Modified red blood cells

Abstract

A method for modifying red blood cells by means of an OaAEP1 enzyme-mediated ligation reaction, wherein red blood cells are coupled to an effector active agent, such as an antibody, via a linker. Also provided are such modified red blood cells and the use thereof, for example for use in effector active agents such as antibodies.

Description

Modified red blood cells

[0001] This application claims priority to and the benefit of PCT application No. PCT / CN2023 / 138465, filed on December 13, 2023, entitled “Modified Red Blood Cells,” the entire contents of which are hereby incorporated by reference. Technical Field

[0002] The present invention relates to modified red blood cells (RBCs), and more particularly to covalently modified RBCs, and their use for the delivery of drugs. Background Art

[0003] Recent developments in drug delivery systems for prolonged drug retention in the treatment of a variety of human diseases have attracted considerable attention. However, many systems still face various challenges and limitations, such as poor stability, unwanted toxicity, and immune responses. Red blood cells (RBCs), the most common cell type in the human body, have been extensively studied for over 30 years as ideal in vivo drug delivery systems due to their unique biological properties, including: (i) a wide range of in vivo circulation; (ii) good biocompatibility as a biomaterial with a long in vivo survival time; (iii) a large surface area to volume ratio; and (iv) the absence of a nucleus, mitochondria, and other organelles.

[0004] RBCs have been developed as drug delivery vehicles by directly encapsulating, non-covalently linking exogenous peptides, or by fusing proteins to RBC surface protein-specific antibodies. Such modified red blood cells have been shown to have limitations in their in vivo applications. For example, encapsulation can damage the cell membrane, thereby affecting the in vivo survival of engineered cells. In addition, the non-covalent connection between polymer particles and red blood cells is easily dissociated, and the payload will quickly degrade in vivo. Therefore, modifying proteins in a covalent and site-specific manner to attach them to red blood cells (e.g., native proteins on the surface of the red blood cell membrane) has become a more reliable option.

[0005] Chemical modification of proteins plays a crucial role in developing tools for studying protein biological properties and producing therapeutic protein conjugates. Applications include in vivo imaging, the generation of drug derivatives, and the direct modification of protein function for screening and therapeutic purposes. Current chemical modification methods mostly rely on modification of cysteine ​​sulfhydryl groups and lysine ε-amines. These methods require extensive optimization for achieving the desired site-specificity for different proteins. Enzyme-catalyzed bioconjugation reactions, which proceed under mild conditions, offer a powerful and highly specific alternative to chemical modification. Recent research has identified numerous enzymes with ligase functions, such as biotin ligases, inteins, subtiligases, sortases, and AEP. Among them, asparaginyl endopeptidase (AEP) is a well-suited ligase for protein modification due to its exceptional catalytic efficiency and extremely short recognition site (tripeptide). AEP is a cysteine ​​protease that cleaves the C-terminus of the recognized asparagine or asparagine residue, forming a thioester intermediate that can be degraded by hydrolysis or attack by a suitable nucleophile to produce the ligation product.

[0006] OaAEP1 is an asparagine endopeptidase isolated from the plant Hedyotis diffusa, which is evolutionarily related to butelase 1. It has the ability to connect the N-terminus and C-terminus of peptide substrates and is easily recombinantly expressed in Escherichia coli and then activated under acidic pH conditions. Moreover, it only needs to recognize a linker unit with only two N-terminal GL residues (NH2-Gly-Leu) and three C-terminal NGL residues (ASN-Gly-Leu-COOH) to efficiently catalyze the connection of two polypeptides / proteins through the two ends in the form of a peptide bond. Given the catalytic properties of this enzyme, it has very broad prospects in many aspects such as protein modification, chemical-enzymatic synthesis and protein labeling.

[0007] In order to provide a more reliable, stable, convenient and low-cost drug (effective active agent) delivery system, the art needs to provide a more efficient method through new tools.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention therefore provides a method for modifying erythrocytes to couple / link them to effector active agents, wherein the method allows the desired effector active agent to be coupled / linked to the erythrocytes via a linker polypeptide, thereby the latter becoming a delivery platform for the effector active agent.

[0010] The effector active agent after being linked to red blood cells has one or more of the following properties, in particular, has one or more of the following improved properties compared to the effector active agent not linked to red blood cells: increased activity, prolonged in vivo half-life, improved in vivo distribution, and / or increased safety.

[0011] In some preferred embodiments, the method comprises two steps (two-step method): (a) coupling the linker polypeptide to the red blood cells; and (b) linking the effector active agent to the linker polypeptide in the presence of a ligase. In some specific embodiments, step (a) is performed before step (b). In some specific embodiments, step (b) is performed before step (a).

[0012] In some specific embodiments, the method includes the steps of: (a) coupling a linker polypeptide to a red blood cell to obtain a red blood cell containing a linker, wherein the linker polypeptide contains an N-terminal recognition sequence of a ligase at its N-terminus; (b) in the presence of a ligase, contacting the red blood cell containing the linker obtained in step (a) with an effector active agent under conditions suitable for the ligation reaction to occur, wherein the effector active agent contains a C-terminal recognition sequence of a ligase, wherein the ligase recognizes the N-terminal recognition sequence and the C-terminal recognition sequence and catalyzes the connection between the two, thereby connecting the red blood cell to the effector active agent to form an RBC-effector active agent conjugate.

[0013] In some embodiments, the ligase is an asparaginyl endopeptidase (AEP), such as OaAEP1, preferably OaAEP1 from Hedyotis diffusa, more preferably OaAEP1 having a C247A substitution, and most preferably OaAEP1 having the amino acid sequence shown in SEQ ID NO:1 and / or OaAEP1 encoded by a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:2.

[0014] In some embodiments, the OaAEP1 is activated; preferably, the activation treatment comprises mixing the OaAEP1 protein with acetic acid / sodium acetate buffer and incubating at 35-40°C (e.g., 37°C); more preferably, the activation treatment comprises mixing 3 mg / ml of OaAEP1 protein with 200 mM acetic acid / sodium acetate (V:V=9:1) buffer at a volume ratio of 1:3, and incubating at 35-40°C (e.g., 37°C) for 4-5 hours.

[0015] In some embodiments, the effector agent comprises a C-terminal recognition sequence for a ligase that is coupled or ligated via an in vitro reaction.

[0016] In some embodiments, the effector agent is a protein and comprises a C-terminal recognition sequence for a ligase at the C-terminus of at least one of its polypeptides. In some specific embodiments, the C-terminal recognition sequence for the ligase contained in the effector protein is connected to the C-terminus of the ligase through a flexible linker (e.g., (GGGGS) n ) is connected to the effector protein molecule body (preferably, its C-terminus). In some specific embodiments, the C-terminal recognition sequence of the ligase contained in the effector protein is coupled or connected through an in vitro reaction. In some specific embodiments, the C-terminal recognition sequence of the ligase contained in the effector protein (and optionally, a linker) is obtained by adding a corresponding coding sequence fragment to the 3' end of the gene sequence of the effector protein and expressing the effector protein containing the C-terminal recognition sequence (and optionally, a linker) connected to the C-terminus of the effector protein molecule body in the cell.

[0017] In some specific embodiments, the C-terminal recognition sequence of the ligase is selected from NGL, NDL, NPL, NAL, NCL, NEL, NFL, NHL, NKL, NIL, NLL, NQL, NRL, NSL, NTL, NVL, NWL, NYL, NSLD, NSLAN or NG, preferably NGL, NPL or NDL.

[0018] In some specific embodiments, the N-terminal recognition sequence of the ligase is selected from DL, PL, AL, CL, EL, FL, HL, KL, IL, LL, QL, RL, SL, TL, VL, WL, YL, SLD, SLAN, DLG, PLG, ALG, CLG, ELG, FLG, HLG, KLG, ILG, LLG, QLG, RLG, SLG, TLG, VLG, WLG, YLG, SLDG, SLANG, DLK, PLK, ALK, CLK, ELK, FLK, HLK, KLK, ILK, LLK, QLK, RLK, SLK, TLK, VLK, WLK, YLK, SLDK, SLANK, G, GG, GL, GGG, GLG, GLK or GGL, preferably GLK.

[0019] In some specific embodiments, the effector agent is an antibody. In some preferred embodiments, the effector agent is a monoclonal antibody or an antigen-binding fragment thereof. In some preferred embodiments, the effector agent is a full-length monoclonal antibody. In some specific embodiments, the antibody is a 4-1BB monoclonal antibody or a PD-1 monoclonal antibody.

[0020] In some more specific embodiments, the 4-1BB monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:4; and / or the light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:5.

[0021] In some more specific embodiments, the PD-1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:8; and / or the light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:9.

[0022] In some specific embodiments, the red blood cells in step (a) are treated with a reducing agent, and the linker polypeptide comprises a maleimide group, whereby the red blood cells are linked to the maleimide group via the reduced thiol group, such that the linker polypeptide is coupled to the red blood cells, wherein the linker polypeptide comprises an N-terminal recognition sequence of a ligase at its N-terminus.

[0023] In some specific embodiments, the reducing agent is selected from one or more of β-mercaptoethanol, DTT, DTE, TCEP and glutathione, preferably TCEP.

[0024] In some specific embodiments, the red blood cells are not genetically modified. In some specific embodiments, the red blood cells are not genetically engineered to express a protein comprising an N-terminal recognition sequence for a ligase. In some preferred embodiments, the red blood cells are natural red blood cells, such as natural human red blood cells.

[0025] In another aspect, there is provided a modified red blood cell (RBC) produced according to the method of the present invention.

[0026] In another aspect, a modified erythrocyte is provided that comprises an effector active agent coupled to its outer surface, wherein the effector active agent is linked to a linker polypeptide and coupled to a native erythrocyte membrane protein via the linker polypeptide, and the amino acid sequence NGL, NDL, NPL, NAL, NCL, NEL, NFL, NHL, NKL, NIL, NLL, NQL, NRL, NSL, NTL, NVL, NWL, NYL, NSLD, NSLAN, or NG, preferably NGL, NPL, or NDL, is comprised at the junction of the effector active agent and the linker polypeptide. In some preferred embodiments, the effector active agent is linked to the N-terminus of the linker polypeptide. In some most preferred embodiments, the connection between the effector active agent and the linker polypeptide is through an asparaginyl endopeptidase (AEP)-mediated ligation reaction, such as OaAEP1, preferably OaAEP1 from Hedyotis diffusa, more preferably OaAEP1 with a C247A substitution, and most preferably OaAEP1 with the amino acid sequence shown in SEQ ID NO: 1 and / or OaAEP1 encoded by a nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO: 2.

[0027] In some embodiments, the OaAEP1 is activated; preferably, the activation treatment comprises mixing the OaAEP1 protein with acetic acid / sodium acetate buffer and incubating at 35-40°C (e.g., 37°C); more preferably, the activation treatment comprises mixing 3 mg / ml of OaAEP1 protein with 200 mM acetic acid / sodium acetate (V:V=9:1) buffer at a volume ratio of 1:3, and incubating at 37°C for 4-5 hours.

[0028] In some embodiments, the linker polypeptide is coupled to a sulfhydryl group of a native red blood cell membrane protein via a maleimide group.

[0029] In some embodiments, the effector active agent, prior to linkage, comprises at its C-terminus an asparaginyl endopeptidase recognition sequence selected from NGL, NDL, NPL, NAL, NCL, NEL, NFL, NHL, NKL, NIL, NLL, NQL, NRL, NSL, NTL, NVL, NWL, NYL, NSLD, NSLAN, or NG, preferably NGL, NPL, or NDL.

[0030] In some embodiments, the effector agent is a protein and is linked to a linker polypeptide at its C-terminus. In some preferred embodiments, the effector agent is an antibody. In some more preferred embodiments, the effector agent is a monoclonal antibody or an antigen-binding fragment thereof. In some more preferred embodiments, the effector agent is a full-length monoclonal antibody.

[0031] In some specific embodiments, the antibody is a 4-1BB monoclonal antibody or a PD-1 monoclonal antibody.

[0032] In some more specific embodiments, the 4-1BB monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:4; and / or the light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:5.

[0033] In some more specific embodiments, the PD-1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:8; and / or the light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:9.

[0034] Therefore, the present invention essentially provides red blood cells carrying 4-1BB antibodies (red blood cell-4-1BB antibody conjugates, or referred to as RBC-Anti-4-1BB or Anti-4-1BB-RBC) and methods for preparing the same.

[0035] The Anti-4-1BB-RBC has one or more of the following properties, in particular, one or more of the following improved properties compared to the corresponding 4-1BB antibody not carried on red blood cells:

[0036] 1) Improve the efficacy of the 4-1BB antibody, for example, for cancers such as non-small cell lung cancer, lymphoma, ovarian cancer, head and neck cancer, breast cancer, gastrointestinal cancer such as colon cancer, colorectal cancer or rectal cancer, etc., with better tumor suppression effect and / or lower effective dose; specifically, for example

[0037] (a) It has a better tumor suppression effect in tumors sensitive to 4-1BB antibodies;

[0038] (b) A lower effective dose in 4-1BB antibody-sensitive tumors;

[0039] 2) It is effective for cancers that are tolerant to or insensitive to 4-1BB antibodies; for example, it has better tumor suppression effects and / or has a lower effective dose for non-small cell lung cancer, lymphoma, ovarian cancer, head and neck cancer, breast cancer, gastrointestinal cancer such as colon cancer, colorectal cancer or rectal cancer that are insensitive to 4-1BB antibody treatment; specifically, for example

[0040] (a) It has a better tumor suppression effect in tumors that are insensitive to 4-1BB antibodies;

[0041] (b) A lower effective dose in tumors insensitive to 4-1BB antibodies;

[0042] 3) It has stronger 4-1BB pathway activation ability. For example, in in vitro cell activity experiments, it has stronger 4-1BB pathway activation ability than the same dose of 4-1BB antibody;

[0043] 4) improving the distribution of 4-1BB antibodies in vivo (e.g., in mice or humans), for example, by enriching a higher proportion of 4-1BB antibodies in the spleen and / or activating more effector CD8+ T cells in the spleen;

[0044] 5) improving the half-life of the 4-1BB antibody in vivo (e.g., in mice or humans), such as the plasma half-life, for example, allowing the 4-1BB antibody to remain in the circulation for a longer time and slowing the rate of decrease in the concentration of the 4-1BB antibody in the plasma, for example, slowing down by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%;

[0045] 6) Activates a higher proportion of CD8+ T cells compared to the corresponding 4-1BB antibody at the same dose;

[0046] 7) Compared with the corresponding 4-1BB antibody at the same dose, it better activates the immune system and immune cells, such as activating more immunotherapy markers. For example, compared with the corresponding 4-1BB antibody not carried on red blood cells, it can more significantly activate the NF-κB, JNK / SAPK and / or p38 MAPK signaling pathways at a lower dose (for example, as shown in the downstream Luciferase fluorescence signal (RFU) assay). Enhancement), indicating that it can better activate the immune system and immune cells;

[0047] 8) It has certain safety in acute toxicity experiments.

[0048] Furthermore, the present invention essentially provides red blood cells carrying PD-1 antibodies (red blood cell-PD1 antibody conjugates, or referred to as RBC-Anti-PD1 or Anti-PD1-RBC) and methods for preparing the same.

[0049] The Anti-PD1-RBC has one or more of the following properties, in particular, one or more of the following improved properties compared to the corresponding PD-1 antibody not carried on red blood cells:

[0050] 1) Improve the efficacy of the PD-1 antibody, for example, for cancers such as lung cancer, pancreatic cancer, breast cancer, gastrointestinal cancer such as colon cancer, colorectal cancer or rectal cancer, etc., with better tumor suppression effect and / or lower effective dose; specifically, for example

[0051] (a) It has a better tumor suppressive effect in cancers sensitive to PD1 antibodies;

[0052] (b) A lower effective dose in cancers sensitive to PD1 antibodies;

[0053] 2) It is effective for cancers that are resistant to or insensitive to PD-1 antibodies; for example, it has better tumor suppression effects and / or has a lower effective dose for lung cancer, pancreatic cancer, breast cancer, gastrointestinal cancer such as colon cancer, colorectal cancer or rectal cancer that are insensitive to PD-1 antibody treatment; specifically, for example

[0054] (a) It has a better tumor suppressive effect in cancers that are insensitive to PD1 antibodies;

[0055] (b) A lower effective dose in cancers that are insensitive to PD1 antibodies;

[0056] 3) having a stronger PD1-PDL1 binding blocking ability, for example, having a stronger PD1-PDL1 binding blocking ability than the same dose of the corresponding PD1 antibody not loaded on red blood cells;

[0057] 4) improving the distribution of PD1 antibodies in vivo (e.g., in mice or humans), for example, by enriching a higher proportion of PD1 antibodies in the spleen and / or activating more effector CD8+ T cells in the spleen;

[0058] 5) improving the half-life of the PD1 antibody in vivo (e.g., in mice or humans), such as the plasma half-life, for example, allowing the PD1 antibody to remain in the circulation for a longer time and slowing the rate of decrease in the concentration of the PD1 antibody in the plasma, for example, slowing down by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%;

[0059] 6) Activate a higher proportion of CD8+ T cells compared to the same dose of the corresponding PD1 antibody;

[0060] 7) Compared with the same dose of the corresponding PD-1 antibody, it better activates the immune system and immune cells, such as activating more immunotherapy markers. For example, compared with the corresponding PD-1 antibody not carried on red blood cells, it can more significantly activate the NFAT signaling pathway at a lower dose (for example, as shown in the downstream Luciferase fluorescence signal (RFU) assay). Enhancement), indicating that it can better activate the immune system and immune cells;

[0061] 8) It has a certain degree of safety in acute toxicity experiments;

[0062] In another aspect, a pharmaceutical composition is provided, comprising the modified erythrocytes of the present invention, and optionally, a pharmaceutically / physiologically acceptable carrier.

[0063] In another aspect, the present invention provides a composition comprising an asparagine endopeptidase, a linker polypeptide comprising an N-terminal recognition sequence of an asparagine endopeptidase, an effector active agent comprising a C-terminal recognition sequence of an asparagine endopeptidase, and optionally a pharmaceutically / physiologically acceptable carrier, wherein the linker polypeptide preferably comprises a maleimide group, wherein the asparagine endopeptidase recognizes the N-terminal recognition sequence and the C-terminal recognition sequence under suitable conditions (e.g., physiological conditions) and catalyzes the connection between the two.

[0064] In some embodiments, the asparagine endopeptidase is OaAEP1, preferably OaAEP1 from Hedyotis diffusa, more preferably OaAEP1 having a C247A substitution, and most preferably OaAEP1 having the amino acid sequence shown in SEQ ID NO: 1 and / or OaAEP1 encoded by a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 2.

[0065] In some embodiments, the OaAEP1 is activated; preferably, the activation treatment comprises mixing the OaAEP1 protein with acetic acid / sodium acetate buffer and incubating at 35-40°C (e.g., 37°C); more preferably, the activation treatment comprises mixing 3 mg / ml of OaAEP1 protein with 200 mM acetic acid / sodium acetate (V:V=9:1) buffer at a volume ratio of 1:3, and incubating at 37°C for 4-5 hours.

[0066] In some embodiments, the C-terminal recognition sequence is selected from NGL, NDL, NPL, NAL, NCL, NEL, NFL, NHL, NKL, NIL, NLL, NQL, NRL, NSL, NTL, NVL, NWL, NYL, NSLD, NSLAN or NG, preferably NGL, NPL or NDL.

[0067] In some specific embodiments, the N-terminal recognition sequence is selected from DL, PL, AL, CL, EL, FL, HL, KL, IL, LL, QL, RL, SL, TL, VL, WL, YL, SLD, SLAN, DLG, PLG, ALG, CLG, ELG, FLG, HLG, KLG, ILG, LLG, QLG, RLG, SLG, TLG, VLG, WLG, YLG, SLDG, SLANG, DLK, PLK, ALK, CLK, ELK, FLK, HLK, KLK, ILK, LLK, QLK, RLK, SLK, TLK, VLK, WLK, YLK, SLDK, SLANK, G, GG, GL, GGG, GLG, GLK or GGL, preferably GLK.

[0068] In some embodiments, after the composition contacts red blood cells in vivo, the linker polypeptide is linked to a sulfhydryl group on the surface of the red blood cell membrane via the maleimide group. In some embodiments, after the composition contacts red blood cells in vivo, the asparagine endopeptidase recognizes the N-terminal recognition sequence and the C-terminal recognition sequence and catalyzes the connection between the linker polypeptide and the effector active agent.

[0069] In another aspect, there is provided use of the modified red blood cells of the present invention in the preparation of a medicament for treatment.

[0070] In another aspect, a method of treatment is provided, comprising administering to an individual in need thereof a modified erythrocyte according to the present invention or a pharmaceutical composition of the present invention.

[0071] In another aspect, a method for increasing the plasma half-life of an effector active agent is provided, comprising the steps of: (a) treating the effector active agent so that it contains a C-terminal recognition sequence for a ligase; and (b) linking the effector active agent to red blood cells by the method described above.

[0072] In another aspect, a method for enhancing the in vivo efficacy of an effector active agent is provided, comprising the steps of: (a) treating the effector active agent so that it contains a C-terminal recognition sequence for a ligase; and (b) linking the effector active agent to red blood cells by the method described above.

[0073] Compared with the red blood cell modification and transformation method derived from stem cells, the red blood cell modification method of the present invention has one or more of the following advantages, such as convenient material collection, simple process flow, and a shorter time is required from peripheral blood collection to the acquisition of modified red blood cells, for example, only 2 days. The active effector covalently attached to the red blood cells of the present invention can be a therapeutic drug (such as a protein drug), which is less damaging to red blood cells than the known hypotonic method, for example, it does not destroy the physiological structure of red blood cells. In some embodiments, the therapeutic drug is an antibody. In some embodiments, the therapeutic drug is a 4-1BB antibody. In some embodiments, the therapeutic drug is a PD-1 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 is the SDS-PAGE electrophoresis identification result of OaAEP1 enzyme purified by Ni2+ affinity chromatography. The marker in the figure is the standard molecular weight ladder marker, and the 30-80kDa molecular weight band is indicated. Lanes 1 to 6 are: whole bacteria ultrasonic supernatant; Ni2+ affinity chromatography ... 2+ Affinity chromatography column flow-through; 50 mM imidazole elution fraction; 100 mM imidazole elution fraction; 250 mM imidazole elution fraction; and 500 mM imidazole elution fraction.

[0075] Figure 2 shows the results of SDS-PAGE electrophoresis analysis of OaAEP1 after self-activation under acidic conditions. The marker in the figure is a standard molecular weight ladder, and the 30-80 kDa molecular weight bands are indicated. Lanes 1-3 respectively represent the OaAEP1 protein before activation; the supernatant of the activated OaAEP1 enzyme; and the precipitate of the activated OaAEP1 enzyme.

[0076] Figure 3 shows the time course results of in vitro catalysis of OaAEP1 enzyme. The numbers above the lanes represent the reaction time (in minutes) experienced by the reaction products in each lane. The His6-EGFP-NGL-Flag band with the largest expected molecular weight is the ligation product.

[0077] In Figure 4, a. The small figure shows the results of different NH2-G small peptide substrates catalyzed by different concentrations of OaAEP1 enzyme; b. The small figure shows the results of different NH2-G small peptide substrates catalyzed by different concentrations of TmgSrtA enzyme.

[0078] Figure 5 shows the in vitro transpeptidation results of TmgSrtA catalyzed by different NH2-G peptide substrates and GFP-YFP. The final concentration of the NH2-G peptide substrate was 100 μM, and the four concentration gradients of YFP (Cp Venus) were 10 μM, 20 μM, 50 μM, and 100 μM, respectively.

[0079] FIG6 shows the results of in vitro transpeptidation of OaAEP1 enzyme based on GFP-YFP, in which the four concentration gradients of Cp Venus are respectively 10 μM, 20 μM, 50 μM and 100 μM in final concentration.

[0080] Figure 7 is a schematic diagram of the coupling of OaAEP1 catalytic antibody and engineered red blood cells, where GLK-MAL is a linker polypeptide GLK containing a maleimide group MAL; NGL is an additional OaAEP1 enzyme recognition sequence (tripeptide fragment) introduced at the C-terminus of the antibody molecule.

[0081] Figure 8 shows the effect of different enzyme dosages on the coupling of 4-1BB-NGL antibodies to red blood cells. Panel A shows the proportion of red blood cells successfully coupled to the antibody after being connected to the antibody by different amounts of enzyme catalyzed by flow cytometry, which is displayed in the scatter plot. The coupling rates (cells in the gate) obtained in the 5 μL, 10 μL, 20 μL and 30 μL groups are 0.58%, 11.0%, 23.3% and 26.4%, respectively; Panel B shows a bar chart constructed from the coupling rate numbers in Panel A.

[0082] Figure 9 shows the results of the 4-1BB-NGL antibody potency test (luciferase reporter gene). The different bars represent 4-1BB antibody-conjugated red blood cells, control red blood cells, and free antibody preparations, respectively. The vertical axis shows the fluorescence intensity signal generated by the luciferase downstream of 4-1BB, reflecting the degree of activation of the 4-1BB pathway.

[0083] FIG10 is a flow cytometry analysis result of the circulation metabolism of 4-1BB-mRBC preparation and CT in C57 mice.

[0084] Figure 11 shows the flow cytometry results of the PD-1-NGL antibody-erythrocyte coupling. The cells within the gate are antibody-modified erythrocytes, and the percentage is shown numerically. CT = control group.

[0085] Detailed Description of the Invention

[0086] I. Definition

[0087] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0088] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0089] As used herein, the singular expressions "a," "an," "the," and "the" encompass plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxyl orientation. It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described, as these may vary depending on the specific circumstances used by those skilled in the art.

[0090] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0091] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0092] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0093] The term "drug loading" refers to the amount of drug loaded per unit weight or per unit volume or per single red blood cell. In this application, the drug loading of red blood cells is generally measured in μg / mL as a dosage unit.

[0094] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to a subject to which the composition is administered. The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, etc., that is administered together with the active substance.

[0095] The term "treatment" refers to clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

[0096] The term "preventing" includes the inhibition of the onset or development of a disease or disorder or symptoms of a particular disease or disorder, for example, the administration of a drug before the onset of signs or symptoms of the disease, particularly in a subject at risk of the disease.

[0097] The terms "patient," "individual," and "subject" refer to any mammal for which the treatments or compositions disclosed herein can be used. Thus, the methods and compositions disclosed herein may have medical and / or veterinary applications. In a preferred form, the mammal is a human.

[0098] The terms "cancer" and "cancerous" refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, solid tumors such as lung cancer (e.g., non-small cell lung cancer), lymphoma, breast cancer, liver cancer, bladder cancer, skin cancer, melanoma, colon cancer, rectal cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, thyroid cancer, glioblastoma, glioma, and hematological tumors such as leukemia and lymphoma.

[0099] The term "effective amount" refers to an amount or dosage of the modified red blood cells or compositions of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as the species of the mammal; weight, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0100] The term "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of the modified erythrocytes or compositions of the present invention can vary according to a variety of factors such as disease state, age, sex, and weight of the individual. Relative to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., uric acid content, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and still more preferably at least about 80% or 90%.

[0101] The term "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0102] The term "effect active agent" as described herein encompasses any substance that is effective in vivo (preferably in a mammal; more preferably in a human body), which may be natural or engineered, including therapeutic agents, cytotoxic agents, recombinant proteins, antibodies, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants or immunostimulants), and the like. In a preferred embodiment, the effect active agent is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof, such as a full-length monoclonal antibody. In a more preferred embodiment, the effect active agent is an anti-(human) PD-1 monoclonal antibody and / or an anti-(human) 4-1BB monoclonal antibody. In a most preferred embodiment, the effector agent is an anti-(human) PD-1 monoclonal antibody and / or an anti-(human) 4-1BB monoclonal antibody, wherein the anti-(human) PD-1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 8, or consists thereof; and / or the light chain comprises the amino acid sequence shown in SEQ ID NO: 9, or consists thereof, and the anti-(human) 4-1BB monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 4, or consists thereof; and / or the light chain comprises the amino acid sequence shown in SEQ ID NO: 5, or consists thereof.

[0103] As used herein, the terms "anti-(human) PD-1 antibody," "anti-(human) PD-1," "(human) PD-1 antibody," "antibody that binds to (human) PD-1," or "antibody that specifically binds to (human) PD-1" refer to an antibody that is a monoclonal antibody and that is capable of binding to (human) PD-1 with sufficient affinity so that the antibody can be used as a therapeutic agent targeting (human) PD-1. In one embodiment, the (human) PD-1 antibody binds to (human) PD-1 with high affinity in vitro or in vivo. Various anti-PD-1 antibodies that can be used for therapy are known in the art, such as commercially available antibodies.

[0104] The terms "anti-(human) 4-1BB antibody", "anti-(human) 4-1BB", "(human) 4-1BB antibody", "antibody that binds to (human) 4-1BB" or "antibody that specifically binds to (human) 4-1BB" as used herein refer to antibodies that are monoclonal antibodies and that can bind to (human) 4-1BB with sufficient affinity so that the antibody can be used as a therapeutic agent targeting (human) 4-1BB. In one embodiment, the (human) 4-1BB antibody binds to (human) 4-1BB with high affinity in vitro or in vivo. A variety of anti-4-1BB antibodies that can be used for treatment are known in the art, such as commercially available antibodies.

[0105] The terms "complete antibody", "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules having the structure of a natural immunoglobulin molecule. In the case of a conventional four-chain IgG antibody, a full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of a heavy chain antibody having only heavy chains and lacking light chains, a full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds.

[0106] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0107] An "antigen-binding fragment" refers to a molecule, other than an intact antibody, that comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; diabodies or fragments thereof; or camelid antibodies.

[0108] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially all proteins or peptides, can be used as an antigen. In addition, the antigen can be derived from recombinant or genomic DNA. In some embodiments, the antigen is PD-1, such as human PD-1. As used herein, the term "epitope" refers to a portion of an antigen (e.g., PD-1) that specifically interacts with an antibody molecule.

[0109] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definitions based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0110] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the "Kabat numbering system" described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0111] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also called the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Herein, the term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL and the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases.

[0112] An "IgG-type antibody" refers to an antibody of the IgG type to which the heavy chain constant region belongs. For example, an IgG4-type antibody refers to an antibody of the IgG4-type, or an IgG1-type antibody refers to an antibody of the IgG1-type.

[0113] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0114] The terms "red blood cell" or "RBC," used interchangeably herein, refer to the most common type of blood cell and the primary carrier of oxygen through the bloodstream and circulatory system in vertebrates to body tissues. The cytoplasm of an RBC is rich in hemoglobin, an iron-containing biomolecule that binds oxygen and gives cells and blood their red color. The cell membrane is composed of proteins and lipids; this structure provides properties essential for physiological cellular function, such as deformability and stability, while also enabling passage through the circulatory system, particularly the capillary network. In humans, mature RBCs are flexible, oval, biconcave discs. They lack a nucleus and most organelles to maximize the space for hemoglobin; they can be considered bags for hemoglobin, with the plasma membrane serving as a sack. Approximately 2.4 million new RBCs are produced every second in adults. RBCs make up approximately 84% of the cells in the human body, numbering 20–30 trillion. Nearly half of the blood volume (40% to 45%) is RBCs. Key protein markers on the RBC surface allow them to circulate in the body for extended periods without being cleared by macrophages, resulting in a long half-life, making them excellent candidates for drug delivery. Mature red blood cells without nuclei do not contain any genetic material and therefore have a good safety profile compared to other gene and cell therapies.

[0115] “Adult natural red blood cells” refer to mature natural red blood cells directly isolated from the blood of animals, especially humans (eg, adults or children).

[0116] As used herein, "blood preparations" or "blood products" are used interchangeably and refer to products prepared from (human) blood for medical use. Blood preparations include whole blood preparations, blood component preparations, plasma preparations, or leukoreduced blood preparations.

[0117] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0118] The term "asparagine endopeptidase (AEP)" as used herein, also known as legumain or delta-secretase, refers to a proteolytic enzyme of the C13 peptidase family (EC 3.4.22.34), which utilizes the thiol group of a cysteine ​​residue as a nucleophile to hydrolyze peptide bonds (hence also known as a cysteine ​​protease), cleaving the C-terminus of the identified aspartic acid or aspartic acid residue to form a thioester intermediate, which can be degraded by hydrolysis or attack by a suitable nucleophile to produce a ligated product. This enzyme was discovered in legumes in 1996, and its homologs have also been found in plants, protozoa, vertebrates (including mammals, such as humans), and worms. In the present invention, the preferred asparaginyl endopeptidase is isolated from the closely related plant Oldenlandia affinis, designated OaAEP1. It exhibits the ability to ligate the N- and C-termini of peptide substrates. Upon recognizing the N- and C-terminal recognition sequences, it connects the two termini via a peptide bond, thereby efficiently catalyzing the ligation of two polypeptides / proteins. In a more preferred embodiment of the present invention, the OaAEP1 has a C247A substitution in its amino acid sequence, most preferably having the amino acid sequence set forth in SEQ ID NO: 1.

[0119] In some specific embodiments of the present invention, the OaAEP1 is obtained by expressing its encoding nucleic acid in a suitable host cell, preferably, the encoding nucleic acid has the sequence shown in SEQ ID NO: 2. In some specific embodiments of the present invention, the OaAEP1 is obtained by introducing a vector containing its encoding nucleic acid into a suitable host cell and expressing it, preferably, the vector has the sequence shown in SEQ ID NO: 3.

[0120] II. Erythrocytes and Modified Erythrocytes

[0121] In one aspect, the present invention provides red blood cells (RBCs) linked to an effector active agent, wherein the effector active agent is linked to the extracellular domain of at least one endogenous membrane protein of the RBC via a linker. In some embodiments, the RBC is modified such that its membrane protein is covalently linked to the linker. In a specific embodiment, the linker covalently links to a free sulfhydryl or amino group of the membrane protein via a maleimide moiety contained therein. In a specific embodiment, the RBC is treated with a reducing agent, such that disulfide bonds in some endogenous membrane proteins on the RBC surface are reduced to free sulfhydryl groups, ready for linking to the maleimide moiety of the linker. In another specific embodiment, the modified endogenous membrane protein of the RBC is linked to a linker comprising a maleimide moiety and an N-terminal recognition sequence for a ligase. In another specific embodiment, the RBC carrying the linker is contacted with an active agent containing a C-terminal recognition motif for the ligase under the action of a ligase, and a coupling reaction occurs, thereby obtaining the RBC linked to the effector active agent. In some specific embodiments, the ligase is AEP, preferably OaAEP1, and more preferably OaAEP1(C247A).

[0122] Unless otherwise specified or clear from the context, when this disclosure refers to red blood cells, it generally refers to mature red blood cells.In certain embodiments, the RBC is a human RBC, such as a human naive RBC.

[0123] In some embodiments, the red blood cells are adult natural red blood cells, preferably adult natural red blood cells from humans. In some embodiments, the RBCs have not been genetically engineered. In some embodiments, the present invention provides red blood cells having an effector active agent coupled thereto via a ligase-mediated reaction. In some embodiments, the coupled effector active agent can be one or more of the effector active agents described herein.

[0124] As used herein, unless otherwise indicated, the terms "coupled," "linked," and "conjugated" have the same meaning when referring to a reaction in which an erythrocyte and an effector active agent are covalently bound together through a series of steps; and an erythrocyte "coupled / linked / conjugated to a linker polypeptide / effector active agent" has the same meaning as a "modified" erythrocyte. Unless otherwise indicated or clear from the context, when the present disclosure refers to a modified erythrocyte, it generally refers to an erythrocyte coupled to an effector active agent, e.g., coupled via a linker, e.g., obtained / prepared by the methods herein by OaAEP1 enzymatic ligation.

[0125] When the effector active agent is linked to the linker, the N-terminal and C-terminal recognition sequences of the ligase undergo cleavage and fusion of amino acid residues, and the fused sequence contains all residues of the N-terminal and C-terminal recognition sequences, but the repeated residues are cleaved. For example, the fused sequence contains NGLG, NDLG, NPLG, NALG, NCLG, NELG, NFLG, NHLG, NKLG, NILG, NLLG, NQLG, NRLG, NSLG, NTLG, NVLG, NWLG, NYLG, NSLDG, NSLANG, NGG, NGLK, NDLK, NPLK, NALK, NCLK, NELK, NFLK, NHLK, NKLK, NILK, NLLK, NQLK, NRLK, NSLK, NTLK, NVLK, NWLK, NYLK, NSLDK, NSLANK or NGK.

[0126] In some embodiments, the present invention contemplates the use of autologous red blood cells isolated from an individual, modified in vitro, and then administered to the individual. In some embodiments, the present invention contemplates the use of immunocompatible red blood cells that have the same blood type (e.g., at least with respect to the ABO blood group system, and in some embodiments, with respect to the D blood group system) or a compatible blood type as the individual to whom the cells will be administered.

[0127] In some embodiments, the effector agent of the invention is an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4.

[0128] In some embodiments, the effector active agent of the present invention is a monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the effector active agent of the present invention is a humanized monoclonal antibody or antibody fragment. In some embodiments, the effector active agent of the present invention is a human antibody or a fragment thereof. In some embodiments, the effector active agent of the present invention is a chimeric antibody or a fragment thereof.

[0129] In one embodiment, the effector agent of the invention is a full-length antibody.

[0130] In one embodiment, the antigen-binding fragment of the effector active agent of the present invention is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody or half antibody. In some embodiments, antigen-binding fragments suitable for use in the present invention include, but are not limited to, VHH, Fv molecules, scFv molecules, Fab molecules, and F(ab')2 molecules.

[0131] In some embodiments, a linker can be used to connect an effector active agent (e.g., one or more antibodies or antigen-binding fragments thereof) to an erythrocyte (e.g., a membrane protein of an erythrocyte). In some embodiments, a linker having a reactive functional group can be used that is used to covalently link to an erythrocyte. In some embodiments, the linker reacts with a nucleophilic group of an erythrocyte membrane protein to form a covalent bond. For example, in some embodiments, a sulfhydryl group (thiol group) of a cysteine ​​residue on an erythrocyte membrane protein can form a chemical bond with a reactive functional group of a linker.

[0132] Nucleophilic groups on erythrocyte membrane proteins include, but are not limited to: (i) N-terminal amino groups, (ii) side chain amino groups, such as lysine, (iii) side chain sulfhydryl groups, such as cysteine, and (iv) sugar hydroxyl or amino groups. Amino, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents including: (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyls, and maleimide groups to form covalent bonds.

[0133] In one embodiment, the linker has a functional group capable of reacting with a sulfhydryl group on a cysteine ​​residue present on an RBC membrane protein to form a covalent bond to the RBC. Non-limiting exemplary reactive functional groups of this type include maleimide, haloacetamide, α-haloacetyl, active esters such as succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydride, acyl chloride, sulfonyl chloride, isocyanate and isothiocyanate, preferably maleimide.

[0134] In some embodiments, the functional group that the linker has can react with the amino group (-NH2) on the lysine in the RBC membrane protein to connect.Non-limiting exemplary such reactive functional groups include but are not limited to active esters, such as NHS esters, HOBt esters, haloformates and acid halides, preferably, N-hydroxysuccinimide esters (NHS esters).

[0135] The linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), p-aminobenzyloxycarbonyl ("PAB"), NHS esters such as N-succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("MCC"). Various linker components are known in the art.

[0136] In some embodiments, the linker can be attached to the red blood cells (RBCs) by reacting with the sulfhydryl groups of free cysteine ​​residues of red blood cell membrane proteins.

[0137] In some specific embodiments, the linker is a polypeptide comprising the amino acid sequence DL, PL, AL, CL, EL, FL, HL, KL, IL, LL, QL, RL, SL, TL, VL, WL, YL, SLD, SLAN, DLG, PLG, ALG, CLG, ELG, FLG, HLG, KLG, ILG, LLG, QLG, RLG, SLG, TLG, VLG, WLG, YLG, SLDG, SLANG, GLK, DLK, PLK, ALK, CLK, ELK, FLK, HLK, KLK, ILK, LLK, QLK, RLK, SLK, TLK, VLK, WLK, YLK, SLDK, SLANK or GK (i.e., N-terminal recognition sequence) and has a modification of the maleimide group. In some specific embodiments, the modification of the maleimide group is located on the epsilon-amino group of the lysine residue. In some specific embodiments, the modification of the maleimide group is to replace the H on the ε-amino group of the lysine residue.

[0138] In some embodiments, the RBCs are natural adult red blood cells obtained by treatment with a thiol reducing agent, and contain cysteine ​​residues containing thiol groups. In some embodiments, the thiol reducing agent is TCEP.

[0139] In some embodiments, RBCs are obtained by a method comprising:

[0140] (i) separating and concentrating red blood cells from (human) whole blood, optionally by filtering out leukocytes;

[0141] (ii) treating red blood cells with a thiol reducing agent (e.g., TCEP);

[0142] (iii) The modified red blood cells are collected and concentrated.

[0143] In some embodiments, the reducing agent treatment in (ii) comprises the steps of:

[0144] The thiol reducing agent is mixed with the concentrated red blood cells, wherein the concentration of the reducing agent is between 0.1 mM and 50 mM, such as 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM, and preferably about 2.5 mM.

[0145] III. Methods for Preparing Effector Agent-Red Blood Cell Conjugates

[0146] The present disclosure also provides a method for preparing an effector active agent-erythrocyte conjugate, comprising the steps of: (a) conjugating a linker polypeptide to an erythrocyte; and (b) linking the effector active agent to the linker polypeptide in the presence of a ligase. In some specific embodiments, step (a) is performed before step (b). In some specific embodiments, step (b) is performed before step (a).

[0147] In some specific embodiments, the method includes the steps of: (a) coupling a linker polypeptide to a red blood cell to obtain a red blood cell containing a linker, wherein the linker polypeptide contains an N-terminal recognition sequence of a ligase at its N-terminus; (b) in the presence of a ligase, contacting the red blood cell containing the linker obtained in step (a) with an effector active agent under conditions suitable for the ligation reaction to occur, wherein the effector active agent contains a C-terminal recognition sequence of a ligase, wherein the ligase recognizes the N-terminal recognition sequence and the C-terminal recognition sequence and catalyzes the connection between the two, thereby connecting the red blood cell to the effector active agent to form an RBC-effector active agent conjugate.

[0148] In some embodiments, the method for preparing the effector active agent-erythrocyte conjugate of the present invention is shown in FIG7 , wherein the linker polypeptide is GLK-mal (a polypeptide having the amino acid sequence GLK and a maleimide group), and the antibody in the figure is added with a coding sequence fragment of NGL at the 3' end of its encoding nucleic acid sequence to express an antibody heavy chain having NGL at the C-terminus. The method comprises:

[0149] (1) Obtaining red blood cells and treating them with a thiol reducing agent (e.g., TCEP);

[0150] (2) covalently linking the sulfhydryl group of the red blood cell membrane surface protein to the maleimide group of the linker polypeptide, thereby coupling the linker polypeptide to the red blood cell;

[0151] (3) In the presence of a ligase, the red blood cells containing the linker obtained in (2) are contacted with the effector active agent, so that the ligase recognizes the N-terminal recognition sequence contained in the linker polypeptide and the C-terminal recognition sequence contained in the effector active agent, and catalyzes the connection between the two, thereby connecting the red blood cells to the effector active agent through the linker polypeptide.

[0152] In some embodiments, the method for preparing the effector active agent-erythrocyte conjugate of the present invention comprises the following steps performed in sequence:

[0153] (1) Obtaining natural red blood cells;

[0154] (2) treating the obtained red blood cells with a thiol reducing agent (e.g., TCEP);

[0155] (3) coupling a linker to an erythrocyte to obtain a linker-modified erythrocyte, wherein the linker is a polypeptide having a maleimide group, preferably a tripeptide, more preferably a tripeptide having a sequence of GLK, and most preferably, the linker is a tripeptide having a sequence of GLK and a maleimide group on a lysine residue (GLK-mal);

[0156] (4) In the presence of a ligase, the red blood cells coupled to the linker are contacted with the effector active agent, so that the ligase recognizes the N-terminal recognition sequence contained in the linker polypeptide and the C-terminal recognition sequence contained in the effector active agent, and catalyzes the connection between the two, thereby connecting the red blood cells to the effector active agent through the linker polypeptide.

[0157] In some specific embodiments, step (1) includes transferring a human blood sample from a healthy subject in a blood collection tube into a blood bag using a disposable syringe, removing leukocytes from the whole blood using a leukocyte filter, and then transferring the leukocyte-removed blood sample from the blood bag into a 50 mL centrifuge tube for centrifugation to collect red blood cells. In some more specific embodiments, the centrifugation parameters are set as follows: centrifugal force 500 g, centrifugation time 5 minutes, ramp rate 9, ramp rate 8, and temperature 25°C.

[0158] In some more specific embodiments, the concentrated red blood cell intermediate is obtained by centrifugation, and the centrifugation parameters are set to a centrifugal force of 500g, a centrifugation time of 3 minutes, an ascending speed of 9, a descending speed of 7, and a temperature of 25°C.

[0159] In some more specific embodiments, the step (1) further comprises, after centrifugation, discarding the upper layer of liquid using a disposable sterile pipette or a 1000 μL pipette, and using an electric pipette to draw up the concentrated red blood cells and add them to a subsequent reaction container.

[0160] In some specific embodiments, step (2) comprises mixing a thiol reducing agent (e.g., TCEP) with red blood cells, wherein the concentration of the reducing agent is between 0.1 mM and 50 mM, such as 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM; preferably about 2.5 mM.

[0161] Preferably, the mixture is mixed, for example, at a temperature in the range of 4-37 (preferably 25-35) degrees Celsius (e.g., about 25-about 30°C, for example, about 30°C), for 0.5-2 hours (e.g., about 1 hour), for example, on a rotary mixer at a speed in the range of 5-15 rpm (e.g., 10 rpm);

[0162] Centrifuge to remove the supernatant, preferably, the centrifugal force is 500-1200 g (e.g., about 800 g), the centrifugation time is 2-10 min (e.g., about 3 minutes), the ascending speed is 1-9 (e.g., about 9) and / or the descending speed is 1-7 (e.g., about 7);

[0163] optionally repeated twice, for example at about 20°C-30°C, for example at about 25°C;

[0164] Optionally, the modified packed red blood cells are collected.

[0165] In some specific embodiments, step (2) includes taking a TCEP stock solution and optionally standing it on ice until it is completely melted. Then, standing it in a biosafety cabinet for 1 minute, taking 5 μL of a 0.5M TCEP stock solution and adding it to a clean, sterile EP tube, then adding PBS (e.g., 495 μL) to 500 μL, and then adding 500 μL of concentrated red blood cells to make a total system of 1000 μL, with a final TCEP concentration of 2.5 mM. After thorough mixing and sufficient reaction (e.g., at a temperature in the range of 25-35 degrees Celsius (e.g., about 25-about 30°C, e.g., about 30°C), mixing for 0.5-2 hours (e.g., about 1 hour), for example, placing it on a rotary mixer at a speed in the range of 5-15 rpm (e.g., 10 rpm)), washing it with PBS to obtain RBCs reduced by TCEP. In some more specific embodiments, the red blood cells and TCEP are completely mixed and reacted by placing it on a rotary mixer at 30°C and turning it at 10 rpm for 1 hour. In some more specific embodiments, the red blood cells are washed, preferably three times, by adding 10 volumes of PBS, resuspending the red blood cells, and centrifuging at 900 g for 4 minutes at 4°C, with the speed reduced to 9 / 8, and removing the PBS supernatant after centrifugation. The modified concentrated red blood cells are optionally collected.

[0166] In some specific embodiments, step (3) includes adding a linker (e.g., GLK-mal) to PBS, mixing uniformly, and then adding concentrated RBC reduced with TCEP, mixing completely and reacting fully (e.g., the temperature is in the range of 25-35 degrees Celsius (e.g., about 25-about 30°C, for example, about 30°C), mixing for 5-30 minutes (e.g., about 15 minutes), for example, placing on a rotary mixer at a speed of 5-15 rpm (e.g., 10 rpm)), and then washing with PBS to obtain linker polypeptide-modified red blood cells RBC (GLK-mal-red blood cells). In some more specific embodiments, the red blood cells and the linker are completely mixed and reacted by placing on a rotary mixer and flipping at 10 rpm for 15 minutes at 30°C. In some more specific embodiments, the red blood cells are resuspended by adding 10 times the volume of PBS, and centrifuged at 900g for 4 minutes at 4°C, with a speed of 9 / 8, and the PBS supernatant is removed after centrifugation to wash the red blood cells, preferably washing 3 times.

[0167] In some more specific embodiments, step (3) includes adding 155 μL of 0.625 mM GLK-MAL to 245 μL of PBS, mixing evenly, adding 400 μL of concentrated RBC reduced with TCEP, mixing completely and reacting sufficiently, and then washing with PBS to obtain linker polypeptide-modified red blood cells (GLK-mal-red blood cells).

[0168] In some specific embodiments, step (4) includes adding activated OaAEP1 enzyme and the effector active agent to be coupled (e.g., antibody, e.g., PD1 antibody and / or 4-1BB antibody) to PBS, diluting evenly, and then adding concentrated RBCs. After preparation, the mixture is thoroughly mixed and reacted (e.g., at a temperature in the range of 25-35 degrees Celsius (e.g., about 25-about 30°C, e.g., about 30°C), for a mixing time of 10-60 minutes (e.g., about 30 minutes), for example, placed on a rotary mixer at a speed in the range of 5-15 rpm (e.g., 10 rpm)) to obtain RBCs coupled with the effector active agent. In some more specific embodiments, the activated OaAEP1 enzyme catalyzes the coupling of the effector active agent having an NGL sequence at the C-terminus to the GLK-mal-red blood cells by placing the mixture on a rotary mixer at 10 rpm and 30°C for 30 minutes. The NGL sequence and the GLK sequence at the N-terminus of the linker polypeptide are recognized by the OaAEP1 enzyme, catalyzing the formation of an NGLK peptide fragment to achieve covalent linkage. In some more specific embodiments, step (4) further comprises washing the red blood cells by adding 10 volumes of PBS, resuspending the red blood cells, and centrifuging at 900 g for 4 minutes at 4°C, reducing the speed to 9 / 8, and removing the PBS supernatant after centrifugation to wash the red blood cells, preferably three times.

[0169] In some more specific embodiments, step (4) comprises adding 20 μL of effector active agent (eg, purified antibody), a total of 30 μL of PBS and OaAEP1 enzyme, and 50 μL of GLK-mal-RBC to the reaction system, mixing thoroughly and reacting fully.

[0170] In some embodiments, the method further comprises adding physiological saline (e.g., a volume of physiological saline equal to the volume of concentrated red blood cells, e.g., 100 μL of physiological saline) and red blood cell preservation solution (e.g., 0.5 times the volume of red blood cell preservation solution, e.g., 50 μL of red blood cell preservation solution) to the red blood cells modified with the effector active agent, and gently pipetting to mix evenly to obtain a red blood cell-effector active agent preparation solution. This solution can be added to a packaging container, and after packaging, the preparation can be stored at 4°C for future use.

[0171] In some embodiments, the effector agent is an antibody, preferably a recombinantly expressed antibody. In some specific embodiments, the coding sequence of the antibody comprises a coding segment of a C-terminal recognition sequence (e.g., NGL), so that the C-terminus of the recombinantly expressed antibody (e.g., the C-terminus of the antibody heavy chain) comprises a C-terminal recognition sequence (e.g., NGL). In some specific embodiments, the antibody further comprises a tag sequence and / or a signal peptide sequence. In some preferred embodiments, the recombinantly expressed antibody is purified and / or ultrafiltered, preferably, the antibody concentration after purification and / or ultrafiltration is approximately ≥5 mg / mL, ≥6 mg / mL, ≥7 mg / mL, ≥8 mg / mL, ≥9 mg / mL, ≥10 mg / mL, preferably ≥10 mg / mL. In some preferred embodiments, the purification is affinity chromatography.

[0172] IV. Effector Agent-Red Blood Cell Conjugates, Such as Antibody-Red Blood Cell Conjugates

[0173] The present disclosure relates to an antibody-erythrocyte conjugate, which comprises an erythrocyte coupled to an antibody or an antigen-binding fragment thereof. Preferably, the antibody or the antigen-binding fragment thereof is coupled to the erythrocyte via a linker, preferably the antibody or the antigen-binding fragment thereof is coupled to a membrane protein on the erythrocyte, for example, the antibody or the antigen-binding fragment thereof is coupled to a membrane protein on the erythrocyte via a linker.

[0174] In some embodiments, the red blood cells are adult natural red blood cells, preferably adult natural red blood cells from a human.

[0175] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are antibodies or antigen-binding fragments thereof that bind to PD-1, particularly human PD-1. Any PD-1 antibody or antigen-binding fragment thereof known to those skilled in the art is suitable for use in the present invention, including but not limited to disclosed antibodies or commercially available antibodies. In a specific embodiment, the PD-1 antibody has a heavy chain amino acid sequence as shown in SEQ ID NO: 8 and a light chain amino acid sequence as shown in SEQ ID NO: 9.

[0176] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is an antibody or antigen-binding fragment thereof that binds to 4-1BB, particularly human 4-1BB. Any 4-1BB antibody or antigen-binding fragment thereof known to those skilled in the art is suitable for use in the present invention, including but not limited to disclosed antibodies or commercially available antibodies. In a specific embodiment, the 4-1BB antibody has a heavy chain amino acid sequence as shown in SEQ ID NO: 4 and a light chain amino acid sequence as shown in SEQ ID NO: 5.

[0177] In one embodiment, the number of antibodies on a single red blood cell can be 10 3 -10 6 between, for example, about 10 4 -10 6 Between or 10 5 -10 6 between, for example, about 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 or 9×10 5 indivual.

[0178] V. Treatment

[0179] In one aspect, the present invention provides pharmaceutical compositions or formulations comprising effector active agent-erythrocyte conjugates. These compositions or formulations may also optionally comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0180] In one aspect, the present invention provides a blood preparation comprising an effector active agent-erythrocyte conjugate of the present invention, such as a human blood preparation. In some embodiments, the human blood preparation of the present invention comprises 10-1000 μg / mL of effector active agent-erythrocyte conjugate, such as 10-500 μg / mL of effector active agent-erythrocyte conjugate, for example, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, or 500 μg / mL or more, or within any range of the values. In some embodiments, the blood preparation of the present invention is a leukopenia blood preparation, i.e., a blood preparation in which leukocytes have been filtered out. In some embodiments, the blood preparation of the present invention is a human leukopenia blood preparation.

[0181] In some embodiments, the blood preparation of the present invention can be an allogeneic blood preparation, such as an allogeneic human blood preparation. In some embodiments, the red blood cells in the blood preparation are from healthy subjects.

[0182] In some embodiments, the blood preparation of the present invention can be an autologous blood preparation, such as an autologous human blood preparation. In some embodiments, the red blood cells in the blood preparation are from the subject to be treated.

[0183] In one aspect, the present invention relates to a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of an effector agent-erythrocyte conjugate of the present invention. In some specific embodiments, the disease is cancer, e.g., a hematological tumor, e.g., a leukemia, e.g., a lymphocytic leukemia, e.g., a T-cell leukemia.

[0184] Any documents cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entirety. Example

[0185] Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner and are not intended to and should not limit the scope of the present invention in any way, and that various modifications may be made by those skilled in the art.

[0186] The experimental equipment and consumables used in the examples are as follows

[0187] Table 1 List of experimental equipment and consumables

[0188] The reagents used in the examples are as follows

[0189] Table 2 List of reagents used in the experiment

[0190] Example 1 Recombinant expression and activation of OaAEP1 enzyme

[0191] Clone the build:

[0192] The amino acid sequence of the OaAEP1 enzyme (carrying a C247A substitution) fused to a 6×His and SUMO dual tag at the N-terminus is shown in SEQ ID NO:1. This amino acid sequence was reverse-translated, and the cDNA coding sequence (shown in SEQ ID NO:2) was determined based on the codon preference of E. coli. The coding sequence was synthesized and cloned into a pET vector (shown in SEQ ID NO:3). Positive constructs were screened and transformed into E. coli BL21(DE3) cells. After verification by sequencing, the constructs were used for OaAEP1 protein expression.

[0193] Culture medium preparation:

[0194] LB medium (Amp + ) : Add 25 g of LB medium powder to 900 mL of double-distilled water, dissolve thoroughly, and dilute to 1 L. Autoclave and sterilize. Before use, add ampicillin antibiotics to a final concentration of 100 μg / mL.

[0195] OaAEP1 enzyme expression and purification

[0196] The recombinant OaAEP1 protein was expressed in the E. coli BL21 (DE3) cells mentioned above, and the expression was induced by 0.4 mM IPTG at 16 °C for 20 h. Subsequently, the cells were ultrasonically disrupted and centrifuged to obtain the supernatant. 2+ Protein purification was performed by affinity chromatography. The specific steps are as follows:

[0197] 1. The OaAEP1 expression strain constructed above was inoculated into 5 mL LB medium (Amp + ) and cultured with shaking at 37°C and 220 rpm overnight;

[0198] 2. Inoculate the activated bacteria into 1L LB medium (Amp + ) at 37°C and 220 rpm for 2-3 h, and monitor the cell density;

[0199] 3. When the OD600 of the culture reached 0.6, IPTG was added to a final concentration of 0.4 mM to induce expression, and the culture was continued at 16°C and 220 rpm for 20 h.

[0200] 4. Centrifuge at 8000 rpm for 4 minutes to collect the cells and discard the supernatant;

[0201] 5. Resuspend the cells in 10 mL of equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, pH 8.0).

[0202] 6. Transfer the resuspended cells to a 50 mL centrifuge tube and place in an ice-water mixture;

[0203] 7. Set the ultrasonic disruptor power to 150W and perform ultrasonic disruption until the bacteria are completely broken;

[0204] 8. Add super nuclease to the sonicated solution and incubate on ice for 30 minutes;

[0205] 9. Centrifuge at 14,000 × g, 4°C for 30 min, remove the supernatant, and filter through a 0.45 μm filter membrane;

[0206] 10. Equilibrate the nickel affinity chromatography column with 10 CV (column volume) of double-distilled water and 10 CV of equilibration buffer, respectively;

[0207] 11. Add the filtered supernatant to the chromatography column and adjust the flow rate to about 1 ml / min;

[0208] 12. Elute the impurities with 10 CV of wash buffer (50 mM Tris-HCl, 150 mM NaCl, 50 mM imidazole, pH 8.0);

[0209] 13. Elute the target protein with 10 CV of elution buffer (50 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH 8.0) and collect the eluted fractions;

[0210] 14. The collected target protein eluate was concentrated by ultrafiltration, and the buffer was replaced with 1× PBS to a concentration of about 3 mg / ml, and frozen in a -80℃ refrigerator.

[0211] The target protein (i.e., expressed enzyme) was identified by SDS-PAGE electrophoresis. As shown in Figure 1, distinct bands corresponding to the apparent molecular weight of OaAEP1, 63.1 kD, were visible in lanes 4 and 5. This indicates that the target protein was eluted using 100 mM and 250 mM imidazole. Low levels of the target protein were observed in the 50 mM imidazole wash (lane 3) and in the stubbornly bound protein eluted with 500 mM imidazole (lane 6).

[0212] OaAEP1 enzyme activation

[0213] 1. Prepare activation buffer: Mix 0.2M sodium acetate and 0.2M glacial acetic acid in a ratio of 1:9 to prepare the activation buffer.

[0214] 2. Mix approximately 3 mg / ml of OaAEP1 protein with activation buffer at a ratio of 1:3 and incubate in a 37°C water bath for 4-5 hours.

[0215] 3. Centrifuge the activated solution at 12000 rpm for 5 min to remove insoluble matter;

[0216] 4. The supernatant from centrifugation is the activated OaAEP1 enzyme. After identification by SDS-PAGE electrophoresis, it is aliquoted and stored in a -80°C refrigerator for future use.

[0217] The SDS-PAGE electrophoresis identification of the activated OaAEP1 enzyme is shown in Figure 2. In the supernatant of the activated OaAEP1 enzyme centrifugation (lane 2), the expected band of about 32 kDa molecular weight was visible, namely the activated OaAEP1 enzyme.

[0218] Example 2 OaAEP1 enzyme in vitro transpeptidation assay

[0219] The catalytic time course of OaAEP1 and different small peptide substrates was analyzed and compared with sortase.

[0220] For both enzymes, the acyl acceptors used three substrate peptides: GV-FLAG peptide (GVGDYKDDDDK), GL-FLAG peptide (GLGDYKDDDDK), and GI-FLAG peptide (GIGDYKDDDDK). The acyl acceptors were based on the EGFP protein, with a 6×His tag added to the N-terminus and the recognition sequences for the two enzymes (NGL and LPETGG), respectively, added to the C-terminus. In the ligation reaction, the performance of the tested enzymes in catalyzing the ligation of the two substrates was evaluated.

[0221] Specifically, for catalytic time course analysis, the reaction system was prepared according to Table 3-1 below. Then, the system was placed at 37°C for different reaction times (0-150 min), and then the stop solution was added to terminate the reaction. The accumulation of the product His6-EGFP-NGL-FLAG was detected by SDS-PAGE electrophoresis.

[0222] Table 3-1 OaAEP1 enzyme-catalyzed time course reaction system (100 μL)

[0223] The results are shown in Figure 3. Comparison at reaction time points of 0, 15, 30, 45, 60, 75, 90, 105, and 150 minutes reveals that the expected ligation product, His6-EGFP-NGL-Flag, reaches its peak accumulation at 30 minutes and maintains this level or slowly decreases thereafter. Correspondingly, the amount of His6-EGFP-NGL, one of the substrates, decreases with prolonged reaction time, as indicated by a fading of the corresponding band.

[0224] Different substrate peptides GV-FLAG peptide (GVGDYKDDDDK), GL-FLAG peptide (GLGDYKDDDDK), and GI-FLAG peptide (GIGDYKDDDDK) were tested.

[0225] The reaction system for the reaction using OaAEP1 enzyme as the catalytic enzyme was prepared according to Table 3-2, wherein the final concentration of different G peptides was 100 μM. The prepared reaction system was placed in a 37°C water bath for 1 hour, 10 μL of the reaction system was taken, 20 μL of 2× loading buffer was added to terminate the reaction, and 10 μL of the reaction system was taken for SDS-PAGE detection.

[0226] Table 3-2 OaAEP1 enzyme-catalyzed reaction systems of different small peptide substrates (100 μL)

[0227] The results are shown in Figure 4, where a. The small figure shows the results of different NH2-G peptide substrates catalyzed by different concentrations of OaAEP1 enzyme, C is the control group without OaAEP1 enzyme, L represents the final concentration of OaAEP1 of 0.1 μM (low concentration), and H represents the final concentration of OaAEP1 of 1.2 μM (high concentration). It can be seen that for different G peptide substrates, OaAEP1 enzyme can catalyze the ligation reaction.

[0228] The reaction was catalyzed by using the sorting enzyme TmgSrtA, which had been subjected to mutation screening and could specifically and efficiently recognize the conserved amino acid sequence LPXTG at its C-terminus. The reaction system was prepared according to Table 3-3, except that the OaAEP1 enzyme was replaced with three concentration gradients (1.5 μM, 3 μM, and 6 μM) of TmgSrtA enzyme, wherein the final concentration of the different G peptides was also 100 μM.

[0229] Table 3-3 Sortase enzyme-catalyzed reaction systems of different small peptide substrates (100 μL)

[0230] The results are shown in Figure 4b. For different NH2-G peptide substrates (GV-FLAG peptide (GVGDYKDDDDK), GL-FLAG peptide (GLGDYKDDDDK) and GI-FLAG peptide (GIGDYKDDDDK) peptides), TmgSrtA sortase also exhibited dose-dependent catalytic reaction ability. At the lowest concentration of 1.5 μM, the catalytic efficiency was low, showing a faint product band. As the amount of enzyme added increased, the yield of the ligation product also increased (the band was denser), but the band difference of the substrate His6-EGFP-LPETGG was not obvious.

[0231] Furthermore, the catalytic efficiency of the sorting enzyme TmgSrtA and the activated OaAEP1 enzyme of this patent on small peptides in vitro was compared, and they were configured according to the systems in Table 4-1 and Table 4-2 below, respectively.

[0232] Table 4-1 Sortase transpeptidase system (100 μL)

[0233] Table 4-2 OaAEP1 transpeptidation system (100 μL)

[0234] The protein used in the reaction system was first diluted with PBS, and then the transpeptidation reaction system was established. At the same time, the sortase enzyme was tested using a small peptide containing NH2-G (G-FLAG-biotin peptide / GV-FLAG peptide / GL-FLAG peptide / GI-FLAG peptide). After the prepared reaction system was allowed to stand in a 37°C water bath for 2 hours, 10 μL of the reaction system was taken and 20 μL of 2× loading buffer was added to terminate the reaction. 10 μL of the reaction system was taken for SDS-PAGE detection. The results are shown in Figures 5 and 6. When the concentration of TmgSrtA was low, only 1.5 μM, its catalytic transpeptidation efficiency was significantly reduced, which is consistent with the results of the previous experiment (Figure 4b). In contrast, OaAEP1 can also effectively catalyze the reaction at the purity shown above using only a concentration approximately 60 times lower than that of TmgSrtA.

[0235] Compared with sortase, OaAEP1 has specific advantages in the catalytic efficiency of peptide ligation.

[0236] Example 3 Preparation of 4-1BB Antibody by Recombinant Expression

[0237] Clone Build

[0238] The amino acid sequence of the heavy chain of the 4-1BB antibody (Anti-4-1BB-NGL) with an NGL motif fused to the C-terminus is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain of the same antibody is shown in SEQ ID NO: 5. The amino acid sequence was reverse translated, and the cDNA coding sequence was determined based on the codon preference of mammalian cells (as shown in SEQ ID NO: 6 and 7, respectively). The coding sequences were synthesized and cloned into the pcDNA3.1 vector, and the positive constructs were screened and transformed into Escherichia coli. After sequencing verification, endotoxin was removed and the plasmid was extracted for expression of the Anti-4-1BB-NGL antibody.

[0239] Protein expression:

[0240] The heavy and light chain expression plasmids of Anti-4-1BB-NGL were transfected into 293F cells at a 1:2 ratio to express the recombinant antibody. Seven days after transfection, the cell culture supernatant was harvested and filtered through a 0.45 μm filter for subsequent purification of the Anti-4-1BB-NGL antibody.

[0241] Liquid preparation:

[0242] Protein A elution buffer: Weigh 7.507 g of glycine and dissolve it in 900 mL of double-distilled water. Adjust the pH of the solution to 3.0 with HCl. Dose the solution to 1000 mL with double-distilled water. Filter through a 0.45 μM filter and set aside.

[0243] Protein A neutralization buffer: Weigh 121.14 g of Tris powder and dissolve it thoroughly in 800 mL of double-distilled water. Adjust the pH of the solution to 8.5 with HCl. Dose the solution to 1000 mL with double-distilled water. Filter through a 0.45 μM filter and set aside.

[0244] Antibody purification:

[0245] 1. Equilibrate the Protein A affinity chromatography column with 10 CV (column volume) of double-distilled water and 10 CV of PBS, respectively, at a flow rate of 1 mL / min.

[0246] 2. Load the sample onto the chromatography column at a flow rate of approximately 1 mL / min and collect the effluent;

[0247] 3. Wash the resin with 30 mL of equilibration buffer at a flow rate of approximately 2 mL / min.

[0248] 4. Elute the antibody with 10-15 mL of elution buffer at a flow rate of approximately 1 mL / min. Collect the eluate containing the target protein and immediately add 1 / 10 the volume of elution buffer to neutralize the solution and adjust the pH to 7.4.

[0249] 5. The eluted protein was concentrated by ultrafiltration and exchanged into PBS buffer solution, and frozen at -20℃ for later use.

[0250] Example 4: Red blood cells linked to 4-1BB antibodies

[0251] Natural red blood cell acquisition

[0252] Open the cap of the blood collection tube and use a disposable syringe to transfer the human blood sample from the healthy subject into a blood bag. Once completed, seal the tube twice with a sterile tube sealer. Leukocytes were removed from the whole blood using a leukocyte filter. Using a 20 mL disposable syringe, transfer the leukocyte-removed blood sample from the blood storage bag into a 50 mL centrifuge tube and centrifuge to collect red blood cells. Centrifugation parameters were: 500 g centrifugation force, 5 minutes of centrifugation, 9 ramps, 8 ramps, and 25°C.

[0253] Centrifuge to obtain the concentrated red blood cell intermediate. The centrifugation parameters are set at 500 g for 3 minutes, a ramp rate of 9, a ramp rate of 7, and a temperature of 25°C. Open the centrifuge tube and discard the supernatant using a disposable sterile pipette or a 1000 μL pipette with a matching tip. Aspirate the concentrated red blood cells using an electric pipette and add them to the reaction vessel.

[0254] Linker peptide modified red blood cells

[0255] The ε-amino group of the carboxyl-terminal lysine in the GLK tripeptide was modified with maleimide (GLK-mal) and used as a linker peptide, which was chemically synthesized by GenScript.

[0256] Prepare an ampoule of TCEP stock solution (purchased from Sigma-Aldrich, Cat. No. 646547) at a labeled concentration of 0.5 M from a -20°C refrigerator and place on ice until the solution in the ampoule is completely thawed. After surface disinfection, place the ampoule in a biosafety cabinet and let it sit for 1 minute. Carefully open the ampoule and add 5 μL of the TCEP stock solution to a clean, sterile EP tube. Add 495 μL of PBS, pipette to mix thoroughly, aspirate with a 1 mL syringe, and filter through a 0.22 μm filter to sterilize. After sterilization, return the solution to a new, clean, sterile EP tube and add 500 μL of concentrated RBCs, bringing the total volume to 1000 μL for a final TCEP concentration of 2.5 mM. Place the tube on a rotary mixer at 10 rpm and 30°C for 1 hour. After the reaction is complete, resuspend the tube in 10 volumes of PBS and centrifuge three times (900 g, 4°C, 4 min, ramp 9 / 8). Remove the PBS supernatant. RBCs reduced by TCEP were obtained.

[0257] Prepare a clean, sterile centrifuge tube and prepare according to the system in Table 5 below. First, add PBS, then add GLK-mal and dilute evenly. Finally, add concentrated RBCs reduced with TCEP. Place on a rotary mixer and rotate at 10 rpm and 30°C for 15 minutes to obtain linker peptide-modified RBCs (GLK-mal-RBCs). After the reaction is complete, resuspend in 10 volumes of PBS and wash by centrifugation three times (900g, 4°C, 4 minutes, ramp rate 9 / 8). Remove the PBS supernatant.

[0258] Table 5 Composition of the reaction system of linker polypeptide modified natural red blood cells

[0259] Conjugated with 4-1BB antibody

[0260] Prepare the antibody conjugation reaction system according to the volumes shown in Table 6 below. First, add PBS, then add activated OaAEP1 enzyme and dilute evenly. Finally, add concentrated RBCs. Once prepared, place the mixture on a rotary mixer at 10 rpm and 30°C for 30 minutes. Activated OaAEP1 enzyme catalyzes the conjugation of Anti-4-1BB-NGL to GLK-mal-erythrocytes. The NGL sequence at the C-terminus of the antibody heavy chain and the GLK sequence at the N-terminus of the linker polypeptide are recognized by the OaAEP1 enzyme, catalyzing the formation of the NGLK peptide fragment and covalently linking them. After the reaction, resuspend the mixture in 10 volumes of PBS and wash it three times by centrifugation (900g, 4°C, 4 minutes, ramp rate 9 / 8). Remove the PBS supernatant.

[0261] Table 6 Composition of the Anti-4-1BB-NGL and GLK-mal-erythrocyte coupling reaction system

[0262] Pipette 100 μL of normal saline and 50 μL of red blood cell preservation solution (normal saline equal to the volume of concentrated red blood cells and 0.5 times the volume of CPDA-1 red blood cell preservation solution (Shandong Weigao Group)), add the antibody-modified red blood cells, and gently pipette to mix evenly to obtain a red blood cell-antibody preparation solution. The red blood cell-antibody preparation solution can be added to the packaging container. After the preparation is packaged, store at 4°C until ready for use.

[0263] A schematic diagram of coupling the OaAEP1 catalytic antibody with the linker polypeptide-modified red blood cells is shown in FIG7 .

[0264] Example 5 Antibody Binding Content Verification

[0265] After the coupling is completed, the content of effector active protein (antibody) on the red blood cells is detected.

[0266] The drug loading detection method is as follows:

[0267] 1. Dilute the 4-1BB antigen (Acro Product No. 41B-H5258) solution to 400 ng / mL with coating solution, add 100 μL / well to a 96-well ELISA plate, cover with sealing film, and incubate at 2°C-8°C overnight;

[0268] 2. Drain the liquid from the plate and add 200 μl / well of washing solution (0.05% PBST). Drain the liquid from the plate and turn the plate upside down on absorbent paper. Pat the plate dry. Repeat this step twice.

[0269] 3. Add 100 μL of blocking solution to each well, cover with sealing film, and incubate on a 25°C constant temperature shaker for 1 hour;

[0270] 4. Drain the liquid from the plate and add 200 μl / well of washing solution (0.05% PBST). Drain the liquid from the plate and turn the plate upside down on absorbent paper. Pat the plate dry. Repeat this step twice.

[0271] 5. Centrifuge 50-100 μL of the red blood cell preparation sample at 900 g for 5 minutes, take 5 μL of the prepared red blood cells from the lower layer of the centrifuge and add them to 95 μL of 1xRIPA solution. Vortex mix for 1 minute; dilute the sample to 4050 times and 12150 times with sample diluent.

[0272] 6. The anti-4-1BB-NGL antibody prepared in Example 3 was used as a standard. Based on the labeled protein concentration of the standard, the standard was first diluted to 2 mg / mL with sample diluent, and then gradually diluted to 1000 ng / mL. Three serial dilutions were then performed in an ELISA plate.

[0273] 7. Take 50 μL of the diluted standard and the sample to be tested and add them to two parallel wells of a 96-well ELISA plate. Cover with a sealing film and incubate at 25°C on a constant temperature shaker for 1 hour.

[0274] 8. Drain the liquid from the plate and add 200 μl / well of washing solution (0.05% PBST). Drain the liquid from the plate and invert the plate onto absorbent paper. Pat the plate dry. Repeat this step 4 times.

[0275] 9. Dilute the secondary antibody Goat pAb to Hu IgG (HRP) (Abcam, ab97225) at 1:10,000 in diluent and add 100 μL to each well. Cover with film and incubate at 25°C with shaking for 1 hour.

[0276] 10. Drain the liquid from the plate and add 200 μl / well of washing solution (0.05% PBST). Drain the liquid from the plate and invert the plate onto absorbent paper. Pat the plate dry. Repeat this step 4 times.

[0277] 11. Add 100 μL of TMB substrate to each well, incubate in the dark for 8 minutes to terminate the reaction.

[0278] 12. Calculate the drug loading based on the OD450nm reading.

[0279] The results are shown in Table 7. As can be seen, in the above coupling reaction, as the amount of enzyme increases, the coupling efficiency of red blood cells and 4-1BB-NGL antibody also increases.

[0280] Table 7 Reaction system of 4-1BB antibody coupled with red blood cells at different enzyme dosages

[0281] The labeling effect of the anti-4-1BB-NGL antibody on the erythrocyte membrane was detected by flow cytometry (Cytoflex, Beckman). The results are shown in Figure 8 (Panel A shows a scatter plot, and Panel B shows a bar graph of the ratio of erythrocytes coupled to the antibody in each group). Similar to the results in Table 7, as the amount of enzyme used in the ligation reaction increases, the coupling efficiency of erythrocytes and 4-1BB-NGL antibodies also increases.

[0282] Example 6 Verification of Antibody Binding Efficacy

[0283] The potency of the 4-1BB antibody-erythrocyte conjugate was determined using a 4-1BB potency assay, as follows: 4E6 of 4-1BB-NGL-conjugated RBC preparation, an RBC-CT negative control, and a 4-1BB-NGL protein positive control were incubated with 1E5 4-1BB luciferase reporter gene-engineered Jurkat cells (4-1BB reporter Jurkat) for 6.5 hours. After adding luciferase substrate, the fluorescence intensity was measured to reflect the activation effect of the RBC preparation on the 4-1BB reporter Jurkat cells.

[0284] The results are shown in Figure 9. Compared with the control group RBC-CT preparation group, the RBC-4-1BB-NGL preparation group and 4-1BB-NGL protein were able to activate the 4-1BB reporter gene transfected in Jurkat cells to express luciferase, and the drug loading was 20 ng / ml (equivalent to 20 ng / 10 10 ) of RBC-4-1BB-NGL preparation and 1900ng / ml of 4-1BB-NGL protein activated 4-1BB reporter jurakt cells at a level comparable to that of 4-1BB-NGL protein.

[0285] Example 7 Verification of Antibody Half-Life Loaded by Red Blood Cells

[0286] The engineered mouse red blood cells coupled with the 4-1BB-NGL antibody and the control mouse red blood cells (CT) were labeled with Far-red dye and injected into C57 mice respectively. Each mouse was injected with 200 μL of concentrated red blood cells through the tail vein. Blood was collected from the submandibular vein 30 minutes after injection (Day 0), on the first day (Day 1), Day 2, Day 5, and Day 7, and the Far-Red positive cell ratio in the peripheral blood of the mice was analyzed by flow cytometry to evaluate the drug metabolism results. The results are shown in the scatter plot of Figure 10. The proportions of the control group measured on the above five days were 8.01%, 8.24%, 6.95%, 6.91% and 6.56%, respectively, while the experimental groups were 8.63%, 7.60%, 7.36%, 4.81% and 5.79%, respectively. That is, the proportions of Far-Red positive cells in each group were similar, with no significant difference, indicating that even if the red blood cells were modified and connected to the 4-1BB-NGL antibody, their vitality and characteristics were still basically unaffected.

[0287] Example 8: Red blood cells linked to PD-1 antibodies

[0288] 8.1 Preparation of PD-1 Antibodies by Recombinant Expression

[0289] Clone Build

[0290] The amino acid sequence of the heavy chain of the PD-1 antibody (Anti-PD-1-NGL) with an NGL motif fused to its C-terminus is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain is shown in SEQ ID NO: 9. The amino acid sequence was reverse translated, and the cDNA coding sequences were determined based on the codon preference of mammalian cells (shown in SEQ ID NOs: 10 and 11, respectively). The coding sequences were synthesized and cloned into the pcDNA3.1 vector. Positive constructs were screened and transformed into Escherichia coli. After sequencing verification, endotoxin was removed and plasmids were extracted for expression of the Anti-PD-1-NGL antibody.

[0291] Protein expression:

[0292] The heavy and light chain expression plasmids of Anti-PD-1-NGL were transfected into 293F cells at a 1:2 ratio to express the recombinant antibody. Cell culture supernatant was harvested 7 days after transfection and filtered through a 0.45 μm filter for subsequent purification of the Anti-PD-1-NGL antibody.

[0293] Liquid preparation:

[0294] Protein A elution buffer: Weigh 7.507 g of glycine and dissolve it in 900 mL of double-distilled water. Adjust the pH of the solution to 3.0 with HCl. Dose the solution to 1000 mL with double-distilled water. Filter through a 0.45 μM filter and set aside.

[0295] Protein A neutralization buffer: Weigh 121.14 g of Tris powder and dissolve it thoroughly in 800 mL of double-distilled water. Adjust the pH of the solution to 8.5 with HCl. Dose the solution to 1000 mL with double-distilled water. Filter through a 0.45 μM filter and set aside.

[0296] Antibody purification:

[0297] 1. Equilibrate the Protein A affinity chromatography column with 10 CV (column volume) of double-distilled water and 10 CV of PBS, respectively, at a flow rate of 1 mL / min.

[0298] 2. Load the sample onto the chromatography column at a flow rate of approximately 1 mL / min and collect the effluent;

[0299] 3. Wash the resin with 30 mL of equilibration buffer at a flow rate of approximately 2 mL / min.

[0300] 4. Elute the antibody with 10-15 mL of elution buffer at a flow rate of approximately 1 mL / min. Collect the eluate containing the target protein and immediately add 1 / 10 the volume of elution buffer to neutralize the solution and adjust the pH to 7.4.

[0301] 5. The eluted protein was concentrated by ultrafiltration and exchanged into PBS buffer solution, and frozen at -20℃ for later use.

[0302] 8.2 Linker Peptide Modification of Red Blood Cells

[0303] Red blood cells were obtained according to Example 4 and modified with a linker polypeptide.

[0304] 8.3 Anti-PD-1-NGL Binding to Red Blood Cells

[0305] Activated OaAEP1 enzyme was used to catalyze the coupling of Anti-PD-1-NGL with the linker-modified erythrocyte-peptide. Referring to Example 4, the reaction system was configured according to the system in Table 8 below. PBS was added first, followed by the OaAEP1 enzyme, and diluted evenly. Finally, the concentrated GLK-mal-RBC obtained above was added. After preparation, the mixture was placed on a rotary mixer and tumbled at 10 rpm and 30°C for 30 minutes.

[0306] Table 8

[0307] After the coupling reaction was completed, the coupling reaction results were detected by flow cytometry. As shown in the flow cytometry scatter plot in Figure 11, the anti-PD-1 antibody was successfully coupled to the red blood cell surface.

[0308] The sequences involved in the present invention are shown in the following table:

Claims

1. A method comprising the steps of: (a) coupling a linker polypeptide to an erythrocyte to obtain an erythrocyte comprising a linker, wherein the linker polypeptide comprises an N-terminal recognition sequence of a ligase at its N-terminus; (b) contacting the red blood cells containing the linker obtained in step (a) with an effector active agent in the presence of a ligase under conditions suitable for a ligation reaction, wherein the effector active agent contains a C-terminal recognition sequence of the ligase; in, The ligase recognizes the N-terminal recognition sequence and the C-terminal recognition sequence and catalyzes the connection between the two, thereby connecting the red blood cells to the effector active agent to form an RBC-effector protein conjugate.

2. The method according to claim 1, wherein the ligase is an asparaginyl endopeptidase (AEP), preferably OaAEP1, more preferably OaAEP1 from the closely related oldlandia affinis, most preferably OaAEP1 having the amino acid sequence shown in SEQ ID NO: 1 and / or OaAEP1 encoded by a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:

2.

3. The method according to claim 2, wherein the OaAEP1 is activated; preferably, the activation treatment comprises mixing 3 mg / ml of OaAEP1 protein with 200 mM acetic acid / sodium acetate (V:V=9:1) buffer in a volume ratio of 1:3, and incubating at 35-40°C for 4-5 hours.

4. The method of claim 1, wherein the C-terminal recognition sequence of the ligase contained in the effector active agent is coupled or connected through an in vitro reaction.

5. The method of claim 1, wherein the effector agent is a protein and comprises a C-terminal recognition sequence for a ligase at the C-terminus of at least one polypeptide thereof.

6. The method of claim 1 or claim 5, wherein the effector agent is an antibody, such as a PD-1 antibody or a 4-1BB antibody.

7. The method of claim 1, wherein the C-terminal recognition sequence of the ligase is selected from NGL, NDL, NPL, NAL, NCL, NEL, NFL, NHL, NKL, NIL, NLL, NQL, NRL, NSL, NTL, NVL, NWL, NYL, NSLD, NSLAN or NG, preferably NGL, NPL or NDL.

8. The method of claim 1, wherein the N-terminal recognition sequence of the ligase is selected from the group consisting of DL, PL, AL, CL, EL, FL, HL, KL, IL, LL, QL, RL, SL, TL, VL, WL, YL, SLD, SLAN, DLG, PLG, ALG, CLG, ELG, FLG, HLG, KLG, ILG, LLG, QLG, RLG, SLG, TLG, VLG, WLG, YLG, SLDG, SLANG, DLK, PLK, ALK, CLK, ELK, FLK, HLK, KLK, ILK, LLK, QLK, RLK, SLK, TLK, VLK, WLK, YLK, SLDK, SLANK, G, GG, GL, GGG, GLG, GLK or GGL, preferably GLK.

9. The method according to claim 1, wherein the red blood cells in step (a) are treated with a reducing agent, and the linker polypeptide comprises a maleimide group, so that the red blood cells are connected to the maleimide group via the reduced thiol group, so that the linker polypeptide is coupled to the red blood cells, wherein the linker polypeptide comprises an N-terminal recognition sequence of a ligase at its N-terminus.

10. The method according to claim 9, wherein the reducing agent is selected from one or more of β-mercaptoethanol, DTT, DTE, TCEP and glutathione, preferably TCEP.

11. The method according to any one of claims 1 to 10, wherein the erythrocytes are not genetically engineered to express a protein comprising an N-terminal recognition sequence for a ligase, and preferably the erythrocytes are natural erythrocytes, such as natural human erythrocytes.

12. Modified red blood cells (RBC) produced according to the method of any one of the preceding claims.

13. A modified red blood cell comprising an effector active agent coupled to its outer surface, wherein the effector active agent is connected to a linker polypeptide and coupled to a natural red blood cell membrane protein via the latter, and comprises an amino acid sequence NGL at the junction of the effector active agent and the linker polypeptide, preferably, the effector active agent is connected to the N-terminus of the linker polypeptide.

14. The modified erythrocyte according to claim 13, wherein the linker polypeptide is coupled to the sulfhydryl group of the native erythrocyte membrane protein via a maleimide group.

15. The modified erythrocyte according to claim 13, wherein the effector active agent is a protein and is linked to a linker polypeptide at its C-terminus, preferably the effector active agent is an antibody, such as a PD-1 antibody or a 4-1BB antibody.

16. A pharmaceutical composition comprising the modified erythrocyte according to any one of claims 12 to 15.

17. Use of the modified erythrocytes according to any one of claims 12 to 15 in the preparation of a medicament for treatment.

18. The method of claim 16 or the use of claim 17, wherein the effector agent is an antibody, such as a PD-1 antibody or a 4-1BB antibody.

19. A method of treatment comprising administering the modified red blood cells according to any one of claims 12-15 or the pharmaceutical composition according to claim 16 to an individual in need thereof.

20. A method for increasing the plasma half-life of an effector active agent, comprising the steps of: (a) treating the effector active agent so that it contains a C-terminal recognition sequence for a ligase; (b) attaching the effector agent to red blood cells by the method of any one of claims 1 to 11.

21. A method for enhancing the in vivo efficacy of an effector active agent, comprising the steps of: (a) treating the effector active agent so that it contains a C-terminal recognition sequence for a ligase; (b) attaching the effector agent to red blood cells by the method of any one of claims 1 to 11.

22. A composition comprising an asparagine endopeptidase, a linker polypeptide comprising an N-terminal recognition sequence of the asparagine endopeptidase, an effector active agent comprising a C-terminal recognition sequence of the asparagine endopeptidase, and optionally a pharmaceutically / physiologically acceptable carrier, wherein: Preferably, the linker polypeptide comprises a maleimide group, wherein the asparagine endopeptidase recognizes the N-terminal recognition sequence and the C-terminal recognition sequence under suitable conditions (eg, physiological conditions) and catalyzes the connection between the two.

23. The composition according to claim 22, wherein the ligase is an asparaginyl endopeptidase (AEP), preferably OaAEP1, more preferably OaAEP1 from the closely related oldlandia affinis, most preferably OaAEP1 having the amino acid sequence shown in SEQ ID NO: 1 and / or OaAEP1 encoded by a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:

2.

24. The composition according to claim 22, wherein the OaAEP1 is activated; preferably, the activation treatment comprises mixing 3 mg / ml of OaAEP1 protein with 200 mM acetic acid / sodium acetate (V:V=9:1) buffer in a volume ratio of 1:3, and incubating at 35-40°C for 4-5 hours.

25. The composition of claim 22, wherein the C-terminal recognition sequence is selected from NGL, NDL, NPL, NAL, NCL, NEL, NFL, NHL, NKL, NIL, NLL, NQL, NRL, NSL, NTL, NVL, NWL, NYL, NSLD, NSLAN or NG, preferably NGL, NPL or NDL.

26. The composition of claim 22, wherein the N-terminal recognition sequence is selected from the group consisting of DL, PL, AL, CL, EL, FL, HL, KL, IL, LL, QL, RL, SL, TL, VL, WL, YL, SLD, SLAN, DLG, PLG, ALG, CLG, ELG, FLG, HLG, KLG, ILG, LLG, QLG, RLG, SLG, TLG, VLG, WLG, YLG, SLDG, SLANG, DLK, PLK, ALK, CLK, ELK, FLK, HLK, KLK, ILK, LLK, QLK, RLK, SLK, TLK, VLK, WLK, YLK, SLDK, SLANK, G, GG, GL, GGG, GLG, GLK or GGL, preferably GLK.

27. The composition according to claim 22, wherein after the composition contacts red blood cells in vivo, the linker polypeptide is linked to the sulfhydryl group on the surface of the red blood cell membrane through the maleimide group.

28. The composition according to claim 22, wherein after the composition contacts red blood cells in vivo, the asparagine endopeptidase recognizes the N-terminal recognition sequence and the C-terminal recognition sequence and catalyzes the connection between the linker polypeptide and the effector active agent.

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