective agent formulation is as follows:

[0140] Acetic acid: 0.2~0.4 g / L

[0141] L-Methionine: 1.0~20g / L

[0142] L-histidine: 1.0~2.0g / L

[0143] Tween-20: 0.2~1.0 g / L

[0144] Sucrose: 50~150g / L

[0145] Under the aforementioned protective conditions, the K1932 antibody can be stored long-term at 2–8°C. Accelerated thermal assays at 37°C showed good stability of K1932. Stability was assessed using purity data as the most direct indicator. The purity of K1932 was determined using 40 mM PBS (containing 0.2 M Na2SO4, pH 6.7) as the mobile phase on a Waters e2695 HPLC system with a TSK3000SWxl (7.8*300 mm) column. The results showed that the purity was not less than 95% within the expected two-year shelf life.

[0146] Example 9: Binding reaction of K1932 antibody with various B lymphoma cells

[0147] Raji, Daudi, and IM-9 are B-cell lymphoma cells with CD19 antigen on their cell surface. The K1932 antibody can specifically bind to this CD19 antigen. In this experiment, Raji, Daudi, and IM-9 cells were used as positive cells, and K562 cells were used as negative controls. The CD19-specific binding activity of the K1932 antibody was detected using flow cytometry (NoveCyte 3130, Aglient). The K1932 antibody was diluted with 0.02 mol / L PBS (pH 7.4, containing 1% BSA) to an initial protein concentration of 18 μg / ml, and then serially diluted 3-fold to 0.305 ng / ml, for a total of 11 dilutions. The cell density was 2.0 × 10⁶ cells / ml. 6 Raji, Daudi, IM-9, and K562 cell suspensions (cells / ml) were added to each well at 100 μl. After mixing, the mixture was incubated at 2–8°C for 60 minutes. Then, a 1:250 dilution of goat anti-mouse IgG Fc-PE was added, and the reaction was carried out at room temperature in the dark for 30 minutes. Fluorescence values ​​were detected by flow cytometry. The flow cytometer was set to FAST, and 20,000 events were measured. The average fluorescence value of cells in each well was measured sequentially. The EC50 of K1932 antibody binding to Raji, Daudi, and IM-9 cells was calculated using GraphPadPrism 5.0 software. 50 The binding activity of different B lymphoma cells to CD19-positive cells with K1932 bispecific antibody was compared. Figure 4 The flow cytometry plots and dose-response curves of the K1932 antibody binding reaction with Raji cells are shown. It can be seen that the binding activity of the K1932 antibody with Raji cells is superior to that of Daudi and IM-9 cells, and the EC50 value is significantly lower. The flow cytometry results are summarized in Table 1. Figure 9The relative binding activity of the K1932 antibody stock solution at different dilution concentrations was demonstrated; the results showed that even at higher dilution factors (e.g., 10,000 times), the K1932 antibody could still maintain high binding activity to the CD19 target.

[0148]

[0149] K562 is a cell type that does not express CD19 membrane antigen. The results show that the Mean PE-H values ​​obtained by reacting different concentrations of K1932 antibody with K562 cells are basically consistent with those of the blank control, indicating that no binding reaction occurs. This proves that K1932 does not specifically bind to CD19 negative cells.

[0150] K1932 antibody binds to Raji, Daudi, and IM-9 cells in EC 50 The values ​​were 465.8 ng / ml, 614.4 ng / ml, and 363.1 ng / ml, or 2.329 × 10⁻⁶ ng / ml, respectively. -9 mol / L, 3.072×10 -9 mol / L, 1.816×10 -9 Based on the reaction trend and fluorescence range analysis, the K1932 antibody showed better binding activity to Raji cells than the K1932 antibody to Daudi and IM-9 cells.

[0151] Example 10: Comparison of binding activity between K1932 antibody and humanized monoclonal antibody CD19 monoclonal antibody K19

[0152] The specific binding activity of K1932 antibody, humanized CD19 monoclonal antibody K19, and Raji was detected by flow cytometry (NoveCyte 3130, Aglient). OKT3 monoclonal antibody was used as a control. K1932 antibody and K19 monoclonal antibody were diluted with 0.02 mol / L PBS (pH 7.4, containing 1% BSA) to an initial protein concentration of 18 μg / ml, and serially diluted 3-fold to 0.305 ng / ml. The cell density was prepared at 2.0 × 10⁶ cells / ml. 6 Raji cell suspension at cells / ml was added to a 96-well cell plate, 100 μl to each well. After mixing, the plate was incubated at 2-8°C for 60 minutes. Then, 1:250 diluted goat anti-mouse IgG Fc-PE was added. After reacting at room temperature in the dark for 30 minutes, the fluorescence value was detected by flow cytometry. The flow cytometer was set to FAST and 20,000 events were measured. The average fluorescence value of cells in each well was measured sequentially to compare the binding activity of K1932 bispecific antibody, humanized CD19 monoclonal antibody K19 and CD19 positive cells. Figure 5The binding activity curves of K1932 antibody and K19 monoclonal antibody with Raji cells were compared. It can be seen that the fitted EC50 value of K1932 antibody is 2.329×10^-9 mol / L, which is comparable to the activity of K19 monoclonal antibody. The flow cytometry results are summarized in Table 2.

[0153]

[0154] The fitting trend analysis showed that the Mean PE-H values ​​obtained from the binding reactions of different concentrations of OKT3 monoclonal antibody with Raji cells were basically consistent, and slightly higher than the results of the blank control, proving that OKT3 monoclonal antibody does not specifically bind to CD19 positive cells.

[0155] ECGs containing K1932 antibody and K19 monoclonal antibody and Raji cells 50 The values ​​were 150.9 ng / ml, 465.8 ng / ml, or 1.006 × 10⁻⁶ ng / ml, respectively. -9 mol / L, 2.329×10 -9 mol / L, K1932 antibody and K19 monoclonal antibody bind to Raji cells, fitting EC 50 The (mol / L) values ​​are all above 10. -9 The calculation results show that the binding activities of K1932 antibody, K19 monoclonal antibody and CD19 positive cell membrane antigen are basically the same.

[0156] Example 11: Binding activity of K1932 antibody, OKT3 and CD3 positive cells

[0157] To test the binding ability of the K1932 antibody to CD3, we performed flow cytometry (FACS) analysis on the obtained bispecific antibodies. The K1932 antibody was diluted with 0.02 mol / L PBS (pH 7.4, containing 1% BSA) to an initial protein concentration of 162 μg / ml, followed by a 3-fold gradient to 0.025 μg / ml. The OKT3 antibody was diluted with 0.02 mol / L PBS (pH 7.4, containing 1% BSA) to an initial protein concentration of 6 μg / ml, followed by a 3-fold gradient to 0.305 ng / ml.

[0158] The cell density was prepared at 5.0 × 10⁻⁶. 6Jurkat cell suspension (cells / ml) was added to each of the above sample wells, 100µl of the cell suspension was added sequentially, mixed well, and incubated at room temperature for 60 minutes. After centrifugation, the supernatant was carefully aspirated and discarded. 50µl of mouse anti-human κ chain monoclonal antibody diluted to 2µg / ml was added to each sample well, mixed well, and incubated at room temperature for 60 minutes. After centrifugation, the supernatant was carefully aspirated and discarded. 50µl of FITC-labeled goat anti-mouse IgG (1:1000) was added to each sample well, mixed well, and incubated at room temperature in the dark for 30 minutes. After centrifugation, the supernatant was carefully aspirated and discarded. 170µl of 0.02mol / L PBS (pH 7.4) was added to each well to resuspend the cells. The flow cytometer was set to a loading volume of 10000 events and a flow rate of Fast. After carefully resuspending and mixing the cells with the pipette tip, the cell suspension was transferred to 0.5ml centrifuge tubes, and the cell fluorescence values ​​were measured sequentially. Compare the binding activity of K1932 bispecific antibody, OKT3 antibody, and CD3-positive cells; Figure 6 The flow cytometry profile of the binding activity of K1932 antibody to CD3-positive cells was presented, showing that its binding affinity to CD3 molecules is significantly weaker than that of OKT3, thereby reducing the risk of off-target T cell activation. The results were then used in GraphPad Prism software to calculate EC50. 50 as follows:

[0159]

[0160] The results above show that the ECG of K1932 antibody... 50 The value is 2.255 × 10 -8 EC50 of OKT3 antibody (mol / L) 50 The value is 1.459 × 10 -9 At mol / L, the binding activity of K1932 antibody to CD3 is only about 1 / 10 of that of OKT3, and the binding ability of K1932 to CD3ε molecules on the surface of T cells is significantly weaker than that of OKT3.

[0161] Example 12: K1932 antibody-mediated T lymphocyte activation assay (CD69 assay)

[0162] CD69 is an early marker of T cell activation. Jurkat cells cultured under normal conditions rarely express CD69. However, when Jurkat cells and Raji cells are co-cultured in an appropriate ratio in the presence of K1932 antibody (a CD3 molecule activator), CD69 can be expressed on the surface of Jurkat cells, and the expression level is positively correlated with the concentration of the stimulant.

[0163] Jurkat cells cultured in 10% FBS 1640 medium were collected by centrifugation. After adjusting the cell concentration, they were mixed with Raji cells. The resulting cell suspension contained 3 × 10⁶ Jurkat cells. 6 cells / ml, Raji cells 3×10 5 Cells / ml were used to inoculate the above cell suspension into 24-well cell culture plates. Serially diluted K1932 antibody was added, and the plates were then incubated at 37°C with 5% CO2 for 18 hours. After centrifugation to remove the supernatant, the cells were mixed with Anti-Hu CD69 PE (clone: ​​FN50, eBioscience) and reacted in the dark for 2 hours. The amount of CD69 expressed by the cells was then measured using flow cytometry. The CD69 fluorescence value was used to determine the K1932 content in the corresponding solution. Figure 7 Flow cytometry plots and dose-response curves of CD69 expression in T lymphocytes stimulated with K1932 antibody are presented. The figures show that K1932 antibody effectively activates T lymphocytes, and CD69 expression is positively correlated with antibody concentration. The quantitative response relationship of CD69 expression after stimulation with different concentrations of K1932 is also observed. Figure 8 The graphs show the CD69 expression levels of the K1932 antibody under T+B cell co-culture, T cell culture alone, and B cell culture alone conditions. The graphs indicate that the K1932 antibody significantly activates CD69 expression only when T cells and B cells co-exist, while T cells or B cells alone cannot achieve the same activation effect. This suggests that the mechanism of action of the K1932 antibody depends on the presence of the CD69 target, further validating its target specificity. EC50 was calculated using GraphPad Prism. 50 Value, dose-response curve fitting constant R 2 The value should not be lower than 0.90. Table 4 shows the results of T lymphocyte activation by different batches of K1932 antibody.

[0164]

[0165] The results in the table above show that, in the presence of Raji cells (B cells), the expression of CD69 molecules on the surface of Jurkat cells stimulated by K1932 antibody is positively correlated with the concentration of K1932. The fitting constant R for the dose-response curve is also shown. 2 Greater than 0.98, EC 50 The value is approximately 4 × 10 -12 mol / L.

[0166] Example 13: K1932 antibody activation of T cells requires co-stimulation by B cells.

[0167] Jurkat cells cultured in 10% FBS 1640 medium were collected by centrifugation, and the cell concentration was adjusted to 3 × 10⁻⁶. 6 cells / ml, for later use; ① The cell suspension after mixing with an equal volume of Raji cells contains 3 × 10 Jurkat cells. 5 cells / ml, Raji cells 3×10 4 ② Adjust the Jurkat cell concentration to 1.5 × 10⁶ cells / ml; 6 ③ Adjust the Raji cell concentration to 1.5 × 10⁻⁶ cells / ml; 6 Cells / ml; The above cell suspension was added to 48-well cell culture plates, followed by serially diluted K1932 antibody, and then cultured in a 5% CO2, 37℃ incubator for 18 hours. After 18 hours, the supernatant was removed by centrifugation, and 486µl of Anti-Human CD69PE (clone: ​​FN50, REF: 12-0699-42, eBioscience) was mixed with 3564µl of 0.02mol / L PBS (pH 7.4, containing 1% BSA). 50µl of the mixture was added to each well, and the mixture was incubated at 2-8℃ in the dark for 120 min. The average fluorescence intensity of CD69 expression in cells was measured using a flow cytometer (NoveCyte 3130, Aglient). The results are shown in Table 5 below. The measured concentrations of K1932 were plotted on the x-axis, and the average fluorescence intensity of cells was plotted on the y-axis to observe the response relationship of CD69 expression after stimulation with different concentrations of K1932 (positive control).

[0168]

[0169] Based on the above data, it can be calculated that the (T+B)Cell+K1932 antibody group corresponds to EC. 50 The value was 692.7 pg / ml, and the T Cell+K1932 antibody group corresponded to EC. 50 The value was 132.9 ng / ml, approximately a 191-fold ratio. In the absence of B cells, the MFI of CD69 was low; even with K1932 at 20 μg / ml, the MFI only reached 20821, about 6 times the background value. Therefore, K1932 alone cannot effectively stimulate T cell activation. Figures 10-12 The study presented the killing curve and related data of CD8+ T cells activated by K1932 antibody against B lymphocytes, showing that it can significantly enhance the tumor killing ability mediated by T cells.

[0170] Example 14: Relative binding activity of K1932 (with CD3E, CD19):

[0171] Assay for CD19-CD3 bispecific antibody binding activity using recombinant human CD3e and CD19 double antigen sandwich method: Recombinant human CD3E (Sino Biologcal Inc., batch number: LC14JA0908) was dissolved in 0.25 ml of water for injection, then diluted to 0.4 μg / ml with carbonate coating buffer. 100 μl was coated into each well of a 96-well ELISA plate and incubated at 37°C for 2 hours, then overnight at 2–8°C. The plate was washed three times with washing buffer and patted dry. Blocking buffer (20 mmol / L PBS-T containing 2% BSA) was added to each well of the ELISA plate (220 μl per well) and incubated at 37°C for 60 minutes. The plate was washed three times with washing buffer and patted dry. The plate was then diluted with sample diluent (20 mmol / L PBS-T containing 1% BSA). Three batches of K1932 antibody stock solution (batch numbers: 20240622, 20240630, 20240708) were pre-diluted to 10 μg / ml according to protein content. The diluted K1932 antibody stock solution was added to the first column of the blocked ELISA plate, starting at 10 μg / ml, and serially diluted 3-fold up to the 11th column, for a total of 11 dilutions. 100 μl was added to each well (2 wells per dilution). After adding the sample, the ELISA plate was incubated at room temperature (22℃~27℃) for 60 minutes, then washed four times with washing buffer and patted dry. Biotin-labeled recombinant human CD19 (0.2 mg / mL) was then added. The solution was diluted 3000 times with horseradish peroxide (g / ml) and added to each well of the ELISA plate, 100 μl per well. After incubation at room temperature (22℃~27℃) for 60 minutes, the plate was washed four times with washing buffer and then patted dry. Next, 100 μl of diluted horseradish peroxide-labeled streptavidin (1:120000) was added to each well of the ELISA plate and incubated at room temperature (22℃~27℃) for 60 minutes. After washing five times with washing buffer, the plate was patted dry. 100 μl of TMB chromogenic solution was added to each well of the ELISA plate and incubated at 37℃ in the dark for 10 minutes. Finally, 50 μl of stop solution (1 mol / L sulfuric acid) was added to each well to terminate the reaction. The A450 value was read using an Infinite M200 PRO ELISA reader, and the EC50 of the K1932 antibody solution in the double-antigen sandwich ELISA reaction was calculated using GraphPad Prism 8 software. 50 The values ​​are shown in Table 6.

[0172]

[0173] Analysis of the results in Table 6 shows that the overall absorbance variation range and curve fitting of the three batches of K1932 antibody stock solutions are basically consistent; and the EC50 of each batch of K1932 antibody is also consistent. 50 The values ​​were 66.06 ng / ml, 63.14 ng / ml, and 64.31 ng / ml, or 3.27 × 10⁻⁶ ng / ml, respectively. -10 mol / L, 3.13×10 -10 mol / L, 3.18×10-10 The results were basically consistent across batches at mol / L.

[0174] Example 15: K1932 antibody-mediated activation of CD8 + T-cell killing assay against Raji cells

[0175] The cytotoxic effect of K1932 antibody on B lymphocytes was detected. PBMCs (donor: Z0645) were resuscitated, and T cells were activated and expanded using IL-2 for 7 days before CD8 cells were isolated. + T cells, CD8 + T cell density was adjusted to 7.5 × 10⁻⁶. 5 cells / mL, adjust the target cell density to 2.5 × 10⁻⁶. 5 40 μL (30,000 cells / mL) of CD8 was added to each well of a 96-well transparent U-shaped plate. + T cells and 40 μL (10,000) of target cells were used at a cell ratio of 3:1. 5× sample solutions were prepared using complete RPMI 1640 medium, with a maximum working concentration of 100 ng / mL, diluted 5-fold, resulting in 9 concentrations. 20 µL of sample solution was added to each well of a 96-well plate seeded with cells, with two replicates for each drug concentration. Cells in the drug-treated 96-well plates were cultured at 37 ℃ and 5% CO2 for 24 hours, followed by Bright-Glo analysis. The Bright-Lite Luciferase Assay System reagent was melted and the cell plate was equilibrated to room temperature for 30 minutes. 100 μL of Bright-Lite Luciferase Assay System solution was added to each well. Cells were lysed by shaking on a track-mounted shaker for 3 minutes. The lysate was transferred to 96-well white impermeable plate (Absin#abs7016), and the Luc signal was read using a multi-mode microplate reader. The target cell killing rate was calculated using the following formula:

[0176] lethality % = ×100%

[0177] Data was analyzed using GraphPad Prism 7.0 software. A nonlinear S-curve regression was used to fit the data to derive the dose-response curve, from which the IC was calculated. 50 The values ​​are shown in Table 7.

[0178]

[0179] As shown in Table 7, the killing effect exhibits an S-shaped curve with increasing antibody concentration. K1932, K193, and Blinatumomab show significant killing effects, reaching 72%, 80%, and 80% respectively at high concentrations after 24 hours. The three antibodies mediate the activation of CD8... + IC50 of T cell killing Raji cells 50 The concentrations were 0.066 ng / ml, 0.014 ng / ml, and 0.007 ng / ml, respectively, which is 3.27 × 10⁻⁶. -13 mol / L, 1.86×10 -13 mol / L, 1.30×10 -13 mol / L. K1932 antibody-mediated activation of CD8 + The T cell killing ability against Raji cells is basically the same as that of Blinatumomab.

[0180] Example 16: K1932 antibody-mediated activation of peripheral blood T cells to kill B cells (PBMC) experiment

[0181] The cytotoxic effect of K1932 antibody-mediated T cell activation on B lymphocytes was detected by flow cytometry (Canto II, BD). Peripheral blood mononuclear cells (PBMCs) from healthy individuals were used as experimental cells, and the cell density was prepared to be 1.8 × 10⁻⁶. 6 Cell suspension at 300 μl / ml was added to each well of a 48-well plate. The plate was then incubated at 37.0°C for at least 1 hour in a CO2 incubator containing 5.0% CO2. Quantitative K1932 antibody was prepared using a serial dilution range of 400 ng / ml to 0.4 pg / ml, with 7 spots for 10-fold serial dilutions. 300 μl of the diluted antibody solution (0.4 pg / ml to 400 ng / ml) was added sequentially to each well of the 48-well plate containing PBMCs. The plate was then incubated at 37.0°C for 18 hours, 24 hours, 42 hours, and 72 hours, respectively. After cell collection, non-specific staining induced by the fluorescent antibody against the Fc receptor was blocked using Human BD FcBlock™, which specifically targets the Fcγ receptor. FVS510 fluorescent antibody was added to stain live and dead cells in PBMCs. CD20-PE-Cy™7 was added to stain the surface of B lymphocytes in PBMCs. Fluorescent antibodies corresponding to CD3-PE, CD4-FITC, CD8-APC-Cy7, and CD69-RB705 were added to stain the surface of T lymphocytes in PBMCs. After cell fixation and perforation, fluorescent antibodies corresponding to granzyme B and perforin were added to stain for intracellular factors. Flow cytometry was used to detect CD3+ in 50,000 PBMCs collected under P2 gate. + T appears CD4+ T cells or CD8 + T cells and in CD69 + Simultaneously, the number of cells positive for at least one cytokine, Granzyme B-BV421 and Perforin-APC, was detected. CD4+ + T cells or CD8 + T cell counts were quantitatively converted to 100,000, and then CD69, Granzyme B-BV421, and Perforin-APC positive cell counts were proportionally converted. EC50 was then calculated using GraphPad Prism. 50 The values ​​and statistical results are shown in Tables 8 and 9. CD20 was analyzed under the P2 gate. + Cells were analyzed, and dead cell populations were circled using live-dead fluorescence (FVS510 fluorescence). The percentage of dead cells was calculated as the B cell killing rate. Statistical results are shown in Table 10.

[0182]

[0183]

[0184]

[0185] Analysis of the results in Tables 8 and 9 shows that the number of CD69-positive cells increases with the increase of K1932 antibody content, while the number of CD8-positive cells also increases. + The number of CD69-positive T cells was significantly higher than that of CD4-positive cells. + T cells all exhibit an S-shaped curve, CD8 + T cell CD69 + Perforin + and CD69 + Granzyme B + The trend is compared to CD4 + The positive trend in T cell activity indicates that K1932 can effectively activate T cells, especially CD8 cells. + T cells.

[0186] Analysis of the results in Table 10 shows that the killing effect begins at high concentrations of 18h and 24h. The killing effect is significant at 42h and 72h as the K1932 antibody content increases. At 72h, the killing effect at high concentrations reaches 95%, showing an S-shaped curve.

[0187] In summary, K1932 antibody-mediated T cell activation is mainly due to CD8. + After T cells are activated, they release granzyme B and perforin to kill B cells.

[0188] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A multifunctional antibody that binds human CD19, CD3, and FcγR, characterized in that, include: 1) It specifically binds to the domain of B lymphocyte membrane antigen CD19, wherein the Fab fragment that binds to CD19 contains a humanized light chain variable region VL and a heavy chain variable region VH. The amino acid sequence of the heavy chain variable region VH is SEQ ID No. 2, and the amino acid sequence of the light chain is SEQ ID No.

3. 2) A single-chain antibody domain that specifically binds to the T cell surface antigen CD3 molecule, wherein the single-chain antibody recognizing the CD3 molecule includes a light chain variable region VL and a heavy chain variable region VH, which are linked by Linker2. The amino acid sequence of the light chain variable region is shown in SEQ ID No. 5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

4. 3) The domain of the FcγR receptor on NK cells is monovalently bound, wherein the structure of the Fc fragment includes CH1, the Hinge Region, CH2 and CH3, and the amino acid sequence is SEQ ID No. 1; The single-chain antibody that recognizes CD3 molecules in the single-chain antibody domain that specifically binds to the T cell surface antigen CD3 molecules is linked to the C-terminus of the CD19 antibody light chain via the linker peptide Linker1. The structure of the multifunctional antibody is as follows: ， Furthermore, the FcγR binding sequence is CH1-Hinge Region-CH2-CH3, and the amino acid sequence is SEQ ID No. 1; The sequences of Linker1 and Linker2 are SEQ ID No.6 and SEQ ID No.7, respectively.

2. A method for preparing a multifunctional antibody as described in claim 1, characterized in that, The cells were prepared using gene recombination technology and expressed in CHO cells using expression vectors from different types of mammalian cells. The CHO cells were cultured in a chemically defined culture medium, and no hormones or animal-derived proteins or their hydrolysates were added during the culture process.

3. The method for preparing a multifunctional antibody according to claim 2, characterized in that, The preparation steps include: The plasmid containing the multifunctional antibody gene was tandemly linearized by digestion with an endonuclease. Positive clones were obtained after transfecting CHO cells, and then cultured in a bioreactor, with the products secreted into the culture supernatant. High-purity multifunctional antibodies were obtained by purification using affinity chromatography and ion exchange chromatography media.

4. The use of the multifunctional antibody according to claim 1 in the preparation of a drug for treating relapsed and refractory B-cell lymphoma, wherein the multifunctional antibody is a T-cell-dependent immunotherapeutic drug that kills B cells.

5. A pharmaceutical composition containing the multifunctional antibody of claim 1.

6. The pharmaceutical composition according to claim 5, wherein the method of use comprises: It is formulated into a liquid preparation and administered via intravenous, subcutaneous, or intramuscular injection.

7. The pharmaceutical composition according to claim 5, wherein the formulation comprises: acetic acid, histidine, methionine, sucrose, and Tween 20.

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