An immunotoxin targeting hematologic malignancies, its preparation method and application

Abstract

This invention relates to an immunotoxin targeting hematologic malignancies and its application. The immunotoxin comprises a toxic molecule and a carrier. The toxic molecule is pumpkin protein or a mutant of pumpkin protein. The carrier contains an antibody, ligand, or polypeptide capable of binding to hematologic malignancies cells. The antibody comprises an antibody fragment or a small molecule antibody. This invention provides an immunotoxin targeting hematologic malignancies for the treatment of these malignancies. The immunotoxin using a pumpkin protein mutant as the toxic molecule exhibits enhanced specificity against hematologic malignancies cells with high target expression, significantly increased activity in killing target cells, and allows for a substantial reduction in dosage and toxicity. In summary, the immunotoxin targeting hematologic malignancies using a pumpkin protein mutant as the toxic molecule offers advantages in both synergistic effect and reduced toxicity, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an immunotoxin targeting hematologic malignancies, its preparation method, and its application. Background Technology

[0002] Hematologic malignancies are a group of diseases of the hematopoietic system, including leukemia, lymphoma, and multiple myeloma, characterized by high malignancy, complex treatment, and poor prognosis. The main hematologic malignancies include various types of leukemia, multiple myeloma (accounting for approximately 10%), and malignant lymphoma.

[0003] Chemotherapy is a common treatment for hematologic malignancies, but traditional chemotherapy drugs lack tumor specificity and can also kill normal cells, causing serious adverse reactions. Furthermore, tumor cells easily develop resistance to these chemotherapy drugs, often leading to chemotherapy failure and relapse in hematologic malignancies. Therefore, seeking more effective and less toxic drugs is a key direction for research in the treatment of hematologic malignancies.

[0004] Over the past two decades, with the development of tumor molecular biology research, many targets highly expressed in hematologic malignancies have been discovered, such as CD19, CD22, CD30, CD33, CD38, CD79B, and BCMA. Among these, acute myeloid leukemia highly expresses CD33, B-lymphoblastic leukemia and malignant lymphoma highly express CD19 and CD22, and multiple myeloma highly expresses BCMA and CD38. Antibodies targeting these targets, in addition to their biological signal blocking and immune effects, are also used as drug delivery carriers due to their high target specificity and long half-life, and can be used to prepare antibody-drug conjugates or immunotoxins.

[0005] Immunotoxins are hybrid molecules with specific cell-killing capabilities, formed by coupling a carrier molecule with guiding ability (such as antibodies, cytokines, etc.) and a cytotoxic molecule with cytotoxic effects (such as plant toxins, bacterial toxins, human protein toxins, etc.). Through the guiding effect of the carrier, the toxic molecule carried by it reaches the target cell, thereby inhibiting protein synthesis in the cell and causing cell death, achieving the effect of specifically killing the target cell without damaging normal tissue. Summary of the Invention

[0006] The purpose of this invention is to provide a chemical cross-linking or genetic engineering method for preparing an immunotoxin by linking a carrier (an antibody or other polypeptide molecule that binds to hematologic malignancies) and pumpkin protein; and to test the application of the immunotoxin.

[0007] The objective of this invention is achieved as follows: an immunotoxin targeting hematologic malignancies, comprising a toxic molecule and a carrier, wherein the toxic molecule is pumpkin protein or a mutant of pumpkin protein; characterized in that: the carrier contains an antibody or ligand or polypeptide capable of binding to hematologic malignancies cells; wherein the antibody comprises an antibody fragment or a small molecule antibody.

[0008] The mutant of pumpkin protein is obtained by point mutation, deletion mutation, or insertion mutation of pumpkin protein. The vector is a monoclonal antibody targeting CD19, CD22, CD33, CD38, and BCMA.

[0009] The immunotoxin is formed by chemically cross-linking pumpkin protein or its mutant with an antibody that can bind to hematologic tumor cells, or by fusing the genes of the two through genetic engineering to express the immunotoxin. The application of an immunotoxin targeting hematologic malignancies for the treatment of hematologic malignancies.

[0010] Specifically, the pumpkin protein mutant CUS245C (whose amino acid sequence is based on the complete sequence of pumpkin protein amino acids disclosed in CN 109206521 A) was chemically cross-linked with monoclonal antibodies targeting CD19, CD22, CD33, CD38, and BCMA to prepare immunotoxins. These immunotoxins were then used to determine their targeted killing effect on hematologic malignancies with high expression of these targets. The immunotoxins using the pumpkin protein mutant as the toxin molecule showed enhanced specificity against hematologic malignancies cells with high target expression, significantly increased cell-killing activity, and allowed for a significant reduction in dosage and toxicity. In summary, immunotoxins targeting hematologic malignancies using the pumpkin protein mutant as the toxin molecule offer the advantages of enhanced efficacy and reduced toxicity, and have broad application prospects. The pumpkin protein mutant CUS245C was designed to improve the efficiency of preparing chemically conjugated immunotoxins, and the experimental results can be extrapolated to immunotoxins (including recombinant immunotoxins) using pumpkin protein or other pumpkin protein mutants as the effective carrier. Attached Figure Description

[0011] Figure 1 For I-CUS 245C SDS-PAGE images of the preparation and purification of [the sample]; among which, (A)I-CUS 245C Preparation results: Lane 1 was marker, lane 2 was innotuzumab; lane 3 was I-CUS 245C Mixture; (B) I-CUS 245C Purification: Lane 1 is for markers, lane 2 is for CUS. 245C Lane 3 is Inotuzumab; Lane 4 is I-CUS245C Mixture; Lane 5 contains the filtrate from ultrafiltration; Lane 6 contains the I-CUS after dialysis. 245C ; Figure 2 I-CUS 245C Inhibitory effects on the proliferation of different cell lines; Among them, A: I-CUS 245C CUS 245C , Inotuzumab, Inotuzumab + CUS 245C Inhibitory effect on the proliferation of different tumor cells (72h); B: I-CUS 245C CUS 245C , Inotuzumab, Inotuzumab + CUS 245C Inhibitory effect on the proliferation of different tumor cells (120h); Figure 3 I-CUS 245C In vivo anti-lymphoma effect; where (A) average body weight of mice; (B) survival rate of mice; Figure 4 Fluorescence intensity of each group of nude mice with lymphoma (representative) in IVIS spectral in vivo imaging; Figure 5 For L-CUS 245C SDS-PAGE images of the preparation and purification of [substance name]; where (A) L-CUS [substance name] is the highest. 245C Preparation results: Lane 1 was the marker, Lane 2 was Loncastuximab, and Lane 3 was L-CUS. 245C Mixture; (B) L-CUS 245C Purification: Lane 1 is for markers, lane 2 is for CUS. 245C Lane 3 is Loncastuximab, and lane 4 is L-CUS. 245C Mixture, lane 5 is over-Ni 2+ L-CUS after column purification 245C Lane 6 contains the filtrate from ultrafiltration, and lane 7 contains the L-CUS after dialysis. 245C ; Figure 6 Immunotoxin L-CUS 245C Inhibitory effects on the proliferation of different cell lines; A: L-CUS 245C CUS 245C , Loncastuximab, Loncastuximab + CUS 245C Inhibitory effect on the proliferation of different tumor cells (72h) B: L-CUS 245C CUS 245C , Loncastuximab, Loncastuximab + CUS 245C Inhibitory effect on the proliferation of different tumor cells (120h) Figure 7 For L-CUS 245C and I-CUS 245C Comparison of cytotoxicity against PBMC cells; Figure 8 For G-CUS 245C SDS-PAGE plot; Lane 1: Marker; Lane 2: Gemtuzumab; Lane 3: G-CUS 245C mixture; Figure 9 For B-CUS 245C SDS-PAGE plot; Lane 1: Marker; Lane 2: Belantab; Lane 3: B-CUS 245C mixture Figure 10 For DA-CUS 245C SDS-PAGE images of the preparation and purification process; a: DA-CUS 245C Preparation results: Lane 1 was Daratumumab; Lane 2 was DA-CUS 245C mixture; b: DA-CUS 245C One purification (removal of CUS) 245C Lane 1 is CUS 245C Lane 2 is Daratumumab; Lane 3 is DA-CUS 245C Mixture; Lane 4 is DA-CUS after ultrafiltration. 245C Lane 5 contains the filtrate from ultrafiltration; Lane 6 contains the DA-CUS after dialysis. 245C ; c, d: DA-CUS 245C Secondary purification (removal of Daratumumab): Lane 1 contains Daratumumab; Lanes 2-5 and Lanes 2-6 contain the collected solutions of 15mM and 150mM imidazole elutions, respectively. e:DA-CUS 245C Average molecular weight identification: Lane 1 is the reduced CUS 245C Lane 2 is the reduced form Daratumumab; Lane 3 is the reduced form DA-CUS.245C .

[0012] Figure 11 Immunotoxin DA-CUS 245C Inhibitory effect on proliferation of different cell lines (MTT assay) a: DA-CUS 245C DA, CUS 245C DA+CUS 245C Inhibition rate curves of RPMI 8226, NCI-H929, U266, and K562 after 72 h of treatment. b: DA-CUS 245C DA, CUS 245C DA+CUS 245C Inhibition rate curves of RPMI 8226, NCI-H929, U266, and K562 after 120 h of treatment. Detailed Implementation

[0013] The present invention will now be described in detail with reference to the accompanying drawings and embodiments: Example 1: CD22-targeting immunotoxin I-CUS 245C Preparation and assay of anti-hematologic malignancy 1. Immunotoxin I-CUS 245C Preparation Methods: Take 6 mL of CD22 monoclonal antibody Inotuzumab (3.91 mg / mL), dialyze using a dialysis tube with a molecular weight cutoff of 8–10 kDa, then add 59 μL of 20 mmol / L SPDP solution (dissolved in DMSO), and stir at room temperature for 30 min. Dialyze the reaction product (D-PDP) overnight with the above buffer solution to remove excess SPDP. Separately, take 3 mL of CUS... 245C (Total 8.76 mg), add an appropriate amount of dithiothreitol (DTT) dissolved in 0.01 mol / L NaAc to bring the final DTT concentration to 0.3 mol / L. Stir the reaction at room temperature for 30 min, then dialyze using an 8-10 kDa dialysis column using the same method as above to remove excess DTT; then combine the dialyzed Inotuzumab with CUS 245C The mixture was placed in a small beaker and stirred at room temperature for 18 h. An appropriate amount of iodoacetamide solution was added to bring the final concentration to 0.1 mol / L. After stirring at room temperature for 30 min, the mixture was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was collected and dialyzed using a dialysis tube with a molecular weight cutoff of 100 kDa. The filtrate after ultrafiltration was dialyzed using a dialysis tube with a molecular weight cutoff of 100 kDa to remove uncoupled CUS from the supernatant. 245CThe supernatant after dialysis was diluted 4-5 times with 20 mmol / L PB buffer and added to a nickel affinity chromatography column (1 mL nickel column medium) at a drop rate of 0.3 mL / min. The column was equilibrated by eluting with approximately 30 mL of 20 mmol / L PB buffer (drop rate of 2 mL / min). 20 mL of 15 mmol / L imidazole elution buffer was then added to each EP tube (drop rate of 0.3 mL / min), collecting approximately 1.2 mL from each tube, for a total of 16 tubes. This step removes unconjugated monoclonal antibodies and trace amounts of immunotoxins. Elution with 150 mmol / L imidazole was then performed, collecting approximately 1.2 mL of elution buffer in 8 EP tubes (drop rate of 0.3 mL / min). The high concentration of imidazole competitively elutes adsorbed immunotoxins. Samples from each tube were subjected to SDS-PAGE, and high-concentration sample tubes were collected based on the electrophoresis results. The samples were then filtered, aliquoted, quantified using BCA, and I-CUS measured. 245C Yield.

[0014] Results: Immunotoxin I-CUS was achieved using the heterobifunctional protein cross-linking agent SPDP via chemical cross-linking. 245C Preparation of SPDP. SPDP achieves the binding of amine and thiol groups through reducible disulfide bonds, while CUS is pretreated with the strong reducing agent DTT. 245C This allows its thiol groups to be fully exposed, ultimately enabling the monoclonal antibody to interact with CUS. 245C Synthesized ITs. After cross-linking, CUS is finally obtained. 245C When cross-linked with Inotuzumab, it yields the immunotoxin I-CUS. 245C CUS 245C The coupling ratios with Inotuzumab are mainly concentrated in 1:1 and 1:2 ( Figure 1 A). The initial coupling product is I-CUS. 245C CUS 245C A mixed solution of innotuzumab and cinnamic acid was prepared. Unreacted innotuzumab was removed by nickel ion affinity chromatography, and unreacted cinnamic acid was removed by a combination of ultrafiltration and dialysis. 245C The final product contains no uncrosslinked Inotuzumab and CUS. 245C I-CUS 245C ( Figure 1 B), whose purity can approach 100%.

[0015] The above results indicate that CD22 monoclonal antibodies Inotuzumab and CUS were successfully prepared using chemical methods. 245C I-CUS conjugated immunotoxin 245C .

[0016] 2. Immunotoxin I-CUS 245CInhibitory effect on the proliferation of hematologic malignancies (MTT assay) Methods: Burkitt's lymphoma cell lines Raji and Daudi, B-cell leukemia cell line Nalm-6, multiple myeloma cell line RPMI 8226, and T-cell leukemia cell line Molt-4 in logarithmic growth phase were seeded into 96-well cell culture plates (15,000, 38,000, 20,000, 13,000, and 18,000 cells per well for the 72-hour proliferation inhibition assay, and half the 72-hour cell number per well for the 120-hour proliferation inhibition assay), with a seeding volume of 100 μL per well. An I-CUS experimental setup was used. 245C CUS 245C , Inotuzumab (Ino), Ino + CUS 245C Four groups. Each drug was prepared in triplicate, resulting in seven concentrations: I-CUS 245C After adding Ino to the 96-well culture plate, the final concentrations were 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 nmol / L; CUS 245C Ino + CUS 245C The study started with 1000 nmol / L and proceeded to six half-dilutions. The negative control group received 100 µL / well of RPMI 1640 medium, while the blank control wells (cell-free) received 100 µL of medium for background zeroing. After incubation under standard conditions for 72 h or 120 h, 20 µL / well of MTT solution was added, followed by 70 µL / well of triplet solution 4 h later. After incubation for 24–48 h, the absorbance (optical density, OD) of each well at 550 nm or 570 nm was measured using a microplate reader. The average values ​​were taken from each group, and the cell proliferation inhibition rate (IR) was calculated for each group, with the cell viability of the negative control group as 100%. Inhibition rate = (1 - OD value of drug group / OD value of control group) × 100%. Dose-response curves were plotted using GraphPad Prism 8 software, and statistical significance was analyzed. The IC50 was then calculated. 50 The experiment was repeated twice.

[0017] Results: Tables 1-10 and Figure 2 The results showed that the killing effect on tumor cell lines with high CD22 expression (Raji, Daudi, Nalm-6) increased with increasing drug concentration, exhibiting a concentration-dependent effect; however, there was no significant effect on cells with low expression. Both high and low expression cells could be treated with CUS. 245C Ino+CUS 245CThe Ino group exhibited no targeting and concentration-dependent cytotoxicity. Furthermore, the Ino group showed no significant efficacy at a concentration of 1 nmol / L, indicating that immunotoxins can achieve a stronger cytotoxic effect within the same concentration range. Ino+CUS 245C Group proliferation inhibition curves and CUS 245C The groups almost completely overlap, exhibiting individual CUS. 245C The role of I-CUS. 245C The targeted killing effect of RPMI 8226 and Molt-4 on non-target cells was not observed; at a concentration of 1 nmol / L, they did not significantly inhibit cell proliferation and did not reach the IC50 threshold. 50 Value. At a concentration of 1 nmol / L, I-CUS 245C After 72 h of drug administration, the inhibition rates against Raji, Daudi, and Nalm-6 were 100%, 79.71%, and 65.52%, respectively. The IC50 values ​​at 72 h were... 50 The inhibition rates were 100%, 100%, and 90.12% respectively when the concentrations were 0.10, 88.4, and 196.9 pmol / L, respectively, and the treatment time was extended to 120 h. The IC50 values ​​at 120 h were... 50 The concentrations were 0.04, 15.8, and 29.3 pmol / L, respectively. The results indicate that at the same effective concentration, immunotoxin I-CUS... 245C The inhibitory effect on the proliferation of tumor cells with high CD22 expression was stronger at 120 h than at 72 h, showing a time-dependent effect of target selection.

[0018] The results indicate that I-CUS 245C It exhibits targeted inhibition of lymphoma cell proliferation, selectively killing target cells that highly express CD22. When acting on the same target cells, the immunotoxin I-CUS... 245C It can kill target cells at extremely low concentrations. The IC50 can be obtained by calculating the targeting index (TI). 50 I-CUS 245C <CUS 245C ≈ Ino+CUS 245C With CUS 245C Compared to the group, I-CUS 245C The group showed significant lethality, with a TI value of over 10,000 (Table 11). Table 1 Immunotoxin I-CUS 245C Inhibitory effect on the proliferation of Raji cells for 72 hours ±S, n=3)

[0020] P<0.01 VS CUS Table 2 Immunotoxin I-CUS 245C Inhibitory effect on the proliferation of Daudi cells for 72 hours ±S, n=3)

[0021] P<0.05 VS CUS Table 3 Immunotoxin I-CUS 245C Inhibitory effect on the proliferation of Nalm-6 cells for 72 hours ±S, n=3)

[0022] P<0.01 VS CUS Table 4 Immunotoxin I-CUS 245C Inhibitory effect on the proliferation of Molt-4 cells for 72 hours ±S, n=3)

[0023] P<0.05 VS CUS P<0.01 VS CUS Table 5 Immunotoxin I-CUS 245C Inhibitory effect on RPMI8226 proliferation over 72 hours ( ±S, n=3)

[0024] P<0.05 VS CUS Table 6 Immunotoxin I-CUS 245C Inhibitory effect on the proliferation of Raji cells over 120 hours ( ±S, n=3)

[0025] P<0.01 VS CUS Table 7 Immunotoxin I-CUS 245C Inhibitory effect on the proliferation of Daudi cells over 120 hours ( ±S, n=3)

[0026] P<0.05 VS CUS Table 8 Immunotoxin I-CUS 245C Inhibitory effect on the proliferation of Nalm-6 cells over 120 hours ( ±S, n=3)

[0027] P<0.01 VS CUS Table 9 Immunotoxin I-CUS 245C Inhibitory effect on the proliferation of Molt-4 cells over 120 hours ±S, n=3)

[0028] P<0.05 VS CUS Table 10 Immunotoxin I-CUS 245C Inhibitory effect on RPMI8226 proliferation over 120 hours ( ±S, n=3)

[0029] P<0.05 VS CUS P<0.05 VS CUS Table 11 I-CUS 245C IC50 at 72 h and 120 h for each cell line 50 (Mean ± SD, n=3)

[0030] Compared with CUS 245C , P<0.01, :P<0.0001 TI: target index = 3. I-CUS 245C Experiment on prolonging the survival of a human lymphoma BALb / c nude mouse blood model Methods: Twenty-eight BALb / c nude mice were randomly divided into four groups of seven each. Cyclophosphamide was injected intraperitoneally once daily at a dose of 150 mg / kg for two consecutive days. Twenty-four hours after the second injection, Raji-Luc III human lymphoma cells transfected with the luciferase gene were collected during the logarithmic growth phase. The cells were washed twice with serum-free RPMI 1640 medium (1000 r / min, 5 min), counted, and the cell suspension density was adjusted to 2.5 × 10⁻⁶. 7 / mL, the above three groups of nude mice were injected via tail vein, with each mouse receiving 200 uL; the remaining group was injected via tail vein with NS as a blank control group. I-CUS was set up in the experimental groups. 245C Group, CUS 245C The control group and the NS group (negative control) were administered I-CUS via tail vein injection on days 3, 12, and 18 after injection of Raji-Luc III cell suspension. 245C 0.5 mg / kg, CUS 245C 0.5 mg / kg and normal saline (NS) were administered at 100 μl / 10g injection volumes. Nude mice were weighed every three days, and mouse luciferase kinetic curves were established using an IVIS imaging system. Tumor growth in nude mice was monitored using an in vivo imaging system on days 0, 3, 6, 12, 15, 18, 21, 27, 33, 39, and 45.

[0031] Results: In terms of weight change ( Figure 3 A) The average body weight of the control group gradually increased over time, and the mice were in good condition; the NS group and CUS group... 245C The average body weight of the mice initially showed an upward trend, then dropped sharply at the onset of the disease until death. From the median time to onset of the disease in each group (Table 12), it can be seen that mice in the Control group did not show any symptoms, while the median time to onset of the disease in the NS group was 14 days; CUS 245C In the group of mice, the median time to onset of illness was prolonged to day 17 after drug administration, with a life extension rate of 25%; among them, I-CUS 245C The 0.5 mg / kg group showed the most significant efficacy, with a median time to onset extended to 27.5 days, resulting in a calculated life extension rate of 112.5%. Meanwhile, according to... Figure 3 B, NS Group and CUS 245C The morbidity rate in mice in all groups was 100%, while in I-CUS... 245C There was one disease-free mouse in the group ( Figure 4 I-CUS can be further explored. 245C The possibility of curing lymphoma.

[0032] The above results demonstrate that I-CUS 245CIt has a significant anti-B lymphoma effect in mice.

[0033] Table 12 Comparison of disease onset time and death time of mice in each experimental group (n=7)

[0034] Example 2: CD19-targeting immunotoxin L-CUS 245C Preparation and assay of anti-hematologic malignancy 1. Immunotoxin L-CUS 245C Preparation and purification Method: Take 3.3 mL of CD19 monoclonal antibody Loncastuximab (5.2 mg / mL), CUS 245C 6.4 mg was cross-linked using 43 μL of 20 mmol / L SPDP (the specific method and steps are the same as in Example 1). Result: CUS 245C The coupling ratios with Loncastuximab are mainly concentrated in 1:1 and 1:2 ( Figure 5 A). The initial coupling product is L-CUS. 245C CUS 245C A mixed solution of Loncastuximab and [unreacted CUS] was prepared. Unreacted Loncastuximab was removed by nickel ion affinity chromatography, and unreacted CUS was removed by a combination of ultrafiltration and dialysis. 245C The final product contains no uncrosslinked Loncastuximab and CUS. 245C L-CUS, an immunotoxin 245C ( Figure 5 B), whose purity can approach 100%.

[0035] The above results demonstrate that CD19 monoclonal antibodies Loncastuximab and CUS were successfully prepared using chemical methods. 245C L-CUS conjugated immunotoxin 245C .

[0036] 2. Immunotoxin L-CUS 245C Inhibitory effect on the proliferation of hematologic malignancies (MTT assay) Methods: Burkitt's lymphoma cell lines Raji and Daudi, B-cell leukemia cell line Nalm-6, multiple myeloma cell line RPMI 8226, and T-cell leukemia cell line Molt-4 in logarithmic growth phase were seeded into 96-well cell culture plates (15,000, 38,000, 20,000, 13,000, and 18,000 cells per well for the 72-hour proliferation inhibition assay, and half the 72-hour cell number per well for the 120-hour proliferation inhibition assay), with a seeding volume of 100 μL per well. L-CUS was used as the experimental setup. 245C CUS 245C Lonca, Lonca + CUS 245C Four groups. Each drug was tested in triplicate, resulting in seven concentrations: L-CUS 245C The final concentrations after addition to the 96-well culture plate were 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 nmol / L; Lonca, Lonca + CUS 245C CUS 245C The study started with 1000 nmol / L and proceeded to six half-dilutions. The negative control group received 100 µL / well of RPMI 1640 medium, while the blank control wells (cell-free) received 100 µL of medium for background zeroing. After incubation under standard conditions for 72 h or 120 h, 20 µL / well of MTT solution was added, followed by 70 µL / well of triplet solution 4 h later. After incubation for 24–48 h, the absorbance (optical density, OD) of each well at 550 nm or 570 nm was measured using a microplate reader. The average values ​​were taken from each group, and the cell proliferation inhibition rate (IR) was calculated for each group, with the cell viability of the negative control group as 100%. Inhibition rate = (1 - OD value of drug group / OD value of control group) × 100%. Dose-response curves were plotted using GraphPad Prism 8 software, and statistical significance was analyzed. The IC50 was then calculated. 50 The experiment was repeated twice.

[0037] Results: Tables 13-20 and Figure 6 The results showed that the killing effect on hematologic malignancy cell lines with high CD19 expression (Raji, Daudi, Nalm-6) increased with increasing drug concentration, exhibiting a concentration-dependent effect; however, there was no significant effect on hematologic malignancy cells with low CD19 expression. Both high- and low-expression cells could be treated with CUS. 245C Lon + CUS 245CThe Lonca group exhibited no targeting and concentration-dependent cytotoxicity. Furthermore, the Lonca group showed no significant efficacy at a concentration of 1 nmol / L, indicating that immunotoxins can achieve stronger cytotoxic effects within the same concentration range. Lonca + CUS 245C Group proliferation inhibition curves and CUS 245C The groups almost completely overlap, exhibiting individual CUS. 245C The function of L-CUS. 245C The targeted killing effect of RPMI 8226 and Molt-4 on non-target cells was not observed; at a concentration of 1 nmol / L, they did not significantly inhibit cell proliferation and did not reach the IC50 threshold. 50 Value. At a concentration of 1 nmol / L, L-CUS 245C After 72 h of administration, the inhibition rates of Raji and Nalm-6 were 93.63% and 58.19%, respectively, and the IC50 at 72 h was [missing data]. 50 The concentrations were 18.09 and 233.70 pmol / L, respectively. With the treatment time extended to 120 h, the inhibition rates were 100% and 90%, respectively. The IC50 values ​​at 120 h were... 50 The concentrations were 4.64 and 45.04 pmol / L, respectively. The results indicate that at the same effective concentration, the immunotoxin L-CUS... 245C The inhibitory effect on the proliferation of CD19-overexpressing tumor cells was stronger at 120 h than at 72 h, showing a time-dependent effect of target selection.

[0038] The results indicate that L-CUS 245C The immunotoxin exhibits targeted inhibition of hematologic malignancy cell proliferation, selectively killing target cells highly expressing CD19. Table 21 shows that when acting on the same target cells, the immunotoxin can kill target cells at extremely low concentrations. The IC50 value can be obtained by calculating the targeting index TI. 50 L-CUS 245C <CUS 245C ≈ Lonca + CUS 245C With CUS 245C Compared to the group, L-CUS 245C The group has a significant killing effect, with a TI value of over 1000.

[0039] Table 13 Immunotoxin L-CUS 245C Inhibitory effect on the proliferation of Raji cells for 72 hours ±S, n=3)

[0040] P<0.001 VS CUS Table 14 Immunotoxin L-CUS245C Inhibitory effect on the proliferation of Nalm-6 cells for 72 hours ±S, n=3)

[0041] P<0.05 VS CUS P<0.001 VS CUS Table 15 Immunotoxin L-CUS 245C Inhibitory effect on the proliferation of Molt-4 cells for 72 hours ±S, n=3)

[0042] P<0.001 VS CUS Table 16 Immunotoxin L-CUS 245C Inhibitory effect on RPMI8226 proliferation over 72 hours ( ±S, n=3)

[0043] P<0.01 VS CUS P<0.001 VS CUS Table 17 Immunotoxin L-CUS 245C Inhibitory effect on the proliferation of Raji cells over 120 hours ( ±S, n=3)

[0044] P<0.05 VS CUS Table 18 Immunotoxin L-CUS 245C Inhibitory effect on the proliferation of Nalm-6 cells over 120 hours ( ±S, n=3)

[0045] P<0.05 VS CUS Table 19 Immunotoxin L-CUS 245C Inhibitory effect on the proliferation of Molt-4 cells over 120 hours ±S, n=3)

[0046] P<0.05 VS CUS Table 20 Immunotoxin L-CUS 245C Inhibitory effect on RPMI8226 cell proliferation over 120 hours ( ±S, n=3)

[0047] P<0.05 VS CUS P<0.05 VS CUS Table 21 L-CUS 245C IC50 at 72 h and 120 h for each cell line 50 (Mean ± SD, n=3)

[0048] Compared with CUS 245C , P<0.01, :P<0.0001 TI: target index = Example 3: Immunotoxin L-CUS 245C and I-CUS 245C Effects on the proliferation of peripheral blood lymphocytes (PBMCs) in normal individuals Methods: PBMC cells were seeded at a density of 45,000 cells / well at a rate of 100 µL / well in 96-well plates and incubated for 15 min to allow the cells to settle evenly at the bottom of the plate. The target drug was then added, and L-CUS was set. 245C CUS 245C and I-CUS 245C Three groups. Each drug was prepared in triplicate, resulting in six concentrations: L-CUS 245C and I-CUS 245C The final concentrations after addition to the 96-well culture plate were 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 nmol / L; CUS 245CThe study started with 1000 nmol / L and proceeded to six half-dilutions. The negative control group received 100 µL of RPMI 1640 medium per well, while the blank control wells (cell-free) received 100 µL of medium for background zeroing. After 72 h of incubation under standard conditions, 20 µL of MTT solution was added per well, followed by 70 µL of triplet solution per well after 4 h. The wells were incubated for 24–48 h before measurement using a microplate reader. The absorbance (OD) values ​​at 550 nm or 570 nm were measured. The average value was calculated after background subtraction, and the cell viability of the negative control group was taken as 100%. The cell proliferation inhibition rate (IR) was calculated using the formula: Inhibition rate = (1 - OD value of drug group / OD value of control group) × 100%. The IC50 was analyzed and calculated using GraphPad Prism 8 software. 50 .

[0049] Result: As Figure 7 As shown in Table 22, L-CUS 245C and I-CUS 245C Concentrations below 1000 nmol / L did not show significant killing effect on PBMC cells, indicating that L-CUS... 245C and I-CUS 245C While killing tumor cells, it does not damage healthy cells, demonstrating the effectiveness of the immunotoxin L-CUS. 245C and I-CUS 245C Superior targeted killing ability.

[0050] Table 22 IC50 of immunotoxins on PBMC cells after 72 h of action 50 Comparison (Mean ± SD, n=3)

[0051] Example 4: CD33-targeting immunotoxin G-CUS 245C Preparation and anti-acute myeloid leukemia activity 1. Immunotoxin L-CUS 245C Preparation and purification Method: Take 10 mg of CD33 monoclonal antibody Gemtuzumab, CUS 245C 3.75 mg was cross-linked with 25 μL of 20 mmol / L SPDP (the specific method and steps are the same as in Example 1). result: Figure 8 The display shows that CUS 245C The coupling ratios with Gemtuzumab were primarily 1:1 and 1:2. The initial coupling product was G-CUS. 245C CUS 245CA mixed solution of Gemtuzumab and GC was prepared. Unreacted Gemtuzumab was removed by nickel ion affinity chromatography, and unreacted CUS was removed by a combination of ultrafiltration and dialysis. 245C The final product contains no uncrosslinked Gemtuzumab and CUS. 245C G-CUS, an immunotoxin 245C Its purity can reach over 95%.

[0052] The above results indicate that CD33 monoclonal antibodies Gemtuzumab and CUS were successfully prepared using chemical methods. 245C G-CUS conjugated immunotoxin 245C .

[0053] 2. Immunotoxin G-CUS 245C Inhibitory effect on the proliferation of hematologic malignancies (MTT assay) Methods: HL-60 and THP-1 acute myeloid leukemia cell lines in logarithmic growth phase were seeded into 96-well cell culture plates at a density of 20,000 cells / 100 µL / well. G-CUS was used as the experimental setup. 245C CUS 245C Gemtuzumab(G), G + CUS 245C Four groups. Each drug was prepared in triplicate, resulting in six concentrations: G-CUS 245C The final concentrations after addition to the 96-well culture plate were 100, 10, 1, 0.1, 0.01, and 0.001 nmol / L; G, G+, and CUS 245C CUS 245C Five half-dilutions were performed, starting with 500 nmol / L. The negative control group received 100 µL / well of RPMI 1640 medium, while the blank control wells (cell-free) received medium for background zeroing. After 120 h of incubation under standard conditions, 20 μL / well of MTT solution was added, followed by 70 μL / well of triple solution 4 h later. After incubation for 24–48 h, the absorbance (optical density, OD) of each well at 550 nm was measured using a microplate reader. The average values ​​were taken from each group, and the cell proliferation inhibition rate (IR) was calculated for each group, with the negative control group cell viability set at 100%. Inhibition rate = (1 - OD value of drug group / OD value of control group) × 100%. Dose-response curves were plotted using GraphPad Prism 8 software, and statistical significance was analyzed. The IC50 was then calculated. 50 The experiment was repeated twice.

[0054] Results: Tables 23-25 ​​show the individual CUS 245CIt only showed inhibitory effects on HL-60 and THP-1 cell lines at high concentrations (>32 nmol / L), and this effect was concentration-dependent. 500 nmol / L CUS 245C The inhibition rates of HL-60 and THP-1 cells after 120 hours of treatment were 79.93% and 75.42%, respectively, with IC50 values ​​of [missing information]. 50 The concentrations were 149.10 nmol / L and 156.44 nmol / L, respectively; in contrast, G-CUS... 245C It can inhibit the proliferation of these two cell lines at extremely low concentrations. 1 nmol / L of G-CUS 245C After 120 hours of treatment, the inhibition rates against HL-60 and THP-1 cells were 91.15% and 68.17%, respectively, with an IC50 concentration of [missing information]. 50 The concentrations were 0.083 nmol / L and 0.033 nmol / L, respectively, with a pharmacodynamic ratio of CUS. 245C The targeted lethality was 1795 and 4740 times stronger, respectively (targeted lethality indices of 1796 and 4741). Meanwhile, the same concentrations of Gemtuzumab and G+ CUS alone... 245C It has no obvious effect.

[0055] The above results indicate that the immunotoxin G-CUS 245C It has a significant targeted killing effect on CD33-overexpressing acute myeloid leukemia HL60 and THP-1 cells.

[0056] Table 23 Inhibitory effect of immunotoxin G-CUS on the proliferation of HL-60 cells ( ±S, n=3)

[0057] P<0.05 VS CUS P<0.01 VS CUS Table 24 Inhibitory effect of immunotoxin G-CUS on the proliferation of THP-1 cells ( ±S, n=3)

[0058] P<0.05 VS CUS P<0.01 VS CUS Table 25 Targeted killing effect of immunotoxin G-CUS on myeloid leukemia cells

[0059] TI stands for Targeted Kill Index, and CUS stands for IC. 50 IC with G-CUS- 50 ratio Compared to CUS, p <0.001 Example 5: BCMA-targeting immunotoxin B-CUS 245C Preparation and anti-multiple myeloma activity 1. Immunotoxin B-CUS 245C Preparation and purification Method: Take 10 mg of CD33 monoclonal antibody Belantab, CUS 245C 3.75 mg was cross-linked with 25 μL of 20 mmol / L SPDP (the specific method and steps are the same as in Example 1). result: Figure 9 The display shows that CUS 245C The coupling ratios with Belantamab were primarily 1:1 and 1:2. The initial coupling product was B-CUS. 245C CUS 245C A mixed solution of Belantamab and β-CUS was prepared. Unreacted Belantamab was removed by nickel ion affinity chromatography, and unreacted CUS was removed by a combination of ultrafiltration and dialysis. 245C The final product contains no uncrosslinked Belantab and CUS. 245C G-CUS, an immunotoxin 245C Its purity can reach over 950%.

[0060] The above results demonstrate that the BCMA monoclonal antibodies Belantab and CUS were successfully prepared using chemical methods. 245C B-CUS conjugated immunotoxin 245C .

[0061] 2. Immunotoxin B-CUS 245C Inhibitory effect on the proliferation of multiple myeloma cells (MTT assay) Methods: Multiple myeloma cell line NCI-H929 in logarithmic growth phase was seeded into 96-well cell culture plates at a density of 20,000 cells / 100 µL / well. B-CUS was used as the experimental setup. 245C CUS 245C Belantab (B), B + CUS 245C Four groups. Each drug was tested in triplicate, resulting in six concentrations: B-CUS 245CThe final concentrations after addition to the 96-well culture plate were 50, 10, 1, 0.1, 0.01, 0.001, and 0.0001 nmol / L; B, B+, CUS 245C CUS 245C The study started with 1000 nmol / L and proceeded to six half-dilutions. The negative control group received 100 µL / well of RPMI 1640 medium, while the blank control wells (cell-free) received medium for background zeroing. After 120 h of incubation under standard conditions, 20 µL / well of MTT solution was added, followed by 70 µL / well of a triplet solution 4 h later. After incubation for 24–48 h, the absorbance (optical density, OD) of each well at 550 nm was measured using a microplate reader. The average values ​​were taken from each group, and the cell proliferation inhibition rate (IR) was calculated for each group, with the negative control group cell viability set at 100%. Inhibition rate = (1 - OD value of drug group / OD value of control group) × 100%. Dose-response curves were plotted using GraphPad Prism 8 software, and statistical significance was analyzed. The IC50 was then calculated. 50 The experiment was repeated twice.

[0062] Results: Tables 26 and 27 show the individual CUS 245C It only showed inhibitory effects on the NCI-H929 cell line at high concentrations (>16 nmol / L), and this effect was concentration-dependent. 250 nmol / L CUS 245C The inhibition rate of cells after 120 hours of action was 87.63%, IC50 50 It was 78.97 nmol / L; in comparison, B-CUS 245C It can inhibit cell proliferation at extremely low concentrations. 1 nmol / L of B-CUS 245C After 120 hours of treatment, the inhibition rate against HL-60 cells was 99.83%, and the IC50 concentration was [missing value]. 50 The concentration was 0.043 nmol / L, and its efficacy was higher than that of CUS. 245C It is 1815 times stronger (targeted lethality index is 1816). Meanwhile, Belantamab and B+CUS at the same concentration... 245C It has no obvious effect.

[0063] The above results indicate that the immunotoxin G-CUS 245C It has a significant targeted killing effect on multiple myeloma cells that overexpress BCMA.

[0064] Table 26 Inhibitory effect of immunotoxin B-CUS on the proliferation of NCI-H929 cells ( ±S, n=3)

[0065] P<0.05 VS CUS P<0.01 VS CUS Table 27 Targeted killing effect of immunotoxin B-CUS on NCI-H929 cells

[0066] TI stands for Targeted Kill Index, and CUS stands for IC. 50 IC with B-CUS 50 ratio Compared to CUS, p <0.001 Example 6: DA-CUS, an immunotoxin targeting CD38 245C Preparation and anti-multiple myeloma activity 1. Immunotoxin CD-CUS 245C Preparation and purification Method: Take 0.5 mL of CD38 monoclonal antibody Daratumumab (DA, 20 mg / mL), CUS 245C 7.45 mg was cross-linked using 16.45 μL of 20 mmol / L SPDP (the specific method and steps are the same as in Example 1). result: Figure 10 The display shows that CUS 245C The conjugation ratios with Daratumumab are mainly concentrated in 1:2 and 1:3 ( Figure 10 a) The initial coupling product is DA-CUS 245C CUS 245C A mixed solution of Daratumumab and [other ingredients]. Unreacted Daratumumab was removed by nickel ion affinity chromatography. Figure 10 c and Figure 10 d) Unreacted CUS is removed by a combination of ultrafiltration and dialysis. 245C ( Figure 10 b and Figure 10 e). The final product contains no uncrosslinked Daratumumab and CUS. 245C DA-CUS, an immunotoxin 245C ( Figure 10 e), its purity can approach 100%.

[0067] The above results demonstrate that CD38 monoclonal antibodies Daratumumab and CUS were successfully prepared using chemical methods.245C DA-CUS conjugated immunotoxin 245C .

[0068] 2. Inhibitory effect of immunotoxin DA-CUS245C on the proliferation of multiple cells (MTT assay) Methods: Human multiple myeloma cell lines NCI-H929, RPMI-8226, and U266, and chronic myeloid leukemia cell line K562, all in logarithmic growth phase, were seeded into 96-well cell culture plates at cell counts of 25,000, 13,000, 8,000, and 3,000 cells / well, respectively, with a seeding volume of 100 μL per well. Four groups were established: DA-CUS245C, CUS245C, DA, and DA + CUS245C. Each drug was prepared in triplicate, with seven concentrations: DA-CUS245C was added to 96-well plates at final concentrations of 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 nmol / L; DA, DA + CUS245C, and CUS245C groups were prepared at 1000 nmol / L, with six half-dilutions. The negative control group received 100 µL of RPMI 1640 medium per well, and the blank control wells (cell-free) received 100 µL of medium for background zeroing. After incubation under standard conditions for 72 h or 120 h, 20 μL of MTT solution was added per well, followed by 70 μL of triplet solution per well after 4 h. After incubation for 24–48 h, the absorbance (optical density, OD) of each well at 550 nm or 570 nm was measured using a microplate reader. The average value of each group was taken, and the cell viability of the negative control group was taken as 100%. The cell proliferation inhibition rate (IR) of each group was calculated according to the formula: Inhibition rate = (1 - OD value of drug group / OD value of control group) × 100%. The dose-response curve was plotted using GraphPad Prism 8 software, and its statistical significance was analyzed. The IC50 was calculated. The experiment was repeated twice.

[0069] Result: From Figure 11 It can be seen that as the concentration increases, DA-CUS 245C The efficacy of the drug against MM cells (NCI-H929, RPMI-8226, U266) was concentration-dependent, while it had no significant effect on non-MM cells (K562). CUS 245C Daratumumab + CUS 245C The efficacy of the CUS group was concentration-dependent on both MM and non-MM cells, while the DA group showed no significant effect. 245C IC50 in the group 72 h after administration 50The concentrations were 40.2, 349.8, 322.5, and 290 nmol / L, respectively. The IC50 concentration at 120 h after drug administration was... 50 The concentrations were 29.9, 239.6, 108.2, and 166.8 nmol / L, respectively, indicating that CUS... 245C The inhibition of cancer cell proliferation exhibited a concentration-dependent, non-target-selective effect, and the efficacy at 120 h was stronger than at 72 h. Daratumumab + CUS 245C Group proliferation inhibition curves and CUS 245C The groups almost completely overlap, exhibiting individual CUS. 245C The role of DA-CUS. In contrast, DA-CUS 245C The inhibitory effect on target cell proliferation was significantly stronger than that of the control group. At a concentration of 1 nmol / L, DA-CUS 245C After 72 h of drug administration, the inhibition rates against H929, RPMI 8226, and U266 were 98%, 90%, and 78%, respectively, and the IC50 at 72 h was [missing value]. 50 The concentrations were 0.91, 7.05, and 5.5 pmol / L, respectively. With an extended treatment time of 120 h, the inhibition rates were 98%, 90%, and 90%, respectively. The IC50 values ​​at 120 h were... 50 The concentrations were 0.46, 6.92, and 1.52 pmol / L, respectively, but there was no inhibitory effect on the proliferation of non-target cells K562, and the IC50 was not reached at a concentration of 1 nmol / L. 50 Values ​​and results for DA-CUS 245C The inhibition of MM cell proliferation is targeted, selectively killing target cells expressing CD38. Table 26 shows that, when acting on the same target cell, compared to CUS... 245C Group, DA-CUS 245C It can achieve IC50 at extremely low concentrations. 50 Its targeting index can reach over 10,000 (P<0.0001).

[0070] Results indicate that DA-CUS 245C The immunotoxin exhibits targeted inhibition of multiple myeloma cell proliferation, selectively killing target cells that highly express CD38. Table 28 shows that when acting on the same target cells, the immunotoxin can kill target cells at extremely low concentrations. The IC50 value can be obtained by calculating the targeting index TI. 50 L-CUS 245C <CUS 245C ≈ Lonca + CUS 245C With CUS 245C Compared to the group, L-CUS 245C The group has a significant killing effect, with a TI value of over 1000.

[0071] Table 28 DA-CUS 245C IC50 values ​​of various cell lines after 72 h or 120 h of treatment 50 (Mean±SD, n=3)

[0072] Compared with CUS 245C , P<0.0001 TI: target index = Although the present invention has been illustrated and described through specific embodiments and alternative methods, it should be understood that various changes and modifications may be made without departing from the spirit and scope of the invention. Therefore, it should be understood that the present invention is not limited in any sense except by the appended claims and their equivalents.

Claims

1. An immunotoxin targeting hematological tumors, comprising a toxic molecule and a carrier, said toxic molecule being a Cucurbitin or a mutant of Cucurbitin; characterized in that: The carrier contains antibodies, ligands, or peptides capable of binding to hematologic tumor cells; wherein the antibody comprises antibody fragments or small molecule antibodies.

2. The immunotoxin of claim 1, characterized in that: The mutant of the pumpkin protein is a mutant obtained by point mutation, deletion mutation or insertion mutation of pumpkin protein.

3. The immunotoxin of claim 1, characterized in that: The vector is a monoclonal antibody targeting CD19, CD22, CD33, CD38, and BCMA.

4. The method of any one of claims 1 to 3, wherein the immunotoxin targeting hematologic tumors is prepared by: Pumpkin protein or its mutants can be linked to a vector through chemical cross-linking or genetic engineering to create chemically conjugated immunotoxins or recombinant immunotoxins. ​ 5. The use of an immunotoxin targeting haematological tumours according to any one of claims 1 to 3, characterized in that: Used to treat hematologic malignancies.

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