DKK1 activity enhanced monoclonal antibody and application thereof

Abstract

The invention provides a DKK1 activity enhanced monoclonal antibody and application thereof, specifically, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises three CDRs, H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises three CDRs, L-CDR1, L-CDR2 and L-CDR3; the amino acid sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are CDR1, CDR2 and CDR3 of a heavy chain variable region as shown in SEQ ID NO: 3; the amino acid sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are CDR1, CDR2 and CDR3 of a light chain variable region as shown in SEQ ID NO: 5.

Description

Technical Field

[0001] The present invention belongs to the field of immunology, and particularly relates to an activity-enhanced monoclonal antibody 3F7 specifically targeting DKK1 protein and applications thereof. Background Art

[0002] Dickkopf-1 (DKK1) is a secreted protein with a molecular weight of approximately 28-30 kDa, primarily expressed in various cell types, including osteoblasts, tumor cells, and various other tissue cells. As a natural inhibitor of the canonical Wnt signaling pathway (Wnt / b-catenin signaling pathway), DKK1 is involved in the pathological processes of various diseases. For example, by inhibiting the Wnt / b-catenin signaling pathway, DKK1 can reduce osteoblast activity and bone matrix synthesis, thereby leading to osteoporosis. Activation of the Wnt / b-catenin signaling pathway is a key mechanism for promoting bone formation and osteocyte proliferation, and therefore, inhibition of DKK1 plays a crucial role in the pathogenesis of osteoporosis. DKK1 expression is elevated in patients with rheumatoid arthritis, and its overexpression can lead to the destruction of articular cartilage and osteocytes, thereby exacerbating joint inflammation and pain. DKK1 also plays a role in the development of neurological diseases, such as stroke, Alzheimer's disease and other neurodegenerative diseases. It may affect the survival and function of neurons by inhibiting the Wnt signaling pathway, thereby promoting neurodegeneration.

[0003] DKK1 expression is elevated in various cancers, including head and neck squamous cell carcinoma, pancreatic cancer, and liver cancer. It may promote tumor progression through multiple mechanisms, including affecting the tumor microenvironment, promoting cancer cell proliferation, inhibiting apoptosis, and participating in tumor cell migration and invasion. Furthermore, DKK1 is associated with chemotherapy resistance in tumors.

[0004] Regarding the development of drugs that inhibit the activity of DKK1 protein, there have been some important advances and representative DKK1 inhibitors. 1) WAY-262611: This is a small molecule DKK1 inhibitor that is used to promote osteogenesis. In a study on an ovariectomized rat model, WAY-262611 was used to induce spinal fusion. Studies have shown that WAY-262611 can enhance Wnt signaling activity and osteogenesis in bone marrow-derived stem / stromal cells (BMSCs) and promote cartilage formation and osteogenesis in vivo. 2) DKN-01: This is an antibody drug that shows good development prospects, especially in patients with high DKK1-expressing tumors or gynecological and malignant esophageal cancers with known Wnt pathway gene mutations.

[0005] While DKK1 expression is upregulated and promotes tumor growth in many types of cancer, studies have also shown that increasing DKK1 levels may have therapeutic effects in certain specific cancer types. For example, in some tumors, abnormal activation of the Wnt signaling pathway promotes cancer development and metastasis, such as colorectal cancer, breast cancer, and prostate cancer. Therefore, in these cases, increasing DKK1 levels or activity may have therapeutic potential to prevent tumor growth by inhibiting the Wnt signaling pathway.

[0006] In addition to its potential role in cancer treatment, increasing the activity or expression of DKK1 in other diseases may also have therapeutic benefits. For example, in some bone overgrowth diseases, such as osteoarthritis and certain forms of osteoarthritis, excessive bone growth may cause pain and limited function. In these cases, inhibiting bone formation by enhancing DKK1 activity may help control the disease. Although DKK1 plays an important role in regulating the Wnt signaling pathway, no drugs specifically targeting DKK1 activity or expression levels are currently under development or on the market.

[0007] Currently, most of the treatments for DKK1 on the market focus on inhibiting its activity, such as the use of anti-DKK1 antibodies or small molecule inhibitors. In cases where precise regulation of the Wnt signaling pathway is required to treat specific diseases, existing drugs, especially small molecule compounds, may not provide sufficient selectivity and effectiveness. In addition, because the Wnt signaling pathway plays an important role in a variety of physiological processes, excessive inhibition of DKK1 may lead to unexpected side effects, such as affecting the regeneration and repair of normal tissues. Existing DKK1 inhibition strategies may find it difficult to balance therapeutic effects and side effects. Finally, existing DKK1 inhibitors are mainly focused on anti-tumor and osteoporosis treatments. However, in certain diseases, such as bone overgrowth diseases or certain central nervous system diseases, it may be necessary to enhance rather than inhibit the activity of DKK1. Summary of the Invention

[0008] The present invention aims to at least solve the technical problems existing in the prior art. To this end, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a DKK1 activity-enhanced antibody or a biologically active fragment derived therefrom that can specifically bind to DKK1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises three CDRs, H-CDR1, H-CDR2, and H-CDR3, and the light chain variable region comprises three CDRs, L-CDR1, L-CDR2, and L-CDR3; the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 are CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 3; and the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 are CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 5.

[0010] In the present invention, the CDRs are defined according to any one or more of the definitions of Kabat, Chothia and ABM.

[0011] According to a specific embodiment of the present invention, the amino acid sequence of the H-CDR1 of the heavy chain variable region is DYSIH (SEQ ID NO: 6), the amino acid sequence of the H-CDR2 of the heavy chain variable region is WINTETGEPTCADDFKG (SEQ ID NO: 7), the amino acid sequence of the H-CDR3 of the heavy chain variable region is DGYYALYAMDY (SEQ ID NO: 8), the amino acid sequence of the L-CDR1 of the light chain variable region is HASQNINVWLN (SEQ ID NO: 9), the amino acid sequence of the L-CDR2 of the light chain variable region is KASNLHT (SEQ ID NO: 10), and the amino acid sequence of the L-CDR3 of the light chain variable region is QQGQSYPFT (SEQ ID NO: 11).

[0012] As a preferred embodiment, the antibody is an animal-derived antibody, a chimeric antibody or a humanized antibody.

[0013] As a preferred embodiment, the antibody is a monoclonal antibody.

[0014] In a second aspect, the present invention provides a polynucleotide encoding the antibody of claim 1 or a biologically active fragment derived from the antibody that is capable of specifically binding to DKK1.

[0015] According to a specific embodiment of the present invention, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0016] In a third aspect, the present invention provides a vector comprising the polynucleotide according to the second aspect of the present invention.

[0017] In a fourth aspect, the present invention provides a cell comprising the polynucleotide according to the second aspect of the present invention or the vector according to the third aspect of the present invention.

[0018] In a fifth aspect, the present invention provides a conjugate comprising an antibody and a conjugated moiety, wherein the antibody is the antibody according to the first aspect of the present invention or a biologically active fragment thereof that can specifically bind to DKK1, and the conjugated moiety is a detectable label.

[0019] In the present invention, the coupling part is a cytotoxin, an immunomodulator, a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

[0020] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the antibody according to the first aspect of the present invention or a biologically active fragment thereof capable of specifically binding to DKK1, or the conjugate according to the fifth aspect of the present invention.

[0021] As a preferred embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients (such as carriers and / or excipients).

[0022] In a seventh aspect, the present invention provides a pharmaceutical preparation comprising the pharmaceutical composition described in the sixth aspect of the present invention.

[0023] In an eighth aspect, the present invention provides the use of the antibody of the first aspect of the present invention or a biologically active fragment thereof capable of specifically binding to DKK1, the polynucleotide of the second aspect of the present invention, the vector of the third aspect of the present invention, the cell of the fourth aspect of the present invention, the conjugate of the fifth aspect of the present invention, the pharmaceutical composition of the sixth aspect of the present invention, or the pharmaceutical preparation of the seventh aspect of the present invention in any of the following: (1) preparing a drug for treating and / or preventing a disease mediated by DKK1;

[0024] (2) preparing a kit for detecting DKK1;

[0025] (3) DKK1 testing for non-diagnostic purposes;

[0026] As a preferred embodiment, the DKK1-mediated diseases include tumors and bone-related diseases.

[0027] As a preferred embodiment, the tumor includes colorectal cancer, breast cancer, and prostate cancer.

[0028] Beneficial effects:

[0029] This invention utilizes hybridoma technology to generate the monoclonal antibody 3F7, which specifically recognizes and enhances the activity of the DKK1 protein. Unlike traditional neutralizing antibodies, this antibody enhances the inhibitory effect of DKK1 on the Wnt / β-catenin signaling pathway. This effect may have therapeutic benefits for diseases associated with abnormal activation of the Wnt / β-catenin signaling pathway, such as cancer, bone-related diseases, tissue repair, and regenerative medicine.

[0030] The antibody of the present invention exhibits a different mechanism of action from the prior art, namely, promoting the function of DKK1 by interacting with the DKK1 protein.

[0031] The antibodies of the present invention offer a novel solution. Their development aims to enhance, rather than inhibit, DKK1 activity, providing more options and possibilities for treating diseases requiring Wnt signaling pathway modulation. The uniqueness of these antibodies lies in their ability to specifically bind to DKK1 and promote its function, which is of significant value in treating diseases that are ineffective with existing technologies. Furthermore, by precisely regulating DKK1 activity, the antibodies of the present invention are expected to reduce side effects during treatment, improving safety and efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The effect of DKK1 protein monoclonal antibody 3F7 on the transcription level of AXIN2 in HEK 293T cells. DETAILED DESCRIPTION

[0033] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0034] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0035] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in this technical field.

[0036] Example

[0037] 1. DKK1 protein immunization of mice: Human DKK1 recombinant protein was used as an immunogen to immunize four BALB / c mice. 23 days after immunization, the tail blood serum test results of the four mice are shown in Table 1. Among them, the serum titer of mouse #1 was the highest, and the titer of recognizing human DKK1 protein reached 1 / 10000, and cell fusion was performed.

[0038] The recombinant human DKK1 (Accession #094907) protein used as the immunogen was expressed and purified by Shanghai Jinan Technology Co., Ltd. (without the signal peptide): TLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQTIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCMRHAMCCPGNYCKNGICVSSDONHFRGEIEETITESFGNDHSTLDGYSRRTTLSSKMYHKGQEGSYCLRSSDCASGLCCARHFWSKICKPVLKEGOVCTKHRRKGSHGLEIFORCYCGEGLSCRIQKDHHOASNSSRLHTCQRH (SEQ ID NO: 1).

[0039] Table 1: Tail blood test results of mice immunized with Human DKK1 protein

[0040]

[0041] Note: NC is the negative control, NaN indicates OD>4.0.

[0042] 2. Primary Cell Fusion Screening: Based on the results of human DKK1 protein identification in the monoclonal cell supernatant, positive clones with an OD > 1.0 were selected. One positive clone was selected from 564 clones in six 96-well plates for secondary screening. The results of the primary clone screening are shown in Table 2.

[0043] Table 2: Cell fusion initial screening test results

[0044]

[0045]

[0046]

[0047] Note: NC is the negative control, PC is 1# mouse heart serum (04 / 18 / 2020), and NaN indicates OD>4.0.

[0048] 3. Cell fusion rescreening: Clone 3F7, which was positive in the initial screening, was rescreened. The rescreening results are shown in Table 3. Clone 3F7, which was positive in the initial screening, was still positive in the rescreening.

[0049] Table 3: Cell fusion rescreening test results

[0050]

[0051] Note: NC is the negative control, PC is 1# mouse heart serum (04 / 18 / 2020), and NaN indicates OD>4.0.

[0052] 4. Specificity Testing of Cell Fusion Clone: ​​Mouse ascites fluid was prepared from the positive clone 3F7, and the ascites fluid was purified using Protein G to obtain a mouse monoclonal antibody. Mouse monoclonal antibody 3F7 was then coated with human and mouse DKK1, DKK2, DKK3, and DKK4 proteins for specificity testing. The test results are shown in Table 4. Mouse monoclonal antibody 3F7 recognized human and mouse DKK1 protein with a sensitivity of 0.005 μg / mL, but did not recognize human and mouse DKK2, DKK3, or DKK4 proteins.

[0053] Table 4: Cell fusion clone specificity detection results

[0054]

[0055] Note: NC is the negative control, NaN indicates OD>4.0.

[0056] 5. Bioactivity detection of DKK1 protein monoclonal antibody 3F7: L cells overexpressing human Wnt3a protein were used for 24 hours of culture. The supernatant was mixed with DMEM medium in an equal ratio (1:1) as conditioned medium (Wnt3a CM) and added to HEK 293T cells. Figure 1 As shown in Figure 2, qPCR detection revealed that the transcription level of AXIN2, a target gene of the Wnt / β-catenin signaling pathway, was significantly increased ( *** P < 0.001), indicating that the pathway activity was increased by Wnt3a CM. If DKK1 protein (200 ng / ml) was added simultaneously with Wnt3a CM, the transcription level of AXIN2 was significantly reduced ( ** P < 0.01). If 3F7 was added at a molar ratio of 5:1 along with DKK1 protein (3F7:DKK1 = 5:1), the transcription level of AXIN2 was significantly lower than that of the control IgG ( * P < 0.05), suggesting that 3F7 can significantly increase the inhibitory effect of DKK1 protein on the Wnt / β-catenin signaling pathway. Figure 1 As shown in the figure, n=3, *P<0.05, One-way ANOVA.

[0057] 6. Gene and amino acid sequencing of DKK1 protein monoclonal antibody 3F7

[0058] After immunization, fusion, monoclonalization, antibody purification, and antibody specificity and bioactivity testing, total RNA from the 3F7 monoclonal antibody cell line was extracted and reverse-transcribed into cDNA. The cDNA was then used as a template for PCR amplification of the heavy and light chain variable regions of the antibody. Total RNA from the 3F7 monoclonal antibody cell line was extracted using the Invitrogen TRIzol reagent kit (15596-026) according to the manufacturer's instructions. VH and VL antibody fragments were then amplified using the Takara PrimeScript™ 1st Strand cDNA Synthesis Kit (Cat#2690A) according to the standard protocol from Biointron Biologi- cals and cloned into the TA / Blunt-Zero Cloning vector. Clones were screened using colony PCR, and at least three positive clones were sequenced.

[0059] The sequencing results are:

[0060] Heavy chain (leader sequence-FR1+CDR1+FR2+CDR2+FR3+CDR3+FR4)

[0061] 1) DNA sequence: 417 bp

[0062] ATGGCTTGGGTGTGGACCTTGCTATTCCTGATGGCAGCTGCCCAAAGTATCCAAGCACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTAGTTATACCTTCACAGACTATTCAATACACTGGGTGATGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATGTGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGAGATGGTTACTACGCCCTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:2) Amino acid sequence: 139aa

[0063] MAWVWTLLFLMAAAQSIQAQIQLVQSGPELKKPGETVKISCKASSYTFTDYSIHWVMQAPGKGLKWMGWINTETGEPTCADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDGYYALYAMDYWGQGTSVTVSS(SEQID NO:3)

[0064] Light chain (leader sequence - FR1 + CDR1 + FR2 + CDR2 + FR3 + CDR3 + FR4)

[0065] 1) DNA sequence: 381bp

[0066] ATGAGGGTCCTTGCTGAGCTCCTGGGGCTGCTGCTGTTCTGCTTTTTAGGTGTGAGATGTGACATCCAGATGAACCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTAAACTGGTACCAGCAGAAACCAGGAAATATTCCT AAACTATTGATCTATAAGGCTTCCAACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTAACCATCAGCAGCCTGCAGCCTGAAGACATTGGCACTTTCTACTGTCAACAGGGTCAAAGTTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA(SEQ ID NO:4)

[0067] 2) Amino acid sequence: 127aa

[0068] MRVLAELLGLLLFCFLGVRCDIQMNQSPSSLSASLGDTITITCHASQNINVWLN WYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIGTFYCQQG QSYPFTFGSGTKLEIK(SEQ ID NO:5)

[0069] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A DKK1 activity-enhancing antibody or a biologically active fragment thereof that is capable of specifically binding to DKK1, wherein: The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDRs, H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises three CDRs, L-CDR1, L-CDR2 and L-CDR3; The amino acid sequences of the H-CDR1, H-CDR2, and H-CDR3 are CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 3; The amino acid sequences of the L-CDR1, L-CDR2, and L-CDR3 are the CDR1, CDR2, and CDR3 of the light chain variable region shown in SEQ ID NO:

5.

2. The antibody according to claim 1 or a biologically active fragment thereof capable of specifically binding to DKK1, wherein The CDRs are defined according to any one or more of the definitions of Kabat, Chothia and ABM.

3. The antibody according to claim 1 or a biologically active fragment thereof capable of specifically binding to DKK1, wherein The amino acid sequence of H-CDR1 in the heavy chain variable region is DYSIH (SEQ ID NO: 6), The amino acid sequence of the H-CDR2 of the heavy chain variable region is WINTETGEPTCADDFKG (SEQ ID NO: 7), The amino acid sequence of H-CDR3 of the heavy chain variable region is DGYYALYAMDY (SEQ ID NO: 8), The amino acid sequence of L-CDR1 of the light chain variable region is HASQNINVWLN (SEQ ID NO: 9), The amino acid sequence of L-CDR2 of the light chain variable region is KASNLHT (SEQ ID NO: 10), and the amino acid sequence of L-CDR3 of the light chain variable region is QQGQSYPFT (SEQ ID NO: 11). Preferably, the antibody is an animal-derived antibody, a chimeric antibody or a humanized antibody.

4. A polynucleotide encoding the antibody according to any one of claims 1 to 3 or a biologically active fragment thereof that is capable of specifically binding to DKK1; Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO:

4.

5. A vector comprising the polynucleotide according to claim 4.

6. A cell comprising the polynucleotide of claim 4 or the vector of claim 5.

7. A conjugate comprising an antibody and a conjugated moiety, wherein: The antibody is the antibody according to any one of claims 1 to 3 or a biologically active fragment thereof that can specifically bind to DKK1, and the coupling portion is a detectable label. Preferably, the coupling moiety is a cytotoxin, an immunomodulator, a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

8. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 3 or a biologically active fragment thereof capable of specifically binding to DKK1, or the conjugate according to claim 7. Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

9. A pharmaceutical preparation comprising the pharmaceutical composition according to claim 8.

10. Use of the antibody according to any one of claims 1 to 3 or a biologically active fragment thereof capable of specifically binding to DKK1, the polynucleotide according to claim 4, the vector according to claim 5, the cell according to claim 6, the conjugate according to claim 7, the pharmaceutical composition according to claim 8, or the pharmaceutical preparation according to claim 9 in any one of the following: (1) preparing a drug for treating and / or preventing a disease mediated by DKK1; (2) preparing a kit for detecting DKK1; (3) DKK1 testing for non-diagnostic purposes; Preferably, the DKK1-mediated diseases include tumors and bone-related diseases. Preferably, the tumor includes colorectal cancer, breast cancer, and prostate cancer.

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