CD33 blocking humanized antibody and application

Abstract

The invention discloses a CD33 blocking humanized antibody and application thereof. 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 as shown in 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 as shown in SEQ ID NO.21. The invention also discloses a preparation method of the CD33 blocking humanized antibody, the CD33 blocking humanized antibody is obtained by carrying out humanized transformation on a CD33 rabbit-derived antibody, and the invention also discloses an application of the CD33 blocking humanized antibody in preparation of drugs for inhibiting liver metastasis of neuroendocrine prostate cancer and small cell lung cancer. The CD33 blocking humanized antibody provided by the invention has high affinity and high specificity to CD33, and has good treatment and inhibition effects on liver metastasis of neuroendocrine prostate cancer and small cell lung cancer.

Description

TECHNICAL FIELD

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

[0002] CD33 is a transmembrane glycoprotein expressed on the surface of myeloid hematopoietic cells, belonging to the Siglec family. Its molecular weight is about 67 kDa, with typical glycosylation modification. CD33 is activated after cross-linking or binding with specific ligands, mainly recruiting and activating SHP-1 and SHP-2 phosphatases through its cytoplasmic immunoreceptor tyrosine inhibitory motif (ITIM), thereby transmitting inhibitory signals. This signal transduction is involved in the regulation of various cell functions, including intracellular calcium mobilization, cell adhesion, leukemia cell apoptosis, myeloid cell differentiation and maturation, and cytokine production. Abnormal expression of CD33 is closely related to the occurrence and development of hematological malignancies such as acute myeloid leukemia (AML), making it an important therapeutic target.

[0003] Liver metastasis is quite common in neuroendocrine neoplasms (NENs). According to statistics, up to 40% to 90% of patients diagnosed with advanced (metastatic) NENs will be accompanied by liver metastasis. For some specific primary sites (such as pancreas and small intestine), the liver is often the first and main site of metastasis. This high metastasis rate makes the liver a key organ for disease progression and prognosis of NENs. Liver metastasis not only directly damages liver function, leading to the risk of failure, but also poses a serious threat to the survival period and quality of life of patients by increasing tumor burden, inducing severe endocrine syndromes, and greatly increasing the complexity and difficulty of treatment. Therefore, for patients diagnosed with NENs, close attention to liver status, early detection and active intervention of liver metastasis are the key to improving prognosis.

[0004] The current treatment dilemma for liver metastasis of neuroendocrine neoplasms is as follows: (1) High metastatic load and surgical limitations. More than 50% of advanced neuroendocrine neoplasms (NENs) have liver metastasis, of which 40% are multifocal and diffuse, and only 10%-20% of patients are suitable for radical resection; the control rate of radiofrequency / embolization therapy for lesions >3 cm is less than 30%.

[0005] (2) Systemic treatment resistance. Targeted drug failure: the objective response rate (ORR) of mTOR inhibitors (everolimus) and anti-angiogenic drugs (sunitinib) is only 9%-12%, and the median progression-free survival (mPFS) is less than 12 months. Chemotherapy bottleneck: the ORR of the streptozotocin + 5-Fu regimen for G3 grade neuroendocrine carcinoma (NEC) is less than 15%, and the incidence of grade 3-4 hematotoxicity is more than 40%.

[0006] (3) Immune therapy "cold tumor" dilemma: T cell infiltration deficiency leads to poor PD-1 inhibitor effect. Tumor-associated macrophages (TAMs) account for >60%, mediate immune suppression. SUMMARY

[0007] To solve the above technical problems, the purpose of the present application is to provide a CD33 blocking humanized antibody which can inhibit the liver metastasis of neuroendocrine cancer including neuroendocrine prostate cancer and small cell lung cancer, and can be used for preparing a safe, effective and specific drug for treating liver metastasis of neuroendocrine cancer.

[0008] The purpose of the present application is achieved by the following technical solutions: In a first aspect, the present application provides a CD33 blocking humanized antibody, which 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 in 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 in SEQ ID NO. 21.

[0009] As some specific embodiments of the present application, the heavy chain further comprises a heavy chain constant region, and the amino acid sequence of the heavy chain constant region is shown in 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 in SEQ ID NO. 25.

[0010] In a second aspect, the present application provides a preparation method of the CD33 blocking humanized antibody as described in any one of the above, comprising 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 selected human FR template skeleton to form the variable region of the preliminary humanized antibody heavy chain and light chain; S3, back mutation: mutating part of the amino acids in the FR skeleton of the preliminary humanized antibody heavy chain and light chain variable region 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: the gene fragments of the codon-optimized humanized antibody heavy chain and light chain variable regions are respectively cloned into expression vectors containing human heavy chain constant regions and light chain constant regions to construct heavy chain and light chain expression plasmids; S6, the heavy chain and light chain expression plasmids are transfected into HEK293F cells for antibody transient expression, and the antibody is obtained after purification.

[0011] As some specific embodiments of the present application, in step S1, the amino acid sequence of the CD33 rabbit-derived monoclonal antibody heavy chain variable region 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.

[0012] As some specific embodiments of the present application, 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.

[0013] As some specific embodiments of the present application, in step S3, the amino acid sequence of the finally determined CD33 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. 21.

[0014] As some specific embodiments of the present application, in step S4, the gene sequence of the codon-optimized CD33 humanized antibody heavy chain variable region is shown in SEQ ID NO. 22, and the gene sequence of the light chain variable region is shown in SEQ ID NO. 23.

[0015] As some specific embodiments of the present application, in step S5, the heavy chain constant region is selected from a human IgG1 heavy chain constant region, and the amino acid sequence is shown in SEQ ID NO. 24; the light chain constant region is selected from a human kappa light chain constant region, and the amino acid sequence is shown in SEQ ID NO. 25.

[0016] In a third aspect, the present application provides a use of the CD33 blocking humanized antibody as described in any one of the above in the preparation of a medicament for inhibiting liver metastasis of neuroendocrine prostate cancer. The CD33 blocking humanized antibody of the present application can inhibit liver metastasis of neuroendocrine tumors.

[0017] In a fourth aspect, the present application provides a use of the CD33 blocking humanized antibody as described in any one of the above in the preparation of a medicament for inhibiting liver metastasis of small cell lung cancer. The CD33 blocking humanized antibody of the present application can inhibit liver metastasis of small cell lung cancer.

[0018] Compared with the prior art, the present application has the following beneficial effects: (1) The application adopts CD33 rabbit-derived antibody to carry out humanization modification, and obtains high affinity and high specificity humanized blocking antibody for CD33.

[0019] (2) The humanized CD33 blocking antibody has good treatment and inhibition effect on liver metastasis of neuroendocrine prostate cancer and small cell lung cancer. BRIEF DESCRIPTION OF DRAWINGS

[0020] Other features, objects and advantages of the application will become more apparent from the following detailed description of non-limiting embodiments, made with reference to the following drawings: Figure 1 Plasmid map of the target expression vector pcDNA3.4-huVH containing human heavy chain constant region CH1-CH3 in Example 1; Figure 2 Plasmid map of the target expression vector pcDNA3.4-huVL containing human light chain constant region CL in Example 1; Figure 3 Plasmid map of the heavy chain expression plasmid pHC-huCD33 of the humanized CD33 antibody constructed in Example 1; Figure 4 Plasmid map of the light chain expression plasmid pLC-huCD33 of the humanized CD33 antibody constructed in Example 1; Figure 5 Western blot result graph of the CD33 humanized antibody in Example 1; wherein M1 is SDS-PAGE Marker, Lane 1 is BSA, and Lane 2-3 are CD33 humanized antibody (reduced / non-reduced); Figure 6 Inhibition result graph of the humanized CD33 antibody and IgG antibody on neuroendocrine prostate cancer tumor liver metastasis in Example 2, wherein a is a tumor morphological change graph of prostate cancer liver metastasis; b and c are HE staining scanning graphs of liver sections of prostate cancer tumor liver metastasis in IgG antibody and CD33 humanized antibody groups respectively; d is a metastatic focus number statistical graph of prostate cancer tumor liver metastasis; Figure 7 Inhibition result graph of the rabbit-derived CD33 antibody and IgG antibody on neuroendocrine prostate cancer liver metastasis in Example 2, wherein a is a tumor morphological change graph of prostate cancer liver metastasis; b is a metastatic focus number statistical graph of prostate cancer liver metastasis; Figure 8Figures of inhibition results of humanized CD33 antibody and IgG antibody on liver metastasis of small cell lung cancer in Example 3, wherein a is a figure of tumor morphological changes of small cell lung cancer; b is a figure of metastatic focus number statistics of small cell lung cancer liver metastasis; c is a figure of HE staining scanning of liver section of small cell lung cancer liver metastasis in the IgG antibody group; d is a figure of HE staining scanning of liver section of small cell lung cancer liver metastasis in the humanized CD33 antibody group; Figure 9 Figures of inhibition results of humanized CD33 antibody and IgG antibody on liver metastasis of small cell lung cancer in Example 3, wherein a is a figure of tumor morphological changes of small cell lung cancer; b is a figure of metastatic focus number statistics of small cell lung cancer liver metastasis; c is a figure of HE staining scanning of liver section of small cell lung cancer liver metastasis in the IgG antibody group; d is a figure of HE staining scanning of liver section of small cell lung cancer liver metastasis in the humanized CD33 antibody group; DETAILED DESCRIPTION

[0021] The application will be described in detail below with specific embodiments. The following examples will help those skilled in the art to further understand the application, but do not limit the application in any form. It should be noted that for those skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made. These are within the scope of the present application.

[0022] Example 1 Preparation of CD33 humanized antibody based on rabbit-derived antibody Take the rabbit-derived CD33 purified antibody, i.e. the HZAB_2 antibody in the applicant's previous patent (authorized announcement number: CN119241711B) as the initial antibody to prepare the CD33 humanized antibody.

[0023] 1.1 Humanized antibody design (1) The rabbit-derived CD33 purified antibody HZAB_2, the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO. 1: QSVDESGGRLVTPGTPLTLGIDLSTNSRYNMNWVRQAPGKGLEWIGVIGGSGSTISPVAKPYYANWAKGRFTISKTSTTVDLKITSPTTEDTARLWDFSFDLWGQDTLVTVSS.

[0024] Among them, the three complementarity determining regions are respectively: CDR1: GIDLSTNS (SEQ ID NO. 2); CDR2: IGGSGST (SEQ ID NO. 3); CDR3: ARLWDF (SEQ ID NO. 4).

[0025] The amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO. 5: AAVLTQTPSPVSAAVGGTVTISCQSVYGNNEQSVYNSDWGQPPKLKASDASTLESGVPSRFRGSGSGTQFTLTISGVQCDDAATYYCASYITDDFGLGGYASTADYAGFGGGTEV.

[0026] Three complementarity determining regions are respectively: CDR1: QSVYGNNE (SEQ ID NO. 6); CDR2: KAS; CDR3: SYITDDF (SEQ ID NO. 7).

[0027] (2) Human framework region (FR) selection In the IMGT human antibody germline gene database, the human FR template with the highest homology to the HZAB_2 antibody is selected for the heavy chain and light chain of the humanized antibody, respectively.

[0028] For the heavy chain of the humanized antibody, the amino acid sequence of the heavy chain variable region of the selected human FR template is shown in SEQ ID NO. 8: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGGINPSNGGTNFNEKFKDRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARERDYRLDYWGQGTLVTVSSGSAST; For the light chain of the humanized antibody, the amino acid sequence of the light chain variable region of the selected human FR template is shown in SEQ ID NO. 9: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKVEIKGGSGSGSGAA.

[0029] (3) CDR grafting The amino acid sequences of the six CDR regions (CDR1, CDR2, CDR3 of the antibody heavy chain variable region; CDR1, CDR2, CDR3 of the antibody light chain variable region) of the aforementioned purified rabbit antibody HZAB_2 are accurately "grafted" onto the selected human FR template framework, respectively, to form the preliminary humanized VH and VL sequences.

[0030] The preliminary humanized antibody heavy chain variable region VH sequence formed is shown in SEQ ID NO. 10: QVQLVQSGAEVKKPGASVKVSCKASGIDLSTNSWVRQAPGQGLEWMGIGGSGSTRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARARLWDFWGQGTLVTVSSGSAST.

[0031] These include: FR1: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO.11), CDR1:GIDLSTNS (SEQ ID NO.2), FR2: WVRQAPGQGLEWMG (SEQ ID NO.12), CDR2: IGGSGST (SEQ ID NO.3), FR3: RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO.13), CDR3: ARLWDF (SEQ ID NO.4), FR4: WGQGTLVTVSSGSAST (SEQ ID NO. 14).

[0032] The preliminary humanized antibody light chain variable region (VL) sequence is shown in SEQ ID NO. 15: DIQMTQSPSSSLSASVGDRVTITCQSVYGNNEWYQQKPGKAPKPLIYKASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSYITDDFFGQGTKVEIKGGSGSGSGAA.

[0033] These include: FR1: DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO.16), CDR1: QSVYGNNE (SEQ ID NO.6), FR2: WYQQKPGKAPKPLIY (SEQ ID NO.17), CDR2: KAS FR3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO.18), CDR3: SYITDDF (SEQ ID NO.7), FR4: FGQGTKVEIKGGSGSGSGAA (SEQ ID NO. 19).

[0034] (4) Reversal mutation 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. huVH:QVQLVQSGAEVKKPGASVKVSCKASGIDLSTNSWVRQAPGQGLEWMGIGGSGSTRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARARLWDFWGQGTLVTLSSGSAST (SEQ ID NO. 20); huVL:DIQMTQSPSSSLSASVGDRVTITCQSVYGNNEWYQQKPGKAPKLLIYKASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSYITDDFFGQGTKVEIKGGSGSGSGAA (SEQ ID NO. 21).

[0035] (5) Sequence optimization 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.

[0036] The codon-optimized gene sequences are shown in SEQ ID NO.22 and SEQ ID NO.23, respectively: huVH: CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCGCGAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCATTGATCTGAGCACCAACAGCTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCATTGGCGGCAGCGGCAGCACCCGCGTGACCCTGACCGTGGATAAAAGCACCAGCACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCGCGCGCCTGTGGGATTTTTGGGGCCAGGGCACCCTGGTGACCCTGAGCAGCGGCAGCGCGAGCACC (SEQ ID NO.22); huVL: GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAGAGCGTGTATGGCAACAACGAATGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAAAGCGAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCAGCTATATTACCGATGATTTTTTTGGCCAGGGCACCAAAGTGGAAATTAAAGGCGGCAGCGGCAGCGGCAGCGGCGCGGCG (SEQ ID NO.23).

[0037] (6) Constant region sequence The constant region of the heavy chain uses the constant region of human IgG1 heavy chain (CH1-CH3), and the amino acid sequence is shown in SEQ ID NO.24: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, 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: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0038] 1.2 Construction of humanized antibody expression vector (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.

[0039] 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.

[0040] 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.

[0041] (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.

[0042] (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).

[0043] 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.

[0044] (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.

[0045] (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.

[0046] 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.

[0047] 1.3 Transient expression and preliminary purification of humanized antibodies 1.3.1 Cell Culture and Transfection: (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%.

[0048] (2) Prepare the DNA-transfection reagent complex: 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.

[0049] 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.

[0050] 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.

[0051] (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.

[0052] 1.3.2 Cultivation and Gains: Six hours or the day after transfection, add feed (expression enhancer such as Valproic Acid) as needed. Collect the culture supernatant after 7 days of culture; Centrifuge at 4000g for 15 minutes at 4℃ to remove cell debris and collect the clear supernatant; 1.3.3 Protein A / G affinity chromatography purification: (1) Equilibrate the Protein A / G affinity chromatography column with 10 column volumes (CV) of PBS.

[0053] (2) Load the clarified cell culture supernatant into the equilibrated column at an appropriate flow rate (1 ml / min).

[0054] (3) Wash the column thoroughly with 15 CV PBS until the baseline stabilizes (A280 is close to zero) to remove unbound contaminants.

[0055] (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).

[0056] (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.

[0057] 1.3.4 Buffer Replacement and Concentration: 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.

[0058] 1.3.5 Western blot validation of humanized antibody expression 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.

[0059] Example 2 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] Example 3 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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 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.20; The light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.

21.

2. The CD33 blocking humanized antibody according to claim 1, 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.24; 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.

3. A method for preparing a CD33-blocking humanized antibody as described in claim 1 or 2, 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 a selected human FR template to form the variable regions of the initial humanized antibody heavy and light chains. S3, Reversal Mutation: Mutate some amino acids in the FR backbone of the variable regions of the heavy and light chains of the preliminary humanized antibody 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.

4. The preparation method according to claim 3, 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.1, and the amino acid sequence of the light chain variable region is shown in 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 in SEQ ID NO.10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

15.

6. The preparation method according to claim 3, 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.20; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

21.

7. The preparation method according to claim 3, 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.22, and the gene sequence of the light chain variable region is shown in SEQ ID NO.

23.

8. The preparation method according to claim 3, characterized in that, In step S5, the heavy chain constant region is selected from the human IgG1 heavy chain constant region, and its amino acid sequence is shown in SEQ ID NO.24; the light chain constant region is selected from the human κ light chain constant region, and its amino acid sequence is shown in SEQ ID NO.

25.

9. The use of a CD33-blocking humanized antibody as described in claim 1 or 2 in the preparation of a medicament for inhibiting liver metastasis of neuroendocrine prostate cancer.

10. The use of a CD33-blocking humanized antibody as described in claim 1 or 2 in the preparation of a drug for inhibiting liver metastasis of small cell lung cancer.

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