Targeted degradable protein and application thereof

Abstract

The invention provides a targeted degradable protein and application thereof, and belongs to the technical field of biological medicine. The amino acid sequence of the targeted degradation protein is SEQ ID NO.1. When the targeted degradation protein is used for treating breast cancer, target protein degradation can be directly mediated, so that the effect of the target protein is weakened fundamentally, the targeted degradation protein only needs to be combined with the target protein with very strong affinity, specific epitopes do not need to be combined, the design difficulty is low, and the number of adaptive targets is large. Therefore, the targeted degradation protein provided by the invention takes the membrane molecule with high expression of tumor specificity as the effect protein, so that the dual effects of tumor targeting and mediated endocytosis 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 the "effector" protein, 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] Glucose-regulated protein 78 (GRP78, also known as BiP) is a member of the highly conserved HSP70 family, which is a 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. In adaptation to these chronic stresses in the tumor microenvironment, cancer cells are observed to upregulate the expression of GRP78 to promote their proliferation, invasion, therapeutic resistance and immune evasion, making GRP78 a prototypical oncogene. 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, which affects the maturation of dendritic cells and impairs the production of pro-inflammatory cytokines to favor resolution of the immune response by generating regulatory T and B cell populations. The release of sGRP78 can infer the strength of chemotherapy-related damage. The inventors' previous studies observed the fact that serum sGRP78 increased in breast cancer patients, and the inventors reported that the levels of some patients further increased after receiving 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 the sensitivity of patients to chemotherapy, to achieve precise stratified treatment. SUMMARY

[0006] One of the purposes of the present application is to provide a targeted degradation protein, the amino acid sequence of which is SEQ ID NO. 1.

[0007] 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. 1 is as shown in SEQ ID NO. 2.

[0008] The third purpose of the present application is to provide the use of the above-mentioned targeted degradation protein in the preparation of a medicament for treating breast cancer.

[0009] Preferably, the targeted degradation protein is the only active ingredient in the medicament.

[0010] More preferably, the medicament is an injection.

[0011] The fourth purpose of the present application is to provide a medicament for treating breast cancer, which contains the above-mentioned targeted degradation protein.

[0012] Preferably, the medicament further contains a clinically acceptable excipient.

[0013] Compared with the prior art, the present application has the following beneficial effects: Traditional extracellular protein targeting mainly uses neutralizing antibodies for blocking. Compared with corresponding neutralizing antibodies, the eTPD therapy developed by the present application has the following characteristics: (1) Traditional neutralizing antibodies directly block the effector region of target proteins and can also enter cells through Fc receptors, but they cannot directly reduce target proteins. The eTPD therapy can directly mediate target protein degradation, thereby weakening the effects of target proteins at the root; (2) The antigen epitope of traditional neutralizing antibodies must be the effector region of target proteins, such as the binding site of target proteins and their receptors. However, it is difficult to design antibodies against this epitope, and 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 target protein degradation, only needs to have strong affinity with target proteins, does not need to bind to specific epitopes, has low design difficulty, and is suitable for many target points; (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 tumors. The eTPD therapy uses tumor-specific high-expression membrane molecules as its effector proteins, thereby achieving the dual effects of tumor targeting and mediating endocytosis. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 A schematic diagram of the enzymatic degradation of sGRP78 in HER2+ cancer by subA in Example 1.

[0015] Figure 2 Binding curves of aHER2 and aHER2-subA on HER2+ BT474 and HER2- MDA-MB-468 cells in Example 1, quantified by anti-His mean fluorescence intensity (MFI).

[0016] Figure 3 Relationship between sICOS concentration in the supernatant of in vitro HER2+ BT474 cell culture and aHER2 / aHER2-subA and chemotherapy in Example 1.

[0017] Figure 4 B cells were co-cultured with BT474 cells and treated with chemotherapy drugs (nab-P), aHER2 or aHER2-subA for 18 hours, and IL10 and PD-L1 were stained in B cells in Example 1.

[0018] Figure 5In Example 1, B cells were co-cultured with BT474 cells and treated with chemotherapy drugs (nab-P), αHER2, or αHER2-subA for 18 hours. B cells were then sorted and co-cultured with naive CD4+ T cells for 2 days, and the proportion of Treg cells was measured.

[0019] Figure 6 BALB / c mice were inoculated with syngeneic 4T1 breast cancer cells carrying human HER2 as in Example 1, and then injected with αHER2 or αHER2-subA. Tumor volume was monitored every 3 days.

[0020] Figure 7 The image shows the survival curve of the mouse in Example 1.

[0021] Figure 8 The weight of the mouse in Example 1. Detailed Implementation

[0022] Example 1: Targeted Degradation of sGRP78 It has been reported that the bacterial serine protease subunit (SubA) can specifically degrade the GRP78 protein. The inventors had previously purified the 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 the receptor on the surface of breast cancer cells. Figure 1 The inventors designed a conjugate that links the C-terminus of SubA to the nanobody, called "αHER2-SubA". Figure 1 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 agarose Resin column. The complete sequences of the constructs are given below: >αHER2-subA amino acid sequence (SEQ ID NO.1) MSALLILALVGAAVVWAEVQLVEKGGGRVQAGGSLRLRCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFRIRRDNAKNTVYLRMRRLKPEDTAVYYCKRFRTAAQGTDYWGQGTRVTVSKGGSGGSGGSGGSMLKILWTYILFLLFISASARAEKPWYFDAIGLTETTMSLTDKNTPVVVSVVDSGVAFIGGLSDSEFAKFSFTQDGSPFPVKKSEALYIHGTAMASLIASRYGIYGVYPHALISSRRVIPDGVQDSWIRAIESIMSNVFLAPGEEKIINISGGQKGVASASVWTELLSRMGRNNDRLIVAAVGNDGADIRKLSAQQRIWPAAYHPVSSVNKKQDPVIRVAALAQYRKGETPVLHGGGITGSRFGNNWVDIAAPGQNITFLRPDAKTGTGSGTSEATAIVSGVLAAMTSCNPRATATELKRTLLESADKYPSLVDKVTEGRVLNAEKAISMFCKKNYIPVRQGRMHHHHHH >alphaHER2-subA nucleic acid sequence (SEQ ID NO. 2) The affinity of aHER2-SubA (Kd = 23.5 nM) to HER2-expressing cancer cells was similar to aHER2 (Kd = 20.3 nM) (see Figure 2 ). Next, ELISA experiments showed that aHER2-SubA significantly reduced sGRP78 in the culture supernatant of human cancer cell lines (see Figure 3 ). Thus, the Nanobody-SubA fusion specifically targets cancer cells to degrade sGRP78.

[0023] Through a series of cell experiments, the inventors tested whether the Nanobody-SubA could selectively reverse sGRP78-dependent regulatory B cell induction. HER2+ breast cancer cells were co-cultured with B cells and treated with a chemotherapy drug (nab-P) and eTPD proteins (aHER2-SubA or aHER2). aHER2-SubA was able to downregulate IL10 and PD-L1 expression in B cells, regardless of chemotherapy (see Figure 4 ). These aHER2-SubA treated B cells were also less potent in inducing Tregs than other groups (see Figure 5 ). Thus, the Nanobody-SubA fusion degrades cancer cell-derived sGRP78 to reverse B cell-induced CTL dysfunction.

[0024] 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 injecting 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 a vehicle control. Treatment with aHER2-SubA resulted in reduced tumor size ( Figure 6 ). aHER2-SubA treated mice had a longer survival time compared to control mice ( Figure 7 ). Treatment with aHER2 alone did not result in attenuated tumor growth or prolonged survival ( Figure 6 ,7). 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 8 ).

[0025] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application, and various modifications and improvements of the present application made by those skilled in the art based on the technical solutions of the present application without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

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

1.

2. A nucleotide sequence encoding the targeted degradation protein of claim 1, characterized in that, The nucleotide sequence encoding the targeted degradation protein, as shown in SEQ ID NO.1, is shown in SEQ ID NO.

2.

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

4. The application according to claim 3, characterized in that, The drug contains a targeted degradation protein as its sole active ingredient.

5. The application according to claim 3 or 4, characterized in that, The drug is an injectable form.

6. A drug for treating breast cancer, characterized in that, The drug contains the targeted degradation protein as described in claim 1.

7. The drug according to claim 6, characterized in that, The drug also contains clinically acceptable excipients.

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