CN118786207A - Modified cells and their uses - Google Patents

Abstract

The present disclosure provides a cell having a substance connected thereto, wherein the substance is connected to at least one membrane protein of the cell through a sortase recognition motif and a linker comprising an N-terminal glycine. Also provided are methods for obtaining modified cells, and uses of modified cells for delivering substances such as drugs and probes.

Description

Modified cells and their uses

Technical Field

The present disclosure generally relates to modified cells, and more particularly to membrane protein modified cells and their use in delivering substances, including drugs and probes, etc., especially for treating and preventing diseases associated with HPV infection.

Background Art

Human papillomavirus infection (HPV infection) is an infection caused by human papillomavirus (HPV), a DNA virus belonging to the family Papillomaviridae. Many HPV infections have no symptoms, and 90% of infections clear up on their own within two years. However, in some cases, HPV infection persists and causes warts or precancerous lesions. Depending on the site of infection, these lesions increase the risk of cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oral cancer, tonsil cancer, or laryngeal cancer.

The vast majority of cervical cancers are caused by HPV; two strains, HPV16 and HPV18, account for 70% of all cases. Nearly 90% of HPV-positive oropharyngeal cancers are associated with HPV16. HPV6 and HPV11 are common causes of genital warts and laryngeal papillomatosis. HPV16 causes 50% of cervical cancers and precancerous cervical lesions. Cervical cancer is the fourth most common cancer in women worldwide. However, for patients with recurrent cervical cancer, treatment options are limited. According to the Global Cancer Epidemiology Statistics Report released by the World Health Organization (WHO), there were approximately 600,000 new cases of cervical cancer and 340,000 deaths from cervical cancer worldwide in 2020, of which approximately 85% of new cases and 90% of deaths occurred in developing countries. In 2020, there were 110,000 new cases of cervical cancer in China, accounting for approximately 18.3% of the world's new cases of cervical cancer, and 60,000 deaths, accounting for approximately 17.6% of the world's deaths from cervical cancer.

Effective anti-tumor immunity in humans is associated with the presence of T cells targeting cancer neoantigens, a class of HLA-bound peptides generated by tumor-specific mutations. Emerging data suggest that the identification of such neoantigens has become a major aspect of immunotherapy in the clinic. Massively parallel whole-exome sequencing has been used to detect all mutations within tumors to predict neoantigens. Neoantigen vaccination can expand existing neoantigen-specific T cell populations and induce new tumor-specific T cells. Although neoantigens have become potential ideal targets for anti-tumor immune responses, many questions remain to be answered before clinical applications can be achieved. The activation of T cells requires MHC molecules to present MHC-restricted peptides to specific T cell receptors. The lack of specific antigen presentation systems is one of the problems with neoantigen vaccination.

In recent years, the development of drug delivery systems for extending drug retention time in the treatment of various human diseases has attracted widespread attention. However, many systems still face various challenges and limitations, such as poor stability, unwanted toxicity and immune response [J.W.Yoo,D.J.Irvine,D.E.Discher,and S.Mitragotri, "Bio-inspired, bioengineered and biomimetic drug delivery carriers," Nat.Rev.Drug Discov.,vol.10,no.7,pp.521-535,2011.]. Red blood cells (RBCs) are the most common cell type in the human body and have been widely studied as ideal in vivo drug delivery systems for more than 30 years due to their unique biological properties, including: (i) wide circulation throughout the body; (ii) good biocompatibility as a biomaterial and long survival time in vivo; (iii) large surface area to volume ratio; (iv) no nucleus, mitochondria and other organelles.

RBCs have been developed as drug delivery vehicles by direct encapsulation, non-covalent attachment of exogenous peptides, or by fusion with RBC surface protein-specific antibodies to install proteins. It has been shown that the in vivo application of such modified RBCs has limitations. For example, encapsulation can destroy the cell membrane, thereby affecting the in vivo survival rate of only engineered cells. In addition, the non-covalent attachment of polymer particles to RBCs is easily dissociated, and the payload will soon degrade in vivo.

Bacterial sortases are transpeptidases that can modify proteins in a covalent and site-specific manner [JM Antos, J. Ingram, T. Fang, N. Pishesha, M.C. Truttmann, and H.L. Ploegh, "Site-Specific Protein Labeling via Sortase-Mediated transpeptidation," 2017.]. Wild-type sortase A (wt SrtA) from Staphylococcus aureus recognizes the LPXTG motif and cleaves between threonine and glycine to form a covalent acylase intermediate between the enzyme and the substrate protein. This intermediate is dissociated by nucleophilic attack by peptides or proteins that typically have three consecutive glycine residues (3×glycine, G ₃ ) at the N-terminus. Previous studies have genetically overexpressed the KELL membrane protein on RBCs, which has an LPXTG motif at its C-terminus, which can be linked to the N-terminus of 3×glycine or G _(n≥3) modified proteins/peptides by using wt SrtA [J. Shi, L. Kundrat, N. Pishesha, A. Bilate, C. Theile, and T. Maruyama, "Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes," pp. 1-6, 2014.]. These drug-carrying RBCs have shown efficacy in treating diseases in animal models. However, this requires the steps of engineering hematopoietic stem cells or progenitor cells (HSPCs) and differentiating these cells into mature RBCs, which greatly limits the application.

Therefore, there remains a need in the art for an improved cell delivery system for treating diseases associated with HPV infection.

Summary of the invention

In a first aspect, the present disclosure provides a cell having a substance attached thereto, wherein the substance is attached to at least one membrane protein of the cell via a sortase recognition motif, and the substance attached to the at least one membrane protein comprises the following structure: AML-Gly _m X _n -MP, wherein A represents the substance, L represents the remainder of the sortase recognition motif after a sortase-mediated reaction, Gly _m represents m glycines, wherein m is preferably 1-5, X _n represents n spacer amino acids, wherein n is preferably 0-10, M represents the remainder of a bifunctional cross-linker after cross-linking, and P represents at least one membrane protein of the cell.

In some embodiments, the bifunctional crosslinker is of the amine-thiol type, preferably maleimidocarboxylic acid (C _2-8 ), for example, 6-maleimidocaproic acid and 4-maleimidobutyric acid.

In some embodiments, the bifunctional cross-linker forms a cross-link between the side chain amino group and at least one exposed sulfhydryl group of at least one membrane protein.

In some embodiments, the sortase recognition motif comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, wherein X is any amino acid.

In some embodiments, the sortase recognition motif comprises an unnatural amino acid at position 5 from the N-terminus to the C-terminus of the sortase recognition motif, wherein the unnatural amino acid is an optionally substituted hydroxycarboxylic acid having the formula _CH2OH- ( _CH2 ) _n -COOH, wherein n is an integer from 0 to 3, preferably n=0.

In some embodiments, the sortase recognition motif comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein * represents an optionally substituted hydroxycarboxylic acid; and X and Y independently represent any amino acid.

In some embodiments, the sortase recognition motif comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S, and LPXT*A, wherein M is preferably LPET*G and * is 2-hydroxyacetic acid.

In some embodiments, L is selected from the group consisting of LPXT, LPXA, LPXS, LPXL, LPXV, LGXT, LAXT, LSXT, NPXT, MPXT, IPXT, SPXT, VPXT, and YPXR, wherein X is any amino acid.

In some embodiments, substance A comprises an exposed sulfhydryl group, preferably an exposed cysteine, more preferably a terminal cysteine, most preferably a C-terminal cysteine.

In some embodiments, the substance comprises an HPV antigen peptide, an optional first peptide linker, and MHC-I from the N-terminus to the C-terminus. In some other embodiments, the substance comprises an HPV antigen peptide, an optional first peptide linker, β2-microglobulin (e.g., human β2-microglobulin), an optional second peptide linker, and an MHC-I heavy chain (preferably an MHC-I heavy chain lacking a transmembrane region and a cytoplasmic region) from the N-terminus to the C-terminus.

In some embodiments, the HPV antigen peptide binds to major histocompatibility complex class I (MHC-I) proteins, such as HLA-A*02:01, HLA-A*02:02, HLA-A*02:06, HLA-A*02:07, and HLA-A*02:11.

In some embodiments, the HPV antigen peptide is an HPV16 antigen peptide, preferably comprising or consisting of an amino acid sequence selected from SEQ ID NO: 9 (YMLDLQPET), SEQ ID NO: 10 (LLMGTLGIV) and SEQ ID NO: 11 (KCLKFYSKI), or functionally equivalent variants thereof.

In some embodiments, the sortase is sortase A (SrtA), e.g., Staphylococcus aureus transpeptidase A, e.g., Staphylococcus aureus transpeptidase A variant (mgSrtA).

In some embodiments, the cells are selected from enucleated cells, preferably erythrocytes.

In a second aspect, the present disclosure provides a method of modifying a cell, comprising:

a) providing a sortase substrate comprising an A-M-L' structure, wherein A represents a substance, L' represents a sortase recognition motif, and M represents a residual portion of a bifunctional cross-linker after cross-linking;

b) (i) providing a peptide having the formula Gly _m X _n -M', wherein Gly _m represents m glycine, m is preferably 1-5; X _n represents n spacer amino acids, n is preferably 0-10; M' represents a bifunctional cross-linking agent;

(ii) treating the cell with a _GlymXn - _M ' peptide, wherein M represents the remaining part of the bifunctional cross-linking agent after cross-linking and P represents at least one membrane protein of the cell, under conditions such that the _{GlymXn -} M' peptide is linked to at least one membrane protein of the cell to generate _{GlymXn -} MP; and

c) contacting the treated cells with a sortase substrate in the presence of a sortase under one or more conditions suitable for the sortase to conjugate the sortase substrate to Gly _m via a sortase-mediated reaction;

a) and b) can be performed simultaneously, or a) can be performed before b), or a) can be performed after b).

In some embodiments, prior to the treating step, the method further comprises the step of pre-treating the cells with a reducing agent to form exposed sulfhydryl groups.

In some embodiments, the bifunctional crosslinker is of the amine-thiol type, preferably maleimidocarboxylic acid (C _2-8 ), for example, 6-maleimidocaproic acid and 4-maleimidobutyric acid.

In some embodiments, _Xn comprises at least one amino acid having a side chain amino group, such as lysine, and preferably, the C-terminal amino acid of _Xn is an amino acid having a side chain amino group.

In some embodiments, the bifunctional cross-linker cross-links the side chain amino group and at least one exposed thiol group of at least one membrane protein.

In some embodiments, the sortase-mediated reaction forms a substance attached to at least one membrane protein of a cell, the substance comprising the following structure: AML-Gly _m X _n -MP, wherein A represents the substance, L represents the remaining portion of the sortase recognition motif after the sortase-mediated reaction, Gly _m represents m glycines, m is preferably 1-5, X _n represents n spacer amino acids, n is preferably 0-10, M represents the remaining portion of the bifunctional cross-linker after cross-linking, and P represents at least one membrane protein of the cell.

In some embodiments, the sortase recognition motif comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, wherein X is any amino acid.

In some embodiments, the sortase recognition motif comprises an unnatural amino acid at position 5 from the N-terminus to the C-terminus of the sortase recognition motif, wherein the unnatural amino acid is an optionally substituted hydroxycarboxylic acid having the formula _CH2OH- ( _CH2 ) _n -COOH, wherein n is an integer from 0 to 3, preferably n=0.

In some embodiments, the sortase recognition motif comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein * represents an optionally substituted hydroxycarboxylic acid; and X and Y independently represent any amino acid.

In some embodiments, the sortase recognition motif comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S, and LPXT*A, wherein M is preferably LPET*G and * is 2-hydroxyacetic acid.

In some embodiments, substance A comprises an exposed sulfhydryl group, preferably an exposed cysteine, more preferably a terminal cysteine, most preferably a C-terminal cysteine.

In some embodiments, the substance comprises an HPV antigen peptide, an optional first peptide linker, and MHC-I from the N-terminus to the C-terminus. In some other embodiments, the substance comprises an HPV antigen peptide, an optional first peptide linker, β2-microglobulin (e.g., human β2-microglobulin), an optional second peptide linker, and an MHC-I heavy chain (preferably an MHC-I heavy chain lacking a transmembrane region and a cytoplasmic region) from the N-terminus to the C-terminus.

In some embodiments, the HPV antigen peptide binds to major histocompatibility complex class I (MHC-I) proteins, such as HLA-A*02:01, HLA-A*02:02, HLA-A*02:06, HLA-A*02:07, and HLA-A*02:11.

In some embodiments, the HPV antigen peptide is an HPV16 antigen peptide, preferably comprising or consisting of an amino acid sequence selected from SEQ ID NO: 9 (YMLDLQPET), SEQ ID NO: 10 (LLMGTLGIV) and SEQ ID NO: 11 (KCLKFYSKI), or functionally equivalent variants thereof.

In some embodiments, the sortase is sortase A (SrtA), e.g., Staphylococcus aureus transpeptidase A, e.g., Staphylococcus aureus transpeptidase A variant (mgSrtA).

In some embodiments, the cells are selected from enucleated cells, preferably erythrocytes.

In a third aspect, the present disclosure provides a cell obtained by the method of the second aspect.

In a fourth aspect, the present disclosure provides a composition comprising the cell of the first and/or third aspect and optionally a physiologically acceptable carrier.

In a fifth aspect, the present disclosure provides methods for diagnosing, treating or preventing diseases associated with HPV infection.

In some embodiments, the disease associated with HPV infection is selected from cervical cancer, anogenital cancer, head and neck cancer, and oropharyngeal cancer.

In a sixth aspect, the present disclosure provides a method of delivering a substance to a subject in need thereof, comprising administering the cell of the first and/or third aspect or the composition of the fourth aspect to the subject.

In a seventh aspect, the present disclosure provides a method of increasing the circulation time or plasma half-life of a substance in a subject, comprising linking the substance to a cell according to the method of the second aspect.

In an eighth aspect, the present invention provides use of the cell of the first and/or third aspect or the composition of the fourth aspect in the preparation of a medicament for diagnosing, treating or preventing a disease associated with HPV infection.

In some embodiments, the disease associated with HPV infection is selected from cervical cancer, anogenital cancer, head and neck cancer, and oropharyngeal cancer.

In some embodiments, the medicament is a vaccine.

In a ninth aspect, there is provided the cell of the first and/or third aspect or the composition of the fourth aspect for use in diagnosing, treating or preventing a disease associated with HPV infection in a subject in need thereof.

In some embodiments, the disease associated with HPV infection is selected from cervical cancer, anogenital cancer, head and neck cancer, and oropharyngeal cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, embodiments of the present disclosure are shown by way of example. It should be clearly understood that the description and the accompanying drawings are only used to illustrate and help understand the present disclosure, and are not intended to be a definition of the limitations of the present invention.

FIG. 1 illustrates an exemplary method for labeling cells with peptides in vitro according to the present disclosure.

FIG. 2 shows the structural formula of the glycine GGGSK-6-MAL linker according to the present disclosure.

FIG. 3 shows that HPV16(YMLDLQPET)-hMHC1 is efficiently conjugated to RBCs in vitro according to an exemplary embodiment of the present disclosure.

FIG. 4 shows the IFN-γ response induced by HPV16(YMLDLQPET)-hMHC1-RBC in patient PBMCs detected by IFN-γ ELISpot method.

FIG5 shows the cytotoxic potential of the immune response generated by HPV16(YMLDLQPET)-hMHC1-RBCs detected by the expression of fluorescein-conjugated MHC tetramers (A) 4-1BB and CD107a (B) on the surface of CD8+ T cells.

FIG6 shows the survival of Caski cells after T cell cytotoxicity assay.

FIG. 7 shows the survival of HPV16-MC38 cells after T cell cytotoxicity assay (A: different time points; B: spleen cells from different mice).

FIG8 shows tumor growth in animals receiving repeated infusions of HPV16(KCLKFYSKI)-mMHC1-RBCs.

FIG. 9 shows the structural formula of 6-maleimidocaproic acid-Leu-Pro-Glu-Thr-2-hydroxyacetic acid-Gly.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe these embodiments. It will be understood, however, that no limitation of the scope of the present disclosure is intended.

In the present disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, preferred methods and materials are described herein. Therefore, the terms defined herein are more fully described by reference to the entire specification.

As used herein, the singular terms a, an, and the include plural references unless the context clearly dictates otherwise.

Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation.

It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending upon the specific context in which it is used by one skilled in the art.

As used herein, in the context of an amino acid sequence, the term "consisting essentially of" refers to the recited amino acid sequence plus an additional 1, 2, 3, 4 or 5 amino acids at the N- or C-terminus thereof.

Unless the context requires otherwise, the terms "includes," "comprising," and "including" or similar terms are intended to mean a non-exclusive inclusion such that the listed elements or features include not only those stated or listed elements but may include other elements or features not listed or stated.

As used herein, the terms "patient," "individual," and "subject" are used in the context of any mammalian recipient of the treatments or compositions disclosed herein. Thus, the methods and compositions disclosed herein may have medical and/or veterinary applications. In a preferred form, the mammal is a human.

As used herein, the term "sequence identity" is intended to include the number of accurate nucleotide or amino acid matches, considering the appropriate comparison using standard algorithms, considering the degree of sequence identical on the comparison window. Therefore, by the following calculation "sequence identity percentage ratio": compare two optimal alignment sequences on the comparison window, determine the number of positions where identical nucleic acid bases (for example, A, T, C, G) occur in the two sequences, to produce the number of matching positions, divide the total number of positions in the comparison window (that is, window size) by the number of matching positions, then multiply the result by 100, and obtain the percentage ratio of sequence identity. For example, "sequence identity" can be understood as representing the "matching percentage ratio" calculated by DNASIS computer program (Windows version 2.5; Available from Hitachi Software Engineering Co., Ltd. in South San Francisco, California, USA).

As used herein, the term "conjugated" or "conjugation" refers to the association of two molecules (eg, two proteins or a protein and a small molecule or other entity) with each other, which are linked by means of direct or indirect covalent or non-covalent interactions.

The inventors have developed a new strategy to modify cells, such as blood cells, especially natural RBCs, with substances (e.g., peptides and/or small molecules) mediated by sortase reactions. This technology can produce cell products by directly modifying natural cells (e.g., RBCs) rather than HSPCs that are limited by resources, and the labeling efficiency is very high. In addition, the modified cells retain their original biological properties well and remain as stable as the natural state. In some embodiments, the labeled red blood cells have the same lifespan as natural RBCs and continue to signal in the circulation for up to 28 days or longer.

cell

In one aspect, the disclosure provides a cell having a substance attached thereto, wherein the substance is attached to at least one membrane protein of the cell via a sortase recognition motif. In order to improve the labeling efficiency of the sortase-mediated reaction, a linker comprising an N-terminal glycine is conjugated to at least one membrane protein of the cell via a bifunctional crosslinker (e.g., an amine-thiol type bifunctional crosslinker), preferably to at least one exposed thiol group of at least one membrane protein.

The present disclosure encompasses various living animal cells, such as mammalian cells, such as various blood cells, including erythrocytes, T cells, B cells, monocytes, NK cells and megakaryocytes. In some embodiments, the animal cell is a mammalian cell, such as a human cell. In some embodiments, the cell is an immune system cell, such as a lymphocyte (such as a T cell or a NK cell) or a dendritic cell. In some embodiments, the cell is a cytotoxic cell. In some embodiments, the cell is a non-immortalized cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a natural cell. In some embodiments, the cell is not genetically engineered to express a polypeptide comprising a sortase recognition sequence, a sequence comprising one or more glycine or alanine at its end, or both.

In some embodiments, the cell is a mature red blood cell (RBC). In certain embodiments, the RBC is a human RBC, such as a human natural RBC. In some embodiments, the RBC is a red blood cell that has not been genetically engineered to express a protein that contains a sortase recognition motif or a nucleophilic receptor sequence. In some embodiments, the RBC is not genetically engineered.

As used herein, the terms "non-engineered", "non-genetically modified" and "non-recombinant" are interchangeable and refer to not being genetically engineered, the absence of genetic modification, etc. Non-engineered membrane proteins encompass endogenous proteins. In certain embodiments, non-genetically engineered erythrocytes do not contain non-endogenous nucleic acids, such as DNA or RNA derived from a vector, from a different species, or containing artificial sequences, such as DNA or RNA introduced artificially. In certain embodiments, non-engineered cells are not intentionally contacted with nucleic acids that can cause heritable genetic changes under conditions suitable for cellular uptake of nucleic acids.

In some embodiments, the cell is not genetically engineered for sorting. A cell is considered "not genetically engineered for sorting" if it is not genetically engineered to express a protein comprising a sortase recognition motif or a nucleophilic receptor sequence in a sortase catalyzed reaction.

In some aspects, the present disclosure provides a cell having a substance attached thereto, wherein the substance is attached to at least one membrane protein of the cell via a sortase recognition motif, and the substance attached to at least one membrane protein comprises the following structure: AML-Gly _m X _n -MP, wherein A represents the substance, L represents the remaining portion of the sortase recognition motif after a sortase-mediated reaction, Gly _m represents m glycines, m is preferably 1-5, X _n represents n spacer amino acids, n is preferably 0-10, M represents the remaining portion of a bifunctional cross-linker after cross-linking, and P represents at least one membrane protein of the cell.

In some embodiments, as described herein, the disclosure provides cells with substances connected thereto. In some embodiments, compositions comprising a plurality of such cells are provided. In some embodiments, at least a selected percentage of cells in the composition is modified, for example, with substances connected to at least one membrane protein of the cell. For example, in some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the cells have substances connected thereto. In some embodiments, the substances connected may be one or more substances described herein.

In some embodiments, the present disclosure provides a cell comprising a substance, wherein the substance is connected to a membrane protein of the cell by using a sortase recognition motif and a linker comprising a terminal glycine, such as an N-terminal glycine (e.g., a linker having a structure of Gly _m X _n , wherein Gly _m represents m glycines, wherein m is preferably 1-5, and X _n represents n spacer amino acids, wherein n is preferably 0-10). In some embodiments, the connected substances can be the same, or the cell can be sorted with a plurality of different substances.

In some embodiments, the substance is linked to a linker comprising a terminal glycine, such as an N-terminal _glycine , via a sortase recognition motif (e.g., a linker having a structure of _GlymXn , wherein _Glym represents m glycines, wherein m is preferably 1-5, and _Xn represents n spacer amino acids, wherein n is preferably 0-10), which is cross-linked to at least one membrane protein of the cell. In some embodiments, the sortase recognition motif can be selected from the group consisting of: LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, wherein X is any amino acid. In some embodiments, the sortase recognition motif may comprise an unnatural amino acid at position 5 from the N-terminal to C-terminal direction of the sortase recognition motif, wherein the unnatural amino acid is an optionally substituted hydroxycarboxylic acid having the formula CH ₂ OH-(CH ₂ ) _n -COOH, wherein n is an integer from 0 to 3, preferably n = 0. In some embodiments, the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein * represents an optionally substituted hydroxycarboxylic acid, and X and Y independently represent any amino acid. In some embodiments, the sortase recognition motif comprising an unnatural amino acid can be selected from the group consisting of: LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S and LPXT*A, wherein M is preferably LPET*G and wherein * is preferably 2-hydroxyacetic acid.

It is understood that after the substance is linked to the membrane protein, the last one or two residues from the 5th position (in the direction from the N-terminus to the C-terminus) of the sortase recognition motif are replaced by the amino acid to which the linkage occurs, as described elsewhere herein. For example, the substance linked to the membrane protein can comprise the structure of AML- _{GlymXn -} MP, wherein L represents the remaining part of the sortase recognition motif after the sortase-mediated reaction, and can be selected from the group consisting of: LPXT, LPXA, LPXS, LPXL, LPXV, LGXT, LAXT, LSXT, NPXT, MPXT, IPXT, SPXT, VPXT and YPXR.

In some embodiments, genetically engineered cells are modified by attaching or conjugating or connecting a sortase substrate to a membrane protein of the cell using a sortase. For example, cells (e.g., RBCs) have been genetically engineered to express any of a variety of products, such as polypeptides or non-coding RNAs, can be genetically engineered to have a deletion of at least a portion of one or more genes, and/or can be genetically engineered to have one or more precise changes in the sequence of one or more endogenous genes. In certain embodiments, such genetically engineered cells are sorted with any of the various substances described herein according to the methods described herein.

In some embodiments, the present disclosure contemplates the use of autologous cells, such as red blood cells, which are isolated from an individual and, after in vitro modification, these isolated cells are administered to the individual. In some embodiments, the present disclosure contemplates the use of red blood cells that have the same immunological compatibility of the blood type as the individual to whom such cells are to be administered (e.g., at least in terms of the ABO blood group system, and in some embodiments, in terms of the D blood group system), or the use of red blood cells that can be of a compatible blood type.

Linker containing terminal glycine

In some embodiments, a linker comprising a terminal glycine, such as an N-terminal _glycine (e.g., a linker having a structure of _GlymXn , wherein _Glym represents m glycines, m is preferably 1-5, and _Xn represents n spacer amino acids, wherein n is preferably 0-10) is attached to at least one membrane protein of a cell via a bifunctional cross-linker.

As used herein, Gly _m refers to m glycines, wherein m is preferably 1-5, such as one, two, three, four or five glycines. As used herein, X _n represents n optional spacer amino acids, which can be any amino acid. In some embodiments, n can be 0-10 or greater, such as 0-5, 1-4 or 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the spacer amino acid can be any natural or non-natural amino acid, such as Gly, Ala, Ser, Lys, Asn, Thr, Glu or Gln. In some embodiments, X _n can be AAS or AASK. In some embodiments, X _n can be GGS or GGSK. In some embodiments, X _n can be LGS or LGSK.

As used herein, the term "bifunctional crosslinker" refers to a reagent designed to link two reactive groups. If a bifunctional crosslinker is designed so that the two reactive groups are the same, it is called a homobifunctional crosslinker; if its two reactive groups are different, it is a heterobifunctional crosslinker. A crosslinker is photoactivatable, photoreactive, photosensitive, or photoactivated if one or both reactive groups of the crosslinker become so only as a result of a photochemical reaction caused by exposing the crosslinker to light of an appropriate wavelength. The present disclosure contemplates the use of various bifunctional crosslinkers capable of crosslinking a linker comprising a terminal glycine to at least one membrane protein of a cell.

In some embodiments, the bifunctional cross-linking agent may include, but is not limited to: (1) zero-length type (e.g., EDC; EDC plus sulfo-NHS; CMC; DCC; DIC; N,N'-carbonyldiimidazole;Woodward's reagent K); (2) amine-thiol type, such as NHS ester-maleimide heterobifunctional cross-linking agent; maleimidocarboxylic acid (C _2-8 ) (e.g., 6-maleimidocaproic acid and 4-maleimidobutyric acid); EMCS; SPDP, LC-SPDP, sulfo-LC-SPDP; SMPT and sulfo-LC-SMPT; SMCC, LC-SMCC and sulfo-SMCC; MBS and sulfo-MBS; SIAB and sulfo-SIAB; SMPB and sulfoSMPB; GMBS and sulfoGMBS; SIAX and SIAXX; SIAC and SIACX; NPIA; (3) homobifunctional NHS ester type (e.g. , DSP; DTSSP; DSS; DST and sulfo-DST; BSOCOES and sulfo-BSOCOES; EGS and sulfo-EGS); (4) homobifunctional imide ester type (e.g., DMA; DMP; DMS; DTBP); (5) carbonyl-thiol type (e.g., KMUH; EMCH; MPBH; M2C2H; PDPH); (6) thiol-reactive type (e.g., DPDPB; BMH; HBVS); (7) thiol-hydroxyl type (e.g., PMPI); or the like.

In some embodiments, the bifunctional cross-linker is an amine-thiol type, preferably a maleimide carboxylic acid (C _2-8 ), for example, 6-maleimidocaproic acid and 4-maleimidobutyric acid. In some other embodiments, the linker comprising a terminal glycine comprises at least one amino acid having a side chain amino group, such as lysine, and preferably, the C-terminal amino acid of _Xn is an amino acid having a side chain amino group, which enables a cross-linking reaction to occur between the side chain amino group and at least one exposed thiol group of at least one membrane protein.

Sortase

Enzymes called "sortases" have been isolated from a variety of Gram-positive bacteria. Sorting enzymes, sortase-mediated transacylation reactions, and their use in protein engineering are well known to those of ordinary skill in the art (see, e.g., PCT/US2010/000274 (WO/2010/087994) and PCT/US2011/033303 (WO/2011/133704)). According to sequence alignment and phylogenetic analysis of 61 sortases in the genome of Gram-positive bacteria, sortases are divided into four categories, named A, B, C, and D (Dramsi S, Trieu-Cuot P, Bierne H, Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res Microbiol. 156 (3): 289-97, 2005). Those skilled in the art can readily assign sortases to the correct class based on their sequence and/or other characteristics, such as those described in Drami et al., supra. As used herein, the term "sortase A" refers to class A sortases, generally designated SrtA in any particular bacterial species, such as SrtA from Staphylococcus aureus (S. aureus) or Streptococcus pyogenes (S. pyogenes).

The term "sortase", also known as transamidase, refers to an enzyme with transamidase activity. The sortase recognizes a substrate comprising a sortase recognition motif (e.g., the amino acid sequence LPXTG). Molecules recognized by the sortase (i.e., comprising the sortase recognition motif) are sometimes referred to herein as "sortase substrates". The sortase can tolerate a variety of changes in the portion near the cleavage site, so as long as the substrate contains a properly exposed sortase recognition motif and has a suitable nucleophilic reagent, multifunctional conjugation of different entities can be achieved. The terms "sortase-mediated transacylation reaction", "sortase-catalyzed transacylation reaction", "sortase-mediated reaction", "sortase-catalyzed reaction", "sortase reaction", "sortase-mediated transpeptidation reaction" and similar terms are used interchangeably herein to refer to such reactions. In terms of sequences recognized by transamidase or sortase, the terms "sortase recognition motif", "sortase recognition sequence" and "transamidase recognition sequence" are used interchangeably herein. The term "nucleophilic acceptor sequence" refers to an amino acid sequence capable of acting as a nucleophile in a reaction catalyzed by a sortase, for example, a sequence comprising an N-terminal glycine (eg, 1, 2, 3, 4 or 5 N-terminal glycines).

The present disclosure encompasses embodiments involving any sortase class known in the art (e.g., sortase A, B, C, or D from any bacterial species or strain). In some embodiments, a sortase A is used, such as SrtA from Staphylococcus aureus. In some embodiments, two or more sortases can be used. In some embodiments, the sortases can utilize different sortase recognition sequences and/or different nucleophilic receptor sequences.

In some embodiments, the sortase is sortase A (SrtA). SrtA recognizes the motif LPXTG, where common recognition motifs are, for example, LPKTG, LPATG, LPNTG. In some embodiments, LPETG is used. However, motifs falling outside the consensus sequence can also be recognized. For example, in some embodiments, the motif comprises A, S, L, or V instead of T at position 4, for example, LPXAG, LPXSG, LPXLG, or LPXVG, for example, LPNAG or LPESG, LPELG, or LPEVG. In some embodiments, the motif comprises A instead of G at position 5, for example, LPXTA, for example, LPNTA. In some embodiments, the motif comprises G or A instead of P at position 2, for example, LGXTG or LAXTG, for example, LGATG or LAETG. In some embodiments, the motif comprises I or M instead of L at position 1, for example, MPXTG or IPXTG, for example, MPKTG, IPKTG, IPNTG, or IPETG. Pishesha et al. 2018 described multiple recognition motifs for sortase A.

In some embodiments, the sortase recognition sequence is LPXTG, wherein X is a standard or non-standard amino acid. In some embodiments, X is selected from D, E, A, N, Q, K, or R. In some embodiments, the recognition sequence is selected from LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, wherein X can be any amino acid, such as those selected from D, E, A, N, Q, K, or R in certain embodiments.

In some embodiments, the sortase can recognize a motif comprising a non-natural amino acid, preferably located at position 5 in the N-terminal to C-terminal direction of the sortase recognition motif. In some embodiments, the non-natural amino acid is a substituted or unsubstituted hydroxycarboxylic acid having the formula CH ₂ OH-(CH ₂ ) _n -COOH, wherein n is an integer from 0 to 5, for example, 0, 1, 2, 3, 4 and 5, and preferably n=0. In some embodiments, the non-natural amino acid is a substituted hydroxycarboxylic acid, and in some other embodiments, the hydroxycarboxylic acid is substituted with one or more substituents selected from halogen, C _1-6 alkyl, C _1-6 haloalkyl, hydroxy, C _1-6 alkoxy and C _1-6 haloalkoxy. The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine, preferably fluorine and chlorine. The term "alkyl" by itself or as part of another substituent refers to a hydrocarbon group of the formula C _n H _2n+1 , wherein n is a number greater than or equal to 1. In some embodiments, the alkyl group that can be used for the present disclosure comprises 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 3 carbon atoms, and even more preferably 1 to 2 carbon atoms. The alkyl group can be straight or branched, and can be further substituted as shown herein. _Cxy alkyl refers to an alkyl group comprising x to y carbon atoms. Suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl and isomers thereof (e.g., n-pentyl, isopentyl) and hexyl and isomers thereof (e.g., n-hexyl, isohexyl). Preferred alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. The term "haloalkyl" refers to an alkyl group having the meaning as defined above, alone or in combination, in which one or more hydrogens are replaced by halogens as defined above. Non-limiting examples of such haloalkyl groups include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1,1,1-trifluoroethyl, etc.

In some embodiments, the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein * represents an optionally substituted hydroxycarboxylic acid, and X and Y independently represent any amino acid. In some embodiments, the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S, and LPXT*A, wherein M is preferably LPET*G, wherein * is preferably 2-hydroxyacetic acid.

In some embodiments, the disclosure contemplates the use of naturally occurring variants of sortase. In some embodiments, the variant is capable of mediating glycine _(n) conjugation, wherein n is preferably 1 or 2. Such variants can be generated by methods such as directed evolution, site-specific modification, etc. A large amount of structural information about sortase (e.g., sortase A enzyme) can be obtained, including NMR or crystal structures of SrtA alone or SrtA bound to a sortase recognition sequence (see, e.g., Zong Y et al., J. Biol Chem. 2004, 279, 31383-31389). The active site and substrate binding pocket of Staphylococcus aureus SrtA have been determined. A person of ordinary skill in the art can generate functional variants by, for example, avoiding deletions or substitutions that would destroy or substantially change the active site or substrate binding pocket of the sortase. In some embodiments, directed evolution on SrtA can be performed by utilizing the FRET (fluorescence resonance energy transfer) selection assay described in Chen et al. Sci. Rep. 2016, 6 (1), 31899. In some embodiments, the functional variants of S. aureus SrtA may be those described in CN106191015A and CN109797194A. In some embodiments, the S. aureus SrtA variant may be a truncated variant, wherein, for example, 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59 or 60) amino acids are removed from the N-terminus (compared to wild-type S. aureus SrtA).

In some embodiments, a functional variant of S. aureus SrtA useful in the present disclosure may be a S. aureus SrtA variant comprising one or more mutations at the following amino acid positions: D124, Y187, E189, and F200, such as D124G, Y187L, E189R, and F200L, and optionally further comprising one or more mutations in P94S/R, D160N, D165A, K190E, and K196T. In certain embodiments, the S. aureus SrtA 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. In some embodiments, the S. aureus SrtA variant removes 25 to 60 (e.g., 25, 30, 35, 40, 45, 50, 55, 59 or 60) amino acids from the N-terminus. In some embodiments, the amino acid positions of the mutations are numbered according to the numbering of wild-type S. aureus SrtA (e.g., as shown in SEQ ID NO: 1). In some embodiments, the full-length nucleotide sequence of wild-type S. aureus SrtA is, for example, as shown in SEQ ID NO: 2.

SEQ ID NO: 1 (full length, GenBank accession number: CAA3829591.1)

1MKKWTNRLMT IAGVVLILVA AYLFSKPHID NYLHDKDKDE KIEQYDKNVK

51EQASKDKKQQ AKPQIPKDKS KVAGYIEIPD ADIKEPVYPG PATPEQLNRG

101VSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNET

151RKYKMTSIRD VKPTDVGVLD EQKGKDKQLT LITCDDYNEK TGVWEKRKIF

201VATEVK

SEQ ID NO: 2 (full length, wild type)

ATGAAAAAATGGACAAATCGATTAATGACAATCGCTGGTGTGGTACTTATCCTAG

TGGCAGCATATTTGTTTGCTAAACCACATATCGATAATTATCTTCACGATAAAGA

TAAAGATGAAAAGATTGAACAATATGATAAAAATGTAAAAGAACAGGCGAGTA

AAGATAAAAAGCAGCAAGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGG

CAGGCTATATTGAAATTCCAGATGCTGATATTAAAGAACCAGTATATCCAGGACC

AGCAACACCTGAACAATTAAATAGAGGTGTAAGCTTTGCAGAAGAAAATGAATC

ACTAGATGATCAAAATATTTCAATTGCAGGACACACTTTCATTGACCGTCCGAAC

TATCAATTTACAAATCTTAAAGCAGCCAAAAAAGGTAGTATTGGTGTACTTTAAAG

TTGGTAATGAAACACGTAAGTATAAAATGACAAGTATAAGAGATGTTAAGCCTA

CAGATGTAGGAGTTCTAGATGAACAAAAAGGTAAAGATAAACAATTAACATTAA

TTACTTGTGATGATTACAATGAAAAGACAGGCGTTTGGGAAAAACGTAAAATCTT

TGTAGCTACAGAAGTCAAA

In some embodiments, compared to wild-type S. aureus SrtA, the S. aureus SrtA variant can comprise one or more mutations at one or more of positions corresponding to positions 94, 105, 108, 124, 160, 165, 187, 189, 190, 196, and 200 of SEQ ID NO: 1. In some embodiments, compared to wild-type S. aureus SrtA, the S. aureus SrtA variant can comprise one or more mutations corresponding to P94S/R, E105K, E108A, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L. In some embodiments, compared to wild-type S. aureus SrtA, the S. aureus SrtA variant may comprise one or more mutations corresponding to D124G, Y187L, E189R, and F200L, and optionally further comprises one or more mutations corresponding to P94S/R, D160N, D165A, K190E, and K196T, and optionally further comprises one or more mutations corresponding to E105K and E108A. In certain embodiments, compared to wild-type S. aureus SrtA, the S. aureus SrtA variant may comprise one or more mutations corresponding to D124G; D124G and F200L; P94S/R, D124G, D160N, D165A, K190E, and K196T; P94S/R, D160N, D165A, Y187L, E189R, K190E 87L, E189R, K190E, K196T, and F200L. In some embodiments, the SrtA variant of Staphylococcus aureus may comprise one or more mutations of P94S/R, E105K, E108A, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L relative to SEQ ID NO: 1. In some embodiments, the S. aureus SrtA variant may comprise D124G, Y187L, E189R, and F200L, and optionally further comprises one or more mutations in P94S/R, D160N, D165A, K190E, and K196T, and optionally further comprises E105K and/or E108A relative to SEQ ID NO: 1. In certain embodiments, the S. aureus SrtA variant may comprise D124G, Y187L, E189R, and F200L, and optionally further comprises one or more mutations in P94S/R, D160N, D165A, K190E, and K196T, and optionally further comprises E105K and/or E108A relative to SEQ ID NO: 1. NO: 1, may contain: 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. In some embodiments, mutations E105K and/or E108A/Q allow the sortase-mediated reaction to be Ca2 ⁺ -independent. In some embodiments, the SrtA variants described herein can remove 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids from the N-terminus. In some embodiments, the amino acid positions of the mutations described above are numbered according to the numbering of the full length of wild-type SrtA (e.g., as shown in SEQ ID NO: 1).

In some embodiments, a functional variant of S. aureus SrtA useful in the present disclosure may be a S. aureus SrtA variant comprising one or more mutations of P94S/R, E105K, E108A/Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L. In certain embodiments, the S. aureus SrtA variant may comprise P94S/R, E105K, E108Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L; or P94S/R, E105K, E108A, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L. In some embodiments, the S. aureus SrtA variant may comprise one or more mutations relative to P94S/R, E105K, E108A/Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L of SEQ ID NO: 1. In certain embodiments, the S. aureus SrtA variant can comprise P94S/R, E105K, E108Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L relative to SEQ ID NO: 1; or P94S/R, E105K, E108A, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L relative to SEQ ID NO: 1. In some embodiments, the S. aureus SrtA variant has 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids removed from the N-terminus. In some embodiments, the amino acid positions of the mutations are numbered according to the numbering of wild-type S. aureus SrtA (eg, as shown in SEQ ID NO: 1).

In some embodiments, the present disclosure encompasses a S. aureus SrtA variant (mg SrtA) comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) identity to the amino acid sequence as shown in SEQ ID No: 3. In some embodiments, SEQ ID NO: 3 is a truncated SrtA, and mutations corresponding to wild-type SrtA are shown below in bold and underlined. In some embodiments, the SrtA variant comprises, consists essentially of, or consists of an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) identity to the amino acid sequence as shown in SEQ ID No:3, and the SrtA variant comprises mutations of P94R/S, D124G, D160N, D165A, Y187L, E189R, K190E, K196T and F200L, and optionally comprises E105K and/or E108A/Q (numbering according to the numbering of SEQ ID NO:1).

SEQ ID NO: 3 (mutations shown in bold and underlined)

In some embodiments, the disclosure provides a nucleic acid encoding a S. aureus SrtA variant, which in some embodiments is shown in SEQ ID NO:4.

SEQ ID NO:4

AAACCACATATCGATAATTATCTTCACGATAAAGATAAAGATGAAAAGATTGAA

CAATATGATAAAAATGTAAAAGAACAGGCGAGTAAAGATAAAAAGCAGCAAGC

TAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGCTATATTGAAATTCC

AGATGCTGATATTAAAGAACCAGTATATCCAGGACCAGCAACACGTGAACAATT

AAATAGAGGTGTAAGCTTTGCAGAAGAAAATGAATCACTAGATGATCAAAATAT

TTCAATTGCAGGACACACTTTTCATTGGCCGTCCGAACTATCAATTTACAAATCTT

AAAGCAGCCAAAAAAGGTAGTATGGGTACTTTAAAGTTGGTAATGAAACACGT

AAGTATAAAATGACAAGTATAAGAAATGTTAAGCCTACAGCTGTAGGAGTTCTA

GATGAACAAAAAGGTAAAGATAAACAATTAACATTAATTACTTGTGATGATCTT

AATCGGGAGACAGGCGTTTGGGAAACACGTAAAATCTTGGTAGCTACAGAAGTC

AAA

In some embodiments, the S. aureus SrtA variant can be a truncated variant, wherein, for example, 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59 or 60) amino acids are removed from the N-terminus (compared to wild-type S. aureus SrtA). In some embodiments, the truncated variant comprises, or consists essentially of, or consists of, an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more, such as 100%) identity to the amino acid sequence as shown in SEQ ID No: 5 or 7. Nucleic acids encoding SEQ ID NOs: 5 and 7 are shown in SEQ ID NOs: 6 and 8 below.

SEQ ID NO: 5 (mutations compared to wt SrtA are shown in bold and underlined)

SEQ ID NO:6

CAAGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGCTATATTGAA

ATTCCAGATGCTGATATTAAAGAACCAGTATATCCAGGACCAGCAACACGTGAA

CAATTAAATAGAGGTGTAAGCTTTGCAGAAGAAAATGAATCACTAGATGATCAA

AATATTTCAATTGCAGGACACACTTTCATTGGCCGTCCGAACTATCAATTTACAA

ATCTTAAAGCAGCCAAAAAAGGTAGTATGTGTGTACTTTAAAGTTGGTAATGAAA

CACGTAAGTATAAAATGACAAGTATAAGAAATGTTAAGCCTACAGCTGTAGGAG

TTCTAGATGAACAAAAAGGTAAAGATAAACAATTAACATTAATTACTTGTGATG

ATCTTAATCGGGAGACAGGCGTTTGGGAAACACGTAAAATCTTGGTAGCTACAG

AAGTCAAA

SEQ ID NO:7 (mutations compared to wt SrtA are shown in bold and underlined)

SEQ ID NO:8

ATGCAAGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGCTATATT

GAAATTCCAGATGCTGATATTAAAGAACCAGTATATCCAGGACCAGCAACACGT

GAACAATTAAATAGAGGTGTAAGCTTTGCAGAAGAAAATGAATCACTAGATGAT

CAAAATATTTCAATTGCAGGACACACTTTCATTGGCCGTCCGAACTATCAATTTA

CAAATCTTAAAGCAGCCAAAAAAGGTAGTATGTGTGTACTTTAAAGTTGGTAATG

AAACACGTAAGTATAAAATGACAAGTATAAGAAATGTTAAGCCTACAGCTGTAG

GAGTTCTAGATGAACAAAAAGGTAAAGATAAACAATTAACATTAATTACTTGTG

ATGATCTTAATCGGGAGACAGGCGTTTGGGAAACACGTAAAATCTTGGTAGCTA

CAGAAGTCAAA

In some embodiments, the sortase A variant may comprise any one or more of the following: an S residue at position 94 (S94) or an R residue at position 94 (R94), a K residue at position 105 (K105), an A residue at position 108 (A108), a Q residue at position 108 (Q108), a G residue at position 124 (G124), an N residue at position 160 (N160), an A residue at position 165 (A165), an R residue at position 189 (R189), an E residue at position 190 (E190), a T residue at position 196 (T196), and an L residue at position 200 (L200) (according to wild-type SrtA, e.g., SEQ ID NO:1), optionally removing about 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59 or 60) amino acids from the N-terminus of wild-type S. aureus SrtA. For example, in some embodiments, the sortase A variant comprises two, three, four or five of the aforementioned mutations relative to wild-type S. aureus SrtA (e.g., SEQ ID NO:1). In some embodiments, the sortase A variant comprises an S residue at position 94 (S94) or an R residue at position 94 (R94), and an N residue at position 160 (N160), an A residue at position 165 (A165), and a T residue at position 196 (T196) relative to wild-type S. aureus SrtA (e.g., SEQ ID NO:1). For example, in some embodiments, the sortase A variant comprises P94S or P94R, and D160N, D165A, and K196T relative to wild-type S. aureus SrtA (e.g., SEQ ID NO: 1). In some embodiments, the sortase A variant comprises an S residue at position 94 (S94) or an R residue at position 94 (94), and an N residue at position 160 (N160), an A residue at position 165 (A165), an E residue at position 190, and a T residue at position 196 relative to wild-type S. aureus SrtA (e.g., SEQ ID NO: 1). For example, in some embodiments, the sortase A variant comprises P94S or P94R, and D160N, D165A, K190E, and K196T relative to wild-type S. aureus SrtA (e.g., SEQ ID NO: 1). In some embodiments, relative to wild-type S. aureus SrtA (e.g., SEQ ID NO: 1), the sortase A variant comprises an R residue at position 94 (R94), an N residue at position 160 (N160), an A residue at position 165 (A165), an E residue at position 190, and a T residue at position 196. In some embodiments, relative to wild-type S. aureus SrtA (e.g., SEQ ID NO: 1), the sortase A variant comprises P94R, D160N, D165A, K190E, and K196T. In some embodiments, the S. aureus SrtA variant can remove 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids from the N-terminus.

In some embodiments, a sortase A variant having a higher transamidase activity than naturally occurring sortase A can be used. In some embodiments, the activity of the sortase A variant is at least about 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, or 200 times that of wild-type S. aureus sortase A. In some embodiments, such sortase variants are used in the compositions or methods of the present disclosure. In some embodiments, the sortase variant comprises any one or more of the following substitutions relative to wild-type S. aureus SrtA: P94S/R, E105K, E108A, E108Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L mutations. In some embodiments, the SrtA variant can have 25-60 (eg, 30, 35, 40, 45, 50, 55, 59, or 60) amino acids removed from the N-terminus.

In some embodiments, the amino acid mutation position is determined by comparing the parent Staphylococcus aureus SrtA (the Staphylococcus aureus SrtA variant described herein is derived from the parent) with the polypeptide of SEQ ID No: 1, that is, the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent Staphylococcus aureus SrtA. Methods for determining the amino acid position corresponding to the mutation position described herein are well known in the art. The identification of the corresponding amino acid residue in another polypeptide can be confirmed by using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably 3.0.0 or higher. Based on the above-mentioned well-known computer programs, it is a routine task for those skilled in the art to determine the amino acid position of the polypeptide of interest described herein.

In some embodiments, the sortase variant may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid mutations. It is well known in the art that conservative amino acid mutations do not substantially affect protein activity.

The use of sortases, such as mg SrtA, to covalently label proteins onto cells has broad prospects in scientific research and clinical applications. However, this may have certain limitations: different types of cells have different types of membrane proteins, and the number of proteins containing N-terminal glycine (e.g., G1 of mg SrtA) is also different. The strategy disclosed herein makes it possible to use various sortases to modify various cells with substances.

Irreversible joints

Since SrtA-mediated protein-cell conjugation is a reversible reaction, in order to improve the efficiency of cell labeling, it would be beneficial to minimize the occurrence of reverse reactions. One solution to increase product yield is to increase the concentration of the reaction substrate, but in practical applications, it may be difficult to reach very high concentrations for large molecular proteins; even if high concentrations can be achieved, high costs may limit the use of this technology. Another solution is to continuously remove products from the reaction system so that the reaction does not stop due to equilibrium, but since the reaction is carried out on cells, product separation may be difficult. The inventors of the present application surprisingly found that for cell labeling, the reverse reaction can be prevented by introducing hydroxyacetyl-like byproducts that are not reverse reaction substrates, thereby making the labeling reaction irreversible.

In order to obtain hydroxyacetyl-like byproducts, the present disclosure contemplates the use of a sortase recognition motif comprising an unnatural amino acid, preferably at position 5 in the N-terminal to C-terminal direction of the sortase recognition motif. In some embodiments, the unnatural amino acid is a substituted or unsubstituted hydroxycarboxylic acid having the formula CH ₂ OH-(CH ₂ ) _n -COOH, wherein n is an integer from 0 to 5, e.g., 0, 1, 2, 3, 4, and 5, and preferably n=0. In some embodiments, the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein * represents an optionally substituted hydroxycarboxylic acid, and X and Y independently represent any amino acid. In some embodiments, the sortase recognition motif comprising an unnatural amino acid can be selected from the group consisting of: LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S, and LPXT*A, wherein M is preferably LPET*G, wherein * is preferably 2-hydroxyacetic acid. In some embodiments, Leu-Pro-Glu-Thr-2-hydroxyacetic acid-Gly (LPET-(2-hydroxyacetic acid)-G) is used as a linker to ensure that the byproduct will make the reaction irreversible.

To introduce an irreversible linker into a substance, in some embodiments, a sortase recognition motif comprising an unnatural amino acid as a linker is chemically synthesized and can be directly conjugated to a substance, such as a protein or polypeptide.

In some embodiments, the sortase recognition motif comprising an unnatural amino acid can be conjugated to a substance by various chemical means to produce the desired sortase substrate. These methods may include chemical conjugation with a bifunctional crosslinker or a bifunctional crosslinking substance (e.g., NHS ester-maleimide heterobifunctional crosslinker) to connect the primary amine group to the reduced thiol group. Other molecular fusions can be formed between the sortase recognition motif and the substance, for example, through a spacer. As used herein, in some embodiments, the term "spacer" refers to the remaining portion of the bifunctional crosslinker after crosslinking the sortase recognition motif and the substance.

Various chemical conjugation methods for connecting bifunctional crosslinkers or spacers can be used in the present disclosure, including but not limited to: (1) zero-length type (e.g., EDC; EDC plus sulfo-NHS; CMC; DCC; DIC; N,N'-carbonyldiimidazole;Woodward's reagent K); (2) amine-thiol type, such as NHS ester-maleimide heterobifunctional crosslinker; maleimidocarboxylic acid ( _C2-8 ) (e.g., 6-maleimidocaproic acid and 4-maleimidobutyric acid); EMCS; SPDP, LC-SPDP, sulfo-LC-SPDP; SMPT and sulfo-LC-SMPT; SMCC, LC-SMCC and sulfo-SMCC; MBS and sulfo-MBS; SIAB and sulfo-SIAB; SMPB and sulfoSMPB; GMBS and sulfoGMBS; SIAX and SIAXX; SIAC and SIACX; NPIA; (3) homobifunctional NHS ester type (e.g. , DSP; DTSSP; DSS; DST and sulfo-DST; BSOCOES and sulfo-BSOCOES; EGS and sulfo-EGS); (4) homobifunctional imide ester type (e.g., DMA; DMP; DMS; DTBP); (5) carbonyl-thiol type (e.g., KMUH; EMCH; MPBH; M2C2H; PDPH); (6) thiol-reactive type (e.g., DPDPB; BMH; HBVS); (7) thiol-hydroxyl type (e.g., PMPI); or the like.

In some embodiments, amine-sulfhydryl type or NHS ester-maleimide heterobifunctional crosslinker is a preferred spacer that can be used in this article. In certain embodiments, maleimide carboxylic acid (C _2-8 ) heterobifunctional crosslinker, for example, 6-maleimidocaproic acid and 4-maleimidobutyric acid are particularly useful spacers for constructing the desired sortase substrate. Maleimide carboxylic acid (C _2-8 ) heterobifunctional crosslinker, for example, 6-maleimidocaproic acid and 4-maleimidobutyric acid can react with exposed sulfhydryl (for example, on exposed cysteine) Michael addition reaction occurs, but the reaction will not occur with unexposed cysteine. In one embodiment, 6-maleimidocaproic acid is introduced into the irreversible linker of the present disclosure to obtain 6-maleimidocaproic acid-Leu-Pro-Glu-Thr-2-hydroxyacetic acid-Gly, as shown in Figure 9. In some embodiments, a cysteine residue is or has been added to the C-terminus of the substance to provide an exposed cysteine.

By using the spacers described herein, in particular maleimidocarboxylic acid (C _2-8 ) heterobifunctional crosslinkers, such as 6-maleimidocaproic acid and 4-maleimidobutyric acid, the inventors have successfully designed linkers with different structures, including bifurcated, trifurcated and multifurcated. According to actual needs, for example, to obtain multimodal treatment, these different linkers can be used to label cells such as RBCs. In the multifurcated structure design of some embodiments, one or more spacers can be connected to the amino group of the N-terminal amino acid and/or the amino group of the lysine side chain, and the same or different substances such as proteins or polypeptides can be connected to one or more spacers. This technology can further expand the types of cell marker substances such as proteins and improve the efficiency of RBC engineering.

Sortase substrates

Substrates suitable for sortase-mediated conjugation can be easily designed. A sortase substrate can comprise a sortase recognition motif and a substance. For example, a substance such as a polypeptide can be modified to comprise a sortase recognition motif at or near the C-terminus, thereby allowing it to act as a substrate for a sortase. The sortase recognition motif need not be located at the very C-terminus of the substrate, but should generally be close enough to be accessible to the enzyme to participate in the sortase reaction. In some embodiments, a sortase recognition motif is considered to be "close to" the C-terminus if there are no more than 5, 6, 7, 8, 9, or 10 amino acids between the most N-terminal amino acid (e.g., L) of the sortase recognition motif and the C-terminal amino acid of the polypeptide. A polypeptide comprising a sortase recognition motif can be modified by incorporating or linking any of a variety of moieties (e.g., peptides, proteins, compounds, nucleic acids, lipids, small molecules, and sugars) thereto.

In some embodiments, the present disclosure provides a sortase substrate comprising the structure AM-L', wherein A represents a substance, M represents one or more optional spacers, and L' represents a sortase recognition motif comprising a non-natural amino acid as described herein. In some embodiments, one or more M is selected from the following types of cross-linkers: (1) zero length type; (2) amine-sulfhydryl type; (3) homobifunctional NHS ester type; (4) homobifunctional imine ester type; (5) carbonyl-sulfhydryl type; (6) sulfhydryl reactive type; and (7) sulfhydryl-hydroxy type; preferably, one or more M is a maleimidocarboxylic acid (C _2-8 ) type heterobifunctional cross-linker, such as 6-maleimidocaproic acid and 4-maleimidobutyric acid, and the substance comprises an exposed sulfhydryl group, preferably an exposed cysteine, more preferably a terminal cysteine, and most preferably a C-terminal cysteine. In some embodiments, when two or more spacers are present, the substance attached to the spacer can be the same or different.

substance

In some embodiments, the substance comprises an HPV antigen peptide. In some other embodiments, the HPV antigen peptide binds to a major histocompatibility complex class I (MHC-I) protein, such as HLA-A*02:01, HLA-A*02:02, HLA-A*02:06, HLA-A*02:07, and HLA-A*02:11.

The term "antigenic peptide" as used herein refers to a peptide that can bind to the peptide binding groove of an MHC molecule (particularly a class I MHC molecule), thereby forming an MHC complex with the MHC molecule. It is well known in the art that the display of antigenic peptides in the MHC complex on the surface of an APC does not usually involve, for example, the entire protein. On the contrary, the antigenic peptide located in the groove is usually a small part of the entire protein. In some embodiments, the antigenic peptide is derived from a pathogen protein, such as a viral protein. In some embodiments, the antigenic peptide is a tumor neoantigen. In some embodiments, the term "antigenic peptide" includes tumor antigenic peptides derived from tumor-specific changes in proteins. Tumor antigenic peptides cover but are not limited to tumor antigens produced by the following reasons: for example, substitutions in protein sequences, frameshift mutations, in-frame deletions, insertions, and tumor-specific overexpression of polypeptides. The term "tumor-specific changes" refers to changes that are not present in normal non-cancerous cells but are found in cancer or tumor cells.

In some embodiments, the substance is a fusion protein, which comprises an antigenic peptide, an optional first peptide linker and MHC-I from N-terminus to C-terminus. In some other embodiments, the substance is a fusion protein, which comprises an antigenic peptide, an optional first peptide linker, β2-microglobulin (i.e., MHC-I light chain), an optional second peptide linker and MHC-I heavy chain (preferably MHC-I heavy chain lacking transmembrane region and cytoplasmic region) from N-terminus to C-terminus. In some embodiments, the first peptide linker and the second peptide linker are independent linkers rich in glycine and/or serine.

The fusion protein described herein can be referred to as "artificial MHC single-chain molecule", which refers to a fusion protein comprising an antigenic peptide, β2-microglobulin and MHC class I heavy chain. In some documents, this fusion protein is also referred to as a "single-chain trimer". Typically, there is a peptide linker between the antigenic peptide and the β2-microglobulin (i.e., MHC-I light chain), and there is a peptide linker between the β2-microglobulin and the MHC class I heavy chain. Therefore, the artificial MHC single-chain molecule can include an antigenic peptide, a first peptide linker, a β2-microglobulin, a second peptide linker and an MHC class I heavy chain from the N-terminus to the C-terminus. In some structures, the artificial MHC single-chain molecule can also include a signal peptide (e.g., a β2-microglobulin signal peptide) at the N-terminus. In some structures, the MHC class I heavy chain portion in the artificial MHC single-chain molecule is a truncated version of the missing transmembrane region and the cytoplasmic region.

In some embodiments, the antigenic peptide is an HPV antigenic peptide. In some other embodiments, the term "HPV antigenic peptide" refers to a tumor antigenic peptide derived from a tumor-specific change in an HPV (such as HPV16 or HPV18) protein. The two main oncogenic proteins of high-risk HPV are E6 and E7. The naming of "E" indicates that these two proteins are early proteins (expressed early in the HPV life cycle). The HPV genome contains six early (E1, E2, E4, E5, E6 and E7) open reading frames (ORFs). After the host cell is infected, the viral early promoter is activated and a polycistronic primary RNA containing all six early ORFs is transcribed. Then, this polycistronic RNA generates multiple mRNA isomers by active RNA splicing. The early transcription of E2 initiates viral E2 regulation because high levels of E2 inhibit transcription. The HPV genome is integrated into the host genome by destroying the ORF of E2, preventing E2 from inhibiting E6 and E7. Therefore, the integration of the viral genome into the host DNA genome will increase the expression of E6 and E7, promoting cell proliferation and the chance of malignant transformation. The degree of E6 and E7 expression is related to the type of cervical lesions that may eventually develop. In some embodiments, the HPV antigen peptide is an E6 or E7 antigen peptide of HPV16 or HPV18. In some embodiments, the HPV antigen peptide is an HPV16-E6 antigen peptide, an HPV16-E7 antigen peptide, an HPV18-E6 antigen peptide or an HPV18-E7 antigen peptide, or a combination thereof. In some embodiments, the HPV antigen peptide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 9 (YMLDLQPET) (derived from human HPV16-E7), SEQ ID NO: 10 (LLMGTLGIV) (derived from human HPV16-E7) and SEQ ID NO: 11 (KCLKFYSKI) (derived from mouse HPV16-E6), SEQ ID NO: 12 (FHNIRGRWTGR), SEQ ID NO: 13 (PYAVCDKCL), SEQ ID NO: 14 (EQQYNKPLC), SEQ ID NO: 15 (RFHNIRGR), SEQ ID NO: 16 (IRGRWTGRCM), SEQ ID NO: 17 (LPQLCTELQT), SEQ ID NO: 18 (MHQKRTAM), SEQ ID NO: 19 (MFQDPQERPR), SEQ ID NO: 20 (NKPLCDLLIRC), SEQ ID NO: 21 (LRREVYDFAFR), SEQ ID NO: 22 (KLPQLCTEL), SEQ ID NO: 23 ( NO:23(TIHDIILECV), SEQ ID NO:24(TIHDIILEC), SEQ ID NO:25(IVYRDGNPYA), SEQ ID NO:26(IVYRDGNPYAV), SEQ ID NO:27(FQDPQERPRKL), SEQ ID NO:28(YRDGNPYAV), SEQ ID NO:29(QLCTELQTTI), SEQ ID NO:30(KISEYRHYC ), SEQ ID NO:31(QQYNKPLCDLL), SEQ ID NO:32(TLHEYMLDLQ), SEQ ID NO:33(PAGQAEPD), SEQ ID NO:34(FCCKCDST), SEQ ID NO:35(LRLCVQSTH), SEQ ID NO:36(CVQSTHVD), SEQ ID NO:37(NIVTFCCK), SEQ ID NO:38(VCPICSQK), SEQ ID NO:39(MLDLQPETTD)、SEQ ID NO:40 (CYEQLNDSSE), SEQ ID NO:41 (TLHEYMLDL), SEQ ID NO:42 (TLEDLLMGTL), SEQ ID NO:43 (YMLDLQPETT), SEQ ID NO:44 (RTLEDLLMGTL), SEQ ID NO:45 (MLDLQPETTDL), SEQ ID NO:46 (MLDLQPETT), SEQ ID NO:47 (GTLGIVCPI), SEQ ID NO:48 (TLEDLLMGT) and SEQ ID NO:49 (RLCVQSTHV), or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 a sequence selected from SEQ ID NOs:9-48.

On the one hand, the disclosure provides red blood cells (RBC) connected with one or more HPV antigen peptides or variants thereof. In some embodiments, RBC comprises more than one (e.g., two, three, four, five or more) polypeptides, and each polypeptide comprises at least one HPV antigen peptide or variants thereof. In some embodiments, cells described herein comprise more than one HPV antigen peptide type, wherein each peptide comprises one HPV antigen peptide, and the HPV antigen peptides are not the same (e.g., HPV antigen peptides can comprise different types of antigen peptides from different oncoproteins of different HPVs, or variants of the same type of HPV antigen peptides). For example, in some embodiments, RBC can comprise a first HPV antigen peptide or variants thereof, and a second HPV antigen peptide.

As used herein, the term "major histocompatibility complex" or "MHC" is a cluster of genes that play a role in controlling cellular interactions responsible for physiological immune responses. There are two main types of MHC molecules, namely MHC class I (referring to "MHC-I", "MHC I" or "MHC1") and MHC class II (referring to "MHC-II", "MHC II" or "MHC2"). In humans, the MHC complex is also called the human leukocyte antigen (HLA) complex. In some embodiments, the MHC complex is HLA class I. In some embodiments, HLA class I can be selected from HLA A0201, HLA A2402, HLA B07, HLA B18, HLA B35 or HLA B44, for example, HLA A0201. In some embodiments, the HPV antigen peptide is bound to an MHC class I protein, or an allelic variant of an MHC class I protein. In some embodiments, the allelic variant has up to about 10 (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions compared to the MHC class I protein. In some embodiments, the allelic variant and the MHC class I protein are of the same serotype. In some embodiments, the allelic variant and the MHC class I protein are of different serotypes.

In some embodiments, the HPV antigen peptide is bound to an MHC class I protein (e.g., HLA-A02, such as HLA-A02:01, HLA-A02:03 (GenBank Accession No.: AAA03604), HLA-A02:05 (GenBank Accession No.: AAA03603), HLA-A02:06 (GenBank Accession No.: CCB78868), HLA-A02:07 (GenBank Accession No.: ACR55712), and HLA-A02:11 (GenBank Accession No.: CAB56609)).

In some embodiments, the substance comprises an HPV antigen peptide and an MHC-I, and in some other embodiments, the MHC-I binds to the C-terminus of the HPV antigen peptide.

In some embodiments, the amino acid sequence of HLA-A*02:01 is shown as follows in SEQ ID NO:50:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGP

EYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLR

GYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCV

EWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRD

GEDQTTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEM

(SEQ ID NO:50)

In some embodiments, the β2-microglobulin used in the present invention is human β2-microglobulin. In some embodiments, the β2-microglobulin has the amino acid sequence shown in SEQ ID NO: 51 as follows:

IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:51)

Methods of modifying cells

The sortase can recognize a specific sortase recognition motif, such as the sequence LPXTG, and connect the LPXTG at the C-terminus of one protein to the G at the N-terminus of another protein through a transpeptidation reaction. This principle can be used to modify a substance of interest so that it can be linked to a linker containing a terminal glycine that is already linked to at least one membrane protein of a cell (e.g., RBC).

In one aspect, the present disclosure provides a method for modifying a cell, comprising: a) providing a sortase substrate comprising an AM-L' structure, wherein A represents a substance, L' represents a sortase recognition motif, and M represents a residual portion of a bifunctional cross-linking agent after cross-linking; b) (i) providing a peptide having the formula _{GlymXn -} M', wherein _Glym represents m glycines, m is preferably 1-5, and _Xn represents n spacer amino acids, n is preferably 0-10; and M' represents a bifunctional cross-linking agent; (ii) treating the cell with the _{GlymXn -} M' peptide under conditions such that the peptide is linked to at least one membrane protein of the cell to generate _{GlymXn -} MP, wherein M represents a residual portion of a bifunctional cross-linking agent after cross-linking, and P represents at least one membrane protein of the cell; and c) contacting the treated cell with the sortase substrate in the presence of a sortase under one or more conditions suitable for the sortase to conjugate the sortase substrate to _Glym through a sortase-mediated reaction. a) and b) can be performed simultaneously, or a) can be performed before b), or a) can be performed after b).

In some embodiments, when sulfhydryl is one of the reactive groups of the bifunctional cross-linking agent to be used, before the treatment step, the step of pre-treating the cells with a reducing agent is performed to form or increase the number of exposed sulfhydryls. The present disclosure contemplates various reducing agents, as long as they can reduce the disulfide bonds within or between surface membrane proteins to expose sulfhydryls. In some embodiments, a reducing agent having no or almost no negative impact on the viability of the treated cells is used. In some embodiments, a reducing agent such as tris (2-carboxyethyl) phosphine hydrochloride (TCEP) or dithiothreitol (DTT) or β-mercaptoethanol can be used, for example, under partial or complete reducing conditions.

It should be understood that one of ordinary skill in the art can select conditions (e.g., optimal temperature, pH, reaction time, concentration) suitable for the sortase to connect the sortase substrate to the linker comprising a terminal glycine according to the properties of the sortase substrate, the type of sortase, etc.

It should also be understood that one of ordinary skill in the art can select appropriate conditions (e.g., optimal temperature, pH, reaction time, concentration) for attaching a linker comprising a terminal glycine to at least one membrane protein of a cell (e.g., a blood cell such as an RBC).

use

In some aspects, the present disclosure provides a method of treating or preventing a disease associated with HPV infection in a subject in need thereof, the method comprising administering to the subject a red blood cell or composition as described herein.

As used herein, the term "disease associated with HPV infection" refers to a disease associated with or caused by HPV infection, such as a disease caused by infection with HPV16 or HPV18. In embodiments, the disease associated with HPV infection is cancer. In some embodiments, the cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer.

As used herein, "treatment" refers to a therapeutic intervention that at least partially ameliorates, eliminates or reduces symptoms or pathological signs after the onset of a pathogen-related disease, disorder or indication. Treatment need not be absolutely beneficial to the subject. Beneficial effects can be determined using any method or criteria known to those of ordinary skill.

As used herein, "prevention" or "prevention" refers to a series of actions that begin before infection or exposure to a pathogen or its molecular components and/or before the onset of symptoms or pathological signs of a disease, disorder or indication to prevent infection and/or alleviate symptoms or pathological signs. It should be understood that such prevention does not need to be absolutely beneficial to the subject. "Preventive" treatment is a treatment administered to a subject who does not show signs of a disease, disorder or indication or only shows early signs, with the purpose of reducing the risk of symptoms or pathological signs of a disease, disorder or indication.

In some embodiments, the methods described herein further comprise administering the conjugated red blood cells to the subject, such as by intravenous administration directly into the circulatory system via injection or infusion.

In some embodiments, the subject receives a single dose of cells during treatment, or receives multiple doses of cells, for example, 2 to 5, 10, 20 or more doses. In some embodiments, the dose or total number of cells can be expressed as cells/kg. For example, the dose can be about 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ cells/kg.

In some embodiments, the course of treatment lasts from about 1 week to 12 months or longer, for example, 1, 2, 3 or 4 weeks or 2, 3, 4, 5 or 6 months. In some embodiments, the subject can be treated about every 2-4 weeks.

One of ordinary skill in the art will appreciate that the number of cells, dosage, and/or dosing interval can be selected based on a variety of factors, such as the subject's weight and/or blood volume, the indication being treated, the subject's response, etc. The exact number of cells required may vary from subject to subject, depending on a variety of factors, such as the species, age, weight, sex, and general condition of the subject, the severity of the disease or condition, the specific cells, the composition and activity of the substance conjugated to the cells, the mode of administration, concurrent therapy, and the like.

Composition

In another aspect, the present disclosure provides a composition comprising a modified cell described herein and optionally a physiologically acceptable carrier, for example in the form of a pharmaceutical composition, a delivery composition, a diagnostic composition, or a kit of parts.

In some embodiments, the composition can include a plurality of cells, such as blood cells, for example, RBCs. In some embodiments, at least a selected percentage of cells in the composition are modified, i.e., conjugated to a substance by the methods described herein. For example, in some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the cells have a substance conjugated thereto. In some embodiments, two or more red blood cells or red blood cell populations conjugated to different substances are included.

In some embodiments, the composition comprises modified cells of the present disclosure, such as red blood cells, wherein the cells are modified with any substance of interest. In some embodiments, the composition comprises an effective amount of cells, for example, up to about 10 ¹⁴ cells, for example, about 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 5×10 ⁵ , 10 ⁶ , 5×10 ⁶ , 10 ⁷ , 5×10 ⁷ , 10 ⁸ , 5×10 ⁸ , 10 ⁹ , 5×10 ⁹ , 10 ¹⁰ , 5×10 ¹⁰ , 10 ¹¹ , 5×10 ¹¹ , 10 ¹² , 5×10 ¹² , 10 ¹³ , 5×10 ¹³ , or 10 ¹⁴ cells. In some embodiments, the number of cells can be between any two of the aforementioned numbers.

As used herein, the term "effective amount" refers to an amount sufficient to achieve a biological response or effect of interest (e.g., reducing one or more symptoms or manifestations of a disease or indication, or modulating an immune response). In some embodiments, the composition administered to a subject comprises up to about 10 ¹⁴ cells, e.g., about 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , or 10 ¹⁴ cells, or any intermediate number or range.

As used herein, the term "physiologically acceptable carrier" refers to a solid or liquid filler, diluent or encapsulating material that can be safely used for systemic administration. Depending on the specific route of administration, a variety of carriers, diluents and excipients well known in the art can be used. These can be selected from sugars, starches, cellulose and derivatives thereof, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as inorganic acid salts, including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water.

Those skilled in the art will appreciate that other variations of the embodiments described herein may also be practiced without departing from the scope of the present invention.Other modifications are therefore possible.

Although the present disclosure has been described and illustrated in an exemplary form with a certain degree of particularity, it should be noted that the description and illustration are provided by way of example only. Many changes may be made to the details of construction and combination and the arrangement of parts and steps. Therefore, such changes are intended to be included in the present invention, and the scope of the present invention is defined by the claims.

Example

Method (see Figure 1)

1. Expression and purification of HPV16-MCH1 protein

HPV16-MHC cDNA was cloned into the pcDNA3.1 vector. The cDNA was mixed with electroporation buffer and placed in an electroporation tank. The vector was electroporated into CHO cells according to the manufacturer's (Ettabiotech) optimized protocol for CHO cells. After 7 days, all supernatants were collected after centrifugation at 14,000 g for 40 minutes at 4°C and filtered through a 0.22 μm filter.

In the Examples, four HPV16-MHC1 fusion proteins were generated.

Fusion protein 1: HPV16-hMHC1-Fc comprises the structure of human β2M signal peptide-HPV16 epitope (SEQ ID NO: 9)-GS linker-human β2M-GS linker-HLA0201-GS linker-human IgG1Fc-Cys. The amino acid sequence and DNA sequence of fusion protein 1 are shown in SEQ ID NOs: 52 and 53.

Fusion protein 2: HPV16-hMHC1-Fc comprises the structure of human β2M signal peptide-HPV16 epitope (SEQ ID NO: 10)-GS linker-human β2M-GS linker-HLA0201-GS linker-human IgG1Fc-Cys. The amino acid sequence and DNA sequence of fusion protein 2 are shown in SEQ ID NOs: 54 and 55.

Fusion protein 3: HPV16-mMHC1-His comprises the structure of mouse β2M signal peptide-HPV16 epitope (SEQ ID NO: 11)-GS linker-mouse β2M-GS linker-mouse H2KB-GS linker-His ₆ -AAC. The amino acid sequence and DNA sequence of fusion protein 3 are shown in SEQ ID NOs: 56 and 57.

Fusion protein 4: HPV16-mMHC1-Fc comprises the structure of mouse β2M signal peptide-HPV16 epitope (SEQ ID NO: 11)-GS linker-mouse β2M-GS linker-mouse H2KB-GS linker-human IgG1 Fc-Cys. The amino acid sequence and DNA sequence of fusion protein 4 are shown in SEQ ID NO: 58 and 59.

After separation from cells by centrifugation and microfiltration, the supernatant was loaded onto an IMAC BestaroseFF column (Bestchrom, Shanghai, China) with Ni ²⁺ ions, which was balanced with binding buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.6). The column was washed with binding buffer and then eluted with elution buffer 1 (20 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.6) until the UV absorbance at 280 nm was stable. Protein was collected with elution buffer 2 (20 mM Tris-HCl, 500 mM NaCl, 100 mM imidazole, pH 7.6).

The protein fraction was then diluted with ddH ₂ O (1:1) and loaded onto a Diamond Mix-A column (Bestchrom, Shanghai, China) equilibrated with binding buffer (10 mM Tris-HCl, 250 mM NaCl, pH 7.6). After washing with binding buffer and eluting with elution buffer 1 (13.3 mM Tris-HCl, 337.5 mM NaCl, pH 7.6), the target protein was eluted with elution buffer 2 (20 mM Tris-HCl, 2000 mM NaCl, pH 7.6) and then concentrated using an Amicon Ultra-15 centrifugal filter device (Millipore, Darmstadt, Germany).

The concentrated protein was loaded onto Chromdex 200 pg (Bestchrom, Shanghai, China) equilibrated with PBS, and the target protein fractions were collected. The protein was concentrated and stored at -80°C.

2. Conjugation of irreversible linkers to HPV16-MHC1 via cysteine coupling

The irreversible linker, 6-maleimidocaproic acid-Leu-Pro-Glu-Thr-2-hydroxyacetic acid-Gly (6-maleimidocaproic acid-LPET-(2-hydroxyacetic acid)-G, 6-Mal-LPET*G), was synthesized with a purity of more than 99%. The reaction was carried out in a total volume of 1 mL in PBS buffer at room temperature for 1 hour while rotating at a speed of 10 rpm. The concentrations of 6-Mal-LPET*G and HPV16-MHC1 protein were 2 mM and 500 μM, respectively. This method used twice the molar amount of irreversible linker compared to HPV16-MHC1 protein. After the reaction, the excess irreversible linker was removed by dialysis and ultrafiltration, and the HPV16-MHC1-6-mal-LPET*G product was collected.

3. Mg SrtA-mediated HPV16-MHC1-6-mal-LEPT*G labeling

3.1 RBC pretreatment

Red blood cells were separated from peripheral blood by density gradient centrifugation. The separated red blood cells were washed 3 times with PBS. The RBCs were then pretreated with 1 mM TCEP for 1 hour at room temperature. The pretreated RBCs were then washed 3 times with PBS. The GGGSK-6-MAL linker (synthesized by Beijing Zhongke Yaguang Biotechnology Co., Ltd.) (see Figure 2) was synthesized with a purity of over 99%. GGGSK-6-MAL was dissolved in phosphate buffer at 37°C to a final concentration of 100 μM. 1×10 ⁹ RBCs were then pretreated with 50 μM GGGSK-6-MAL at 37°C for 30 minutes. The pretreated RBCs were then washed 3 times with PBS. The pretreated RBCs need to be used immediately and cannot be stored for a long time.

3.2 Conjugation of GGGSK-6-MAL-RBC to HPV16-MHC1-6-mal-LPET*G via a Mg-sortase-mediated reaction

The reaction was carried out in PBS buffer in a total volume of 200 μL at room temperature for 2 hours while rotating at 10 rpm. The concentration of GGGSK-6-MAL-RBC in the reaction was 1×10 ⁹ /mL. The concentration of mg SrtA (SEQ ID NO: 5) was 10 μM, and the concentration of HPV16-MHC1-6-mal-LEPT*G substrate was 25 μM. After the reaction, the labeling efficiency of RBC was analyzed using Beckman Coulter CytoFLEX LX.

4. Interferon γ (IFN γ ) ELISpot method

The human IFN-γ ELISpot kit (Mabtech) was used according to the manufacturer's instructions. For ex vivo ELISpot, peripheral blood mononuclear cells (PBMCs) isolated from HPV16 ⁺ patients were plated in duplicate at 2×10 ⁵ cells per well. Modified RBCs were plated at 10 ⁷ cells per well. HLA-A2-restricted HPV16 E6/E7-derived peptides were added to the ELISpot wells at a concentration of 20 μg/mL. The plates were incubated at 37°C for 48 hours. Secreted IFNγ was detected by biotin-labeled anti-IFNγ mAb, and the reaction was developed using streptavidin-HRP and the pigment reagent BCIP/NBT. The number of T cells specifically secreting IFNγ was calculated.

5. Cytotoxic potential of HPV16-MHC1-RBC immune response in vitro

This study used HLA-A*0201-positive, HPV-16-positive human cervical cancer cell line and HPV16-MC38 cell line (transduced to express the E6 and E7 oncogenes of HPV-16).

The CaSki cells (CRL-1550TM) obtained from ATCC integrated the HPV-16 genome. Peripheral blood mononuclear cells (PBMCs) from HPV16 ⁺ patients were incubated with HPV16 (YMLDLQPET) -MHC1-RBC (PBMC: engineered RBC = 1: 50) at 37 ° C for 72 hours. After stimulation, CD8 ⁺ T cell isolation kit (Stem cell company) was used to enrich CD8 ⁺ T cells in the patient's PBMC. The isolated CD8 ⁺ T cells were co-cultured with target cells CaSki for 72 hours (effector cells: target cells = 100: 1). The cytotoxic potential of the immune response generated by HPV16 (YMLDLQPET) -MHC1-RBC was detected by the expression of 4-1BB, CD107a and fluorescein-conjugated MHC tetramers on the surface of ^CD8 + T cells and the survival rate of CaSki cells.

Splenocytes from mice carrying HPV16-MC38 tumors were incubated with HPV16 (KCLKFYSKI)-mMHC1-RBC (splenocytes: engineered RBC = 1:50) at 37 ° C for 72 hours. After stimulation, CD8 ⁺ T cells were enriched from splenocytes using a CD8 ⁺ T cell isolation kit (Stem cell company). The isolated CD8 ⁺ T cells were co-cultured with target cells HPV16-MC38 for 72 hours (effector cells: target cells = 100: 1). The cytotoxic potential of the immune response generated by HPV16 (KCLKFYSKI)-mMHC1-RBC was detected by the expression of CD107a and fluorescein ^- conjugated MHC tetramers on the surface of CD8 + T cells and the survival rate of HPV16-MC38 cells.

6. HPV16-MHC1-RBC therapy in solid tumor models

In immunotherapy, 10 ⁵ HPV16-MC38 cells were first injected subcutaneously (sc) in the left posterior abdomen of mice on D0 day. The mice were divided into 2 groups: (1) control RBC treatment group; (2) HPV16 (KCLKFYSKI) -mMHC1-RBC treatment group. HPV16 (KCLKFYSKI) -mMHC1-RBC was administered on days 1, 4, 7, 10, 13, and 16. Body weight and tumor volume were measured every three days. Tumor volume was calculated using the empirical formula V = 1/2 × [(shortest diameter) ² × (longest diameter)]. After 4 weeks of treatment, the mice were sacrificed and the tumors were weighed and processed for IHC analysis.

result

Labeling efficiency

We characterized the efficiency of mg SrtA-mediated HPV16(YMLDLQPET)-hMHC1 labeling on RBC membranes. The conjugation efficiency was detected by incubating the labeled RBCs with anti-Fc tag antibodies conjugated with PE and analyzing them using flow cytometry. The results in Figure 3 show that more than 99% of natural human RBCs were labeled with HPV16(YMLDLQPET)-hMHC1 by mg SrtA in vitro. In contrast, in the mock control group without mg SrtA enzyme, no significant Fc tag signal was detected on the surface of human RBCs. Compared with the efficiency of mg SrtA-mediated HPV16(YMLDLQPET)-hMHC1 on RBCs without GGGSK-mal pretreatment, the labeling efficiency after pretreatment was also significantly higher.

Immunogenicity

The immunogenicity of HPV16(YMLDLQPET)-hMHC1-RBC was evaluated in peripheral blood by IFN-γ ELISpot. HPV16(YMLDLQPET) peptide, control RBC and HPV16(YMLDLQPET)-hMHC1-RBC were analyzed in 3 HPV16 ⁺ cervical cancer patients and 2 healthy donors. As shown in Figure 4, peripheral immune responses to HPV16(YMLDLQPET)-hMHC1-RBC were detected in patients, but almost no immune responses to HPV16(YMLDLQPET)-hMHC1-RBC were detected in the peripheral blood of healthy donors. In summary, the HPV16(YMLDLQPET)-hMHC1-RBC stimulation that led to the generation of T cell responses was strong and specific.

Cytotoxic potential

In the presence of HPV16(YMLDLQPET)-hMHC1-RBC, the surface expression of 4-1BB, CD107a and fluorescein-conjugated MHC tetramers was measured to determine the cytotoxic phenotype of neoantigen-specific CD8 ⁺ T cells in peripheral blood. Figure 5 shows the surface expression of 4-1BB, CD107a and MHC tetramers of patient CD8+ T cells co-cultured with HPV16(YMLDLQPET)-hMHC1-RBC.

The cytotoxicity assay is based on the survival of Caski target cells co-cultured with pre-treated patient PBMCs stimulated with HPV16(YMLDLQPET)-hMHC1-RBCs. The data in Figure 6 show that HPV16(YMLDLQPET)-hMHC1-RBCs generate cytotoxic T cells that kill cancer cells.

The cytotoxicity assay is based on the survival of HPV16(KCLKFYSKI)-MC38 target cells co-cultured with pre-treated splenocytes from HPV16-MC38 tumor-bearing mice. The data in Figure 7 demonstrate that HPV16(KCLKFYSKI)-mMHC1-RBCs generate cytotoxic T cells that kill cancer cells.

Tumor growth

In the therapeutic study, as shown in FIG8 , animals that received repeated infusions of HPV16(KCLKFYSKI)-mMHC1-RBCs had a significant delay in tumor growth (p<0.01).

Although specific embodiments of the present invention have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the present invention. On the contrary, the words used in the description are descriptive rather than restrictive words, and it is understood that various changes can be made without departing from the spirit and scope of the present invention.

Claims (39)

1. A cell having a substance attached thereto, wherein the substance is attached to at least one membrane protein of the cell by a sortase recognition motif, and the substance attached to the at least one membrane protein comprises the structure: A-M-L-Gly _mX_n -M-P, wherein A represents a substance, L represents the remainder of the sortase recognition motif after sortase-mediated reaction, gly _m represents M glycine, wherein M is preferably 1-5, X _n represents n spacer amino acids, wherein n is preferably 0-10, M represents the remainder of the bifunctional cross-linker after cross-linking, and P represents at least one membrane protein of the cell.

2. The cell according to claim 1, wherein the bifunctional crosslinking reagent is an amine-thiol, preferably maleimide carboxylic acid (C _2-8), such as 6-maleimide hexanoic acid and 4-maleimide butanoic acid.

3. Cell according to claim 1 or 2, wherein X _n comprises at least one amino acid having a side chain amino group, such as lysine, and preferably the C-terminal amino acid of X _n is an amino acid having a side chain amino group.

4. The cell of claim 3, wherein M crosslinks the side chain amino group with at least one exposed thiol group of the at least one membrane protein.

5. The cell of any one of claims 1-4, wherein the sortase recognition motif comprises, or consists essentially of, or consists of an amino acid sequence selected from LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS and LPXTA, wherein X is any amino acid.

6. The cell of any one of claims 1-4, wherein the sortase recognition motif comprises an unnatural amino acid at position 5 in the N-terminal to C-terminal direction of the sortase recognition motif, wherein the unnatural amino acid is an optionally substituted hydroxycarboxylic acid having the formula CH ₂OH-(CH₂)_n -COOH, where N is an integer from 0to 3, preferably N = 0.

7. The cell of claim 6, wherein the sortase recognition motif comprises, or consists essentially of, or consists of an amino acid sequence selected from LPXT Y, LPXA, Y, LPXS, Y, LPXL, Y, LPXV, Y, LGXT, Y, LAXT, Y, LSXT, Y, NPXT, Y, MPXT, Y, IPXT, Y, SPXT, Y, VPXT, and YPXR, Y, wherein x represents an optionally substituted hydroxycarboxylic acid; and X and Y independently represent any amino acid.

8. The cell of claim 7, wherein the sortase recognition motif comprises, or consists essentially of, or consists of an amino acid sequence selected from LPXT*G、LPXA*G、LPXS*G、LPXL*G、LPXV*G、LGXT*G、LAXT*G、LSXT*G、NPXT*G、MPXT*G、IPXT*G、SPXT*G、VPXT*G、YPXR*G、LPXT*S and LPXT a, wherein M is preferably LPET x G, is 2-hydroxyacetic acid.

9. The cell of any one of claims 1-8, wherein L is selected from LPXT, LPXA, LPXS, LPXL, LPXV, LGXT, LAXT, LSXT, NPXT, MPXT, IPXT, SPXT, VPXT and YPXR and X is any amino acid.

10. The cell according to any one of claims 1-9, wherein the substance a comprises an exposed thiol group, preferably an exposed cysteine, more preferably a terminal cysteine, most preferably a C-terminal cysteine.

11. The cell of any one of claims 1-10, wherein the substance comprises HPV antigenic peptides.

12. The red blood cell of claim 11, wherein the HPV antigenic peptide binds to major histocompatibility complex class I (MHC-I) proteins, such as HLA-A 02:01, HLA-A 02:02, HLA-A 02:06, HLA-A 02:07, and HLA-A 02:11.

13. The red blood cell according to claim 11 or 12, wherein the HPV antigenic peptide is an HPV16 antigenic peptide, preferably comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID No. 9 (YMLDLQPET), SEQ ID No. 10 (LLMGTLGIV) and SEQ ID No. 11 (KCLKFYSKI), or a functionally equivalent variant thereof.

14. The cell of any one of claims 1-13, wherein the sortase is sortase a (SrtA), e.g., staphylococcus aureus transpeptidase a variant (mgSrtA).

15. The cell according to any one of claims 1-14, wherein the cell is selected from enucleated cells, preferably erythrocytes.

16. A method of modifying a cell, the method comprising:

a) Providing a sortase substrate comprising an a-M-L 'structure, wherein a represents a substance, L' represents a sortase recognition motif, and M represents the remainder of the bifunctional cross-linking agent after cross-linking;

b) (i) providing a peptide having the formula Gly _mX_n -M', wherein Gly _m represents M glycine, M preferably being 1-5; x _n represents n spacer amino acids, n preferably being 0 to 10; m' represents a bifunctional crosslinking agent;

(ii) Treating a cell with Gly _mX_n -M 'peptide under conditions that allow linking of the Gly _mX_n -M' peptide to at least one membrane protein of the cell to produce Gly _mX_n -M-P, wherein M represents the remainder of the bifunctional cross-linking agent after cross-linking and P represents the at least one membrane protein of the cell; and

C) Contacting the treated cells with a sortase substrate in the presence of a sortase under one or more conditions suitable for the sortase to conjugate the sortase substrate to Gly _m by a sortase-mediated reaction;

a) And b) may be carried out simultaneously, either a) before b), or a) after b).

17. The method of claim 16, wherein prior to the treating step, the method further comprises the step of pre-treating the cells with a reducing agent to form exposed sulfhydryl groups.

18. The method according to claim 16 or 17, wherein the bifunctional crosslinking reagent is an amine-thiol, preferably a maleimide carboxylic acid (C _2-8), such as 6-maleimide caproic acid and 4-maleimide butyric acid.

19. The method according to any one of claims 16-18, wherein X _n comprises at least one amino acid having a side chain amino group, such as lysine, and preferably the C-terminal amino acid of X _n is an amino acid having a side chain amino group.

20. The method of claim 19, wherein the bifunctional crosslinking reagent crosslinks the side chain amino groups and at least one exposed thiol group of the at least one membrane protein.

21. The method of any one of claims 16-20, wherein the sortase-mediated reaction forms a substance that is linked to at least one membrane protein of the cell, the substance comprising the structure: A-M-L-Gly _mX_n -M-P, wherein A represents a substance, L represents the remainder of the recognition motif of the sortase after sortase-mediated reaction, gly _m and X _n are as defined in claim 16, M represents the remainder of the bifunctional crosslinking reagent after crosslinking, and P represents at least one membrane protein of the cell.

22. The method of any one of claims 16-21, wherein the sortase recognition motif comprises, or consists essentially of, or consists of an amino acid sequence selected from LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS and LPXTA, wherein X is any amino acid.

23. The method of any one of claims 16-21, wherein the sortase recognition motif comprises an unnatural amino acid at position 5 in the N-terminal to C-terminal direction of the sortase recognition motif, wherein the unnatural amino acid is an optionally substituted hydroxycarboxylic acid having the formula CH ₂OH-(CH₂)_n -COOH, where N is an integer from 0 to 3, preferably N = 0.

24. The method of claim 23, wherein the sortase recognition motif comprises, or consists essentially of, or consists of an amino acid sequence selected from LPXT Y, LPXA, Y, LPXS, Y, LPXL, Y, LPXV, Y, LGXT, Y, LAXT, Y, LSXT, Y, NPXT, Y, MPXT, Y, IPXT, Y, SPXT, Y, VPXT, and YPXR, Y, wherein x represents an optionally substituted hydroxycarboxylic acid; and X and Y independently represent any amino acid.

25. The method of claim 24, wherein the sortase recognition motif comprises, or consists essentially of, or consists of an amino acid sequence selected from LPXT*G、LPXA*G、LPXS*G、LPXL*G、LPXV*G、LGXT*G、LAXT*G、LSXT*G、NPXT*G、MPXT*G、IPXT*G、SPXT*G、VPXT*G、YPXR*G、LPXT*S and LPXT a, wherein M is preferably LPET x G, is 2-hydroxyacetic acid.

26. The method of any one of claims 16-25, wherein the substance comprises HPV antigenic peptides.

27. The method of claim 26, wherein the HPV antigenic peptide binds to major histocompatibility complex class I (MHC-I) proteins, such as HLA-A 02:01, HLA-A 02:02, HLA-A 02:06, HLA-A 02:07, and HLA-A 02:11.

28. The method of claim 26 or 27, wherein the HPV antigenic peptide is an HPV16 antigenic peptide, preferably comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID No. 9 (YMLDLQPET), SEQ ID No. 10 (LLMGTLGIV) and SEQ ID No. 11 (KCLKFYSKI), or a functionally equivalent variant thereof.

29. The method of any one of claims 16-28, wherein the sortase is sortase a (SrtA), e.g., staphylococcus aureus transpeptidase a variant (mgSrtA).

30. The method according to any one of claims 16-28, wherein the cells are selected from enucleated cells, preferably erythrocytes.

31. A cell obtained by the method of any one of claims 16-30.

32. A composition comprising the cell of any one of claims 1-15 and 31 and optionally a physiologically acceptable carrier.

33. A method for diagnosing, treating or preventing a disease associated with HPV infection in a subject in need thereof, the method comprising administering to the subject the cell of any one of claims 1-15 and 31 or the composition of claim 32.

34. The method of claim 33, wherein the disease associated with HPV infection is selected from cervical cancer, anogenital cancer, head and neck cancer, and oropharyngeal cancer.

35. A method of delivering a substance to a subject in need thereof, the method comprising administering to the subject the cell of any one of claims 1-15 and 31 or the composition of claim 32.

36. A method of increasing the circulation time or plasma half-life of a substance in a subject, the method comprising attaching the substance to a cell according to the method of any one of claims 16-30.

37. Use of a cell according to any one of claims 1-15 and 31 or a composition according to claim 32 in the manufacture of a medicament for the diagnosis, treatment or prevention of a disease associated with HPV infection.

38. The use according to claim 37, wherein the disease associated with HPV infection is selected from cervical cancer, anogenital cancer, head and neck cancer and oropharyngeal cancer.

39. The use of claim 37 or 38, wherein the medicament is a vaccine.