CD33-blocking antibody, CD33-blocking humanized antibody and use thereof

Abstract

Provided are a CD33-blocking antibody, a CD33-blocking humanized antibody and the use thereof. The CD33-blocking antibody is HZAB_1 or HZAB_2. The HZAB_2 comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 3 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 4. The HZAB_1 and HZAB_2 antibodies can enhance the phagocytic efficiency of macrophages against colorectal neuroendocrine carcinoma cells and small cell lung cancer cells, thereby delaying the progression of colorectal neuroendocrine carcinoma and small cell lung cancer. Also provided is a CD33-blocking humanized antibody obtained by performing humanization on the HZAB_2 antibody. The CD33-blocking humanized antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 30 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 31. The humanized CD33-blocking antibody can inhibit liver metastasis of neuroendocrine prostate cancer and liver metastasis of small cell lung cancer.

Description

A CD33 blocking antibody and a blocking humanized antibody and their uses Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a CD33 blocking antibody and a humanized blocking antibody, and their uses. Background Technology

[0002] As early as the beginning of the 20th century, Paul Ehrlich proposed the concept of "magic bullets," which can be seen as an early idea for ADCs (antibody-drug conjugates). However, it was not until the 1950s, when Mathe first conjugated anti-mouse leukocyte immunoglobulin with methotrexate for the treatment of leukemia, that the research on ADCs truly began.

[0003] CD33 is a membrane glycoprotein expressed in hematopoietic cells. It is a 67 kDa glycosylated transmembrane protein belonging to the Siglec family. Activated upon cross-linking or ligand binding, it mediates inhibitory signaling and regulates intracellular calcium mobilization, cell adhesion, leukemia cell apoptosis, myeloid cell maturation, and cytokine production, and is associated with the occurrence and development of certain hematologic malignancies.

[0004] In 2000, Gemtuzumab ozogamicin (GO), a CD33 antibody-drug conjugate, was developed by combining a humanized anti-CD33 monoclonal antibody with the DNA intercalation agent calicheamicin (CLM). It became the first FDA-approved ADC for the treatment of CD33-positive acute myeloid leukemia (AML). However, due to the lack of significant improvement in treatment efficacy compared to traditional chemotherapy and serious safety concerns shown in post-marketing Phase III studies, GO was withdrawn from the market in 2010.

[0005] Anti-CD33 monoclonal antibodies (such as Gemtuzumab Ozogamicin) were first used to treat acute myeloid leukemia (AML). Their antibody-drug conjugates (ADCs) combine CD33 antibodies with cytotoxins to achieve more precise therapeutic effects. CD33 antibody clone 10C8 can be used to treat hepatitis B.

[0006] The treatment of small cell lung cancer (SCLC) and colorectal neuroendocrine carcinoma (LCNEC) faces multiple challenges. SCLC is known for its high malignancy, rapid progression, and early metastasis, resulting in limited treatment options and generally poor prognosis. Although relatively sensitive to initial treatment, SCLC patients often develop resistance rapidly, limiting the effectiveness of second-line and subsequent therapies. Furthermore, the treatment of SCLC faces challenges related to drug safety, particularly the potential side effects of immunotherapy, such as pneumonia and colitis.

[0007] For colorectal neuroendocrine carcinoma, due to its rarity, treatment strategies lack support from large-scale clinical trials. Treatment regimens are usually referenced for SCLC and non-small cell lung cancer (NSCLC), but the results are not ideal. In addition, the heterogeneity of LCNEC complicates the prediction of treatment response, and treatment options for advanced or metastatic LCNEC are very limited, usually relying on chemotherapy.

[0008] Therefore, exploring a highly specific monoclonal antibody that can be used to prepare safe, effective, and highly specific drugs for the treatment of small cell lung cancer and colorectal neuroendocrine carcinoma has become an urgent problem to be solved.

[0009] CD33 is a transmembrane glycoprotein expressed on the surface of myeloid hematopoietic cells, belonging to the sialic acid-binding immunoglobulin-like lectin (Siglec) family. Its molecular weight is approximately 67 kDa, and it exhibits typical glycosylation modifications. CD33 is activated upon cross-linking or binding to specific ligands, primarily recruiting and activating phosphatases such as SHP-1 and SHP-2 via its cytoplasmic immunoreceptor tyrosine inhibitory motif (ITIM), thereby transmitting inhibitory signals. This signaling pathway participates in regulating various cellular functions, including intracellular calcium ion mobilization, cell adhesion, leukemia cell apoptosis, myeloid cell differentiation and maturation, and cytokine production. Abnormal CD33 expression is closely related to the occurrence and development of hematologic malignancies such as acute myeloid leukemia (AML), making it an important therapeutic target.

[0010] Liver metastasis is quite common in neuroendocrine tumors (NENs). Statistics show that 40% to 90% of patients diagnosed with advanced (metastatic) NENs have liver metastases. For NENs originating in certain primary sites (such as the pancreas or small intestine), the liver is often the first and primary site of metastasis. This high metastasis rate makes the liver a key organ in the progression and prognosis of NENs. Liver metastasis not only directly impairs liver function, leading to the risk of liver failure, but also poses a serious threat to patient survival and quality of life by increasing tumor burden, inducing severe endocrine syndromes, and significantly increasing the complexity and difficulty of treatment. Therefore, for patients diagnosed with NENs, close monitoring of liver status and early detection and active intervention of liver metastases are crucial for improving prognosis.

[0011] The current challenges in treating liver metastases from neuroendocrine tumors are as follows:

[0012] (1) High metastatic burden and surgical limitations. More than 50% of advanced neuroendocrine tumors (NENs) metastasize to the liver, of which 40% are multifocal and diffuse, and only 10%-20% of patients are suitable for radical resection; radiofrequency / embolization therapy has a control rate of less than 30% for lesions >3cm.

[0013] (2) Systemic therapy resistance. Targeted therapy failure: The objective response rate (ORR) of mTOR inhibitors (everolimus) and anti-angiogenic drugs (sunitinib) is only 9%-12%, with a median progression-free survival (mPFS) of <12 months. Chemotherapy bottleneck: The streptozotocin + 5-FU regimen has an ORR of <15% for G3 grade neuroendocrine carcinoma (NEC), and the incidence of grade 3-4 hematologic toxicity is >40%.

[0014] (3) The dilemma of "cold tumor" immunotherapy: The lack of T cell infiltration leads to poor efficacy of PD-1 inhibitors. Tumor-associated macrophages (TAMs) account for >60% and mediate immunosuppression. Summary of the Invention

[0015] This invention provides a CD33-blocking antibody to block the immunosuppressive effect of CD33. It also provides a CD33-blocking humanized antibody that can inhibit liver metastasis of neuroendocrine carcinomas, including neuroendocrine prostate cancer and small cell lung cancer, and can be used to prepare a safe, effective, and highly specific drug for treating liver metastasis of neuroendocrine carcinomas.

[0016] The objective of this invention is achieved through the following technical solution:

[0017] In a first aspect, the present invention provides a CD33 blocking antibody, wherein the CD33 blocking antibody is HZAB_1 or HZAB_2;

[0018] The HZAB_1 includes a heavy chain and a light chain; the heavy chain includes a heavy chain variable region, and the three complementary determinant regions of the heavy chain variable region are as follows:

[0019] CDR1: GFSLISYH (SEQ ID NO.5);

[0020] CDR2: IYTNGSA (SEQ ID NO.6);

[0021] CDR3: VRGIDL (SEQ ID NO.7);

[0022] And / or, the light chain includes a light chain variable region, the three complementary determinants of which are:

[0023] CDR1: QSVYNNHD(SEQ ID NO.8);

[0024] CDR2: YAS;

[0025] CDR3: LGVYDDDADTA (SEQ ID NO.9);

[0026] The HZAB_2 includes a heavy chain and a light chain. The heavy chain includes a variable region, and the three complementary determinant regions of the variable region are:

[0027] CDR1:GIDLSTNS(SEQ ID NO.10);

[0028] CDR2: IGGSGST (SEQ ID NO.11);

[0029] CDR3: ARLWDF (SEQ ID NO.12);

[0030] And / or, the light chain includes a light chain variable region, the three complementary determinants of which are:

[0031] CDR1: QSVYGNNE (SEQ ID NO.13);

[0032] CDR2: KAS;

[0033] CDR3: SYITDDF (SEQ ID NO. 14).

[0034] As some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of HZAB_1 is shown in SEQ ID NO.1;

[0035] And / or, the amino acid sequence of the light chain variable region of HZAB_1 is shown in SEQ ID NO.2.

[0036] As some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of HZAB_2 is shown in SEQ ID NO.3;

[0037] And / or, the amino acid sequence of the light chain variable region of HZAB_2 is shown in SEQ ID NO.4.

[0038] As some specific embodiments of the present invention, the CD33 blocking antibody is obtained by immunization with recombinant CD33 protein, the amino acid sequence of which is shown in SEQ ID NO.15.

[0039] As some specific embodiments of the present invention, the recombinant CD33 protein comprises the CD33 protein as shown in SEQ ID NO.17, and a flag tag with an amino acid sequence as shown in SEQ ID NO.16.

[0040] Secondly, the present invention provides the application of a CD33 blocking antibody in the preparation of a drug for treating colorectal cancer.

[0041] This invention also provides the application of a CD33 blocking antibody in the preparation of a drug for treating small cell lung cancer.

[0042] Thirdly, the present invention provides a CD33 blocking humanized antibody, wherein the CD33 blocking humanized antibody comprises a heavy chain and a light chain;

[0043] The heavy chain includes a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.30;

[0044] The light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.31.

[0045] As some specific embodiments of the present invention, the heavy chain further includes a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO.34;

[0046] And / or, the light chain further includes a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.35.

[0047] Fourthly, the present invention provides a method for preparing a CD33 blocking humanized antibody as described in any of the preceding claims, comprising the following steps:

[0048] S1. Using CD33 rabbit monoclonal antibody as the initial antibody, define its CDR region, and select the human FR template with the highest homology to the CD33 rabbit monoclonal antibody from the database.

[0049] S2, CDR transplantation: The CDR region of the CD33 rabbit monoclonal antibody is transplanted onto the backbone of the selected human FR template to form the variable regions of the initial humanized antibody heavy and light chains;

[0050] S3, Reversal Mutation: Some amino acids in the FR backbone of the preliminary humanized antibody heavy chain and light chain variable regions are mutated to obtain the final determined heavy chain variable region and light chain variable region of the CD33 humanized antibody.

[0051] S4. Sequence optimization: Codon optimization is performed on the heavy chain variable region and light chain variable region of the CD33 humanized antibody obtained in step S3 to synthesize the optimized gene sequence.

[0052] S5. Expression vector construction: Gene fragments of the codon-optimized humanized antibody heavy chain and light chain variable regions were cloned into expression vectors containing human heavy chain constant regions and light chain constant regions, respectively, to construct heavy chain and light chain expression plasmids.

[0053] S6. Transfect the heavy chain and light chain expression plasmids into HEK293F cells for transient antibody expression, and then purify them to obtain the antibody.

[0054] As some specific embodiments of the present invention, in step S1, the amino acid sequence of the heavy chain variable region of the CD33 rabbit monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0055] As some specific embodiments of the present invention, in step S2, the amino acid sequence of the preliminary humanized antibody heavy chain variable region is shown in SEQ ID NO.20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.25.

[0056] As some specific embodiments of the present invention, in step S3, the amino acid sequence of the heavy chain variable region of the finally determined CD33 humanized antibody is shown in SEQ ID NO.30; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.31.

[0057] As some specific embodiments of the present invention, in step S4, the gene sequence of the heavy chain variable region of the codon-optimized CD33 humanized antibody is shown in SEQ ID NO.32, and the gene sequence of the light chain variable region is shown in SEQ ID NO.33.

[0058] As some specific embodiments of the present invention, in step S5, the heavy chain constant region is selected from the human IgG1 heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO.34; the light chain constant region is selected from the human κ light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.35.

[0059] Fifthly, the present invention provides the use of the CD33-blocking humanized antibody as described in any of the preceding claims in the preparation of a medicament for inhibiting liver metastasis of neuroendocrine prostate cancer. The CD33-blocking humanized antibody of the present invention can inhibit liver metastasis of neuroendocrine tumors.

[0060] Sixthly, the present invention provides the use of the CD33-blocking humanized antibody as described in any of the preceding claims in the preparation of a medicament for inhibiting liver metastasis of small cell lung cancer. The CD33-blocking humanized antibody of the present invention can inhibit liver metastasis of small cell lung cancer tumors.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] (1) This invention provides a high-affinity and high-specificity blocking antibody against CD33, which has a specific blocking effect on CD33 protein;

[0063] (2) The CD33 blocking antibody provided by the present invention can improve the phagocytic efficiency of macrophages on colorectal neuroendocrine cancer cells and small cell lung cancer cells, slow down the progression of colorectal neuroendocrine cancer and small cell lung cancer, and inhibit the growth of colorectal neuroendocrine cancer cells and small cell lung cancer cells in vivo.

[0064] (3) The present invention uses CD33 rabbit-derived antibody for humanization modification to obtain a humanized blocking antibody with high affinity and high specificity against CD33;

[0065] (4) The humanized CD33 blocking antibody of the present invention has a good therapeutic and inhibitory effect on liver metastasis of neuroendocrine prostate cancer and small cell lung cancer. Attached Figure Description

[0066] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0067] Figure 1 shows the Western blot protein electrophoresis diagrams of the two antibodies (HZAB_1 and HZAB_2) obtained by immunizing rabbits with CD33 antigen in Example 1; the left image is the protein electrophoresis diagram of HZAB_1 (Lane M is the marker, Lane 1 is BSA, Lane 2 is the HZAB_1 reducing antibody, and Lane 3 is the HZAB_1 non-reducing antibody); the right image is the protein electrophoresis diagram of HZAB_2 (Lane M is the marker, Lane 1 is BSA, Lane 2 is the HZAB_2 reducing antibody, and Lane 3 is the HZAB_2 non-reducing antibody).

[0068] Figure 2 shows the validation results of CD33 antibody against colorectal neuroendocrine carcinoma and small cell lung cancer in Examples 2 and 3. In Figure 2, a and ad represent the effects of CD33 antibody on the phagocytic effect of NCI-H82 (a,b) and COLO 320DM (c,d) tumor cells, respectively. In Figure 2, e and j represent the effects of CD33 antibody on the growth of NCI-H82 (e,f,g) and COLO 320DM (h,i,j) tumor cells in mice.

[0069] Figure 3 shows the plasmid map of the target expression vector pcDNA3.4-huVH containing the human heavy chain constant region CH1-CH3 in Example 4;

[0070] Figure 4 shows the plasmid map of the target expression vector pcDNA3.4-huVL containing the human light chain constant region CL in Example 4.

[0071] Figure 5 shows the plasmid map of the heavy chain expression plasmid pHC-huCD33 of the humanized CD33 antibody constructed in Example 4;

[0072] Figure 6 shows the plasmid map of the light chain expression plasmid pLC-huCD33 of the humanized CD33 antibody constructed in Example 4;

[0073] Figure 7 shows the Western blot results of the CD33 humanized antibody in Example 4; where Lane M1 is the SDS-PAGE Marker, Lane 1 is BSA, and Lanes 2-3 are the CD33 humanized antibody (reduced / non-reduced);

[0074] Figure 8 shows the inhibition results of humanized CD33 antibody and IgG antibody on liver metastasis of neuroendocrine prostate cancer in Example 5. In this figure, a is a diagram of the morphological changes of prostate cancer liver metastasis; b and c are HE staining scans of liver sections of prostate cancer liver metastasis in the IgG antibody and CD33 humanized antibody groups, respectively; d is a statistical diagram of the number of metastatic lesions of prostate cancer liver metastasis.

[0075] Figure 9 shows the inhibition results of rabbit-derived CD33 antibody and IgG antibody on liver metastasis of neuroendocrine prostate cancer in Example 5. In this figure, a is a graph showing the morphological changes of prostate cancer liver metastasis; b is a graph showing the number of metastatic lesions of prostate cancer liver metastasis.

[0076] Figure 10 shows the inhibition results of humanized CD33 antibody and IgG antibody on liver metastasis of small cell lung cancer in Example 6. Among them, a is a diagram of tumor morphological changes in small cell lung cancer; b is a statistical diagram of the number of metastatic lesions in liver metastasis of small cell lung cancer; c is an HE staining scan of liver sections of small cell lung cancer liver metastasis in the IgG antibody group; d is an HE staining scan of liver sections of small cell lung cancer liver metastasis in the humanized CD33 antibody group.

[0077] Figure 11 shows the inhibition results of rabbit-derived CD33 antibody and IgG antibody on liver metastasis of small cell lung cancer in Example 6. In this figure, a is a graph of tumor morphological changes in liver metastasis of small cell lung cancer; b is a statistical graph of the number of metastatic lesions in liver metastasis of small cell lung cancer. Detailed Implementation

[0078] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0079] Example 1

[0080] 1. Dissolve the recombinant CD33 protein in sterile 0.85% NaCl solution or PBS (pH 7.2), with a volume of 250 μl per rabbit. The total amount of antigen varies depending on the properties of the antigen, with 100 μg of protein antigen per rabbit being appropriate.

[0081] 2. Inject a mixture of a pre-set dose of immunogen (100 μg of recombinant CD33 protein per rabbit) and Freund's complete adjuvant (FCA) subcutaneously at multiple points on the back of the rabbits, 0.1 ml at each point, for a total of 4-6 points.

[0082] The immunogen used was recombinant CD33 protein, which includes CD33 protein and a flag tag. The amino acid sequence of CD33 protein is shown in SEQ ID NO.17, the amino acid sequence of flag tag is shown in SEQ ID NO.16, and the amino acid sequence of recombinant CD33 protein is shown in SEQ ID NO.15.

[0083] 3.3 weeks later, a second immunization was performed, using Freund's incomplete adjuvant (FIA) mixed with CD33 protein for booster immunization, in the same manner as the initial immunization.

[0084] 4. Blood should be drawn starting 2-3 weeks after the initial immunization, and then every 3 weeks thereafter, using the ear vein blood collection method, collecting 2-5 ml of blood each time.

[0085] 5. After blood collection, place the blood sample in a centrifuge and centrifuge at 10,000 rpm for 10 minutes. Collect the supernatant, which is the serum containing antibodies.

[0086] 6. Use ELISA to detect the titer and specificity of antibodies in serum. Add 100 μL of detection antibody solution to each well and incubate at room temperature for 2 hours (500 rpm).

[0087] 7. Purification of antibodies from serum by protein A / G affinity chromatography. Protein A affinity chromatography columns have become widely used affinity columns for purifying antibodies, and can separate and purify antibodies of various mammalian subtypes or genetically engineered recombinant proteins containing antibody Fc fragments from ascites fluid, serum, and cell culture supernatants or cell extracts.

[0088] Two antibodies were obtained, designated HZAB_1 and HZAB_2, respectively. Figure 1 shows the results of protein electrophoresis of these two antibodies. The left image is the protein electrophoresis diagram of HZAB_1, where Lane M is the SDS-PAGE marker, Lane 1 is BSA (9.00 μg), Lane 2 is the HZAB_1 reducing antibody, and Lane 3 is the HZAB_1 non-reducing antibody; the right image is the protein electrophoresis diagram of HZAB_2, where Lane M is the SDS-PAGE marker, Lane 1 is BSA (9.00 μg), Lane 2 is the HZAB_2 reducing antibody, and Lane 3 is the HZAB_2 non-reducing antibody.

[0089] HZAB_1 contains a light chain variable region and a heavy chain variable region. The heavy chain variable region includes three complementarity-determining regions, CDR1, CDR2, and CDR3, whose respective amino acid sequences are shown below:

[0090] CDR1: GFSLISYH (SEQ ID NO.5);

[0091] CDR2: IYTNGSA (SEQ ID NO.6);

[0092] CDR3: VRGIDL (SEQ ID NO.7).

[0093] The amino acid sequence of the heavy chain variable region (SEQ ID NO.1) is as follows:

[0094] The light chain variable region includes three complementarity-determining regions, CDR1, CDR2, and CDR3, whose amino acid sequences are shown below:

[0095] CDR1: QSVYNNHD(SEQ ID NO.8);

[0096] CDR2: YAS;

[0097] CDR3: LGVYDDDADTA (SEQ ID NO. 9).

[0098] The amino acid sequence of the light chain variable region (SEQ ID NO.2) is as follows:

[0099] HZAB_2 contains a light chain variable region and a heavy chain variable region. The heavy chain variable region includes three complementarity-determining regions, CDR1, CDR2, and CDR3, whose respective amino acid sequences are shown below:

[0100] CDR1:GIDLSTNS(SEQ ID NO.10);

[0101] CDR2: IGGSGST (SEQ ID NO.11);

[0102] CDR3: ARLWDF (SEQ ID NO. 12).

[0103] The amino acid sequence of the heavy chain variable region (SEQ ID NO.3) is as follows:

[0104] The light chain variable region includes three complementarity-determining regions, CDR1, CDR2, and CDR3, whose amino acid sequences are shown below:

[0105] CDR1: QSVYGNNE (SEQ ID NO.13);

[0106] CDR2: KAS;

[0107] CDR3: SYITDDF (SEQ ID NO. 14).

[0108] The amino acid sequence of the light chain variable region (SEQ ID NO.4) is as follows:

[0109] Example 2 – CD33 antibody can improve the phagocytic efficiency of macrophages against colorectal neuroendocrine cancer cells and small cell lung cancer cells.

[0110] 1. BMDM induction

[0111] Six-week-old C57 mice were sacrificed and disinfected by soaking in 75% alcohol. The femur and tibia were removed, and the surface muscle tissue was stripped away as much as possible. (From this point onwards, the cell culture process will proceed in the cell culture chamber hood.) The cells were soaked and rinsed in 75% alcohol for 10 seconds, then rinsed in PBS for 10 seconds, repeating this cycle three times. The ends of the bones were cut open with sterile scissors. Using a syringe (a 20ml syringe can be used, but with a 1ml syringe needle), PBS was drawn up to flush out the bone marrow cells. Approximately 10ml of PBS was used to flush one bone. The cells were incubated at 1500 rpm for 5 minutes. The cells were then lysed with Rhodopsin and centrifuged. The cells were washed again with PBS. Bone marrow cells from one mouse (4 bones) were resuspended in 10ml of 1640 (serum-free, antibiotics not required) and cultured in a 10cm dish for 8 hours. The culture medium was recovered, and the cells at the bottom of the dish were washed with PBS (within 8 hours, the stromal cells are firmly attached to the bottom of the dish, and the bone marrow cells will not adhere to the supernatant). The supernatant was discarded after centrifugation. The cell pellet was resuspended in 10 ml of 1640 medium (containing 10% FBS and 10 ng / ml M-CSF), spread onto a 10 cm dish, and the medium was changed on day 4 (at which point the cells had adhered and grown). After culturing for 7 days, BMDM (mouse bone marrow-derived macrophages) were formed.

[0112] 2. Antibody treatment

[0113] Experimental group: CD33 antibody (HZAB_2) prepared in Example 1 was added to the culture medium of differentiated and mature macrophages and incubated for 2 hours at a concentration of 5 μg / ml;

[0114] Control group: IgG antibody was added to the culture medium of differentiated and mature macrophages and incubated for 2 hours at a concentration of 5 μg / ml;

[0115] 3. Incubation and co-culture, cell collection and analysis

[0116] Macrophages from the experimental and control groups were co-cultured with colorectal neuroendocrine cancer cell line COLO 320DM and small cell lung cancer cell line NCI-H82, respectively, and the phagocytic ratio of macrophages to tumor cells was detected by flow cytometry.

[0117] As shown in Figure 2a, Figure 2a is a flow cytometry diagram of macrophages co-cultured with the NCI-H82 cell line, and Figure 2b is a statistical graph of the results of Figure 2a. According to Figure 2a and Figure 2b, it can be seen that compared with the control group IgG, the efficiency of macrophage phagocytosis of the NCI-H82 cell line after treatment with CD33 antibody was significantly improved.

[0118] Figure 2c shows the flow cytometry diagram of macrophages co-cultured with the COLO 320DM cell line, and Figure 2d shows the statistical results of Figure 2c. According to Figure 2c and Figure 2d, it can be seen that compared with the control group IgG, the COLO 320DM cell line was significantly improved in terms of phagocytosis efficiency by macrophages after treatment with CD33 antibody.

[0119] The above results indicate that CD33 antibodies can enhance the phagocytic efficiency of macrophages against colorectal neuroendocrine cancer cells and small cell lung cancer cells.

[0120] Example 3 – Therapeutic use of CD33 monoclonal antibody HZAB_2 in colorectal neuroendocrine carcinoma and small cell lung cancer

[0121] 1. Establishment of animal experimental groups

[0122] C57 / B6J mice were selected as the model animals to ensure compliance with ethical requirements. Mice were randomly divided into two groups: a control group and a CD33 antibody (HZAB_2) treatment group.

[0123] Tumor cell transplantation: Colorectal neuroendocrine cancer cell line COLO 320DM and small cell lung cancer cell line NCI-H82 were orthotopically transplanted into C57 mice subcutaneously. The surgery was performed under sterile conditions, and the weight and general condition of the mice after transplantation were recorded.

[0124] 2. CD33 antibody therapy

[0125] Three days after surgery, mice were randomly divided into a control group (IgG) and a 1M1D3 antibody treatment group (experimental group). The dosage and frequency of administration were 10 mg / kg, twice a week, for 3 weeks, during which the health status of the mice was observed regularly. Four weeks after inoculation, the mice were euthanized, and tumor tissue was collected. The treatment effect was evaluated by observing the size of the tumor. The results are shown in Figure 2, ej.

[0126] Figure 2e shows the in vivo treatment of small cell lung cancer cell line NCI-H82 in the experimental group and the control group. In Figure 2e, the tumor morphology changes are shown, the mouse weight is shown, and the tumor weight is shown. According to the results, the small cell lung cancer tumors treated with CD33 antibody were significantly smaller than those treated with IgG antibody in the control group.

[0127] Figure 2 shows the in vivo treatment of colorectal neuroendocrine cancer cell line COLO 320DM in the experimental group and the control group. In Figure 2, h represents the tumor morphology changes, i represents the mouse weight statistics, and j represents the tumor weight statistics. According to the results, the colorectal neuroendocrine tumors treated with CD33 antibody were significantly smaller than those treated with IgG antibody in the control group.

[0128] The above results indicate that CD33 antibodies can significantly slow the progression of colorectal neuroendocrine carcinoma and small cell lung cancer.

[0129] Example 4 - Preparation of CD33 humanized antibody based on rabbit antibody

[0130] HZAB_2 antibody from the purified rabbit CD33 antibody was used as the initial antibody to prepare humanized CD33 antibody.

[0131] 1.1 Humanized Antibody Design

[0132] (1) The amino acid sequence of the rabbit CD33 purified antibody HZAB_2, whose heavy chain variable region (VH) is shown in SEQ ID NO.3:

[0133] The three complementary determinant regions are as follows:

[0134] CDR1:GIDLSTNS(SEQ ID NO.10);

[0135] CDR2: IGGSGST (SEQ ID NO.11);

[0136] CDR3: ARLWDF (SEQ ID NO. 12).

[0137] The amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO.4:

[0138] The three complementary determinant regions are as follows:

[0139] CDR1: QSVYGNNE (SEQ ID NO.13);

[0140] CDR2: KAS;

[0141] CDR3: SYITDDF (SEQ ID NO. 14).

[0142] (2) Selection of Human Frame Region (FR)

[0143] In the IMGT human antibody germline gene database, human FR templates with the highest homology to HZAB_2 antibody were selected for the heavy and light chains of the humanized antibody.

[0144] For the humanized antibody heavy chain, the amino acid sequence of the variable region of the selected human FR template heavy chain is shown in SEQ ID NO.18:

[0145] For the humanized antibody light chain, the amino acid sequence of the variable region of the selected human FR template light chain is shown in SEQ ID NO.19:

[0146] (3) CDR transplantation

[0147] The amino acid sequences of the six CDR regions (CDR1, CDR2, and CDR3 of the antibody heavy chain variable region and CDR1, CDR2, and CDR3 of the antibody light chain variable region) of the aforementioned rabbit-derived purified antibody HZAB_2 were precisely "transplanted" onto the backbone of the selected human FR template to form preliminary humanized VH and VL sequences.

[0148] The preliminary humanized antibody heavy chain variable region VH sequence is shown in SEQ ID NO.20:

[0149] These include:

[0150] FR1: QVQLVQSGAEVKKPGASVKVSCKAS(SEQ ID NO.21),

[0151] CDR1: GIDLSTNS (SEQ ID NO.10),

[0152] FR2: WVRQAPGQGLEWMG(SEQ ID NO.22),

[0153] CDR2: IGGSGST (SEQ ID NO.11),

[0154] FR3: RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO.23),

[0155] CDR3: ARLWDF(SEQ ID NO.12),

[0156] FR4: WGQGTLVTVSSGSAST (SEQ ID NO. 24).

[0157] The preliminary humanized antibody light chain variable region (VL) sequence is shown in SEQ ID NO. 25:

[0158] These include:

[0159] FR1:DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO.26),

[0160] CDR1: QSVYGNNE (SEQ ID NO.13),

[0161] FR2:WYQQKPGKAPKPLIY(SEQ ID NO.27),

[0162] CDR2: KAS

[0163] FR3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO.28),

[0164] CDR3: SYITDDF (SEQ ID NO.14),

[0165] FR4: FGQGTKVEIKGGSGSGSGAA (SEQ ID NO. 29).

[0166] (4) Reversal mutation

[0167] To maximize the binding affinity of the original antibody, targeted mutations were performed on certain amino acids in the humanized antibody FR to obtain the final amino acid sequences of huVH and huVL. The final amino acid sequences of huVH and huVL are shown in SEQ ID NO.30 and SEQ ID NO.31, respectively.

[0168] (5) Sequence optimization

[0169] The final huVH and huVL codons were optimized to adapt to the preferences of the target expression host cell (CHO cell), improve expression efficiency, and synthesize the optimized gene sequence.

[0170] The codon-optimized gene sequences are shown in SEQ ID NO.32 and SEQ ID NO.33, respectively:

[0171] (6) Constant region sequence

[0172] The heavy chain constant region was selected from the human IgG1 heavy chain constant region (CH1-CH3), and its amino acid sequence is shown in SEQ ID NO.34:

[0173] The light chain constant region was selected from the human κ light chain constant region (CL), and its amino acid sequence is shown in SEQ ID NO.35:

[0174] 1.2 Construction of humanized antibody expression vector

[0175] (1) The synthesized huVH gene fragment, huVL gene fragment, target expression vector containing the human heavy chain constant region CH1-CH3, and target expression vector containing the human light chain constant region CL were double-digested with the selected restriction endonuclease.

[0176] The plasmid map of the target expression vector pcDNA3.4-huIgG1-CH containing the human heavy chain constant region CH1-CH3 is shown in Figure 3, and the plasmid map of the target expression vector pcDNA3.4-huκ-CL containing the human light chain constant region CL is shown in Figure 4.

[0177] Restriction endonucleases AgeI and SalI were used to double digest the huVH gene fragment and pcDNA3.4-huIgG1-CH, while restriction endonucleases EcoRI and BsiWI were used to double digest the huVL gene fragment and pcDNA3.4-huκ-CL.

[0178] (2) Gel electrophoresis and recovery: The enzyme digestion products were subjected to agarose gel electrophoresis, and the huVH, huVL fragments and linearized vector fragments were recovered.

[0179] (3) Ligation: The recovered huVH fragment was ligated into a linearized vector containing the human heavy chain constant region CH1-CH3; the recovered huVL fragment was ligated into a linearized vector containing the human light chain constant region CL (or a bicistronic vector was used).

[0180] The ligation system (20 μl) includes: 50-100 ng of vector fragment, insert fragment (molar ratio 3:1), 2 μl of 10×T4 DNA Ligase Buffer, 1 μl (400 U) of T4 DNA Ligase, and ddH2O to make up the difference. Ligate overnight at 16°C.

[0181] (4) Transformation and screening: The ligation products were transformed into DH5α competent cells and plated on LB agar plates containing ampicillin. The plates were incubated overnight at 37°C. Single colonies were picked, cultured in small quantities, and then colony PCR or plasmid digestion was performed to verify positive clones.

[0182] (5) Plasmid extraction and sequencing verification: Extract the DNA of positive clone plasmids and send them to a sequencing company to verify whether the inserted huVH and huVL gene sequences are correct.

[0183] Finally, the heavy chain expression plasmid pHC-huCD33 and the light chain expression plasmid pLC-huCD33 of the humanized CD33 antibody were obtained. The plasmid map of the heavy chain expression plasmid pHC-huCD33 is shown in Figure 5, and the plasmid map of the light chain expression plasmid pLC-huCD33 is shown in Figure 6.

[0184] 1.3 Transient expression and preliminary purification of humanized antibodies

[0185] 1.3.1 Cell Culture and Transfection:

[0186] (1) One day before transfection, HEK293F cells in the logarithmic growth phase were seeded in shake flasks or culture plates at a cell density of approximately 1–2 × 10⁻⁶ cells / year. 6 The cell / ml ratio and volume are determined according to requirements (30ml in this example). On the day of transfection, ensure cell viability >95%.

[0187] (2) Prepare the DNA-transfection reagent complex:

[0188] a. Mix the heavy chain expression plasmid (pHC-huCD33) and the light chain expression plasmid (pLC-huCD33) at a mass ratio of 1:1 and dilute them in Opti-MEM™ serum-reduced medium. The total amount of plasmid DNA is optimized according to the system. In this example, it is 1 μg DNA / ml culture system.

[0189] b. Take the transfection reagent PEI Max, with a mass ratio of 3:1 to plasmid DNA, and dilute it in an equal volume of Opti-MEM™ serum-reduced medium. Incubate at room temperature for 5 minutes.

[0190] c. Add the diluted transfection reagent dropwise to the diluted plasmid DNA solution, mix gently, and let stand at room temperature for 15-30 minutes to form a complex.

[0191] (3) Add the DNA-transfection reagent complex dropwise and evenly to the cell culture, and gently shake to mix. Return the cells to the incubator (37℃, 8% CO2, 120rpm) and continue culturing.

[0192] 1.3.2 Cultivation and Gains:

[0193] Six hours or the day after transfection, add feed (expression enhancer such as Valproic Acid) as needed;

[0194] Collect the culture supernatant after 7 days of culture;

[0195] Centrifuge at 4000g for 15 minutes at 4℃ to remove cell debris and collect the clear supernatant;

[0196] 1.3.3 Protein A / G affinity chromatography purification:

[0197] (1) Equilibrate the Protein A / G affinity chromatography column with 10 column volumes (CV) of PBS.

[0198] (2) Load the clarified cell culture supernatant into the equilibrated column at an appropriate flow rate (1 ml / min).

[0199] (3) Wash the column thoroughly with 15CV PBS until the baseline stabilizes (A280 is close to zero) to remove unbound contaminating proteins.

[0200] (4) Elute the bound antibody with elution buffer (0.1M Glycine-HCl, pH 3.0) and collect the elution peak (usually the fraction with a significant increase in A280).

[0201] (5) Immediately add an appropriate amount of neutralization buffer (1 / 10 volume of 1M Tris-HCl, pH 9.0) to the collected acidic eluent, mix gently, and quickly adjust the pH back to neutral (~7.0-7.4) to avoid the antibody being inactivated in an acidic environment for a long time.

[0202] 1.3.4 Buffer Replacement and Concentration:

[0203] After neutralization, the antibody solution is placed in a dialysis bag or concentrated using an ultrafiltration tube (molecular weight cutoff MWCO 30kDa or 100kDa), and dialyzed overnight at 4°C with a large amount of PBS buffer or the buffer is replaced by ultrafiltration. The antibody concentration is determined (A280 method, IgG extinction coefficient is calculated as 1.4), aliquoted, and stored at -80°C for later use.

[0204] 1.3.5 Western blot validation of humanized antibody expression

[0205] Figure 7 shows the Western blot results of the CD33 humanized antibody; Lane M1 is the SDS-PAGE marker, Lane 1 is BSA, and Lanes 2-3 are the CD33 humanized antibody (reduced / unreduced). As can be seen from Figure 7, the molecular weight of the CD33 humanized antibody is approximately 180 kDa. After reduction, there are two bands, at 55 kDa and 25 kDa respectively.

[0206] Example 5

[0207] 1. Human neuroendocrine prostate cancer cells LASCPC-01 were implanted into mice via the tail vein. After intraperitoneal injection of IgG antibody and CD33 humanized antibody obtained in Example 4, it was found that CD33 humanized antibody could significantly inhibit liver metastasis of neuroendocrine prostate cancer tumors in mice.

[0208] Figure 8a shows the morphological changes of prostate cancer liver metastases in mice after intraperitoneal injection of IgG antibody and humanized CD33 antibody. It can be seen that injection of humanized CD33 antibody can inhibit liver metastasis of neuroendocrine prostate cancer tumors in mice.

[0209] Figure 8d shows the statistical chart of the number of liver metastases in prostate cancer. It can be seen that the CD33 humanized antibody can significantly inhibit the number of liver metastases in prostate neuroendocrine carcinoma in mice.

[0210] Figure 8b and c show HE-stained scans of typical liver metastases from the IgG antibody group and the CD33 humanized antibody group. It can be seen that the CD33 humanized antibody significantly inhibited the pathological progression of liver metastases from prostate neuroendocrine carcinoma in mice.

[0211] 2. Human neuroendocrine prostate cancer cells LASCPC-01 were implanted into mice via the tail vein. After intraperitoneal injection of IgG antibody and rabbit-derived CD33 antibody HZAB_2 from Example 1, it was found that the rabbit-derived CD33 antibody could not significantly inhibit liver metastasis of prostate cancer in mice.

[0212] Figure 9a shows the tumor morphological changes in prostate cancer liver metastases after intraperitoneal injection of IgG antibody and rabbit-derived CD33 antibody in mice. It can be seen that the rabbit-derived CD33 antibody cannot significantly inhibit liver metastases of prostate cancer in mice.

[0213] Figure 9b shows the statistical chart of the number of liver metastases in prostate cancer. It can be seen that the CD33 rabbit-derived antibody cannot significantly inhibit the number of liver metastases in neuroendocrine prostate cancer cells in mice. The inhibitory effect of the CD33 rabbit-derived antibody on liver metastases of neuroendocrine prostate cancer is not significantly different from that of the IgG antibody.

[0214] Example 6

[0215] 1. Human small cell lung cancer cells NCI-H82 were seeded into mice via the tail vein. After intraperitoneal injection of IgG antibody and CD33 humanized antibody obtained in Example 4, it was found that CD33 humanized antibody could significantly inhibit liver metastasis of small cell lung cancer in mice.

[0216] Figure 10a shows the morphological changes of small cell lung cancer liver metastases in mice after intraperitoneal injection of IgG antibody and humanized CD33 antibody. It can be seen that injection of humanized CD33 antibody can inhibit liver metastasis of small cell lung cancer tumors in mice.

[0217] Figure 10 shows HE-stained scans of typical liver metastases from the IgG antibody group and the CD33 humanized antibody group, respectively. It can be seen that the CD33 humanized antibody significantly inhibits the pathological progression of liver metastases from small cell lung cancer in mice.

[0218] Figure 10b shows the statistical chart of the number of liver metastases in small cell lung cancer. It can be seen that the CD33 humanized antibody can significantly inhibit the number of liver metastases in mice with small cell lung cancer.

[0219] 2. Human small cell lung cancer cells NCI-H82 were seeded into mice via the tail vein. After intraperitoneal injection of IgG antibody and rabbit-derived CD33 antibody HZAB_2 from Example 1, it was found that the rabbit-derived CD33 antibody could not significantly inhibit liver metastasis of small cell lung cancer in mice.

[0220] Figure 11a shows the tumor morphology changes in liver metastases of small cell lung cancer after intraperitoneal injection of IgG antibody and rabbit-derived CD33 antibody in mice. It can be seen that the rabbit-derived CD33 antibody cannot significantly inhibit liver metastases of small cell lung cancer in mice.

[0221] Figure 11b shows the statistical chart of the number of liver metastases in small cell lung cancer. It can be seen that the CD33 rabbit-derived antibody cannot significantly inhibit the number of liver metastases in small cell lung cancer in mice.

[0222] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A CD33 blocking antibody, characterized in that, The CD33 blocking antibody is HZAB_2; The HZAB_2 includes a heavy chain and a light chain. The heavy chain includes a variable region, and the three complementary determinant regions of the variable region are: CDR1: GIDLSTNS; CDR2: IGSGST; CDR3: ARLWDF; And / or, the light chain includes a light chain variable region, the three complementary determinants of which are: CDR1: QSVYGNNE; CDR2: KAS; CDR3: SYITDDF.

2. The CD33 blocking antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of HZAB_2 is shown in SEQ ID NO.3; And / or, the amino acid sequence of the light chain variable region of HZAB_2 is shown in SEQ ID NO.

4.

3. The use of a CD33 blocking antibody as described in claim 1 or 2 in the preparation of a drug for treating colorectal cancer.

4. The use of a CD33 blocking antibody as described in claim 1 or 2 in the preparation of a drug for treating small cell lung cancer.

5. A CD33 blocking humanized antibody, characterized in that, The CD33 blocking humanized antibody includes a heavy chain and a light chain; The heavy chain includes a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.30; The light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.

31.

6. The CD33 blocking humanized antibody according to claim 5, characterized in that, The heavy chain also includes a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO.34; And / or, the light chain further includes a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.

35.

7. A method for preparing a CD33-blocking humanized antibody as described in claim 5 or 6, characterized in that, Includes the following steps: S1. Using CD33 rabbit monoclonal antibody as the initial antibody, define its CDR region, and select the human FR template with the highest homology to the CD33 rabbit monoclonal antibody from the database. S2, CDR transplantation: The CDR region of the CD33 rabbit monoclonal antibody is transplanted onto the backbone of the selected human FR template to form the variable regions of the initial humanized antibody heavy and light chains; S3, Reversal Mutation: Some amino acids in the FR backbone of the preliminary humanized antibody heavy chain and light chain variable regions are mutated to obtain the final determined heavy chain variable region and light chain variable region of the CD33 humanized antibody. S4. Sequence optimization: Codon optimization is performed on the heavy chain variable region and light chain variable region of the CD33 humanized antibody obtained in step S3 to synthesize the optimized gene sequence. S5. Expression vector construction: Gene fragments of the codon-optimized humanized antibody heavy chain and light chain variable regions were cloned into expression vectors containing human heavy chain constant regions and light chain constant regions, respectively, to construct heavy chain and light chain expression plasmids. S6. Transfect the heavy chain and light chain expression plasmids into HEK293F cells for transient antibody expression, and then purify them to obtain the antibody.

8. The preparation method according to claim 7, characterized in that, In step S1, the amino acid sequence of the heavy chain variable region of the CD33 rabbit monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

9. The preparation method according to claim 7, characterized in that, In step S2, the amino acid sequence of the preliminary humanized antibody heavy chain variable region is shown in SEQ ID NO.20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

25.

10. The preparation method according to claim 7, characterized in that, In step S3, the amino acid sequence of the heavy chain variable region of the finally determined CD33 humanized antibody is shown in SEQ ID NO.30; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

31.

11. The preparation method according to claim 7, characterized in that, In step S4, the gene sequence of the heavy chain variable region of the codon-optimized CD33 humanized antibody is shown in SEQ ID NO.32, and the gene sequence of the light chain variable region is shown in SEQ ID NO.

33.

12. The preparation method according to claim 7, characterized in that, In step S5, the heavy chain constant region is selected from the human IgG1 heavy chain constant region, whose amino acid sequence is shown in SEQ ID NO.34; the light chain constant region is selected from the human κ light chain constant region, whose amino acid sequence is shown in SEQ ID NO.

35.

13. The use of a CD33-blocking humanized antibody as described in claim 5 or 6 in the preparation of a medicament for inhibiting liver metastasis of neuroendocrine prostate cancer.

14. The use of a CD33-blocking humanized antibody as described in claim 5 or 6 in the preparation of a drug for inhibiting liver metastasis of small cell lung cancer.

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