Improved sortase

Abstract

A sortase variant having improved catalytic activity and stability. Specifically, compared with a parent sortase (SEQ ID NO: 2), the mutant further comprises a mutant M155V / L200F or M155V / K1616P, and is numbered according to SEQ ID NO: 1. The present invention also relates to a use of the sortase mutant.

Description

Improved sortase Technical Field

[0001] The present application relates to a sortase variant with improved catalytic activity and stability, specifically a sortase mutant further comprising the mutations M155V / L200F or M155V / K162P relative to the parent sortase (SEQ ID NO: 2), numbered according to SEQ ID NO: 1. The present invention also relates to uses of the sortase mutant. Background Art

[0002] Bacterial sortases are transpeptidases that can modify proteins in a site-specific manner. The most widely used sortase is wild-type sortase A (SrtA, shown in SEQ ID NO: 1) from Staphylococcus aureus. It recognizes the motif LPXTG and cleaves the peptide bond between threonine and glycine to form an enzyme-substrate (thioester) intermediate. This intermediate is then nucleophilically attacked and disintegrated, allowing the formation of a new peptide bond between the substrate and the target, thereby achieving labeling or conjugation. Therefore, sortases are widely used in protein engineering (e.g., site-directed modification and site-directed ligation).

[0003] Wild-type SrtA contains 206 amino acid residues, and due to its recognition of specific motifs, its substrate spectrum is limited. In addition, since the ligation reaction catalyzed by SrtA is reversible, there are problems such as low catalytic activity and slow catalytic rate, thus generally requiring higher concentrations of polypeptide substrates and larger enzyme dosages. Although various efforts have been reported to improve the performance of sortases, such as blocking the reverse reaction of sortases (Yamamura, Y. et al. reported introducing a β-hairpin near the substrate recognition site to induce a β-hairpin structure near the ligation site to enhance protein ligation mediated by sortase A (Chem. Commun. 47 (2011) 4742-4744)); modifying sortases to enhance their reaction kinetics and / or change their substrate preference (US 9,267,127), etc., there is still a demand in the art to improve or enhance the activity and stability of SrtA.

[0004] Numerous attempts have been made to develop red blood cells (RBCs) as drug delivery vehicles. However, existing RBC modifications have limitations for in vivo applications. For example, the modification process disrupts the cell membrane, thus affecting the in vivo survival of engineered RBCs. Improving the RBC payload without compromising its integrity and function, thereby increasing drug concentration at the target site and enhancing its therapeutic efficacy, remains a pressing challenge.

[0005] The present application solves the above-mentioned problem to a certain extent by providing a sorting enzyme with improved performance.

[0006] SUMMARY OF THE INVENTION

[0007] In a first aspect, the present invention provides a sortase mutant, whose amino acid sequence is at least 90% identical to the parent SEQ ID NO: 2, and comprises one or more mutations selected from M155V, L200F, and K162P.

[0008] In a specific embodiment, the sortase mutant is at least 95% identical to the parent SEQ ID NO: 2 and comprises the mutations M155V / L200F or M155V / K162P.

[0009] In a specific embodiment, the sortase mutant comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0010] In a second aspect, the present invention provides a polynucleotide molecule encoding the sortase mutant of the first aspect.

[0011] In a third aspect, the present invention provides a vector comprising the polynucleotide molecule of the second aspect.

[0012] In a fourth aspect, the present invention provides a cell comprising the polynucleotide molecule of the second aspect or the vector of the third aspect. In a specific embodiment, the cell is a eukaryotic cell.

[0013] In a fifth aspect, the present invention provides a method for coupling an active molecule to an erythrocyte, comprising contacting the sortase mutant of the first aspect with the erythrocyte.

[0014] In a specific embodiment, the cell membrane surface of the erythrocyte comprises a sortase recognition motif. In another specific embodiment, the cell membrane surface of the erythrocyte comprises a linker, and the linker comprises a sortase receptor motif. In another specific embodiment, the active molecule comprises a sortase recognition motif. In another specific embodiment, the sortase recognition motif is, for example, the amino acid sequence LPXTG, wherein X is a standard or non-standard amino acid (e.g., X is selected from D, E, A, N, Q, K, or R). Preferably, the motif recognized by the sortase A is LPETG (SEQ ID NO: 11). In another embodiment, the sortase recognition motif can be modified to increase its recognition efficiency. Preferably, LPETG is modified to increase its affinity for the sortase, for example, by adding a G to the C-terminus of the recognition sequence. Preferably, the sortase recognition motif is LPETGG (SEQ ID NO: 6) or LPETGGK (SEQ ID NO: 7). Optionally, the recognition motif further comprises an Abz / Lys (DNP) modification.

[0015] In a sixth aspect, the present invention provides use of the sortase mutant of the first aspect in directed coupling of molecules. In a specific embodiment, the present invention provides use of the sortase mutant of the first aspect in directed coupling of active molecules to the surface of erythrocyte membranes.

[0016] In a seventh aspect, the present invention provides a kit comprising the sortase mutant of the first aspect, or the polynucleotide molecule of the second aspect, or the vector of the third aspect. Optionally, the kit may further comprise auxiliary materials, such as a buffer, a preservative, instructions, and the like.

[0017] In a specific embodiment, the present invention provides a kit comprising the red blood cells disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 shows the catalytic activity curves of different mutants under non-heat-treated conditions and the results of the reaction rate changes with the concentration of SrtA substrate (SrtA peptide), wherein (1) represents the catalytic activity curve of SEQ ID NO: 2 and Vmax, Km and R 2 (2) shows the catalytic activity curve of mutant M155V / K162P (SEQ ID NO: 4) and Vmax, Km and R 2 (3) shows the catalytic activity curve of mutant M155V / L200F (SEQ ID NO: 3) and Vmax, Km and R 2(4) represents the change in reaction rate of the mutant shown in SEQ ID NO: 2, mutant M155V / K162P (SEQ ID NO: 4) and mutant M155V / L200F (SEQ ID NO: 3) at different SrtA peptide concentrations after no heat treatment.

[0019] Figure 2 shows the catalytic activity curves and reaction rate changes of different mutants after incubation at 40°C for 2 hours, including: (1) catalytic activity curve and Vmax, Km and R2 values ​​of the mutant shown in SEQ ID NO: 2 after incubation at 40°C for 2 hours; (2) catalytic activity curve and Vmax, Km and R2 values ​​of mutant M155V / K162P (SEQ ID NO: 4) after incubation at 40°C for 2 hours; (3) catalytic activity curve and Vmax, Km and R2 values ​​of mutant M155V / L200F (SEQ ID NO: 3) after incubation at 40°C for 2 hours; (4) changes in reaction rates of the mutant shown in SEQ ID NO: 2, mutant M155V / K162P (SEQ ID NO: 4) and mutant M155V / L200F (SEQ ID NO: 3) at different SrtA substrate concentrations after heating treatment at 40°C for 2 hours.

[0020] Figure 3 shows the flow cytometric detection results of the coupling efficiency of PD1 antibodies and red blood cells under the catalysis of Sortase-ct (SEQ ID NO: 2) and SrtA-V2 enzyme (SEQ ID NO: 3), wherein the negative control peak represents the flow cytometric result of RBC without antibody coupling; the RBC-Sortase-ct peak represents the coupling efficiency of PD1 antibodies and red blood cells under the catalysis of SEQ ID NO: 2; and the RBC-Sortase-mut peak represents the coupling efficiency of PD1 antibodies and red blood cells under the catalysis of SEQ ID NO: 3.

[0021] Figure 4 shows the flow cytometric detection results of the coupling efficiency of anti-PD1 antibodies and red blood cells under the catalysis of Sortase-ct (SEQ ID NO: 2) and SrtA-V1 enzyme (SEQ ID NO: 4), wherein the negative control peak represents the flow cytometric result of RBC without antibody coupling; the RBC-Sortase-ct peak represents the coupling efficiency of anti-PD1 antibodies and red blood cells under the catalysis of SEQ ID NO: 2; and the RBC-Sortase-mut peak represents the coupling efficiency of anti-PD1 antibodies and red blood cells under the catalysis of SEQ ID NO: 4.

[0022] Figure 5 shows the time course of in vitro transpeptidation reactions catalyzed by different mutants under different NH2-G small peptide substrates, as well as the results of in vitro transpeptidation catalysis at different enzyme concentrations, wherein (a) shows the time course of transpeptidation catalyzed by mutant (SEQ ID NO: 2), M155V / L200F (SEQ ID NO: 3) and M155V / K162P (SEQ ID NO: 4) at different reaction times; (b) shows the results of transpeptidation catalyzed by mutant (SEQ ID NO: 2), M155V / L200F (SEQ ID NO: 3) and M155V / K162P (SEQ ID NO: 4) under different concentration conditions.

[0023] Detailed Description of the Invention

[0024] I. Definition

[0025] In order to promote an understanding of the principles of the invention, reference will now be made to the embodiments shown in the accompanying drawings and described in detail. However, it should be understood that this is not intended to limit the scope of the invention.

[0026] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein can be used to practice the present invention, but preferred methods and materials are described herein. Therefore, the terms defined herein are more fully described by the specification as a whole.

[0027] As used herein, the singular expressions "a," "an," 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 this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the specific context used by those skilled in the art.

[0028] Unless the context requires otherwise, the terms "comprises," "comprising," and "containing" or similar terms are intended to mean a non-exclusive inclusion, whereby a list of elements or features includes not only those elements mentioned or listed, but may include additional elements or features that are not listed or mentioned.

[0029] The terms "individual" or "subject" are used interchangeably and refer to a mammal with which the methods or compositions disclosed herein can be used. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Thus, the methods and compositions disclosed herein may have medical and / or veterinary applications. In a preferred form, the mammal is a human.

[0030] As used herein, term " sequence identity " refers to, with regard to the same degree of sequence on comparison window, after using standard algorithm to carry out suitable comparison, the nucleotide of complete matching or amino acid number.Therefore, " sequence identity percentage ratio " is calculated in the following manner: on comparison window, compare the sequence of two optimal comparisons, determine the quantity of the position that identical nucleic acid base (for example, A, T, C, G) occurs in two sequences to produce the quantity of matching position, the quantity of matching position is divided by the position sum (i.e. window size) in comparison window, and result is multiplied by 100 to produce sequence identity percentage ratio.For example, " sequence identity " can be understood as representing by DNASIS computer program (Windows version 2.5; Can obtain from Hitachi Software Engineering Co., Ltd. in South San Francisco, California, USA) " matching percentage ratio " calculated.

[0031] In the context of polypeptides, the term "mutation" refers to the replacement of at least one amino acid residue in a parent amino acid sequence with a different amino acid residue. The one or more replacement residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) or "non-naturally occurring amino acid residues." Non-naturally occurring amino acid residues refer to residues other than naturally occurring amino acid residues that are capable of covalently binding to one or more adjacent amino acid residues in a polypeptide chain, such as norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs.

[0032] The term "amino acid deletion" refers to the removal of at least one amino acid residue at a predetermined position in an amino acid sequence.

[0033] The term "amino acid substitution" refers to replacing at least one amino acid residue in a predetermined parent amino acid sequence with a different amino acid residue. The one or more replacement residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) or "non-naturally occurring amino acid residues."

[0034] In the context of polypeptides, the term "position" refers to the position of an amino acid residue in the amino acid sequence of a polypeptide. In any case, the positions are numbered sequentially, with the first amino acid residue being numbered 1.

[0035] 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.

[0036] 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.

[0037] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0038] 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.

[0039] 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%.

[0040] 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.

[0041] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0042] II. Red blood cells (RBC)

[0043] In the human body, red blood cells (RBCs) are the most numerous blood cells in the circulation. Unlike other blood cells, RBCs lack a nucleus and are flexible, allowing them to change shape to fit into human blood vessels. Their primary function in the body is to supply oxygen. Key protein markers on the surface of RBCs allow them to circulate in the body for extended periods without being cleared by macrophages, resulting in a long half-life. This property makes them excellent candidates for drug carriers. Mature RBCs without nuclei do not contain any genetic material, and therefore have a good safety profile compared to other gene and cell therapies.

[0044] 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.

[0045] In some embodiments, RBCs are not genetically engineered. In some embodiments, the present invention provides red blood cells having an active agent conjugated thereto by a sortase-mediated reaction. In one embodiment, the active agent includes a binding agent, a therapeutic agent, or a detection agent, including, for example, a protein, an antibody, or a functional antibody fragment thereof, an antigen such as a tumor antigen, an MHC-peptide complex, a drug such as a small molecule drug (e.g., an anti-tumor agent, such as a chemotherapeutic agent), an enzyme (e.g., a functional metabolic enzyme such as UOX, or a therapeutic enzyme), a hormone, a cytokine, a growth factor, an antimicrobial agent, a probe, a ligand, a receptor, an immune tolerance-inducing peptide, a targeting moiety, a prodrug, or any combination thereof. In one embodiment, the active agent is an anti-PD1 antibody. In one embodiment, the active agent can be connected to a sortase receptor motif via a sortase recognition motif. In a specific embodiment, the active agent is a peptide molecule. In one embodiment, the active agent is an anti-PD1 antibody. In one embodiment, the membrane protein on the surface of the red blood cell comprises a sortase recognition motif. In another embodiment, the membrane protein on the surface of the red blood cell comprises a sortase receptor motif.

[0046] A "sortase receptor motif" refers to a motif comprising oligoglycine that acts as an amine nucleophile to attack the intermediate formed between the sortase and the recognition motif, and ultimately covalently couples to the recognition motif. In one embodiment, the sortase receptor motif is located at the N-terminus or C-terminus of the polypeptide. In one embodiment, the receptor motif comprises Gly-[Gly]n- (SEQ ID NO: 5), where n = 0-5.

[0047] A "sortase recognition motif" refers to a polypeptide that forms a thioester bond with a sortase molecule after cleavage by the sortase molecule. In one embodiment, the sortase recognition motif comprises LPXTG or LPXTA (SEQ ID NO: 12), where X is any amino acid. In one embodiment, sortase cleavage occurs between T and G / A.

[0048] 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.

[0049] In some embodiments, the present invention provides a method for increasing the drug loading capacity of red blood cells by using an improved sortase variant, comprising contacting red blood cells with an active agent and causing a conjugation reaction under the action of the improved sortase of the present application, thereby improving the drug loading capacity of red blood cells.

[0050] In one embodiment, the present invention provides red blood cells with a higher drug loading. By using the improved sortase of the present application, the red blood cells are contacted with the active agent and a conjugation reaction occurs, thereby obtaining red blood cells with a higher drug loading. In one embodiment, the red blood cells contain a sortase recognition motif on their cell membrane surface, and the active agent contains a sortase receptor motif. In one embodiment, the active agent is an anti-PD1 antibody. In one embodiment, the anti-PD1 antibody contains a sortase receptor motif and is connected to the RBC via a linker containing a sortase recognition motif on the red blood cell membrane surface. In one embodiment, the red blood cells are modified so that their membrane proteins are covalently linked to the linker.

[0051] III. Sortase

[0052] Sortase refers to an enzyme that catalyzes a transpeptidation reaction that conjugates a first molecule to a second molecule via transamidation. Sortase recognizes a specific motif (e.g., the amino acid sequence LPXTG (SEQ ID NO: 10)), and molecules containing the sortase recognition motif are referred to herein as "sortase substrates." In the context of this application, the term sortase includes full-length sortase (e.g., full-length naturally occurring sortase), active sortase fragments, and modified sortase variants.

[0053] The sortases of the present invention encompass all known types of sortases, such as sortase A, sortase B, sortase C, and sortase D. In one embodiment, the sortase is sortase A. The term "sortase A" refers to a class A sortase from any bacterial species or strain, commonly referred to as SrtA. In one embodiment, sortase A is Staphylococcus aureus sortase A. In another embodiment, sortase A is sortase A from Streptococcus pyogenes (S. pyogenes).

[0054] The motif commonly recognized by sortase A is the short peptide LPXTG, where X is a standard or non-standard amino acid (e.g., X is selected from D, E, A, N, Q, K, or R). Common recognition motifs include LPKTG (SEQ ID NO: 13), LPATG (SEQ ID NO: 14), and LPNTG (SEQ ID NO: 15). In some embodiments, the motif recognized by sortase A is LPETG. In one embodiment, the sortase recognition motif can be modified to improve its recognition efficiency. Preferably, LPETG is modified to increase its affinity for the sortase, for example, by adding a G to the C-terminus of the recognition sequence, such that the modified sequence is LPETGG.

[0055] In addition, sortase A can also recognize other motifs, for example, a motif in which position 4 of the sortase A recognition motif contains "A", "S", "L", or "V" instead of "T". In some embodiments, position 5 of the motif contains "A" instead of "G". In some embodiments, position 2 of the motif contains "G" or "A" instead of "P". In some embodiments, position 1 of the motif contains "I" or "M" instead of "L". Pishesha et al. 2018 describe various recognition motifs for sortase A.

[0056] Sortase A can achieve covalent conjugation of two entities in vitro by recognizing substrates containing a recognition motif. Specifically, sortase A specifically recognizes substrates containing the motif LPXTG and cleaves between the threonine and glycine residues in the motif, forming a substrate thioester acyl-enzyme intermediate. Amine nucleophiles from the oligoglycine-containing entity then attack this intermediate, causing its decomposition, resulting in the covalent attachment of the oligoglycine-containing entity to the substrate containing the recognition motif and regeneration of sortase A. Motifs that can undergo amine nucleophile attack reactions with sortase recognition motifs are also referred to herein as sortase receptor motifs and typically contain oligoglycine sequences.

[0057] The specific catalytic activity of sortases has been exploited for a variety of protein modification and bioconjugation purposes. With sortase A, natural and synthetic functional entities can be introduced into entities labeled with the LPXTG sequence, including polymers containing oligoglycine (e.g., PEG), fluorophores, vitamins (e.g., biotin and folic acid), lipids, carbohydrates, nucleic acids, synthetic peptides, and proteins (e.g., GFP). Furthermore, Westlake Biopharmaceutical Technology (Hangzhou) Co., Ltd. has successfully conjugated a variety of active substances to erythrocytes by engineering the LPXTG motif onto erythrocyte membranes and leveraging the properties of sortase, thereby transforming erythrocytes into advantageous drug carriers and achieving corresponding therapeutic functions.

[0058] IV. Sortase mutants

[0059] In some embodiments, the present invention contemplates the use of naturally occurring variants of sortase. Extensive structural information on sortase A is available in the prior art, including NMR or crystal structures of SrtA alone or in combination with a sortase recognition sequence (see, for example, Zong Y et al. J. Biol Chem. 2004, 279, 31383-31389). Recombinant techniques can be used to generate sortase fragments with transamidation activity. For example, the fragment lacks the N-terminal transmembrane region required for non-catalytic activity, such as the 25-60 amino acids at the N-terminus of the sortase.

[0060] In some embodiments, the sequence of full-length wild-type S. aureus SrtA is shown in SEQ ID NO: 1 (full length, GenBank accession number: CAA3829591.1):

[0061] In some embodiments, a functional variant of Staphylococcus aureus sortase A useful in the present invention can be a Staphylococcus aureus sortase A variant comprising, for example, one or more mutations at amino acid positions D124, Y187, E189, and F200, D124G, Y187L, E189R, and F200L, and optionally further comprising one or more mutations at amino acid positions P94S / R, D160N, D165A, K190E, and K196T (numbered according to the sequence shown in SEQ ID NO: 1). In certain embodiments, the S. aureus sortase variant may comprise D124G; D124G and F200L; P94S / R, D124G, D160N, D165A, K190E, and K196T; P94S / R, D160N, D165A, Y187L, E189R, K190E, and K196T; P94S / R, D124G, D160N, D165A, Y187L, E189R, K190E, and K196T; D124G, Y187L, E189R, and F200L; or P94S / R, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L (according to SEQ ID NO: 1). The sequence is numbered as shown in NO: 1).

[0062] In one embodiment, the sortase A mutant is a soluble truncated sortase A lacking a transmembrane region, for example lacking 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) amino acids at the N-terminus. In one embodiment, the sortase A mutant comprises a fragment from about position 60 to about position 206 of wild-type Staphylococcus aureus SrtA (numbered according to the sequence shown in SEQ ID NO: 1). In one embodiment, mutations at relevant sites are further performed on the basis of the truncated SrtA to obtain the mutant shown in SEQ ID NO: 2 (numbered according to the sequence shown in SEQ ID NO: 1):

[0063] In the present application, amino acid mutation position is determined by comparing with SEQ ID NO:1. The method for determining the amino acid position corresponding to mutation position as herein described is well known in the art. The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) performed in the Needle program (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277, preferably version 3.0.0 or higher) of the EMBOSS software package can be used to determine the corresponding amino acid residue in another polypeptide. Based on the above-mentioned well-known computer program, those skilled in the art can routinely determine the amino acid position of a polypeptide of interest as herein described.

[0064] The inventors have found that further modification of one or more positions M155, K162 and L200 (numbered according to the sequence shown in SEQ ID NO: 1) of the sortase mutant shown in SEQ ID NO: 2 will enhance the stability and catalytic activity of sortase A.

[0065] Thus, the present invention provides a sortase A mutant comprising an amino acid sequence homologous to the amino acid sequence of wild-type sortase A (SEQ ID NO: 1) or a mutant of SEQ ID NO: 2. In one embodiment, the sortase mutant may further comprise one or more additional mutations. For example, the sortase mutant may have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1, or the sortase mutant may have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the mutant comprises one or more mutations selected from M155, K162, and L200 in SEQ ID NO: 1 or SEQ ID NO: 2, for example, one or more mutations selected from M155V, K162P, or L200F (numbered according to the sequence shown in SEQ ID NO: 1).

[0066] In one embodiment, further modification of one or both of positions M155, K162, and L200 of the amino acid sequence of SEQ ID NO: 1 or 2 enhances the stability and catalytic activity of sortase A (numbering according to the sequence shown in SEQ ID NO: 1). In one embodiment, modification of positions M155 and K162 of the amino acid sequence of SEQ ID NO: 1 or 2 enhances the stability and catalytic activity of sortase A (numbering according to the sequence shown in SEQ ID NO: 1). In one embodiment, further modification of positions M155 and L200 of the amino acid sequence of SEQ ID NO: 1 or 2 enhances the stability and catalytic activity of sortase A (numbering according to the sequence shown in SEQ ID NO: 1).

[0067] In one embodiment, introducing the double mutations M155V and L200F into SEQ ID NO: 1 or 2 enhances the stability and catalytic activity of sortase A (numbering is based on the sequence shown in SEQ ID NO: 1, wherein the expression M155V refers to the substitution / mutation of amino acid residue M at position 155 of SEQ ID NO: 1 or 2 with amino acid residue V, and the expression L200F refers to the substitution / mutation of amino acid residue L at position 200 of SEQ ID NO: 1 or 2 with amino acid residue F). In one embodiment, the sortase mutant obtained by making the above mutations into SEQ ID NO: 2 has the following sequence:

[0068] In one embodiment, the introduction of modifications M155V and K162P into SEQ ID NO: 1 or 2 enhances the stability and catalytic activity of sortase A (numbering is based on the sequence shown in SEQ ID NO: 1, wherein the expression M155V refers to the substitution / mutation of amino acid residue M at position 155 of SEQ ID NO: 1 or 2 to amino acid residue V, and the expression K162P refers to the substitution / mutation of amino acid residue K at position 162 of SEQ ID NO: 1 or 2 to amino acid residue P). In one embodiment, the sortase mutant obtained by making the above mutations to SEQ ID NO: 2 has the following sequence:

[0069] In some embodiments, the catalytic activity of the sortase A mutant (e.g., as set forth in SEQ ID NO:3 or SEQ ID NO:4) is increased by at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or even more relative to the parent enzyme (e.g., SEQ ID NO:1 or 2).

[0070] In some embodiments, the stability of the sortase A mutant (e.g., as set forth in SEQ ID NO: 3 or SEQ ID NO: 4) is improved by at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or even more relative to the parent (e.g., SEQ ID NO: 1 or 2). In a specific embodiment, the stability is thermostability.

[0071] In some embodiments, the maximum activation rate (Vmax) of the sortase A mutant (e.g., as set forth in SEQ ID NO:3 or SEQ ID NO:4) is increased by at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or even more relative to the parent (e.g., SEQ ID NO: 1 or 2).

[0072] In some embodiments, the kinetic constant (Km) value of the sortase A mutant (e.g., as set forth in SEQ ID NO:3 or SEQ ID NO:4) is at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or even more, relative to the parent enzyme (e.g., SEQ ID NO: 1 or 2).

[0073] In some embodiments, the sortase A mutant (e.g., as set forth in SEQ ID NO:3 or SEQ ID NO:4) increases drug loading of red blood cells by at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or even more, relative to the parent enzyme (e.g., SEQ ID NO: 1 or 2).

[0074] In some embodiments, the sortase mutant can further comprise any one or more of the following mutations relative to the parent (e.g., SEQ ID NO: 1): P94S / R, E105K, E108A, E108Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L mutations (numbered according to the sequence shown in SEQ ID NO: 1).

[0075] In some embodiments, the sortase mutants obtained in the present application may also contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative amino acid mutations that do not significantly affect enzyme function / activity. Conservative amino acid mutations that do not significantly affect protein activity are well known in the art.

[0076] V. Sortase Activity Assay

[0077] Methods for assaying sortase activity are known in the art.

[0078] The sortase reaction can be carried out in any convenient container (e.g., any solid or semi-solid support such as a microcentrifuge tube, microtiter plate, glass slide, silicon wafer, filter, multichannel chip, etc.). The reaction is carried out at any convenient temperature at which the sortase reaction can be carried out, for example, at a temperature between about 15°C and 50°C, preferably at a temperature between 30°C and 40°C, and more preferably at a temperature between about 23°C and 37°C. In one embodiment, the sortase reaction is carried out at a temperature of about 37°C. In another embodiment, the sortase reaction is carried out at room temperature (i.e., about 20°C to 25°C).

[0079] In the sortase reaction, the molar ratio of enzyme to substrate containing the recognition motif is, for example, between 1:100, such as between about 1:10, between 1:5, between 1:4, between 1:3, and between 1:2. In other words, in some embodiments, the concentration of the substrate containing the recognition motif in the reaction mixture is about 10 μM to about 10 mM, such as about 100 μM to about 1 mM, about 100 μM to about 5 mM, about 200 μM to about 1 mM, about 200 μM to about 800 μM, or about 400 μM to about 600 μM.

[0080] In some embodiments, the concentration of the substrate containing the sortase receptor sequence is between about 1 μM and about 500 μM, such as about 15 μM to about 150 μM, about 25 μM to about 100 μM, about 40 μM to about 60 μM.

[0081] In some embodiments, the concentration of the sortase in the reaction system is about 1 μM to about 500 μM, for example, about 15 μM to about 150 μM, about 25 μM to about 100 μM, or about 40 μM to about 60 μM.

[0082] In some embodiments, the reaction mixture comprises a buffer. Those skilled in the art are familiar with a variety of buffers that can be used for the methods reported herein. In some embodiments, the reaction is carried out between pH 6-8.5, for example, between pH 6-8, between pH 6-7.5, between pH 6.5-8.5, between pH 7-8.5, between pH 7.5-8.5, between pH 7.0-8.5. In some embodiments, the reaction is carried out between pH 7.3-7.8. Example

[0083] The present invention is further illustrated by the following examples; however, it should be understood that the examples are described in an illustrative rather than a limiting sense and that various modifications may be made by those skilled in the art.

[0084] Unless expressly stated to the contrary, the present invention will be practiced using conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill of the art. The experimental materials used, unless otherwise specified, are commercially available. Where specific techniques or conditions are not specified in the examples, the experiments were performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the corresponding product specifications. Where the manufacturer of the reagents or instruments used is not specified, all are conventional products that can be purchased through regular channels.

[0085] Example 1. Experimental materials

[0086] 1.1 Mutants of the present invention

[0087] Based on the sortase mutant disclosed in SEQ ID NO: 2, mutations were performed on it according to conventional methods to obtain the mutants shown in Table 1:

[0088] Table 1. Mutants obtained in this experiment

[0089] 1.2 Culture medium

[0090] (1) Seed culture medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl;

[0091] (2) High-density fermentation medium 1: 5 g / L KH2PO4, 10 g / L K2HPO4·3H2O, 5 g / L (NH4)2SO4, 1 g / L anhydrous citric acid, 1 g / L MgSO4·7H2O, 10 g / L yeast extract, 50 g / L glycerol, 0.1 g / L vitamin B1, and 1 mL / L trace elements;

[0092] (6) Trace elements: 100 g·L-1Fe(III) citrate, 18 g·L -1 ZnCl3, 14.64 g·L - 1 MnSO4·H2O, 0.75g·L -1 CuSO4·5H2O, 2g·L -1 Na2MoO4·2H2O、2g·L -1 CaCl2.2H2O, 3.0g·L -1 H3BO3, 2.5g·L -1 CoCl2.6H2O, 2.5g·L-1NiSO4.6H2O and 100mL HCl.

[0093] 1.3 Experimental Reagents

[0094] Table 2 Drugs involved in this experiment

[0095] 1.4 Main solutions and reagents

[0096] Enzyme and substrate preparation method: Sortase A was diluted with PBS to a 10μM 1000μl working solution; SrtA peptide was prepared into a 1mM stock solution with water and the pH was adjusted to 7-8 with NaOH; GL peptide was prepared into a 4mM stock solution with PBS and the pH was adjusted to 7-8 with NaOH.

[0097] Example 2. Construction, expression and purification of sortase mutants

[0098] 2.1 Strain construction

[0099] The nucleotide sequence encoding the sortase mutant (as shown in SEQ ID NO: 3 or SEQ ID NO: 4) was constructed into the pET-28a(+) expression vector to obtain the plasmid pET-28a(+)-Sortase, which was then transformed into E. coli BL21(DE3) competent cells and cultured statically on a 37°C LB plate for 12 hours. Colonies were then picked, the nucleic acid from the colonies was extracted, and PCR verification was performed. Positive transformants were picked and transferred to a test tube containing LB medium at 37°C, shaken at 220 rpm, and the plasmid was extracted and sequenced for verification. Strains with correct sequencing were inoculated and subcultured and the strains were preserved.

[0100] 2.2 Seed liquid culture

[0101] The positive single clone strain obtained in Section 2.1 was streaked and cultured for 8-10 h. A clone was picked and inoculated into a 250 mL shake flask containing 25 mL of LB medium and cultured at 37°C and 220 rpm for 8-10 h.

[0102] 2.3 250 mL shake flask culture

[0103] Inoculate 50 mL of high-density fermentation medium in a 250 mL shake flask with the secondary seed solution obtained in Section 2.2 at a 2% inoculum level. Incubate at 37°C, 220 rpm, and shake. After 2-3 hours, add IPTG and induce protein production at 30°C, 220 rpm.

[0104] 2.4 Cell disruption

[0105] After induction, collect the bacterial cells from Section 2.3 by centrifugation and resuspend them in cell lysis buffer to an appropriate volume. Disrupt the cells using a high-pressure homogenizer at 700-900 psi for multiple cycles until the cell suspension becomes clear. Recover the corresponding sortase mutant (as shown in SEQ ID NO: 3 or SEQ ID NO: 4).

[0106] Example 3. Functional identification of sortase mutants

[0107] 1. Sortase A-mediated catalytic reaction

[0108] (1) The 4 mM stock solution of SrtA recognition substrate was diluted 2-fold with water to form 8 working concentrations, namely 500 / 250 / 125 / 62.5 / 31.25 / 15.625 / 7.8125 / 3.90625 μM;

[0109] (2) The catalytic reaction system was configured according to Table 3 below:

[0110] Table 3 Reaction system

[0111] The fluorescent peptide was synthesized by GenScript with the sequence LPETGGK and contained Abz / Lys(DNP) modification. The nucleophilic attack peptide was synthesized by GenScript with the sequence GLGDYKDDDDK (SEQ ID NO: 17).

[0112] (3) According to the catalytic reaction system, 50 μl of GL peptide and 40 μl of one of the eight concentration gradients of SrtA recognition substrate were added to the corresponding positions of a 96-well plate in triplicate;

[0113] (4) Set the microplate reader temperature to 30°C and perform the pre-warm program for 5 minutes to allow the reaction system temperature to reach equilibrium;

[0114] (5) Add enzyme to initiate the reaction and perform kinetic fluorescence monitoring, with ex at 320 nm and em at 420 nm, reading every 23 s for a total of 10 min. Set the vibrating plate before the first reading and between each reading.

[0115] (6) The fluorescence enhancement rate and substrate concentration were fitted to the Michaelis-Menten equation, and nonlinear fitting was performed using the Michaelis-Menten equation in Origin 2018 to obtain the Km and Vmax values ​​for comparison of relative catalytic efficiency.

[0116] In this series of catalytic reactions, SEQ ID NO: 2 was used as a control to compare the catalytic activities of the mutants obtained in the present application comprising the paired mutations M155V / L200F or M155V / K162P.

[0117] 2. Experimental Results

[0118] 2.1 The fluorescence enhancement rates of the mutants are shown in Tables 4-6 below, and the fluorescence enhancement rate curve is shown in Figure 1.

[0119] Table 4: SEQ ID NO:2

[0120] Table 5: SrtA-V1 (SEQ ID NO:4)

[0121] Table 6: SrtA-V2 (SEQ ID NO: 3)

[0122] From the above results, it can be seen that compared with SEQ ID NO: 2, the reaction enzyme speed of the new mutants SrtA-V1 and SrtA-V2 is significantly improved, which can greatly improve the enzyme reaction efficiency, shorten the reaction time, and thus save the amount of enzyme used.

[0123] 2.2 Vmax and Km values ​​of each mutant

[0124] The following table lists the maximum rate (Vmax) and kinetic constant (Km) of different mutants in catalytic reactions

[0125] Table 7 Vmax and Km values ​​of different mutants

[0126] As can be seen from Table 7, compared with the parent SEQ ID NO: 2, the maximum reaction rate and kinetic constant of the further modified sortase mutants are significantly improved, and the improvement may even exceed 1 or 2 orders of magnitude.

[0127] 2.3 Improved thermal stability of mutants

[0128] In order to detect the thermal stability of the obtained mutants, the stability was tested by incubating the sortase at 40°C for 2 h and detecting its catalytic activity. The results are shown in Tables 8-10 and Figure 2 below.

[0129] Table 8: SEQ ID NO:2

[0130] Table 9: SrtA-V1 (SEQ ID NO:4)

[0131] Table 10: SrtA-V2 (SEQ ID NO: 3)

[0132] The Vmax and Km values ​​of the mutants after incubation at 40°C for 2 h are shown in Table 11. As can be seen, the mutants obtained in the present application have improved stability relative to the parent SEQ ID NO: 2. After incubation at 40°C for 2 h, the maximum reaction rate and kinetics of the mutants are often still 1-2 orders of magnitude higher than those of the parent.

[0133] Table 11 Vmax and Km values ​​of different mutants

[0134] Example 4. Sortase mutants increase drug loading capacity of erythrocytes

[0135] This example studies the improvement of the drug loading capacity of erythrocyte-conjugated drugs containing a sortase recognition marker by the mutants (SEQ ID NO: 3 and SEQ ID NO: 4).

[0136] Red blood cells were isolated from the peripheral blood of C57 / B6 mice (Shanghai Jihui Laboratory Animal Breeding Co., Ltd.) using density gradient centrifugation. The isolated red blood cells were rinsed three times with PBS and then pretreated with 5 mM tris(2-carboxyethyl)phosphine (TCEP, Sigma) at 30°C for 1 hour. TCEP treatment reduces the extracellular disulfide bonds in red blood cell membrane proteins, resulting in free sulfhydryl groups on the red blood cell membrane surface. The pretreated red blood cells were washed three times with PBS and reacted with a linker containing the receptor motif (GGGSK(6-Mal)-NH2) (SEQ ID NO: 16).

[0137] In the presence of the sortase mutant obtained in this application, 1×10 9 10 μM of RBC containing the linker was reacted with an anti-PD1 antibody containing the sortase recognition motif LPETG. In the conjugation reaction, the concentration of the sortase mutant was 10 μM and the concentration of the anti-PD1 antibody was 25 μM.

[0138] The amino acid sequence of the heavy chain of the anti-PD1 antibody - LPETG is shown below:

[0139] The amino acid sequence of the light chain of the anti-PD1 antibody is shown below:

[0140] The labeling effect of anti-PD1 antibodies on erythrocyte membranes was monitored by flow cytometry (Cytoflex, Beckman), thereby characterizing the efficiency of anti-PD1 antibody coupling to erythrocytes. The results are shown in Figures 3-4. It shows that compared with Sortase-ct (SEQ ID NO: 2), the coupling efficiency of anti-PD1 antibodies and erythrocytes catalyzed by SrtA-V2 (SEQ ID NO: 3, Figure 3) and SrtA-V1 (SEQ ID NO: 4, Figure 4) is improved, and its MFI value, i.e., mean fluorescence intensity, is enhanced.

[0141] In addition, the amount of anti-PD1 antibodies carried by red blood cells under the catalysis of different sortase enzymes was detected by Fc Elisa.

[0142] Specifically, PD1 is coated on an ELISA test plate through routine ELISA enzyme-linked immunosorbent assay (RIPA) operation, and the red blood cell membrane is lysed to release the anti-PD1 antibody. The lysate is added to the ELISA test plate coated with PD1 at an appropriate dilution, incubated to allow the anti-PD1 antibody to bind to PD1, and then a secondary detection antibody conjugated with HRP is added to bind to the Fc end of the anti-PD1 antibody. The reaction substrate (TMB) of HRP is added for color development, and the light absorbance of each sample is detected at a wavelength of 450nm and compared with the light absorbance of the standard, thereby obtaining the drug loading of the anti-PD1 antibody conjugated on the red blood cells.

[0143] The results are shown in Table 12. It can be seen that the sortase mutant obtained in the present application significantly increases the drug loading capacity of red blood cells compared with the parent, and is therefore more conducive to delivering the corresponding drug to the target in the body through red blood cells.

[0144] Table 12 Drug loading of erythrocyte-coupled anti-PD1 antibodies under different sortase enzyme catalysis

[0145] Example 5. Comparison of the catalytic time course of sortase mutants under different small peptide substrates and in vitro transpeptidation at different enzyme concentrations

[0146] This example studies the time course of in vitro catalytic transpeptidation reactions of mutants (SEQ ID NO: 3 and SEQ ID NO: 4) containing different NH2-G peptide acceptor motifs, as well as the efficiency of in vitro catalytic transpeptidation at different enzyme concentrations. The accumulation of the ligation product His6-EGFP-LPRT-G-peptide reflects the efficiency of catalytic transpeptidation of different mutants.

[0147] For the study of the catalytic time course, the system was configured according to Table 13, and the system was placed at 37°C for different reaction times (0-120 min). The results are shown in Figure 5-a. Compared with SEQ ID NO: 2, the mutants (SEQ ID NO: 3 and SEQ ID NO: 4) can quickly accumulate more ligation products within 15 min of the reaction. Among them, the mutant (SEQ ID NO: 3) reached the highest accumulation amount at 30 min under the conditions of two NH2-G small peptide substrates (GV peptide and GL peptide), and continued to maintain this level thereafter. The mutant (SEQ ID NO: 4) reached the highest accumulation at 30 min (GV peptide) and 45 min (GL peptide), respectively.

[0148] For the catalytic efficiency of different enzyme concentrations, the system was configured according to Table 14. After the configured reaction system was allowed to stand in a 37°C water bath for 2 h, 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 subjected to SDS-PAGE analysis. The results are shown in Figure 5-b. The accumulation of the ligation product was positively correlated with the increase in enzyme concentration. The mutants M155V / L200F (SEQ ID NO: 3) and M155V / K162P (SEQ ID NO: 4) had improved transpeptidation efficiency relative to SEQ ID NO: 2, and the accumulation of the product was higher than that of SEQ ID NO: 2 under low enzyme concentration conditions.

[0149] Table 13 Mutant time course reaction system (100 μL)

[0150] Table 14. Reaction system of different enzyme concentrations of mutants (100 μL)

Claims

1. A sortase mutant, whose amino acid sequence is at least 90% identical to the parent SEQ ID NO: 2 and comprises one or more mutations selected from M155V, L200F, K162P, wherein the amino acid positions are numbered according to SEQ ID NO:

1.

2. The sortase mutant of claim 1, which has at least 95% identity to the parent SEQ ID NO: 2 and comprises the mutations M155V / L200F or M155V / K162P, wherein the amino acid positions are numbered according to SEQ ID NO:

1.

3. The sortase mutant according to claim 1 or 2, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4. A polynucleotide molecule encoding the sortase mutant according to any one of claims 1 to 3. A vector comprising the polynucleotide molecule of claim 4.

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

7. A method for coupling an active molecule to erythrocytes, comprising contacting the sortase mutant according to any one of claims 1 to 3 with erythrocytes.

8. The method of claim 7, wherein the cell membrane surface of the erythrocyte comprises a sortase receptor motif, and the active molecule comprises a recognition motif of the sortase.

9. The method of claim 8, wherein the sortase recognition motif is selected from the amino acid sequence LPXTG, wherein X is a standard or non-standard amino acid (e.g., X is selected from D, E, A, N, Q, K or R), preferably, the motif recognized by the sortase A is LPETG.

10. The method of claim 9, wherein the sortase recognition motif is modified to have improved affinity with the sortase, preferably, the sortase recognition motif is LPETGG, LPETGGK, and optionally, the recognition motif further contains Abz / Lys(DNP) modification.

11. Use of the sortase mutant according to any one of claims 1 to 3 in directed coupling of molecules.

12. Use of the sortase mutant according to any one of claims 1 to 3 for directed coupling of active molecules to the surface of erythrocyte membrane.

13. A red blood cell carrying an active molecule, wherein the active molecule is an anti-PD1 antibody. 14 . A kit comprising the sortase mutant according to any one of claims 1 to 3 , or the polynucleotide molecule according to claim 4 , or the vector according to claim 5 .

15. A kit comprising the red blood cells of claim 13.

Images