Recombinant complement protein and use thereof in the preparation of an anti-leukemia drug

Abstract

The present application relates to a kind of recombinant complement protein and the application in the preparation of anti-leukemia drugs, belong to biological medicine technical field;The present application discloses that single drug treatment, i.e. injection recombinant C1QBP protein has significant effect in the treatment of acute leukemia, can significantly inhibit the proliferation of leukemia cells in hematopoietic system bone marrow, spleen, peripheral blood, while inhibiting the infiltration of leukemia cells in extramedullary organs (skin, etc.), thereby reducing the whole body tumor load, significantly prolonging the survival period of leukemia mouse.Recombinant C1QBP protein is combined with clinical first-line chemotherapy regimen, has synergistic effect, compared with single use chemotherapy drug has stronger effect of inhibiting the proliferation of leukemia cells, can significantly prolong the survival period of leukemia mouse, has played the effect of attenuated and synergistic effect.The present application first proves that complement C1Q binding protein has the effect of treating leukemia, and provides preparation method and combined drug regimen, has higher clinical conversion prospect and application value.

Description

Technical Field

[0001] This invention relates to a recombinant complement protein and its application in the preparation of anti-leukemia drugs, belonging to the field of biomedical technology. Background Technology

[0002] Complement is a group of proteins composed of more than 30 components that, upon activation, possess enzymatic activity and are found in the serum and tissue cells of healthy humans and animals. Complement is activated through the classical, alternative, and lectin pathways, forming the membrane attack complex. Complement-mediated cytolytic effects are an important component of the innate immune system. C1Q is a component of complement 1 (C1), the initiating factor of the classical complement pathway in innate immunity. It binds to antigen-antibody complexes and plays a crucial role in the classical complement activation pathway. The complement C1Q binding protein, C1QBP, has been reported in the literature as p33, p32, gC1qR, HABP1, or C1QBP. C1QBP exists in a pre-protein form containing 282 amino acid residues. After synthesis, its N-terminal 73 residues are hydrolyzed to form the mature p33 protein containing 209 amino acids. The mature protein has high charge and acidity, with an isoelectric point of 4.15. The main ligands of the reported p33 protein include C1q, hyaluronic acid, calreticulin, CD44, integrins, PKC, splicing factors ASF / SF2, and several microbial proteins. C1QBP is involved in functions including mitochondrial metabolism and dynamics, apoptosis, splicing, immune responses, inflammation, and regulates several cellular signaling pathways.

[0003] C1QBP (NCBI Gene ID 708, Unirot Protein Database Registry Number Q07021) is typically located in mitochondria, but can also be found in the cytoplasm, nucleus, and cytoplasmic membrane, and can be secreted extracellularly. A series of studies have found that this protein is expressed on the cell membrane surface. For example, the earliest identified and purified p33 protein was obtained from Raji cell membrane components. Flow cytometry and confocal microscopy analysis using specific antibodies against C1QBP (mAb 60.11 or 74.5.2) showed that many cell types, including Raji and A549, exhibited C1QBP-specific signals in their cell membranes. Previous analysis of RNA-seq sequencing data from large AML public databases (TCGA and BEAT datasets) and AML samples collected by our research group showed that C1QBP is highly expressed in AML (acute myelocytic leukemia) and is associated with poor prognosis, as evidenced by a significantly shortened survival in the high C1QBP expression group. Therefore, drugs developed targeting C1QBP have great potential for application in the treatment of AML.

[0004] Leukemia is a malignant disease originating from hematopoietic stem cells, characterized by the excessive proliferation and impaired differentiation of abnormal white blood cells (leukemia cells) in the bone marrow, leading to the suppression of normal hematopoietic function. Based on disease progression rate and cell type, leukemia can be divided into acute leukemia (such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML)) and chronic leukemia (such as chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Its pathogenesis involves multiple factors, including genetic mutations, epigenetic alterations, abnormal signaling pathways, and dysregulation of the microenvironment. Common causative factors include radiation, chemical exposure, viral infections, and genetic susceptibility. Treatment strategies for leukemia vary depending on the type and stage. Acute leukemia typically involves high-intensity chemotherapy, targeted therapy, and hematopoietic stem cell transplantation. In recent years, immunotherapy (such as CAR-T cell therapy) has shown significant efficacy in some ALL patients. Chronic leukemia relies more heavily on targeted drugs (such as tyrosine kinase inhibitors, TKIs) and immunotherapy to control disease progression and prolong survival. Despite advancements in treatment methods, some patients still face challenges such as drug resistance, relapse, and treatment-related complications.

[0005] Acute myeloid leukemia (AML) is a hematologic malignancy caused by the excessive proliferation of immature granulocytes due to impaired differentiation of hematopoietic cells, accounting for approximately 80% of all acute leukemia cases. The incidence of AML increases with age, with a median age at diagnosis of 67 years. The incidence is 1 / 100,000 in patients under 30 years old and as high as 17 / 100,000 in those over 75 years old. With the accelerating aging of my country's population, AML is becoming one of the malignant tumors affecting the health of adults, especially the elderly. AML can be divided into several different subtypes, M0-M7, based on cell morphology and histochemical characteristics. Among them, M5 acute monocytic leukemia is characterized by a high relapse rate and extramedullary invasion, poor response to chemotherapy, and a long-term disease-free survival rate of less than 30%. In recent years, with the deepening research into the pathogenesis of AML, some novel targeted therapies have been successfully applied clinically. However, a considerable number of AML patients still relapse shortly after remission and develop resistance to these drugs. Therefore, in-depth research into the pathogenesis of AML and the development of novel targeted therapies are necessary and of great clinical significance (Blood. 2023, 141(7): 766-786; Nat Med. 1997, 7(3), 730-737). Thus, finding an effective targeted drug to treat leukemia, and one that can be combined with currently used clinical medications, is an urgent problem to be solved. Summary of the Invention

[0006] The purpose of this invention is to solve the technical problem of how to obtain an effective drug and drug combination for the treatment of leukemia.

[0007] To address the aforementioned problems, this invention provides a recombinant complement C1Q binding protein, namely C1QBP, the amino acid sequence of which is shown in SEQ ID No. 1.

[0008] SEQ ID No. 1:

[0009] 1-LHTDGDKAFVDFLSDEIKEERKIQKHKTLPKMSGGWELELNGTEAKLVRKVAGEKITVTFNINNSIPPTFDGEEEPSQGQKVEEQEPELTSTPNFVVEVIKNDDG KKALVLDCHYPEDEVGQEDEAESDIFSIREVSFQSTGESEWKDTNYTLNTDSLDWALYDHLMDFLADRGVDNTFADELVELSTALEHQEYITFLEDLKSFVKSQ-209

[0010] The present invention provides an expression vector comprising the nucleotide sequence corresponding to the amino acid sequence of the recombinant complement C1Q binding protein C1QBP described above.

[0011] The present invention provides a transformant comprising the above-described expression vector; the transformant is prepared by transforming the above-described expression vector into a host cell to obtain the transformant, wherein the host cell includes Escherichia coli, CHO cells or 293 cells.

[0012] This invention provides a method for preparing recombinant C1QBP protein, the method comprising constructing a nucleotide sequence encoding C1QBP protein; introducing the nucleotide sequence into an expression vector; transforming the expression vector into a suitable host cell; and then inducing the host cell to express recombinant C1QBP protein.

[0013] This invention provides the use of recombinant complement C1Q binding protein C1QBP in the preparation of a drug for treating leukemia, wherein the leukemia includes all FABs and all subtypes of acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia according to the WHO classification.

[0014] Preferably, the application is the preparation of a drug for treating leukemia by inhibiting intramedullary leukemia cells and / or inhibiting extramedullary infiltrating leukemia cells.

[0015] The present invention provides the use of a pharmaceutical composition in the preparation of a medicament for treating leukemia, the pharmaceutical composition comprising C1QBP protein, and any one or a combination of other chemotherapeutic drugs and / or targeted drugs.

[0016] Preferably, the dosage form of the drug includes tablets, powders, granules, capsules, oral liquids, injections, or sustained-release formulations.

[0017] This invention provides a modified recombinant complement C1Q binding protein C1QBP. The C1QBP protein and its mutants are modified by means of any one or a combination of at least two of horseradish peroxidase, alkaline phosphatase, biotin, fluorescein isothiocyanate, Cy3 or Cy5.

[0018] This invention provides a conjugated recombinant complement C1Q binding protein C1QBP, wherein the C1QBP protein and its mutants are conjugated, and the conjugated portion includes a detectable marker, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof, and the protein portion and the conjugated portion are conjugated by a chemical bond or a linker.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention discloses the significant therapeutic effect of single-agent therapy, namely injection of recombinant C1QBP protein, on acute leukemia. Specifically, recombinant C1QBP protein significantly inhibits the proliferation of leukemia cells in the bone marrow, spleen, and peripheral blood of the hematopoietic system, while also inhibiting the infiltration of leukemia cells into extramedullary organs (such as skin), thereby reducing systemic tumor burden and significantly prolonging the survival of leukemia mice. This effect has been observed in CDX (cell line-derived xenograft) models constructed from various human leukemia cell lines and PDX (patient-derived xenografts) models derived from leukemia patients, demonstrating excellent reproducibility.

[0021] More importantly, the recombinant C1QBP protein, when used in combination with first-line clinical chemotherapy regimens (daunorubicin + cytarabine), exhibits a synergistic effect, demonstrating a stronger inhibitory effect on leukemia cell proliferation than chemotherapy alone, significantly prolonging the survival of leukemia mice, and playing a role in reducing toxicity and enhancing efficacy.

[0022] This invention is the first to demonstrate that complement C1Q binding protein has a therapeutic effect on leukemia, and provides a method for its preparation and a combination therapy regimen, which has high clinical translation prospects and application value. Attached Figure Description

[0023] Figure 1 This is a diagram showing the expression and purification of recombinant C1QBP protein.

[0024] Figure 2 Figure showing the effects of recombinant C1QBP protein administration to the spleen and extramedullary tissues of leukemia cells in the Molm13 leukemia mouse model (NSG mouse);

[0025] Figure 3 Figure showing the results of flow cytometry analysis of C1QBP protein inhibiting the proliferation of Molm13 leukemia cells in vivo.

[0026] Figure 4 Staining diagram showing the effect of recombinant C1QBP protein inhibiting the infiltration of leukemia cells into the liver and spleen in the Molm13 leukemia mouse model.

[0027] Figure 5 Figure showing the results of flow cytometry analysis of C1QBP protein inhibiting the proliferation of MV4 leukemia cells in vivo.

[0028] Figure 6 Staining diagram showing the infiltration of leukemia cells into the liver and spleen in a mouse model of MV4 leukemia where recombinant C1QBP protein inhibits leukemia.

[0029] Figure 7 Figure showing the results of recombinant C1QBP protein inhibiting the infiltration of leukemia cells into the bone marrow, spleen, and liver in a C1498 leukemia mouse model;

[0030] Figure 8 The figure shows the results of recombinant C1QBP protein inhibiting the growth of leukemia cells in a Molm13 nude mouse model;

[0031] Figure 9 The figure shows the results of recombinant C1QBP protein inhibiting the growth of leukemia cells in a PDX model;

[0032] Figure 10 The figure shows the in vivo results of C1QBP protein dose-dependent inhibition of leukemia cell proliferation;

[0033] Figure 11 The figure shows the in vivo efficacy of chemotherapy drugs combined with C1QBP protein in inhibiting leukemia.

[0034] Figure 12 Figure showing that the combination of chemotherapy drugs and C1QBP protein significantly prolongs the survival of mice. Detailed Implementation

[0035] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0036] The purpose of this invention is to disclose a recombinant human complement C1Q-binding protein (C1QBP), which can be used to prepare drugs for treating leukemias including acute myeloid leukemia (AML). The C1QBP's classification identifier, or Gene ID, is 708 in the NCBI international nucleotide sequence database. This protein can be used in combination with first-line chemotherapy regimens for AML in clinical practice, reducing the toxic side effects of chemotherapy drugs while enhancing therapeutic efficacy.

[0037] Specifically, in a first aspect, the present invention provides a recombinant complement C1Q binding protein, namely C1QBP, the amino acid sequence of which is shown in SEQ ID No. 1. SEQ ID No. 1:

[0038] 1-LHTDGDKAFVDFLSDEIKEERKIQKHKTLPKMSGGWELELNGTEAKLVRKVAGEKITVTFNIN NSIPPTFDGEEEEPSQGQKVEEQEPELTSTPNFVVEVIKNDDGKKALVLDCHYPEDEVGQEDEAESDIFS IREVSFQSTGESEWKDTNYTLNTDSLDWALYDHLMDFLADRGVDNTFADELVELSTALEHQEYITFLED LKSFVKSQ-209

[0039] Secondly, this invention provides a recombinant complement C1Q-binding protein, namely C1QBP, encoding the first aspect of this invention. In this document, the polynucleotide can be in DNA or RNA form, preferably DNA. The DNA form includes natural cDNA and synthetic cDNA, and the DNA can be the coding strand or the template strand. Those skilled in the art can readily obtain the nucleic acid molecule polynucleotide or fragments thereof encoding the mutants of this invention using conventional techniques such as PCR, recombinant methods, or synthetic methods. Once these sequences are obtained, they can be cloned into vectors, transformed or transfected into appropriate cells, and then proliferated using conventional host cells to isolate a large number of nucleic acid molecules.

[0040] Thirdly, this invention provides an expression vector, which includes expression vectors and cloning vectors, referring to bacterial plasmids, phage particles, yeast plasmids, plant cell viruses, animal viruses, and various other viral vectors commonly used in the art. The vectors applicable to this invention include, but are not limited to: vectors for expression in bacteria (prokaryotic expression vectors), vectors for expression in yeast (such as Pichia pastoris vectors, Hansenula polymorpha vectors, etc.), baculovirus vectors for expression in insect cells, vectors for expression in mammalian cells (vaccinia virus vectors, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, etc.), plant virus vectors for expression in plants, and various vectors for expression in mammalian mammary glands. In short, any plasmid and vector can be used as long as it can stably replicate in the host cell. Preferred expression vectors contain selection marker genes, such as bacterial ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, streptomycin resistance genes, and chloramphenicol resistance genes; yeast neomycin resistance genes and Zeocin resistance genes, yeast defect selection markers such as His, Leu, and Trp; and eukaryotic neomycin resistance genes, Zeocin resistance genes, dihydrofolate reductase genes, and fluorescent protein marker genes. In a specific embodiment of the present invention, the commercially available pSUMO3 plasmid is used.

[0041] Fourthly, this invention provides a method for preparing recombinant complement C1Q binding protein, namely C1QBP, comprising the following steps: culturing the above-mentioned transformant, expressing and purifying it from the culture to obtain recombinant complement C1Q binding protein. The main steps of recombinant expression of C1QBP protein are: constructing a nucleic acid sequence encoding C1QBP protein; ligating it into a pSUMO3 expression vector; transforming the expression vector into appropriate host cells and inducing expression. Specific details include:

[0042] (1) The nucleic acid sequence encoding C1QBP was amplified by PCR and cloned into the pSUMO3 expression vector.

[0043] It should be understood that those skilled in the art can synthesize corresponding nucleotide sequences based on codon degeneracy and expression preferences in different species, clone them into appropriate expression vectors, and transform them into corresponding hosts. For example, a nucleic acid sequence corresponding to the amino acid sequence shown in SEQ ID NO. 1 can be synthesized, cloned into the pET-20b-SUMO3 expression vector, and transformed into BL21(DE3) competent cells.

[0044] (2) By culturing the above-transformed host cells, the expression of C1QBP protein with SUMO3 tag was induced, SUMO3-C1QBP was isolated and purified, the SUMO3 tag was removed, and then C1QBP was obtained by FPLC purification.

[0045] Fifthly, in practical applications, the recombinant C1QBP protein can be structurally and sequentially modified using known protein modification techniques to obtain the desired properties. Modification of the recombinant C1QBP protein includes:

[0046] (1) Marker protein

[0047] Proteins can be labeled for easy detection. Labeling methods include fluorescent labeling, chemiluminescent labeling, radioactive labeling, enzyme-linked labeling, biotin / avidin labeling, magnetic bead labeling, or nanoparticle labeling.

[0048] Preferably, the purified C1QBP protein is modified, and the modification conjugate used for the modification includes any one or a combination of at least two of horseradish peroxidase, alkaline phosphatase, biotin, fluorescein isothiocyanate, Cy3 or Cy5, and Cy5.5.

[0049] Preferably, the purified C1QBP protein is modified, and the modification conjugate used includes compounds with the following cytotoxic effects:

[0050] a) Traditional chemotherapy drugs such as methotrexate, vinblastine and doxorubicin are used as cytotoxic loads.

[0051] b) Tubulin inhibitors include the following categories: ① Tubulin polymerization enhancers, such as the β-subunit auristatin compounds MMAE (monomethyl auristatin E) and MMAF (monomethyl auristatin F) that act on α and β tubulin dimers. ② Tubulin polymerization inhibitors, such as maytansine compounds DM1 and DM4. ③ Eribuli compounds, namely natural products of polyether macrolides such as Halichondrin B; ④ Tubulysins, natural anti-mitotic peptides isolated from slime mold culture media; ⑤ Cryptophycin-1 and cryptophycin-25; ⑥ Kinesin (KSP / EG5 / KIF11) inhibitors, etc.

[0052] c) DNA damaging agents that act on the cell cycle: Topoisomerase I inhibitors, such as DXd (DX-8951 derivative); Enediyne; PDB (Pyrrolo[2,1-c][1,4]benzodiazepines), a pyrrolobenzodiazepine compound found in Streptomyces; Duocarmycin A, a strong DNA alkylating agent isolated from Streptomyces;

[0053] d) RNA-targeting payloads: RNA splicing inhibitors (Thailanstatin and its analogues) and RNA polymerase II inhibitors (Amatoxins).

[0054] e) Radionuclides, including radioisotopes such as I-131, Y-90, Lu-177 and Re-188, Bi-213 and At-211. For example, tiuxetan (Y-90), which emits only beta rays, is already used clinically.

[0055] (2) Sequence and structural modification

[0056] The sequence and structure of recombinant C1QBP proteins can be modified, including adding signal peptides to enable their expression in specific spaces and times, and reducing amino acids and domains that have no effect on function to reduce protein size.

[0057] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the recombinant C1QBP protein of the first aspect of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can treat leukemia. Therefore, the pharmaceutical composition of the present invention can be safely administered over a long period. In this document, a pharmaceutically acceptable carrier refers to a non-toxic filler, stabilizer, diluent, adjuvant, or other pharmaceutical excipient. According to prior art, the pharmaceutical composition can be formulated into various dosage forms according to the therapeutic purpose and route of administration. Preferably, the composition is in unit dose form, such as a lyophilized form, tablet, capsule, powder, emulsion, injection, or spray. More preferably, the pharmaceutical composition is in an injectable dosage form, such as a lyophilized powder for injection. It is also preferred that the pharmaceutical composition is a liquid formulation, such as one that may contain a buffer solution (e.g., phosphate buffer, Tris-HCl buffer).

[0058] This invention discloses the use of recombinant complement C1Q binding protein C1QBP in the preparation of drugs for leukemia, wherein the leukemia includes all subtypes of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocyticle leukemia (CLL), and chronic myelogenous leukemia (CML) according to their respective FAB classifications and WHO classifications. The FAB classifications refer to the French-American-British classification system, and the WHO classification refers to the World Health Organization's classification of hematologic malignancies, updated to the fifth edition of the classification (WHO-HAEM5) in 2022.

[0059] This invention discloses the use of recombinant C1QBP protein in the preparation of drugs for leukemia, wherein the use includes inhibitory effects on intramedullary leukemia and inhibitory effects on extramedullary infiltrating leukemia.

[0060] Seventhly, the use of the recombinant protein, the conjugate, and the pharmaceutical composition in the preparation of a leukemia treatment drug. The leukemia treatment drug includes a combination of chemotherapy drugs and / or small molecule targeted drugs.

[0061] In this article, chemotherapy drugs for treating leukemia include:

[0062] Common chemotherapy drugs include cytarabine (Ara-C), doxorubicin, daunorubicin, methotrexate, etoposide, cyclophosphamide, vincristine, 6-mercaptopurine (6-MP), fludarabine, and cladribine. Glucocorticoids include prednisone and dexamethasone. Targeted therapies include tyrosine kinase inhibitors (TKIs), such as imatinib (Gleevec), dasatinib (Sprycel), nilotinib (Tasigna), bosutinib (Bosulif), ponatinib (Iclusig), FLT3 inhibitors midostaurin (Rydapt) and gilteritinib (Xospata), BCL-2 inhibitor venetoclax (Venclexta), and IDH1 / IDH2 inhibitors ivonib (Tibsovo) and enasidenib (Idhifa). CD20 monoclonal antibodies include rituximab (Rituxan) and ofatumumab (Arzerra). CD22 monoclonal antibody Inotuzumab ozogamicin (Besponsa). CD33 monoclonal antibody Gemtuzumab ozogamicin (Mylotarg). JAK inhibitor Ruxolitinib (Jakafi). PI3K inhibitor Idelalisib (Zydelig). Nuclear export inhibitor Selinexor (Xpovio). Immunotherapy drugs CAR-T cell therapy including tesarenxel (Kymriah) and brexusabtagene autoleucel (Tecartus). Bispecific antibody Blinatumomab (Blincyto). Arsenic trioxide (Trisenox) and interferon alfa.

[0063] Example 1

[0064] Expression and purification of recombinant C1QBP protein;

[0065] The following is a method for producing recombinant human C1QBP protein, including the following steps:

[0066] 1. Induction of SUMO3-C1QBP protein expression. E. coli BL21(DE3) competent cells transformed with the pET-20b-SUMO3-C1QBP vector were screened for positive transformations using ampicillin-resistant plates. Single colonies of the selected positive recombinant bacteria were inoculated into LB medium and cultured at 37°C with shaking for 16 h. Then, 5% of these colonies were transferred to fresh 2×YT medium. The expression conditions, including temperature, IPTG concentration, and induction time, were optimized. The study confirmed that an OD value of 0.8, induction with 0.5 mM IPTG at 20°C for 16 h, and lysis by sonication (100 W, 2 s sonication, 2 s interval) were the optimal conditions for soluble expression of the C1QBP protein.

[0067] 2. Removal of the SUMO3 tag: The SUMO3 tag is removed using the SUMO protease (SENP2): Purified SUMO3-C1QBP protein is added to the SENP2 protease, and the enzymatic digestion reaction is carried out under appropriate conditions. This invention found that 12 hours of digestion yields the best results, completely removing the SUMO3 tag without damaging the integrity of the target protein (e.g., ...). Figure 1 A). After the reaction was terminated, SDS-PAGE electrophoresis and Coomassie brilliant blue staining were performed to detect the target protein and demonstrate protein yield and purity.

[0068] 3. Further purification of C1QBP protein using molecular sieves. The C1QBP protein solution with the SUMO3 tag removed was collected and further purified from the culture medium according to molecular weight using a Superdex 200 Increase 10 / 300GL (GE Healthcare, GE28-9909-44) size exclusion column and AKTA system, following the manufacturer's instructions. The equilibration buffer was phosphate-buffered saline (pH 7.5). Fractions eluted from the Superdex 200 size exclusion column were collected and Western blot analysis was performed using an anti-C1QBP (24474-1-AP, Proteintech) antibody, confirming the acquisition of high-purity and high-concentration C1QBP protein (e.g., [missing information]). Figure 1 B).

[0069] The vectors, host bacteria, etc. described in the present invention can be obtained commercially. For example, pET-20b and BL21(DE3) are purchased from Novagen. Operations such as the design, synthesis, and cloning of genes involved in the present invention, the construction of expression vectors, DNA sequence analysis and identification, and the separation and purification of expression products can be carried out according to the techniques known in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY, 1989).

[0070] Example 2 Preparation of C1QBP protein conjugate;

[0071] Prepare the reducing buffer: Dissolve TCEP (Tris-2-carboxyethyl-phosphine) and DTPA (diethylenetriaminepentaacetic acid) in PBS. The concentrations of the two substances in the reducing buffer are 0.26 mM and 2 mM, respectively.

[0072] Protein reduction: Mix 20 mg / mL C1QBP (in PBS buffer) with the reducing buffer at a volume ratio of 1:1, and stir and react at 25 °C for 2 h.

[0073] Prepare the small molecule drug solution: Dissolve the small molecule toxins MC-VC-PAB-MMAE, MC-VC-PAB-MMAF, or MC-MMAF in DMSO (dimethyl sulfoxide) to a final concentration of 10 mM.

[0074] Conjugation: Add 25% DMSO to the reduced protein, and then slowly add the small molecule drug solution according to the molar ratio of small molecule drug to C1QBP of 8 for conjugation. Stir and react at 25 °C for 1 h. Finally, dialyze the conjugated protein in PBS to remove the unconjugated small molecule drugs for later use.

[0075] Example 3 Observation of the in vivo efficacy of injecting C1QBP protein to inhibit leukemia (Molm13-NSG model);

[0076] Experimental animals: 6-8-week-old female NSG immunodeficient mice, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. License number: SCXK (Su)-0008. Feeding environment: Central Laboratory Animal House of Shanghai Jiao Tong University School of Medicine (SPF level).

[0077] Experimental procedure: After 5 days of adaptation in a closed SPF-level environment, healthy mice were selected and injected with human AML cell line Molm13 (5×10 6 cells / mouse) via the tail vein, and the animals were randomly grouped. The dosing dose was 5 mg / kg, once a week. The control group was injected with an equal volume of PBS. The experimental endpoint was the death of mice in the control group due to leukemia. After the mice reached the experimental endpoint and were sacrificed, tests were conducted in three aspects: ① The spleens and extramedullary infiltrations (subcutaneous infiltration foci) of the mice were photographed to compare the spleen size and the number of subcutaneous infiltration foci. ② Femoral bone marrow and spleen cells were taken for flow cytometry detection. By labeling human myeloid leukemia markers CD45 (hCD45) and CD33 (hCD33), the proliferation of leukemia cells was judged; ③ Liver and spleen tissues were taken for HE and IHC (antibody: hCD45) staining to judge the growth and infiltration of human leukemia cells in the hematopoietic organs of mice. As Figure 2 shown in the gross photographs, injection of C1QBP protein (treatment group) significantly reduced the spleen size (as shown in Figure 2 Figure A in Figure 2 ) and the number of subcutaneous infiltration foci (as shown in Figure 3 Figure B in Figure 4 ). The results of flow cytometry in Figure 4 showed that injection of C1QBP protein (treatment group) significantly reduced the proportion of human leukemia cells (hCD45+hCD33+ positive cells) in the bone marrow and spleen.

[0078] Example 4 Observation of the in vivo efficacy of injecting C1QBP protein to inhibit leukemia (MV4 model);

[0079] Experimental animals: 6-8-week-old female NSG immunodeficient mice, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. License number: SCXK (Su)-0008. Breeding environment: Central Laboratory Animal House of Shanghai Jiao Tong University School of Medicine (SPF level).

[0080] Experimental procedure: After 5 days of adaptation in a closed SPF-level environment, healthy mice were selected and injected with human AML cell line MV4 (1×10 6The animals were randomly grouped after injecting 5×10 cells per mouse with C1498 leukemia cell line. The dosage of the drug was 5 mg / kg, once a week. The control group was injected with an equal volume of PBS. The end point of the experiment was the death of the mice in the control group due to leukemia. After the mice were sacrificed at the end point of the experiment, femoral bone marrow and spleen cells were taken for flow cytometry detection. By labeling human myeloid leukemia markers CD45 (hCD45) and CD33 (hCD33), the proliferation of leukemia cells was judged. Figure 5 Flow cytometry results showed that injecting C1QBP protein (treatment group) significantly reduced the proportion of human leukemia cells (hCD45+hCD33+ positive cells) in the bone marrow and spleen, indicating that injecting C1QBP protein (treatment group) significantly inhibited the in vivo proliferation of leukemia cells. Figure 6 Pathological indexes showed that the number of infiltrated human leukemia cells (such as Figure 6 hCD45+ positive cells in the IHC staining of Figure B) in the liver and spleen was significantly reduced.

[0081] Example 5 Observation of the in vivo efficacy of injecting C1QBP protein in inhibiting leukemia (C1498 model);

[0082] Experimental animals: Female C57BL / 6 mice, 6-8 weeks old, were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. License number: SCXK (Su)-0008. Breeding environment: Central Laboratory Animal House of Shanghai Jiao Tong University School of Medicine (SPF level).

[0083] Experimental procedure: After the experimental mice were acclimatized in a clean-level closed environment for 5 days, healthy mice were selected and injected with C1498 leukemia cell line (5×10 5 cells per mouse) via the tail vein. The animals were randomly grouped. The dosage of the drug was 5 mg / kg, once a week. The control group was injected with an equal volume of PBS. The end point of the experiment was the death of the mice in the control group due to leukemia. After the mice were sacrificed at the end point of the experiment, femoral bone marrow and spleen cells were taken for flow cytometry detection. By labeling human myeloid leukemia markers CD45 (hCD45) and CD33 (hCD33), the proliferation of leukemia cells was judged. As Figure 7 shown in Figure A of [reference], flow cytometry results showed that injecting C1QBP protein (treatment group) significantly reduced the proportion of leukemia cells in the bone marrow and spleen, indicating that injecting C1QBP protein (treatment group) significantly inhibited the in vivo proliferation of leukemia cells. As Figure 7 shown in Figure B of [reference], the gross liver index showed that the liver volume and weight were significantly reduced, indicating that the infiltration of leukemia cells into the liver was significantly inhibited.

[0084] Example 6 Observation of the in vivo efficacy of injecting C1QBP protein in inhibiting leukemia (Molm13-nude mouse model);

[0085] Experimental animals: Female nude mice, 6 - 8 weeks old, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. License number: SCXK(Su)-0008. Breeding environment: Central Laboratory Animal House of Shanghai Jiao Tong University School of Medicine (SPF level).

[0086] Experimental procedures: After the experimental mice were acclimated in a closed SPF - level environment for 5 days, healthy mice were selected and injected with the human AML cell line Molm13 (1×10 6 cells / mouse) via the tail vein, and the animals were randomly grouped. The dosing dose was 5 mg / kg, once a week. The control group was injected with an equal volume of PBS. The experimental endpoint was the death of mice in the control group due to leukemia. After the experimental endpoint mice were sacrificed, photos were taken to show the size of the tumors formed by leukemia (such as Figure 8 ). The results showed that C1QBP protein significantly inhibited the growth of leukemia cells in vivo.

[0087] Example 7 Observation of the in - vivo efficacy of injecting C1QBP protein to inhibit leukemia (PDX model);

[0088] Experimental animals: Female NSG immunodeficient mice, 6 - 8 weeks old, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. License number: SCXK(Su)-0008. Breeding environment: Central Laboratory Animal House of Shanghai Jiao Tong University School of Medicine (SPF level).

[0089] Experimental procedures: After the experimental mice were acclimated in a closed SPF - level environment for 5 days, healthy mice were selected and injected with bone marrow mononuclear cells from AML patients (5×10 6 cells / mouse) via the tail vein, and the animals were randomly grouped. The dosing dose was 5 mg / kg, once a week. The control group was injected with an equal volume of PBS. The experimental endpoint was the death of mice in the control group due to leukemia. After the experimental endpoint mice were sacrificed, the spleens and extramedullary infiltrations (subcutaneous infiltration foci) of the mice were taken for photos to compare the spleen size and the number of subcutaneous infiltration foci. Femoral bone marrow and spleen cells were taken for flow cytometry detection. By labeling human myeloid leukemia markers CD45 (hCD45) and CD33 (hCD33), the proliferation of leukemia cells was judged. As Figure 9 shown in Figure A of, injecting C1QBP protein (treatment group) significantly reduced the spleen size and the number of subcutaneous infiltration foci, Figure 9 and as shown in Figure B of the flow cytometry results in, injecting C1QBP protein (treatment group) significantly reduced the proportion of human leukemia cells (hCD45 + hCD33 + positive cells) in the bone marrow and spleen. The results indicated that injecting C1QBP protein (treatment group) significantly inhibited the in - vivo proliferation of leukemia cells and their infiltration into organs.

[0090] Example 8 Observation of the in - vivo efficacy of injecting C1QBP protein with dose - dependent inhibition of leukemia;

[0091] Experimental animals: 6 - 8 - week - old female NSG immunodeficient mice, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. License number: SCXK(Su) - 0008. Feeding environment: Central Laboratory Animal House of Shanghai Jiao Tong University School of Medicine (SPF level).

[0092] Experimental procedure: After the experimental mice were adapted in a closed SPF - level environment for 5 days, healthy mice were selected and injected with human AML cell line Molm13 (1×10 6 cells / mouse) via the tail vein. The animals were randomly divided into 4 groups. The control group was injected with an equal volume of PBS, and the treatment groups were given different doses of C1QBP protein injection. The experimental endpoint was the death of mice in the control group due to leukemia. After the experimental endpoint mice were sacrificed, femoral bone marrow and spleen cells were taken for flow cytometry detection. By labeling human myeloid leukemia markers CD45 (hCD45) and CD33 (hCD33), the proliferation of leukemia cells was judged. Figure 10 Flow cytometry results showed that with the increase in the dose of C1QBP protein injection, the proportion of leukemia infiltration decreased significantly, indicating that C1QBP protein could inhibit the in - vivo proliferation of leukemia in a dose - dependent manner.

[0093] Example 9 Observation of the in - vivo efficacy of the combination of chemotherapy drugs and C1QBP protein in inhibiting leukemia;

[0094] Experimental animals: 6 - 8 - week - old female NSG immunodeficient mice, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. License number: SCXK(Su) - 0008. Feeding environment: Central Laboratory Animal House of Shanghai Jiao Tong University School of Medicine (SPF level).

[0095] Experimental procedure: After the experimental mice were adapted in a closed SPF - level environment for 5 days, healthy mice were selected and injected with human AML cell line Molm13 (1×10 6 cells / mouse) via the tail vein. The animals were randomly divided into 4 groups: PBS, daunorubicin (chemotherapy group), C1QBP (protein group), and daunorubicin + C1QBP (protein + chemotherapy combined group). The dosing doses were as follows: protein dose 5 mg / kg, once a week; daunorubicin dose 10 mg / kg, once a day for 5 consecutive days. The control group was injected with an equal volume of PBS. The experimental endpoint was the death of mice in the control group due to leukemia. After the experimental endpoint mice were sacrificed, femoral bone marrow and spleen cells were taken for flow cytometry detection. By labeling human myeloid leukemia markers CD45 (hCD45) and CD33 (hCD33), the proliferation of leukemia cells was judged. As Figure 11Flow cytometry results showed that C1QBP protein injection (treatment group) significantly reduced the proportion of human leukemia cells (hCD45+hCD33+ positive cells) in the bone marrow and spleen, with the combination chemotherapy group showing the most significant inhibitory effect. This indicates that C1QBP protein injection (treatment group) combined with chemotherapy can synergistically inhibit the in vivo proliferation of leukemia cells. Figure 12 The study showed survival data, demonstrating that C1QBP protein injection alone significantly prolonged the survival of leukemia mice, and that combination with chemotherapy drugs further prolonged survival. These results indicate that C1QBP protein, along with other chemotherapy drugs, can synergistically inhibit the in vivo growth of leukemia cells.

[0096] This invention demonstrates that monotherapy with recombinant C1QBP protein significantly enhances the treatment of acute leukemia. Specifically, recombinant C1QBP protein significantly inhibits the proliferation of leukemia cells in the bone marrow, spleen, and peripheral blood of the hematopoietic system, while also suppressing their infiltration into extramedullary organs (such as skin), thereby reducing systemic tumor burden and significantly prolonging the survival of leukemia-affected mice. This effect has been observed in CDX (cell line-derived xenograft) models constructed from various human leukemia cell lines and PDX (patient-derived xenografts) models, exhibiting excellent reproducibility. More importantly, the combination of recombinant C1QBP protein with first-line clinical chemotherapy (daunorubicin + cytarabine) shows a synergistic effect, exhibiting a stronger inhibitory effect on leukemia cell proliferation and significantly prolonging the survival of leukemia-affected mice compared to chemotherapy alone, thus demonstrating a synergistic effect and reducing toxicity.

[0097] In the attached diagram, Figure 1 Figure A shows the expression and purification of recombinant C1QBP protein. Figure A shows the expression of C1QBP protein after transformation of the pSUMO3-C1QBP expression vector into BL21 competent bacteria, induction with IPTG, and purification of the lysate by Ni-NTA followed by SENP2 digestion. Protein purity was then determined by SDS-PAGE electrophoresis and Cognizine staining. Figure B shows the further purification of the digested protein sample using Superdex 200 molecular sieves, with the peak positions corresponding to fractions collected for Western blot detection of C1QBP.

[0098] Figure 2 Figure 1 shows the effects of recombinant C1QBP protein on the spleen and extramedullary tissue in a Molm13 leukemia mouse model (NSG mouse). Figure A compares the spleen size of the control group (injected with PBS) and the treatment group (injected with C1QBP protein). Figure B compares the subcutaneous infiltration foci of the control group (injected with PBS) and the treatment group (injected with C1QBP protein).

[0099] Figure 3 The image shows the results of flow cytometry analysis of the inhibition of Molm13 leukemia cell proliferation by C1QBP protein. Single-cell suspensions were prepared from bone marrow and spleen cells of Molm13 leukemia mice in the control group (injected with PBS) and the treatment group (injected with C1QBP protein), labeled with humanCD45 (hCD45) and humanCD33 (hCD33) antibodies, and the proportion of leukemia cell infiltration was detected by flow cytometry.

[0100] Figure 4 Figure 1 shows the results of recombinant C1QBP protein inhibiting leukemia cell infiltration in the liver and spleen of a Molm13 leukemia mouse model. Figure A shows HE staining, indicating leukemia cell infiltration in the liver and spleen of control (PBS injection) and treatment (C1QBP protein injection) Molm13 leukemia mice. Figure B shows immunohistochemical (IHC) staining using hCD45 antibody, indicating leukemia cell infiltration in the liver and spleen.

[0101] Figure 5 The image shows the results of flow cytometry analysis of the inhibition of MV4 leukemia cell proliferation by C1QBP protein. Single-cell suspensions of bone marrow and spleen cells from MV4 leukemia mice in the control group (injected with PBS) and the treatment group (injected with C1QBP protein) were prepared, labeled with hCD45 and hCD33 antibodies, and the proportion of leukemia cell infiltration was detected by flow cytometry.

[0102] Figure 6 Figure 1 shows the results of recombinant C1QBP protein inhibiting leukemia cell infiltration in the liver and spleen of a mouse model of MV4 leukemia. Figure A shows HE staining, indicating leukemia cell infiltration in the liver and spleen of MV4 leukemia mice in the control group (injected with PBS) and the treatment group (injected with C1QBP protein). Figure B shows immunohistochemical (IHC) staining using hCD45 antibody, indicating leukemia cell infiltration in the liver and spleen.

[0103] Figure 7 Figure 1 shows the results of C1QBP protein inhibiting the infiltration of leukemia cells into the bone marrow, spleen, and liver in a mouse model of C1498 leukemia. Figure A shows the in vivo proliferation of C1498 leukemia cells inhibited by C1QBP protein as detected by flow cytometry. Figure B shows gross photographs and a comparison of liver weight between the control group (injected with PBS) and the treatment group (injected with C1QBP protein) of C1498 leukemia mice.

[0104] Figure 8 This image illustrates the inhibition of leukemia cell growth by C1QBP protein in a Molm13 nude mouse model. Molm13 leukemia cells were injected into nude mice, followed by intravenous administration of C1QBP protein. The tumor formation of leukemia cells was observed at the experimental endpoint.

[0105] Figure 9 Figure showing the results of recombinant C1QBP protein inhibiting the growth of leukemia cells in the PDX model.

[0106] A PDX model of leukemia was constructed by injecting primary bone marrow cells from leukemia patients into NSG mice. Then, C1QBP protein was injected intravenously to treat the mice. The size of the spleen and the number of subcutaneous infiltrates were observed (as shown in Figure A). The proportion of leukemia cells in the bone marrow and spleen was detected by flow cytometry (as shown in Figure B).

[0107] Figure 10 The figure shows the in vivo results of C1QBP protein dose-dependent inhibition of leukemia cell proliferation. After Molm13 leukemia mice were treated with intravenous injection of different doses of C1QBP protein, the proportion of leukemia cells (hCD45+hCD33+) infiltrating in the bone marrow and spleen was detected by flow cytometry.

[0108] Figure 11 This figure shows the in vivo efficacy of chemotherapy drugs combined with C1QBP protein in inhibiting leukemia. Molm13 leukemia cells were injected into NSG mice to create a leukemia model. The mice were then treated with PBS, daunorubicin (chemotherapy group), C1QBP (protein group), and daunorubicin + C1QBP (protein + chemotherapy combination group), respectively. The experimental endpoint was determined by flow cytometry to detect the proportion of leukemia cells in the bone marrow (Figure A) and spleen (Figure B).

[0109] Figure 12 The figure shows that the combination of chemotherapy drugs and C1QBP protein significantly prolonged the survival of mice. Molm13 leukemia cells were injected into NSG mice to establish the leukemia model. The mice were then treated with PBS, daunorubicin (chemotherapy group), C1QBP (protein group), and daunorubicin + C1QBP (protein + chemotherapy combination group), and the survival time of the mice was recorded.

[0110] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The use of a recombinant complement C1Q binding protein C1QBP in the preparation of a drug for treating leukemia, said drug inhibiting the infiltration of leukemia cells into bone marrow and extramedullary tissues and organs.

2. The use of a pharmaceutical composition in the preparation of a medicament for the synergistic treatment of leukemia, characterized in that, The pharmaceutical composition includes recombinant complement C1Q binding protein C1QBP, and other chemotherapeutic agents, the other chemotherapeutic agents being daunorubicin.

3. The application according to any one of claims 1-2, characterized in that, The drug is in the form of an injection.

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