CN114478772A

CN114478772A - Engineered immune cells, nanogels, and preparation methods and applications thereof

Info

Publication number: CN114478772A
Application number: CN202111677244.XA
Authority: CN
Inventors: 郑毅然; 陈星烨
Original assignee: Suzhou University
Current assignee: Suzhou Yiran Biotechnology Co ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-05-13
Anticipated expiration: 2041-12-31
Also published as: CN114478772B

Abstract

The invention discloses an engineered immune cell, a nanogel, a preparation method and an application thereof, wherein the preparation method comprises the steps of crosslinking a PD-1 monoclonal antibody and a connecting agent with redox response to synthesize the nanogel, carrying out dibenzocyclooctyne modification on the surface of the nanogel, and then combining the nanogel with a T cell marked by azide through click chemical reaction to lead NG anchor points to be positioned on the surface of the T cell. The spatiotemporal co-existence of PD-1 mab with ACT T cells and drug release triggered by T cell activation can significantly increase the effective dose of PD-1 mab in TME. In a mouse subcutaneous solid tumor model, the combination of cell surface anchored PD-1 mab and T cells inhibited tumor growth by 96%.

Description

Engineered immune cells, nanogels, and preparation methods and applications thereof

Technical Field

The invention relates to an engineered immune cell, nanogel, and a preparation method and application thereof.

Background

Tumor-infiltrating T cells isolated from living tissues or genetically engineered peripheral blood lymphocytes from patients recognize tumor antigens specifically via T Cell Receptors (TCR) or Chimeric Antigen Receptors (CAR), respectively. In Adoptive Cell Therapy (ACT), these tumor-specific cells are activated in vitro, proliferated, and then returned to the patient to kill the cancer cells. ACT, as typified by CAR-T therapy, currently achieves promising clinical efficacy in certain hematologic cancer patients, but its efficacy against solid tumors remains unsatisfactory. Among these, the immunosuppressive Tumor Microenvironment (TME) is one of the major challenges of ACT to treat solid tumors.

In TME, tumor cells use various mechanisms such as secretion of soluble suppressors or direct physical contact to inhibit T cell function. One of the most effective methods is to up-regulate the expression of the tumor cell's own programmed cell death ligand 1 (PD-L1). PD-L1 binds to programmed cell death receptor 1(PD-1) expressed by Cytotoxic T Lymphocytes (CTLs) thereby "hijacking" the immune checkpoint pathway and inhibiting the anti-tumor function of T cells. Immune checkpoint inhibitors can reverse immunosuppression from tumor cells, thereby enhancing the therapeutic efficacy of exogenous ACT cells and endogenous tumor-specific CTLs.

Monoclonal antibodies against PD-1 or PD-L1 (aPD1 and aPDL1) are currently being widely used in clinical studies in order to enhance the function of endogenous CTLs. Although immune checkpoint inhibitors achieve a long-lasting anti-tumor effect in a subset of patients, the overall response rate of patients to ICI is low and the overall survival rate of patients remains low. For exogenous ACT T cells, although immune checkpoint inhibitors may be effective in prolonging their function, clinical therapeutic efficacy is modest. In addition, the use of immune checkpoint inhibitors at high doses or for long periods of time may lead to systemic toxic side effects, causing severe adverse reactions in the gastrointestinal tract, endocrine glands, skin and liver, etc. organ systems. Some patients develop resistance to immune checkpoint inhibitors or develop autoimmune diseases even after long-term administration.

There have been studies attempting to improve the therapeutic effect of CAR-T cells by genetically engineering CAR-T cells to autocrine immune checkpoint inhibitors or to replace the intracellular inhibitory domain of the T cell PD-1 receptor with a proliferation-enhancing domain. However, CAR-T cells may proliferate uncontrollably in a patient, creating a safety hazard.

Biomaterials have been used to increase the local concentration of immune checkpoint inhibitors in the TME, increasing the therapeutic effect while reducing the adverse side effects of non-tumor sites. Immune checkpoint inhibitors, as represented by PD-1 mab, can be transported in "cargo" form to the vicinity of TME or tumor via different delivery platforms. In addition, immune checkpoint inhibitors can also serve as ligands for enhancing targeting of the nanoparticles, as an important component of a vehicle or as an integral part of a combination therapy. However, these approaches primarily enhance endogenous T cell function rather than enhance the anti-tumor efficacy of ACT T cells. Furthermore, the mechanism by which immune checkpoint inhibitors are released from these delivery platforms is not linked to the activation and function of ACT T cells, and cannot function at the time and location where T cells most require drug support.

Recently, the use of T cells as drug carriers has attracted interest. Nanoparticles coupled to the surface of T cells may also enter the TME when ACT T cells infiltrate into the tumor tissue. The method allows the adjuvant drug to coexist with ACT T cell in space-time, increases the effective dosage of the drug and enhances the anti-tumor function of the T cell. Immunomodulatory drugs such as the cytokine interleukin 15 and small molecule drugs have been conjugated to the surface of T cells and have improved efficacy in preclinical studies.

Disclosure of Invention

In one aspect, the present invention provides a method for preparing a nanogel, which is characterized by comprising the following steps:

1) preparing a connecting agent solution;

2) and adding the connecting agent solution obtained in the step 1) into the monoclonal antibody solution for mixing reaction to obtain the nanogel.

In some embodiments, the monoclonal antibody comprises PD-1 monoclonal antibody, CTLA-4 monoclonal antibody, TNF α monoclonal antibody.

In some embodiments, the linker comprises DTSSP or a molecule with chemical functional groups at both ends that are capable of reacting with an antibody.

In some embodiments, the molar ratio of the monoclonal antibody to the linking agent is (1: 25-1: 200); the reaction time is 1-2 hours.

In some embodiments, further comprising step 3), adding DBCO-PEG-NHS solution to the nanogel obtained in step 2), and performing reaction on a shaker to obtain DBCO-aPD 1-NGs.

In some embodiments, further comprising step 3), adding a solution of DSPE-PEG-NHS to the nanogel obtained in step 2), and reacting on a shaker to obtain DSPE-aPD 1-NGs.

In another aspect, the invention also provides the nanogel prepared by the preparation method of the nanogel.

In another aspect, the invention further provides an engineered immune cell, wherein the DBCO-aPD1-NGs are combined with azide-labeled engineered immune cells through a click chemistry reaction, so that the nanogel is anchored on the surface of the engineered immune cell.

In some embodiments, the engineered immune cell is prepared by a method comprising:

1) preparing a DSPE-PEG-N3 solution;

2) adding the engineering immune cell re-suspension into the DSPE-PEG-N3 solution obtained in the step 1) for mixing reaction to obtain an engineering immune cell with the surface connected with DSPE-PEG-N3;

3) and re-suspending the engineered immune cells with the DSPE-PEG-N3 attached to the surface in a culture medium, adding the DBCO-aPD1-NGs into the culture medium for incubation, and collecting the engineered immune cells with the DBCO-aPD1-NGs bound to the surface after the incubation is finished.

In another aspect, the invention also provides an engineered immune cell, wherein the DSPE-aPD1-NGs are combined with the engineered immune cell by co-incubation, so that the nanogel is anchored on the surface of the engineered immune cell.

1) resuspending the engineered immune cells in a culture medium;

2) adding DSPE-aPD1-NGs into the culture medium in the step 1) for incubation, and collecting the engineered immune cells with the DSPE-aPD1-NGs bound on the surfaces after the incubation is finished.

In some embodiments, the engineered immune cell comprises a T cell, an NK cell, or a macrophage.

In another aspect, the invention also provides an inhibitor comprising an engineered immune cell as described above.

In another aspect, the invention also provides a pharmaceutical composition comprising the engineered immune cell described above and a pharmaceutically acceptable excipient.

In another aspect, the use of the engineered immune cells described above, the inhibitors described above, and the pharmaceutical compositions described above in ACTs using T cell receptor T cells or chimeric antigen receptor T cells to inhibit the growth of solid tumors.

The invention discloses a method for anchoring nanogel (aPD1-NGs) formed by polymerizing an immune checkpoint inhibitor PD1 monoclonal antibody on the surface of an ACT T cell, so that the ACT and the immune checkpoint inhibitor can play a synergistic effect, and the treatment effect of solid tumors is improved. We cross-linked PD-1 mab with redox linker 3,3' -dithiobis (succinimidyl propionate sulfonate) (DTSSP) to form nanoparticles, which were then surface modified (fig. 1A). We explored two approaches to increase NG drug loading on the T cell surface. The method comprises the following steps: the NGs with surface modified diacyl lipid function are inserted into cell membranes through diacyl lipids and anchored to the surfaces of T cells; the second method comprises the following steps: dibenzocyclooctyne (DBCO) -modified NGs are first inserted onto the cell surface with diacyl lipids bearing an azido functionality, and then anchored to the cell surface by click chemistry reaction with azide. With the second modification, the amount of PD-1 mAb loaded on each million T cells can be as much as 4. mu.g. After intravenous injection, these T cells carrying the NGs backpack circulate in the body and infiltrate the tumor along with the NGs. In TME, adoptive T cells were activated by tumor antigens, up-regulating the cell surface reduction potential, resulting in disulfide bond cleavage in DTSSP, releasing PD1 mab from NGs to combat immunosuppression (fig. 1B). In the mouse subcutaneous melanoma tumor model, the aPD1-NG backpack can help T cells significantly inhibit tumor growth. The average tumor weight of the T cell backpack group was only 18% of the T cell helper treated group treated with equal amounts of free PD-1 mab. This study illustrates a method of using ACT in conjunction with an immune checkpoint inhibitor. This technique can complement existing genetic engineering and material delivery methods, and can also be used as an independent therapeutic approach to improve the therapeutic efficacy of ACT in solid tumors. Furthermore, this approach can also be widely applied to ACT treatment strategies using naive T cells, TCR-T cells or CAR-T cells.

Drawings

FIG. 1 is a schematic representation of the Nanogel (NG) synthesis protocol and the enhanced efficacy of cell surface NGs "back pack" in conjunction with adoptive T cell therapy;

wherein, (A) PD-1 monoclonal antibody and DTSSP cross-link and synthesize NGs, then surface modification NGs, (B) two kinds of methods of anchoring NGs on the surface of T cell, activated after ACT T cell meets tumor antigen in TME, trigger PD-1 monoclonal antibody to release, enhance curative effect.

FIG. 2 is a representation of NGs and optimization of the anchoring of NGs to the surface of T cells;

wherein (a) the particle size of free PD-1 monoclonal antibodies, NGs, DSPE surface-modified NGs (DSPE-aPD1-NGs) and DBCO surface-modified NGs (DBCO-aPD1-NGs) in Dynamic Light Scattering (DLS) and transmission electron microscopy (TEM, scale 100 nm); optimizing the time and the molar ratio of the nanogel reaction; (B-C) effect of incubation time on efficiency of insertion of fluorescently labeled diacyl lipid (DSPE-PEG-FITC) into T cell membrane; washing away unbound polymer and analyzing the T cells by flow cytometry; shown are a histogram of the Mean Fluorescence Intensity (MFI) of T cells (B) and a trend of MFI over time (C); (D-E) Effect of feed molar ratio of DBCO-PEG-NHS on coupling efficiency of DBCO-aPD1FITC-NGs to T cells; washing away unbound NGs and analyzing T cells with a flow cytometer; shown are the T cell fluorescence histogram and the percentage of T cells bound to NGs (D); quantitative T cell MFI analysis (E); (F-G) influence of feed molar ratio of DSPE-PEG-NHS on coupling efficiency of DSPE-aPD1FITC-NGs to T cells; as shown are the T cell fluorescence histogram and the percentage of T cells bound to NGs (F); quantitative analysis of T cell MFI (G).

FIG. 3 shows the anchoring of NGs to the surface of T cells;

wherein (A) CD8 under confocal microscope⁺T cells, T cells with fluorescently labeled NGs and DSPE-aPD1^APC-NGs binding; (B) confocal microscopy CD8⁺T cells, DSPE-PEG-N3-inserted T cells, and DBCO-aPD1^APCImages of NGs-bound T cells and T cells inserted DSPE-PEG-N3 and then bound to DBCO-aPD1 APC-NGs; NGs, red; the cell membrane was stained with DiI, green; nuclei were stained by Hoechst33342, blue; the scale bar is 10 μm.

FIG. 4 is a graph showing that DBCO-aPD1-NGs can be loaded onto the surface of T cells more efficiently than DSPE-aPD 1-NGs;

wherein (A-B) untreated CD8⁺T cells or T cells inserted with azide-modified diacyl lipid DSPE-PEG-N3 were conjugated with DBCO-aPD1FITC-NGs, and untreated T cells were combined with equal doses of DSPE-aPD1^FITC-NGs binding; washing away unbound NGs and analyzing T cells with a flow cytometer; as shown are the fluorescence histogram of NGs on T cells and the percentage of T cells bound to NGs (a); quantitative analysis of T cell MFI (B); (C) will be 5X 10⁶CD8+ T cells modified with DSPE-PEG-N3 and varying amounts of DBCO-aPD1^FITC-NGs co-incubation. After the NGs are anchored, unbound NGs are washed away, T cells are lysed, and the cell surface is quantified using an enzyme reader aPD1^FITCThe number of (2); (D) the cracking surface is combined with DBCO-aPD1^FITCDifferent numbers of T cells of NGs, bound aPD1 quantified with a microplate reader^FITCThe number of the cells.

FIG. 5 shows that NGs can release PD-1 monoclonal antibody in response to the artificial reduction environment and the increase of cell surface reduction potential induced by TCR signal;

wherein (A-B) after 12 hours of treatment with 10mM GSH solution, DLS (A) and TEM (b) the particle size of the NGs detected; scale bar 100 nm; (C) aPD1^APC-NGs are treated in dialysis bags (MWCO 300kDa) with PBS (black curve), 1mM GSH solution (green curve) or 10mM GSH solution (red curve) for 24 hours; measuring the fluorescence of the sample in the container at different time points by a microplate reader; shown is the cumulative percentage of aPD1 released in the NGs; (D) measurement of immature CD8 by the WST-1 method⁺T cells and activated OT-1 CD8⁺Cell surface reduction potential of T cells; (E) CD8+ T cells and DBCO-aPD1 with DSPE-PEG-N3 inserted on the surface^FITC-NGs binding; removing unbound NGs and then analyzing the T cells bound or unbound to NGs by flow cytometry; after 1 day of activation of the NGs-bound T cells, analysis was again performed by flow cytometry; the sample fluorescence histogram on T cells is shown, where the MFI of T cells is indicated; (F) the MFI of T cells was quantified either after NGs binding (Day 0) or immediately after 1 Day activation signaling (Day 1).

FIG. 6 is a graph showing that anchoring of NGs to the cell surface has no negative effect on T cell function in vitro;

wherein (A) T cells, DSPE-PEG-N3 inserted T cells, DBCO-aPD1-NGs combined T cells in vitro culture for 2 days; t cells were stained with LIVE/DEAD fixed-DEAD Cell Stain Kit on day 1 (left) and day 2 (right) and analyzed by flow cytometry; t cell survival for each group is shown; (B) flow analysis of expression of PD-L1 after treatment of B16-OVA cells with 0ng/mL IFN-. gamma. (black curve), 10ng/mL IFN-. gamma. (blue curve) and 20ng/mL IFN-. gamma. (red curve); (C-D) inoculating B16-OVA cells onto a 96-well plate, and culturing in DMEM containing 10ng/mL IFN-. gamma.; after 24 hours, activated OT-1 CD8⁺T cells, T cells mixed with free PD-1 monoclonal antibody and T cells bound with aPD1-NGs (DBCO-aPD1-NGs @ T-N3) were added to B16-OVA cells at different effective target ratios (E/T); after 24h of co-incubation, tumor cell lysis (C) was observed under a bright field microscope (magnification 100X, scale 100 μm) and the killing effect of T cells was determined using LDH cytotoxicity kit (D).

FIG. 7 is a graph showing that PD-1 monoclonal antibody anchored to the cell surface enhances the efficacy of adoptive T cell therapy in a mouse subcutaneous tumor model;

among them, C57BL/6 mice were inoculated subcutaneously with B16-OVA tumor cells (2X 10)⁵Individual cells) 6 days later, mice were cleared of lymphocytes with cyclophosphamide (100mg/kg, i.p.); adoptive T cell therapy was performed on mice every other day. Intravenous PBS, 5X 10⁶Activated OT-1 CD8+ T cells (T cells), T cells mixed with free PD-1 mab (T + free aPD1), and T cells with equal amounts of PD-1 mab bound to the cell surface as a "backpack" of NGs (DBCO-aPD1-NGs @ T-N3); tumor size and body weight of each group of mice were monitored every 2 days; at the end of the experiment, mice were sacrificed and tumors were removed; n is 5; (A) in vivo treatment experiment timeline; (B) mouse tumor growth curve; (C) photographs of tumors for each treatment group; (D) quantitatively analyzing the tumor weight of each group; (E) body weight curves for each group of mice; (F) HE stained sections of heart, liver, spleen, lung, and kidney, scale 50 μm.

Detailed Description

The present invention generally discloses that immune checkpoint inhibitors anchored to the cell surface enhance the therapeutic efficacy of adoptive immune cell therapies on solid tumors.

Adoptive Cell Therapy (ACT) has achieved promising results in certain hematological malignancies. However, ACT remains poorly effective against solid tumors, primarily due to the immunosuppressive Tumor Microenvironment (TME). In TME, tumor cells inhibit the function of ACT T cells through immune checkpoint pathways such as PD-1/PD-L1. Although immune checkpoint inhibitors, such as PD-1 mab, may be effective against immunosuppression, the synergistic effect of PD-1 mab on ACT is still not ideal. The use of high doses of PD-1 mab causes systemic toxicity, and T cells with intracellular PD-1 blocking function may cause safety problems due to their uncontrolled proliferation in the patient. Presented herein is a method of anchoring Nanogel (NG) composed of PD1 mab to the surface of T cells prior to adoptive T cell injection into the body to enhance the therapeutic efficacy of ACT in solid tumors. The PD-1 monoclonal antibody and a connecting agent with redox response are crosslinked to synthesize nanogel, the surface of the nanogel is modified by Dibenzocyclooctyne (DBCO), and then the nanogel is combined with T cells marked by azide through click chemistry reaction, so that NG anchors on the surfaces of the T cells. The spatiotemporal co-existence of PD-1 mab with ACT T cells and drug release triggered by T cell activation can significantly increase the effective dose of PD-1 mab in TME. In a mouse subcutaneous solid tumor model, the combination of cell surface anchored PD-1 mab and T cells inhibited tumor growth by 96%. More importantly, the mean tumor weight of the cell surface anchored PD-1 mab and T cell groups was only 18% of that of the treated group of equal amounts of free PD-1 mab and T cells. The technique can be widely applied to ACTs using T cell receptor T cells (TCR-T) or chimeric antigen receptor T cells (CAR-T) to improve the treatment outcome of patients with solid tumors. In addition, the method can also provide new ideas and technical references for developing future cell surface nano-drug carriers and cell surface anchoring technologies.

The embodiment discloses a preparation method of nanogel, which comprises the following steps:

1) preparing a connecting agent solution; wherein the linking agent comprises DTSSP or a molecule with chemical functional groups at two ends capable of reacting with the antibody;

2) adding the connecting agent solution obtained in the step 1) into the monoclonal antibody solution for mixing reaction to obtain nanogel; wherein the monoclonal antibody comprises PD-1 monoclonal antibody, CTLA-4 monoclonal antibody and TNF alpha monoclonal antibody, and the molar ratio of the monoclonal antibody to the linking agent is (1: 25-1: 200); the reaction time is 1-2 hours;

in a preferred embodiment, the method further comprises a step 3) of adding a DBCO-PEG-NHS solution to the nanogel obtained in the step 2) and carrying out a reaction on a shaker to obtain DBCO-aPD 1-NGs.

In a preferred embodiment, the method further comprises a step 3) of adding a DSPE-PEG-NHS solution to the nanogel obtained in the step 2) and carrying out a reaction on a shaker to obtain DSPE-aPD 1-NGs.

The embodiment also discloses the nanogel prepared by the preparation method of the nanogel.

The embodiment also discloses an engineered immune cell, wherein DBCO-aPD1-NGs are combined with the azide-labeled engineered immune cell through a click chemistry reaction, so that the nanogel is anchored on the surface of the engineered immune cell.

In a preferred embodiment, the engineered immune cell is prepared by the following method:

1) preparing DSPE-PEG-N3 solution;

The embodiment also discloses an engineered immune cell, wherein the DSPE-aPD1-NGs are combined with the engineered immune cell through co-incubation, so that the nanogel is anchored on the surface of the engineered immune cell.

1) resuspending the engineered immune cells in a culture medium;

It should be noted that: according to research, the general nanogel cannot be adsorbed on the engineered immune cells, and can be adsorbed on the engineered immune cells after being modified by DBCO and DSPE.

In preferred embodiments, the engineered immune cells comprise T cells, NK cells, or macrophages.

The medium includes a conventional medium or physiological saline as long as it does not contain serum.

Also disclosed in this example are inhibitors, including the engineered immune cells described above.

The embodiment also discloses a pharmaceutical composition, which comprises the T cell and pharmaceutically acceptable auxiliary materials.

The term "pharmaceutically acceptable adjuvant" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by genomic AR,19th ed. pennsylvania: mach publishing g Company,1995), and include, but are not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants; ionic strength enhancers include, but are not limited to, sodium chloride.

Use of the engineered immune cells, the inhibitors and the pharmaceutical compositions described above in ACTs using T cell receptor T cells (TCR-T) or chimeric antigen receptor T cells (CAR-T) to inhibit the growth of solid tumors. Wherein the solid tumor comprises melanoma, lung cancer, breast cancer, and liver cancer.

Material

PD1, CD3, CD28 monoclonal antibodies were purchased from Bioxcell (Lebanon, NH, USA). Ovalbumin peptide (257-264) was purchased from Invivogen (San Diego, Calif., USA). Murine IL-2and IL-7 were purchased from Peprotech (Rocky Hill, NJ, USA). EasySep^TMmouse CD8⁺T cell isolation kit was purchased from StemCell Techologies (Vancouver, BC, Canada). Ficoll-Paque Plus was purchased from Cytiva (Pharmacia, Uppsala, Sweden). LIVE/DEAD Fixable Cell Stain Kit was purchased from Invitrogen (Carlsbad, Calif., USA). Anti-PD-L1 APC (clone 10F.9G2) was purchased from Biolegend (San Diego, Calif., USA). 3,3' -dithiobis (sulfosuccinimidyl propionate) (DTSSP) was purchased from Abcam (Cambridge, UK). DBCO-PEG-NHS (4 repeating units of PEG) was purchased from BroadPharm (San Diego, Calif., USA). Distearoyl phosphatidylethanolamine (DSPE) includes DSPE-PEG-N3(molecular weight, MW, of PEG ═ 2000), DSPE-PEG-NHS (MW of PEG ═ 2000) and DSPE-PEG-FITC (MW of PEG ═ 2000) available from position Biotech (Shanghai, China). Protein labeling kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). WST-1cell promotion assay kit, Lactate Dehydrogene (LDH) cytoxicity assay kit, DiI, Hoechst33342 and RIPALysis buffer were purchased from Beyotime (Shanghai, China). The abbreviations and full names of the above materials are shown in the following Table 1

TABLE 1

Mouse and cell lines

C57BL/6 female mice (6 weeks old) were purchased from Cavens (Changzhou, China). OT-1 mice were purchased from Shanghai Model organics Center (Shanghai, China) and bred by themselves. All mice were housed in SPF grade rooms with 5 mice per cage and adequate feed and drinking water. The breeding rooms are kept in light and dark circulation, each 12 hours (7: 00 am-7: 00 pm) and the room temperature is 25 +/-1 ℃. All animal protocols were reviewed and approved by the institutional animal care and use committee of suzhou university. The B16-OVA cell line was purchased from ATCC (Rockville, Md., USA) and cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS).

Preparation and characterization of Nanogels (NGs)

Example S1:

a certain amount of DTSSP was precisely weighed and dissolved in PBS to prepare a solution of 5 mg/ml. Adding DTSSP into PD-1 monoclonal antibody solution according to the molar ratio of 1:200 (antibody: cross-linking agent), uniformly mixing, and then adding 1M NaHCO into the reaction system according to the proportion of 10% (volume/volume)₃The pH of the solution was adjusted to about 8.2. The reaction was carried out for 2h at room temperature on a four-dimensional rotator, and then centrifuged to remove excess PD-1 mAb and DTSSP.

The process parameters and corresponding nanogel particle sizes of the examples S2-12 (see also FIG. 2A) are specifically shown in Table 2 below, and the examples S2-12 are different from the example S1 in aPD1: DTSSP and difference in reaction time;

TABLE 2

S2	aPD1：DTSSP	Reaction time (hours)	Nanogel particle size (nm)
				S3	1：25	1	10
S4	1：50	1	13
				S5	1：100	1	12
S6	1：150	1	122
				S7	1：200	1	130
S8	1：25	2	12
				S9	1：50	2	13
S10	1：100	2	135
				S11	1：150	2	160
S12	1：200	2	107

Example T1:

if the surface of the nanogel is modified by DBCO, DBCO-PEG-NHS is dissolved in DMSO with the concentration of 10mg/mL, the solution is added into NG solution according to the molar ratio of 1:30 (aPD1: DBCO-PEG-NHS), a proper amount of PBS solution is added to reduce the concentration of the DMSO to 1%, and the mixture is reacted on a shaker at room temperature for 5 hours and then incubated at 4 ℃ overnight.

The process parameters and corresponding nanogel particle sizes for examples T2-T6 (see also fig. 2D-E) are specifically shown in table 3 below, and examples T2-T6 differ from example T1 by aPD1: differences in DBCO-PEG-NHS;

TABLE 3

T2	aPD1：DBCO-PEG-NHS	Binding rate of nanogel to T cell (%)
			T3	1:5	64
T4	1:10	91
			T5	1:20	99
T6	1:30	99

Example V1:

If the surface of the nanogel is modified by DSPE, DSPE-PEG-NHS is dissolved in absolute ethyl alcohol, the concentration is 10mg/ml, the solution is added into NGs solution according to the molar ratio of 1:30 (aPD1: DSPE-PEG-NHS), a proper amount of PBS solution is added to reduce the concentration of the absolute ethyl alcohol to 1%, and the solution is reacted on a shaker at room temperature for 5 hours and then incubated at 4 ℃ overnight.

The process parameters and corresponding nanogel particle sizes of examples V2-V7 (see also fig. 2F-G) are specifically shown in table 4 below, and examples V2-V7 differ from example V1 by aPD1: differences in DBCO-PEG-NHS;

TABLE 4

V2	aPD1：aPD1:DSPE-PEG-NHS	Binding rate of nanogel to T cell (%)
			V3	1：2	94
V4	1：5	96
			V5	1：10	100
V6	1：20	100
			V7	1：30	100

After the reaction, the reaction product was collected in an ultrafiltration tube (Amicon Ultra, MWCO 100kDa), and excess DBCO-PEG-NHS or DSPE-PEG-NHS was washed off by PBS ultrafiltration. To prepare fluorescently labeled PD-1 mAb, the PD-1 mAb was labeled with Fluorescein Isothiocyanate (FITC) or Allophycocyanin (APC) using a commercially available protein labeling kit (Thermo Fisher Scientific, USA) for NGs quantification and visualization. The NGs were characterized for Particle Size and morphology using dynamic light scattering (90Plus Particle Size Analyzer, Brookhaven, USA) and transmission electron microscopy.

Kinetics of release of NGs

The kinetics examination is carried out on the PD-1 monoclonal antibody released in vitro by the NGs by adopting a dialysis bag method, and the release conditions are as follows:

1. PBS solution

2. PBS solution containing 1mM GSH

3. PBS solution containing 10mM GSH

To study the in vitro release kinetics of PD-1 mAb in NGs, APC-labeled PD-1 mAb (aPD 1) was used^APC) To prepare 300. mu.g of NGs. 100 μ l of aPD1 was taken^APCFluorescence intensity (Ex 620nm, Em 670nm) of NGs in 96-well black-bottom plates was measured with a microplate reader (M1000 Pro, TECAN, Switzerland). Within a certain range, aPD1^APCFluorescence intensity of (2) and aPD1^APCIs proportional to the concentration of (D), and 300. mu.g of aPD1 is calculated^APC-total fluorescence intensity of NGs. 300 μ g of aPD1 was added to dialysis bags (MWCO. RTM. 300k Da) fastened at one end^APCNGs, while the opening is tightened, and then placed in 15ml centrifuge tubes containing 5ml of different release media. The centrifuge tube was placed in a constant temperature shaker, released at 37 ℃ for 24h at 100rpm, and 100. mu.l of release medium was aspirated from the centrifuge tube at 1, 2, 4, 6, 8, 12, and 24h, respectively, into a black matrix 96-well plate, and then supplemented with 100. mu.l of fresh release medium at the same temperature. Finally, the fluorescence intensity of 100. mu.l of the release medium at different time points was measured by a microplate reader, and the cumulative percentage release of PD-1 mab was calculated from the fluorescence intensity.

aPD1-NGs without fluorescent labels were treated with 10mM GSH for 12 hours, and then the NGs were characterized for Particle Size and morphology using dynamic light scattering (90Plus Particle Size Analyzer, Brookhaven, USA) and transmission electron microscopy.

Activation and culture of T cells

Using EasysepTM CD8⁺T cell isolation kit (StemCell Technologies, Vancouver, BC, Canada) immature CD8 was isolated from spleen of C57BL/6 mice⁺T cells. CD8⁺The T cells were resuspended in RPMI 1640 medium supplemented with murine IL-2(10ng/mL) and IL-7(1ng/mL), and then CD8 was added⁺T cell suspension added to the bottom with anti boundCD3 (1. mu.g/mL) and anti-CD28 (5. mu.g/mL) in 6-well plates. After 3 days incubation at 37 ℃ the medium was changed and the dead cells were removed by centrifugation on a Ficoll-Pague Plus gradient, CD8⁺T cells were further cultured in RPMI 1640 medium containing murine IL-2(10 ng/mL). The growth density of T cells is maintained at 0.75-2X 10⁶The solution is changed every two days between each mL.

Splenocytes were extracted from the spleen of OT-1 mice, red blood cells were removed from the splenocytes using red blood cell lysate, and the remaining cells were resuspended in OVA-containing suspension_257-264peptide (1. mu.g/mL), murine IL-2(10ng/mL) and IL-7(1ng/mL) in RPMI 1640 medium at a cell density of 2X 10⁶one/mL. After 3 days incubation at 37 ℃ the medium was changed, dead cells were removed by centrifugation on a Ficoll-Pague Plus gradient and the resulting CD8 was collected⁺T cells were cultured in RPMI 1640 medium containing murine IL-2(10ng/mL) and IL-7(10ng/mL) for 1 day and then used in vitro experiments, or for 2 days and then used in vivo animal experiments.

NGs anchored on the surface of T cells

Immature or activated T cells were resuspended in PBS buffer at a cell density of 4X 10⁶one/mL. Accurately weighing a certain amount of DSPE-PEG-N₃Dissolving in anhydrous ethanol to obtain 20mg/ml solution, each 1 × 10⁶50 μ g DSPE-PEG-N was added to each T cell₃Adding a proper amount of PBS solution to reduce the concentration of absolute ethyl alcohol in the incubation system to 1 percent, incubating for 1 hour at 37 ℃, and gently mixing once every 15 minutes. 700g centrifugation to remove excess DSPE-PEG-N₃The obtained product has DSPE-PEG-N connected to the surface₃T cell (T-N) of (2)₃). After centrifugation 3 times, the T cells were resuspended in serum-free RPMI 1640 medium at a cell density of 5X 10⁶Per mL, according to 10⁶An amount of 80. mu.g DBCO-aPD1-NGs per cell DBCO-aPD1-NGs was added to the cell suspension and incubated at 37 ℃ for 1.5 hours, gently mixed every 20 minutes. 700g centrifugation removed unbound DBCO-aPD1-NGs from the supernatant and T cells with DBCO-aPD1-NGs bound to their surface were collected (DBCO-aPD1-NGs @ T-N)₃) The cells were resuspended in PBS solution for further experiments.

To obtain T cells with DSPE-aPD1-NGs bound to their surfaces, the T cells were heavySuspended in serum-free RPMI 1640 medium at a cell density of 5X 10⁶one/mL. According to each 10⁶80 μ g of DSPE-aPD1-NGs per cell to the cell suspension DBCO-aPD1-NGs were added and incubated at 37 ℃ for 1.5 hours, gently mixed every 20 minutes. Unbound DSPE-aPD1-NGs were removed from the supernatant by centrifugation, T cells (DSPE-aPD1-NGs @ T) with DSPE-aPD1-NGs bound to their surface were collected and resuspended in PBS solution for further experiments.

aPD1 observed with Confocal Laser Scanning Microscope (CLSM)^APCNGs are anchored to the surface of T cells. T cells were stained with Hoechst33342 for 10 min, centrifuged to remove excess Hoechst33342, stained with DiI for 20 min, then centrifuged and washed 3 times with PBS, and finally visualized and imaged with DSEP-aPD1 using CLSM (LSM710, Zeiss, German)^APC-NGs @ T and DBCO-aPD1^APC-NGs@T-N₃。

Drug loading assay for T cell surface NGs

aPD1 labeled with FITC (aPD 1)^FITC) Preparation of DBCO-aPD1^FITC-NGs and DSPE-aPD1^FITCNGs to compare the drug loading of the two nanoparticles on the T cell surface. After NGs are bound to T cells, NG fluorescent signals on T cells are detected using a flow cytometer (FACS Aria III, BD Biosciences, USA), thereby evaluating the binding efficiency of NGs to T cells. A fixed number of NGs-bound T cells were lysed using RIPA lysate and fluorescence intensity was measured using a microplate reader, pass aPD1^FITCCalculation of T cell surface aPD1 from Standard Curve^FITCThe drug loading amount of (1).

Evaluation of NGs cytotoxicity in vitro

CD8⁺T cells with DSPE-PEG-N embedded on the cell surface₃CD8 (1)⁺T cells (T-N)₃) And CD8 with DBCO-aPD1-NGs bound to the cell surface⁺T cells (DBCO-aPD1-NGs @ T-N)₃) The cells were cultured in RPMI 1640 medium containing murine IL-2(10ng/mL) for 1 day and 2 days, respectively. T cells were stained using the LIVE/DEAD Fixable Cell Stain Kit, and then assessed for T Cell activity using flow cytometry. At the same time, the number of T cells was counted to examine the effect of surface-anchored NGs on T cell proliferation.

In vitro tumor killing experiment

And (3) adopting a lactate dehydrogenase detection method to investigate the influence of the NGs on the tumor killing function of the T cells. The experimental procedure was as follows:

(1) taking B16-OVA cells growing logarithmically according to 1X 10⁴Each well was seeded at a density of 100. mu.L of DMEM medium (containing 10ng/mL IFN-. gamma.) per well in a 96-well plate, and a T cell non-administration group and a whole killer group were set as controls and cultured at 37 ℃ for 24 hours.

(2) And after 24 hours of culture, observing the state and the density of the cells under a microscope, wherein the density reaches 70%, and carrying out the next experiment. Carefully discard the original culture medium, and add activated OT-1 CD8 at different effective target ratios (E/T)⁺T cells, T cells spiked with free aPD1, and T cells surface-bound aPD1-NGs (DBCO-aPD1-NGs @ T-N)₃) mu.L of RPMI 1640 medium (containing 10ng/mL murine IL-2) per well.

(3) After 24 hours of incubation, the morphology of B16-OVA cells was observed by a bright field microscope (magnification 100X), and the T cell killing effect was examined according to the instructions of the lactate dehydrogenase cytotoxicity assay kit.

In vivo therapeutic experiments

To examine the therapeutic effect of designed NGs in vivo, B16-OVA cells were dissolved in PBS, matrigel was added at 1:1, and tumor cells were inoculated subcutaneously at a density of 2X 10 in C57BL/6 mice⁵One/only. After 6 days of inoculation, tumor-bearing mice were intraperitoneally injected with cyclophosphamide at a dose of 100mg/kg to eliminate lymphocytes in the mice. The mice were randomly divided into the following 4 groups of 5 mice each.

(1) PBS group

(2)CD8⁺T cell group

(3) T + free aPD1

(4)DBCO-aPD1-NGs@T-N₃Group of

After 1 day, 5X 10⁶Activated OT-1 CD8⁺T cells, T cells mixed with free PD-1 mAb, and T cells with equal amounts of aPD1-NGs bound to their surface were injected tail vein into mice. Each group of mice was tested every two days for tumor size and body weight. At the end of the experiment, each group of mice was euthanized, tumors were removed, photographed and weighed.

Statistical analysis

All experiments were statistically analyzed using GraphPad Prism software. All numerical values and errors are mean ± s.d., unless otherwise specified. Except for special instructions, two-sided t-tests were used for comparisons between the two data sets, one-way analysis of variance (ANOVA) was used for comparisons of the data sets at a single time point, and two-way analysis of variance was used for tumor growth curves. P < 0.05; p < 0.01; p < 0.001; p < 0.0001.

As a result:

aPD1-NGs preparation and characterization

The connecting agent DTSSP contains disulfide, and n-hydroxysuccinimide (NHS) functional groups at two ends of the disulfide can react with amine groups in the antibody, so that the PD-1 monoclonal antibody and the DTSSP can be crosslinked to form NGs. The particle size of the free PD-1 mAb was about 11nm and the particle size of the NGs was about 109nm as measured by Dynamic Light Scattering (DLS) (FIG. 2A, left). The particle size results show that PD-1 monoclonal antibody and DTSSP are successfully crosslinked to form NGs.

In order to allow NGs to anchor to the T cell surface, we functionally modify NGs. Different molar ratios of DBCO-PEG-NHS or DSPE-PEG-NHS were conjugated to NGs via amide reactions. DBCO-modified NGs (DBCO-aPD1-NGs) and DSPE-functionalized NGs (DSPE-aPD1-NGs) were slightly larger than the particle size of the NGs, 124nm and 162nm, respectively (FIG. 2A, center and right). The reason why the larger particle size of DSPE-aPD1-NGs compared to DBCO-aPD1-NGs is probably due to the longer PEG chains in DSPE-PEG-NHS. Transmission Electron Microscopy (TEM) imaging showed NGs of about 95nm, DBCO-aPD1-NGs of about 115nm, DSPE-aPD1-NGs of about 150nm, and electron micrographs matching the values measured by dynamic light scattering (FIG. 2A).

Binding of NGs to T cell surfaces

To explore the optimal time required for DSPE to insert into T cell membranes, DSPE-PEG-FITC was incubated with T cells at 37 ℃ for 0.5, 1, 2, 4h, and unbound DSPE-PEG-FITC was washed off by centrifugation. FITC fluorescence intensity on the surface of the T cell is detected in a flow mode, and insertion efficiency of DSPE on a T cell membrane is evaluated. Flow results showed that significant fluorescent signal was detected on all T cells after only 0.5h incubation (fig. 2B). The Mean Fluorescence Intensity (MFI) of T cells increased with prolonged binding time, indicating more insertion of DSPE-PEG-FITC (FIGS. 2B-C). The trend of the increase in fluorescence intensity was stable after 2h, probably due to saturation of the inserted lipids in the cell membrane (FIGS. 2B-C). The influence of the overall treatment time and the DSPE membrane insertion process on the activity of the T cells is comprehensively considered, and the incubation time of 1h is selected in the subsequent experiment.

The DSPE-PEG-N is prepared by the method₃Embedding into the surface of T cells. Take aPD1^FITCDBCO functionalized NGs (DBCO-aPD 1) are prepared by different feeding molar ratios of DBCO-PEG-NHS^FITCNGs) to explore its pair DBCO-aPD1^FITC-effect of the efficiency of the anchorage of NGs on the surface of T cells. DBCO-aPD1^FITCNGs bind to T cell surfaces by click reaction of cell surface azides with DBCO groups on NGs. Centrifuging to remove unbound DBCO-aPD1^FITCNGs, flow analysis of the efficiency of their anchorage on the surface of T cells.

Streaming results display, aPD1^FITCDBCO-aPD1 obtained by crosslinking with DBCO-PEG-NHS at a molar ratio of 1:5^FITCNGs can anchor to more than 60% of T cells and bind to more than 90% of T cells at a molar ratio of 1: 10. Further increasing the reaction molar ratio to 1:20 and 1:30, DBCO-aPD1^FITCThe binding rate of NGs to T cells almost reaches 100%. While only a small fraction of NGs that were not modified with DBCO anchored to the T cell surface, probably due to PD1 expressed on T cells, some NGs bound to T cells through PD1-aPD1 interactions (fig. 2D). MFI on the surface of T cells showed DBCO-aPD1 produced at a molar ratio of 1:30^FITCNGs have the highest binding rate, with 8-fold higher number of NGs anchored on T cell surface than unmodified NGs (fig. 2E). DSPE-aPD1 modified by different molar ratios^FITCThe binding Effect of NGs to T cells with DBCO-aPD1^FITCNGs are similar. When aPD1^FITCWhen the molar ratio of DBCO-PEG-NHS to DBCO-PEG-NHS is more than 1:5, more than 90 percent of T cells can be mixed with DSPE-aPD1^FITCNGs binding (fig. 2F). MFI on the surface of T cells shows that increasing the molar ratio increases DSPE-aPD1^FITCBinding efficiency of NGs to T cell surface (fig. 2G).

Confocal laser scanningMicroscopic (CLSM) images showed that NGs fluorescence (red) co-localized with the DiI (green) signal of the membrane dye, indicating that NGs are anchored to the T cell membrane rather than being endocytosed by the T cell (fig. 3). Furthermore, DSPE-aPD1^APCThe fluorescent signal of NGs is significantly higher than that of NGs, which indicates that T cell membrane and DSPE-aPD1^APCStronger binding of NGs (fig. 3A). For absence of DSPE-PEG-N₃Labeled T cells or NGs without DBCO modification, observed weak NGs fluorescence on T cells. In contrast, DSPE-PEG-N₃After insertion into T cells, DBCO-aPD1 was observed^APC-NGs fluorescence. Thus, this also demonstrates that most DBCO-aPD1^APCNGs do bind to T cells via a click reaction of DBCO with azide.

Drug loading comparison of different modification methods

NGs can be bound to the T cell surface by two different modification methods. For DBCO-aPD1^FITCNGs, reaction of DSPE-PEG-N₃Inserting T cell membrane, and click reacting to obtain DBCO-aPD1^FITCCoupling of NGs in situ to the surface of T cells to obtain DBCO-aPD1^FITC-NGs@T-N₃. For DSPE-aPD1^FITCNGs anchored directly to the T cells by the hydrophobic long alkyl chain of the DSPE, giving DSPE-aPD1^FITC-NGs@T。

To compare the coupling efficiencies of the two processes, DBCO-aPD1 was prepared from the same batch of NGs^FITCNGs (1:30) and DSPE-aPD1^FITC-NGs (1: 30). Equal amount of DBCO-aPD1^FITC-NGs and DSPE-aPD1^FITCNGs in equal numbers of T cells or T-N, respectively₃Cells were co-incubated for 1.5 h. Unbound NGs were washed off by centrifugation and T cell MFI was detected using flow cytometry to assess the binding efficiency of both modification methods. The results show that DBCO-aPD1^FITC-NGs and T-N₃The cell combination efficiency is as high as 99.7 percent, and the cell combination efficiency is not higher than that of DSPE-PEG-N₃The embedded T cells bound only 23% of the efficiency, which is probably due to the expression of PD1 on T cells (fig. 4A). Although DSPE-aPD1^FITCThe binding efficiency of NGs to T cells was 98.4%, slightly lower than that of DBCO-aPD1^FITC-NGs and T-N₃Binding efficiency of cells, but in terms of T cell MFI, DBCO-aPD1^FITC-NGThe binding efficiency of s is DSPE-aPD1 ^FITC2 times that of NGs (FIGS. 4A-B).

To calculate the amount of PD-1 mAb anchored to the surface of T cells, the surface was lysed using RIPA lysate to anchor aPD1^FITC-T cells of NGs, and then FITC fluorescence readings are determined with a microplate reader. According to free aPD1^FITCThe concentration of T cell surface bound aPD1 was calculated from the constructed standard curve. Respectively mixing 100-300 μ g DBCO-aPD1^FITC-NGs and DSPE-aPD1^FITCNGs added to T cells, the amount of aPD1 bound to the T cell surface increased with increasing concentration of NGs, but the trend of increase gradually leveled off (fig. 4C). When the amount of NGs administered was fixed at 300 μ g, the amount of aPD1 anchored on the T cell surface correlated strongly with the number of T cell lysates, indicating that the method was reliable for quantitative analysis of loaded aPD 1. The slope of the fitted curve indicates DBCO-aPD1^FITCNGs may be in every 10 th⁶Each T cell was loaded with 4. mu.g of aPD1, ratio DSPE-aPD1^FITC-NGs are about 2 times higher (fig. 4D).

In vitro release of NGs

Since the PD-1 monoclonal antibody is formed by crosslinking with a DTSSP linker containing a disulfide bond, NGs will decompose and release the PD-1 monoclonal antibody under reducing conditions. Dynamic light scattering and transmission electron microscopy showed a significant reduction in the particle size of NGs in a 10mM Glutathione (GSH) -induced reducing environment (fig. 5A-B). A substantial portion of the particle size was matched to the particle size of the free PD-1 mab, indicating that NGs can release PD-1 mab under reducing conditions (fig. 5A). aPD1^APCRelease studies of NGs in 10mM GSH solution indicated about 70% aPD1^APCIsolated from NGs within 12h and released as free antibody (fig. 5C). On the other hand, in a low reducing environment (1mM GSH solution), aPD1 in 12h^APCRelease only 17%, aPD1 within 24h in PBS solution^APCLess than 5% (fig. 5C). These results indicate that NGs have good stability under normal physiological conditions and release PD-1 mab under redox conditions.

To examine whether NGs "back-wraps" release PD-1 monoclonal antibodies in the face of changes in redox activity on the cell surface, we measured both unactivated T cells and activated T cells using the WST-1 methodSurface reduction potential of (2). The experimental results show that OVA_257-264Polypeptide-activated OT-1T cell surface reduction potential vs. unactivated CD8⁺T cells increased 3-fold more (fig. 5D). DBCO-aPD1^FITCAfter NGs bind to the T cell surface, more than 80% of NGs fluorescence disappears within 1 day under the action of activation signals (fig. 5E-F), indicating that aPD1 in the NGs "backpack" can be released when T cells are activated.

The NGs anchored on T cells do not affect the in vitro functions of the cells

Packaging NGs in OT-1 CD8⁺After T cell surface, T cells were expanded in medium containing cytokines for 2 days. Flow results show that DSPE-PEG-N₃Insertion into the cell membrane, or coupling of DBCO-aPD1-NGs to azide-labeled T cells, did not affect T cell activity and proliferation (FIG. 6A).

The OT-1 CD8 supported by PD-1 monoclonal antibody or NGs' knapsack⁺T cells were co-incubated with B16-OVA tumor cells to assess whether coupling of NGs to the cell surface would affect the killing ability of T cells in vitro. Pretreatment of B16-OVA cells with IFN- γ upregulated the expression of PD-L1 (fig. 6B), thereby producing immunosuppressive effects on T cells by tumor cells. Tumor cells grew normally in wells without the addition of T cells (fig. 6C). When the effective target ratio (E/T) is 20:1, T cells accumulate on and lyse tumor cells, leaving visible gaps between tumor cells. The T cells supported by the free PD-1 monoclonal antibody and DBCO-aPD1-NGs backpack have stronger killing effect on the tumor, and the gap area between the tumor cells is larger and more obvious (FIG. 6C). At an E/T ratio of 5:1, not too many voids were observed in all groups, indicating that T cells were less soluble in the tumor at this E/T ratio. Similarly, Lactate Dehydrogenase (LDH) cytotoxicity experiments showed that activated T cells have a weak tumor killing capacity due to immune checkpoint inhibition without support by PD-1 mab. aPD 1-NGs-bearing T cells had similar killing efficiency compared to free PD-1 mab-supported T cells (fig. 6D), probably because both PD-1 mab and free PD-1 mab released by NGs were available to T cells in co-culture systems. The results also show that the formation of NGs is paired with PThe biological activity of the D-1 monoclonal antibody has no obvious influence.

PD1 monoclonal antibody NGs anchored on cell surface for improving curative effect of adoptive T cell therapy

To test the antitumor effect of aPD 1-NGs-bound T cells in vivo, we inoculated C57Bl/6J mice with B16-OVA cells subcutaneously. The mice were cleared of lymphocytes 6 days after tumor inoculation, and were treated with adoptive cell therapy, i.v. PBS, activated OT-1 CD8⁺Equal amounts of PD-1 mAbs NGs anchored to the surface of T cells, T cells and free PD-1 mAbs, and T cells and free PD-1 mAbs (FIG. 7A). When mice were given tumor-specific cytotoxic T cells, tumor growth was significantly slowed. Free PD-1 mAb together with T cells can further retard tumor growth. More importantly, the group of PD-1 mabs anchored to the surface of T cells inhibited tumor growth significantly more than the equivalent dose of free PD-1 mab in combination with T cells (fig. 7B). At the end of the experiment, tumors were extirpated and weighed. DBCO-aPD1-NGs @ T-N₃The mean tumor weight of the groups was only 18% of the free PD-1 mab group and 9% of the pure T cell group (fig. 7C-D). The hematoxylin-eosin stained sections of the mouse body weight profile and major tissues such as heart, liver, spleen, lung and kidney also showed that anchoring of PD-1 mab to the surface of T cells did not cause significant toxicity in mice (FIGS. 7E-F).

Compared with the random insertion of DSPE-aPD1-NGs into cell membranes, DBCO-aPD1-NGs and DSPE-PEG-N are utilized₃The pre-labeled T cell click-binding approach may allow the nanogel to be more efficiently coupled to the T cell surface. We speculate that DSPE-aPD1^FITCThe larger size of NGs may affect the intercalation of lipid duplexes into the cell membrane. In contrast, DSPE-PEG-N₃Can be more efficiently inserted into a membrane, T-N₃With DBCO-aPD1^FITCIn-situ click reactions of NGs are fast and efficient, resulting in more aPD1^FITC-NGs bind to T cells.

Oligonucleotides, small molecule drugs and chemical-loaded liposomes can be modified and anchored to the cell surface by spontaneous insertion of diacyl lipids into the cell membrane. This study explored how to use diacyl lipids to anchor protein drugs such as mabs to the cell surface. In contrast, coupling nanocarriers to the cell surface through chemical reactions or ligand-receptor interactions, spontaneous membrane insertion of diacyl lipids does not alter cell membrane proteins, with less impact on cell signal transduction and function. Furthermore, by membrane insertion, T cells can be loaded with up to 4 μ g of PD1 mab, comparable to the amount of cytokine drug anchored on T cells by ligand-receptor interaction. Thus, membrane insertion of diacyl lipids may provide a more cost-effective alternative to existing methods of anchoring protein drugs to the surface of T cells.

The method for anchoring the PD-1 monoclonal antibody nanogel on the surface of the T cell is considered to improve the treatment effect through the following mechanisms: 1) when NGs-loaded ACT T cells infiltrated the tumor, more PD1 mab could enter the tumor. The spatiotemporal co-existence of immune checkpoint inhibitors and ACT cells also allowed PD1 mab to provide support for T cells when needed. 2) In TME, the release of immune checkpoint inhibitors is triggered by the activation of ACT T cells after encounter with tumor antigens. The tumor responsive release reduces leakage of immune checkpoint inhibitor in circulation in vivo and increases the effective concentration of immune checkpoint inhibitor in the TME. 3) Anchoring PD1 mab NGs to the ACT T cell surface minimizes PD-1 mab loss due to the reticuloendothelial system.

The curative effect of ACT in a mouse subcutaneous tumor model is obviously improved by a method of combining drug release and T cell activation by cooperating ACT and an immune checkpoint inhibitor in a NGs 'backpack' mode. This technique can also be extended to the treatment of common solid tumors, such as colon cancer and breast cancer. It may also help to further develop T cell packs and cell surface anchoring techniques.

We show a strategy to increase the therapeutic efficacy of Adoptive Cell Therapy (ACT) in solid tumors by anchoring PD1 mab to the surface of T cells prior to adoptive transfer. T cell activation-responsive NGs are formed by cross-linking PD-1 monoclonal antibodies with a linker containing disulfide bonds followed by surface modification. We tested two methods of anchoring NGs to the surface of T cells. One method is that firstly, the azido diacyl lipid is inserted into the labeled T fine membraneCells, then through the bioorthogonal click chemistry reaction DBCO-aPD1-NGs anchored to T cells. Alternatively, DSPE-aPD1-NGs are directly bound to the T cell surface by membrane insertion methods. Although DBCO-aPD1-NGs and DSEP-aPD1-NGs are capable of anchoring greater than 98% of T cells in vitro, DBCO-aPD1-NGs are present every 10⁶The loading capacity on each T cell can reach 4 mu g. The anchorage of NGs to the cell surface has no negative effects on the survival, proliferation and in vitro antitumor activity of T cells. In the mouse subcutaneous melanoma model, DBCO-aPD1-NGs co-exist with ACT T cells spatio-temporally and trigger the release of PD-1 mab in TME, enabling NGs anchored to the cell surface to significantly inhibit tumor growth. The mean tumor weight of the cell surface anchored NGs group was only 18% of the free PD-1 mab group at the same dose and 9% of the pure T cell group. The method can be widely applied to adoptive cell therapy using TCR-T or CAR-T cells, and provides theoretical basis and technical support for further developing cell surface nano-drug carriers and cell surface anchoring technology.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims

1. A preparation method of nanogel is characterized by comprising the following steps:

1) preparing a connecting agent solution;

2. The method of claim 1, wherein the monoclonal antibody comprises PD-1 monoclonal antibody, CTLA-4 monoclonal antibody, or TNF α monoclonal antibody.

3. The method of claim 2, wherein the linker comprises DTSSP or a molecule having a chemical functional group at both ends thereof capable of reacting with an antibody.

4. The method for preparing nanogel according to claim 1, wherein the molar ratio of the monoclonal antibody to the linker is (1: 25-1: 200); the reaction time is 1-2 hours.

5. The method for preparing nanogel according to any one of claims 1 to 4, further comprising a step 3) of adding a DBCO-PEG-NHS solution to the nanogel obtained in the step 2) and reacting the solution on a shaker to obtain DBCO-aPD 1-NGs.

6. The method for preparing nanogel according to any one of claims 1 to 4, further comprising a step 3) of adding a DSPE-PEG-NHS solution to the nanogel obtained in the step 2) and reacting the solution on a shaker to obtain DSPE-aPD 1-NGs.

7. A nanogel prepared by the method for preparing a nanogel according to any one of claims 1 to 6.

8. An engineered immune cell, wherein the DBCO-aPD1-NGs of claim 5 are combined with azide-labeled engineered immune cells by click chemistry, such that the nanogel is anchored to the surface of the engineered immune cell.

9. The engineered immune cell of claim 8, wherein the engineered immune cell is prepared by the following method:

1) preparing a DSPE-PEG-N3 solution;

10. An engineered immune cell, wherein the DSPE-aPD1-NGs of claim 6 are co-incubated with the engineered immune cell in combination with an anchor for the nanogel on the surface of the engineered immune cell.

11. The engineered immune cell of claim 10, wherein the engineered immune cell is prepared by the method comprising:

1) resuspending the engineered immune cells in a culture medium;

12. The engineered immune cell of any one of claims 8 to 11, wherein the engineered immune cell comprises a T cell, an NK cell, or a macrophage.

13. An inhibitor comprising the engineered immune cell of any one of claims 8 to 12.

14. A pharmaceutical composition comprising the engineered immune cell of any one of claims 8 to 12 and a pharmaceutically acceptable excipient.

15. Use of an engineered immune cell according to any one of claims 8 to 12, an inhibitor according to claim 13 and a pharmaceutical composition according to claim 14 in adoptive cell therapy using T cell receptor T cells or chimeric antigen receptor T cells to inhibit the growth of solid tumors.