CN114686428A

CN114686428A - Cell-polypeptide conjugate and preparation method and application thereof

Info

Publication number: CN114686428A
Application number: CN202011609724.8A
Authority: CN
Inventors: 胡俊; 杨哲; 周丽红; 高建伟
Original assignee: Beijing Cominghealth Bio Tec Co ltd
Current assignee: Beijing Cominghealth Bio Tec Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-07-01
Anticipated expiration: 2040-12-30
Also published as: CN114686428B

Abstract

The invention provides a cell-polypeptide conjugate and a preparation method and application thereof, and particularly provides a cell-polypeptide conjugate which comprises an immune effector cell and a polypeptide which is covalently connected to the surface of the immune effector cell membrane and specifically binds gp 96. The cell-polypeptide conjugate can remarkably improve the killing effect of NK cells on tumor cells (especially liver cancer cells or triple negative breast cancer cells).

Description

Cell-polypeptide conjugate and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, and particularly relates to a cell-polypeptide conjugate, a preparation method thereof and application thereof in tumor resistance.

Background

Triple Negative Breast Cancer (TNBC) refers to a breast cancer in which Estrogen Receptor (ER), Progesterone Receptor (PR) and human epidermal growth factor receptor 2(Her-2) are negative, and accounts for about 15% -20% of the pathological types of breast cancer. The triple negative breast cancer has poor prognosis and great harm, no corresponding targeted medicine exists clinically at present, and only the radiotherapy and the chemotherapy have certain curative effect.

NK cells, an important component of innate immunity, have a direct killing effect on tumor cells and exert immunoregulatory functions by secreting cytokines and chemokines. Unlike T cells, NK cells exert killing activity without the need for tumor cells to express tumor antigens, and are more potent in killing tumor cells that express low or no MHC molecules.

Adoptive reinfusion of allogeneic NK cells has been used in clinical trials for the treatment of blood-based tumors (lymphomas, acute leukemias, etc.) and a variety of solid tumors (advanced gastric and colorectal cancers, liver metastases and childhood relapse of gastrointestinal tumors, refractory neuroblastoma, etc.), with good safety, without causing graft-versus-host disease (GVHD), but with only moderate therapeutic effect. Possible causes are lack of targeting of NK cells and insufficient tumor infiltration. In order to improve the targeted killing activity of NK cells, the current research mainly constructs CAR-NK cells targeting tumor antigens through viral vectors, but the CAR vector transfection efficiency of primary NK cells is still low even if a lentivirus infection method is adopted; the virus vector has certain potential safety hazard; the CAR-NK preparation period is long and the cost is high.

Therefore, the development of a non-transfection approach to provide a method for specifically targeted killing of NK cells of triple negative breast cancer is a current focus of research.

Disclosure of Invention

The cell-polypeptide conjugate is obtained by modifying the surface of an NK cell membrane with a specific gp 96-targeted polypeptide, and the killing effect of the NK cell on tumor cells (particularly liver cancer cells or triple negative breast cancer cells) can be remarkably improved.

Cells

The invention provides a cell-polypeptide conjugate comprising an immune effector cell and a polypeptide that specifically binds gp96 covalently attached to the surface of the immune effector cell membrane.

The immune effector cell is a T cell, an NK cell or a macrophage. In some embodiments, the effector cell is an NK cell.

One skilled in the art will appreciate that both primary cells obtained from human tissue and existing cell lines are suitable for engineering to obtain the cell-polypeptide conjugates of the invention. For example, in some embodiments, the NK cells can be derived from primary cells obtained from a subject (e.g., a human).

In some embodiments, the primary NK cell population may be obtained from a sample of a mammalian subject (e.g., a human subject). The sample or source may be cord blood, bone marrow, or peripheral blood.

In some aspects, NK cells expressing one or more NK cell-specific markers are isolated from the population of cells. Methods for the isolation and identification of NK cells are well known in the art and include those discussed in Da hl berg et al, Frontiers in Immunology, Vol.6, No. 605, pp.1-18 (2015).

In some embodiments, NK cells can be identified as those expressing typical human NK cell markers (e.g., KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, CD56, and CD 161).

In some embodiments, NK cells may be selectively enriched using positive or negative selection. For example, NK cells that do not express CD3 can be selected by exposing a mixture of cells to an immobilized anti-CD 3 antibody and removing unbound cells. According to some embodiments of the invention, the NK cells comprise CD56+ CD3 "cells. According to some embodiments of the invention, the NK cells comprise CD56+ CD16+ CD 3-cells.

In some embodiments, the NK cell comprises an NK cell line. In some aspects, the NK cell line allows for the production of a greater number of cells without having to expand a small number of NK cells derived from the subject. Cell lines also have the advantage of being well characterized.

In some embodiments, the cell line is a clonal cell line. In some embodiments, the cell line is derived from a patient having NK cell leukemia or lymphoma. In some embodiments, the NK cell line comprises NK-92, NKYS, KHYG-1, NKL, NKG, SNK-6 or IMC-1.

NK-92 is an NK-like cell line that is initially isolated from the blood of subjects with large granular lymphoma and subsequently proliferated in cell culture. The NK-92 cell line has been described (Gong et al, 1994; Klingemann, 2002). NK-92 cells have a CD3-/CD56+ phenotype unique to NK cells. They express all known NK cell activating receptors except CD16, but lack all known NK cell inhibitory receptors except NKG2A/CD94 and ILT2/LIR1 (expressed at low levels). Furthermore, unlike polyclonal NK cells isolated from blood, NK-92 is a clonal cell line that expresses these receptors in a consistent manner, both in type and cell surface concentration. Similarly, when administered therapeutically to a human subject, NK-92 cells are not immunogenic and do not elicit immune rejection. In fact, NK-92 cells are well tolerated in humans and have no known deleterious effects on normal tissues.

In some embodiments, the immune effector cell has attached to its surface a polypeptide that specifically binds gp96, said polypeptide being selected from the group consisting of:

1) a polypeptide having or comprising the amino acid sequence set forth in SEQ ID No.1 (i.e., P37);

2) a polypeptide having one or more amino acid residue substitutions, deletions, additions or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions, additions or any combination thereof) as compared to SEQ ID No.1, which polypeptide still specifically binds gp 96; preferably, the substitutions are conservative substitutions; and the combination of (a) and (b),

3) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, 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%, at least 99% or 100% sequence identity compared to SEQ ID No. 1.

In some embodiments, the surface of the immune effector cell is modified with an azide group, and the polypeptide is linked to the azide group on the surface of the immune effector cell via a linker, wherein the linker is selected from DBCO, DIBO, and BCN.

Cell population

In another aspect, the invention provides a cell population comprising a plurality of cell-polypeptide conjugates as described above.

In some embodiments, the cell-polypeptide conjugate comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, 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%, at least 99%, or 100% of the total number of the population of cells.

Composition comprising a metal oxide and a metal oxide

In another aspect, provided herein are compositions comprising the cell-polypeptide conjugates. The compositions include pharmaceutical compositions and formulations for administration (e.g., for adoptive cell therapy). In some embodiments, the cell-polypeptide conjugate is formulated with a pharmaceutically acceptable carrier.

In some embodiments, the composition comprises a cell-polypeptide conjugate or cell population as described above, and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers can include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and The like, compatible with pharmaceutical administration (Gennaro,2000, Remington: The science and practice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia, Pa.). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as non-volatile oils, may also be used. Supplementary active compounds may also be incorporated into the compositions. The pharmaceutical carrier should be one suitable for NK cells, such as saline solution, dextrose solution, or a solution comprising human serum albumin.

In some embodiments, the number of cells in the composition provides a cell-polypeptide conjugate in a therapeutically effective amount. In some embodiments, the amount is an amount that reduces the severity, duration, and/or symptoms associated with cancer in the animal. In certain other embodiments, a therapeutically effective amount is a dose of cells that results in at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% reduction in the growth or spread of cancer in a patient or animal administered the composition relative to the growth or spread of cancer in a patient (or animal) or group of patients (or animals) not administered the composition described herein. In some embodiments, the composition comprises a dose of cell-polypeptide conjugate that is from or from about 10⁵To about 10¹²Individual cell, or about 10⁵To about 10⁸Individual cell, or about 10⁶To about 10¹²Individual cell, or about 10⁸To about 10¹¹Individual cell, or about 10⁹To about 10¹⁰And (4) cells. In some embodiments, the composition comprises greater than 10⁵About 10⁶About 10⁷About 10⁸About 10⁹About 10¹⁰About 10¹¹Or about 10¹²Single cell or greater than about 10⁵About 10⁶About 10⁷About 10⁸About 10⁹About 10¹⁰About 10¹¹Or about 10¹²And (4) cells.

In some embodiments, the volume of the composition is at least or at least about 10mL, 50mL, 100mL, 200mL, 300mL, 400mL, or 500mL, such as from or about 10mL to 500mL, 10mL to 200mL, 10mL to 100mL, 10mL to 50mL, 50mL to 500mL, 50mL to 200mL, 50mL to 100mL, 100mL to 500mL, 100mL to 200mL, or 200mL to 500mL, inclusive. In some embodiments, the composition has at least or at least about 1 x 10⁵Individual cell/mL, 5X 10⁵Individual cell or cellmL、1×10⁶Individual cell/mL, 5X 10⁶ 1X 10 cells/mL⁷Individual cell/mL, 5X 10⁷Individual cell/mL or 1X 10⁸Cell density of individual cells/mL. In some embodiments, the cell density of the composition is between or about 1 × 10⁵Individual cell/mL to 1X 10⁸ 1X 10 cells/mL⁵cell/mL to 1X 10⁷ 1X 10 cells/mL⁵cell/mL to 1X 10⁶ 1X 10 cells/mL⁶Individual cell/mL to 1X 10⁷ 1X 10 cells/mL⁶cell/mL to 1X 10⁸ 1X 10 cells/mL⁶cell/mL to 1X 10⁷Individual cell/mL or 1X 10⁷cell/mL to 1X 10⁸Between individual cells/mL, inclusive.

Depending on the method of engineering NK cells, it may be necessary to culture the NK cells to expand them before formulating them into a composition for administration. In some embodiments, the method of producing a composition comprising engineered NK cells comprises culturing or incubating the engineered NK cells, such as expanding the cells to a therapeutically effective amount prior to administering the NK cells to an individual in need thereof.

In some embodiments, the enriched NK cells (typically > 99% CD3 negative and > 85% CD56+) are expanded in vitro before or after engineering the cells.

In some embodiments, the subject-derived expanded primary cells may be expanded and/or cultured prior to the engineered modification. In some embodiments, the engineered primary cells may be cultured and/or expanded after engineering and prior to administration to a patient.

Techniques for the in vitro isolation and large-scale expansion of NK cells are known. An exemplary procedure is described in U.S. patent application publication No. 2014/0086890, which is incorporated herein by reference in its entirety. One of ordinary skill in the art can identify additional methods for expanding NK cells, for example as described in Childs et al, Hematol. the expression Program, 2013:234-246, 2013, which is incorporated herein by reference in its entirety.

The expression of markers for NK cells that were not expanded and expanded (e.g., CD56, CD16, TRAIL, FasL, NKG2D, LFA-1, perforin, and granzymes A and B) can be analyzed by flow cytometry. In some examples, the expression of one or more markers is measured at baseline and >10 days after in vitro amplification. Chromium release assays can be used to assess cytotoxicity of fresh and expanded NK cells against cancer cell targets. One of ordinary skill in the art can identify other methods of assessing NK cell populations (e.g., purity), viability, and/or activity.

In some embodiments, the NK cells may be cultured using feeder cells, or in the presence of cytokines, which enhance their growth and/or activation. As used herein, "culturing" includes providing the chemical and physical conditions (e.g., temperature, gas) and growth factors required for NK cell maintenance. In one embodiment, culturing the NK cells comprises providing conditions for NK cells to proliferate. Examples of chemical conditions that can support NK cell proliferation include, but are not limited to, buffers, nutrients, serum, vitamins and antibiotics as well as cytokines and other growth factors typically provided in the growth (i.e., culture) medium. In one embodiment, the NK medium is, for example, L500. The culture may be supplemented with amino acids, antibiotics, and/or cytokines to promote optimal viability, proliferation, function, and/or survival.

In some embodiments, culturing the population of cells comprising engineered NK cells is performed without a feeder layer or feeder cells. In some of these embodiments, the engineered NK cells may be cultured with growth factors. According to some embodiments, the at least one growth factor comprises a growth factor selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL-15, IL-12, and IL-21. According to some embodiments, the at least one growth factor is IL-2 or IL-2 and IL-15. According to some embodiments, the at least one growth factor is only IL-2.

In some embodiments, the composition further comprises a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and/or glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures (e.g., ultra-low temperatures), e.g., at temperatures ranging from-40 ℃ to-150 ℃, such as or about 80 ℃ ± 6.0 ℃.

In some embodiments, the engineered NK cells can be stored at ultra-low temperatures prior to administration to a patient. Engineered NK cells can also be stored at ultra-low temperatures after isolation from a mammalian subject and prior to genetic engineering. For example, lymphocytes or another source of engineered NK cells can be isolated, stored at ultra-low temperatures, and then processed to produce engineered NK cells. Alternatively, lymphocytes or another source of engineered NK cells can be isolated, treated to produce engineered NK cells, and then stored at ultra-low temperatures.

A typical method for small scale storage at ultra-low temperatures is described, for example, in us patent No. 6,0168,991. For small scale, cells can be passed through a low density suspension (e.g., at a concentration of about 200X 10) in pre-cooled 5% Human Albumin Serum (HAS)⁶/mL) was stored at ultra low temperature. An equal amount of 20% DMSO may be added to the HAS solution. An aliquot of the mixture can be placed into a vial and frozen overnight in an ultra-low temperature chamber at about-80 ℃.

In some embodiments, the cryopreserved NK cells are prepared for administration by thawing. In some cases, the NK cells may be administered to the subject immediately after thawing. In such embodiments, the composition is used without any further treatment. In other cases, the NK cells are further processed after thawing, such as by resuspension with a pharmaceutically acceptable carrier, incubation with an activator or stimulator, or activated washed and resuspended in a pharmaceutically acceptable buffer prior to administration to the subject.

Use or method of treatment

In another aspect, the invention provides the use of a cell-polypeptide conjugate or cell population as hereinbefore described in the manufacture of a medicament or agent for:

(1) inhibiting tumor cell proliferation;

(2) inhibiting tumor cell growth;

(3) inhibiting tumor cell invasion;

(4) promoting tumor cell apoptosis; and/or

(5) Killing tumor cells.

In some embodiments, the tumor cell is a breast cancer cell (preferably a triple negative breast cancer cell) or a liver cancer cell.

In another aspect, the invention provides the use of a cell-polypeptide conjugate or cell population as described hereinbefore in the manufacture of a medicament for the treatment and/or prophylaxis of a tumour, for example breast cancer (preferably triple negative breast cancer) or liver cancer.

In another aspect, the present invention provides a method for the treatment and/or prevention of a tumor comprising the step of administering to a subject in need thereof an effective amount of a cell-polypeptide conjugate or cell population as described hereinbefore.

In some embodiments, the individual is a mammal, e.g., a human. In some embodiments, the individual has cancer. In some embodiments, the method comprises administering 1 x 10 to the individual⁸～1×10¹⁰Cells/m²。

In some embodiments, the cell-polypeptide conjugate or population of cells is allogeneic to the individual. In some embodiments, the cell-polypeptide conjugate or cell population is autologous to the individual.

The engineered NK cells may be administered to a subject by any convenient route, including parenteral routes, such as subcutaneous, intramuscular, intravenous, and/or epidural routes of administration.

Tumors described herein include, but are not limited to: brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, stomach cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, cancer of the female genital tract, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma. In some embodiments, the tumor is a breast cancer, e.g., a triple negative breast cancer. In some embodiments, the tumor is liver cancer.

Tumor cells described herein include, but are not limited to: brain tumor cells, lung cancer cells, squamous cell carcinoma cells, bladder cancer cells, stomach cancer cells, ovarian cancer cells, peritoneal cancer cells, pancreatic cancer cells, breast cancer cells, head and neck cancer cells, cervical cancer cells, endometrial cancer cells, rectal cancer cells, liver cancer cells, kidney cancer cells, esophageal adenocarcinoma cells, esophageal squamous cell carcinoma cells, prostate cancer cells, female genital tract cancer cells, carcinoma-in-situ cells, lymphoma cells, neurofibroma cells, thyroid cancer cells, bone cancer cells, skin cancer cells, brain cancer cells, colon cancer cells, testicular cancer cells, gastrointestinal stromal tumor cells, prostate tumor cells, mast cell tumor cells, multiple myeloma cells, melanoma cells, glioma cells, or sarcoma cells. In some embodiments, the tumor cell is a breast cancer cell, e.g., a triple negative breast cancer cell. In some embodiments, the tumor is liver cancer.

In certain embodiments, the breast cancer cell is MDA-MB-231.

In certain embodiments, the liver cancer cell is HepG 2.

Methods of producing cell-polypeptide conjugates or cell populations

Based on the interdisciplinary development of glycometabolism approach, biochemistry and the like, the method is widely applied to the field of preclinical basic research and shows a wider biomedical application prospect. The azido modified mannose (Ac4Mannaz) can be effectively taken by cells, an azido bond is expressed on the cell surface of the azido modified mannose through a sugar metabolism way, and the azido bond can be combined with different small molecules with DBCO terminals in a click chemistry way and used for functional modification of the cell surface.

In this regard, the present invention further provides a method of producing a cell-polypeptide conjugate or cell population as hereinbefore described comprising the steps of:

(1) providing an immune effector cell;

(2) co-culturing the immune effector cells and azide-modified mannose to obtain immune effector cells with surfaces modified with azide groups;

(3) coupling the polypeptide which is modified with a connecting group at the end and specifically binds to gp96 to the immune effector cell obtained in the step (2) by a click chemistry method (such as a copper-free click chemistry method) to obtain the cell-polypeptide conjugate or the cell population;

wherein the linking group is selected from DBCO, DIBO and BCN.

In some embodiments, the methods are characterized by one or more of the following:

a) the concentration of the immune effector cells in the step (1) is 2-2.5 multiplied by 10⁶cells/mL, preferably 2.5X 10⁶cell/mL;

b) the azide-modified mannose of step (2) is Ac4Mannaz, preferably, the concentration thereof is 40-50. mu.M (e.g., 45. mu.M);

c) the culture time in the step (2) is 24-48 hours (preferably 36 hours);

d) after the culture in the step (2) is finished, processing the cells by adopting the following method to obtain the immune effector cells with the surface modified with the azide groups: 350-450 Xg (e.g. 400 Xg) centrifuging at room temperature for 5-10 minutes (e.g. 5 minutes), discarding the supernatant, and washing with physiological saline for 1-3 times;

e) the step (3) comprises the following operations: resuspending the cells obtained in step (2) in a medium (e.g., L500) to a concentration of 2-2.5X 10⁶Adding 4-5 mu M (preferably 4 mu M) of polypeptide which is modified with a connecting group at the end and specifically binds to gp96 into the cells/mL for co-incubation for 2-3 hours (preferably 3 hours); preferably, after the incubation is finished, centrifuging at 350-450 Xg (e.g. 400 Xg) for 5-10 minutes (e.g. 5 minutes) at room temperature, discarding the supernatant, and washing with physiological saline for 1-3 times to obtain the cell-polypeptide conjugate or cell population; further preferably, the obtained cell-polypeptide conjugate or cell population is resuspended to a concentration of 1-5X 10 with physiological saline⁷cells/mL for use;

f) the ratio of the polypeptide to the immune effector cells in the step (3) is 20-50ug of polypeptide: 1*10⁶A cell.

In some embodiments, when the linker is DBCO and the polypeptide is P37, the structure of the gp 96-specific binding polypeptide end-modified with a linker is as follows:

methods for synthesizing polypeptides are known in the art and the P37 of the present invention can be synthesized based on sequence information. P37 and DBCO or DBCO with fluorescein or other linking groups are further linked.

In some embodiments, the copper-free click chemistry occurs on DBCO and immune effector cell surface modified-N₃In the meantime.

Definition of terms

Molecular biology, microbiology, and recombinant DNA techniques that may be used in the present invention are within the skill of the art. These techniques have been explained fully in the literature. See, for example: sambrook, Fritsch & Maniatis, Molecular cloning: a Laboratory Manual, (1982); DNA Cloning, A Practical Approach Volumes I & II, D.N. Glover. 1985; oligonucleotide Synthesis, m.j.gait ed.1984; nucleic Acid Hybridization, B.D.Hames & S.J.Higgins eds.1985; transformation and transformation, B.D.Hames & S.J.Higgins eds.1984; animal Cell Culture, r.i. freshney, ed.1986; immobilised Cells And Enzymes, IRL Press, 1986; BPerbal, A Practical Guide To Molecular Cloning,1984 based thereon, the terms appearing herein are defined as follows.

As used herein, the term "gp 96 protein" refers to a heat shock protein (also known as GRP94) of about 96kD molecular weight that is present in the endoplasmic reticulum of eukaryotic cells. The amino acid sequence of gp96 protein is known to those skilled in the art and is found in various public databases (e.g., GenBank database, Genbank Accession NO. AY040226). An exemplary amino acid sequence of the wild-type gp96 protein is shown in SEQ ID NO. 2. Thus, in the present invention, when referring to the sequence of gp96 protein, it is described using the sequence shown in SEQ ID NO. 2. However, it is understood by those skilled in the art that mutations or variations (including, but not limited to, substitutions, deletions and/or additions) can be naturally occurring or artificially introduced in SEQ ID NO.2 without affecting the biological properties of the gp96 protein. Thus, when referring to the gp96 protein, the invention is intended to include the polypeptide shown in SEQ ID No.2 as well as natural or artificial variants thereof which retain the biological properties of the gp96 protein.

The P37 polypeptide described herein is a polypeptide that binds to gp96 protein. The polypeptide contains an alpha-helical sequence, can be specifically combined with gp96, blocks the rearrangement and conformation change of internal motifs of gp96 molecules, and further interferes the interaction of cell membrane gp96 and HER-2, uPAR or ER-a 36. The amino acid sequence is shown in SEQ ID NO. 1. When referring to P37, the invention is intended to encompass P37 and variants thereof, which "variants" refer to polypeptides whose amino acid sequence is one or more (e.g., 1-10 or 1-5 or 1-3) amino acid differences (e.g., conservative amino acid substitutions) or at least 60%, at least 70%, 80%, 85%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95%, 96%, 97%, 98%, 99% or 100% identity compared to the amino acid sequence of P37, and which have the same function as the PIBC, which "function" may be one or more of the following functions: (i) capable of specifically binding to gp96, (ii) capable of blocking motif rearrangements and conformational changes within gp96 molecules, (iii) capable of interfering with the interaction of envelope gp96 with HER-2, uPAR or ER-a 36.

As used herein, the term "identity" is used to refer to the match in sequence between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of E.Meyers and W.Miller (Compout.appl biosci., 4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48: 444-.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological activity of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl Acad. set USA 94:412-417(1997), which are incorporated herein by reference).

As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.

As used herein, the terms "about" and "approximately" should be understood by those skilled in the art and will vary to some extent depending on the context in which they are used. If the meaning is not clear to a person skilled in the art from the context of the term application, "about" means a deviation of not more than plus or minus 10% of the stated particular value or range.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

As used herein, the term "autologous" refers to cells or tissues derived from within or taken from the individual's own tissues. For example, in autologous transfer or transplantation of NK cells, the donor and recipient are the same person.

As used herein, the term "allogeneic" refers to cells or tissues that belong to or are obtained from the same species, but are genetically different, and thus, in some cases, immunologically incompatible. Typically, the term "allogeneic" is used to define cells that are transplanted from a donor to a recipient of the same species.

As used herein, the term "expression" refers to the process of transcription (e.g., transcription into mRNA or other RNA transcript) of a polynucleotide from a DNA template and/or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. The transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include splicing of the mRNA.

As used herein, the term "composition" refers to any mixture of two or more products, substances or compounds (including cells or antibodies). It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof. The formulations are typically in a form that allows the biological activity of the active ingredient (e.g., antibody) to be effective.

As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than an active ingredient that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, preservatives, and the like.

As used herein, the term "individual" or "subject" is a mammal, e.g., a bovine, equine, porcine, canine, feline, rodent, primate; among these, particularly preferred subjects are humans.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

The amount of the drug administered to the subject depends on the type and severity of the disease or condition and the characteristics of the subject, such as general health, age, sex, body weight and tolerance to the drug, as well as on the type of formulation and mode of administration of the drug, and the period or interval of administration. One skilled in the art will be able to determine the appropriate dosage based on these and other factors.

In some embodiments, the engineered NK cells are administered to the individual shortly after isolation and engineering of the NK cells. In some embodiments, the engineered NK cells are administered to the individual within 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days of isolation and engineering.

Drawings

FIG. 1 is a graph showing the results of in vitro amplification of NK cell purity (CD3-CD56+) in example 1.

FIG. 2 is a graph showing the results of NK cell modification of P37 polypeptide in example 2.

FIG. 3 is a graph showing the results of NK-Ac4Mann-P37 and NK cell in vitro killing of triple-negative breast cancer cell line MDA-MB-231 in example 3.

FIG. 4 is a graph showing the results of NK-Ac4Mann-P37 and NK cell in vitro killing of the hepatoma cell line HepG2 in example 4.

FIG. 5 is a graph showing the results of NK-Ac4Mann-P37 and NK cells and polypeptide killing triple-negative breast cancer cell line MDA-MB-231 in vivo in example 5.

FIG. 6 is a graph showing the results of NK-Ac4Mann-P37 and NK cells and polypeptides killing the hepatoma cell line HepG2 in vivo in example 6.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory press, 1989, and f.m. ausubel et al, eds. molecular biology laboratory guidelines, 3 rd edition, John 5Wiley & Sons, inc., 1995.

The gp96 protein and the sequence information of P37 related to the present invention are shown in the following table.

Sequence information Profile

Example 1 in vitro expansion of NK cells

Human peripheral blood mononuclear cells were purchased from AllCells, inc, under product catalog number PB 004; easy sep^TMHuman CD3 Positive Selection Kit II, available from STEMCELL corporation under product catalog # 17851; human cytokines IL-2, IL21, IL15 and IL12 were purchased from shanghai, inshore biotechnology limited under the respective catalog numbers: c013, CC45, C016 and CI 58. SuperCultureL500 lymphocyte serum-free medium was purchased from Shenzhenjn Davidae, bioengineering, Inc., under the catalog number: DKW-SCL 5. DPBS was purchased from Thermo Fisher Scientific, Inc. under the catalog number: A1285601. flow-through antibodies Anti-Human CD3 FITC and Anti-Human CD56 APC were purchased from Thermo Fisher Scientific under catalog numbers 11-0038-42 and 17-0567-41, respectively.

1.1 cell concentration was adjusted to 1X 10 with DPBS⁸cells/mL, transferred to a sterile 5mL flow tube, and Easysep added as a 150. mu.l/mL sample^TMHuman CD3 Positive Selection Cocktail II in Human CD3 Positive Selection Kit II, and mixing, standing at room temperature for 3 min.

1.2 Easysep^TMRapidSphere in Human CD3 Positive Selection Kit II^TM50100 mixing them uniformly according toAdd 90. mu.l/mL of sample to the flow tube of step 1.1, mix well and let stand at room temperature for 3 min.

1.3 the volume of the cell suspension in the 5mL sterile flow tube of step 2.2 was made up to 2.5mL with DPBS and gently mixed.

1.4 Place the 5mL sterile flow tube in a magnet at room temperature for 3min, pick up the magnet and pour the cells into a 15mL sterile centrifuge tube.

1.5 remove 5mL of sterile flow tube from magnet, add 2.5mL of DPBS to the 5mL of sterile flow tube, and resuspend the cells.

1.6 placing 1.5 step 5mL sterile flow tube in magnet and standing at room temperature for 3min, taking up magnet, pouring cells into 15mL sterile centrifuge tube in step 1.4, supplementing the cell suspension volume to 5mL with DPBS, and mixing well. 20 μ l of the cell suspension was counted in a 1.5mL EP tube.

1.7 based on the results of the 1.6-step counting, the cell concentration was adjusted to 1X 10 with L500 medium⁶cells/mL, IL-2 was added to a final concentration of 1000IU/mL, and IL21, IL15 and IL12 were added to a final concentration of 50ng/mL and mixed well.

1.8 cells were incubated at 37 ℃ with 5.0% CO at saturation humidity₂And continuing culturing in the incubator.

1.9 phenotypic testing was performed at day 14 of culture.

As a result, the purity of the in vitro amplified NK cells (CD3-CD56+) was more than 95% as shown in FIG. 1.

Example 2 optimization of NK-Ac4Mann-P37 cell modification efficiency conditions

Ac4ManNA was purchased from Merck, catalog number 900917. DBCO-P37-FITC was synthesized by Nanjing King Shirui Biotech, Inc.

2.1 Using L500 Medium, the NK cell concentration was adjusted to 2X 10⁶Or 2.5X 10⁶cells/mL.

2.2 Add Ac4Mannaz to a final concentration of 40, 45 or 50. mu.M, respectively, 5% CO at 37 ℃₂Incubate in incubator for 24, 36 or 48 hours, respectively.

2.3 after the end of incubation, 400 Xg at room temperature for 5 minutes, abandoning the supernatant, using L500 medium to cell selection to a concentration of 1X 10⁶cells/mL, DBCO-P37-FITC was added to final concentrations of 4 or 5. mu.M, respectively, and 5% CO was added at 37 ℃ respectively₂Incubate in incubator for 2 or 3 hours.

2.4 after the incubation, 400 Xg room temperature centrifugation for 5 minutes, abandon the supernatant, saline after washing twice, using saline heavy suspension NK-Ac4Mann-P37-FITC, and flow detection.

The modification efficiency is shown in Table 1, and the modification efficiency is greatly different under different conditions, wherein the concentration of NK cells is 2.5 multiplied by 10⁶cells/mL, Ac4Mannaz at a concentration of 45. mu.M was added, incubated for 36 hours, then DBCO-P37-FITC at a concentration of 4. mu.M was added, and finally incubated for 3 hours with the best results, with greater than 95% of NK cells modified with P37 polypeptide as shown in FIG. 2.

TABLE 1 modification efficiency of NK cells under different conditions

Example 3 NK-Ac4Mann-P37 cell Activity assay for killing triple negative breast cancer MDA-MB-231 cells in vitro

DBCO-P37 was synthesized by Nanjing King Shirui Biotech, Inc. CFSE dye was purchased from Thermo Fisher Scientific (Cat. C34554). Propidium Iodide (PI) was purchased from Thermo Fisher Scientific, catalog No.: 00-6990-50.

3.1 Trypsin digestion of MDA-MB-231 cells, adjusting the cell suspension concentration to 2X 10⁵CSFE was added to the cells/mL to a final concentration of 1. mu.M, and the cells were protected from light at room temperature for 20 min.

3.2 centrifuge and collect the cells (1000rpm, 5min), resuspend the cells in serum-free medium and mix well. At 37 ℃ in the dark for 5 min. Cells were harvested by centrifugation and washed twice with serum-free medium. Resuspend with complete medium, count, add 5 million cells per 96 wells.

3.3 NK cells were prepared according to example 1 as control cells, and NK-Ac4Mann-P37 cells were prepared according to the preparation method of example 2, in which DBCO-P37-FITC polypeptide was replaced with DBCO-P37 polypeptide, and NK-Ac4Mann-P37 cells were prepared as experimental cells.

3.4 mu.L of NK-Ac4Mann-P37 cells and a control cell suspension or an equal volume of medium (blank) were added to a 96-well U plate at different effect-to-target ratios (E: T ═ 0:1, 2.5:1, 5:1, 10:1) and incubated in an incubator at 37 ℃ for 4-6 h.

3.5 cells were harvested, suspended in 400. mu.l PBS, stained with Propidium Iodide (PI) marker (added 2min before loading), and flow assayed.

The results are shown in fig. 3, where the effective target ratio is 10:1, the NK-Ac4Mann-P37 and the NK cell have killing activity in vitro, and compared with the NK cell, the killing activity of the NK-Ac4Mann-P37 cell is obviously improved (P is less than 0.01), and the killing efficiency is increased by 94.8%.

Example 4 detection of the Activity of NK-Ac4Mann-P37 cells to kill liver cancer HepG2 cells in vitro

DBCO-P37 was synthesized by Nanjing King-Smiry Biotechnology Ltd. CFSE dye was purchased from Thermo Fisher Scientific (Cat. C34554). Propidium Iodide (PI) was purchased from Thermo Fisher Scientific, catalog No.: 00-6990-50.

4.1 Trypsin digestion of HepG2 cells, adjusting the cell suspension concentration to 1X 10⁵CSFE was added to the cells/mL to a final concentration of 1. mu.M, and the cells were protected from light at room temperature for 20 min.

4.2 centrifuge and collect the cells (1000rpm, 5min), resuspend the cells in serum-free medium and mix well. At 37 ℃ in the dark for 5 min. Cells were collected by centrifugation and washed twice with serum-free medium. Resuspend with complete medium, count, add 3 million cells per 96 wells.

4.3 preparation of NK cells according to example 1 as control cells, and NK-Ac4Mann-P37 cells according to the preparation method of example 2, wherein DBCO-P37-FITC polypeptide was replaced with DBCO-P37 polypeptide, and NK-Ac4Mann-P37 cells were prepared as experimental cells;

4.4 mu.L of NK-Ac4Mann-P37 cells and a control cell suspension or an equal volume of medium (blank) were added to a 96-well U plate at different effect-to-target ratios (E: T ═ 0:1, 2.5:1, 5:1, 10:1) and incubated in an incubator at 37 ℃ for 4-6 h.

4.5 cells were harvested, suspended in 400. mu.l PBS, stained with Propidium Iodide (PI) marker (added 2min before loading), and flow assayed.

The results are shown in fig. 4, where the effective target ratio is 10:1, the NK-Ac4Mann-P37 and the NK cell have killing activity in vitro, and compared with the NK cell, the killing activity of the NK-Ac4Mann-P37 cell is obviously improved (P is less than 0.05), and the killing efficiency is improved by 78.2%.

Example 5 detection of NK-Ac4Mann-P37 cell Activity against triple negative Breast cancer in vivo

5.1 Breast cancer cells MDA-MB-231 cultured to logarithmic growth phase are inoculated subcutaneously into BALB/c nude mice (Beijing Wintonlihua laboratory animal technology Co., Ltd.), each nude mouse is inoculated with 1000 ten thousand cells, a transplanted tumor model is established, and then the nude mice are subjected to 3 passages in vivo for tumor inoculation experiment.

5.2 BALB/c nude mice were randomized into 3 groups of 5 mice each by the time the tumor grew to 100mm3, and were treated as follows, with day 1 being assigned on day 1:

NK-Ac4Mann-P37 panel: the prepared NK-Ac4Mann-P37 cells (prepared in reference example 2) were suspended in 0.9% physiological saline and treated by intravenous injection for a total of two weeks with a cell count of 1X 107 per injection and a volume of 200. mu.l administered once a week;

NK group: the prepared NK cells (prepared in reference example 1) were suspended in 0.9% physiological saline and were treated by intravenous injection, the number of cells per injection being 1X 10⁷Volume 200 μ Ι, once weekly for a total of two weeks of treatment;

polypeptide group: was administered intravenously at a dose of 5mg/kg once a day.

Control group: treatment was performed intravenously in PBS buffer at the same volume as the NK-Ac4Mann-P37 treatment group, once weekly for a total of two weeks.

5.3 Secondary tumor volumes were measured weekly, nude mice sacrificed after two weeks of treatment, tumor weights were weighed and tumor inhibition rates calculated.

The formula for calculating the tumor inhibition rate is as follows: (tumor volume of control mice-volume of tumor of polypeptide mice)/tumor volume of control mice x 100%.

The results of tumor suppression rates for NK-Ac4Mann-P37, the NK treated group and the polypeptide group are shown in FIG. 5. The result shows that the NK-Ac4Mann-P37, the NK cell and the polypeptide can effectively inhibit the growth of the breast cancer tumor caused by the breast cancer cell MDA-MB-231, the treatment effect of the NK-Ac4Mann-P37 group is better (P <0.01), and compared with the NK cell and the polypeptide alone, the inhibition of the NK-Ac4Mann-P37 on the growth of the tumor is improved by 169% and 118%.

Example 6 detection of NK-Ac4Mann-P37 cell anti-hepatoma Activity in vivo

6.1 liver cancer cells HepG2 cultured to logarithmic growth phase were subcutaneously inoculated into BALB/c nude mice (animal technology Co., Ltd., Wei Tonglihua, Beijing), each was inoculated with 1000 ten thousand cells to establish a transplanted tumor model, and then used for tumor grafting experiments after 3 passages in nude mice.

6.2 wait until the tumor grows to 100mm³BALB/c nude mice were randomly divided into 3 groups of 5 mice each, and the following treatment treatments were performed, with day 1 being assigned as day 1:

NK-Ac4Mann-P37 panel: the prepared NK-Ac4Mann-P37 cells (prepared in reference example 2) were suspended in 0.9% physiological saline and treated by intravenous injection for a total of two weeks, with the number of cells per injection being 1X 107, the volume being 200. mu.l, administered once per week;

6.3 Secondary tumor volumes were measured weekly, nude mice sacrificed after two weeks of treatment, tumor weights were weighed and tumor inhibition rates calculated.

The results of tumor suppression rates for NK-Ac4Mann-P37, the NK treated group and the polypeptide group are shown in FIG. 6. The result shows that the NK-Ac4Mann-P37 and the NK cells can effectively inhibit the tumor growth caused by the hepatoma cell HepG2, the treatment effect of the NK-Ac4Mann-P37 group is better (P <0.05), and compared with the single NK cell, the inhibition of the NK-Ac4Mann-P37 on the tumor growth is improved by 114%.

Although specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that, based upon the overall teachings of the disclosure, various modifications and alternatives to those details could be developed and still be encompassed by the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A cell-polypeptide conjugate comprising an immune effector cell and a polypeptide that specifically binds gp96 covalently attached to the surface of the immune effector cell membrane.

2. The cell-polypeptide conjugate of claim 1, wherein the polypeptide is selected from the group consisting of:

1) a polypeptide having or comprising the amino acid sequence shown in SEQ ID No. 1;

3. The cell-polypeptide conjugate of claim 1 or 2, wherein the immune effector cell is a T cell, an NK cell, or a macrophage; NK cells (e.g., NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6 or IMC-1) are preferred.

4. The cell-polypeptide conjugate of any one of claims 1-3, wherein the surface of said immune effector cell is modified with an azide group and said polypeptide is linked to said azide group on the surface of said immune effector cell via a linking group, wherein said linking group is selected from the group consisting of DBCO, DIBO, and BCN.

5. A cell population comprising a plurality of cell-polypeptide conjugates of any one of claims 1-4; preferably, the cell-polypeptide conjugate comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, 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%, at least 99%, or 100% of the total number of the population of cells.

6. A composition comprising the cell-polypeptide conjugate of any one of claims 1-4 or the cell population of claim 5, and a pharmaceutically acceptable carrier;

preferably, the carrier is selected from saline solution, dextrose solution or 5% human serum albumin;

preferably, the concentration of cells in the composition is 1X 10⁵～1×10⁸cell/mL;

preferably, the composition comprises a cryoprotectant.

7. Use of the cell-polypeptide conjugate of any one of claims 1-4 or the cell population of claim 5 in the preparation of a medicament or agent for:

(1) inhibiting tumor cell proliferation;

(2) inhibiting tumor cell growth;

(3) inhibiting tumor cell invasion;

(4) promoting tumor cell apoptosis; and/or

(5) Killing tumor cells;

preferably, the tumor cell is a breast cancer cell (preferably a triple negative breast cancer cell) or a liver cancer cell.

8. Use of the cell-polypeptide conjugate of any one of claims 1-4 or the cell population of claim 5 in the manufacture of a medicament for the treatment and/or prevention of a tumor, such as breast cancer (preferably triple negative breast cancer) or liver cancer.

9. A method of preparing the cell-polypeptide conjugate of any one of claims 1-4 or the cell population of claim 5, comprising the steps of:

(1) providing immune effector cells, such as NK cells derived from human peripheral blood mononuclear cells or NK92 cell line;

(3) coupling the polypeptide which is specifically combined with gp96 and is modified with a connecting group at the end to the immune effector cells obtained in the step (2) through a click chemistry method to obtain the cell-polypeptide conjugate or cell population;

wherein the linking group is selected from DBCO, DIBO and BCN.

10. The method of claim 8, characterized by one or more of the following:

c) the culture time in the step (2) is 24-48 hours (preferably 36 hours);

d) after the culture in the step (2) is finished, processing the cells by adopting the following method to obtain the immune effector cells with the surface modified with the azide groups: centrifuging at the room temperature of 350-450 Xg for 5-10 minutes, discarding the supernatant, and washing with physiological saline for 1-3 times;

e) the step (3) comprises the following operations: resuspending the cells obtained in step (2) in a medium (e.g., L500) to a concentration of 2-2.5X 10⁶Adding 4-5 mu M (preferably 4 mu M) of polypeptide which is modified with a connecting group at the end and specifically binds to gp96 to the cells/mL for incubation for 2-3 hours (preferably 3 hours); preferably, after the incubation is finished, centrifuging at 350-450 Xg for 5-10 minutes at room temperature, discarding the supernatant, and washing with physiological saline for 1-3 times to obtain the cell-polypeptide conjugate or cell population; further preferably, the resulting cell-polypeptide conjugate or cell population is resuspended to a concentration of 1-5X 10 with physiological saline⁷cells/mL for use;

f) the feeding proportion of the polypeptide and the immune effector cells in the step (3) is 20-50ug of polypeptide: 1*10⁶A cell.