Targeted protein degradation and uses thereof

Abstract

The application provides a targeted degradation protein and application thereof, and belongs to the technical field of biological medicine. The amino acid sequence of the targeted degradation protein is at least one of SEQ ID NO. 3, 5, 9, 11, 13, 15 and 17. When the breast cancer is treated by using the targeted degradation protein, the targeted protein can be directly mediated to be degraded, so that the effect of the targeted protein is weakened at the root, the targeted degradation protein only needs to have strong affinity with the targeted protein, does not need to combine with a specific epitope, has low design difficulty and is suitable for many target points. It can be seen that the targeted degradation protein provided by the application takes a tumor-specific high-expression membrane molecule as an effector protein, so that the double effects of tumor targeting and endocytosis mediation are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a targeted degradation protein and application thereof. BACKGROUND

[0002] Extracellular soluble proteins are the core of various cell functions and the targets of many drugs, but there is currently a lack of suitable methods to block extracellular proteins. Therefore, developing strategies to degrade extracellular proteins has special significance for both basic research and therapeutic intervention purposes.

[0003] Compared with traditional small molecule inhibitor-based therapies, an ideal treatment should have low toxicity and high efficacy. The following features help improve overall efficacy; such as not completely dependent on continuous target binding; can completely or partially limit the function of the target protein; and the target cell cannot weaken the efficacy by overexpression of the target protein, natural ligand competition or limiting binding. Targeted protein degradation is a rapidly developing field in drug discovery, which provides a novel therapeutic mechanism as a supplement to traditional drug models, and can solve challenging targets or increase the therapeutic potential of currently used drugs.

[0004] Existing targeted protein degradation focuses more on intracellular proteins and membrane proteins, and less on extracellular soluble proteins. Existing extracellular targeted protein degradation (eTPD) strategies mostly use bispecific molecules to simultaneously bind soluble target proteins and "effector" proteins, so that the target protein is endocytosed into the cell with the "effector" protein, and then degraded in the lysosome. Traditional "effector" proteins include membrane E3 ubiquitin ligases (named PROTAB), membrane cytokine receptors (named KineTAC), membrane glycan receptors (named LYTAC), and transferrin receptors (named TransTAC). However, the effectiveness of these methods is limited by the expression level of "effector" proteins, such as the ASGPR molecule targeted by LYTAC is mainly highly expressed in the liver, but the expression level in tumors is low, making it difficult to take effect. Therefore, the current technology cannot cover all diseases, and the development of "effector" proteins that are overexpressed in different diseases and tissues will greatly expand the indications of eTPD and improve the targeting specificity.

[0005] The inventors' previous studies identified soluble ICOSL (sICOSL) as a chemotherapy-induced soluble factor mediating CTL dysfunction. Through multi-omics analysis of clinical samples and biological experiments in cells and mouse models, a previously undescribed EZH2-DPP4-sICOSL-ICOS pathway was discovered that regulates cancer-CTL communication, tumor immune escape, and chemotherapy immunotherapy failure. The use of EZH2 and ADAM10 / 17 inhibitors only reduced chemotherapy-induced sICOSL but did not affect its baseline levels. Moreover, these drugs are not tissue-specific and can cause serious side effects.

[0006] Glucose-regulated protein 78 (GRP78, also known as BiP) is a member of the highly conserved HSP70 family that is central regulator of endoplasmic reticulum homeostasis by playing a key role in folding, transport and quality control of nascent protein chains. This endoplasmic reticulum resident molecule is upregulated under stress conditions, including hypoxia, nutrient deprivation, environmental or genetic perturbations, to provide cytoprotection. To adapt to these chronic stresses in the tumor microenvironment, cancer cells upregulate the expression of GRP78 to promote their proliferation, invasion, therapy resistance and immune escape, making GRP78 a prototypical onco-gene. Upregulation of GRP78 leads to its escape from the endoplasmic reticulum and translocation to the extracellular space. The soluble form of GRP78 (sGRP78) has been widely accepted as an immunomodulatory molecule that favors resolution of the immune response by generating regulatory T and B cell populations, affecting dendritic cell maturation and impairing pro-inflammatory cytokine production. The release of sGRP78 can infer the strength of chemotherapy-related damage. The inventors' previous studies observed the fact that sGRP78 is increased in the serum of breast cancer patients, and the inventors reported that the levels further increased in some patients after several rounds of neoadjuvant therapy, and the higher the "sGRP78 index", the worse the therapeutic response. Subsequent functional experiments revealed that chemotherapy-induced sGRP78 exacerbated the immunosuppressive tumor microenvironment by reshaping the plasticity of B cells. The sGRP78 index is worth developing as a new predictive marker to identify patient sensitivity to chemotherapy, enabling precise stratified treatment.

[0007] HERV K (human endogenous retrovirus K) is a class of ancient viral sequences that have lost the ability to replicate during evolution but still exist in the human genome. The expression of HERV K has been confirmed in various tumors, and their role in tumor development has attracted widespread attention. In the tumor immune microenvironment, HERV K can affect the interaction between tumor and immune cells through various mechanisms. For example, certain protein products of HERV K can be involved in regulating the proliferation, invasion and metastasis of tumor cells. In addition, the expression of HERV K can be related to tumor immune escape, which can help tumor cells evade the surveillance and attack of the immune system by affecting the expression of tumor cell surface antigens. The expression of HERV K can also be related to the inflammatory response in the tumor microenvironment. Some studies have shown that the activation of HERV K can be related to tumor-related inflammation and immune suppression, which can affect the function of immune cells, including T cells, B cells and natural killer (NK) cells. The inventors' previous studies found that HERV K is released by tumor cells into the extracellular and mediates immune escape.

[0008] Basic research evidence suggests that TGFβ is a potential therapeutic target in some malignancies, and several anti-TGFβ drugs have been explored in clinical studies. Several different ways of blocking TGFβ signaling have been developed, including the use of monoclonal neutralizing antibodies to TGFβ ligands and receptors; double antibodies targeting TGFβ / PD-L1; antisense oligonucleotides; TGFβ-related vaccines; and receptor kinase inhibitors. However, the process of translating the knowledge of the basic molecular mechanisms of TGFβ in the role of malignant tumors into clinical applications has been relatively slow. The pro-cancer and anti-cancer effects of TGFβ and its pleiotropic role in regulating cell and tissue homeostasis make the rapid clinical development of anti-TGFβ drugs extremely challenging. In addition, TGFβ plays an important role in the development of cardiovascular system organs and the remodeling process after cardiac injury. As an anticancer therapy, systemic inhibition of TGF-β signaling can affect the development and function of the heart, which can be a major challenge in research. Preclinical studies have shown that animals receiving TGF-βRI inhibitors AZ12601011 and AZ12799734 have an increased incidence of heart valve lesions. Cardiotoxicity is not limited to small molecule drugs. A recent study reported that a pan-TGFβ monoclonal neutralizing antibody was associated with an increased risk of bleeding and cardiotoxicity in mice and monkeys. In addition, an anti-TGF-βRII antibody LY3022859 is being studied in patients with advanced solid tumors (N=14), and despite the fact that patients were given antihistamines and corticosteroids in advance to prevent sudden conditions, the trial was discontinued due to lack of clinical efficacy and cytokine storm side effects. This paper attempts to develop a targeted protein degradation therapy to reverse the immune escape mediated by TGFβ.

[0009] Vascular endothelial growth factor (VEGF) plays an important role in the tumor immune microenvironment. It is mainly secreted by tumor cells, stromal cells such as macrophages, endothelial cells and fibroblasts, and has multiple functions in the tumor microenvironment, including promoting angiogenesis, increasing vascular permeability, and regulating the recruitment and activity of immune cells, thereby mediating tumor cell escape from immune attack. Conventional treatment methods targeting VEGF include anti-VEGF antibodies (such as Bevacizumab), VEGF receptor tyrosine kinase inhibitors (such as Sorafenib and Sunitinib), and the efficacy of these treatments has been clinically proven, but there are still problems, on the one hand is the drug resistance produced by long-term use, on the other hand is the off-target vascular side effects of many tumors, including hypertension, proteinuria, bleeding, thrombosis, etc.

[0010] Soluble NKG2DL (sNKG2DL) is a mechanism by which tumor cells evade NK cell attack. In the tumor microenvironment, tumor cells can shed NKG2DL from the tumor cell surface to become sNKG2DL through proteolysis mediated by ADAM family metalloproteases or certain matrix metalloproteases (MMPs), or mediate the secretion of sNKG2DL through exosomes, resulting in elevated levels of sNKG2DL. sNKG2DL binds and blocks NKG2D, thereby inhibiting NK cell activity, thereby helping tumor cells evade immune surveillance. Existing treatment methods targeting the NKG2D / NKG2DL axis include using small molecule inhibitors, antibodies or fusion proteins to regulate the expression of NKG2DL on the surface of tumor cells, and CAR-T / NK cell adoptive therapy based on the axis, etc. Excessive sNKG2DL can block NKG2D, rendering these treatments ineffective, and is an important drug resistance mechanism.

[0011] Galectin 3 is a β-galactoside binding type of agglutinin, which is synthesized by cells and released into the extracellular, and plays a variety of biological functions. Galectin 3 can directly regulate tumor cell adhesion, migration, angiogenesis and interaction with immune cells. In the tumor immune microenvironment, Galectin 3 can interact with a variety of immune cells in the tumor microenvironment, affecting their functional status. For example, Galectin 3 can affect the activation, proliferation and differentiation of T cells, B cells and dendritic cells by binding to the sugar chain structure on the surface of immune cells, which may inhibit the killing effect of these immune cells on tumor cells. In addition, Galectin 3 can also capture cytokines in the tumor microenvironment, such as interferon-γ (IFN-γ), reduce the spread of these cytokines, thereby reducing their concentration gradient in the tumor, affecting the infiltration and activation of immune cells. This effect may help tumor cells escape immune surveillance and attack. Previous studies targeting Galectin 3 mostly use small molecule inhibitors, which have poor blocking effect, cannot completely block the effect of Galectin 3, and have poor targeting and easy to produce off-target side effects. SUMMARY

[0012] The present application explores the "effector" protein that can be used in cancer, and discloses a target for mediating endocytosis and degradation in tumors.

[0013] One of the purposes of the present application is to provide a targeted degradation protein, the amino acid sequence of which is at least one of SEQ ID NO. 3, 5, 9, 11, 13, 15, and 17.

[0014] The second purpose of the present application is to provide a nucleotide sequence encoding the above-mentioned targeted degradation protein. The nucleotide sequence encoding the targeted degradation protein with the amino acid sequence as shown in SEQ ID NO. 3 is as shown in SEQ ID NO. 4; the nucleotide sequence encoding the targeted degradation protein with the amino acid sequence as shown in SEQ ID NO. 5 is as shown in SEQ ID NO. 6; the nucleotide sequence encoding the targeted degradation protein with the amino acid sequence as shown in SEQ ID NO. 9 is as shown in SEQ ID NO. 10; the nucleotide sequence encoding the targeted degradation protein with the amino acid sequence as shown in SEQ ID NO. 11 is as shown in SEQ ID NO. 12; the nucleotide sequence encoding the targeted degradation protein with the amino acid sequence as shown in SEQ ID NO. 13 is as shown in SEQ ID NO. 14; the nucleotide sequence encoding the targeted degradation protein with the amino acid sequence as shown in SEQ ID NO. 15 is as shown in SEQ ID NO. 16; and the nucleotide sequence encoding the targeted degradation protein with the amino acid sequence as shown in SEQ ID NO. 17 is as shown in SEQ ID NO. 18.

[0015] The third object of the present application is to provide the use of the above-mentioned targeted protein degradation in the preparation of a product for treating breast cancer.

[0016] Preferably, the product is a medicament.

[0017] More preferably, the medicament has the targeted protein degradation as the only active ingredient.

[0018] More preferably, the medicament is an injection.

[0019] The fourth object of the present application is to provide a product for treating breast cancer, which contains the above-mentioned targeted protein degradation.

[0020] Preferably, the product is a medicament.

[0021] More preferably, the medicament further contains a clinically acceptable excipient.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] Traditional blocking of extracellular proteins mainly uses neutralizing antibodies. Compared with corresponding neutralizing antibodies, the targeted protein degradation (eTPD) therapy developed in the present application has the following characteristics:

[0024] (1) Traditional neutralizing antibodies directly block the effector region of the target protein and can also enter the cell through the Fc receptor part, but they cannot directly reduce the target protein. The eTPD therapy can directly mediate the degradation of the target protein, thereby weakening the effect of the target protein at the root;

[0025] (2) The antigen epitope of traditional neutralizing antibodies must be the effector region of the target protein, such as the binding site of the target protein and its receptor. However, it is difficult to design antibodies against this epitope, and secondly, some target proteins have complex effects and may interact with many receptors, making it difficult to design corresponding neutralizing antibodies. The eTPD therapy directly mediates the degradation of the target protein, only needs to have a strong affinity for the target protein, does not need to bind to a specific epitope, has low design difficulty, and is suitable for many target points;

[0026] (3) Many target proteins are expressed in many tissues and have complex physiological functions. However, traditional neutralizing antibodies do not have tissue targeting, mainly bind and neutralize target proteins in the periphery, and thus produce many side effects outside the tumor. The eTPD therapy uses tumor-specific high-expression membrane molecules as its effector protein, thereby achieving the dual effects of tumor targeting and mediating endocytosis. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1Schematic of the enzymatic degradation of sICOSL in HER2+ cancer by DPP4 targeting in Example 1.

[0028] Figure 2 SDS-PAGE run under non-reducing conditions to show oligomers of the original protein for the results of the detection of the secretion of aHER2-DPP4 from HEK293T cells by anti-His WB analysis in Example 1.

[0029] Figure 3 Quantification by anti-His mean fluorescence intensity (MFI) for the binding curves of aHER2 and aHER2-DPP4 on HER2+ BT474 and HER2- MDA-MB-468 cells in Example 1.

[0030] Figure 4 Relationship of sICOSL concentration in the supernatant of in vitro HER2+ BT474 cell culture with aHER2 / aHER2-DPP4 and chemotherapy in Example 1.

[0031] Figure 5 Relationship of sICOSL concentration in the supernatant of in vitro HER2- MDA-MB-468 cell culture with aHER2 / aHER2-DPP4 and chemotherapy in Example 1.

[0032] Figure 6 Tumor antigen-activated CD8+ T cells were co-cultured with control and ICOSLKO BT474 cells and treated with aHER2 or aHER2-DPP4 for 18 hours, and ICOS, granzyme B and IFNy were stained in CD8+ T cells in Example 1.

[0033] Figure 7 BALB / c mice were inoculated with syngeneic 4T1 breast cancer cells carrying human HER2 and then injected with aHER2 or aHER2-DPP4 in Example 1. Tumor volume was monitored every 3 days.

[0034] Figure 8 Mouse survival curve in Example 1.

[0035] Figure 9 Body weight of mice in Example 1.

[0036] Figure 10 Histopathology of mice in Example 1.

[0037] Figure 11 sICOSL concentration in the interstitial fluid and serum of 4T1 breast tumors after treatment in Example 1.

[0038] Figure 12Quantification of histograms and representative contour plots shown in Example 1 display the positive rate of CD8+ T cell infiltration and activation markers granzyme B, LAG3, and IFNy in CD8+ T cells.

[0039] Figure 13 Schematic of targeting sGRP78 for enzymatic degradation in HER2+ cancer by subA in Example 2.

[0040] Figure 14 Binding curves of aHER2 and aHER2-subA on HER2+ BT474 and HER2- MDA-MB-468 cells in Example 2, quantified by mean fluorescence intensity (MFI) of anti-His.

[0041] Figure 15 Relationship of sICOSL concentration in in vitro HER2+ BT474 cell culture supernatant with aHER2 / aHER2-subA and chemotherapy in Example 2.

[0042] Figure 16 B cells were co-cultured with BT474 cells and treated with chemotherapy drug (nab-P), aHER2, or aHER2-subA for 18 hours, and stained for IL10 and PD-L1 in B cells in Example 2.

[0043] Figure 17 B cells were co-cultured with BT474 cells and treated with chemotherapy drug (nab-P), aHER2, or aHER2-subA for 18 hours in Example 2. Sorted B cells were co-cultured with naive CD4+ T cells for 2 days, and Treg cell proportion was measured.

[0044] Figure 18 BALB / c mice were inoculated with syngeneic 4T1 breast cancer cells carrying human HER2, then injected with aHER2 or aHER2-subA in Example 2. Tumor volume was monitored every 3 days.

[0045] Figure 19 Mouse survival curve in Example 2.

[0046] Figure 20 Body weight of mice in Example 2.

[0047] Figure 21 Schematic of targeting sERVK for enzymatic degradation in HER2+ cancer by ERVK binding sequence in Example 3.

[0048] Figure 22 Relationship of sERVK concentration in in vitro HER2+ BT474 and HER2- MDA-MB-468 cell culture supernatant with TPD treatment in Example 3.

[0049] Figure 23 Figure 6. Tumor volume in mice treated with aHER2-hlgGlFc1 or aHER2-hlgGlFc1-aTGFp in Example 4.

[0050] Figure 24 Figure 7. Survival curve of mice in Example 4.

[0051] Figure 25 Figure 8. TGFp1 concentration in 4T1 breast tumor interstitial fluid and serum after treatment in Example 4.

[0052] Figure 26 Figure 9. Schematic of enzymatic degradation of TGFp by TGFp-binding sequences targeted in HER2+ cancer in Example 4.

[0053] Figure 27 Figure 10. Relationship of VEGF concentration in culture supernatant of HER2+ BT474 and HER2- MDA-MB-468 cells in vitro to TPD treatment in Example 5.

[0054] Figure 28 Figure 11. Tumor volume in mice treated with aHER2-hlgGlFc1 or aHER2-hlgGlFc1-aVEGF in Example 5.

[0055] Figure 29 Figure 12. Survival curve of mice in Example 5.

[0056] Figure 30 Figure 13. VEGF concentration in 4T1 breast tumor interstitial fluid and serum after treatment in Example 5.

[0057] Figure 31 Figure 14. Schematic of enzymatic degradation of VEGF by VEGF-binding sequences targeted in HER2+ cancer in Example 5.

[0058] Figure 32 Figure 15. Relationship of VEGF concentration in culture supernatant of HER2+ BT474 and HER2- MDA-MB-468 cells in vitro to TPD treatment in Example 5.

[0059] Figure 33 Figure 16. Tumor volume in mice treated with aHER2-hlgGlFc1 or aHER2-hlgGlFc1-aVEGF in Example 5.

[0060] Figure 34 Survival curve for mice in Example 5.

[0061] Figure 35 VEGFA concentration in 4T1 breast tumor interstitial fluid and serum after treatment in Example 5.

[0062] Figure 36 Schematic of targeting sNKG2DL for enzymatic degradation in HER2+ cancer by NKG2D sequence in Example 6.

[0063] Figure 37 In vitro, MICA (one of the NKG2DLs) concentration in HER2+ BT474 and HER2- MDA-MB-468 cell culture supernatant in relation to TPD treatment in Example 6.

[0064] Figure 38 BALB / c mice inoculated with syngeneic 4T1 breast cancer cells carrying human HER2 were then injected with aHER2-hlgGlFc1 or aHER2-hlgGlFc1-NKG2D. Tumor volume was monitored every 3 days in Example 6.

[0065] Figure 39 Survival curve for mice in Example 6.

[0066] Figure 40 sMICA concentration in 4T1 breast tumor interstitial fluid and serum after treatment in Example 6.

[0067] Figure 41 Schematic of targeting sGal3 for enzymatic degradation in HER2+ cancer by sGal3 binding sequence in Example 7.

[0068] Figure 42 BALB / c mice inoculated with syngeneic 4T1 breast cancer cells carrying human HER2 were then injected with aHER2-hlgGlFc1 or aHER2-hlgGlFc1-aGal3. Tumor volume was monitored every 3 days in Example 7.

[0069] Figure 43 Survival curve for mice in Example 7. DETAILED DESCRIPTION

[0070] Example 1 Targeted degradation of sICOSL

[0071] This example sought to develop a drug capable of directly degrading sICOSL within the tumor. Given that DPP4 is the key degrading enzyme of sICOSL, a genetic fusion of the DPP4 enzyme and an antibody against a breast cancer cell surface receptor was created (aDPP4-anti-HER2) (Figure 1). Figure 1). To avoid degradation of membrane-bound ICOSL in FcR-expressing cells (such as dendritic cells and B cells), nanobodies were applied instead of Fc-containing whole antibodies Figure 1 ). Therefore, the inventors designed a conjugate of DPP4 C-terminus to anti-HER2 nanobody, aHER2, i.e. aHER2-DPP4. The aHER2 and aHER2-DPP4 constructs were cloned into pSecTag2A vector, respectively, and transiently transfected into 293 cells. The next day, the medium was changed to serum-free medium, and the supernatant was collected continuously, followed by purification using Ni-NTA Agarose Resin column. The complete sequences of the constructs are given below.

[0072] > aHER2 amino acid sequence (SEQ ID NO. 1)

[0073] MSALLILALVGAAVVWAEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKHHHHHH

[0074] > aHER2 nucleic acid sequence (SEQ ID NO. 2)

[0075] ATGAGCGCTCTGCTGATTCTCGCACTGGTTGGAGCAGCCGTCGTGTGGGCTGAGGTGCAGCTGGTGGAGAAGGGTGGTGGCAGAGTGCAGGCTGGTGGCTCTTTGAGACTGCGCTGTGCTGCAAGCGGTATTACCTTCTCCATCAACACCATGGGCTGGTATAGACAGGCTCCAGGTAAGCAGAGAGAACTCGTGGCTCTGATCTCCAGCATTGGTGATACCTATTACGCCGACTCCGTGAAGGGACGGTTCAGAATCAGGCGAGATAACGCCAAGAACACAGTGTATCTGCGGATGAGAAGGCTGAAGCCAGAAGATACCGCTGTGTACTATTGTAAACGGTTTAGAACTGCCGCTCAGGGTACAGACTATTGGGGCCAGGGCACAAGAGTGACAGTGAGCAAGCATCATCACCACCACCACTAG

[0076] > aHER2-DPP4 amino acid sequence (SEQ ID NO. 3)

[0077] MSALLILALVGAAVVWAMEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKGGSGGSGGSGGSNKGTDDATADSRKTYTLTDYLKNTYRLKLYSLRWISDHEYLYKQENNILVFNAEYGNSSVFLENSTFDEFGHSINDYSISPDGQFILLEYNYVKQWRHSYTASYDIYDLNKRQLITEERIPNNTQWVTWSPVGHKLAYVWNNDIYVKIEPNLPSYRITWTGKEDIIYNGITDWVYEEEVFSAYSALWWSPNGTFLAYAQFNDTEVPLIEYSFYSDESLQYPKTVRVPYPKAGAVNPTVKFFVVNTDSLSSVTNATSIQITAPASMLIGDHYLCDVTWATQERISLQWLRRIQNYSVMDICDYDESSGRWNCLVARQHIEMSTTGWVGRFRPSEPHFTLDGNSFYKIISNEEGYRHICYFQIDKKDCTFITKGTWEVIGIEALTSDYLYYISNEYKGMPGGRNLYKIQLSDYTKVTCLSCELNPERCQYYSVSFSKEAKYYQLRCSGPGLPLYTLHSSVNDKGLRVLEDNSALDKMLQNVQMPSKKLDFIILNETKFWYQMILPPHFDKSKKYPLLLDVYAGPCSQKADTVFRLNWATYLASTENIIVASFDGRGSGYQGDKIMHAINRRLGTFEVEDQIEAARQFSKMGFVDNKRIAIWGWSYGGYVTSMVLGSGSGVFKCGIAVAPVSRWEYYDSVYTERYMGLPTPEDNLDHYRNSTVMSRAENFKQVEYLLIHGTADDNVHFQQSAQISKALVDVGVDFQAMWYTDEDHGIASSTAHQHIYTHMSHFIKQCFSLPDIHHHHHH

[0078] > alphaHER2-DPP4 nucleic acid sequence (SEQ ID NO. 4)

[0079]

[0080] aHER2-DPP4 remained in a homodimeric form ( Figures 1-2 ), in which form DPP4 exhibits enzymatic activity. aHER2-DPP4 bound significantly to BT474 (high HER2 expression) but not to MDA-MB-468 (no HER2 expression) Figure 3 ). The effective dissociation constant (Kd = 21.5 nM) of aHER2-DPP4 was comparable to that of the pure nanobody (Kd = 16.5 nM), indicating that the fusion did not affect the affinity to the target cells Figure 3

[0081] Next, the inventors tested the targeting activity of the conjugate in a variety of human cancer cell lines and immune cells, which differ in their HER2 expression levels. 1 nM aHER2-DPP4 almost completely eliminated the release of soluble ICOSL by HER2+ cancer cells, whether or not treated with paclitaxel Figure 4 ). There was no discernible loss of sICOSL on HER2- cancer cells Figure 5 Thus, the nanobody-DPP4 fusion specifically targets cancer cells to degrade sICOSL. Through a series of cellular experiments, the inventors tested whether the nanobody-DPP4 could selectively reverse sICOSL-dependent CTL dysfunction. HER2+ breast cancer cells were treated with aHER2-DPP4 or aHER2 and co-cultured with CTLs. aHER2-DPP4 upregulated ICOS, IFNy and GZMB expression by CTLs Figure 6 ). Consistently, aHER2-DPP4 had little effect on CTLs when co-cultured with ICOSL knock-out cell lines Figure 6 Thus, the nanobody-DPP4 fusion degrades cancer cell-derived sICOSL to reverse CTL dysfunction.

[0082] To assess the targeting efficacy of aHER2-DPP4 in vivo, the inventors turned to a previously validated mouse model of HER2-positive breast cancer. This model involves injection of 4T1 cells stably expressing human HER2 (hHER2) and GFP into the mammary fat pad of mice, followed by i.p. injection of aHER2, an equimolar dose of aHER2-DPP4 (10 mg / kg q3d) or vehicle control. Treatment with aHER2-DPP4 led to a reduction in tumor size Figure 7 ). After 32 days, all mice treated with vehicle reached a tumor burden that required euthanasia, whereas mice treated with aHER2-DPP4 survived for up to 62 days Figure 8 Treatment with aHER2 alone did not result in attenuated tumor growth or prolonged survival Figure 7 ​8). In terms of safety, mice treated with αHER2 or αHER2-DPP4 did not experience weight loss during the experiment, indicating that the treatment was well tolerated. Figure 9 No non-targeted toxicity was detected in the liver, spleen, kidneys, and lungs. Figure 10 ).

[0083] To analyze intratumoral sICOSL degradation and immune infiltration in HER2-treated animals, a group of animals were treated with the vector, αHER2, or αHER2-DPP4 as described above and euthanized on day 10 post-implantation. ELISA analysis of the αHER2-DPP4-treated animals showed a significant reduction in intratumoral sICOSL with minimal impact on serum sICOSL, confirming that αHER2-DPP4 selectively depletes intratumoral sICOSL in vivo. Figure 11 In animals treated with the vector or αHER2 control, both intratumoral and serum sICOSL levels remained unchanged. Figure 11 Next, the inventors analyzed the immune composition within the tumor and found that αHER2-DPP4 treatment increased CD8+ T cell infiltration into the tumor. Figure 12 Notably, CD8+ T cells in animals treated with the conjugate also showed significantly increased levels of ICOS, granzyme B, and perforin. Figure 12 These data support the regulation of the tumor immune microenvironment following αHER2-DPP4 treatment, consistent with the importance of sICOSL signaling in the tumor microenvironment. In conclusion, targeted degradation of tumor-derived sICOSL limits breast cancer progression.

[0084] Example 2: Targeted Degradation of sGRP78

[0085] It has been reported that a bacterial serine protease subunit (SubA) can specifically degrade the GRP78 protein. The inventors had previously purified recombinant SubA protein and verified its ability to degrade sGRP78 in vitro and in vivo. However, recombinant SubA lacks tissue specificity and may cause serious side effects. Therefore, the inventors sought to develop a drug that can directly degrade sGRP78 within tumors, thereby creating a genetic fusion of the SubA enzyme and an antibody targeting a receptor on the surface of breast cancer cells. Figure 13 The inventors designed a conjugate that links the SubAC terminus to the nanobody, called "αHER2-SubA". Figure 13 The αHER2 and αHER2-subA constructs were cloned into the pSecTag2A vector, transiently transferred into 293 cells, and the medium was changed to serum-free medium the next day. The supernatant was continuously collected and purified using a Ni-NTA garose Resin column. The complete sequences of the constructs are given below:

[0086] > alphaHER2-subA amino acid sequence (SEQ ID NO. 5)

[0087] MSALLILALVGAAVVWAEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKGGSGGSGGSGGSMLKILWTYILFLLFISASARAEKPWYFDAIGLTETTMSLTDKNTPVVVSVVDSGVAFIGGLSDSEFAKFSFTQDGSPFPVKKSEALYIHGTAMASLIASRYGIYGVYPHALISSRRVIPDGVQDSWIRAIESIMSNVFLAPGEEKIINISGGQKGVASASVWTELLSRMGRNNDRLIVAAVGNDGADIRKLSAQQRIWPAAYHPVSSVNKKQDPVIRVAALAQYRKGETPVLHGGGITGSRFGNNWVDIAAPGQNITFLRPDAKTGTGSGTSEATAIVSGVLAAMTSCNPRATATELKRTLLESADKYPSLVDKVTEGRVLNAEKAISMFCKKNYIPVRQGRMHHHHHH

[0088] > alphaHER2-subA nucleic acid sequence (SEQ ID NO. 6)

[0089]

[0090] The affinity of aHER2-SubA (Kd= 23.5 nM) to HER2-expressing cancer cells was similar to aHER2 (Kd= 20.3 nM) (see Figure 14 ). Next, ELISA experiments showed that aHER2-SubA significantly reduced sGRP78 in culture supernatants of human cancer cell lines (see Figure 15 ). Thus, the Nanobody-SubA fusion specifically targets cancer cells to degrade sGRP78.

[0091] Through a series of cellular experiments, the inventors tested whether the Nanobody-SubA fusion could selectively reverse sGRP78-dependent regulatory B cell induction. HER2+ breast cancer cells were co-cultured with B cells and treated with chemotherapy (nab-P) and eTPD proteins (aHER2-SubA or aHER2). aHER2-SubA downregulated IL10 and PD-L1 expression in B cells regardless of chemotherapy (see Figure 16 ). These aHER2-SubA-treated B cells were also less potent in inducing Tregs than other groups (see Figure 17 ). Thus, the Nanobody-SubA fusion degrades cancer cell-derived sGRP78 to reverse B cell-induced CTL dysfunction.

[0092] To assess the targeting efficacy of aHER2-SubA in vivo, the inventors turned to a previously validated HER2-positive breast cancer mouse model. This model involves injection of 4T1 cells stably expressing human HER2 (hHER2) and GFP into the mammary fat pad of mice, followed by intraperitoneal (i.p.) injection of aHER2, an equimolar dose of aHER2-SubA (10 mg / kg q3d), or vehicle control. Treatment with aHER2-SubA resulted in reduced tumor size ( Figure 18 ). aHER2-SubA-treated mice had a longer survival time compared to control mice ( Figure 19 ). Treatment with aHER2 alone did not result in attenuated tumor growth or prolonged survival ( Figure 18 , 19). In terms of safety, mice treated with aHER2 or aHER2-SubA did not experience weight loss during the experiment, indicating that the treatment was well tolerated ( Figure 20 ).

[0093] Example 3 Targeted degradation of sERVK

[0094] The inventors utilize HER2 as effector protein, targeting tumor area and mediating endocytosis of target protein; the other end targets sERVK to assist its endocytosis degradation; and remove ADCC / ADCP / CDC effect with human IgG1 Fc segment as a bridge design drug Figure 21 ) respectively. The αHER2-hIgG1 Fc1 and αHER2-hIgG1 Fc1-αERVK constructs were cloned into pSecTag2A vector, transiently transfected into 293 cells, and the next day the medium was changed to serum-free medium. After continuous collection of supernatant, Ni-NTA Agarose Resin column was used for purification. ELISA experiments found that it can degrade sERVK in the supernatant of HER2+ cancer cells Figure 22

[0095] The complete sequence of the construct is given below:

[0096] > αHER2-hIgG1 Fc1 amino acid sequence (SEQ ID NO. 7) MSALLILALVGAAVVWAMEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

[0097] > αHER2-hIgG1 Fc1 nucleic acid sequence (SEQ ID NO. 8)

[0098] ​

[0099] > alphaHER2-hlgGl Fc1-alphaERVK Amino Acid Sequence (SEQ ID NO. 9)

[0100] MSALLILALVGAAVVWAMEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWWMNWVKQRPEQGLEWIGRIDPYDSETHYNQKFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCASLYYYGISLWGQGTLVTVSGGGGSGGGGSGGGGSGD VVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGESPKLLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCLQATHFPWTFGGGTKLEIKHHHHHH

[0101] > alphaHER2-hlgGl Fc1-alphaERVK Nucleic Acid Sequence (SEQ ID NO. 10)

[0102]

[0103] To evaluate the targeting efficacy of aHER2-hIgGlFcl-aERVK in vivo, the inventors turned to a previously validated mouse model of HER2-positive breast cancer. This model involves injection of 4T1 cells stably expressing human HER2 (hHER2) and GFP into the mammary fat pad of mice, followed by intraperitoneal (i.p.) injection of aHER2-hIgGlFcl, an equimolar dose of aHER2-hIgGlFcl-aERVK (10 mg / kg q3d), or vehicle control. Treatment with aHER2-hIgGlFcl-aERVK resulted in a reduction in tumor size ( Figure 23 ) compared to control mice. Mice treated with aHER2-hIgGlFcl-aERVK had a longer survival time ( Figure 24 ) compared to control mice. Treatment with aHER2 alone did not result in a reduction in tumor growth or prolonged survival ( Figure 23 , 24). ELISA analysis of aHER2-hIgGlFcl-aERVK treated animals showed a significant reduction in sERVK in the tumor interstitial fluid, with little effect on serum sERVK, confirming that aHER2-hIgGlFcl-aERVK selectively depletes sERVK in the tumor in vivo ( Figure 25 ).

[0104] Example 4 Targeted degradation of TGFp

[0105] The inventors designed a drug that targets tumor area and mediates endocytosis of target protein using HER2 as effector protein; targets TGFp at the other end to mediate its endocytosis degradation; and uses human IgGl Fc segment that removes ADCC / ADCP / CDC effect as a bridge design ( Figure 26 ). aHER2-hIgGlFcl and aHER2-hIgGlFcl-aTGFp constructs were cloned into pSecTag2A vector respectively, transiently transfected into 293 cells, and the next day the medium was changed to serum-free medium. After continuous collection of supernatant, the inventors purified the supernatant using Ni-NTA Agarose Resin column. ELISA experiments showed that it can degrade TGFpl in the supernatant of HER2+ cancer cells ( Figure 27 ).

[0106] The complete sequence of the construct is given below:

[0107] > aHER2-hIgGlFcl-aTGFp amino acid sequence (SEQ ID NO. 11)

[0108] MSALLILALVGAAVVWAMEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQRFKGRVTITADESTSTTYMELSSLRSEDTAVYYCASTLGLVLDAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAPGIPDRFSGSGSGTDFTTISRLEPEDFAVYYCQQYADSPITFGOGTRLEIKHHHHHH

[0109] > alpha HER2-hlgGl Fc1-alpha TGF beta nucleic acid sequence (SEQ ID NO. 12)

[0110]

[0111] To evaluate the targeting efficacy of aHER2-hlgGlFcl-aTGFp in vivo, the inventors turned to a previously validated mouse model of HER2-positive breast cancer. This model involves injection of 4T1 cells stably expressing human HER2 (hHER2) and GFP into the mammary fat pad of mice, followed by intraperitoneal (i.p.) injection of aHER2-hlgGlFcl, an equimolar dose of aHER2-hlgGlFcl-aTGFp (10 mg / kg q3d), or vehicle control. Treatment with aHER2-hlgGlFcl-aTGFp resulted in reduced tumor size ( Figure 28 ) compared to control mice. Mice treated with aHER2-hlgGlFcl-aTGFp had longer survival times ( Figure 29 ) than control mice. Treatment with aHER2 alone did not result in attenuated tumor growth or prolonged survival ( Figure 28 , 29). ELISA analysis of aHER2-hlgGlFcl-aTGFp-treated animals showed a significant reduction in TGFpi in the tumor interstitial fluid, with little effect on serum TGFp, confirming that aHER2-hlgGlFcl-aTGFp selectively depletes TGFpi in the tumor in vivo ( Figure 30 ).

[0112] Example 5 Targeted degradation of VEGF

[0113] The inventors designed a drug that targets tumor regions using HER2 as the effector protein, mediates endocytosis of the target protein; targets VEGF on the other end to assist its endocytic degradation; and uses a human IgGl Fc segment that removes ADCC / ADCP / CDC effects as a bridge design ( Figure 31 ). The aHER2-hlgGlFcl and aHER2-hlgGlFcl-aVEGF constructs were cloned into the pSecTag2A vector, transiently transfected into 293 cells, and the next day the medium was changed to serum-free medium. The supernatant was collected continuously and purified using a Ni-NTA Agarose Resin column. ELISA experiments initially verified that they can degrade VEGFA in the supernatant of HER2+ cancer cells ( Figure 32 ).

[0114] The complete sequence of the construct is given below:

[0115] > aHER2-hlgGlFcl-aVEGF amino acid sequence (SEQ ID NO. 13)

[0116] MSALLILALVGAAVVWAMEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSGDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKHHHHHH

[0117] > alpha HER2-hlgGl Fc1-alpha VEGF nucleic acid sequence (SEQ ID NO. 14)

[0118]

[0119] To evaluate the targeting efficacy of aHER2-hIgGlFcl-aVEGF in vivo, the inventors turned to a previously validated mouse model of HER2-positive breast cancer. This model involves injection of 4T1 cells stably expressing human HER2 (hHER2) and GFP into the mammary fat pad of mice, followed by intraperitoneal (i.p.) injection of aHER2-hIgGlFcl, an equimolar dose of aHER2-hIgGlFcl-aVEGF (10 mg / kg q3d), or vehicle control. Treatment with aHER2-hIgGlFcl-aVEGF resulted in a reduction in tumor size ( Figure 33 ). Mice treated with aHER2-hIgGlFcl-aVEGF had a longer survival time than control mice ( Figure 34 ). Treatment with aHER2 alone did not result in a reduction in tumor growth or prolonged survival ( Figure 33 , 34). ELISA analysis of aHER2-hIgGlFcl-aVEGF-treated animals showed a significant reduction in VEGF in the tumor interstitial fluid, with little effect on serum sVEGF, confirming that aHER2-hIgGlFcl-aVEGF selectively depletes intratumoral VEGFA in vivo ( Figure 35 ).

[0120] Example 6 Targeted degradation of sNKG2DL

[0121] The inventors designed a drug that utilizes HER2 as the effector protein to target tumor region and mediate endocytosis of the target protein; the other end targets sNKG2DL to assist its endocytosis degradation; and a human IgGl Fc segment that removes ADCC / ADCP / CDC effect as the bridge design ( Figure 36 ). The aHER2-hIgGlFcl and aHER2-hIgGlFcl-NKG2D constructs were cloned into pSecTag2A vector, respectively, and transiently transfected into 293 cells. The next day, the medium was changed to serum-free medium, and the supernatant was collected continuously, followed by purification using Ni-NTA Agarose Resin column. ELISA experiments initially verified that it can degrade sNKG2DL in the supernatant of HER2+ cancer cells ( Figure 37 ).

[0122] The complete sequence of the construct is given below:

[0123] > aHER2-hIgGlFcl-NKG2D amino acid sequence (SEQ ID NO. 15)

[0124] MSALLILALVGAAVVWAMEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVHHHHHH

[0125] > alpha HER2-hlgGl Fc1 -NKG2D nucleic acid sequence (SEQ ID NO. 16)

[0126]

[0127] To evaluate the targeting potency of aHER2-hIgGlFc1-NKG2D in vivo, the inventors turned to a previously validated mouse model of HER2-positive breast cancer. This model involves injection of 4T1 cells stably expressing human HER2 (hHER2) and GFP into the mammary fat pad of mice, followed by i.p. injection of aHER2-hIgGlFc1, an equimolar dose of aHER2-hIgGlFc1-NKG2D (10 mg / kg q3d), or vehicle control. Treatment with aHER2-hIgGlFc1-NKG2D resulted in a reduction in tumor size ( Figure 38 ) compared to control mice. Mice treated with aHER2-hIgGlFc1-NKG2D had a longer survival time ( Figure 39 ) than control mice. Treatment with aHER2 alone did not result in a reduction in tumor growth or prolonged survival ( Figure 38 , 39). ELISA analysis of aHER2-hIgGlFc1-NKG2D-treated animals showed a significant reduction in sNKG2DL in the tumor interstitial fluid, with little effect on serum sNKG2DL, confirming that aHER2-hIgGlFc1-NKG2D selectively depletes sNKG2DL in the tumor in vivo ( Figure 40 ).

[0128] Example 7 Targeted degradation of sGalactin 3

[0129] The inventors designed a drug that utilizes HER2 as the effector protein to target tumor regions and mediate endocytosis of the target protein; the other end targets sGal3 to assist its endocytosis and degradation; and a human IgGl Fc segment that removes the ADCC / ADCP / CDC effect as the bridge design ( Figure 41 ). The aHER2-hIgGlFc1 and aHER2-hIgGlFc1-aGal3 constructs were cloned into the pSecTag2A vector, respectively, and transiently transfected into 293 cells. The next day, the medium was changed to serum-free medium, and the supernatant was continuously collected for purification using Ni-NTA Agarose Resin columns. The complete sequence of the construct is given below.

[0130] > aHER2-hIgGlFc1-aGal3 amino acid sequence (SEQ ID NO. 17)

[0131] MSALLILALVGAAVVWAMEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGFLGGVRGVDGQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWWMNWVKQRPEQGLEWIGRIDPYDSETHYNQKFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCASLYYYGISLWGQGTLVTVSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGESPKLLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCLQATHFPWTFGGGTKLEIKHHHHHH*

[0132] >αHER2-hIgG1Fc1-αGal3 nucleic acid sequence (SEQ ID NO.18)

[0133]

[0134] To assess the targeting efficacy of aHER2-hIgGlFc1-aERVK in vivo, the inventors turned to a previously validated mouse model of HER2-positive breast cancer. This model involves injection of 4T1 cells stably expressing human HER2 (hHER2) and GFP into the mammary fat pad of mice, followed by intraperitoneal (i.p.) injection of aHER2-hIgGlFc1, an equimolar dose of aHER2-hIgGlFc1-aGal3 (10 mg / kg q3d), or vehicle control. Treatment with aHER2-hIgGlFc1-aGal3 resulted in a reduction in tumor size ( Figure 42 ) compared to control mice. Mice treated with aHER2-hIgGlFc1-aGal3 had a longer survival time ( Figure 42 ) compared to control mice. Treatment with aHER2 alone did not result in attenuated tumor growth or prolonged survival ( Figure 42 , 43). In terms of safety, mice treated with aHER2-hIgGlFc1 or aHER2-hIgGlFc1-aERVK did not experience weight loss over the course of the experiment, indicating that the treatments were well tolerated.

[0135] The above-described embodiments are merely illustrative of the preferred ways of practicing the present application and do not limit the scope of the present application. Any modifications and improvements made to the technical solutions of the present application by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of the claims of the present application.

Claims

1. A targeted protein degradation, characterized in that, The amino acid sequence of the targeted degradation protein is SEQ ID NO.

3.

2. A nucleotide sequence encoding the targeted degrading protein of claim 1, wherein, The nucleotide sequence encoding the targeted degradation protein with the amino acid sequence shown in SEQ ID NO. 3 is shown in SEQ ID NO.

4.

3. The targeted degradation protein of claim 1 for use in the preparation of a medicament for treating breast cancer.

4. Use according to claim 3, characterized in that, The targeted degradation protein is the only active ingredient in the medicament.

5. Use according to claim 3 or 4, characterized in that, The medicament is an injection.

6. A medicament for treating breast cancer, characterized by, The medicament contains the targeted degradation protein as claimed in claim 1.

7. The medicament according to claim 6, characterized in that, The medicament also contains a clinically acceptable excipient.

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