A CD33 blocking humanized antibody and its uses

Abstract

This invention discloses a CD33-blocking humanized antibody and its uses. The CD33-blocking humanized antibody comprises a heavy chain and a light chain; the heavy chain includes a variable region, the amino acid sequence of which is shown in SEQ ID NO. 20; the light chain includes a variable region, the amino acid sequence of which is shown in SEQ ID NO. 21. This invention also discloses a method for preparing the CD33-blocking humanized antibody, obtained by humanizing a rabbit CD33 antibody. Furthermore, it discloses the application of the CD33-blocking humanized antibody in the preparation of drugs for inhibiting liver metastasis of neuroendocrine prostate cancer and small cell lung cancer. The CD33-blocking humanized antibody of this invention exhibits high affinity and high specificity for CD33, and demonstrates excellent therapeutic and inhibitory effects on liver metastasis of neuroendocrine prostate cancer and small cell lung cancer.

Description

Technical Field

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

[0002] 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.

[0003] 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 and 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.

[0004] The current challenges in treating liver metastases from neuroendocrine tumors are as follows:

[0005] (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.

[0006] (2) Systemic therapy resistance. Targeted drug 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%.

[0007] (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%, mediating immunosuppression. Summary of the Invention

[0008] To address the aforementioned technical problems, the present invention aims to provide a CD33-blocking humanized antibody that can inhibit liver metastasis of neuroendocrine cancers, 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 cancers.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] In a first aspect, the present invention provides a CD33 blocking humanized antibody, wherein the CD33 blocking humanized antibody comprises a heavy chain and a light chain;

[0011] The heavy chain includes a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.20;

[0012] The light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.21.

[0013] 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.24;

[0014] And / or, the light chain further includes a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.25.

[0015] In a second aspect, the present invention provides a method for preparing a CD33 blocking humanized antibody as described in any of the above claims, comprising the following steps:

[0016] 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.

[0017] S2, CDR transplantation: The CDR region of the CD33 rabbit monoclonal antibody is transplanted onto the backbone of a selected human FR template to form the variable regions of the initial humanized antibody heavy and light chains.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5.

[0023] 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.10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.15.

[0024] 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.20; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.21.

[0025] 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.22, and the gene sequence of the light chain variable region is shown in SEQ ID NO.23.

[0026] 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.24; 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.25.

[0027] Thirdly, 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.

[0028] Fourthly, 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.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) 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.

[0031] (2) 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

[0032] 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:

[0033] Figure 1 The plasmid map of the target expression vector pcDNA3.4-huVH containing the human heavy chain constant region CH1-CH3 in Example 1;

[0034] Figure 2 The plasmid map of the target expression vector pcDNA3.4-huVL containing the human light chain constant region CL in Example 1;

[0035] Figure 3 The plasmid map of the heavy chain expression plasmid pHC-huCD33 of the humanized CD33 antibody constructed in Example 1;

[0036] Figure 4 The plasmid map of pLC-huCD33, the light chain expression plasmid of the humanized CD33 antibody constructed in Example 1;

[0037] Figure 5 The image shows the Western blot results of the CD33 humanized antibody in Example 1; where M1 is the SDS-PAGE Marker, Lane 1 is BSA, and Lanes 2-3 are the CD33 humanized antibody (reduced / non-reduced).

[0038] Figure 6The image shows the inhibition results of humanized CD33 antibody and IgG antibody on liver metastasis of neuroendocrine prostate cancer in Example 2. In the image, 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.

[0039] Figure 7 The graph shows the inhibition results of rabbit-derived CD33 antibody and IgG antibody on liver metastasis of neuroendocrine prostate cancer in Example 2. In the graph, a is a graph of tumor morphological changes in liver metastasis of prostate cancer; b is a graph of the number of metastatic lesions in liver metastasis of prostate cancer.

[0040] Figure 8 The images show the inhibition results of humanized CD33 antibody and IgG antibody on liver metastasis of small cell lung cancer in Example 3. In the images, a is a graph showing the changes in tumor morphology of small cell lung cancer; b is a graph showing 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; and d is an HE staining scan of liver sections of small cell lung cancer liver metastasis in the humanized CD33 antibody group.

[0041] Figure 9 The graph shows the inhibition results of rabbit-derived CD33 antibody and IgG antibody on liver metastasis of small cell lung cancer in Example 3. In the graph, a is a graph of tumor morphological changes in liver metastasis of small cell lung cancer; b is a graph of the number of metastatic lesions in liver metastasis of small cell lung cancer. Detailed Implementation

[0042] 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 various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0043] Example 1: Preparation of CD33 Humanized Antibody Based on Rabbit Antibody

[0044] The rabbit-derived CD33 purified antibody, namely the HZAB_2 antibody in the applicant's previous patent (authorization announcement number: CN119241711B), was used as the initial antibody to prepare a humanized CD33 antibody.

[0045] 1.1 Humanized Antibody Design

[0046] (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.1:

[0047] QSVDESGGRLVTPGTPLTLGIDLSTNSRYNMNWVRQAPGKGLEWIGVIGGSGSTSPVAKPYYANWAKGRFTISKTSTTVDLKITSPTTEDTARLWDFSFDLWGQDTLVTVSS.

[0048] The three complementary determinant regions are as follows:

[0049] CDR1:GIDLSTNS (SEQ ID NO.2);

[0050] CDR2: IGGSGST (SEQ ID NO.3);

[0051] CDR3: ARLWDF (SEQ ID NO. 4).

[0052] The amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO.5:

[0053] AAVLTQTPSPVSAAVGGTVTISCQSVYGNNEQSVYNSDWGQPPKLKASDASTLESGVPSRFRGSGSGTQFTLTISGVQCDDAATYYCASYITDDFGLGGYASTADYAGFGGGTEV.

[0054] The three complementary determinant regions are as follows:

[0055] CDR1: QSVYGNNE (SEQ ID NO.6);

[0056] CDR2: KAS;

[0057] CDR3: SYITDDF (SEQ ID NO. 7).

[0058] (2) Selection of human framing region (FR)

[0059] 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.

[0060] 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. 8:

[0061] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGGINPSNGGTNFNEKFKDRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARERDYRLDYWGQGTLVTVSSGSAST;

[0062] 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. 9:

[0063] DIQMTQSPSSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKVEIKGGSGSGSGAA.

[0064] (3) CDR transplantation

[0065] 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.

[0066] The preliminary humanized antibody heavy chain variable region VH sequence is shown in SEQ ID NO.10:

[0067] QVQLVQSGAEVKKPGASVKVSCKASGIDLSTNSWVRQAPGQGLEWMGIGGSGSTRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARARLWDFWGQGTLVTVSSGSAST.

[0068] These include:

[0069] FR1: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO.11),

[0070] CDR1:GIDLSTNS (SEQ ID NO.2),

[0071] FR2: WVRQAPGQGLEWMG (SEQ ID NO.12),

[0072] CDR2: IGGSGST (SEQ ID NO.3),

[0073] FR3: RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO.13),

[0074] CDR3: ARLWDF (SEQ ID NO.4),

[0075] FR4: WGQGTLVTVSSGSAST (SEQ ID NO. 14).

[0076] The preliminary humanized antibody light chain variable region (VL) sequence is shown in SEQ ID NO. 15:

[0077] DIQMTQSPSSSLSASVGDRVTITCQSVYGNNEWYQQKPGKAPKPLIYKASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSYITDDFFGQGTKVEIKGGSGSGSGAA.

[0078] These include:

[0079] FR1: DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO.16),

[0080] CDR1: QSVYGNNE (SEQ ID NO.6),

[0081] FR2: WYQQKPGKAPKPLIY (SEQ ID NO.17),

[0082] CDR2: KAS

[0083] FR3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO.18),

[0084] CDR3: SYITDDF (SEQ ID NO.7),

[0085] FR4: FGQGTKVEIKGGSGSGSGAA (SEQ ID NO. 19).

[0086] (4) Reversal mutation

[0087] 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.20 and SEQ ID NO.21, respectively.

[0088] huVH:QVQLVQSGAEVKKPGASVKVSCKASGIDLSTNSWVRQAPGQGLEWMGIGGSGSTRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARARLWDFWGQGTLVTLSSGSAST (SEQ ID NO. 20);

[0089] huVL:DIQMTQSPSSSLSASVGDRVTITCQSVYGNNEWYQQKPGKAPKLLIYKASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSYITDDFFGQGTKVEIKGGSGSGSGAA (SEQ ID NO. 21).

[0090] (5) Sequence optimization

[0091] 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.

[0092] The codon-optimized gene sequences are shown in SEQ ID NO.22 and SEQ ID NO.23, respectively:

[0093] huVH:CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAAACCGGGCGCGAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCATTGATCTGAGCACCAACAGCTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCATTGGCGGCAGCGGCAGCAC CCGCGTGACCCTGACCGTGGATAAAAGCACCAGCACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCGCGCGCCTGTGGGATTTTTGGGGCCAGGGCACCCTGGTGACCCTGAGCAGCGGCAGCGCGAGCACC (SEQ ID NO.22);

[0094] huVL: GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAGAGCGTGTATGGCAACAACGAATGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAAAGCGAGCGGCGTGCCGAGCCG CTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCAGCTATATTACCGATGATTTTTTTGGCCAGGGCACCAAAGTGGAAATTAAAGGCGGCAGCGGCAGCGGCAGCGGCGCGGCG (SEQ ID NO.23).

[0095] (6) Constant region sequence

[0096] 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.24:

[0097] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,

[0098] The light chain constant region was selected from the human κ light chain constant region (CL), and the amino acid sequence is shown in SEQ ID NO.25:

[0099] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0100] 1.2 Construction of humanized antibody expression vector

[0101] (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.

[0102] The plasmid map of the target expression vector pcDNA3.4-huIgG1-CH containing the human heavy chain constant region CH1-CH3 is shown below. Figure 1 As shown, the plasmid map of the target expression vector pcDNA3.4-huκ-CL containing the human light chain constant region CL is as follows. Figure 2 As shown.

[0103] 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.

[0104] (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.

[0105] (3) Ligation: The recovered huVH fragment is ligated into a linearized vector containing the human heavy chain constant region CH1-CH3; the recovered huVL fragment is ligated into a linearized vector containing the human light chain constant region CL (or a bicistronic vector is used).

[0106] The ligation system (20 μl) includes: 50-100 ng of vector fragment, insert fragment (molar ratio 3:1), 2 μl of 10× T4 DNALigase Buffer, 1 μl of T4 DNA Ligase (400 U), and ddH2O to make up the volume. Ligate overnight at 16°C.

[0107] (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.

[0108] (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.

[0109] Finally, the heavy chain expression plasmid pHC-huCD33 and the light chain expression plasmid pLC-huCD33, representing the humanized CD33 antibody, were obtained. The plasmid map of the heavy chain expression plasmid pHC-huCD33 is shown below. Figure 3 As shown, the plasmid map of the light chain expression plasmid pLC-huCD33 is as follows. Figure 4 As shown.

[0110] 1.3 Transient expression and preliminary purification of humanized antibodies

[0111] 1.3.1 Cell Culture and Transfection:

[0112] (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%.

[0113] (2) Prepare the DNA-transfection reagent complex:

[0114] 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 in Opti-MEM. TM The total amount of plasmid DNA in the serum-reduced culture medium is optimized according to the system. In this example, it is specifically a culture system of 1 μg DNA / ml.

[0115] 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. TM Incubate in serum-reduced medium at room temperature for 5 minutes.

[0116] 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.

[0117] (3) Add the DNA-transfection reagent complex dropwise to the cell culture and shake gently. Return the cells to the incubator (37℃, 8% CO2, 120rpm) and continue culturing.

[0118] 1.3.2 Cultivation and Gains:

[0119] Six hours or the day after transfection, add feed (expression enhancer such as Valproic Acid) as needed.

[0120] Collect the culture supernatant after 7 days of culture;

[0121] Centrifuge at 4000g for 15 minutes at 4℃ to remove cell debris and collect the clear supernatant;

[0122] 1.3.3 Protein A / G affinity chromatography purification:

[0123] (1) Equilibrate the Protein A / G affinity chromatography column with 10 column volumes (CV) of PBS.

[0124] (2) Load the clarified cell culture supernatant into the equilibrated column at an appropriate flow rate (1 ml / min).

[0125] (3) Wash the column thoroughly with 15 CV PBS until the baseline stabilizes (A280 is close to zero) to remove unbound contaminants.

[0126] (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).

[0127] (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.

[0128] 1.3.4 Buffer Replacement and Concentration:

[0129] The neutralized 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.

[0130] 1.3.5 Western blot validation of humanized antibody expression

[0131] As attached Figure 5 The image shows the Western blot results of the CD33 humanized antibody; where M1 is the SDS-PAGE marker, Lane 1 is BSA, and Lanes 2-3 are the CD33 humanized antibody (reduced / non-reduced). Figure 5 It can be seen that the molecular weight of the CD33 humanized antibody is approximately 180 kDa, and after reduction, it has two bands, at 55 kDa and 25 kDa respectively.

[0132] Example 2

[0133] 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 1, it was found that CD33 humanized antibody could significantly inhibit liver metastasis of neuroendocrine prostate cancer tumors in mice.

[0134] Figure 6 Figure a 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.

[0135] Figure 6 The graph in section d represents the statistical number of liver metastases in prostate cancer. It can be seen that the CD33 humanized antibody significantly inhibited the number of liver metastases in prostate neuroendocrine carcinoma in mice.

[0136] Figure 6 Images b and c show 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 inhibited the pathological progression of liver metastases from prostate neuroendocrine carcinoma in mice.

[0137] 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.

[0138] Figure 7 Figure a shows the morphological changes of prostate cancer liver metastases in mice after intraperitoneal injection of IgG antibody and rabbit-derived CD33 antibody. It can be seen that the rabbit-derived CD33 antibody could not significantly inhibit liver metastases of prostate cancer in mice.

[0139] Figure 7 Figure b shows a 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.

[0140] Example 3

[0141] 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 1, it was found that CD33 humanized antibody could significantly inhibit liver metastasis of small cell lung cancer in mice.

[0142] Figure 8 Figure a 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.

[0143] Figure 8 Images c and d are 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.

[0144] Figure 8 Figure b shows a 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 small cell lung cancer in mice.

[0145] 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 obtained in a previous patent, it was found that the rabbit-derived CD33 antibody could not significantly inhibit liver metastasis of small cell lung cancer in mice.

[0146] Figure 9 Figure a 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 could not significantly inhibit liver metastases of small cell lung cancer in mice.

[0147] Figure 9 Figure b 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.

[0148] 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 humanized antibody, characterized in that, The CD33 blocking humanized antibody comprises a heavy chain and a light chain; The heavy chain comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 20; The light chain comprises a light chain variable region, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

21.

2. The CD33 blocking humanized antibody of claim 1, wherein, The heavy chain further comprises a heavy chain constant region, and the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO. 24; And / or, the light chain further comprises a light chain constant region, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO.

25.

3. A method for producing the CD33 blocking humanized antibody as claimed in claim 1 or 2, characterized in that, The method comprises the following steps: S1, taking a CD33 rabbit-derived monoclonal antibody as an initial antibody, defining the CDR region thereof, and selecting a human FR template with the highest homology with the CD33 rabbit-derived monoclonal antibody in a database; S2, CDR grafting: grafting the CDR region of the CD33 rabbit-derived monoclonal antibody to the framework of the selected human FR template to form a preliminary variable region of a humanized antibody heavy chain and a light chain; S3, back mutation: mutating part of the amino acids in the FR framework of the preliminary variable region of the humanized antibody heavy chain and light chain to obtain a final determined heavy chain variable region and a light chain variable region of the CD33 humanized antibody; S4, sequence optimization: codon optimizing the heavy chain variable region and the light chain variable region of the CD33 humanized antibody obtained in step S3 to synthesize an optimized gene sequence; S5, expression vector construction: cloning the gene fragments of the codon-optimized humanized antibody heavy chain and light chain variable regions 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, transfecting the heavy chain and light chain expression plasmids into HEK293F cells for antibody transient expression, and obtaining the antibody after purification.

4. The production method according to claim 3, characterized by, In step S1, the amino acid sequence of the heavy chain variable region of the CD33 rabbit-derived monoclonal antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

5.

5. The preparation method according to claim 3, characterized in that, In step S2, the amino acid sequence of the preliminary humanized antibody heavy chain variable region is shown as SEQ ID NO. 10, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

15.

6. The preparation method according to claim 3, characterized in that, In step S4, the gene sequence of the codon-optimized CD33 humanized antibody heavy chain variable region is shown as SEQ ID NO. 22, and the gene sequence of the light chain variable region is shown as SEQ ID NO.

23.

7. The preparation method according to claim 3, characterized in that, In step S5, the heavy chain constant region is selected to be a human IgG1 heavy chain constant region, and the amino acid sequence thereof is shown as SEQ ID NO. 24; the light chain constant region is selected to be a human kappa light chain constant region, and the amino acid sequence thereof is shown as SEQ ID NO.

25.

8. Use of the CD33 blocking humanized antibody of claim 1 or 2 in the preparation of a medicament for inhibiting neuroendocrine prostate cancer liver metastasis.

9. Use of the CD33 blocking humanized antibody of claim 1 or 2 in the preparation of a medicament for inhibiting small cell lung cancer liver metastasis.

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