Polypeptide specifically binding to erythrocyte membrane or mutant thereof

Abstract

A polypeptide specifically binding to an erythrocyte membrane or a mutant thereof. The polypeptide or mutant thereof can be fused with various recombinant proteins. By using the binding property of the polypeptide or mutant thereof to erythrocytes and dynamically balancing the binding to and releasing from an erythrocyte membrane, the sustained release of a drug can be achieved, thereby prolonging the circulation half-life of the drug.

Description

A polypeptide or mutant thereof that specifically binds to the red blood cell membrane Technical Field

[0001] This invention discloses a polypeptide. Background Technology

[0002] The half-life of a drug is a crucial parameter describing its transport, metabolism, and excretion processes within a living organism. It reflects the rate at which the drug is eliminated from the body and determines the frequency and dosage of administration, making it an effective tool for measuring drug efficacy. Drugs with short half-lives experience a rapid decline in blood concentration, requiring more frequent dosing; conversely, drugs with long half-lives exhibit a slower decline in blood concentration, reducing the frequency of administration and maintaining stable drug concentrations for more effective disease control. The development of long-acting drug technologies not only reduces patient treatment costs by lowering dosages, thus reducing nursing costs and safety risks, but also improves patient compliance and treatment outcomes. For example, the long-acting formulations of growth hormone and GLP-1 receptor (glucagon-like peptide-1 receptor) agonists have not only provided patients with a more convenient treatment experience but have also significantly improved safety and efficacy. Currently, methods to increase the circulation of protein drugs mainly focus on three strategies: 1) increasing the apparent hydrodynamic radius of the molecule to reduce the glomerular filtration rate, for example, by chemically coupling polyethylene glycol (PEG) or other water-soluble polymers (such as XTEN); 2) regulating the balance between binding and release to plasma proteins to slowly release the drug and thus prolong its half-life, for example, by chemically coupling fatty acid chains (such as smegglutinin), which prolongs the half-life by binding to serum albumin through fatty acid chains; 3) fusion with the Fc of antibodies (such as abatacept), which not only increases the molecular volume and reduces renal clearance, but also prolongs the half-life by utilizing the protective effect mediated by FcRn (Fc receptor), with a plasma half-life reaching 10 days.

[0003] Mature red blood cells circulate in the human body for up to 120 days, making them a potential excellent carrier for extending drug half-life. Furthermore, red blood cells are the most numerous cells in the blood, lack a nucleus, can be reinfused from other cells, and possess extremely high biocompatibility, complete degradation, and the ability to escape and tolerate immune irrations. Based on these characteristics, many studies are currently focused on drug development and delivery, but significant breakthroughs have not yet been achieved. There are two main strategies for extending half-life using red blood cells: 1) using physicochemical methods to encapsulate drugs within red blood cells, and 2) enriching drugs on the surface of red blood cells. Many studies have been conducted on methods for encapsulating drugs within red blood cells, including hypotonic methods, chemical methods, endocytosis, and lipid fusion methods. The main mechanism involves actively or passively introducing the drug into the red blood cell under hypotonic conditions or through physical compression, followed by repairing the red blood cell membrane under hypertonic conditions to encapsulate the drug; the drug then enters the human circulation and is released using the permeability of the red blood cell membrane. This method causes significant damage to red blood cells and is difficult to maintain normal red blood cell circulation lifespan. Methods of enriching drugs on the surface of red blood cells are relatively gentle, such as using nanopolymer particles to attach them to the surface of red blood cells. These attached nanoparticles circulate with the red blood cells and gradually detach, thereby prolonging the drug's effect to some extent (Nanoscale. 2019, 11:1636–46; Acta Pharmacol Sin 2021,42:1040–1054). In addition, there is considerable research on red blood cell membrane-specific binding, such as targeting complement receptor type 1 (CR1), also known as the C3b receptor or C3b / C4b receptor, with approximately 50–1400 CR1 molecules per red blood cell. Using antibody-targeted CR1 antibodies fused with therapeutic proteins can significantly increase the drug's half-life by binding to red blood cells. For example, Sergei Zaitsev et al. fused a CR1 antibody with tPA, which increased the drug's half-life while reducing side effects (Blood. 2006,108:1895–902). However, because the copy number of CR1 on erythrocytes is relatively low, it carries fewer drugs. Furthermore, as an immune adhesion receptor, targeting CR1 can affect the important functions of erythrocytes in carrying, transporting, and clearing circulating immune complexes. B3p (Band 3 protein), also known as anion exchanger 1 (SLC4A1), is also mainly expressed on the surface of erythrocytes, with approximately 1.2 million copies, accounting for about 25% of erythrocyte cell membrane proteins. Balthasar JP et al. used a fusion of a nanobody targeting b3p and an antibody targeting TNF-α to significantly prolong the half-life in mice while exhibiting good safety (Int J Mol Sci. 2022 Dec 28:475.).However, erythrocyte b3p plays an important mechanical role in supporting the biconcave disc shape of erythrocytes through physical connections with ankyrin and the cytoskeleton network, and regulates the pH value within erythrocytes. It is directly related to physiological functions such as the elasticity, plasticity, and maintenance of gas exchange area of ​​erythrocytes. Targeting b3p may cause changes in the physical properties of erythrocytes and shorten their lifespan.

[0004] Phage display technology uses genetic engineering to express exogenous peptide or protein libraries on the surface of phages. Through screening based on whether or not the phages bind to target proteins anchored on the solid surface, and after 3-5 rounds of washing and enrichment, the proportion of phages specifically recognizing target molecules is gradually increased, ultimately yielding peptides or proteins that specifically bind to the target molecules. Jeffrey A. Hubbell and Stephan Kontos described using intact red blood cells as targets to screen phage libraries in several publications (Mol Pharm. 2010 Dec 6, 2141-7; Chem Soc Rev. 2012 Apr 7, 41:2686-95; Curr Opin Immunol. 2015 Aug, 35:80-8) and PCT / US2011 / 047078, obtaining a series of peptides. Using intact red blood cells as targets for phage library screening is problematic because cell-based targets are difficult to solidify, and red blood cells are prone to rupture during screening, leading to contamination from hemoglobin in the cytoplasm. The aforementioned patents and literature describe a peptide named ERY1 that specifically binds to mouse red blood cells but not to human red blood cells. In mouse models, ERY1 conjugation to antigen proteins prolongs the serum half-life of the antigen and weakens its immunogenicity. However, because ERY1 cannot bind to human red blood cells, there is a gap in translational medicine. Furthermore, using specific membrane proteins of red blood cells as targets, recombinant proteins are expressed and purified using genetic engineering methods, such as targeting CR1 (complement receptor 1) and GYPA (glycophorin-A), to screen for peptides targeting them. However, this method ignores the unique characteristics of red blood cells, which exhibit relatively unique post-translational modifications for all protein molecules, including phosphorylation, acetylation, methylation, glycosylation, and ubiquitination. Technical issues

[0005] To extend the half-life of a drug by targeting erythrocytes, several conditions must be met: 1) The binding to erythrocytes must be reversible, meaning the affinity cannot be too high; the drug must dissociate from the erythrocyte membrane into a free state to exert its effect; 2) High specificity, meaning it does not bind to other blood cells (such as lymphocytes) and will not cross the vascular endothelium to enter tissues due to binding to other cells. Furthermore, the binding to erythrocytes should ideally not affect their function. The binding site is not necessarily an amino acid sequence on a membrane protein; it can also be a specific post-translational modification or combination of proteins.

[0006] Therefore, the objective of this invention is to provide a polypeptide that conforms to the above description and can specifically bind to the erythrocyte membranes of mice and humans. When fused with a recombinant protein drug, the peptide's erythrocyte-binding property prolongs the drug's circulating half-life. Technical solutions

[0007] To achieve the above objectives, the present invention first provides a polypeptide or a mutant thereof that specifically binds to the erythrocyte membrane, the amino acid sequence of which is shown in SEQ ID NO.1, and the polypeptide is named RHM-6 in the present invention.

[0008] The amino acid sequence of the mutant polypeptide is either a mutant in which any amino acid residue at any position is mutated to alanine, or a truncated mutant in which amino acid residues at positions 8-9 are deleted, based on the amino acid sequence shown in SEQ ID NO.1.

[0009] To further optimize RHM-6, this invention conducted site-directed mutagenesis studies on nine amino acids in the sequence. First, it was found that removing the two amino acids at the C-terminus had little impact on the binding function to human erythrocytes. Subsequently, alanine (Ala, A) substitution mutations were performed on each amino acid pair. The study revealed that three mutants—V2A (replacing valine V at the N-terminal second amino acid with alanine A), D4A (replacing aspartic acid D at the N-terminal fourth amino acid with alanine A), and W6A (replacing tryptophan W at the N-terminal sixth amino acid with alanine A)—significantly improved the binding affinity to human erythrocytes.

[0010] In a preferred embodiment, the amino acid sequence of the mutant is selected from any one of SEQ ID NO.2-10.

[0011] Among them, the mutant with the amino acid sequence shown in SEQ ID NO.2 is named RHM-6 (L1A), the mutant with the amino acid sequence shown in SEQ ID NO.3 is named RHM-6 (V2A), the mutant with the amino acid sequence shown in SEQ ID NO.4 is named RHM-6 (E3A), the mutant with the amino acid sequence shown in SEQ ID NO.5 is named RHM-6 (D4A), the mutant with the amino acid sequence shown in SEQ ID NO.6 is named RHM-6 (L5A), the mutant with the amino acid sequence shown in SEQ ID NO.7 is named RHM-6 (W6A), the mutant with the amino acid sequence shown in SEQ ID NO.8 is named RHM-6 (I7A), and the mutant with the amino acid sequence shown in SEQ ID NO.9 is named RHM-6 (V9A); the truncated mutant with the amino acid residues at positions 8-9 deleted as shown in SEQ ID NO.10 is named RHM-6 (delGV).

[0012] In a more preferred embodiment, the red blood cells include human red blood cells, mouse red blood cells, rat red blood cells, and cynomolgus monkey red blood cells.

[0013] In this invention, biotin is conjugated to the N-terminus of the synthesized polypeptides for subsequent detection. The synthesized polypeptides were incubated in vitro with fresh human and mouse erythrocytes, and the binding of the selected polypeptides to intact erythrocytes was further confirmed by flow cytometry (FACS) and detection with fluorescently labeled streptomycin (e.g., Streptavidin-PE). Among them, polypeptide RHM-6 can bind to human and mouse erythrocytes with similar affinity (Kd value between 20 and 40 μM), indicating weak affinity. RHM-6 can bind not only to human and mouse erythrocytes, but also to rat erythrocytes, cynomolgus monkey erythrocytes, and beagle dog erythrocytes. However, RHM-6 did not show significant binding to circulating lymphocytes, neutrophils, NK cells, etc., nor to common mouse and human tumor cell lines. Sequence homology analysis of RHM-6 revealed no sequence identical to its full length in known protein sequence databases; similar sequences were found to be bacterial enzymes or partial sequences of unknown proteins (approximately 10% of the total protein sequence).

[0014] In this invention, to further understand the binding site of RHM-6 on the erythrocyte membrane, liquid chromatography-mass spectrometry (LC-MS) was performed: Biotin-RHM-6 was bound to streptavidin beads (SA beads), and then to human erythrocyte protein extracts. Components in the erythrocyte membrane extract specifically bound to RHM-6 were separated and purified, and then analyzed and identified using LC-MS. The identified proteins mainly included Ephrin type-A receptor 2 (EPHA2), MANSC domain-containing protein 4 (MANSC4), Beta-1,4-galactosyltransferase 1 (B4GALT1), Long-chain fatty acid transport protein 6 (SLC27A6), Transferrin receptor protein 2 (TFR2), Fer-1-like protein 6 (FER1L6), and Membrane protein FAM174A (FAM174A). Although these proteins are not erythrocyte-specific, no specific binding of RHM-6 to other cell types was observed. Therefore, it is hypothesized that these proteins undergo specific post-translational modifications on erythrocytes, including phosphorylation, acetylation, methylation, glycosylation, and ubiquitination. These findings also demonstrate that using erythrocyte membrane extracts, rather than recombinant proteins, as screening targets is a more effective strategy.

[0015] In a preferred embodiment, the polypeptide or its mutant specifically binds to one or more of the following on the erythrocyte membrane: Ephrin type-A receptor 2, MANSC domain-containing protein 4, Beta-1,4-galactosyltransferase 1, Long-chain fatty acid transport protein 6, Transferrin receptor protein 2, Fer-1-like protein 6, and Membrane protein FAM174A.

[0016] After co-incubation with RHM-6, no disruption of erythrocyte membrane integrity was observed, as evidenced by no significant difference in intracellular 2,3-DPG (2,3-diphosphoglycerate) and free hemoglobin compared to native erythrocytes (results not shown). In mouse experiments, no change in erythrocyte half-life was observed after co-incubation with RHM-6. Therefore, RHM-6 itself does not cause alterations in erythrocyte function.

[0017] Secondly, the present invention provides a fusion protein containing the above-mentioned polypeptide or its mutant.

[0018] In a preferred embodiment, the fusion protein further contains a biologically functional molecule or a biomarker.

[0019] In a more preferred embodiment, the biological functional molecule or biomarker is an antibody, enzyme, nucleic acid, or peptide. In a specific embodiment of the present invention, the antibody is a recombinant monoclonal antibody against trinitrophenyl (TNP) (ATNP-hIgG4), and its fusion protein with the peptide or its mutant is named as follows: ATNP-hIgG4-RHM-6, ATNP-hIgG4-RHM-6(L1A), ATNP-hIgG4-RHM-6(V2A), ATNP-hIgG4-RHM-6(E3A), ATNP-hIgG4-RHM-6(D4A), ATNP-hIgG4-RHM-6(L5A), ATNP-hIgG4-RHM-6(W6A), ATNP-hIgG4-RHM-6(I7A), ATNP-hIgG4-RHM-6(V9A), and ATNP-hIgG4-RHM-6 (delGV).

[0020] In another specific embodiment, the biological functional molecule is a human growth factor, and the fusion protein is named "hGH-RHM-6".

[0021] Furthermore, the present invention provides a conjugate, which is a biological functional molecule or biomarker conjugated with the above-mentioned polypeptide or its mutant.

[0022] In a preferred embodiment, the biological functional molecule or biomarker is an antibody, enzyme, nucleic acid, or polypeptide.

[0023] In one specific embodiment of the invention, a free cysteine ​​(Cys) residue is added to the N-terminus of RHM-6, and biotin is simultaneously coupled to facilitate subsequent detection. Under the mediation of the amine-thiol crosslinking agent sulfur-SMCC, Biotin-Cys-RHM-6 is randomly coupled to the lysine residues on hGH (human growth hormone) (hGH has 9 lysine residues), and the conjugate is called hGH-RHM-6×n. After mass spectrometry analysis, each hGH carries 2-3 RHM-6 residues.

[0024] According to the findings of this invention, a pharmaceutically effective strategy for prolonging the half-life of biofunctional molecules or biomarkers is provided. This strategy comprises fusing or conjugating a "erythrocyte-binding moiety" that specifically binds to patient erythrocytes (i.e., the erythrocyte membrane-binding polypeptide disclosed in this invention) with a pharmacologically active protein molecule, such as a recombinant protein, antibody, or antibody fragment. For example, in this invention, RHM-6 is fused to the C-terminus of an antibody heavy chain, or to the C-terminus of a recombinant protein such as growth hormone (GH), or RHM-6 is conjugated to growth hormone. First, the ability of the fusion protein and conjugate to bind to erythrocytes was tested in vitro using flow cytometry. Then, the fusion protein or conjugate was intravenously injected into mice, and the half-life of the fusion protein or conjugate in the mice was tested. This invention found that both can prolong the half-life.

[0025] Finally, this invention provides a method for screening peptides that specifically bind to erythrocyte membranes. Erythrocytes, as a special cell type, exhibit relatively unique post-translational modifications for all protein molecules, including phosphorylation, acetylation, methylation, and glycosylation. Extracts from erythrocyte membranes retain these unique post-translational modifications, enabling the screened peptides to specifically recognize protein molecules on erythrocyte membranes, even if those molecules are also expressed on other cell types. However, some peptides screened using existing methods only recognize protein molecules on erythrocytes. For example, MANSC domain-containing protein 4 is expressed in various cells and tissues, including squamous epithelial cells, neurons, and trophoblasts, possibly due to differences in glycosylation at positions 407 and 435. This invention utilizes a sequential screening method. For example, mouse erythrocyte membranes are first used as targets, and the resulting subgroup is then screened using human erythrocyte membranes as targets, and vice versa, to obtain peptides that can bind to both mouse and human erythrocyte membranes. The method includes the following steps:

[0026] (1) Prepare a mixed linear peptide library with a length of 8-15 amino acids, a linear peptide library with a length of 12 amino acids, and a peptide library based on the mixed linear peptide library with a length of 8-15 amino acids, excluding non-specific sequences that affect screening.

[0027] (2) Using mouse erythrocyte membrane as the solid phase and the peptide library obtained in step (1) as the mobile phase, the screened phages were named the primary sublibrary; then, using human erythrocyte membrane as the solid phase and the primary sublibrary as the mobile phase, the screened phages were named the secondary sublibrary; and

[0028] Using human erythrocyte membrane as the solid phase and the peptide library obtained in step (1) as the mobile phase, the selected phages were named the primary sublibrary; then using mouse erythrocyte membrane as the solid phase and the primary sublibrary as the mobile phase, the selected phages were named the secondary sublibrary.

[0029] (3) Use immunological methods to identify positive phage clones and obtain candidate phages that can bind to both mouse and human erythrocyte membranes simultaneously;

[0030] (4) The polypeptide sequence of the positive clone was determined by sequencing.

[0031] In the method provided by the present invention, the target is selected as erythrocyte membrane extract, which fully removes all components inside the cell, but retains all possible unique protein post-translational modifications of erythrocytes, and has a greater probability of obtaining peptides that specifically bind to erythrocytes.

[0032] In step (1), three different artificially synthesized phage libraries were used for screening, including a mixed linear peptide library of 8–15 amino acids (Lib P), a linear peptide library of 12 amino acids (Lib P Trimer), and the Lib P-QY peptide library. The Lib P peptide library screens for multiple different target proteins. If the same peptide repeatedly appears as a positive peptide in the library, it indicates that the peptide may bind to different target proteins due to non-specific adhesion and is considered a non-specific sequence, thus being removed from Lib P. Therefore, the Lib P-QY peptide library is a peptide library reconstructed based on the Lib P peptide library, excluding non-specific sequences that affect screening, thus improving the specificity for the target. All three libraries are fully synthetic peptide libraries constructed using the Kunkel mutation method. Kunkel mutation is an experimental method for studying gene mutations (Methods Mol Biol. 2012, 907:195–209). The principle is to use the error amplification mechanism of DNA polymerase to introduce mutations under specific in vitro conditions, thereby changing the sequence of the target gene. The Kunkel mutation efficiency used in this invention can reach 100%, which helps to ensure the diversity of the library and thus improve the success rate of screening.

[0033] In step (2), to facilitate translational medicine and obtain peptides capable of binding to both mouse and human erythrocyte membranes, multiple rounds of screening were conducted using two strategies: First, mouse erythrocyte membranes were used as the solid phase and a phage library as the mobile phase; the screened phages were named the primary sub-library. Then, human erythrocyte membranes were used as the solid phase and the primary sub-library as the mobile phase; the screened phages were named the secondary sub-library. The secondary sub-library was then tested using phage ELISA to identify positive phage clones, which were then amplified. Similarly, the primary sub-library was first screened using human erythrocyte membranes as the solid phase, and the secondary sub-library was then screened using mouse erythrocyte membranes as the solid phase; positive phage clones were then identified using phage ELISA. This cross-screening strategy yielded candidate phages capable of binding to both mouse and human erythrocyte membranes.

[0034] In step (3), the immunological method is ELISA. Human and mouse red blood cell membranes are used to coat ELISA plates, and empty phages are used as negative controls. The affinity of the obtained phage clones is semi-quantitatively detected and sorted, and phage clones with high affinity and binding to both human and mouse red blood cell membranes are selected.

[0035] In step (4), the polypeptide sequence of the positive clone was determined by PCR. After screening three phage libraries, 12 single polypeptide sequences were obtained. Beneficial effects

[0036] In summary, the peptides screened using the method for peptides that specifically bind to the erythrocyte membrane in this invention can be fused with protein drugs for expression. When the drug is injected into peripheral blood, its reversible binding to erythrocytes, using erythrocytes as a carrier, prolongs the drug's plasma half-life. The peptide fragments are small and lack specific functions, thus not affecting the drug's efficacy. Furthermore, the peptides of this invention can bind to both human and mouse erythrocytes with similar affinity; therefore, the drug's performance in mouse models can be used to predict its performance in humans, demonstrating excellent medical translational characteristics. Attached Figure Description

[0037] Figure 1. Detection of the binding affinity of phage-screened peptides to human erythrocytes, mouse erythrocytes, and HEK293 cells;

[0038] Figure 2. RHM-6 peptide binds specifically to erythrocytes but not to non-erythrocytes;

[0039] Figure 3. Affinity detection of RHM-6 peptide with human and mouse erythrocytes;

[0040] Figure 4. RHM-6 and mutant fusion expression with antibody, and detection of binding affinity to erythrocytes;

[0041] Figure 5. Serum half-life of RHM-6 fused with antibody in mice;

[0042] Figure 6. RHM-6 fusion expression or conjugation with recombinant protein hGH and its binding affinity to erythrocytes. Embodiments of the present invention

[0043] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0044] Example 1. Screening for polypeptide sequences that specifically bind to mouse and human erythrocyte membranes.

[0045] Using a cell membrane extraction kit (Beijing Xinquan Yongshuo Technology Co., Ltd., catalog number XQ-P1203), membrane proteins were extracted from mouse and human erythrocytes according to the instructions. A brief description is as follows: Take EDTA-anticoagulated blood from humans or mice, centrifuge at 500 g for 5 minutes to remove plasma and intermediate layer cells. Add twice the volume of 1×PBS (2 times the volume of erythrocytes), mix well, centrifuge at 500 g for 5 minutes, remove the supernatant, and repeat this washing process three times. Add 500 μL of CER to every 100 μL of erythrocyte pellet, vortex to resuspend, incubate on ice for 2 minutes, and then manually homogenize 20-30 times in an ice-cold glass homogenizer. Take 600 μL of the lysate, centrifuge at 800 g at 4 ℃ for 5 minutes, collect the supernatant (the supernatant is the cell membrane-cytoplasm mixture), add 1 / 10 of MER (60 μL), mix well, and incubate on ice for 5 minutes. Centrifuge at 14000 rpm, 4 ℃ for 30 minutes, remove the supernatant, and collect the precipitate, which is the cell membrane component. Resuspend in cell lysis buffer (Beyotime, product number P0013) to a concentration of 2×10⁻⁶. 7 A solution of / μL.

[0046] The screening of phage libraries was commissioned to Wuhan Shengshi Junlian Technology Biotechnology Co., Ltd. The phage libraries included a mixed linear peptide library of 8-15 amino acids (Lib P peptide library), a linear peptide library of 12 amino acids (Lib P Trimer peptide library), and a Lib P-QY peptide library. Taking the mixed linear peptide library of 8-15 amino acids (Lib P peptide library) as an example, screening was first performed on human erythrocyte membranes, and then on mouse erythrocyte membranes. The process is briefly described below:

[0047] 1. Coating: Human erythrocyte membrane extract was mixed with coating buffer (1×PBS, pH 7.2) at a volume ratio of 1:39 to prepare a cell membrane extract solution. 100 μL was applied per well to an ELISA plate (Thermofisher, catalog number 442404) and incubated overnight at 4 °C. The plate was washed three times with PBST (1×PBS, pH 7.2, 0.05% Tween 20). Wells not coated with human erythrocyte membranes served as negative controls for subsequent enrichment and identification in step 7.

[0048] 2. Blocking: Block the ELISA plate with 1% PVA (polyvinyl alcohol, Sigma, catalog number 363170, prepared with double distilled water), 100 μL / well, and incubate at room temperature for two hours. Wash four times with PBST.

[0049] 3. Binding: The Lib P peptide library was diluted to 1×10⁻⁶ with 1×PBS at pH 7.2. 13 Add 100 μL of pfu / mL to each well, incubate at room temperature for two hours, and then wash 8 times with PBST.

[0050] 4. Elution: Add 100 mM HCl at a rate of 100 μL / well to the above ELISA plate to elute the bound phages.

[0051] 5. Neutralization: Neutralize the eluted phage solution with 1 M Tris-HCl pH 8.0 (Solarbio, catalog number T1150) at a rate of 100 μL / well.

[0052] 6. Amplification: Using 100 μL of the neutralized phage described above, infect 1 mL of NEB with an OD600 of 0.8. ® 5-alpha F' Iq E. coli competent cells (NEB, catalog number C2992I) were cultured by shaking (220 rpm, 37 ℃, 1 h); then M13K07 helper phage (NEB, catalog number N0315S) was added, and cultured by shaking (220 rpm, 37 ℃, 1 h), before being transferred to 40 mL of 2YT / Carb / Kan medium (2x YT medium (Starmetre, catalog number M0009), 50 μg / mL carbenicillin (BBI, catalog number A600469-0005), 25 μg / mL kanamycin (BBI, catalog number A600286-0025)) and cultured overnight at 37 ℃ for expansion.

[0053] 7. Enrichment Identification: After amplification, if the number of phages screened by the human erythrocyte membrane is more than 5 times that of the negative control (wells not coated with human erythrocyte membrane serve as the negative control), the screening is considered successful. If the enrichment factor is insufficient, the phages amplified in step 6 are precipitated with PEG8000 / NaCl (200 g PEG8000, 146 g NaCl, and double-distilled water to 1 L), purified, and subjected to a second round of screening. Steps 1-7 are repeated until the enrichment factor exceeds 5 times.

[0054] The Lib P peptide library underwent six rounds of screening targeting human erythrocyte membranes (phage library screening typically involves 3-5 rounds; a sixth round was conducted because an enrichment trend was observed in the fifth round, with an enrichment fold approaching 5-fold). This resulted in a successfully enriched primary sublime library. Then, mouse erythrocyte membrane extract was used to coat well plates, and the primary sublime library underwent screening steps 1-7. Successfully enriched secondary sublime libraries were obtained. In this experiment, the primary sublime library underwent four rounds of screening to obtain successfully enriched secondary sublime libraries.

[0055] After successful enrichment, single clones were randomly selected from the secondary sub-library and subjected to phage ELISA to further determine whether they were positive clones. ELISA plates coated with 1% BSA (Sigma, catalog number A1933) served as negative controls. Clones with an OD value greater than or equal to 2 compared to the negative control (based on the ratio of the OD value bound to the erythrocyte membrane extract) were defined as positive clones. Positive clones were selected, and their DNA fragments were amplified and sequenced using universal primers. The phage ELISA procedure is briefly described as follows: positive phage clones were inoculated, cultured overnight, saturated, and then the phages were collected. Phages were added to wells coated with erythrocyte membrane extract (1% BSA-coated ELISA plates were used as negative controls), incubated, washed, and developed with anti-M13 HRP antibody (Sino Biological, catalog number 11973-MM05T-H) and TMB (Solarbio, catalog number PR1200). The absorbance at 450 nm was measured using a Thermo Fisher MLtiskan FC microplate reader.

[0056] DNA fragments from positive clones were amplified and sequenced. The sequencing results were analyzed, and single sequences with complete sequencing, no terminator mutations, and no double peaks were selected (a polypeptide region with one or more amino acid differences was defined as a single sequence), as shown in Tables 1 and 2. No polypeptide sequences binding to human or mouse erythrocyte membranes were found in the Lib P Trimer polypeptide library. For human erythrocyte membrane extracts only, the Lib P polypeptide library screened 14 single-sequence polypeptides (named RH); for mouse erythrocyte membrane extracts only, it screened 6 single-sequence polypeptides (named RM). Positive clones from the primary sub-library screened first for human erythrocyte membrane extracts and then for mouse erythrocyte membrane extracts were named (RHM); positive clones from the primary sub-library screened first for mouse erythrocyte membrane extracts and then for human erythrocyte membrane extracts were named (RMH). These combined yielded a total of 8 single-sequence polypeptides.

[0057] Table 1. Peptides screened from the Lib P peptide library

[0058]

[0059] Eleven peptides that bind to human and mouse erythrocyte membrane extracts were screened from the Lib P-QY peptide library. As shown in Table 2, RHM-6, RHM-108, and RHM-112 were peptides screened from both the Lib P and Lib P-QY phage libraries. RHM-6, in particular, was repeatedly screened from both phage libraries.

[0060] Table 2. Peptides screened from the Lib P-QY peptide library

[0061]

[0062] Based on the semi-quantitative results of phage ELISA, empty phages were used as negative controls and 1% BSA was used as blank controls. Phages with higher readings than the negative controls were selected and listed in descending order of affinity for human erythrocyte membranes, as shown in Table 3.

[0063] Table 3. Peptides that bind to both human and mouse erythrocyte membranes (sorted by affinity from highest to lowest)

[0064]

[0065] Example 2. Flow cytometry detection of peptide binding to fresh intact red blood cells of mice and humans

[0066] The 12 peptide sequences in Table 3 were synthesized by Genscript Biotech Inc. During synthesis, biotin was conjugated to the N-terminus, and a stock solution (collectively referred to as Biotin Peptide) with PBS or DMSO was prepared according to the peptide's solubility for subsequent detection.

[0067] The procedure for obtaining human and mouse erythrocytes is briefly described below: Select 10-12 week old SPF-grade female Balb / C mice. Disinfect under anesthesia, draw cardiac blood, and transfer it to K2-EDTA-coated centrifuge tubes to prevent coagulation. Store at 4°C. 0.5-0.8 mL of whole blood can be obtained from each mouse. Centrifuge at 500 g for 5 minutes at 4°C, and label the hematocrit (red, lower layer) and plasma (yellow, upper layer) levels on the tube. Slowly and thoroughly aspirate the plasma and intermediate precipitate (buffy coat) using a micropipette. Add bleach to the aspirated liquid and discard it into biohazardous waste. Transfer 0.1 mL of hematocrit to a 1.5 mL centrifuge tube, add 1 mL of PBS pH 7.4 solution, cap, and invert several times to mix. Centrifuge at 500 g for 5 minutes at 4°C, aspirate the supernatant, and discard it. Repeat the PBS washing step 3-4 times. After washing, add 100 μL to 1000 μL of PBS and store at 4 degrees Celsius until use.

[0068] Obtain 2-3 mL of blood from a healthy, anonymous blood donor via the antecubital vein. After skin disinfection, use a disposable vacuum blood collection device operated by a professional. Allow the blood to flow slowly along the tube wall, remove the tube at the marked point, and quickly invert it several times to mix and prevent coagulation. Centrifuge the blood at 500 g and 4°C for 5 minutes, and mark the hematocrit (red, lower layer) and plasma (yellow, upper layer) levels on the tube. Slowly and thoroughly aspirate the plasma and buffy coat using a micropipette. Add bleach to the aspirated liquid and discard it into biohazardous waste. Transfer 0.5 mL of hematocrit to a 15 mL centrifuge tube, add 5 mL of PBS pH 7.4 solution, cap, and invert several times to mix. Centrifuge at 500 g and 4°C for 5 minutes, aspirate the supernatant, and discard it. Repeat the PBS washing step 3-4 times. Add 1000 μL of PBS to the washed red blood cells and store at 4°C for later use.

[0069] HEK293 cells (human embryonic kidney cells) were used as a negative control for flow cytometry analysis. The specific procedure is as follows:

[0070] Dilute the 10 mg / mL stock concentration of Biotin Peptide to 20 μg / mL with PBS, and take 1×10 6Human red blood cells (hRBCs) or mouse red blood cells (mRBCs), 1×10 5 HEK293 was incubated with 20 μg / mL Biotin Peptide at room temperature for 20 minutes, washed twice with PBS, stained with Streptavidin-PE (BioLegend, product number 405204, 200-fold dilution), and detected by flow cytometry (Beijing Cenglang Biotechnology Co., Ltd., model MateCyte) and analyzed using NovoExpress software. The results (Figure 1) showed that these 12 peptides had different affinities for hRBC, mRBC, and HEK293.

[0071] The positivity rates of these 12 peptides on the three cell types are shown in Table 4. Three peptides—RHM-6, RHM-112, and RHM-106—showed strong binding affinity to both hRBCs and mRBCs (positivity rate > 70%). Of these three peptides, only RHM-6 showed weak binding affinity to HEK293. RHM-6 was selected for further investigation.

[0072] Table 4 shows the flow cytometry analysis of the binding of 12 peptides to hRBC, mRBC, and HEK293.

[0073]

[0074] Example 3. Flow cytometry detection of the binding of Biotin-RHM-6 peptide to other cells

[0075] Flow cytometry was used to detect whether Biotin-RHM-6 bound to rat erythrocytes, cynomolgus monkey erythrocytes (IPHASE Biotechnology Co., Ltd., catalog number 082B107.11), and beagle dog erythrocytes (Nanjing Senbeijia Biotechnology Co., Ltd., catalog number SBJ-RBC-BD002). Rat erythrocytes were obtained as in Example 2 (mouse erythrocytes). 1×10⁻⁶ erythrocytes were collected from each rat. 6 Red blood cells from rats, cynomolgus monkeys, and beagle dogs were washed twice with PBS, incubated with 20 μg / mL Biotin-RHM-6 at room temperature for 20 min, washed twice with PBS, and then incubated with Streptavidin-PE (BioLegend, catalog number 405204, 200-fold dilution) at room temperature for 20 min. After washing twice with PBS, the cells were analyzed by flow cytometry (Beijing Cenglang Biotechnology Co., Ltd., model MateCyte) and NovoExpress software. The results (top row of Figure 2) showed that Biotin-RHM-6 could bind to red blood cells from rats, cynomolgus monkeys, and beagle dogs, in addition to binding to mouse and human red blood cells.

[0076] To detect whether Biotin-RHM-6 binds to other blood cells, mouse spleen cells were extracted, and the binding of Biotin-RHM-6 was detected by flow cytometry, similar to the procedure described above.

[0077] The process of isolating and preparing mouse spleen lymphocytes is briefly described as follows: Mice were euthanized by vertebral dislocation. The lower abdomen of the mice was disinfected with alcohol swabs. The abdomen was aseptically opened, and the spleen was removed from the abdominal cavity and placed in a culture dish containing PBS buffer. Excess tissue was washed and removed. The spleen was cut into three segments. Using sterile forceps, the spleen was placed on a 200-mesh nylon sieve and then placed in a small petri dish containing 5 mL of fresh PBS buffer. The spleen was then ground using a 5 mL syringe plunger. The nylon sieve was removed, and the cell suspension was collected in a 15 mL centrifuge tube. The cells were centrifuged at 2000 rpm for 5 minutes at 4 °C, and the supernatant was discarded. The spleen cells were resuspended in 5 mL of erythrocyte lysis buffer. The cells were thoroughly dispersed using a pipette and incubated at room temperature (on ice) for 5 minutes. Then, the cells were centrifuged at 2000 rpm for 5 minutes at 4 °C, and the supernatant was discarded. The cells were resuspended in 1 mL of PBS buffer and transferred to a 1.5 mL centrifuge tube for later use.

[0078] The flow cytometry method is briefly described as follows: Take 2×10 5 Mouse spleen cells were incubated with 20 μg / mL Biotin-RHM-6 at room temperature for 20 minutes. After washing twice with PBS, they were then treated with ER780 anti-mouse CD45 (Elabscience, E-AB-F1136S, 50-fold dilution), Streptavidin-PE (BioLegend, 405204, 200-fold dilution), PE-Cy7 anti-mouse CD3 (Elabscience, E-AB-F1103UH, 200-fold dilution), FITC anti-mouse CD19 (Elabscience, E-AB-F0986UC, 500-fold dilution), or ER780 anti-mouse CD45 (Elabscience, E-AB-F1136S, 50-fold dilution), PE-Cy7 anti-mouse NK1.1 (Elabscience, E-AB-F1136S, 50-fold dilution), PE-Cy7 anti-mouse NK1.1 (Elabscience, E-AB-F1136S, 50-fold dilution). The binding of Biotin-RHM-6 to various cell types was detected using assays such as E-AB-F0987H (50-fold dilution) and Streptavidin-PE (BioLegend, 405204, 200-fold dilution). The results, as shown in the middle row of Figure 2, indicate that Biotin-RHM-6 did not bind non-specifically to mouse spleen cells, nor to mouse T cells or B cells, and showed virtually no binding to NK cells.

[0079] Flow cytometry was used to detect whether Biotin-RHM-6 specifically binds to CT26 (mouse colon cancer cells), Jurkat (human T-lymphocytic leukemia cells), MC38 (mouse colon cancer cells), NK-92MI (natural killer cells from human malignant non-Hodgkin lymphoma patients), and HEK293 (human embryonic kidney cells). 1×10⁻⁶ cells were used. 5 Cells were incubated with 20 μg / mL Biotin-RHM-6 at room temperature for 20 min, washed twice with PBS, and then incubated with Streptavidin-PE (BioLegend, catalog number 405204, 200-fold dilution) at room temperature for 20 min. After washing twice with PBS, the cells were analyzed by flow cytometry (Beijing Cenglang Biotechnology Co., Ltd., model MateCyte) and NovoExpress software. The results (bottom row of Figure 2) showed that Biotin-RHM-6 did not bind non-specifically to CT26, Jurkat, MC38, NK-92MI, and HEK293 cells.

[0080] In summary, Biotin-RHM-6 specifically binds to mouse erythrocytes, human erythrocytes, rat erythrocytes, cynomolgus monkey erythrocytes, and beagle dog erythrocytes. However, it does not show significant binding to circulating lymphocytes, neutrophils, NK cells, or common mouse and human tumor cell lines.

[0081] Example 4. Flow cytometry detection of the affinity of Biotin-RHM-6 for mouse and human erythrocytes.

[0082] Based on the amino acid sequence (WMVLPWLPGTLD) of ERY1 in the literature (Mol Pharm. 2010 Dec 6, 2141-7), Genscript Biotech Inc. was commissioned to synthesize the peptide. Biotin was conjugated to the N-terminus of the ERY1 peptide during synthesis and served as the control group in this invention. It has been reported (PCT / US2011 / 047078) that ERY1 specifically binds to mouse erythrocytes but not to human erythrocytes. In mouse models, conjugation of ERY1 to the antigen protein prolongs the serum half-life of the antigen and weakens its immunogenicity. Therefore, this invention uses it as a control experiment.

[0083] The 10 mg / mL stock concentration of Biotin-RHM-6 and Biotin-ERY1 was diluted to 250 μg / mL with PBS, and then serially diluted to the following concentrations: 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.95, 0.97, 0.49, and 0.24 μg / mL. The 11 dilutions of the peptide (0.24–250 μg / mL) were then mixed with 2 × 10⁻⁶ ppm of PBS. 5 Human or mouse erythrocytes were incubated with the peptide (room temperature, 20 min). Human or mouse erythrocytes not incubated with the peptide served as a negative control. After washing twice with PBS, Streptavidin-PE (BioLegend, catalog number 405204, 200-fold dilution) was added and incubated at room temperature for 20 min for staining. After washing twice more with PBS, flow cytometry (Beijing Cenglang Biotechnology Co., Ltd., model MateCyte) was used for detection. The mean fluorescence intensity (MFI) in the PE channel after different concentrations of the peptide bound to human or mouse erythrocytes was analyzed using NovoExpress software. Plotting peptide concentration on the x-axis and MFI in the PE channel on the y-axis, the concentration was logarithmized using GraphPad Prism 9.4.1, and then analyzed using Log(agonist) vs. response—Variable slope (four parameters). The results (Figure 3) showed that Biotin-RHM-6 could bind to human and mouse erythrocytes with similar affinity, with Kd values ​​of 20.7 μM and 37.77 μM, respectively. Kd is the dissociation constant, reflecting the affinity of a molecule for a target site; a smaller value indicates stronger affinity. Biotin-ERY1 was detected using the same method as Biotin-RHM-6, but exhibited completely different characteristics: Biotin-ERY1 did not bind to human erythrocytes, but only to mouse erythrocytes, with a Kd value of 2892 μM, far lower than the affinity of Biotin-RHM-6.

[0084] Example 5. Analysis of RHM-6 bound to erythrocyte membrane surface proteins using liquid chromatography-mass spectrometry.

[0085] Quantitative proteomics is a method for identifying and quantifying all proteins in a complex system. It can be used to screen and identify differentially expressed proteins among samples. Liquid chromatography-mass spectrometry (LC-MS) is a commonly used method in quantitative proteomics. The basic principle of LC-MS protein analysis is as follows: First, the protein sample is digested into short peptide fragments by trypsin. Then, after chromatographic separation, the fragments enter a mass spectrometer. The ion source of the mass spectrometer ionizes the short peptide sample, giving the peptides a charge. By recording the mass-to-charge ratio and charge of the ionized peptides, the relative molecular mass of the peptide is determined. Then, target peptides are screened using a high-energy collision cell to obtain a series of fragment ions (such as β-ions and γ-ions generated by amide bond breakage). By matching these fragment ion data with known databases, the accurate identification of protein or peptide molecules is ultimately achieved. In this invention, to identify the target protein that RHM-6 binds to on the erythrocyte membrane, an immunoprecipitation method was first used to separate the protein bound to RHM-6 from the erythrocyte membrane extract. Then, liquid chromatography-mass spectrometry was used to identify the protein bound to RHM-6. The method is briefly described below:

[0086] 1. Sample preparation

[0087] 1.1. Extract human erythrocyte membrane proteins, following the same procedure as in Example 1.

[0088] 1.2. Removal of components that non-specifically bind to human erythrocyte membrane extract and streptavidin beads (SA-beads).

[0089] 1.2.1. Cleaning the magnetic beads: Take 20 μL of SA-beads magnetic beads (Beyotime, P2151) and wash them three times with 500 μL of 1×TBS (20 mM Tris, 0.13 M NaCl, pH 7.6) according to the instructions. Then place the washed magnetic beads on a magnetic rack to collect them.

[0090] 1.2.2. Removal of components from human erythrocyte membrane extract that nonspecifically bind to SA-beads: 1.6 × 10 9 The membrane proteins produced by erythrocytes were diluted to 150 μL with PBST (1×PBS, pH 7.2, 0.05% Tween 20), and the magnetic beads were resuspended in this solution. After incubation at room temperature for 30 min, the beads were placed on a magnetic rack and the supernatant was collected.

[0091] 1.3. Combination of SA-beads with Biotin-RHM-6

[0092] 1.3.1. Cleaning the magnetic beads: Same as step 1.2.1.

[0093] 1.3.2. SA-beads binding with Biotin-RHM-6: Biotin-RHM-6 at a storage concentration of 10 mg / mL was diluted to 50 μg / mL with PBST. 200 μL of the washed magnetic beads was resuspended and incubated at room temperature for 30 min. The magnetic beads were washed four times with PBST.

[0094] 1.3.3. SA-beads (after binding with Biotin-RHM-6) were conjugated with human erythrocyte membrane extract: The beads from step 1.3.2 were resuspended in 50 μL of the supernatant from step 1.2.2, incubated at room temperature for 30 min, and then washed four times with PBST. After washing twice more with PBS, the supernatant was discarded, and the magnetic beads were stored at -80°C.

[0095] 2. Identification by Liquid Chromatography-Mass Spectrometry (LC-MS)

[0096] 2.1. Sample enzymatic digestion

[0097] 2.1.1. Preparation of TCEP lysis buffer: 1% DOC (Sodium deoxycholate, Sigma-Aldrich, catalog number 30970-100G), 10 μM TCEP (Tris (2-carboxyethyl) Phosphine hydrochloride, Sigma-Aldrich, catalog number C4706-10G), 40 mM CAA (2-Chloracetamide, Sigma-Aldrich, catalog number 22790-250G-F), 100 mM Tris-HCl (Sangon Biotech, catalog number A610195-0500), 1 mM PMSF (Phenylmethanesuonyl fluoride, Sigma, catalog number 78830-5G), pH 8.5.

[0098] 2.1.2. Sample Processing

[0099] After thawing the beads, 60 μL of TCEP lysis buffer was added to the sample, and the mixture was incubated at 95 °C and 1500 rpm for 10 min. Once the sample had cooled to room temperature, 1 μg of trypsin (Promega, catalog number V5280) was added, and the mixture was digested overnight at 37 °C. Subsequently, 6 μL of 10% formic acid aqueous solution (Fisher Chemical, catalog number A117-50) was added and the mixture was incubated for 3 min. After centrifugation, the supernatant was collected. The supernatant was desalted using a C18 desalting column and then concentrated to a dry powder state using an Eppendorf Concentrator Plus centrifuge, ready for loading.

[0100] 2.2. Data Acquisition

[0101] The experimental samples were separated using an EASY-nLC 1200 (Thermo Fisher) high-performance liquid chromatography system with a flow rate of nanoliters. Mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was a mixture of 0.1% formic acid and 80% acetonitrile (Fisher Chemical, catalog number A955-4). First, the column was equilibrated with 100% mobile phase A. Then, the enzymatically digested peptides of the sample were delivered to the sample column via an autosampler, and subsequently separated by the analytical column at a flow rate of 600 nL / min.

[0102] Peptide samples were separated by analytical column chromatography and analyzed using a Q Exactive HF-X (Thermo Fisher) mass spectrometer. The detection mode was positive ion mode, with a precursor ion scan range of 300-1400 m / z, a primary mass spectrometry resolution of 120,000 at 200 m / z, an AGC (Automatic Gain Control) target of 3e6, a maximum IT of 30 ms, and a dynamic exclusion time of 12.0 s. The mass-to-charge ratio of peptides and peptide fragments was acquired as follows: 60 fragment spectra were acquired after each full scan (MS2 scan) using a higher-energy collisional dissociation (HCD) fragmentation mode with a collision energy of 27%, an isolation window of 1.6 m / z, and a secondary mass spectrometry resolution of 7,500 at 200 m / z.

[0103] 2.3. Protein Identification and Quantitative Analysis

[0104] The raw mass spectrometry data were RAW files, and the iProteome one-stop data analysis cloud platform was used for qualitative and quantitative analysis via database search. The database search parameters were set as follows:

[0105] Enzyme: Trypsin

[0106] Fixed modifications: Variable modifications: Carbamidomethyl (C), Oxidation (M), Acetyl (Protein N-term)

[0107] Missed Cleavage: 2

[0108] Peptide Mass Tolerance: 20 ppm

[0109] Fragment Mass Tolerance: 50 mmu

[0110] The components of RHM-6 that bind to the human erythrocyte membrane were identified by LC-MASS, as shown in Table 5.

[0111] Table 5. Components of RHM-6 that bind to human erythrocyte membranes

[0112]

[0113] Example 6. Flow cytometry detection of ATNP-hIgG4-RHM-6 mutant and the binding of ATNP-hIgG4-RHM-6 to human erythrocytes (LVEDLWIGV)

[0114] To investigate the amino acid residues in the RHM-6 polypeptide sequence that have a key impact on the binding function of human erythrocytes, a total of 9 RHM-6 mutants were designed. Specifically, each amino acid in the RHM-6 sequence (amino acid and nucleic acid sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 11, respectively) was mutated to alanine, resulting in a single-site alanine substitution mutation, named RHM-6 (L1A) (amino acid and nucleic acid sequences as shown in SEQ ID NO. 2 and SEQ ID NO. 12, respectively), RHM-6 (V2A) (amino acid and nucleic acid sequences as shown in SEQ ID NO. 3 and SEQ ID NO. 13, respectively), RHM-6 (E3A) (amino acid and nucleic acid sequences as shown in SEQ ID NO. 4 and SEQ ID NO. 14, respectively), RHM-6 (D4A) (amino acid and nucleic acid sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 15, respectively), RHM-6 (L5A) (amino acid and nucleic acid sequences as shown in SEQ ID NO. 6 and SEQ ID NO. 16, respectively), RHM-6 (W6A) (amino acid and nucleic acid sequences as shown in SEQ ID NO. 7 and SEQ ID NO. 17, respectively), and RHM-6... (I7A) (amino acid sequence and nucleic acid sequence are shown in SEQ ID NO.8 and SEQ ID NO.18, respectively), RHM-6 (V9A) (amino acid sequence and nucleic acid sequence are shown in SEQ ID NO.9 and SEQ ID NO.19, respectively); the 8th and 9th amino acids were removed to form a truncation mutation, named RHM-6 (delGV) (amino acid sequence and nucleic acid sequence are shown in SEQ ID NO.10 and SEQ ID NO.20, respectively). The nucleic acid sequences of the above 9 different mutants were fused with the C-terminal nucleic acid sequence of the antibody heavy chain for expression. In this invention, the antibody selected was the recombinant monoclonal antibody ATNP-hIgG4 against trinitrophenyl (TNP) (Immunochemistry.1969,6(2):163-174). Due to its high specificity in binding to TNP and lack of affinity for most proteins, it is often used as an isotype control for antibodies.The fusion proteins were named ATNP-hIgG4-RHM-6, ATNP-hIgG4-RHM-6(L1A), ATNP-hIgG4-RHM-6(V2A), ATNP-hIgG4-RHM-6(E3A), ATNP-hIgG4-RHM-6(D4A), ATNP-hIgG4-RHM-6(L5A), ATNP-hIgG4-RHM-6(W6A), ATNP-hIgG4-RHM-6(I7A), ATNP-hIgG4-RHM-6(V9A), and ATNP-hIgG4-RHM-6 (delGV). These 10 fusion proteins (each containing an Fc tag) were recombinantly expressed and purified. The purified proteins were then subjected to binding experiments with human erythrocytes. Since ATNP-hIgG4 itself does not bind to human erythrocytes, the binding of the fusion proteins to human erythrocytes is achieved through RHM-6 or its mutants. In this invention, the fusion protein was analyzed one by one using flow cytometry. It was found that the removal of the two amino acids at the C-terminus had little effect on the binding function of human erythrocytes. Subsequently, alanine (Ala) substitution mutations were performed on each amino acid. The study found that except for the mutation of lysine (L) at the N-terminus 1 position and valine (V) at the N-terminus 9 position to alanine (A) which did not improve affinity, the other mutations could enhance the affinity for human erythrocytes. The mutations of valine (V) at the N-terminus 2 position, aspartic acid (D) at the N-terminus 4 position, and tryptophan (W) at the N-terminus 6 position were particularly significant.

[0115] The experimental procedure is briefly described as follows:

[0116] 1. Plasmid construction:

[0117] Suzhou Junji Biotechnology Co., Ltd. was commissioned to synthesize plasmid DNA, which was then cloned into the pTT5 universal vector (Transient expression and purification of chimeric heavy chain antibodies. Protein Expression and Purification. 2009, 65(1):77-82). Based on the pTT5 plasmid, the multiple cloning site was modified and named pJSV. It was used to express 10 fusion proteins, namely ATNP-hIgG4-RHM-6, ATNP-hIgG4-RHM-6 (L1A), ATNP-hIgG4-RHM-6 (V2A), ATNP-hIgG4-RHM-6 (E3A), ATNP-hIgG4-RHM-6 (D4A), ATNP-hIgG4-RHM-6 (L5A), ATNP-hIgG4-RHM-6 (W6A), ATNP-hIgG4-RHM-6 (I7A), ATNP-hIgG4-RHM-6 (V9A), and ATNP-hIgG4-RHM-6 (delGV). For specific procedures, refer to "Molecular Cloning: A Laboratory Manual". The plasmid should first be transformed into TOP10, sequenced, preserved, and cultured. Prepare the plasmid according to the procedures outlined in the "Qiagen Mini-prep Kit" and "Qiagen Endofree Maxi-prep Kit".

[0118] 2. Transient expression of the recombinant antibody ATNP-hIgG4-RHM-6 and its mutant in HEK293 mammalian cells (human embryonic kidney cells). Using a 30 mL transfection system as an example, 30 μg of plasmid was added to 1.5 mL of Opti-MEM (Thermo Fisher, catalog number 11058021), vortexed, and incubated at room temperature for 5 min. Simultaneously, 150 μg of PEI (Thermo Fisher, catalog number 919012) was added to 1.5 mL of Opti-MEM, gently inverted, and then added to the above plasmid solution. After gently inverting and incubating at room temperature for 20 min, the mixture was transferred to 30 mL of HEK293 cells. The cells were then incubated in a 5% CO2 incubator at 37 °C with shaking. 24 hours after transfection, add 600 μL of 50×KT-Feed (final concentration 1×, Zhuhai Kerry Biotechnology) and 180 μL of 500 mM VPA (final concentration 1 mM, MercK, catalog number P4543). 96 hours after transfection, centrifuge to collect the cell supernatant.

[0119] 3. Protein purification: Ezfast AT protein A 1 mL (Borglon) was used to perform affinity purification of the recombinant protein in the supernatant.

[0120] 4. Purified recombinant ATNP-hIgG4-RHM-6 variant and its biotinylation and flow cytometry detection:

[0121] First, the purified ATNP-hIgG4-RHM-6 and its mutants were biotinylated for subsequent detection with fluorescently labeled streptomycin (e.g., Streptavidin-PE). ATNP-hIgG4-RHM-6 and its mutants were diluted to 0.5 mg / mL. 100 μL of each was added to 2.56 μL of 10 mM sulfo-Biotin-NHS ester (Bio-Lab, GS4320), mixed well, and incubated at room temperature for 1 hour. Then, 5 μL of 1 M Tris-HCl (pH 8.0) was added to each, mixed well, and incubated at room temperature for 5 min. After replacing the buffer in the PD SpinTrap G-25 column (Cytiva, catalog number 28918004) with 1×PBS pH 7.2, the ATNP-hIgG4-RHM-6 mutant and ATNP-hIgG4-RHM-6 were desalted, and Biotin-ATNP-hIgG4-RHM-6 mutant and Biotin-ATNP-hIgG4-RHM-6 were successfully prepared with a concentration of 0.474 mg / mL and a volume of 108 μL.

[0122] Biotinylated ATNP-hIgG4-RHM-6 and its mutants were incubated with fresh red blood cells to detect the binding ability of various mutants to human red blood cells: 20 μL of biotinylated ATNP-hIgG4-RHM-6 and its mutants (concentration 0.474 mg / mL) were incubated with 1 μL of human red blood cells (concentration 1×10⁻⁶). 6 The cells were incubated at room temperature for 1 hour, washed twice with PBS, resuspended in 20 μL of Streptavidin-PE (BioLegend, product number 405204, 200-fold dilution), and incubated at room temperature for 20 min. After washing twice with PBS and resuspending in PBS, the cells were then analyzed and processed using flow cytometry (Beijing Cenglang Biotechnology Co., Ltd., model MateCyte). The mean fluorescence intensity (MFI) of human erythrocytes binding to ATNP-hIgG4-RHM-6 and its mutants was analyzed and calculated using Novo Express software.

[0123] The results (Figure 4, Table 6) showed that ATNP-hIgG4-RHM-6 and its mutants were well distinguishable from negative cell populations after binding to human erythrocytes, indicating that RHM-6 and its mutants can directly fuse to the C-terminus of the protein without affecting the binding of RHM-6 to erythrocytes. Removing the two amino acids at the C-terminus of the RHM-6 peptide has little effect on its binding function to human erythrocytes. The average fluorescence intensity of ATNP-hIgG4-RHM-6(V2A), ATNP-hIgG4-RHM-6(D4A), and ATNP-hIgG4-RHM-6(W6A) after binding to human erythrocytes is stronger than that of ATNP-hIgG4-RHM-6. The average fluorescence intensity of ATNP-hIgG4-RHM-6(E3A), ATNP-hIgG4-RHM-6(L5A), and ATNP-hIgG4-RHM-6(V9A) after binding to human erythrocytes is slightly stronger than that of ATNP-hIgG4-RHM-6. The average fluorescence intensity of ATNP-hIgG4-RHM-6(delGV), ATNP-hIgG4-RHM-6(L1A), and ATNP-hIgG4-RHM-6(I7A) after binding to human erythrocytes is slightly lower than that of ATNP-hIgG4-RHM-6.

[0124] Table 6. Mean fluorescence intensity of human erythrocytes after binding to ATNP-RHM-6 mutant and ATNP-RHM-6

[0125]

[0126] Example 7. Evaluation of the half-life of antibody drug fused with RHM-6 in mice

[0127] To investigate whether the fusion expression of antibody drugs with RHM-6 can prolong their half-life in animals, this experiment was conducted. Female C57BL6J mice aged 6–8 weeks (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into two groups: G1 and G2. Group G1 received a single intravenous infusion of ATNP-hIgG4-RHM-6 at 350 μg / kg, while group G2 received a single intravenous infusion of ATNP-hIgG4 at 350 μg / kg. Two days prior to intravenous infusion, anticoagulated blood was collected as a negative control. The day of intravenous infusion was designated as day 0. On days 0, 2, 5, 7, 9, 14, 21, 28, and 35, 50 μL of EDTA-anticoagulated blood was collected from each mouse in each group (blood collection time for day 0 was 5 min after intravenous infusion). The collected anticoagulated blood was diluted 2-fold with PBSA-5 (PBS pH 7.2, 5% BSA) and 10 mM EDTA, and then frozen at -80℃. The levels of ATNP-hIgG4-RHM-6 and ATNP-hIgG4 in mouse blood were detected using an anti-human IgG4 ELISA kit (Solepro, catalog number SEKH-0208) to determine the pharmacokinetics of ATNP-hIgG4-RHM-6 and ATNP-hIgG4 in mice. The results are shown in Figure 5. The half-life of ATNP-hIgG4-RHM-6 in vivo is as long as 20 days, while the half-life of ATNP-hIgG4 in vivo is 17 days.

[0128] Animal experiments suggest that fusion expression of RHM-6 on antibodies can prolong their half-life in mice. This provides a strategy for improving the efficacy, safety, and cost-effectiveness of antibody drugs.

[0129] Example 8. Evaluation of the half-life of human growth hormone (hGH) fusion expression with RHM-6 and chemical conjugation with RHM-6 in mice.

[0130] hGH has been proven to be an effective treatment for growth hormone deficiency (GHD), aiming to restore longitudinal growth and improve quality of life. Compared to short-acting daily growth hormone formulations, long-acting weekly formulations significantly reduce the frequency of injections in children, effectively improving treatment adherence and quality of life. Currently, four long-acting growth hormones have been approved for marketing globally: Jinsai Zeng (PEGylated modification) from Changchun High & New Technology Industries Group's subsidiary Jinsai Pharmaceutical, Novo Nordisk's Sogroya (fatty acid chain modification), Ascendis' Skytrofa (inert methoxy polyethylene glycol carrier), and Pfizer's Ngenla (human chorionic gonadotropin C-terminal peptide fragment fusion). There are approximately 5 million people with short stature in my country, but only 5% receive treatment. While long-acting formulations eliminate the pain of daily injections, the treatment cost is high; for example, Jinsai Zeng costs over 100,000 yuan annually, while Skytrofa costs approximately 200,000 yuan per year overseas. Long-acting, affordable growth hormone is the future trend.

[0131] In this invention, RHM-6 is directly fused to the C-terminus of hGH (sequence Genebank ID: NM_000515.5), a simple process. However, considering that each hGH molecule only fuses with one RHM-6, resulting in weak affinity for erythrocytes, a chemical conjugation strategy was also employed to ensure that each hGH molecule has multiple RHM-6 molecules, thereby increasing affinity for erythrocytes. hGH fused with RHM-6 and RHM-6 chemically conjugated were intravenously injected into mice to investigate whether RHM-6 could prolong the half-life of hGH in animals.

[0132] 1. Preparation of hGH-RHM-6 and hGH-RHM-6×n protein samples:

[0133] The RHM-6 fusion-expressed hGH (hGH-RHM-6) was prepared by Shanghai Baiying Biotechnology Co., Ltd. To facilitate recombinant expression and purification, a histidine tag (HIS tag) was added between hGH and RHM-6 (amino acid sequence as shown in SEQ ID NO.22, nucleic acid sequence as shown in SEQ ID NO.21; in this invention, unless otherwise stated, hGH-RHM-6 refers to a fusion protein with a 6-histidine tag added between hGH and RHM-6). Similar to the expression and purification process of ATNP-IgG4-RMH-6 in Example 6, after plasmid synthesis and transient expression in HEK293 cells, purification was performed using a HiTrap chelated HP column (Cytiva, catalog number 29051021) according to the manufacturer's instructions. The purified protein was then fluorescently labeled using a Sulfo-NHS-Cy5 (Maclean, catalog number S849951) for subsequent flow cytometry detection.

[0134] The RHM-6 chemically conjugated hGH (hGH-RHM-6×n) was synthesized by Genscript Biotech Inc. Biotin-Cys-RHM-6 (sequence GSCGGSGLVEDLWIGV). A free cysteine ​​(C) residue was added to the N-terminus of RHM-6, and biotin was simultaneously conjugated for convenient subsequent detection. Under the mediation of the amine-thiol crosslinking agent sulfo-SMCC (sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate, Yisheng Biotech, catalog number 20332ES25), Biotin-Cys-RHM-6 was randomly conjugated to lysine residues on hGH (hGH has 9 lysine residues), and the conjugate was named hGH-RHM-6×n. Mass spectrometry analysis showed that each hGH carried 2-3 RHM-6 residues. The steps are briefly described as follows: hGH was prepared by Shanghai Baiying Biotechnology Co., Ltd., and a histidine tag (HIS tag) was fused to the C-terminus for easy purification. In this invention, all recombinant hGH were fused with an HIS tag at the C-terminus.

[0135] hGH at a concentration of 1 mg / mL was incubated with sulfur-SMCC (Yisheng Biotechnology, catalog number 20332ES25) at a molar ratio of 1:20 in PBS pH 7.2 at room temperature for 1 hour. After removing free small molecules with a desalting column PD SpinTrap G-25 (Cytiva, catalog number 28918004), it was mixed with Biotin-Cys-RHM-6 dissolved in 3 M guanidine hydrochloride (Sinopharm Chemical Reagent Co., Ltd., catalog number 50-01-1) at a molar ratio of 1:10. After standing at room temperature for 0.5 hours, free small molecules and unreacted free Biotin-Cys-RHM-6 were removed with PD SpinTrap G-25.

[0136] 2. Flow cytometry detection of the binding of hGH-RHM-6 and hGH-RHM-6×n to erythrocytes

[0137] Take 20 μL of hGH-RHM-6 (concentration 0.64 mg / mL) and 1 μL of mRBC (concentration 1×10⁻⁶). 6The cells were incubated at room temperature for 20 min with 20 μL of Streptavidin-PE (BioLegend, product number 405204, 200-fold dilution) for 20 min, washed twice with PBS, resuspended in PBS, and then incubated at room temperature for 20 min. The cells were washed twice with PBS, resuspended in PBS, and then analyzed using flow cytometry (Beijing Cenglang Biotechnology Co., Ltd., model MateCyte). The results are shown in Figure 6 (bottom). The hGH-RHM-6 bound to erythrocytes showed good differentiation from the negative cell population, indicating that hGH-RHM-6 can bind to mRBCs.

[0138] hGH-RHM-6×n contains biotin, so its binding to erythrocytes can be directly detected using Streptavidin-PE. Take 20 μL of hGH-RHM-6×n (0.3 mg / mL) and 1 μL of erythrocytes (1×10⁻⁶ ppm). 6 The cells were incubated at room temperature for 20 min with 20 μL of Streptavidin-PE (BioLegend, product number 405204, 200-fold dilution) for 20 min, washed twice with PBS, resuspended in PBS, and then incubated at room temperature for 20 min. The cells were washed twice with PBS, resuspended in PBS, and then analyzed using flow cytometry (Beijing Cenglang Biotechnology Co., Ltd., model MateCyte). The results are shown in Figure 6 (top). The hGH-RHM-6×n bound to erythrocytes showed good differentiation from the negative cell population. This indicates that hGH-RHM-6×n can bind to mRBCs. Because the staining methods used to detect the binding of hGH-RHM-6×n and hGH-RHM-6 to erythrocytes are different, the binding strength of hGH-RHM-6×n and hGH-RHM-6 to erythrocytes cannot be directly compared.

[0139] 3. Evaluation of half-life in mice

[0140] Twenty-four 6-8 week old female C57BL6J mice were ordered from Beijing Vital River Laboratory Animal Technology Co., Ltd., and randomly divided into three groups: G1, G2, and G3. Each group of mice was further divided into four subgroups, with two mice in each subgroup. Blood was collected in rotation according to the blood collection time points. hGH-RHM-6×n, hGH-RHM-6, and hGH were intravenously infused into mice in groups G1, G2, and G3 at 300 μg / kg, respectively. Anticoagulated blood was collected two days before the intravenous infusion as a negative control. The time point of intravenous infusion was recorded as 0 min. Blood was collected from four groups of mice at 2 min, 4 min, 6 min, 8 min, 15 min, 30 min, 1 hour, 2 hour, 4 hour, 8 hour, 16 hour, and 24 hours. 20 μL of anticoagulated blood was collected from the outer canthus of each mouse. The collected anticoagulated blood was diluted twice with PBSA-5 (PBS pH 7.2, 5% BSA) and 10 mM EDTA, and then frozen at -80℃. The level of hGH in mouse blood was detected using a human growth hormone kit (Solepro, catalog number SEKH-0196) to determine the pharmacokinetics of hGH-RHM-6×n, hGH-RHM-6, and hGH in mice. The half-life of hGH-RHM-6×n in mice was the longest at 10.3 min, followed by hGH-RHM-6 at 8.0 min, while hGH had the shortest half-life at 7.2 min. hGH fusion expression of RHM-6 or chemically conjugated RHM-6 can prolong the half-life in mice. Furthermore, the more RHM-6 molecules on each hGH molecule, the longer the half-life in animals. This provides a strategy for improving the efficacy, safety, and economic feasibility of human growth hormone. Industrial applicability

[0141] This invention discloses a polypeptide or its mutant that specifically binds to the erythrocyte membrane. The polypeptide or its mutant that specifically binds to the erythrocyte membrane can be used for industrial preparation and has industrial applicability.

Claims

1. A polypeptide or a mutant thereof that specifically binds to the erythrocyte membrane, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1. The amino acid sequence of the mutant of the polypeptide is a mutant in which other amino acid residues at any position are mutated to alanine based on the amino acid sequence shown in SEQ ID NO.1, or a truncated mutant in which amino acid residues at positions 8-9 are deleted.

2. The polypeptide or its mutant according to claim 1, characterized in that, The amino acid sequence of the mutant is selected from any one of SEQ ID NO.2-SEQ ID NO.

10.

3. The polypeptide or its mutant according to claim 1 or 2, characterized in that, The red blood cells include human red blood cells, mouse red blood cells, rat red blood cells, and cynomolgus monkey red blood cells.

4. The polypeptide or its mutant according to claim 3, characterized in that, The polypeptide or its mutants specifically bind to one or more of the following on the erythrocyte membrane: Ephrin type-A receptor 2, MANSC domain-containing protein 4, Beta-1,4-galactosyltransferase 1, Long-chain fatty acid transport protein 6, Transferrin receptor protein 2, Fer-1-like protein 6, and Membrane protein FAM174A.

5. A fusion protein comprising the polypeptide of claim 1 or 2 or a mutant thereof.

6. The fusion protein according to claim 5, characterized in that, The fusion protein also contains biological functional molecules or biomarkers.

7. The fusion protein according to claim 6, characterized in that, The biological functional molecules or biomarkers are antibodies, enzymes, nucleic acids, or polypeptides.

8. The fusion protein according to claim 7, characterized in that, The polypeptide is a human growth factor.

9. A conjugate, characterized in that, The conjugate is a biological functional molecule or biomarker that is conjugated with the polypeptide or its mutant as described in claim 1 or 2.

10. The conjugate according to claim 9, characterized in that, The biological functional molecules or biomarkers are antibodies, enzymes, nucleic acids, or polypeptides.

11. A method for screening peptides that specifically bind to the erythrocyte membrane, characterized in that, The method includes the following steps: (1) Prepare a mixed linear peptide library with a length of 8-15 amino acids, a linear peptide library with a length of 12 amino acids, and a peptide library based on the mixed linear peptide library with a length of 8-15 amino acids, excluding non-specific sequences that affect screening. (2) Using mouse erythrocyte membrane as the solid phase and the peptide library obtained in step (1) as the mobile phase, the screened phages were named the primary sublibrary; then, using human erythrocyte membrane as the solid phase and the primary sublibrary as the mobile phase, the screened phages were named the secondary sublibrary; and Using human erythrocyte membrane as the solid phase and the peptide library obtained in step (1) as the mobile phase, the selected phages were named the primary sublibrary; then using mouse erythrocyte membrane as the solid phase and the primary sublibrary as the mobile phase, the selected phages were named the secondary sublibrary. (3) Use immunological methods to identify positive phage clones and obtain candidate phages that can bind to both mouse and human erythrocyte membranes simultaneously; (4) The polypeptide sequence of the positive clone was determined by sequencing.

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