A cbln1 humanized antibody and uses thereof

Abstract

This invention discloses a humanized CBLN1 antibody and its uses. The humanized CBLN1 antibody comprises 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. 7; the light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO. 18. The use of the humanized CBLN1 antibody in the preparation of a drug for inhibiting prostate cancer metastasis is also disclosed. Furthermore, an antibody composition comprising the humanized CBLN1 antibody and a PD1 antibody is disclosed, and its use in the preparation of a drug for treating immunosuppressive prostate cancer is also disclosed. The neurocerebellar peptide CBLN1 humanized antibody of this invention has a more significant function in slowing the progression of prostate cancer and can significantly improve the response rate of prostate cancer to immunotherapy αPD1 antibody, thereby improving the treatment effect for patients with immunosuppressive prostate cancer.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a humanized CBLN1 antibody and its uses. Background Technology

[0002] Studies have shown that neural tissue plays a crucial role in tumor initiation, proliferation, and invasion / metastasis, and surgical resection or pharmacological blocking of nerves can significantly inhibit tumor progression. Currently, there are few neuropharmaceuticals used in tumor treatment, and most target classical neurotransmitter signaling axes, leading to numerous side effects in early clinical trials. Neuropeptides, however, possess advantages such as diversity and good tissue specificity, making them a promising option for targeted tumor therapy. Currently, there are no studies exploring the potential role of neuropeptides in immunotherapy tolerance and tumor metastasis.

[0003] CBLN1, a neurocerebellar peptide secreted by cerebellar granule cells, is a highly homologous secreted protein found in both humans and mice. It plays a crucial role in synapse formation, functional maintenance, and neural network construction by forming a supercomplex with proteins such as neural adhesion molecules. We found a positive correlation between intratumoral innervation and the inhibitory nature of the immune microenvironment, and CBLN1 protein was significantly enriched in patients unresponsive to immunotherapy. Furthermore, the objective response rate (ORR) of immune checkpoint therapy was low in patients with metastatic castration-resistant prostate cancer (mCRPC).

[0004] Currently, there are no studies on CBLN1 in the context of immunotherapy resistance and metastatic malignant tumors, and there are no CBLN1 humanized antibodies on the market with functional blocking effects. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a humanized CBLN1 antibody and its uses. Based on clinical needs in tumor treatment, CBLN1 has been found to be significantly enriched in patients resistant to immunotherapy and to promote tumor metastasis, making it a potential target for improving the response rate of immune checkpoint therapy and treating tumor metastasis. By using genetic engineering techniques to obtain a humanized CBLN1 antibody, and through mouse prostate cancer xenograft treatment experiments, the present invention provides a novel use of the humanized CBLN1 antibody ZH003 in improving the response rate of tumor immune checkpoint therapy and treating tumor metastasis (taking prostate cancer as an example).

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a humanized antibody against CBLN1, comprising a heavy chain and a light chain;

[0008] The heavy chain includes a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.7;

[0009] The light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.18.

[0010] As some specific embodiments of the present invention, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.15.

[0011] As some specific embodiments of the present invention, the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.25.

[0012] 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.16.

[0013] Furthermore, the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO.17.

[0014] As some specific embodiments of the present invention, the light chain further includes a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.26.

[0015] Furthermore, the nucleotide sequence of the light chain constant region is shown in SEQ ID NO.27.

[0016] In a second aspect, the present invention provides the use of the humanized CBLN1 antibody as described in any of the preceding claims in the preparation of a medicament for inhibiting prostate cancer metastasis.

[0017] Thirdly, the present invention provides an antibody composition comprising the humanized CBLN1 antibody described in any one of the preceding claims, and a PD1 antibody.

[0018] Fourthly, the present invention provides the use of the antibody composition described above in the preparation of a medicament for treating prostate cancer.

[0019] This invention involves intraperitoneal injection of the CBLN1 monoclonal antibody ZH003 into humanized tumor-bearing mice, followed by analysis of tumor size and mass to clarify the potential use of the humanized CBLN1 antibody ZH003 in enhancing the response rate of tumor immune checkpoint therapy. Furthermore, humanized mice were injected with a prostate cancer cell line into their hearts, followed by intraperitoneal injection of the humanized CBLN1 antibody ZH003. In vivo imaging of small animals was used to analyze the metastatic lesions, clarifying the potential use of the humanized CBLN1 antibody ZH003 in treating tumor metastases.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The humanized antibody ZH003 for neurocerebellar peptide CBLN1 of the present invention has the effect of specifically recognizing neurocerebellar peptide CBLN1 and has a strong affinity for neurocerebellar peptide CBLN1.

[0022] (2) This invention provides the possibility of CBLN1 as an effective target for improving the response rate of immunotherapy for prostate cancer and anti-tumor metastasis. Compared with the mouse CBLN1 antibody, the humanized CBLN1 antibody ZH003 of this invention has a more significant function of slowing down the progression of prostate cancer and can significantly improve the response rate of prostate cancer to immunotherapy αPD1 antibody, which can improve the treatment effect of immunosuppressive prostate cancer patients.

[0023] (3) The humanized antibody ZH003 of the neurocerebellar peptide CBLN1 of the present invention can inhibit the metastasis of prostate cancer and has shown a new use in the treatment of tumor metastasis. Attached Figure Description

[0024] 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:

[0025] Figure 1 This is an electrophoresis result of the quality test of the humanized CBLN1 antibody ZH003 in Example 1;

[0026] Figure 2 This is a diagram showing the results of the humanized antibody ZH003 and the initial mouse antibody specifically recognizing the CBLN1 protein in Example 2;

[0027] Figure 3 The image shows the synergistic effect of the humanized CBLN1 antibody ZH003 and the mouse antibody with the immunotherapy PD1 antibody in Example 3; the left image is a tumor morphology image, and the right image is a tumor weight statistics image.

[0028] Figure 4 The figure shows the mouse experimental results of the inhibition of tumor metastasis by the humanized CBLN1 antibody ZH003 and the mouse antibody in Example 3. Detailed Implementation

[0029] 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.

[0030] Example 1: Preparation of CBLN1 humanized antibody ZH003

[0031] 1. Initial murine antibody

[0032] Using the CBLN1 monoclonal antibody ZH-DT7792 from patent CN119264251B as the initial murine antibody, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.1:

[0033] EVQLQQSGAEVVRSGASVELSCTASGFNIKDYFMHWVKQRPEQGLEWIGWIDPANGNTKYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNAFHYGHYGRGYAVDYWGQGISVTVSS;

[0034] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2:

[0035] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSSEGTDFTLKISRVEAEDLGVYFCSQNTHVPWTFGSGTKLEIK.

[0036] The heavy and light chain sequences were input into AlphaFold2 to construct the antibody structure in multimer mode. The antibody model was then input into GROMACS for energy minimization, and a new antibody structure was output. The amino acid sequences in the antibody structure model were renumbered using the Kabat nomenclature scheme.

[0037] 2. Select human antibody Germline gene

[0038] The variable region sequences of the heavy and light chains from mouse sources were distinguished as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 according to the Kabat nomenclature scheme.

[0039] Antibody Germline templates were obtained using the intact variable region, FR1+FR2+FR3+FR4, and CDR1+CDR2+CDR3, respectively. Three sets of antibody Germline templates were obtained from the heavy chain and light chain variable regions, respectively.

[0040] Mouse antibody heavy chain FR1+FR2+FR3+FR4 sequence (SEQ ID NO.3):

[0041] EVQLQQSGAEVVRSGASVELSCTASMHWVKQRPEQGLEWIGWKYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCWGQGISVTVSS;

[0042] Mouse antibody heavy chain CDR1+CDR2+CDR3 sequence (SEQ ID NO.4):

[0043] GFNIKDYFIDPANGNTNAFHYGHYGRGYAVDY;

[0044] Mouse antibody light chain FR1+FR2+FR3+FR4 sequence (SEQ ID NO.5):

[0045] DVVMTQTPLSLPVSLGDQASISCRSSLHWYLQKPGQSPKLLIYNRFSGVPDRFSGSSEGTDFTLKISRVEAEDLGVYFCFGSGTKLEIK;

[0046] Mouse antibody light chain CDR1+CDR2+CDR3 sequence (SEQ ID NO.6):

[0047] QSLVHSNGNTYKVSSQNTHVPWT.

[0048] 3. CDR porting

[0049] Under the same Kabat nomenclature scheme, the CDR of the murine antibody was transplanted to the CDR position of the corresponding human template Germline to construct a basic humanization framework.

[0050] 4. Generate a reverse mutation sequence

[0051] Based on the key amino acid positions reported in the paper Foote J, Winter G. Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 1992 Mar 20;224(2):487-99. doi: 10.1016 / 0022-2836(92)91010-m. PMID: 1560463, some murine amino acids were selectively retained to maintain the stability and binding activity of the CDR conformation, thus forming an optimized humanized framework.

[0052] 5. Energy calculation and sequence selection

[0053] All heavy and light chain combinations generated from the above two steps were systematically evaluated and compared with the initial murine CBLN1 monoclonal antibody ZH-DT7792 structure. First, the energy change was assessed based on each differential amino acid. Amino acids meeting the energy change requirements were selected and their combinations optimized, ultimately choosing the sequence corresponding to the most stable structure. A balance was struck between energy and humanization level to finally select candidate sequences.

[0054] The final selected humanized CBLN1 monoclonal antibody ZH003 sequence is shown below:

[0055] (1) Heavy chain

[0056] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7:

[0057] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYFIHWVRQAPGQGLEWIGWIDPANGNTKYDPKFQGRATITADTSASTAYMELSSLRSEDTAVYYCNAFHYGHYGRGYAVDYWGQGTLVTVSS;

[0058] The three complementary determinant regions are as follows:

[0059] CDR1:GFNIKDYF (SEQ ID NO.8),

[0060] CDR2: IDPANGNT (SEQ ID NO.9),

[0061] CDR3: NAFHYGHYGRGYAVDY (SEQ ID NO.10);

[0062] The four skeletal regions are as follows:

[0063] FR1: QVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO.11),

[0064] FR2: IHWVRQAPGQGLEWIGW (SEQ ID NO.12),

[0065] FR3:KYDPKFQGRATITADTSASTAYMELSSLRSEDTAVYYC (SEQ ID NO.13),

[0066] FR4: WGQGTLVTVSS (SEQ ID NO.14);

[0067] The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.15:

[0068] CAGGGTGCAGCTGGTGCAGAGCGGAGCAGAAGTGAAGAAGCCCGGAGCTTCCGTGAAGGTGTCTTGCAAGGCCTCCGGCTTCAACATCAAGGACTACTTCATCCATTGGGTCCGACAGGCTCCAGGACAGGGACTCGAGTGGATCGGTTGGATCGACCCAGCCAACGGCAACACCAAGTACGACCC CAAGTTCCAGGGCAGAGCTACCATCACCGCCGATACCTCCGCTTCTACCGCTTACATGGAGCTGAGCTCTCTGCGGAGCGAGGATACCGCCGTGTACTATTGCAACGCCTTCCACTACGGCCACTACGGCCGAGGCTACCGCCGTGGACTATTGGGGCCAGGGAACCCTGGTGACAGTGTCTTCC.

[0069] The heavy chain constant region is the heavy chain constant region of human IgG1 antibody, and its amino acid sequence is shown in SEQ ID NO.16:

[0070] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0071] The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO.17:

[0072] GCTAGCACCAAGGGCCCTAGCGTGTTTCCTCTGGCCCCTTCTAGCAAGTCTACAAGCGGCGGAACAGCCGCTCTGGGTTGCCTGGTGAAAGACTACTTCCCCGAGCCAGTGACCGTGTCTTGGAATAGCGGAGCCCTGACCAGCGGAGTGCATACCTTTCCAGCCGTGCTGCAGAGCTCAGGACTGTACAGCCTGTCCAGCGTGGTGACAGTGCCTTCTAGCAGCCTGGGCACACAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACTTGCCCTCCTTGTCCAGCTCCAGAACTGCTGGGAGGACCAAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGACTTGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAGGTCAAGTTCAATTGGTACGTGGACGGAGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTGCCAGCCCCCATCGAGAAGACCATCAGCAAGGCTAAGGGCCAGCCTAGAGAACCTCAGGTGTACACCCTGCCCCCTAGCAGAGACGAGCTGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAATCTAACGGCCAGCCAGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCCGCTGGCAGCAGGGCAACGTGTTCTCTTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCAGGAAAG。

[0073] (2) Light chain

[0074] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.18:

[0075] DVVMTQSPLSLPVTLGQQASISCRSSQSLVHSNGNTYLHWFQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQNTHVPWTFGQGTKVEIK;

[0076] The three complementary determinant regions are as follows:

[0077] CDR1: QSLVHSNGNTY (SEQ ID NO.19),

[0078] CDR2: KVS

[0079] CDR3: SQNTHVPWT (SEQ ID NO.20);

[0080] The four skeletal regions are as follows:

[0081] FR1: DVVMTQSPLSLPVTLGQQASISCRSS (SEQ ID NO.21),

[0082] FR2: LHWFQQRPGQSPRLLIY (SEQ ID NO.22),

[0083] FR3: NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC (SEQ ID NO.23),

[0084] FR4: FGQGTKVEIK (SEQ ID NO.24);

[0085] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.25:

[0086] GACGTCGTGATGACCCAGTCCCCTCTGTCTCTGCCAGTGACACTGGGACAGCAGGCTTCTATCAGCTGCAGATCCTCCCAGTCCCTGGTGCACTCCAACGGCAACACCTACCTGCATTGGTTCCAGCAGAGACCAGGCCAGTCTCCTAGGCTGCTGATCTACAAGGTG TCCAACCGGTTCTCCGGCGTGCCAGACAGATTTTCCGGCTCTGGATCCGGCACCGACTTCACCCTGAAGATCTCTAGAGTGGAGGCAGAAGACGTGGGCGTGTACTTCTGCTCCCAGAACACCCACGTGCCTTGGACATTCGGCCAGGGAACCAAGGTGGAGATCAAG.

[0087] The constant region of the light chain is the constant region of the human κ-type light chain, and its amino acid sequence is shown in SEQ ID NO.26:

[0088] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0089] The nucleotide sequence of the light chain constant region is shown in SEQ ID NO.27:

[0090] AGAACCGTGGCCGCCCCTAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAAAGCGGAACAGCCAGCGTCGTCTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAAGTCCAGTGGAAGGTGGACAACGCTCTGCAGAGCGGCAACTCTCAGGA GAGCGTGACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCTTGCGAGGTCACACACCAGGGACTGTCTAGCCCAGTGACCAAGAGCTTCAACCGCGGCGAGTGT.

[0091] 6. Express and purify antibodies

[0092] (1) Construction of humanized antibody expression vector:

[0093] The light and heavy chain amino acid sequences of the humanized antibody were optimized using the MaxCodon™ Optimization Program (V13) codon optimization software. The antibody light and heavy chain genes were synthesized as a whole and inserted into the expression vector pcDNA3.4 by double enzyme digestion (enzyme sites: EcoRI / BamHI). The successful construction of the expression vector was confirmed by enzyme digestion and sequencing. The vector was then transformed into the JM108 cloning strain, and plasmids were extracted.

[0094] (2) Transformation and culture of expression vectors:

[0095] Heavy and light chain expression plasmids containing the humanized antibody gene were added to prepared JM108 competent cells. After heat shock transformation, the transformed competent cells were added to LB liquid medium and incubated at 37°C with shaking for approximately 30 min. The cultured competent cells were then removed, and a portion of the suspension was spread onto LB agar plates containing ampicillin, and incubated overnight at 37°C. Single clones were picked from fresh culture plates and cultured in LB liquid medium at 37°C and 200 rpm for 8 h. The cultured cells were then inoculated at a 1 / 500 ratio into LB liquid medium and incubated overnight at 37°C. The cultured bacterial suspension was collected, centrifuged, and the supernatant was discarded for plasmid extraction to obtain the target plasmid.

[0096] (3) Antibody purification and identification

[0097] Transfected 293 cells in suspension culture. Cell seeding density was 2.0 × 10⁻⁶. 6 Cells / mL were cultured in suspension in an incubator. On the day of transfection, cells grew to 3.0-5.0 × 10⁶ cells / mL. 6 The cells / mL were diluted with fresh 293 cell culture medium to a density of 3.0 × 10⁻⁶ cells / mL. 6 cells / mL.

[0098] Perform transfection. Add DNA (light chain to heavy chain plasmid ratio of 1:1) and polyethyleneimine transfection reagent, mix well, and incubate at 37°C. Add the DNA-transfection reagent mixture to the cells to be transfected, and incubate at 37°C with 110 rpm and 8% CO2.

[0099] 24 hours after transfection, 5% feed was added for continued culture. Approximately 4-6 days post-transfection, the cell culture was harvested, centrifuged, and the supernatant or cells were collected. The supernatant was filtered through a 0.22 μm filter, dialyzed to buffer at 4°C, and then purified using a Protein A column. Samples with relatively high purity were collected and dialyzed into PBS buffer solution containing 5% Glycerol at 10 mM and pH 7.4. The samples were then aliquoted and frozen at -80°C.

[0100] The quality of the purified antibody was detected by SDS-PAGE gel electrophoresis, and the results are as follows: Figure 1 As shown. Figure 1 In the diagram, Lane M is the SDS-PAGE marker, Lane 5 is BSA, and Lanes 6 and 7 are the reduced and non-reduced humanized antibodies against ZH003, respectively. The results indicate that the ZH003 antibody has high purity.

[0101] Example 2: Identification of CBLN1 humanized antibody ZH003

[0102] Recombinant human CBLN1 protein: In the previous patent CN119264251B, the full-length sequence of human CBLN1 protein was used as the target sequence. By adding a signal peptide and a C-His tag, and using HEK293 / CHO as the expression host cell, the recombinant CBLN1 protein was obtained through codon optimization and gene synthesis, expression, and purification. Its amino acid sequence is shown in SEQ ID NO.28.

[0103] MLGVLELLLLGAAWLAGPARGQNETEPIVLEGKCLVVCDSNPTSDPTGTALGISVRSGSAKVAFSAIRSTNHEPSEMSNRTMIIYFDQVLVNIGNNFDSERSTFIAPRKGIYSFNFHVVKVYNRQTIQVSLMLNGWPVISAFAGDQDVTREAASNGVLIQMEKGDRAYLKLERGNLMGGWKYSTFSGFLVFPLHHHHHH.

[0104] Dilute the above-mentioned human CBLN1 recombinant protein to 2 μg / ml with coating buffer (carbonate buffer, pH 9.6) and coat overnight. Discard the coating solution, wash the plate three times with PBST, 200 μL each time, and blot dry. Add 200 μL of 5% skim milk powder to each well as blocking solution and incubate at 37 ℃ for 1 h.

[0105] Add 100 μL of sample to each well (ZH003 purified antibody and initial mouse antibody were diluted according to a concentration gradient; initial mouse antibody ZH-DT7792 was used as a positive control, and PBS was used as a negative control), with two replicates, and incubate at 37 ℃ for 1 h. After washing the plate three times, add 100 μL of mAb-D-HRP working solution (1:2000, pre-diluted with blocking buffer) to each well and incubate at 37 ℃ for 1 h. After washing the plate five times, add 100 μL of TMB substrate solution to each well and react at room temperature in the dark for 7–10 min. Add 50 μL of 0.64 M H2SO4 stop solution to each well and immediately read the absorbance at 450 nm.

[0106] The results are as follows Figure 2 As shown, this indicates that ZH003 has a high affinity for the CBLN1 protein.

[0107] Example 3: Therapeutic applications of ZH003 antibody in slowing prostate cancer metastasis and improving immunotherapy response rate.

[0108] 1. Human prostate cancer cells (PC3) were subcutaneously inoculated into humanized mice (a highly immunodeficient mouse model of functional human hematopoietic-immune system reconstructed in vivo, which can simulate human immune response and disease mechanisms at the in vivo level). When the subcutaneous tumor was visibly palpable (approximately 10 days after inoculation), the mice were randomly divided into a control IgG group, an αPD1 immunotherapy group, a mouse antibody ZH-DT7792 + αPD1 treatment group, and a ZH003 antibody + PD1 antibody treatment 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 for evaluation by observing tumor size and mass.

[0109] The results are as follows Figure 3 As shown, the left image is a tumor morphology diagram, and the right image is a tumor weight statistics diagram. Figure 3 The results showed that in a humanized mouse tumor-bearing model, the humanized antibody ZH003 exhibited a more significant effect in slowing the progression of immunosuppressive prostate cancer than the mouse antibody, and could significantly improve the response rate of immunosuppressive prostate cancer to the immunotherapy αPD1 antibody. Clinically, prostate cancer patients respond poorly to immunotherapy αPD1, with almost no response. However, the humanized CBLN1 antibody ZH003 of this invention can exert a synergistic effect with PD1, which can improve the treatment effect of immunosuppressive prostate cancer patients.

[0110] 2. Human prostate cancer cells (PC3-luciferase) were injected cardiacally into humanized mice. One week after inoculation, the mice were randomly divided into a control IgG group, a mouse antibody ZH-DT7792 treatment group, and a ZH003 antibody treatment group. The dosage and frequency of administration were 10 mg / kg, twice a week, for 3 weeks. The health status of the mice was observed regularly during this period. In vivo imaging was performed on the mice 3 weeks after inoculation, and the treatment effect was evaluated based on the intensity of the imaging signal. The results are as follows: Figure 4 As shown, this indicates that ZH003 can slow down and inhibit the metastasis of prostate cancer in a humanized mouse metastasis model.

[0111] 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 CBLN1 humanized antibody, characterized in that, 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. 7; 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.

18.

2. The CBLNl 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.

16.

3. The CBLNl humanized antibody of claim 1, wherein, 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.

26.

4. Use of the CBLN1 humanized antibody according to any one of claims 1-3 in the preparation of a drug for inhibiting the metastasis of prostate cancer.

5. An antibody composition comprising the CBLN1 humanized antibody according to any one of claims 1-3, and a PD1 antibody.

6. Use of the antibody composition according to claim 5 in the preparation of a drug for treating immunosuppressive prostate cancer.

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