WO2023134573A1

WO2023134573A1 - Modified cells and uses thereof

Info

Publication number: WO2023134573A1
Application number: PCT/CN2023/070977
Authority: WO
Inventors: Xiaofei GAO; Xiaoqian NIE; Yang YUAN; Yanjie HUANG; Xuan Liu; Yun GE
Original assignee: Westlake Therapeutics (Shanghai) Co., Limited
Priority date: 2022-01-12
Filing date: 2023-01-06
Publication date: 2023-07-20

Abstract

Provided is a cell having an agent linked thereto, wherein the agent is linked to at least one membrane protein of the cell via a linker comprising a N-terminal glycine through sortase recognition motif. Also provided is a method for obtaining the modified cell, as well as the use of the modified cells for delivering agents such as drugs and probes.

Description

Modified Cells and Uses Thereof

TECHNICAL FIELD

The present disclosure relates generally to modified cells, and more particularly to membrane protein-modified cells and use of the same for delivering agents including drugs and probes and the like, and particularly the use of the same for treating and preventing an HPV infection associated disease.

BACKGROUND

Human papillomavirus infection (HPV infection) is an infection caused by human papillomavirus (HPV) , a DNA virus from the Papillomaviridae family. Many HPV infections cause no symptoms and 90%resolve spontaneously within two years. However, in some cases, an HPV infection persists and results in either warts or precancerous lesions. These lesions, depending on the site affected, increase the risk of cancer of the cervix, vulva, vagina, penis, anus, mouth, tonsils, or throat.

Nearly all cervical cancer is due to HPV; two strains, HPV16 and HPV18, account for 70%of cases. HPV16 is responsible for almost 90%of HPV-positive oropharyngeal cancers. HPV6 and HPV11 are common causes of genital warts and laryngeal papillomatosis. HPV-16 has caused 50%of cervical cancers and pre-cancerous cervical lesions. Cervical cancer is the fourth most common cancer among women globally. However, there are limited treatment options for patients with recurrent cervical carcinoma. 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 in the world in 2020, and approximately 85%of new cases and 90%of the deaths occurred in developing countries. In 2020, there were 110,000 new cases of cervical cancer in China, accounting for about 18.3%of the new cases of cervical cancer in the world, and there were 60,000 deaths, accounting for approximately 17.6%of the world’s deaths from cervical cancer.

Effective anti-tumor immunity in humans has been associated with the presence of T cells directed at cancer neoantigens, a class of HLA-bound peptides that arise from tumor-specific mutations, and emerging data suggest that recognition of such neoantigens is a major factor in the activity of clinical immunotherapies. Massively parallel whole exome sequencing has been used to detect all mutations within the tumor to predict neoantigens. Vaccination with neoantigens can expand pre-existing neoantigen specific T cell populations and induce new cancer specific T cells. Although neoantigens have emerged as potentially ideal targets for anti-tumor immune responses, many questions remain to be answered before clinical applications can become a reality. T cell activation needs MHC molecules to present MHC restricted peptides to specific T cell receptors. Lack of a specific antigen presenting system is one of the problems of neoantigen vaccination.

Recent development in drug delivery systems for prolonging drug retention time in treating a variety of human diseases has attracted much attention. However, many of the systems still suffer from various challenges and limitations such as poor stability, unwanted toxicity, and immune responses [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) , the most common cell type in the human body, have been widely investigated as an ideal in vivo drug delivery system for over three decades due to their unique biological properties, including: (i) widespread circulation range throughout the body; (ii) good biocompatibility as a biological material with long in vivo survival time; (iii) large surface to volume ratio; and (iv) no nucleus, mitochondria, and other cellular organelles.

RBCs have been developed as drug delivery carriers by direct encapsulation, noncovalent attachment of foreign peptides, or through installation of proteins by fusion to antibodies specific for RBC surface proteins. It has been demonstrated that such modified RBCs have limitations for applications in vivo. For instance, encapsulation will disrupt cell membranes, which subsequently affects in vivo survival rates of engineered cells. In addition, the non-covalent attachment of polymeric particles to RBCs dissociates readily, and the payloads will be degraded quickly in vivo.

Bacterial sortases are transpeptidases capable of modifying proteins in a covalent and site-specific manner [J.M. 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 from Staphylococcus aureus (wt SrtA) recognizes an LPXTG motif and cleaves between threonine and glycine to form a covalent acyl-enzyme intermediate between the enzyme and the substrate protein. This intermediate is resolved by a nucleophilic attack by a peptide or protein typically having three consecutive glycine residues (3 × glycines, G ₃) at the N-terminus. Previous studies have genetically overexpressed a KELL membrane protein having an LPXTG motif at its C-terminus on RBCs, which can be attached to the N terminus of 3 × glycines-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 RBCs carrying drugs have shown efficacy in treating diseases in animal models. However, this requires the steps of engineering hematopoietic stem or progenitor cells (HSPCs) and differentiating these cells into mature RBCs, which significantly limit the application.

Accordingly, there is still a need in the art for an improved cell delivering system for treating an HPV infection associated disease.

SUMMARY

In a first aspect, the disclosure provides a cell having an agent linked thereto, wherein the agent is linked to at least one membrane protein of the cell via a sortase recognition motif, and the agent linked to at least one membrane protein comprises a structure of A-M-L-Gly _mX _n-M-P, in which A represents the agent, L represents the residual part of a sortase recognition motif after a sortase-mediated reaction, Gly _m represents m glycines with m preferably being 1-5, X _n represents n spacing amino acids with n preferably being 0-10, M represents the residual part of the bifunctional crosslinker after crosslinking, and P represents the at least one membrane protein of the cell.

In some embodiments, the bifunctional crosslinker is an amine-sulfhydryl type, preferably maleimido carbonic acid (C _2-8) , e.g., 6-Maleimidohexanoic acid and 4-Maleimidobutyric acid.

In some embodiments, the bifunctional crosslinker crosslinks said side chain amino group and at least one exposed sulfhydryl of the at least one membrane protein.

In some embodiments, the sortase recognition motif comprises, or 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 located at position 5 from the direction of N-terminal to C-terminal of the sortase recognition motif, wherein the unnatural amino acid is an optionally substituted hydroxyl carboxylic acid having a formula of CH ₂OH- (CH ₂) _n-COOH, with n being an integer from 0 to 3, and preferably n = 0.

In some embodiments, the sortase recognition motif comprises, or 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 the optionally substituted hydroxyl carboxylic acid, and X and Y independently represent any amino acid.

In some embodiments, the sortase recognition motif comprises, or 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 preferably is LPET*G with *being 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, with X being any amino acid.

In some embodiments, the agent A comprises an exposed sulfydryl, preferably an exposed cysteine, more preferably a terminal cysteine, and most preferably a C-terminal cysteine.

In some embodiments, the agent, from N-terminus to C-terminus, comprises an HPV antigenic peptide, an optional first peptide linker, and an MHC-I. In some further embodiments, the agent, from N-terminus to C-terminus, comprises an HPV antigenic peptide, an optional first peptide linker, a β2-microglobulin (such as human β2-microglobulin) , an optional second peptide linker, and an MHC-I heavy chain (preferably the MHC-I heavy chain lacking transmembrane region and cytoplastic region) .

In some embodiments, the HPV antigenic peptide is bound 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.

In some embodiments, the HPV antigenic peptide is an HPV16 antigenic peptide and preferably comprises or consists 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.

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

In some embodiments, the cell is selected from the enucleated cells, preferably the red blood cells.

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

a) Providing a sortase substrate that comprises a structure of A-M-L’, in which A represents the agent, L’ represents the sortase recognition motif, and M represents the residual part of the bifunctional crosslinker after crosslinking;

b) (i) providing a peptide having the formula Gly _mX _n-M’, in which Gly _m represents m glycines with m preferably being 1-5, and X _n represents n spacing amino acids with n preferably being 0-10, and M’ represents the bifunctional crosslinker;

(ii) treating a cell with the Gly _mX _n-M’ peptide under a condition to link the Gly _mX _n-M’ peptide to at least one membrane protein of the cell to produce Gly _mX _n-M-P, in which M represents the residual part of the bifunctional crosslinker after crosslinking, P represents the 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 the Gly _m by a sortase-mediated reaction;

a) and b) can be carried out at the same time, or a) before b) , or a) after b) .

In some embodiments, before the treating step, the method further comprises a step of pretreating the cell with a reducing agent to form an exposed sulfhydryl.

In some embodiments, 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.

In some embodiments, the sortase-mediated reaction forms an agent linked to at least one membrane protein of the cell, comprising a structure of A-M-L-Gly _mX _n-M-P, in which A represents the agent, L ¹represents the residual part of a sortase recognition motif after the sortase-mediated reaction, Gly _m represents m glycines with m preferably being 1-5, X _n represents n spacing amino acids with n preferably being 0-10, M represents the residual part of the bifunctional crosslinker after crosslinking, and P represents the at least one membrane protein of the cell.

In some embodiments, the sortase recognition motif comprises, or 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 the optionally substituted hydroxyl carboxylic acid; and X and Y independently represent any amino acid.

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

In a fourth aspect, the 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 disclosure provides a method for diagnosing, treating, or preventing an HPV infection associated disease.

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

In a sixth aspect, the disclosure provides a method of delivering an agent 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 disclosure provides a method of increasing the circulation time or plasma half-life of an agent in a subject, comprising attaching the agent to a cell according to the method of the second aspect.

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

In some embodiments, the medicament is a vaccine.

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

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, embodiments of the present disclosure are illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and as an aid to understanding the present disclosure, and are not intended as a definition of the limits of the invention.

Fig. 1 illustrate an exemplary process for labeling cells with peptide in vitro according to the present disclosure.

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

Fig. 3 shows efficient conjugation of HPV16 (YMLDLQPET) -hMHC1 on RBC in vitro according to one illustrative embodiment of the present disclosure.

Fig. 4 shows HPV16 (YMLDLQPET) -hMHC1-RBCs elicited IFN-γ responses in patients’ PBMCs as measured by IFN-γ ELISpot assay.

Fig. 5 shows cytotoxic potential of HPV16 (YMLDLQPET) -hMHC1-RBCs-generated immune responses as measured by the expression of the fluorochrome-conjugated MHC Tetramer (A) 4-1BB and CD107a (B) on CD8+ T cell surface.

Fig. 6 shows survival of the Caski cells after T cell cytotoxicity assay.

Fig. 7 shows survival of the HPV16-MC38 after T cell cytotoxicity assay (A: different time point; B: Splenocytes from different mice) .

Fig. 8 shows tumor growth in animals receiving repeated transfusion of HPV16 (KCLKFYSKI) -mMHC1-RBCs.

Fig. 9 shows the structural formula of 6-Maleimidohexanoic 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 the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.

In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings as generally understood by a person skilled in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined herein are more fully described by reference to the Specification as a whole.

As used herein, the singular terms "a, " "an, " and "the" include the plural reference unless the context clearly indicates otherwise.

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

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 context in which they are used by those of skills in the art.

As used herein, the term “consisting essentially of” in the context of an amino acid sequence is meant the recited amino acid sequence together with additional one, two, three, four, or five amino acids at the N-or C-terminus.

Unless the context requires otherwise, the terms “comprise, ” “comprises, ” and “comprising, ” or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.

As used herein, the terms “patient, ” “individual, ” and “subject” are used in the context of any mammalian recipient of a treatment or composition disclosed herein. Accordingly, the methods and composition 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 meant to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size) , and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) .

As used herein, the term “conjugated” or “conjugation” refers to an association of two molecules, for example, two proteins or a protein and a small molecule or other entity, with one another in a way that they are linked by a direct or indirect covalent or non-covalent interaction.

The inventors have herein developed a new strategy to modify cells, e.g., blood cells, especially natural RBCs, with an agent, e.g., peptides and/or small molecules through a sortase-mediated reaction. The technology allows for producing cell products by directly modifying natural cells such as RBCs instead of HSPCs, which are limited by their resources, at a very high labelling efficiency. Also, the modified cells preserve their original biological properties well and remain as stable as their native state. In some embodiments, the labelled red blood cells have the same lifespan as native RBCs and sustained signals in circulation for up to 28 days or longer.

Cells

In one aspect, the present disclosure provides a cell having an agent linked thereto, wherein the agent is linked to at least one membrane protein of the cell via a sortase recognition motif. To increase the labelling efficiency of a sortase-mediated reaction, a linker comprising an N-terminal glycine is conjugated, via a bifunctional crosslinker such as an amine-sulfhydryl type bifunctional crosslinker, to at least one membrane protein of a cell, preferably to at least one exposed sulfhydryl of the at least one membrane protein.

Contemplated in the present disclosure are various living animal cells, such as mammalian cells, e.g., various blood cells, including red blood cells, T cells, B cells, monocytes, NK cellsn and megakaryocytes. In some embodiments the animal cell is a mammalian cell, e.g., a human cell. In some embodiments the cell is an immune system cell, e.g., lymphocytes (e.g., a T cell or NK cell) or dendritic cells. 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 glycines or alanines at its terminus, 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 comprising a sortase recognition motif or a nucleophilic acceptor sequence. In some embodiments the RBC has not been genetically engineered.

The terms “non-engineered, “non-genetically modified, ” and “non-recombinant” as used herein are interchangeable and refer to not being genetically engineered, absence of genetic modification, etc. Non-engineered membrane proteins encompass endogenous proteins. In certain embodiments, a non-genetically engineered red blood cell does not contain a non-endogenous nucleic acid, e.g., DNA or RNA that originates from a vector, from a different species, or that comprises an artificial sequence, e.g., DNA or RNA that was introduced artificially. In certain embodiments, a non-engineered cell has not been intentionally contacted with a nucleic acid that is capable of causing a heritable genetic alteration under conditions suitable for uptake of the nucleic acid by the cells.

In some embodiments, the cells have not been genetically engineered for sortagging. A cell is considered “not genetically engineered for sortagging” if the cell has not been genetically engineered to express a protein comprising a sortase recognition motif or a nucleophilic acceptor sequence in a sortase-catalyzed reaction.

In some aspects, the present disclosure provides a cell having an agent linked thereto, wherein the agent is linked to at least one membrane protein of the cell via a sortase recognition motif, and the agent linked to the at least one membrane protein comprises a structure of A-M-L-Gly _mX _n-M-P, in which A represents the agent, L represents the residual part of a sortase recognition motif after a sortase-mediated reaction, Gly _m represents m glycines with m preferably being 1-5, X _n represents n spacing amino acids with n preferably being 0-10, M represents the residual part of the bifunctional crosslinker after crosslinking, and P represents the at least one membrane protein of the cell.

In some embodiments, the present disclosure provides a cell having an agent linked thereto as described herein. In some embodiments, a composition comprising a plurality of such cells is provided. In some embodiments, at least a selected percentage of the cells in the composition are modified, e.g., by having an agent linked 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 an agent linked thereto. In some embodiments, the linked agent may be one or more of the agents described herein.

In some embodiments, the present disclosure provides a cell that comprises an agent linked to the 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 _mX _n, wherein Gly _m represents m glycines with m preferably being 1-5, and X _n represents n spacing amino acids with n preferably being 0-10) . In some embodiments, the agents linked may be the same or the cell may be sortagged with a plurality of different agents.

In some embodiments, the agent is linked via a sortase recognition motif to a linker comprising a terminal glycine such as an N-terminal glycine (e.g. a linker having a structure of Gly _mX _n, wherein Gly _m represents m glycines with m preferably being 1-5, and X _n represents n spacing amino acids with n preferably being 0-10) that is crosslinked to at least one membrane protein of a cell. In some embodiments, the sortase recognition motif may 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 located at position 5 from the direction of N-terminal to C-terminal of the sortase recognition motif, wherein the unnatural amino acid is an optionally substituted hydroxyl carboxylic acid having a formulae of CH ₂OH- (CH ₂) _n-COOH, with n being an integer from 0 to 3, 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 the optionally substituted hydroxyl carboxylic 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 preferably is LPET*G with *preferably being 2-hydroxyacetic acid.

It can be understood that after the agent is linked to the membrane protein, the last one or two residues from 5 ^th position (from the direction of N-terminal to C-terminal) of the sortase recognition motif is replaced by the amino acid on which the linkage occurs, as described elsewhere herein. For example, the agent linked to the membrane protein may comprise a structure of A-M-L-Gly _mX _n-M-P, in which L represents the residual part of a sortase recognition motif after a sortase-mediated reaction, and may 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 using sortase to attach or conjugate or link a sortase substrate to the membrane protein of the cells. The cell (e.g., RBCs) may, for example, have been genetically engineered to express any of a wide variety of products, e.g., polypeptides or noncoding RNAs, may be genetically engineered to have a deletion of at least a portion of one or more genes, and/or may be genetically engineered to have one or more precise alterations in the sequence of one or more endogenous genes. In certain embodiments, such a genetically engineered cell is sortagged with any of the various agents described herein according to the method described herein.

In some embodiments, the present disclosure contemplates using autologous cells e.g., red blood cells, that are isolated from an individual to whom such isolated cells, after being modified in vitro, are to be administered. In some embodiments, the present disclosure contemplates using immuno-compatible red blood cells that are of the same blood group as an individual to whom such cells are to be administered (e.g., at least with respect to the ABO blood type system and, in some embodiments, with respect to the D blood group system) or may be of a compatible blood group.

A linker comprising a 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 Gly _mX _n, wherein Gly _m represents m glycines with m preferably being 1-5, and X _n represents n spacing amino acids with n preferably being 0-10) is linked via a first bifunctional crosslinker to at least one membrane protein of the cell.

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

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

In some embodiments, the bifunctional crosslinker may include but are 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-sulfhydryl type such as an NHS ester-maleimide heterobifunctional crosslinker; Maleimido carbonic acid (C _2-8) (e.g., 6-Maleimidohexanoic 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 sulfo-SMPB; GMBS and sulfo-GMBS; SIAX and SIAXX; SIAC and SIACX; NPIA; (3) homobifunctional NHS esters type (e.g., DSP; DTSSP; DSS; DST and Sulfo-DST; BSOCOES and Sulfo-BSOCOES; EGS and Sulfo-EGS) ; (4) homobifunctional imidoesters type (e.g., DMA; DMP; DMS; DTBP) ; (5) carbonyl-sulfydryl type (e.g., KMUH; EMCH; MPBH; M2C2H; PDPH) ; (6) sulfhydryl reactive type (e.g., DPDPB; BMH; HBVS) ; (7) sulfhydryl-hydroxy type (e.g., PMPI) ; or the like.

In some embodiments, the bifunctional crosslinker is an amine-sulfhydryl type, preferably maleimido carbonic acid (C _2-8) , e.g., 6-Maleimidohexanoic acid and 4-Maleimidobutyric acid. In some further 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 X _n is an amino acid having a side chain amino group, which enables the crosslinking reaction to occur between the side chain amino group and at least one exposed sulfhydryl of the at least one membrane protein.

Sortase

Enzymes identified as “sortases” have been isolated from a variety of Gram-positive bacteria. Sortases, 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) ) . Sortases have been classified into 4 classes, designated A, B, C, and D, based on sequence alignment and phylogenetic analysis of 61 sortases from Gram-positive bacterial genomes (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 a sortase to the correct class based on its sequence and/or other characteristics such as those described in Drami, et al., supra. The term “sortase A” as used herein refers to a class A sortase, usually named SrtA in any particular bacterial species, e.g., SrtA from S. aureus or S. pyogenes.

The term “sortase, ” also known as transamidases, refers to an enzyme that has transamidase activity. Sortases recognize substrates comprising a sortase recognition motif, e.g., the amino acid sequence LPXTG. A molecule recognized by a sortase (i.e., comprising a sortase recognition motif) is sometimes termed a “sortase substrate” herein. Sortases tolerate a wide variety of moieties in proximity to the cleavage site, thus allowing for the versatile conjugation of diverse entities so long as the substrate contains a suitably exposed sortase recognition motif and a suitable nucleophile is available. The terms “sortase-mediated transacylation reaction, ” “sortase-catalyzed transacylation reaction, ” “sortase-mediated reaction, ” “sortase-catalyzed reaction, ” “sortase reaction, ” “sortase-mediated transpeptide reaction, ” and like terms are used interchangeably herein to refer to such a reaction. The terms “sortase recognition motif, ” “sortase recognition sequence, ” and “transamidase recognition sequence, ” with respect to sequences recognized by a transamidase or sortase, are used interchangeably herein. The term “nucleophilic acceptor sequence” refers to an amino acid sequence capable of serving as a nucleophile in a sortase-catalyzed reaction, e.g., a sequence comprising an N-terminal glycine (e.g., 1, 2, 3, 4, or 5 N-terminal glycines) .

The present disclosure encompasses embodiments relating to any of the sortase classes known in the art (e.g., a sortase A, B, C, or D from any bacterial species or strain) . In some embodiments, sortase A is used, such as SrtA from S. aureus. In some embodiments two or more sortases may be used. In some embodiments the sortases may utilize different sortase recognition sequences and/or different nucleophilic acceptor sequences.

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

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 may be any amino acids, such as those selected from D, E, A, N, Q, K, or R in certain embodiments.

In some embodiments, the sortase may recognize a motif comprising an unnatural amino acid, preferably located at position 5 from the direction of N-terminal to C-terminal of the sortase recognition motif. In some embodiments, the unnatural amino acid is a substituted or unsubstituted hydroxyl carboxylic acid having a formulae of CH ₂OH- (CH ₂) _n-COOH, with n being an integer from 0 to 5, e.g., 0, 1, 2, 3, 4, and 5, and preferably n = 0. In some embodiments, the unnatural amino acid is a substituted hydroxyl carboxylic acid and in some further embodiments, the hydroxyl carboxylic acid is substituted by one or more substituents selected from halo, C _1-6 alkyl, C _1-6 haloalkyl, hydroxyl, C _1-6 alkoxy, and C _1-6 haloalkoxy. The term “halo” or “halogen” means fluoro, chloro, bromo, or iodo, and preferred are fluoro and chloro. The term "alkyl" by itself or as part of another substituent refers to a hydrocarbyl radical of Formula C _nH _2n+1 wherein n is a number greater than or equal to 1. In some embodiments, alkyl groups useful in the present disclosure comprise from 1 to 6 carbon atoms, preferably from 1 to 4 carbon atoms, more preferably from 1 to 3 carbon atoms, still more preferably 1 to 2 carbon atoms. Alkyl groups may be linear or branched and may be further substituted as indicated herein. C _x-y alkyl refers to alkyl groups which comprise from x to y carbon atoms. Suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, tert-butyl, pentyl and its isomers (e.g. n-pentyl, iso-pentyl) , and hexyl and its isomers (e.g. n-hexyl, iso-hexyl) . Preferred alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, and tert-butyl. The term "haloalkyl" alone or in combination, refers to an alkyl radical having the meaning as defined above, wherein one or more hydrogens are replaced with a halogen as defined above. Non-limiting examples of such haloalkyl radicals include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1, 1, 1-trifluoroethyl, and the like.

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 the optionally substituted hydroxyl carboxylic 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 preferably is LPET*G with *preferably being 2-hydroxyacetic acid.

In some embodiments, the present disclosure contemplates using a variant of a naturally occurring sortase. In some embodiments, the variant is capable of mediating a glycine _(n) conjugation, with n preferably being 1 or 2. Such variants may be produced through processes such as directed evolution, site-specific modification, etc. Considerable structural information regarding sortase enzymes, e.g., sortase A enzymes, is available, including NMR or crystal structures of SrtA alone or 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 S. aureus SrtA have been identified. One of ordinary skill in the art can generate functional variants by, for example, avoiding deletions or substitutions that would disrupt or substantially alter the active site or substrate binding pocket of a sortase. In some embodiments, directed evolution on SrtA can be performed by utilizing the FRET (Fluorescence Resonance Energy Transfer) -based selection assay described in Chen, et al. Sci. Rep. 2016, 6 (1) , 31899. In some embodiments, a functional variant of S. aureus SrtA may be those described in CN106191015A and CN109797194A. In some embodiments, the S. aureus SrtA variant can be a truncated variant with, e.g., 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) amino acids being removed from N-terminus (as compared to the 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 on amino acid positions of D124, Y187, E189, and F200 of D124G, such as Y187L, E189R, and F200L and optionally further comprising one or more mutations of 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 variants have 25 to 60 (e.g., 25, 30, 35, 40, 45, 50, 55, 59, or 60) amino acids being removed from the N-terminus. In some embodiments, the mutated amino acid positions above are numbered according to the numbering of a wild type S. aureus SrtA, e.g., as shown in SEQ ID NO: 1. In some embodiments, the full-length nucleotide sequence of the wild type S. aureus SrtA is shown as in e.g., SEQ ID NO: 2.

SEQ ID NO: 1 (full length, GenBank Accession No.: CAA3829591.1)

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

In some embodiments, as compared to a wild type S. aureus SrtA, the S. aureus SrtA variant may comprise one or more mutations at one or more of the positions corresponding to 94, 105, 108, 124, 160, 165, 187, 189, 190, 196, and 200 of SEQ ID NO: 1. In some embodiments, as compared to a wild type S. aureus SrtA, the S. aureus SrtA variant may comprise one or more mutations corresponding to P94S/R, E105K, E108A, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L. In some embodiments, as compared to a 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 one or more mutations corresponding to E105K and E108A. In certain embodiments, as compared to a wild type S. aureus SrtA, the S. aureus SrtA variant may comprise mutations corresponding to 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 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 of 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, relative to SEQ ID NO: 1, 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 allows the sortase-mediated reaction to be Ca ²⁺ independent. In some embodiments, the S. aureus SrtA variants as described herein may have 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids being removed from N-terminus. In some embodiments, the mutated amino acid positions above are numbered according to the numbering of a full length of a wild type S. aureus 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 of P94S/R, E105K, E108A/Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L relative to SEQ ID NO: 1. In certain embodiments, the S. aureus SrtA variant may 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 variants have 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids being removed from N-terminus. In some embodiments, the mutated amino acid positions above are numbered according to the numbering of a wild type S. aureus SrtA, e.g., as shown in SEQ ID NO: 1.

In some embodiments, the present disclosure contemplates a S. aureus SrtA variant (mg SrtA) comprising or 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 higher) identity to an amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, SEQ ID NO: 3 is a truncated SrtA and the mutations corresponding to wild type SrtA are shown in bold and underlined below. In some embodiments, the SrtA 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 higher) identity to an amino acid sequence as set forth in SEQ ID NO: 3 and comprises the mutations of P94R/S, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L and optionally E105K and/or E108A/Q (numbered according to the numbering of SEQ ID NO: 1) .

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

In some embodiments, the present disclosure provides a nucleic acid encoding the S. aureus SrtA variant, and in some embodiments the nucleic acid is set forth in SEQ ID NO: 4.

SEQ ID NO: 4

In some embodiments, the S. aureus SrtA variant can be a truncated variant with, e.g., 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) amino acids being removed from the N-terminus (as compared to the 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 higher, such as 100%) identity to an amino acid sequence as set forth in SEQ ID NO: 5 or 7. The nucleic acids encoding SEQ ID NOs: 5 and 7 are set forth in SEQ ID NOs: 6 and 8 below.

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

SEQ ID NO: 6

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

SEQ ID NO: 8

In some embodiments, a 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 (Q 108) , 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) (numbered according to the numbering of a wild type SrtA, e.g., SEQ ID NO: 1) , optionally with about 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids being removed from N-terminus of the wild type S. aureus SrtA. For example, in some embodiments a sortase A variant comprises two, three, four, or five of the afore-mentioned mutations relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) . In some embodiments a sortase A variant comprises an S residue at position 94 (S94) or an R residue at position 94 (R94) , and also an N residue at position 160 (N160) , an A residue at position 165 (A165) , and a T residue at position 196 (T196) relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) . For example, in some embodiments, a sortase A variant comprises P94S or P94R, and also D160N, D165A, and K196T relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) . In some embodiments a sortase A variant comprises an S residue at position 94 (S94) or an R residue at position 94 (R94) , and also 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 a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) . For example, in some embodiments a sortase A variant comprises P94S or P94R, and also D160N, D165A, K190E, and K196T relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) . In some embodiments a 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 relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) . In some embodiments a sortase comprises P94R, D160N, D165A, K190E, and K196T relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) . In some embodiments, the S. aureus SrtA variants may have 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids being removed from N-terminus.

In some embodiments, a sortase A variety having higher transamidase activity than a naturally occurring sortase A may be used. In some embodiments the activity of the sortase A variety is at least about 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, or 200 times as high as that of wild type S. aureus sortase A. In some embodiments such a sortase variant is used in a composition or method of the present disclosure. In some embodiments a sortase variant comprises any one or more of the following substitutions relative to a 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 may have 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59 or 60) amino acids being removed from N-terminus.

In some embodiments, the amino acid mutation positions are determined by an alignment of a parent S. aureus SrtA (from which the S. aureus SrtA variant as described herein is derived) with the polypeptide of SEQ ID NO: 1, i.e., the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent S. aureus SrtA. Methods for determining an amino acid position corresponding to a mutation position as described herein is well known in the art. 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 version 3.0.0 or later. Based on above well-known computer programs, it is routine work for those skilled in the art to determine the amino acid position of a polypeptide of interest as 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. Conservative amino acid mutations that will not substantially affect the activity of a protein are well known in the art.

Using sortase, e.g., mg SrtA to covalently label proteins onto cells has broad prospects in scientific research and clinical applications. However, this may have certain constraints: different types of cells have different types of membrane proteins, and the number of proteins containing N-terminal glycine (s) (e.g., G1 for mg SrtA) is also different. The strategy of the present disclosure allows the possibility of using various sortases to modify various cells with agents.

Irreversible linkers

Since a SrtA-mediated protein-cell conjugation is a reversible reaction, to improve the efficiency of cell labeling, it would be beneficial to minimize the occurrence of reverse reactions. One solution to increase the product yield is to increase the concentration of the reaction substrates, but it may be difficult to achieve a very high concentration for macromolecular proteins in practical applications; and even if the high concentration could be reached, the high cost may limit the use of this technology. Another solution is to continuously remove the products from the reaction system so that the reaction will not stop due to equilibrium, but since the reaction is carried out on the cell, product separation may be difficult. The inventors of the present application found that surprisingly for cell labelling, the reverse reaction can be prevented by introducing a hydroxyacetyl-like byproduct that is not a substrate for the reverse reaction, thus rendering the labeling reaction irreversible.

To obtain a hydroxyacetyl-like byproduct, the present disclosure contemplates using a sortase recognition motif comprising an unnatural amino acid, preferably located at position 5 from the direction of N-terminal to C-terminal of the sortase recognition motif. In some embodiments, the unnatural amino acid is a substituted or unsubstituted hydroxyl carboxylic acid having a formulae of CH ₂OH- (CH ₂) _n-COOH, with n being an integer from 0 to 5, e.g., 0, 1, 2, 3, 4 and 5, preferably with 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 the optionally substituted hydroxyl carboxylic 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 a 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 preferably is LPET*G with *preferably being 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 would make the reaction irreversible.

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

In some embodiments, the sortase recognition motif comprising an unnatural amino acid can be conjugated to an agent by various chemical means to generate a desired sortase substrate. These methods may include chemical conjugation with a bifunctional crosslinker or a bifunctional cross-linking agent such as, e.g., an NHS ester-maleimide heterobifunctional crosslinker to connect a primary amine group with a reduced thiol group. Other molecular fusions may be formed between the sortase recognition motif and the agent, for example through a spacer. As used herein, in some embodiments, the term “spacer” refers to the residual part of a bifunctional crosslinker after crosslinking the sortase recognition motif and the agent.

Various chemical conjugation means for attaching the bifunctional crosslinker or spacer 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-sulfhydryl type such as an NHS ester-maleimide heterobifunctional crosslinker; Maleimido carbonic acid (C _2-8) (e.g., 6-Maleimidohexanoic 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 sulfo-SMPB; GMBS and sulfo-GMBS; SIAX and SIAXX; SIAC and SIACX; NPIA; (3) homobifunctional NHS esters type (e.g., DSP; DTSSP; DSS; DST and Sulfo-DST; BSOCOES and Sulfo-BSOCOES; EGS and Sulfo-EGS) ; (4) homobifunctional imidoesters type (e.g., DMA; DMP; DMS; DTBP) ; (5) carbonyl-sulfydryl type (e.g., KMUH; EMCH; MPBH; M2C2H; PDPH) ; (6) sulfhydryl reactive type (e.g., DPDPB; BMH; HBVS) ; (7) sulfhydryl-hydroxy type (e.g., PMPI) ; or the like.

In some embodiments, an amine-sulfhydryl type or an NHS ester-maleimide heterobifunctional crosslinker is a preferred spacer that can be used herein. In certain embodiments, a maleimido carbonic acid (C _2-8) heterobifunctional crosslinker such as 6-Maleimidohexanoic acid and 4-Maleimido butyric acid are particularly useful spacers for the construction of desired sortase substrates. The maleimido carbonic acid (C _2-8) heterobifunctional crosslinker, such as 6-Maleimidohexanoic acid and 4-Maleimido butyric acid, can undergo a Michael addition reaction with an exposed sulfhydryl group, e.g., on an exposed cysteine, but this reaction will not occur with an unexposed cysteine. In one embodiment, 6-Maleimidohexanoic acid was introduced in the irreversible linker of the present disclosure, to obtain 6-Maleimidohexanoic acid-Leu-Pro-Glu-Thr-2-hydroxyacetic acid-Gly, as shown in Fig. 9. In some embodiments, a cysteine residue is or has been added to the C-terminal of the agent to provide an exposed cysteine.

By using the spacers as described herein, especially maleimido carbonic acid (C _2-8) heterobifunctional crosslinkers, such as 6-Maleimidohexanoic acid and 4-Maleimido butyric acid, the inventors successfully designed linkers with different structures, including double forks, triple forks, and multiple forks. These different linkers can be used to label cells such as RBCs according to actual needs, for example to obtain multi-modal therapeutics. In the multi-fork structure design of some embodiments, one or more spacers can be linked to the amino group of the N-terminal amino acid and/or the amino group of the side chain of lysine, and the same or different agents like proteins or polypeptides can be linked to the one or more spacers. This technology could further expand the variety of agents, like proteins, for cell labeling and improve the efficiency of RBC engineering.

Sortase substrates

Substrates suitable for a sortase-mediated conjugation can readily be designed. A sortase substrate may comprises a sortase recognition motif and an agent. For example, an agent such as a polypeptide can be modified to include a sortase recognition motif at or near the C-terminus, thereby allowing it to serve as a substrate for sortase. The sortase recognition motif need not be positioned at the very C-terminus of a substrate but should typically be sufficiently accessible by the enzyme to participate in the sortase reaction. In some embodiments, a sortase recognition motif is considered to be “near” a C-terminus if there are no more than 5, 6, 7, 8, 9, or 10 amino acids between the most N-terminal amino acid in the sortase recognition motif (e.g., L) and the C-terminal amino acid of the polypeptide. A polypeptide comprising a sortase recognition motif may be modified by incorporating or attaching any of a wide 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 a structure of A-M-L’, in which A represents an agent, M represents one or more optional spacers, and L’ represents a sortase recognition motif comprising an unnatural amino acid as set forth herein. In some embodiments, the one or more M is selected from the group consisting of the following types of crosslinkers: (1) zero-length type; (2) amine-sulfhydryl type; (3) homobifunctional NHS esters type; (4) homobifunctional imidoesters type; (5) carbonyl-sulfydryl type; (6) sulfhydryl reactive type; and (7) sulfhydryl-hydroxy type; preferably the one or more M is a maleimido carbonic acid (C _2-8) type heterobifunctional crosslinker, such as 6-Maleimidohexanoic acid and 4-Maleimidobutyric acid, and the agent comprises an exposed sulfydryl, preferably an exposed cysteine, more preferably a terminal cysteine, most preferably a C-terminal cysteine. In some embodiments, when two or more spacers are present, the agents linked to the spacers can be the same or different.

Agents

In some embodiments, the agent comprises an HPV antigenic peptide. In some further embodiments, the HPV antigenic peptide is bound 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 which is capable of binding to the peptide-binding cleft of an MHC molecule (especially a class I MHC molecule) , and thus forming an MHC complex with the MHC molecule. As is well known in the art, the presentation of the antigenic peptide in the MHC complex on the surface of an APC generally does not involve, e.g., a whole protein. Rather, an antigenic peptide located in the cleft is typically a small fragment of the whole protein. In some embodiments, the antigenic peptide is derived from a pathogen protein, such as a virus protein. In some embodiments, the antigenic peptide is a cancer neoantigen. In some embodiments, the term “antigenic peptide” includes tumor antigenic peptides which are derived from tumor-specific changes in proteins. Tumor antigenic peptides encompass, but are not limited to, tumor antigens arising from, for example, substitution in the protein sequence, frame shift mutation, in-frame deletion, insertion, and tumor-specific overexpression of polypeptides. The term “tumor-specific changes” refers to changes that are not present in a normal non-cancerous cell but is found in cancer or tumor cells.

In some embodiments, the agent is a fusion protein which, from N-terminus to C-terminus, comprises an antigenic peptide, an optional first peptide linker, and an MHC-I. In some further embodiments, the agent is a fusion protein which, from N-terminus to C-terminus, comprises an antigenic peptide, an optional first peptide linker, a β2-microglobulin (namely, an MHC-I light chain) , an optional second peptide linker, and an MHC-I heavy chain (preferably the MHC-I heavy chain lacking transmembrane region and cytoplastic region) . In some embodiments, the first peptide linkers and the second peptide linker are independently a linker rich in Gly and/or Ser.

Herein the fusion protein can be called as “an artificial MHC single chain molecule, ” which refers to a fusion protein comprising an antigenic peptide, a beta-2 microglobulin, and an MHC class I heavy chain. Such fusion proteins are also called “single chain trimer” in some literatures. Generally, there is a peptide linker between the antigenic peptide and the beta-2 microglobulin (namely, an MHC-I light chain) , and a peptide linker between the beta-2 microglobulin and the MHC class I heavy chain. An artificial MHC single chain molecule therefore may comprise from N-terminus to C-terminus an antigenic peptide, a first peptide linker, a beta-2 microglobulin, a second peptide linker, and an MHC class I heavy chain. In some configurations, an artificial MHC single chain molecule can further comprise a signal peptide at the N-terminus (e.g., beta-2 microglobulin signal peptide) . In some configurations, the MHC class I heavy chain moiety in the artificial MHC single chain molecule is a truncated version, lacking the transmembrane region and the cytoplastic region.

In some embodiments, the antigenic peptide is an HPV antigenic peptide. In some further embodiments, the term “an HPV antigenic peptide” refers to an antigenic peptide which is derived from tumor-specific changes in proteins of HPVs such as HPV16 or HPV18. The two primary oncoproteins of high-risk HPV types are E6 and E7. The “E” designation indicates that these two proteins are early proteins (expressed early in the HPV life cycle) . The HPV genome comprise, six early (E1, E2, E4, E5, E6, and E7) open reading frames (ORF) . After the host cell is infected, the viral early promoter is activated and a polycistronic primary RNA containing all six early ORFs is transcribed. This polycistronic RNA then undergoes active RNA splicing to generate multiple isoforms of mRNAs. Viral early transcription of E2 initiates viral E2 regulation, as high E2 levels repress transcription. HPV genomes integrate into the host genome by disruption of the E2 ORF, preventing E2 repression of E6 and E7. Thus, viral genome integration into the host DNA genome increases E6 and E7 expression to promote cellular proliferation and the chance of malignancy. The degree to which E6 and E7 are expressed is correlated with the type of cervical lesion that can ultimately develop. In some embodiments, the HPV antigenic peptide is an E6 or E7 antigenic peptide of HPV16 or HPV18. In some embodiments, the HPV antigenic peptide is an HPV16-E6 antigenic peptide, an HPV16-E7 antigenic peptide, an HPV18-E6 antigenic peptide, or an HPV18-E7 antigenic peptide, or a combination thereof. In some embodiments, the HPV antigenic peptide comprises or consists of an amino acid 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 (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 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 the group consisting of SEQ ID NOs: 9-48.

In one aspect, the present disclosure provides a red blood cell (RBC) having one or more HPV antigenic peptides or a variant thereof linked thereto. In some embodiments, the RBC comprises more than one (e.g., two, three, four, five, or more) polypeptides, each comprising at least one HPV antigenic peptide or a variant thereof. In some embodiments, the cells described herein comprise more than one type of HPV antigenic peptides, wherein each peptide comprises an HPV antigenic peptide, and wherein the HPV antigenic peptides are not the same (e.g., the HPV antigenic peptides may comprise different types of antigenic peptides from different oncoproteins of different HPVs, or variants of the same type of HPV antigenic peptide) . For example, in some embodiments, the RBC may comprise a first HPV antigenic peptide or a variant thereof, and a second HPV antigenic peptide.

As used herein, the term “Major Histocompatibility Complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. There are two major types of MHC molecules, namely MHC class I (referred to as “MHC-I, ” “MHC I, ” or “MHC1” ) and MHC class II (referred to as “MHC-II, ” “MHC II, ” or “MHC2” ) . In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. In some embodiments, the MHC complex is HLA class I. In some embodiments, HLA class I may be selected from HLA A0201, HLA A2402, HLA B07, HLA B18, HLA B35, or HLA B44, e.g., HLA A0201. In some embodiments, the HPV antigenic peptide is bound to an MHC class I protein, or an allelic variant of the MHC class I protein. In some embodiments, the allelic variant has up to about 10 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions when compared to the MHC class I protein. In some embodiments, the allelic variant is the same serotype as the MHC class I protein. In some embodiments, the allelic variant is a different serotype than the MHC class I protein.

In some embodiments, the HPV antigenic peptide is bound to an MHC class I protein (such as HLA-A02, for example HLA-A*02: 01, HLA-A*02: 03 (GenBank Accession No.: AAA03604) , HLA-A*02: 05 (GenBank Accession No.: AAA03603) , HLA-A*02: 06 (GenBank Accession No.: CCB78868) , HLA-A*02: 07 (GenBank Accession No.: ACR55712) , and HLA-A*02: 11 (GenBank Accession No.: CAB56609) .

In some embodiments, the agent comprises the HPV antigenic peptide and MHC-I, and in some further embodiments, the MHC-I is bound to the C-terminus of the HPV antigenic peptide.

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

In some embodiments, the beta-2 microglobulin used in the present invention is a human beta-2 microglobulin. In some embodiments, the beta-2 microglobulin has an amino acid sequence as shown in SEQ ID NO: 51 below:

Methods for modifying Cells

Sortase can recognize specific sortase recognition motifs, like sequence LPXTG, and connect the LPXTG at the C-terminus of a protein with the G at the N-terminus of another protein through a transpeptidation reaction. This principle can be used to modify an agent of interest so that the agent can be attached to a linker comprising a terminal glycine that has been linked to at least one membrane protein of a cell, such as an RBC.

In an aspect, the present disclosure provides a method for modifying a cell, comprising: a) Providing a sortase substrate that comprises a structure of A-M-L’, in which A represents the agent, L’ represents the sortase recognition motif, and M represents the residual part of the bifunctional crosslinker after crosslinking; b) (i) providing a peptide having the formula Gly _mX _n-M’, in which Gly _m represents m glycines with m preferably being 1-5, and X _n represents n spacing amino acids with n preferably being 0-10, and M’ represents the bifunctional crosslinker; (ii) treating a cell with the Gly _mX _n-M’ peptide under a condition to link the Gly _mX _n-M’ peptide to at least one membrane protein of the cell to produce Gly _mX _n-M-P, in which M represents the residual part of the bifunctional crosslinker after crosslinking, P represents the 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 the Gly _m by a sortase-mediated reaction. a) and b) can be carried out at the same time, or a) before b) , or a) after b) .

In some embodiments, before the treating step, a step of pre-treating the cell with a reducing agent is performed to form or increase the number of exposed sulfhydryls, when sulfhydryl is one of the reactive groups of the bifunctional crosslinker to be used. The present disclosure contemplates various reducing agents as long as they are capable of reducing the disulfide linkages within or between surface membrane proteins so as to expose sulfhydryl. In some embodiments, reducing agents that would have no or little negative effect on the viability of the treated cells are used. In some embodiments, a reducing agent such as tris (2-carboxyethyl) phosphine hydrochloride (TCEP) or dithiothreitol (DTT) or β-mercaptoethanol can be used, e.g., under partial or total reducing conditions.

It would be appreciated that those of ordinary skill in the art are able to select conditions (e.g., optimal temperature, pH, reaction time, concentration) suitable for the sortase to attach the sortase substrate to a linker comprising a terminal glycine according to the nature of sortase substrate, the type of sortase, and the like.

It would be also appreciated that those of ordinary skill in the art are able to select suitable conditions (e.g., optimal temperature, pH, reaction time, concentration) for linking a linker comprising a terminal glycine to at least one membrane protein of a cell, e.g., blood cells such as RBCs.

Uses

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

As used herein, the term a “HPV infection associated disease” refers to a disease associated with or caused by an HPV infection, such as HPV16 or HPV18 infection. In an embodiment, an HPV infection associated disease 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, “treating, ” “treat, ” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates, or reduces a symptom or pathological sign of a pathogen-associated disease, disorder, or condition after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

As used herein, “preventing, ” “prevent, ” or “prevention” refers to a course of action initiated prior to infection by, or exposure to, a pathogen or molecular components thereof and/or before the onset of a symptom or pathological sign of the disease, disorder, or condition, so as to prevent infection and/or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder, or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder, or condition.

In some embodiments, the method as described herein further comprises administering the conjugated red blood cells to a subject, e.g., directly into the circulatory system, e.g., intravenously, by injection or infusion.

In some embodiments, a subject receives a single dose of cells, or receives multiple doses of cells, e.g., between 2 and 5, 10, 20, or more doses, over a course of treatment. In some embodiments a dose or total cell number may be expressed as cells/kg. For example, a dose may be about 103, 104, 105, 106, 107, 108 cells/kg.

In some embodiments a course of treatment lasts for about 1 week to 12 months or more e.g., 1, 2, 3, or 4 weeks or 2, 3, 4, 5 or 6 months. In some embodiments a subject may be treated about every 2-4 weeks.

One of ordinary skills in the art will appreciate that the number of cells, doses, and/or dosing interval may be selected based on various factors such as the weight, and/or blood volume of the subject, the condition being treated, response of the subject, etc. The exact number of cells required may vary from subject to subject, depending on factors such as the species, age, weight, sex, and general condition of the subject, the severity of the disease or disorder, the particular cell (s) , the identity and activity of agent (s) conjugated to the cells, mode of administration, concurrent therapies, and the like.

Composition

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

In some embodiments, the composition may comprise a plurality of cells such as blood cells, e.g., RBCs. In some embodiments, at least a selected percentage of the cells in the composition are modified, i.e., having an agent conjugated thereto by the method as 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 an agent conjugated thereto. In some embodiments, two or more red blood cells or red blood cell populations conjugated with different agents are included.

In some embodiments, the composition comprises modified cells of the present disclosure, such as blood red cells, wherein the cells are modified with any agent of interest. In some embodiments, a composition comprises an effective amount of cells, e.g., up to about 10 ¹⁴ cells, e.g., 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 may range between any two of the afore-mentioned numbers.

As used herein, the term “an 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 condition or modulating an immune response. In some embodiments a 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 intervening number or range.

As used herein, the term “aphysiologically acceptable carrier” means a solid or liquid filler, diluent, or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, diluents, and excipients well known in the art may be used. These may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water.

It will be appreciated by those skilled in the art that other variations of the embodiments described herein may also be practiced without departing from the scope of the invention. Other modifications are therefore possible.

Although the disclosure has been described and illustrated in exemplary forms with a certain degree of particularity, it is noted that the description and illustrations have been made by way of example only. Numerous changes in the details of construction and combination and arrangement of parts and steps may be made. Accordingly, such changes are intended to be included in the invention, the scope of which is defined by the claims.

EXAMPLES

Methods (see Fig. 1)

1. Expression and purification of HPV16-MHC1 protein

HPV16-MHC cDNA was cloned in pcDNA3.1 vectors. cDNA and Electroporation Buffer were mixed and then placed into the electroporation cuvette. The vectors were electroporated into CHO cells following manufacturer protocols (Ettabiotech) that were optimized for CHO cells. After 7 days, all supernatants were collected by centrifuging at 14000g for 40 min at 4℃ and filtered by 0.22 μM filter.

Four HPV16-MHC1 fusion proteins produced in the Examples.

Fusion protein 1: HPV16-hMHC1-Fc comprising a structure of human β2M signal peptide-HPV16 epitope (SEQ ID NO: 9) -GS linker-human β2M-GS linker-HLA0201-GS linker-human IgG1 Fc-Cys. The amino acid sequence and DNA sequence of Fusion protein 1 are shown in SEQ ID NO: 52 and 53.

Fusion protein 2: HPV16-hMHC1-Fc comprising a structure of human β2M signal peptide-HPV16 epitope (SEQ ID NO: 10) -GS linker-human β2M-GS linker-HLA0201-GS linker-human IgG1 Fc-Cys. The amino acid sequence and DNA sequence of Fusion protein 2 are shown in SEQ ID NO: 54 and 55.

Fusion protein 3: HPV16-mMHC1-His comprising a 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 NO: 56 and 57.

Fusion protein 4: HPV16-mMHC1-Fc comprising a 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 being separated from cells by centrifugation and microfiltration, the supernatant was loaded onto the IMAC Bestarose FF column (Bestchrom, Shanghai, China) with Ni ²⁺ ion equilibrated with binding buffer (20 mM Tris-HCl, 500 mM NaCl, pH7.6) . The column was washed by the binding buffer and then eluted by elution buffer 1 (20 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH7.6) until UV absorbance at 280 nm became stable. The protein was collected with elution buffer 2 (20 mM Tris-HCl, 500 mM NaCl, 100 mM imidazole, pH7.6) .

The protein fraction was then diluted with ddH ₂O (1: 1) , and loaded onto Diamond Mix-Acolumn (Bestchrom, Shanghai, China) equilibrated with binding buffer (10 mM Tris-HCl, 250 mM NaCl, pH7.6) . After being washed by the binding buffer and eluted by elution buffer 1 (13.3 mM Tris-HCl, 337.5 mM NaCl, pH7.6) , the target protein was eluted with elution buffer 2 (20 mM Tris-HCl, 2000 mM NaCl, pH7.6) , and then concentrated with Amicon Ultra-15 Centrifugal Filter Unit (Millipore, Darmstadt, Germany) .

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

2. Irreversible linker conjugation to HPV16-MHC1 by cysteine coupling

Irreversible linker, 6-Maleimidohexanoic Acid-Leu-Pro-Glu-Thr-2-hydroxyacetic acid-Gly (6-Maleimidohexanoic Acid-LPET- (2-hydroxyacetic acid) -G, 6-Mal-LPET*G) , was synthesized with more than 99%purity. Reactions were performed in a total volume of 1 mL at room temperature for 1 hr in PBS buffer while being rotated 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 a two-fold molar excess of irreversible linker to HPV16- MHC1 protein. After the reaction, the HPV16-MHC1-6-mal-LPET*G products were collected by removal of excess irreversible linker via dialysis and ultrafiltration.

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

3.1 RBC pretreatment

Red blood cells were separated from peripheral blood by density gradient centrifugation. The separated red blood cells were washed with PBS for 3 times. Then the RBCs were pretreated with 1 mM TCEP for 1 hour at room temperature. Then the pretreated RBCs were washed with PBS for 3 times. GGGSK-6-MAL linker (Synthesized by Beijing Scilight Biotechnology Led. Co. ) (see Fig. 2) was synthesized with more than 99%purity. GGGSK-6-MAL was dissolved in phosphate buffer at 37℃ to a final concentration of 100μM. Then 1×109 RBCs were pretreated with 50 μM GGGSK-6-MAL for 30 mins at 37℃. Then the pretreated RBCs were washed with PBS for 3 times. The pretreated RBCs needed to be used immediately and could not be stored for a long time.

3.2 GGGSK-6-MAL -RBC conjugated with HPV16-MHC1-6-mal-LPET*G via mg sortase mediated reaction

Reactions were performed in a total volume of 200 μL at room temperature for 2 hrs in PBS buffer while being rotated at a speed of 10 rpm. The concentration of GGGSK-6-MAL -RBCs in the reaction was 1×109 /mL. The concentration of mg SrtA (SEQ ID NO: 5) was 10 μM and the concentration of HPV16-MHC1-6-mal-LPET*G substrate was 25μM. After the reaction, the labeling efficiency of RBCs was analyzed by Beckman Coulter CytoFLEX LX.

4. Interferon γ (IFNγ) ELISpot Assay

Human IFN-γ ELISpot Kit (Mabtech) was used according to manufacturer’s instructions. For ex vivo ELISpots, Peripheral blood mononuclear cells (PBMC) isolated from HPV16 ⁺ patients were plated in duplicate with 2 × 10 ⁵ cells per well. Modified RBCs were plated with 10 ⁷ cells per well. HLA-A2 restricted HPV16 E6/E7-derived peptides were added to ELISpot wells at 20 μg/mL. Plates were incubated for 48h at 37℃. Secreted IFNγ was detected by biotinylated anti-IFNγ mAb, and the reaction was developed with streptavidin-HRP and the color reagent BCIP/NBT. The number of specific IFNγ-secreting T cells was calculated.

5. Cytotoxic potential of HPV16-MHC1-RBCs-generated immune responses in vitro

The HLA-A*0201-positive, HPV-16-positive human cervical carcinoma cell line and HPV16-MC38 (transduced to express the E6, E7 oncogene of HPV-16) cell line were used in this study.

CaSki, obtained from ATCC (CRL-1550TM) , was HPV-16 genome integrated. PBMCs from HPV16 ⁺ patients were incubated with HPV16 (YMLDLQPET) -MHC1-RBCs (PBMCs: engineered RBCs=1: 50) for 72h at 37℃. After the stimulation, the patient PBMCs were enriched for CD8 ⁺ T cells using the CD8 ⁺ T Cell isolation kit (Stem cell) . Isolated CD8 ⁺T cells were co-cultured with target CaSki cells for 72h (effector: target=100: 1) . Cytotoxic potential of HPV16 (YMLDLQPET) -MHC1-RBCs-generated immune responses was measured by the expression of 4-1BB, CD107a and fluorochrome-conjugated MHC tetramer on CD8+ T cell surface and the survival of CaSki cells.

Splenocytes from HPV16-MC38 tumor bearing mice were incubated with HPV16 (KCLKFYSKI) -mMHC1-RBCs (splenocytes: engineered RBCs=1: 50) for 72h at 37℃. After the stimulation, the splenocytes were enriched for CD8 ⁺ T cells using the CD8 ⁺ T Cell isolation kit (Stem cell) . Isolated CD8 ⁺ T cells were co-cultured with target HPV16-MC38 cells for 72h (effector: target=100: 1) . Cytotoxic potential of HPV16 (KCLKFYSKI) -mMHC1-RBCs-generated immune responses was measured by the expression of CD107a and fluorochrome-conjugated MHC Tetramer on CD8+ T cell surface and the survival of HPV16-MC38 cells.

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

For immunotherapy, the mice were first injected s. c. in the left rear flank with 10 ⁵ HPV16-MC38 cells on D0. The mice were divided into 2 groups: (1) Control RBCs therapy; (2) HPV16 (KCLKFYSKI) -mMHC1-RBCs therapy. The HPV16 (KCLKFYSKI) -mMHC1-RBCs were administered on

Day

1, 4, 7, 10, 13, 16. Body weight and tumor volume were measured every three days. The tumor volume was calculated using the empirical formula V = 1/2 × [ (shortest diameter) ² × (the longest diameter) ] . After 4 weeks of treatment, the mice were killed and the tumors were weighed and processed for IHC analysis.

Results

Labeling efficiency

We characterized the efficacy of mg SrtA-mediated labeling of HPV16 (YMLDLQPET) -hMHC1 on RBC membranes. The conjugation efficacy was detected by incubating the labeled RBCs with PE-conjugated anti Fc tag antibody and analyzed by flow cytometry. The results in Fig. 3 showed that > 99%of natural human RBCs were HPV16 (YMLDLQPET) -hMHC1-labeled by mg SrtA in vitro. In contrast, no significant Fc tag signal was detected on the surface of human RBCs by the mock control group without mg SrtA enzyme. The labeling efficacy was also significantly higher than the efficacy of mg SrtA-mediated labeling of HPV16 (YMLDLQPET) -hMHC1 on RBCs that were not pre-treated with GGGSK-mal.

Immunogenicity

The immunogenicity of HPV16 (YMLDLQPET) -hMHC1-RBCs was evaluated in the peripheral blood by IFN-γ ELISpot assay. HPV16 (YMLDLQPET) peptides, control RBCs, and HPV16 (YMLDLQPET) -hMHC1-RBCs were analyzed across 3 HPV16 ⁺cervical cancer patients and 2 healthy donors. As shown in Fig. 4, peripheral immune responses reactive against HPV16 (YMLDLQPET) -hMHC1-RBCs were detected in patients, but the immune response to HPV16 (YMLDLQPET) -hMHC1-RBCs in the periphery of healthy donors were rarely detected. In conclusion, the stimulation with HPV16 (YMLDLQPET) -hMHC1-RBCs resulted in the generation of T cell responses that were intense and specific.

Cytotoxic potential

The cytotoxic phenotype of neoantigen-specific CD8 ⁺ T cells in the peripheral blood was determined by the surface expression of 4-1BB, CD107a, and fluorochrome-conjugated MHC tetramer, when assayed in the presence of the HPV16 (YMLDLQPET) -hMHC1-RBCs. Fig. 5 showed the surface expression of 4-1BB, CD107a, and MHC tetramer on patients’ CD8 T cells that were co-cultured with HPV16 (YMLDLQPET) -hMHC1-RBCs.

The cytotoxicity assay was based on the survival of Caski target cells co-cultured with pre-treatment patient PBMCs stimulated with HPV16 (YMLDLQPET) -hMHC1-RBCs. The data in Fig. 6 demonstrated that HPV16 (YMLDLQPET) -hMHC1-RBCs generated cytotoxic T cells that kill cancers.

Th cytotoxicity assay was based on the survival of HPV16 (KCLKFYSKI) -MC38 targets cells co-cultured with pre-treatment Splenocytes from HPV16-MC38 tumor bearing mice. The data in Fig. 7 demonstrated that HPV16 (KCLKFYSKI) -mMHC1-RBCs generated cytotoxic T cells that kill cancers.

Tumor growth

In therapeutic studies, animals receiving repeated transfusion of HPV16 (KCLKFYSKI) -mMHC1-RBCs had significantly delayed tumor growth (p＜0.01) , as shown in Fig. 8.

While embodiments of the invention have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation and it is understood that various changes may be made without departing from the spirit and scope of the invention.

Claims

A cell having an agent linked thereto, wherein the agent is linked to at least one membrane protein of the cell via a sortase recognition motif, and the agent linked to the at least one membrane protein comprises a structure of A-M-L-Gly _mX _n-M-P, in which A represents the agent, L represents the residual part of a sortase recognition motif after a sortase-mediated reaction, Gly _m represents m glycines with m preferably being 1-5, X _n represents n spacing amino acids with n preferably being 0-10, M represents the residual part of the bifunctional crosslinker after crosslinking, and P represents the at least one membrane protein of the cell.
The cell of claim 1, wherein the bifunctional crosslinker is an amine-sulfhydryl type, preferably maleimido carbonic acid (C _2-8) , e.g., 6-Maleimidohexanoic acid and 4-Maleimidobutyric acid.
The cell of 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.
The cell of claim 3, wherein M crosslinks said side chain amino group and at least one exposed sulfhydryl of the at least one membrane protein.
The cell of any of claims 1-4, wherein the sortase recognition motif comprises, or 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.
The cell of any of claims 1-4, wherein the sortase recognition motif comprises an unnatural amino acid located at position 5 from the direction of N-terminal to C-terminal of the sortase recognition motif, wherein the unnatural amino acid is an optionally substituted hydroxyl carboxylic acid having a formulae of CH ₂OH- (CH ₂) _n-COOH, with n being an integer from 0 to 3, and preferably n = 0.
The cell of claim 6, wherein the sortase recognition motif comprises, or 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 the optionally substituted hydroxyl carboxylic acid; and X and Y independently represent any amino acid.
The cell of claim 7, wherein the sortase recognition motif comprises, or 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 preferably is LPET*G with *being 2-hydroxyacetic acid.
The cell of any of claims 1-8, wherein L is selected from the group consisting of LPXT, LPXA, LPXS, LPXL, LPXV, LGXT, LAXT, LSXT, NPXT, MPXT, IPXT, SPXT, VPXT, and YPXR, X being any amino acid.
The cell of any of claims 1-9, wherein the agent A comprises an exposed sulfydryl, preferably an exposed cysteine, more preferably a terminal cysteine, most preferably a C-terminal cysteine.
The cell of any of claims 1-10, wherein the agent comprises an HPV antigenic peptide.
The red blood cell of claim 11, wherein the HPV antigenic peptide is bound 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 red blood cell of claim 11 or 12, wherein the HPV antigenic peptide is an HPV16 antigenic peptide and preferably comprises or consists 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.
The cell of any of claims 1-13, wherein the sortase is a Sortase A (SrtA) such as a Staphylococcus aureus transpeptidase A, e.g., Staphylococcus aureus transpeptidase A variant (mgSrtA) .
The cell of any of claims 1-14, wherein the cell is selected from the enucleated cells, preferably the red blood cells.
A method for modifying a cell, comprising:

a) Providing a sortase substrate that comprises a structure of A-M-L’, in which A represents the agent, L’ represents the sortase recognition motif, and M represents the residual part of the bifunctional crosslinker after crosslinking;

b) (i) providing a peptide having the formula Gly _mX _n-M’, in which Gly _m represents m glycines with m preferably being 1-5, and X _n represents n spacing amino acids with n preferably being 0-10, and M’ represents the bifunctional crosslinker;

(ii) treating a cell with the Gly _mX _n-M’ peptide under a condition to link the Gly _mX _n-M’ peptide to at least one membrane protein of the cell to produce Gly _mX _n-M-P, in which M represents the residual part of the bifunctional crosslinker after crosslinking, P represents the 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 the Gly _m by a sortase-mediated reaction;

a) and b) can be carried out at the same time, or a) before b) , or a) after b) .
The method of claim 16, wherein before the treating step, the method further comprises a step of pretreating the cell with a reducing agent to form an exposed sulfhydryl.
The method of claim 16 or 17, wherein the bifunctional crosslinker is an amine-sulfhydryl type, preferably maleimido carbonic acid (C _2-8) , e.g., 6-Maleimidohexanoic acid and 4-Maleimidobutyric acid.
The method of any 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.
The method of claim 19, wherein the bifunctional crosslinker crosslinks said side chain amino group and at least one exposed sulfhydryl of the at least one membrane protein.
The method of any of claims 16-20, wherein the sortase-mediated reaction forms an agent linked to at least one membrane protein of the cell, comprising a structure of A-M-L-Gly _mX _n-M-P, in which A represents the agent, L represents the residual part of a sortase recognition motif after the sortase-mediated reaction, Gly _m and X _n are as defined in claim 16, M represents the residual part of the bifunctional crosslinker after crosslinking, and P represents the at least one membrane protein of the cell.
The method of any of claims 16-21, wherein the sortase recognition motif comprises, or 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.
The method of any of claims 16-21, wherein the sortase recognition motif comprises an unnatural amino acid located at position 5 from the direction of N-terminal to C-terminal of the sortase recognition motif, wherein the unnatural amino acid is an optionally substituted hydroxyl carboxylic acid having a formula of CH ₂OH- (CH ₂) _n-COOH, with n being an integer from 0 to 3, preferably n = 0.
The method of claim 23, wherein the sortase recognition motif comprises, or 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 the optionally substituted hydroxyl carboxylic acid; and X and Y independently represent any amino acid.
The method of claim 24, wherein the sortase recognition motif comprises, or 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 preferably is LPET*G with *being 2-hydroxyacetic acid.
The method of any of claims 16-25, wherein the agent comprises an HPV antigenic peptide.
The method of any of claim 26, wherein the HPV antigenic peptide is bound 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 method of any of claims 26 or 27, wherein the HPV antigenic peptide is an HPV16 antigenic peptide and preferably comprises or consists 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.
The method of any of claims 16-28, wherein the sortase is a Sortase A (SrtA) such as a Staphylococcus aureus transpeptidase A, e.g., Staphylococcus aureus transpeptidase A variant (mgSrtA) .
The method of any of claims 16-28, wherein the cell is selected from the enucleated cells, preferably the red blood cells.
A cell obtained by the method of any of claims 16-30.
A composition comprising the cell of any of claims 1-15 and 31 and optionally a physiologically acceptable carrier.
A method for diagnosing, treating, or preventing an HPV infection associated disease in a subject in need thereof, comprising administering the cell of any of claims 1-15 and 31 or the composition of claim 32 to the subject.
The method of claim 33, wherein the HPV infection associated disease is selected from the group consisting of cervical cancer, anogenital cancer, head and neck cancer, and oropharyngeal cancer.
A method of delivering an agent to a subject in need thereof, comprising administering the cell of any of claims 1-15 and 31 or the composition of claim 32 to the subject.
A method of increasing the circulation time or plasma half-life of an agent in a subject, comprising attaching the agent to a cell according to the method of any of claims 16-30.
Use of the cell of any of claims 1-15 and 31 or the composition of claim 32 in the manufacture of a medicament for diagnosing, treating, or preventing an HPV infection associated disease.
The use of claim 37, wherein the HPV infection associated disease is selected from the group consisting of cervical cancer, anogenital cancer, head and neck cancer, and oropharyngeal cancer.
The use of claim 37 or 38, wherein the medicament is a vaccine.