A CD33 blocking antibody and its application

Abstract

The present invention discloses a CD33 blocking antibody, which is HZAB_1 or HZAB_2; the amino acid sequence of the heavy chain variable region of HZAB_1 is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of HZAB_1 is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of HZAB_2 is shown in SEQ ID NO.3; the amino acid sequence of the light chain variable region of HZAB_2 is shown in SEQ ID NO.4. HZAB_1 and HZAB_2 antibodies can improve the phagocytic efficiency of macrophages on colorectal neuroendocrine cancer cells and small cell lung cancer cells, and slow down the progression of colorectal neuroendocrine cancer and small cell lung cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a CD33 blocking antibody and application thereof. Background Art

[0002] As early as the beginning of the 20th century, Paul Ehrlich proposed the concept of "magic bullet", which can be regarded as an early conception of ADC (antibody-drug conjugate). However, it was not until the 1950s that Mathe first used anti-mouse leukocyte immunoglobulin conjugated with methotrexate for the treatment of leukemia, which marked the beginning of ADC research.

[0003] CD33 is a membrane glycoprotein expressed in hematopoietic cells. It is a 67KD glycosylated transmembrane protein belonging to the Siglec family. It is activated after cross-linking or ligand binding, mediates inhibitory signals, regulates intracellular calcium mobilization, cell adhesion, leukemia cell apoptosis, myeloid cell maturation and cytokine production, and is related to the occurrence and development of certain blood tumors.

[0004] In 2000, the CD33 antibody-drug conjugate Gemtuzumab ozogamicin (GO), which is composed of a humanized anti-CD33 monoclonal antibody and a DNA intercalator calicheamicin (CLM), became the first ADC drug approved by the US FDA for the treatment of CD33-positive acute myeloid leukemia (AML). However, GO was withdrawn from the market in 2010 because the post-marketing Phase III study showed that it did not significantly improve the treatment effect compared with traditional chemotherapy and had serious safety issues.

[0005] Anti-CD33 monoclonal antibodies (such as Gemtuzumab Ozogamicin) were first used to treat acute myeloid leukemia (AML). Its antibody-drug conjugates (ADCs) combine CD33 antibodies and 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 dilemmas. 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 drug resistance rapidly, limiting the efficacy of second-line and subsequent treatments. In addition, the treatment of SCLC also faces challenges in drug safety, especially the side effects that may be caused by immunotherapy, such as pneumonia and colitis.

[0007] Due to its rarity, treatment strategies for colorectal neuroendocrine carcinoma lack large-scale clinical trial support, and usually refer to the treatment regimens of SCLC and non-small cell lung cancer (NSCLC), but the results are not ideal. In addition, the heterogeneity of LCNEC makes the prediction of treatment response complicated, and the treatment options for advanced or metastatic LCNEC are very limited, usually chemotherapy-based.

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

[0009] The present invention provides a CD33 blocking antibody to block the immunosuppressive effect of CD33. The purpose of the present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides a CD33 blocking antibody, wherein the CD33 blocking antibody is HZAB_1 or HZAB_2;

[0011] The HZAB_1 comprises a heavy chain and a light chain; the heavy chain comprises a heavy chain variable region, and the three complementary determining regions of the heavy chain variable region are:

[0012] CDR1: GFSLISYH (SEQ ID NO.5);

[0013] CDR2: IYTNGSA (SEQ ID NO.6);

[0014] CDR3: VRGIDL (SEQ ID NO.7);

[0015] And / or, the light chain comprises a light chain variable region, and the three complementarity determining regions of the light chain variable region are:

[0016] CDR1: QSVYNNHD (SEQ ID NO.8);

[0017] CDR2: YAS;

[0018] CDR3: LGVYDDDADTA (SEQ ID NO.9);

[0019] The HZAB_2 comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region, and the three complementary determining regions of the heavy chain variable region are:

[0020] CDR1:GIDLSTNS (SEQ ID NO.10);

[0021] CDR2: IGGSGST (SEQ ID NO.11);

[0022] CDR3: ARLWDF (SEQ ID NO.12);

[0023] And / or, the light chain comprises a light chain variable region, and the three complementarity determining regions of the light chain variable region are:

[0024] CDR1: QSVYGNNE (SEQ ID NO.13);

[0025] CDR2: KAS;

[0026] CDR3: SYITDDF (SEQ ID NO. 14).

[0027] 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;

[0028] And / or, the amino acid sequence of the light chain variable region of the HZAB_1 is as shown in SEQ ID NO.2.

[0029] 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;

[0030] And / or, the amino acid sequence of the light chain variable region of the HZAB_2 is as shown in SEQ ID NO.4.

[0031] As some specific embodiments of the present invention, the CD33 blocking antibody is obtained by immunization with recombinant CD33 protein.

[0032] The amino acid sequence of the recombinant CD33 protein is shown in SEQ ID NO.15.

[0033] 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.

[0034] In a second aspect, the present invention provides a use of a CD33 blocking antibody in the preparation of a drug for treating colorectal cancer.

[0035] The present invention also provides an application of a CD33 blocking antibody in the preparation of a drug for treating small cell lung cancer.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The present invention provides a blocking antibody with high affinity and high specificity for CD33, which has a specific blocking effect on CD33 protein;

[0038] (2) The CD33 blocking antibodies 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 the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0040] Figure 1 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 figure is the protein electrophoresis diagram of HZAB_1 (Lane M is Marker, Lane 1 is BSA, Lane 2 is HZAB_1 reduced antibody, Lane 3 is HZAB_1 non-reduced antibody); the right figure is the protein electrophoresis diagram of HZAB_2 (Lane M is Marker, Lane 1 is BSA, Lane 2 is HZAB_2 reduced antibody, Lane 3 is HZAB_2 non-reduced antibody);

[0041] Figure 2 The figure shows the validation results of CD33 antibody against colorectal neuroendocrine carcinoma and small cell lung cancer in Examples 2 and 3, wherein: Figure 2 Figures ad show the effect of adding CD33 antibody on the phagocytic effect of NCI-H82 (a, b) and COLO 320DM (c, d) tumor cells. Figure 2 Figures e and j show the effects of CD33 antibody on the growth of NCI-H82 (e, f, g) and COLO 320DM (h, i, j) tumor cells in mice. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0043] Example 1

[0044] 1. Dissolve the recombinant CD33 protein in sterile 0.85% NaCl solution or PBS (pH 7.2) at a volume of 250 μl / rabbit. The total amount of antigen varies according to the nature of the antigen. The protein antigen is preferably 100 μg / rabbit.

[0045] 2. Subcutaneously inject a mixture of a preset dose of immunogen (a total of 100 μg recombinant CD33 protein per rabbit) and Freund's complete adjuvant (FCA) at multiple points on the back of the rabbit, 0.1 ml per point, for a total of 4-6 points.

[0046] The immunogen used is recombinant CD33 protein, which includes CD33 protein and a flag tag. The amino acid sequence of the CD33 protein is shown in SEQ ID NO.17, the amino acid sequence of the flag tag is shown in SEQ ID NO.16, and the amino acid sequence of the recombinant CD33 protein is shown in SEQ ID NO.15.

[0047] 3. After 3 weeks, the second immunization was performed using Freund's incomplete adjuvant (FIA) mixed with CD33 protein for booster immunization, using the same method as the initial immunization.

[0048] 4. Blood collection began 2-3 weeks after the initial immunization, and then every 3 weeks using the ear vein blood collection method, collecting 2-5 ml of blood each time.

[0049] 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.

[0050] 6. Use ELISA to detect the titer and specificity of antibodies in the serum. Add 100 μL of detection antibody solution to each well and incubate at room temperature for 2 hours (500 rpm).

[0051] 7. Purify antibodies from serum by protein A / G affinity chromatography. Protein A affinity chromatography columns have become widely used affinity columns for purifying antibodies. They can separate and purify antibodies of different subtypes of various mammals or genetically engineered recombinant proteins containing antibody Fc fragments from ascites, serum, cell culture supernatant or cell extracts.

[0052] Two antibodies were obtained, named HZAB_1 and HZAB_2. Figure 1The results of protein electrophoresis of these two antibodies are shown in the figure on the left. The left figure is the protein electrophoresis of HZAB_1, where Lane M is SDS-PAGE Marker, Lane 1 is BSA (9.00μg), Lane 2 is HZAB_1 reduced antibody, and Lane 3 is HZAB_1 non-reduced antibody; the right figure is the protein electrophoresis of HZAB_2, where Lane M is SDS-PAGE Marker, Lane 1 is BSA (9.00μg), Lane 2 is HZAB_2 reduced antibody, and Lane 3 is HZAB_2 non-reduced antibody.

[0053] HZAB_1 contains a light chain variable region and a heavy chain variable region. The heavy chain variable region includes three complementary determining regions CDR1, CDR2 and CDR3, and their respective amino acid sequences are shown below:

[0054] CDR1: GFSLISYH (SEQ ID NO.5);

[0055] CDR2: IYTNGSA (SEQ ID NO.6);

[0056] CDR3: VRGIDL (SEQ ID NO. 7).

[0057] The amino acid sequence of the heavy chain variable region (SEQ ID NO.1) is:

[0058] QSVEESGGRLVTPGGFSLISYHWVRQAPGKGLEWIGIIYTNGSAANWAKGRFTISKTSSTTVDLKMTGLTVRGIDLARGGVYNSGRSFWGQGTLVTVSSWGQGTLVTVSS.

[0059] The light chain variable region includes three complementary determining regions CDR1, CDR2 and CDR3, and their respective amino acid sequences are shown below:

[0060] CDR1: QSVYNNHD (SEQ ID NO.8);

[0061] CDR2: YAS;

[0062] CDR3: LGVYDDDADTA (SEQ ID NO. 9).

[0063] The amino acid sequence of the light chain variable region (SEQ ID NO.2) is:

[0064] ALVMTQTPASVSAAVGGTVTIQSVYNNHDNNDLSWYQQKPGQPPKLLIYYASASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCLGVYDDDADTANNVLAFGGGTEVVVK.

[0065] HZAB_2 comprises a light chain variable region and a heavy chain variable region. The heavy chain variable region includes three complementary determining regions CDR1, CDR2 and CDR3, and their respective amino acid sequences are shown below:

[0066] CDR1:GIDLSTNS (SEQ ID NO.10);

[0067] CDR2: IGGSGST (SEQ ID NO.11);

[0068] CDR3: ARLWDF (SEQ ID NO. 12).

[0069] The amino acid sequence of the heavy chain variable region (SEQ ID NO.3) is:

[0070] QSVDESGGRLVTPGTPLTLGIDLSTNSRYNMNWVRQAPGKGLEWIGVIGGSGSTSPVAKPYYANWAKGRFTISKTSTTVDLKITSPTTEDTARLWDFSFDLWGQDTLVTVSS.

[0071] The light chain variable region includes three complementary determining regions CDR1, CDR2 and CDR3, and their respective amino acid sequences are shown below:

[0072] CDR1: QSVYGNNE (SEQ ID NO.13);

[0073] CDR2: KAS;

[0074] CDR3: SYITDDF (SEQ ID NO. 14).

[0075] The amino acid sequence of the light chain variable region (SEQ ID NO.4) is:

[0076] AAVLTQTPSPVSAAVGGTVTISCQSVYGNNEQSVYNSDWGQPPKLKASDASTLESGVPSRFRGSGSGTQFTLTISGVQCDDAATYYCASYITDDFGLGGYASTADYAGFGGGTEV.

[0077] Example 2 - CD33 antibodies can enhance the phagocytic efficiency of macrophages against colorectal neuroendocrine cancer cells and small cell lung cancer cells

[0078] 1. BMDM induction

[0079] Take six-week-old C57 mice, kill them, and soak them in 75% alcohol for disinfection. Take out the femur and tibia, and try to peel off the surface muscle tissue and peel it clean. (From here on, operate in the cell room hood) Soak and rinse in 75% alcohol for 10 seconds, rinse in PBS for 10 seconds, and cycle 3 times. Cut the two ends of the bone with sterilized scissors, use a syringe (you can use a 20 ml syringe, replace it with a 1 ml syringe needle) to absorb PBS, and rinse out the bone marrow cells. One bone is rinsed with about 10 ml PBS. 1500r, 5 min. Lyse the red blood cells with lysate, centrifuge; wash again with PBS. Resuspend the bone marrow cells of one mouse (4 bones) in 10ml 1640 (without serum, antibiotics are not required), spread on a 10cm dish and culture for 8h. Recover the culture medium, and then rinse the cells at the bottom of the dish with PBS (interstitial cells have been firmly attached to the bottom of the dish within 8h, and bone marrow cells will not adhere to the wall in the supernatant culture medium), centrifuge and discard the supernatant. The cell pellet was resuspended in 10 ml 1640 medium (containing 10% FBS, 10 ng / ml M-CSF), spread to a 10 cm dish, and the medium was changed on the 4th day (at which time the cells have grown adherently), and cultured for 7 days to form BMDM (mouse bone marrow-derived macrophages).

[0080] 2. Antibody treatment

[0081] Experimental group: The CD33 antibody (HZAB_2) prepared in Example 1 was added to the culture medium of differentiated mature macrophages and incubated for 2 hours at a concentration of 5 μg / ml;

[0082] Control group: IgG antibody was added to the culture medium of differentiated mature macrophages and incubated for 2 hours at a concentration of 5 μg / ml;

[0083] 3. Incubation and co-culture, cell collection and analysis

[0084] The macrophages in the experimental group and the control group were co-cultured with the colorectal neuroendocrine carcinoma cell line COLO 320DM and the small cell lung cancer cell line NCI-H82, respectively, and the phagocytic ratio of macrophages to tumor cells was detected by flow cytometry.

[0085] like Figure 2 As shown in the ad, Figure 2 Middle a is a flow cytometric image of macrophages co-cultured with NCI-H82 cell line. Figure 2 b is Figure 2 The result statistics of a in the figure are as follows: Figure 2 A and Figure 2 In middle b, it can be seen that compared with the control group IgG, the efficiency of NCI-H82 cell line phagocytosis by macrophages was significantly improved after treatment with CD33 antibody.

[0086] Figure 2 Middle c is the flow cytometric image of macrophages co-cultured with COLO 320DM cell line. Figure 2 In d Figure 2 The result statistics of c in the figure are as follows: Figure 2 Medium c and Figure 2 In middle d, it can be seen that compared with the control group IgG, the efficiency of COLO 320DM cell line phagocytosis by macrophages was significantly improved after treatment with CD33 antibody.

[0087] The above results indicate that CD33 antibodies can enhance the phagocytic efficiency of macrophages on colorectal neuroendocrine carcinoma cells and small cell lung cancer cells.

[0088] Example 3 - Therapeutic Use of CD33 Monoclonal Antibody HZAB_2 in Colorectal Neuroendocrine Carcinoma and Small Cell Lung Cancer

[0089] 1. Establishment of animal experimental group

[0090] C57 / B6J mice were selected as model animals to ensure compliance with ethical requirements. The mice were randomly divided into two groups: a control group and a CD33 antibody (HZAB_2) treatment group.

[0091] Tumor cell transplantation: The colorectal neuroendocrine carcinoma cell line COLO 320DM and the small cell lung cancer cell line NCI-H82 were orthotopically transplanted subcutaneously into C57 mice. The operation was performed under a sterile environment, and the weight and general condition of the mice after transplantation were recorded.

[0092] 2. CD33 antibody therapy

[0093] Three days after surgery, the 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. The health of the mice was regularly observed during this period. The mice were euthanized 4 weeks after inoculation, and tumor tissues were obtained to evaluate the therapeutic effect by observing the tumor size. Figure 2 As shown in ej.

[0094] Figure 2 In the figure, eg, the in vivo treatment of the small cell lung cancer cell line NCI-H82 by the experimental group and the control group, among which, Figure 2 Figure e in the middle is a diagram showing changes in tumor morphology. Figure 2 The middle f is the statistical chart of mouse weight. Figure 2The middle g is a statistical graph of tumor weight. According to the results, the small cell lung cancer tumors treated with CD33 antibodies were significantly reduced compared to those treated with IgG antibodies in the control group.

[0095] Figure 2 hj in the middle are the in vivo treatment of the colorectal neuroendocrine cancer cell line COLO 320DM in the experimental group and the control group, among which, Figure 2 The middle h is the diagram of tumor morphological changes. Figure 2 The i in the middle is the statistical chart of mouse weight. Figure 2 The j in the middle is a statistical graph of tumor weight. According to the results, the colorectal neuroendocrine tumors treated with CD33 antibodies were significantly reduced compared to those treated with IgG antibodies in the control group.

[0096] The above results indicate that CD33 antibodies can significantly slow the progression of colorectal neuroendocrine carcinoma and small cell lung cancer.

[0097] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may 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 comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region, and the three complementary determining regions of the heavy chain variable region are: CDR1:GIDLSTNS; CDR2: IGGSGST; CDR3:ARLWDF; The light chain includes a light chain variable region, and the three complementarity determining regions of the light chain variable region 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; The amino acid sequence of the light chain variable region of HZAB_2 is shown in SEQ ID NO.

4.

3. Use of the CD33 blocking antibody according to claim 1 or 2 in the preparation of a drug for treating colorectal neuroendocrine cancer.

4. Use of the CD33 blocking antibody according to claim 1 or 2 in the preparation of a drug for treating small cell lung cancer.

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