Multifunctional antibody combined with human CD19, CD3 and Fc gamma R

Abstract

The invention relates to the technical field of biological medicine, in particular to a multifunctional antibody K1932 combined with human CD19, CD3 and Fc gamma R. The multifunctional antibody is composed of a bivalent Fab fragment capable of specifically recognizing CD19 on the surface of B lymphocyte, a bivalent single-chain antibody capable of recognizing CD3 molecules on the surface of T lymphocyte and a human immunoglobulin Fc structural domain combined with an Fc receptor, the single-chain antibody capable of recognizing the CD3 molecules is connected with the C tail end of a Fab light chain through a connecting peptide Linker 1, and the human immunoglobulin Fc structural domain combined with the Fc receptor is connected with a human immunoglobulin Fc structural domain combined with the Fc receptor. The heavy chain and the light chain of the CD3 single-chain antibody are connected through flexible Linker 2; the biomacromolecule formed by the three-function structural domain is a highly-oriented immunotherapy drug, is suitable for injection administration, can guide T cells to attack CD19 positive B lymphocytes and combine the Fc structural domain to Fc gamma R, and can adjust the in-vivo half-life period of the biomacromolecule and reduce the unnecessary influence on normal cells at the same time.

Description

Technical Field

[0001] The present 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 Art

[0002] Pre-B cell is a histological and embryological term published in 2014. Its Chinese name is pre-B cell and its English name is pre-B cell. It is defined as differentiated from progenitor B cells and accounts for about 5% of nucleated cells in adult bone marrow. It has completed heavy chain gene rearrangement, but light chain gene rearrangement has not yet begun. It is large in morphology, does not express membrane immunoglobulin, and can express CD19 molecules. B cell lymphoma is a solid tumor that occurs in B cells of the blood system. It includes Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL). It has many types, including classical Hodgkin's lymphoma and nodular lymphocyte-dominant Hodgkin's lymphoma, which are now considered to be tumors originating from B cells. The five most common B-cell non-Hodgkin lymphomas are diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma / chronic lymphocytic leukemia, and mantle cell lymphoma (MCL), accounting for about three-quarters of non-Hodgkin lymphoma. Non-Hodgkin lymphoma is a group of malignant tumors that originate from lymphoid tissue and spread throughout the body. Its morbidity and mortality rate rank fifth among malignant tumors. Most NHLs originate from B lymphocytes (B-NHL).

[0003] Lymphoma is one of the most common malignant tumors. Acute lymphoblastic leukemia (ALL) is an aggressive hematological malignancy that originates from B-line or T-line lymphocyte progenitor cells and is caused by abnormal differentiation of hematopoietic stem cells. ALL is common in children and adolescents and is relatively rare in adults. Most ALL cases are B-cell and Philadelphia chromosome (Ph) negative. The first-line treatment for ALL is combined chemotherapy, including induction remission and post-remission treatment (consolidation and maintenance). Induction remission treatment drugs include vincristine, glucocorticoids, L-asparaginase and anthracyclines. Commonly used regimens in the consolidation and intensive phase include high-dose cytarabine, high-dose methotrexate, etc. Standard maintenance therapy is based on 6-mercaptopurine and methotrexate. Intrathecal injection of methotrexate, cytarabine and dexamethasone is required during treatment. Patients with poor prognostic factors should actively consider allogeneic hematopoietic stem cell transplantation (allo-HSCT) after the first complete remission. In general, children and adolescents have a higher complete remission rate after treatment than adults, but the complete remission (CR) rate in adult studies can also reach 85% or more. ALL is generally considered a curable malignant disease. Despite this, nearly 50% of adult patients with pre-B-cell ALL and up to 20% of children with pre-B-cell ALL will eventually relapse or develop primary resistance. Ph-positive ALL is a rare ALL subtype, accounting for about 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 remission rate of Ph-positive ALL has improved and is similar to that of Ph-negative ALL patients, but the duration of remission and relapse-free survival are still not ideal. For relapsed and refractory adult B-cell ALL, the NCCN guidelines recommend regimens including belintoquinone, inotuzumab, ozogamicin, CAR-T products, and combination chemotherapy regimens based on products such as cytarabine, alkylating agents, or fludarabine / clofarabine. There is no unified opinion on the treatment of refractory relapsed Ph-negative ALL in China, and combination chemotherapy is still the main recommendation. Relapsed and refractory Ph-positive patients should be tested for BCR-ABL mutations, and TKI replacement can be considered in the salvage treatment plan. Other treatment principles are the same as those for Ph-negative patients. Once a patient relapses, the prognosis is extremely poor. Before belintoquinone was launched, the median survival time of adult patients with relapsed and refractory ALL was about 3-6 months, the CR rate of single-agent chemotherapy was only 8% (95% CI: 4%-14%), and the CR rate of combination chemotherapy was about 25%-46%. Older 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 and refractory Ph-negative ALL treated in 14 centers in China between 2005 and 2014 were retrospectively observed. The CR+CRh rate was about 31%, and the median duration of CR / CRh was 2.7 months. The CR+CRh rate of the first salvage treatment was 41%, the second salvage was 24%, and the late salvage treatment was only 17%. In China, the CAG regimen containing aclarubicin is different from the treatment regimen in Western countries. It can achieve a CR rate of about 50% in patients with relapsed and refractory ALL, but the median overall survival (OS) is only about 3 months. Therefore, patients with relapsed and refractory ALL have a great clinical need for new therapeutic drugs and treatment methods that can effectively improve their remission rate and prolong their survival time.

[0004] The development of genetic engineering technology has made it possible to research and develop bispecific antibodies and multispecific antibodies. More than ten types of bispecific genetic engineering antibodies for treating tumors have been developed, mainly in the form of antibody fragments, tandem single-chain antibodies, miniature bifunctional antibodies, single-chain miniature bifunctional antibodies, bispecific trivalent molecules, bispecific tetravalent molecules, trifunctional antibodies, etc. Although there are many molecular forms of bifunctional antibodies reported in the basic research stage, few of them can enter clinical research.

[0005] There are currently two bispecific antibody products approved for marketing internationally. One is the Catumaxomab antibody developed by TrionPharma, which targets the tumor surface antigen EpCAM and the T cell surface receptor CD3; the other is the Blinatumomab antibody jointly developed by Micromet and Amgen, which targets CD19 and CD3. Both achieve the purpose of treating tumors by activating and recruiting killer T cells.

[0006] Catumaxomab belongs to the Triomab technology platform, which is composed of mouse IgG2a targeting tumors and rat IgG2b targeting human CD3ε. It activates monocytes, macrophages, stellate cells and NK cells through Fcγ receptors to achieve trifunctional antibody activity. Catumaxomab is the first bispecific antibody approved for marketing, and it has very obvious shortcomings, mainly reflected in the complex production process of the Triomab platform antibody technology, which is difficult to control, has low yield, difficult purification and poor stability; in addition, heterologous antibodies have the problem of immunogenicity.

[0007] Blinatumomab is a bispecific antibody based on the BiTE technology platform, which is composed of two single-chain antibodies containing variable regions connected by polypeptides. Unlike the Triomab platform antibody, the BiTE platform antibody is mass-produced by Chinese hamster ovary (CHO) cells, and the BiTE antibody only contains two binding domains, one with high affinity targeting cancer cell surface antigens (such as CD19), and the other with lower affinity targeting CD3. Clinical trials have shown 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. So far, genetically engineered bispecific antibodies have achieved a breakthrough in the immunotherapy of malignant tumors.

[0008] At present, bispecific antibody molecules targeting human CD19 and CD3 have shown significant effects in animal models or some limited clinical trial studies. Depending on the platform technology used, the effects of its action show great differences. There are many reports on prokaryotic expression systems in the literature. This expression system is fast and simple to operate, but the effects of the obtained bispecific antibody molecules are always unsatisfactory, and the stability is also poor. It is easy to form polymers and completely lose activity, and it needs to be cryopreserved at ultra-low temperatures or made into freeze-dried powder. Another method of producing bispecific antibodies by covalently coupling monoclonal antibodies through hybridoma technology and chemical reactions generally has low biological activity and must be used at higher doses to exert a certain effect.

[0009] The markers on the surface of lymphocytes have been generally considered as targets for autoimmune diseases such as B-cell lymphoma and B-cell disorders. The markers on the surface of B lymphocytes include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, CD81-CD86, etc. Currently, monoclonal antibody drugs have been developed for molecules with high expression 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 (rituximab, etc.) have become the first choice for the treatment of non-Hodgkin's lymphoma and have been most widely used worldwide. It is a well-known fact that acute lymphoblastic leukemia (ALL) and many other B lymphocyte malignancies do not express CD20, or express CD20 at a low level. Only about half of non-Hodgkin's lymphoma patients respond to CD20-controlled immunotherapy. CD19 is an important membrane antigen related to B lymphocyte differentiation, activation, proliferation and antibody production. It is the best marker for diagnosing B lymphocyte tumors (leukemia, lymphoma) and identifying B lymphocytes. CD19 is a specific marker on the surface of B lymphocytes. It belongs to the immunoglobulin superfamily and is related to B cell activation and signal transduction. It is expressed in pre-B lymphocytes, immature B lymphocytes, mature B lymphocytes, and activated B lymphocytes, but not in lymphoid pluripotent stem cells and other tissues. Most NHLs originate from B lymphocytes. More than 95% of B cell NHLs express CD19 antigens, and CD19 antigens are relatively exposed. There is no free CD19 in human serum, so CD19 can be used as a target for the treatment of B cell lymphoma. The CD19 molecule is a marker on the surface of B cells that is more widely present than CD20. It is a receptor expressed on the surface of B cells. It belongs to the immunoglobulin superfamily. Its ligands and related molecules are: CR2 (CD21), TAPA-1 (CD81), Leu-13, PI-3K, Vav, lyn and fyn. CD19 is an important signal transduction molecule that regulates the growth, activation and differentiation of B lymphocytes. CD19 can regulate signal responses and plays an important role in regulating the signal threshold of B lymphocyte antigen receptors or other surface receptors. CD19 is a pan-B cell membrane glycoprotein expressed from the early stage of pre-B cell development to terminal differentiation, which can regulate the development and function of B lymphocytes. The expression of CD19 has been identified in most lymphoid tumors, most non-Hodgkin lymphomas (NHLs) and leukemias including chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL) and Waldenstrom's macroglobulinemia (WM).The expression of CD19 runs through the entire life cycle of B lymphocytes, from primitive B cells, pre-B cells, early developed B cells, mature B cells, plasma cells developed from mature B cells, and malignant B lymphoma cells. Most tumor cells derived from B lymphocytes, such as pre-B acute lymphoblastic leukemia, chronic B lymphocytic leukemia, prolymphocytic leukemia, non-Hodgkin's lymphoma, hairy cell leukemia, common acute lymphocytic leukemia and some non-acute lymphoblastic leukemia, multiple myeloma, plasma cell tumors, etc., all 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 subtypes, namely CD3δ, CD3ε and CD3γ. The molecular weight of CD3δ and CD3ε is 20kD, and the molecular weight of CD3γ is 26kD. It is expressed on the surface of T lymphocytes, thymocytes and NK cell membranes. It is expressed by 61% to 85% on normal peripheral blood lymphocytes and 60% to 85% on 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 (TCR), and is the main membrane antigen that transmits TCR signals bound to peptides / MHC. TCR is essential for cell surface expression, antigen recognition and signal transduction. CD3 is a specific molecule on the surface of T lymphocytes, through which T lymphocytes with killing effects can be recruited. Monoclonal antibodies to CD3 can induce or prevent T lymphocyte activation. In the presence of anti-CD28 antibody or IL-2, anti-CD3 antibody can induce apoptosis of T lymphocytes. CD3 is one of the best markers for mature T lymphocytes in peripheral blood. The determination of CD3+T lymphocytes is of great significance for the evaluation of immunodeficiency diseases (T lymphocyte deficiency), leukemia, and lymphoma (T lymphocyte type) typing diagnosis. 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 of severe autoimmune diseases to remove T lymphocytes. U.S. Patent No. 4,361,549 describes a mouse hybrid cell line for the production of monoclonal antibody OKT3 against antigens found in normal human T cells and skin T lymphoma cells. In addition, U.S. Patent No. 5,885,573 describes the transfer of mouse OKT3 into a human antibody framework to construct a humanized monoclonal antibody in order to reduce its immunogenicity when used in humans and reduce the probability of human anti-mouse antibody (HAMA) reactions. OKT3 is the first mouse monoclonal drug approved by the US FDA in 1986 for the treatment of acute rejection of organ transplantation. It is also the first monoclonal antibody drug approved for use by a government drug administration authority in the world. The main drawback of mouse OKT3 monoclonal antibody therapy is T cell activation and HAMA response caused by cytokine release due to cross-linking between T cells and FcγR-bearing cells. After more than 10 years of use in 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 immunopotentiators to stimulate T cell activation and proliferation. Anti-CD3 monoclonal antibodies, anti-CD28 antibodies or interleukin-2 are used in combination in in vitro cell culture to induce T cell proliferation. OKT3 is further used alone or as a component of a bispecific antibody to target cytotoxic T cells to tumor cells and virus-infected cells.So far, the use of antibodies as agents for recruiting T cells has been hampered by several findings. First, natural or engineered antibodies with high T cell affinity often do not activate the T cells to which they bind; second, natural or engineered antibodies with low T cell affinity are often poor or ineffective in eliciting T cell-mediated cell lysis, so it is very important to select an anti-CD3 mAb with appropriate affinity.

[0011] The uptake, processing, and presentation of antigens by FcRs, followed by the activation of T cells, are key processes in adaptive immunity. Effective recognition of antigens by T cells requires first taking up the antigens, processing them, and then presenting them on the MHC molecules on the cell surface. A key step in antigen presentation is the transport of foreign cargo to intracellular compartments where the antigens can be processed and loaded onto MHC molecules. FcγR plays an important role in antigen uptake, processing, and presentation.

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

[0013] Traditional bispecific antibodies have low specificity and may have unnecessary effects on normal cells while attacking leukemia cells. There is a certain probability that nonspecific immune responses will be triggered during treatment. Due to the lack of high specificity, these antibodies may attack normal cells, not just leukemia cells, which leads to the so-called "nonspecific toxicity", that is, normal tissues are damaged, resulting in poor treatment effects and requiring higher doses to achieve therapeutic effects, thereby increasing patient discomfort and risks. At the same time, traditional bispecific antibodies have defects in the selection of specific affinities or linked effectors, which may lead to over-activation of T cells or other adverse reactions. If the affinity of the bispecific antibodies selected is too high or the linked effectors are too strong, they may lead to over-activation of T cells, which may trigger strong immune responses, including cytokine release syndrome, and reduce the safety of treatment. Therefore, there is an urgent need for a multifunctional antibody drug that can provide highly targeted treatment while binding in a low-affinity manner. Summary of the invention

[0014] To this end, the present invention provides a multifunctional antibody that binds to human CD19, CD3 and FcγR to overcome the problems of low specificity of bispecific antibodies in the prior art, unnecessary effects on normal cells, and defects in the selection of specific affinity or binding effectors.

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

[0016] 1) A bivalent domain specifically binding to the B lymphocyte membrane antigen CD19, wherein the Fab fragment binding to CD19 comprises a humanized light chain variable region VL, the amino acid sequence of which is SEQ No. 2, and a heavy chain variable region VH, the amino acid sequence of which is SEQ No. 3;

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

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

[0019] The single-chain antibody that recognizes the CD3 molecule in the bivalent single-chain antibody domain that specifically binds to the T cell surface antigen CD3 molecule is connected to the C-terminus of the CD19 antibody light chain through a hydrophilic connecting peptide;

[0020] The multifunctional antibody structure is:

[0021]

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

[0023] The sequences of the humanized CD19 binding domains 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 is in the form of ScFv, which is directed against 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 of 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 a multifunctional antibody, which is prepared by gene recombination technology, using different types of mammalian cell expression vectors, preferably, expressing in CHO cells, and culturing the CHO cells using a chemically defined medium, without adding hormones and various animal-derived proteins or their hydrolysates during the culture process.

[0029] Furthermore, the preparation steps include:

[0030] The plasmids containing the multifunctional antibody gene were connected in series and linearized by endonuclease digestion;

[0031] After transfection of CHO cells, positive clones were obtained, which were then cultured in a bioreactor, and the products were secreted into the culture supernatant;

[0032] The multifunctional antibody is obtained by purifying using ion exchange chromatography media or affinity chromatography combined with ion exchange chromatography.

[0033] The multifunctional antibody provided by the present invention is a therapeutic drug for treating relapsed and refractory B-cell lymphoma. The multifunctional antibody is a T cell-dependent immunotherapeutic drug for killing tumor cells.

[0034] The invention also discloses a pharmaceutical composition containing the multifunctional antibody.

[0035] Furthermore, the method of using the pharmaceutical composition includes: preparing a liquid preparation and administering the composition by subcutaneous or intramuscular injection.

[0036] Furthermore, the pharmaceutical composition formula comprises: 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 multifunctional antibody with a novel antibody structure, which simultaneously targets CD3, CD19 and FcγR.

[0039] The innovative mechanism of action of this antibody is to use the patient's own cytotoxic T cells to attack malignant B cells. B lymphocytes bind to the anti-CD19 part of K1932, T cells bind to the anti-CD3 part, and Fc binds to monocytes, macrophages, stellate cells, and NK cells, which can regulate the half-life of biomacromolecules in vivo while reducing unnecessary effects on normal cells. This feature of K1932 enables it to temporarily connect malignant cells and T cells, thereby inducing T cells to kill 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 produced by K1932-activated T cells is very similar to the natural cytotoxic T cell response.

[0040] The present invention is a multifunctional antibody targeting CD19, CD3 and FcγR, through which the human body's own T cells can be used to kill CD19-positive tumor cells to achieve the purpose of treating lymphoma and leukemia.

[0041] The molecular structure of K1932 is fundamentally different from other specific antibodies that have entered the clinical research stage. It has the advantage of better targeting and uses low-affinity CD3ε-ScFv as a binding molecule to connect effectors and T cells. Even if each K1932 molecule contains two CD3 binding sites, it cannot activate T cells like OKT3 due to its low concentration. In terms of molecular structure, the molecular structure of K1932 uses low-affinity CD3ε-ScFv as a connecting effector, and its affinity for binding to T cells is weak. It helps prevent over-activation of T cells, thereby reducing adverse reactions, while ensuring preferential binding to the CD19 target. It binds to FcγR to attract effector cells to play a role.

[0042] The invention solves the problems existing in the prior art that the bispecific antibodies have low specificity, may have unnecessary effects on normal cells, and have defects in the selection of specific affinity or connection effectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Constructing a map for the gene co-expression vector of the present invention;

[0044] Figure 2 This is a graph showing the electrophoresis detection results of the plasmid linearization of the present invention (1% agarose gel);

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

[0046] Figure 4 The K1932 antibody of the present invention is a flow cytometric analysis of the site-specific binding reaction with Raji, Daudi, IM-9, and K562 cell CD19 and a dose-response curve;

[0047] Figure 5 The flow cytometric profiles and dose-response curves of the site-specific binding of the K1932 antibody, K19 monoclonal antibody, and OKT3 monoclonal antibody to Raji cell CD19;

[0048] Figure 6 The K1932 antibody, OKT3 and CD3 site binding reaction flow cytometric profile and dose-response curve of the present invention;

[0049] Figure 7 The K1932 antibody of the present invention activates T lymphocytes to express CD69 flow cytometry and a dose-response curve;

[0050] Figure 8 The CD69 expression reaction curve of the K1932 antibody of the present invention reacting with T+B cells, T cells, and B cells;

[0051] Fig. 9 is the relative binding activity of the K1932 antibody stock solution of the present invention;

[0052] Fig.10 The K1932 antibody of the present invention mediates the activation of CD8 + T cells kill B cells;

[0053] Fig.11 The results of the K1932 antibody-mediated T cell activation test (PBMC) of the present invention;

[0054] Fig.12 This is the curve of K1932 antibody of the present invention mediating the activation of T lymphocytes to kill B lymphocytes. DETAILED DESCRIPTION

[0055] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0057] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0058] Supplement some of the contents of the drawings in the description of the drawings in the specification:

[0059] Figure 4 :

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

[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: PE channel fluorescence intensity

[0071] Figure 6 :

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

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

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

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

[0076] Figure 7 :

[0077] Mean PE-H(CD69): Average PE channel fluorescence intensity (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 PE channel fluorescence intensity (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] Fig.10 :Activated CD8+ T+Raji-Luc2 killing curve (activate CD8 + T cell + Raji-Luc2 killing curve)

[0088] Killing percentage (%): Killing percentage (%)

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

[0090] k1932 Stock Solution: K1932 stock solution

[0091] k193 Reference Solution: K193 reference solution

[0092] Blinatumomab: Blinatumomab antibody

[0093] Fig.11 :

[0094] CD69 expression curve

[0095] CD4 + CD69:CD4 + CD69 expression on T cells

[0096] CD8 + CD69:CD8 + CD69 expression on T cells

[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] Fig.12 :

[0104] Killing percentage (%): 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 amino acid sequence of Fcγ is SEQ No.1, and the components constituting the sequence are CH1-Hinge Region-CH2-CH3. The sequence can form an interchain disulfide bond at the cysteine-proline-proline-cysteine ​​sequence. The structure formed can bind to the FcγR receptors of monocytes, macrophages, stellate cells and NK cells, and exert the effector functions of cell killing and antigen presentation. The preferred sequence is derived from human IgG1, and the sequence can also use IgG2, IgG3, IgG4 or other appropriate artificial mutation sequences. The core function of this design is to form an interchain disulfide bond and be able to bind to the FcγR receptor. Different sequences can have different affinities and different dominant target cells, thereby exerting effector functions.

[0109] The domains that bind to CD19 are SEQ No.2 and sequence SEQ No.3. Among them, the SEQ No.2 component is VH, and the SEQ No.3 component is VL-Cκ. SEQ No.2 and SEQ No.3 constitute the region that binds to CD19, namely the Fab domain. Other natural antibodies or artificial antibody variable regions that bind to CD19 can be applied to the present invention. Different artificial mutations can adjust the binding force with CD19, and different binding forces can adjust the molecular targeting ability. Strong binding force with CD19 means strong targeting ability, and vice versa, weak action ability. The sequence used in the present invention has been tested and has relatively strong binding force. For specific data, please refer to the subsequent test results of the present invention. It should be noted that there is no disulfide bond between SEQ No.2 and SEQ No.3, and the C-terminal amino acid of the SEQ No.2 component is connected to the N-terminal amino acid of SEQ No.1 through a peptide bond; a pair of disulfide bonds are formed between the Cκ part of the SEQ No.3 component and the CH1 of the SEQ No.1 component. There is no covalent bond between the VL part of the SEQ No.3 component and SEQ No.2.

[0110] The domains that bind to CD3 are SEQ No.4 and SEQ No.5. SEQ No.4 and SEQ No.5 are connected by a linker peptide chain. The domains of SEQ No.4 and SEQ No.5 are combined together 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, so that the molecule of the present invention has the function of targeting T cells. After binding to CD3, T cells can be activated. The design of the present invention weakens the binding to CD3, which can reduce the occurrence of accidental activation of T cells, that is, reduce off-target activation of T cells. Of course, the design of SEQ No.4 and SEQ No.5 to CD3 binding can also adjust affinity by artificial mutation. The present invention includes two designs, one is that SEQ No.4 and SEQ No.5 are located downstream of the Cκ sequence of SEQ No.3, effectively forming steric hindrance, and the other optimizes the CDR sequence of SEQ No.4 and SEQ No.5, so that the CD3 binding force is appropriate and the activation of T cells in off-target (CD19) conditions is reduced. The present invention is designed by combining two technical considerations to achieve good drug efficacy. The present invention subsequently conducted a CD3 binding activity test, which can be seen in the following examples.

[0111] SEQ No.4 and SEQ No.5 are a kind of SvFc single-chain antibody. The cooperation of the two sequence fragments forms a structural domain that binds to CD3. In order to provide flexible adjustment space for SEQ No.4 and SEQ No.5, the present invention optimizes and designs two linkers (Linker1 and Linker2). The sequence of Linker1 is SEQ No.6: Gly Ser Thr Ser Gly Ser Gly Lys ProGly Ser Gly Glu Gly Ser Thr Lys Gly Gly Ser. The sequence of Linker2 is SEQ No.7: Gly Gly GlyGly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser. The design of Linker1 sequence focuses on factors such as sequence length, hydrophilicity, peptide chain secondary structure, and steric hindrance with adjacent peptide chains; the design of Linker2 sequence focuses on providing flexibility for SEQ No. 5 so that SEQ No. 5 can be functionally combined with SEQ No. 4. The combination of glycine and serine is a classic flexible linker. The present invention selects the (GlyGlyGlyGlySer)3 combination based on experiments. This combination does not affect the activity of SEQ No. 4 and SEQ No. 5, does not affect the binding with CD3, and the resulting binding force meets the design standards.

[0112] As mentioned above, the overall molecular design is divided into three functional domains, namely, the Fc peptide chain that binds to the Fc receptor, the domain that binds to CD19, and the domain that binds to CD3. In order to achieve the connection of the three parts of the polypeptide chain and facilitate the subsequent expression and purification of proteins, two polypeptide chains are specifically designed in combination: SEQ No. 2 is fused in series with SEQ No. 1, and the fused sequence is SEQ No. 8; SEQ No. 3, SEQ No. 4, and SEQ No. 5 are fused in series, where SEQ No. 3 and SEQ No. 4 are connected by Linker 1, and SEQ No. 4 and SEQ No. 5 are connected by Linker 2, and the fused sequence is SEQ No. 9.

[0113] Example 2: Optimizing codons

[0114] The two polypeptide chains designed in Example 1 were codon optimized to achieve the purpose of being suitable for expression in CHO cells. The target gene amino acid sequence SEQ No. 8 was optimized to have a nucleotide sequence of SEQ No. 10. The target gene amino acid sequence SEQ No. 9 was optimized to have a nucleotide sequence of SEQ No. 11.

[0115] Example 3: Construction of cloning plasmid

[0116] After double digestion of the expression vector with restriction endonucleases HindIII / EcoRI, it was dephosphorylated with alkaline phosphatase to obtain a nucleotide sequence fragment with sticky ends; the gene sequence SEQ No.10 was connected to the 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 connected using TaKaRa DNA Ligation Kit LONG, transformed into DH5α and positive clones were screened. After the plasmid was transformed into Escherichia coli DH5α, 3 single colonies were randomly selected for expansion culture, and the plasmid was extracted using a commercial kit. Double digestion with restriction endonucleases HindIII / EcoRI was performed to identify the correct connection. The construction of the first peptide chain was completed and named plasmid p21; Figure 1 The construction map of the gene co-expression vector is shown, wherein SEQ No.10 and SEQ No.11 are nucleotide sequences encoding the multifunctional antibody, corresponding to the DNA sequences of the heavy chain and light chain, respectively; SEQ No.8 and SEQ No.9 are the amino acid sequences after translation of 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 are obtained by translation of nucleotide sequences (SEQ No.10 and SEQ No.11), wherein 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] The same method was used to construct a sequence containing SEQ No. 11 into another vector plasmid. Double enzyme digestion confirmed that the connection was correct, and the second peptide chain was constructed and named plasmid p345.

[0120] Example 4: Concatenation of plasmid p21 and plasmid p345 to construct a tandem expression plasmid

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

[0122] Example 5 Preparation of plasmid for transfection of CHO cells

[0123] The positive clones in Example 4 were inoculated into 300 ml 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 Bomade Gene Technology Co., Ltd.); the plasmid was linearized with endonuclease PvuI (TaKaRa), extracted and purified using phenol / chloroform / isoamyl alcohol, and ethanol precipitated and redissolved in sterile TE Buffer (TaKaRa). The results of agarose gel detection of plasmid p1932 are shown in Figure 1. Figure 2 As shown, Figure 2It includes: Lane M: DNA Marker (10000 bp, 7000 bp, 4000 bp, 2000 bp, 1000 bp, 500 bp and 250 bp), wherein, Lane 1: complete circular plasmid, Lane 2: product after plasmid was digested with PvuI, Lane 3: product after plasmid was digested with PvuI, extracted and purified; Figure 2 It includes a complete circular plasmid (lane 1), a product after PvuI digestion (lane 2), and a purified linear plasmid product (lane 3). Electrophoresis detection shows that the digestion reaction is complete and the target band is consistent with the expected result.

[0124] Lane 1: intact circular plasmid;

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

[0126] Lane 3: The product after the plasmid was digested with PvuI and extracted and purified.

[0127] After the enzyme digestion reaction was completed, a DNA fragment was obtained, the size of which was about 11200bp, and its migration speed in agarose gel electrophoresis was slower than that of the plasmid. This single enzyme digestion reaction was complete, and the target band was consistent with the expected result.

[0128] Example 6 Construction of engineered cell lines

[0129] Establishment and screening of stable clones: In a sterile laminar flow bench, the perforation voltage of the gene pulse generator Xcell (Bio-Rad) was set to 300 V, 900 μF single pulse, and infinite resistance. A disposable electric shock cup with a gap of 4 mm (Bio-Rad) was taken out, and 40 μg of linearized plasmid DNA (100 μl) and 0.7 ml of CHO-K1 cell suspension (1.43×10 7cells / ml), the linearized plasmid p1932 was directly transfected into CHO-K1 cells by electrofection, the cells in the electroporation cup were transferred to a triangular culture flask, 30 ml of CD-CHO culture medium was added, and the cells were cultured on a shaker at 36-37°C and 5% CO2 at 135 rpm. After culturing for 24 hours, the cells were collected by low-speed centrifugation and replaced with CD-CHO culture medium containing 50 μM MSX (without glutamine), the cells were transferred into a 96-well flat-bottom culture plate by limiting dilution, the culture plate was placed in an incubator at 37°C and 10% CO2, and observed under an inverted microscope, the monoclonal cell wells were marked, and then the positive cells in the 96-well, 24-well and 6-well cell culture plates were screened in turn by ELISA (Recombinant Human CD3E protein + expression product double antibody Fc + goat anti-human IgG-HRP), the monoclonal strains with higher expression levels were screened, and continuous subculture and detection were performed to finally obtain cell clones with high expression of the target gene; according to the loading protein The expression level of the recombinant protein in the culture supernatant was detected by HPLC column of A gel, 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 inoculated into a 2L or 3L triangular flask containing 500ml CD-CHO culture medium, the breathable bottle cap was screwed tightly, and the cells were cultured on a shaker at 36-37°C and 5% CO2 at 130-140 rpm for 3-6 days, and then transferred to a bioreactor for culture. Reactor culture conditions: initial inoculation density of the reactor: 500,000-2,000,000 cells per milliliter, culture temperature 32-37°C, pH 6.5-7.5, stirring speed 50-120 r / min, dissolved oxygen 50%±20%, and intermittent feeding (feeding such as sugar, culture medium or alkali solution) was used during the culture period, the culture time was 12-18 days, and the cells were harvested when the viable cell density dropped to between 60% and 70%.

[0132] The harvested culture fluid is centrifuged at 10000r / min for 30 minutes to remove insoluble particles such as cells or use a deep filter to remove cells and cell debris, and the cell culture supernatant is collected. In order to ensure that the process is under aseptic operation, a 0.45μm clarification filter can usually be used to filter the culture supernatant. The filtered intermediate product can be stored at 2-8°C or directly enter the next purification process. Protein A affinity chromatography is a common method for antibody purification. It utilizes the characteristics of the presence of Fc domains in antibodies. The K1932 peptide chain contains Fc domains. After experimental testing, the Fc part of the K1932 structure can be effectively bound to the affinity gel containing protein A. The present invention can use protein A type or protein G type affinity gel for affinity purification. Let us take protein A as an example to illustrate. The above-mentioned harvested liquid after filtration is purified by 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 displayed a value of A. 280 After that, the mobile phase was replaced with a dissociation buffer (0.1 M citric acid buffer, acetic acid buffer, or glycine buffer, pH 3.0-4.5) for elution and dissociation. 280 The elution peak was collected by absorbance value.

[0133] The affinity chromatography solution was incubated at room temperature for 60 minutes to inactivate the virus, and then 1M Tris neutralizing solution was added to neutralize to a pH of 7.2. Purified water was then added to dilute and adjust the solution ionic strength to meet the requirements of the next anion exchange chromatography step.

[0134] The filler used for anion exchange chromatography was DEAE Sepharose 6FF. The intermediate product of low pH incubation and virus removal was adjusted to pH 7.2 and diluted with pure water and then directly passed through the anion exchange chromatography column. 280 The flow-through liquid is collected according to the absorbance value until the absorbance value drops close to the baseline level and then sampling is stopped. This is the anion exchange chromatography liquid.

[0135] The liquid after the anion exchange chromatography was loaded onto a ChromaX TM The Baron CHT II hydroxyapatite column was equilibrated and 70 mM phosphate solution (pH 6.8) was used as the dissociation solution. The UV detector A 280 When the absorbance value rises, start collecting the effluent from the chromatography column, and stop collecting samples when the absorbance value drops close to the baseline level. The purity of the target protein can reach more than 99%; Figure 3The SEC-HPLC spectrum of the purity detection of K1932 antibody is shown. It can be seen from the figure that the single peak of the sample indicates that the purity reaches the designed standard.

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

[0137] The K1932 antibody purification process described in this embodiment is a combination of technologies, which is intended to illustrate the process of obtaining K1932 with biological functions. 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, which are within the scope of protection of the present invention.

[0138] Example 8: K1932 stock solution stabilizer formula

[0139] K1932 is an innovative molecular design, including three functional domains, structurally including antibody Fab segment, Fc segment and ScFv single-chain antibody. In order to maintain molecular stability, it is designed with reference to the general design principles of antibody stabilizers. There are many options for buffer systems, such as phosphate buffer system, citrate buffer system, acetic acid-sodium acetate buffer system, etc. The present invention compares a variety of options, preferably, an acetic acid-histidine buffer system is selected, pH 5.2 ± 0.2. Lysine, arginine, glycine, glutamine, etc. can be selected for the stability of nitrogen-hydrogen bonds and oxygen-hydrogen bonds. Preferably, L-methionine is added. Carbohydrate substances are polyhydroxy protective agents, and various combinations of monosaccharides, disaccharides or other polyhydroxy alcohols can be selected, such as glucose, trehalose, sucrose, lactose, maltose, sorbitol, etc. The present invention preferably selects sucrose as a protective agent component. The function of the surfactant is to reduce the adsorption of the container to the protein and has a good dispersing ability to provide protection for the target protein. The surfactants available include Tween-20, Tween-80, Poloxamer 188, etc. Tween-20 is preferred in the present invention after testing. The protective agent formula formed is as follows:

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

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

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

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

[0144] Sucrose: 50-150 g / L

[0145] Under the above protective agent conditions, K1932 antibody can be stored for a long time at 2-8°C. The 37°C thermal acceleration test showed that K1932 had good stability. The purity data is the most direct observation indicator of stability. The K1932 purity detection method is: 40mM PBS (containing 0.2M Na2SO4, pH6.7) is used as the mobile phase, and the antibody purity analysis is performed on the Waters e2695 HPLC using a TSK3000SWxl (7.8*300mm) column. The test results show that within the expected two-year validity period, the purity is not less than 95%.

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

[0147] Raji, Daudi, and IM-9 are B lymphoma cells with CD19 antigen on their cell surface. K1932 antibody can specifically bind to CD19 antigen on the cell surface. The experiment used Raji, Daudi, and IM-9 as positive cells and K562 cells as negative control cells. Flow cytometry (NoveCyte 3130, Aglient) was used to detect the CD19 site-specific binding activity of K1932 antibody. 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 times to 0.305 ng / ml, for a total of 11 dilutions. The cell density was prepared to be 2.0×10 6 Add 100 μl of Raji cell, Daudi cell, IM-9 cell and K562 cell suspension of 10 cells / ml to each well, mix well and incubate at 2-8°C for 60 minutes, add 1:250 dilution of goat anti-mouse IgG Fc-PE, react at room temperature in the dark for 30 minutes, detect fluorescence value by flow cytometer, set the flow cytometer to FAST, measure 20,000 events, measure the average fluorescence value of cells in each well in turn, and calculate the EC value of K1932 antibody binding to Raji, Daudi and IM-9 cells by GraphPadPrism 5.0 software 50 Values ​​were used to compare the binding activity of different B lymphoma cells to K1932 bispecific antibody CD19-positive cells; Figure 4 The flow cytometric profile and dose-response curve of the binding reaction of K1932 antibody to Raji cells are shown, from which it can be seen that the binding activity of K1932 antibody to Raji cells is better than that to Daudi and IM-9 cells, and the EC50 value is significantly lower; the results of flow cytometry are summarized in Table 1; Fig. 9The relative binding activity of K1932 antibody stock solution at different dilution concentrations was demonstrated; the results showed that even at higher dilution multiples (such as 10,000 times), K1932 antibody could still maintain a high binding activity with the CD19 target.

[0148] Table 1 Summary of the results of K1932 antibody's specific binding reaction with Raji, Daudi, IM-9 cells CD19 and its reaction with K562 cells

[0149]

[0150]

[0151] K562 is a cell that does not express CD19 membrane antigen. The measurement results show that the Mean PE-H value obtained by reacting different concentrations of K1932 antibody with K562 cells is basically consistent with the blank control, and no binding reaction occurs, proving that K1932 has no specific binding with CD19-negative cells.

[0152] EC binding of K1932 antibody to Raji, Daudi, and IM-9 cells 50 The values ​​were 465.8 ng / ml, 614.4 ng / ml, 363.1 ng / ml, or 2.329×10 -9 mol / L、3.072×10 -9 mol / L、1.816×10 -9 mol / L. From the analysis of reaction trend and fluorescence range, the binding activity of K1932 antibody to Raji cells was better than that to Daudi and IM-9 cells.

[0153] Example 10: Comparison of binding activity between K1932 antibody and humanized monoclonal antibody CD19 monoclonal antibody K19

[0154] Flow cytometry (NoveCyte 3130, Aglient) was used to detect the specific binding activity of K1932 antibody, humanized CD19 monoclonal antibody K19 and Raji. 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 then serially diluted 3 times to 0.305 ng / ml. The cell density was 2.0×10 6cells / ml Raji cell suspension was added to a 96-well cell plate, 100 μl was added to each well, mixed and incubated at 2-8°C for 60 minutes, and 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 the cells in each well was measured in turn to compare the binding activity of K1932 bispecific antibody and humanized CD19 monoclonal antibody K19 with CD19-positive cells; Figure 5 The binding activity curves of K1932 antibody and K19 monoclonal antibody to Raji cells were compared, from which it can be seen that the fitted EC50 value of K1932 antibody is 2.329×10^-9mol / L, which is equivalent to the activity of K19 monoclonal antibody; the results of flow cytometry determination are summarized in Table 2.

[0155] Table 2 Summary of the results of site-specific binding of K19 monoclonal antibody, K1932 antibody, OKT3 monoclonal antibody and Raji cell CD19

[0156]

[0157]

[0158] From the fitting trend analysis, the Mean PE-H values ​​obtained from the binding reaction of different concentrations of OKT3 monoclonal antibody with Raji cells were basically consistent, and slightly higher than the blank control results, proving that OKT3 monoclonal antibody had no specific binding with CD19 positive cells.

[0159] EC binding of K1932 antibody and K19 monoclonal antibody to Raji cells 50 The values ​​were 150.9 ng / ml, 465.8 ng / ml, or 1.006×10 -9 mol / L、2.329×10 -9 mol / L, K1932 antibody, K19 monoclonal antibody binding to Raji cells, fitted EC 50 (mol / L) values ​​are all 10 -9 From the calculation results, it can be seen that the binding activities of K1932 antibody and K19 monoclonal antibody with CD19 positive cell membrane antigen are basically the same.

[0160] Example 11: Binding activity of K1932 antibody and OKT3 to CD3-positive cells

[0161] To test the ability of K1932 antibody to bind to CD3, we performed flow cytometry analysis (FACS) on the obtained bispecific antibodies. K1932 antibody was diluted with 0.02mol / L PBS (pH7.4, containing 1% BSA) to a starting protein concentration of 162μg / ml, followed by a 3-fold gradient to 0.025μg / ml. OKT3 antibody was diluted with 0.02mol / L PBS (pH7.4, containing 1% BSA) to a starting protein concentration of 6μg / ml, followed by a 3-fold gradient to 0.305ng / ml.

[0162] The cell density was 5.0 × 10 6 cells / ml of Jurkat cell suspension, add 100μl of cell suspension to the above sample wells in turn, mix and react at room temperature for 60 minutes, centrifuge and carefully aspirate the supernatant and discard. Add 50μl of mouse anti-human κ chain monoclonal antibody diluted to 2μg / ml to each sample well, mix and react at room temperature for 60 minutes, centrifuge and carefully aspirate the supernatant and discard. Add 50μl of FITC-labeled goat anti-mouse IgG (1:1000) to each sample well, mix and react at room temperature in the dark for 30 minutes, centrifuge and carefully aspirate the supernatant and discard. Add 170μl of 0.02mol / L PBS (pH7.4) to each well and resuspend the cells in the well. Set the flow cytometer gate loading volume to 10000events, the flow rate to Fast, and carefully blow the cells with the tip of the gun to suspend and mix them. Then transfer the cell suspension to a 0.5ml centrifuge tube and measure the cell fluorescence value in turn. Compare the binding activities of K1932 bispecific antibody and OKT3 antibody to CD3-positive cells; Figure 6 The flow cytometric profile of the binding activity of K1932 antibody to CD3-positive cells was displayed, indicating that its binding ability to CD3 molecules was significantly weaker than that of OKT3, thereby reducing the risk of off-target activation of T cells; the measurement results were brought into GraphPad Prism software to calculate the EC 50 as follows:

[0163] Table 3 Results of binding activity of K1932 antibody and OKT3 to CD3 positive cells

[0164]

[0165] From the above results, it can be seen that the EC of K1932 antibody 50 The value is 2.255×10 -8 mol / L, EC of OKT3 antibody 50 The value is 1.459×10 -9 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 molecule on the surface of T cells is significantly weaker than that of OKT3.

[0166] Example 12: K1932 antibody-mediated T lymphocyte activation test (CD69 assay)

[0167] The CD69 molecule is an early marker of T cell activation. Jurkat cells cultured under normal conditions rarely express CD69 molecules. When Jurkat cells and Raji cells are co-cultured in an appropriate ratio and in the presence of K1932 antibodies (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 stimulus.

[0168] Jurkat cells cultured in 10% FBS1640 medium were collected by centrifugation, and the cell concentration was adjusted and mixed with Raji cells. The mixed cell suspension contained 3×10 Jurkat cells. 6 cells / ml, Raji cells 3×10 5 cells / ml, the cell suspension was inoculated into a 24-well cell culture plate, a serial dilution of K1932 antibody was added, and then cultured in a 5% CO2, 37°C incubator for 18 hours, the supernatant was centrifuged and 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 determined by flow cytometry, and the CD69 fluorescence value Mean PE-H corresponded to the content of K1932 in the solution. Figure 7 The flow cytometric profile and dose-response curve of CD69 expression on T lymphocytes after stimulation with K1932 antibody are shown. It can be seen from the figure that K1932 antibody can effectively activate T lymphocytes, and CD69 expression is positively correlated with antibody concentration. The quantitative response relationship of CD69 expression after cells were stimulated with K1932 at various concentrations was observed. Figure 8 The CD69 expression response curve of K1932 antibody under the conditions of T+B cell co-culture, T cell single culture, and B cell single culture is shown; it can be seen from the figure that only when T cells and B cells coexist, K1932 antibody can significantly activate the expression of CD69, while T cells or B cells alone cannot achieve the same activation effect; this indicates that the mechanism of action of K1932 antibody depends on the presence of CD19 target, further verifying its targeting specificity; EC was calculated using GraphPad Prism 50 value, dose response curve fitting constant R 2 It should not be less than 0.90. The results of different batches of K1932 antibody activating T lymphocytes are shown in Table 4.

[0169] Table 4 Summary of the results of K1932 antibody activated T lymphocytes expressing CD69

[0170]

[0171] From the results in the above table, it can be seen 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 of the dose-response curve 2 Greater than 0.98, EC 50 The value is about 4×10 -12 mol / L.

[0172] Example 13: K1932 antibody activation of T cells requires co-stimulation of B cells

[0173] Jurkat cells cultured in 10% FBS1640 medium were collected by centrifugation and the cell concentration was adjusted to 3 × 10 6 cells / ml, for later use; ① The cell suspension mixed with equal volume of Raji cells contained 3×10 Jurkat cells 5 cells / ml, Raji cells 3×10 4 ②Adjust the concentration of Jurkat cells to 1.5×10 6 cells / ml; ③Adjust the Raji cell concentration to 1.5×10 6 cells / ml; the cell suspensions were added to 48-well cell culture plates, and then serially diluted K1932 antibodies were added, and then cultured in a 5% CO2, 37°C incubator for 18 hours. After 18 hours, the supernatant was removed by centrifugation, 486μl Anti-Human CD69PE (clone: ​​FN50, REF: 12-0699-42, eBioscience) was mixed with 3564μl 0.02mol / LPBS (pH7.4, containing 1% BSA), 50μl of the mixture was added to each well, and after 120 minutes of reaction at 2-8°C in the dark, the average fluorescence intensity of CD69 expressed by cells was measured using a flow cytometer (NoveCyte 3130, Aglient). The results are shown in Table 5 below. The measured concentrations of K1932 were used as the horizontal axis, and the average fluorescence intensity of the cells was used as the vertical axis to plot the reaction relationship of the CD69 expression amount after the cells were stimulated by each concentration of K1932 (positive control).

[0174] Table 5: Mean fluorescence intensity of CD69

[0175] K1932 antibody content (pg / ml) T+B T cells B cells 0.2 4061 3344 4816 2 3966 3418 4685 20 4650 3468 4951 200 14611 3425 4630 2000 41297 3691 4726 20000 52096 5990 4166 200000 53612 12029 4746 2000000 53848 17385 4380 20000000 51110 20821 4638 Blank control 3311 3311 3311 <![CDATA[R 2 ]]> 0.9999 0.9976 / <![CDATA[EC 50 (pg / ml)]]> 692.7 132933 / <![CDATA[EC 50 (mol / L)]]> <![CDATA[3.27×10 -12 ]]> <![CDATA[9.43×10 -10 ]]> /

[0176] From the above data, we know that the (T+B)Cell+K1932 antibody group corresponds to EC 50 The value is 692.7pg / ml, and the TCell+K1932 antibody group corresponds to EC 50The value is 132.9ng / ml, which is about 191 times the ratio. In the absence of B cells, the MFI of CD69 is low. Even when K1932 reaches 20μg / ml, the MFI can only reach 20821, which is about 6 times the basic background value. Therefore, K1932 alone cannot stimulate T cells to activate well; combined with Figure 10-12 , showing the killing curve and related data of CD8+T cells activated by K1932 antibody on B lymphocytes, it can be seen that it can significantly enhance the tumor killing ability mediated by T cells.

[0177] Example 14: Relative binding activity of K1932 (with CD3E, CD19):

[0178] The binding activity of CD19-CD3 bispecific antibody was determined by the 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, and then diluted to 0.4 μg / ml with carbonate coating solution, coated in a 96-well ELISA plate, 100 μl per well, placed at 37°C for 2 hours, and placed in a refrigerator at 2-8°C overnight; washed 3 times with washing solution and patted dry; blocking solution (20 mmol / L PBS-T containing 2% BSA) was added to each well of the ELISA plate, 220 μl per well, and placed at 37°C for 60 minutes; washed 3 times with washing solution and patted dry; PBS-T) pre-diluted 3 batches of K1932 antibody stock solutions (batch numbers: 20240622, 20240630, 20240708) to 10 μg / ml according to the protein content, added the diluted K1932 antibody stock solution to the first column of the blocked ELISA plate, starting from 10 μg / ml, and diluted 3-fold to the 11th column, with a total of 11 dilutions, 100 μl per well (2 wells for each dilution); after the addition of the sample, the ELISA plate was reacted at room temperature (22°C to 27°C) for 60 minutes, washed 4 times with washing solution, and patted dry; biotin-labeled recombinant human CD19 (0.2m g / ml) was diluted 3000 times and added to each well of the ELISA plate, 100 μl per well, reacted at room temperature (22℃~27℃) for 60 minutes, washed 4 times with washing solution and patted dry; diluted horseradish peroxide-labeled streptavidin (1:120000) was added to each well of the ELISA plate, 100 μl per well, reacted at room temperature (22℃~27℃) for 60 minutes, then washed 5 times with washing solution and patted dry; TMB color development solution was added to each well of the ELISA plate, 100 μl per well, reacted at 37℃ in the dark for 10 minutes; finally, 50 μl stop solution (1 mol / L sulfuric acid) was added to each well to terminate the reaction. The A450 value was read with infinite M200 PRO ELISA instrument, and the EC value of K1932 antibody solution in double antigen sandwich ELISA reaction was calculated using GraphPad Prism 8 software. 50 The results are shown in Table 6.

[0179] Table 6 Results of relative binding activity determination by ELISA of three batches of K1932 antibody stock solutions

[0180]

[0181] From the results in Table 6, it can be seen that the overall absorbance value change range and curve fitting of the three batches of K1932 antibody stock solution samples are basically consistent; and the EC 50 The values ​​were 66.06 ng / ml, 63.14 ng / ml, and 64.31 ng / ml, or 3.27 × 10 -10mol / L、3.13×10 -10 mol / L、3.18×10 -10 mol / L, the results between batches were basically consistent.

[0182] Example 15: K1932 antibody mediated activation of CD8 + T cell killing test on Raji cells

[0183] Detect the killing effect of K1932 antibody on B lymphocytes. Resuscitated PBMC (donor: Z0645) cells, activated and expanded T cells with IL-2 for 7 days, and then isolated CD8 + T cells, CD8 + The T cell density was adjusted to 7.5 × 10 5 cells / mL, and the target cell density was adjusted to 2.5×10 5 cells / mL, 40 μL (30,000) of CD8 + T cells and 40 μL (10,000) target cells, with a cell ratio of 3:1. Use complete RPMI1640 medium to prepare 5× sample solution, the highest working concentration of the sample is 100 ng / mL, 5-fold dilution, 9 concentrations; add 20 μL of sample solution to each well of the 96-well plate inoculated with cells, and set two replicates for each drug concentration. The cells in the 96-well plate with drug addition were placed at 37°C and 5% CO2 for 24 hours, and then Bright-Glo analysis was performed. Melt the Bright-Lite Luciferase Assay System reagent and equilibrate the cell plate to room temperature for 30 minutes. Add 100 μL of Bright-Lite Luciferase Assay System solution to each well. Shake on an orbital shaker for 3 minutes to lyse the cells. Transfer the lysate solution to a 96-well white impermeable flat-bottom plate (Absin#abs7016), read the Luc signal with a multifunctional microplate reader, and calculate the target cell killing rate. The killing rate calculation formula is as follows:

[0184]

[0185] The data were analyzed using GraphPad Prism 7.0 software, and nonlinear S-curve regression was used to fit the data to obtain the dose-effect curve, from which the IC 50 The results are shown in Table 7.

[0186] Table 7 K1932 antibody-mediated activation of CD8 + Results of T cell killing test on Raji cells

[0187]

[0188] From the results in Table 7, it can be seen that as the antibody content increases, the killing effect presents an S-shaped curve. K1932, K193, and Blinatumomab have obvious killing effects, and the killing effects at high concentrations after 24 hours reach 72%, 80%, and 80%, respectively. + IC of T cells killing Raji cells 50 They are 0.066ng / ml, 0.014ng / ml, and 0.007ng / ml, or 3.27×10 -13 mol / L、1.86×10 -13 mol / L、1.30×10 -13 mol / L. K1932 antibody mediated activation of CD8 + The killing ability of T cells against Raji cells is basically the same as that of Blinatumomab.

[0189] Example 16: K1932 antibody-mediated activation of peripheral blood T cells to kill B cells (PBMC)

[0190] Flow cytometry (Canto II, BD) was used to detect the killing effect of K1932 antibody-mediated activated T cells on B lymphocytes. PBMCs from healthy human peripheral blood were used as experimental cells and the cell density was 1.8×10 6 Cell suspension of 10 cells / ml was added to a 48-well plate, 300μl per well, and the 48-well cell culture plate with the added sample was placed in a carbon dioxide incubator containing 5.0% CO2 and incubated at 37.0℃ for more than 1h. The dilution range of the quantitative K1932 antibody was 400ng / ml~0.4pg / ml, and 10-fold gradient dilution was performed at 7 points. The diluted antibody solution (0.4pg / ml~400ng / ml) was added to each cell well of the 48-well cell culture plate where PBMC had been added, and 300μl was added to each well. The 48-well cell culture plate with the added sample was placed in a carbon dioxide incubator containing 5.0% CO2 and incubated at 37.0℃ for 18h, 24h, 42h, and 72h. After collecting the cells, HumanBD Fc Block specifically targeting Fcγ receptors was used. TM Block the non-specific staining caused by fluorescent antibody Fc receptor. Add FVS510 fluorescent antibody to stain the dead and live cells in PBMC, and add CD20-PE-Cy TM7. The surface of B lymphocytes in PBMCs was stained, and the 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 the cells were fixed and permeated, the fluorescent antibodies corresponding to granzyme B and perforin were added to stain the intracellular factors. The CD3 + T appears CD4 + T cells or CD8 + T cells and CD69 + The number of cells positive for at least one cytokine detected simultaneously: Granzyme B-BV421 and Perforin-APC. + T cells or CD8 + The number of T cells was quantitatively converted to 100,000, and then the number of CD69, Granzyme B-BV421, and Perforin-APC positive cells were proportionally converted, and EC was calculated using GraphPad Prism. 50 The statistical results are shown in Tables 8 and 9. Under the P2 gate, CD20 + Cells were collected and the dead cell population was circled using live-dead fluorescence (FVS510 fluorescence), and the percentage of dead cells was calculated as the B cell killing rate. The statistical results are shown in Table 10.

[0191] Table 8 Results of K1932 antibody-mediated peripheral blood T cell activation test (PBMC) - CD4 + T

[0192]

[0193] *Represents the hook effect at a high concentration point, and the result of the previous concentration point is used for forced leveling.

[0194] Table 9 Results of K1932 antibody-mediated peripheral blood T cell activation test (PBMC) - CD8 + T

[0195]

[0196]

[0197] *Represents the hook effect at a high concentration point, and the result of the previous concentration point is used for forced leveling.

[0198] Table 10 Results of the K1932 antibody-mediated killing test of human peripheral blood PBMC on B cells

[0199]

[0200] According to the results in Tables 8 and 9, the number of CD69 positive cells increased with the increase of K1932 antibody content, and the number of CD8 + The number of CD69 positive T cells was significantly higher than that of CD4 + T cells all show an S-shaped curve, CD8 + T cell CD69 + Perforin + and CD69 + Granzyme B + The trend is higher than CD4 + The trend of T cells is good, indicating that K1932 can activate T cells well, especially CD8 + T cells.

[0201] According to the results in Table 10, the killing effect began to appear at high concentrations at 18h and 24h, and the killing effect was obvious at 42h and 72h as the K1932 antibody content increased. The killing effect at the high concentration point at 72h reached 95%, showing an S-shaped curve.

[0202] In summary, K1932 antibody-mediated T cell activation is mainly CD8 + After T cells are activated, they release granzyme B and perforin to kill B cells.

[0203] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle 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 fall within the protection scope of the present invention.

Claims

1. A multifunctional antibody that binds to human CD19, CD3 and FcγR, characterized in that: include: 1) a bivalent domain specifically binding to the B lymphocyte membrane antigen CD19, wherein the Fab fragment binding to CD19 comprises a humanized light chain variable region VL, the amino acid sequence of which is SEQ No. 2, and a heavy chain variable region VH, the amino acid sequence of which is SEQ No. 3; 2) a bivalent single-chain antibody domain that specifically binds to a T cell surface antigen CD3 molecule, wherein the single-chain antibody that recognizes the CD3 molecule comprises a light chain VL and a heavy chain VH, which are connected by Linker1 SEQ No. 6 and Linker2 SEQ No. 7, respectively; 3) a domain that monovalently binds to the FcγR receptor of NK cells, wherein the structure of the Fc fragment includes CH1, hinge region, CH2 and CH3, and the amino acid sequence is SEQ No. 1; The single-chain antibody that recognizes the CD3 molecule in the bivalent single-chain antibody domain that specifically binds to the T cell surface antigen CD3 molecule is connected to the C-terminus of the CD19 antibody light chain through a hydrophilic connecting peptide; The multifunctional antibody structure is: Furthermore, the FcγR binding sequence is CH1-Hinge Region-CH2-CH3, and the amino acid sequence is SEQ No. 1; The sequences of the humanized CD19 binding domains are SEQ No. 2 and SEQ No. 3; The amino acid sequences of the humanized CD3 single-chain antibody are SEQ No. 4 and SEQ No. 5; The sequences of Linker1 and Linker2 are shown in SEQ No.6 and SEQ No.

7.

2. A multifunctional antibody that binds to human CD19, CD3 and FcγR according to claim 1, characterized in that: The single-chain antibody structure that recognizes the CD3 molecule is in the form of ScFv and is directed against human CD3ε, including humanized CD3-specific antibodies of OKT3, X35-3, WT31, WT32, SPv-T3b, TR-66, 11D8, 12F6, M-T301, SMC2 and F101.

01.

3. A multifunctional antibody that binds to human CD19, CD3 and FcγR according to claim 2, characterized in that: The sequence of FcR is the γ chain.

4. The multifunctional antibody binding to human CD19, CD3 and FcγR according to claim 3, characterized in that: The one that binds to CD3 is a single-chain antibody, and the one that binds to CD19 is a humanized antibody.

5. A method for preparing a multifunctional antibody, characterized in that: It is prepared by genetic recombination technology, using different types of mammalian cell expression vectors and expressed in CHO cells. The CHO cells are cultured in a chemically defined medium, and no hormones or proteins from various animal sources or their hydrolysates are added during the culture process.

6. The method for preparing a multifunctional antibody according to claim 5, characterized in that: The preparation steps include: The plasmids containing the multifunctional antibody gene were connected in series and linearized by endonuclease digestion; After transfection of CHO cells, positive clones were obtained, which were then cultured in a bioreactor, and the products were secreted into the culture supernatant; Affinity chromatography and ion exchange chromatography media are used for purification to obtain high-purity multifunctional antibodies.

7. The multifunctional antibody according to claim 1 is a therapeutic drug for treating relapsed, refractory B-cell lymphoma or refractory autoimmune disease, and the multifunctional antibody is an immunotherapeutic drug that kills B cells in a T cell-dependent manner.

8. A pharmaceutical composition comprising the multifunctional antibody according to claim 1.

9. The pharmaceutical composition according to claim 8, wherein the method of use comprises: It is made into a liquid preparation and administered by intravenous, subcutaneous or intramuscular injection.

10. The pharmaceutical composition according to claim 8, wherein the formula comprises: acetic acid, histidine, methionine, sucrose, and Tween 20.

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