CN115887493A - Application of NK (Natural killer) cells, NK cell reinfusion preparation and combined preparation - Google Patents
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Abstract
The invention provides an application of NK cells, an NK cell reinfusion preparation and a combined preparation, wherein NK cell reinfusion can prevent cytomegalovirus infection, eliminate cytomegalovirus infection and promote immune reconstitution of NK cells, is safe and well-tolerated, does not have serious side effects related to infusion, does not cause virus-resistant strains by adopting NK cell reinfusion, does not cause a series of drug side effects such as obvious bone marrow inhibition and renal function damage, and has mature and stronger functions of NK cell phenotypes reconstructed in vivo of patients after NK cell reinfusion.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of NK cells, an NK cell reinfusion preparation and a combined preparation.
Background
Following hematopoietic stem cell transplantation or solid tumor transplantation, the most common complication is Cytomegalovirus (CMV) infection, accounting for nearly 50% of transplant-related deaths. Since the number and function of early immune cells are not reconstructed to normal level after transplantation, cytomegalovirus infection is easy to occur, the death risk related to transplantation is increased, and the transplantation result is poor. The antiviral drugs have limited prevention and treatment effects, and long-term application of the antiviral drugs can cause virus-resistant strains, and can cause a series of drug side effects such as obvious bone marrow suppression, renal function damage and the like. Prevention and treatment of CMV infection is therefore still a major challenge to be addressed at present.
Disclosure of Invention
In order to solve the problems in the background art, the invention provides an application of NK cells and an NK cell reinfusion preparation, so as to prevent cytomegalovirus infection or eliminate cytomegalovirus infection or promote the immune reconstitution of NK cells.
In a first aspect, the invention provides a use of NK cells for preparing a preparation for preventing cytomegalovirus infection, wherein the preparation for preventing cytomegalovirus infection comprises a first preparation; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 7 /kg, said first formulation is for use in the prevention of cytomegalovirus infection.
Alternatively, the first formulation is administered two times each 20 days and 27 days after hematopoietic stem cell transplantation.
Optionally, the agent for preventing cytomegalovirus infection further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M 2 The second formulation is administered after administration of the first formulation, the second formulation being for aiding the first formulation in preventing cytomegalovirus infection.
In a second aspect, the present invention provides a use of NK cells in the preparation of a cytomegalovirus-clearing formulation comprising a first formulation; the first formulation comprises in vitro expanded NK cells, the first formulationThe content of NK cells in the preparation is not less than 1 × 10 7 /kg, said first formulation is used to clear cytomegalovirus infections.
Alternatively, the first formulation is administered two times each 20 days and 27 days after hematopoietic stem cell transplantation.
Optionally, the agent for clearing cytomegalovirus infection further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M 2 The second agent is administered after administration of the first agent, the second agent being used to aid the first agent in clearing cytomegalovirus infections.
In a third aspect, the present invention provides the use of NK cells in the preparation of an immune reconstitution promoting formulation, said immune reconstitution promoting formulation comprising a first formulation; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 7 /kg, said first formulation is for promoting immune reconstitution of NK cells.
Alternatively, the first formulation is administered two times each 20 days and 27 days after hematopoietic stem cell transplantation.
Optionally, the agent that promotes immune reconstitution further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M 2 And a second formulation for administration after administration of the first formulation, the second formulation for assisting the first formulation in promoting immune reconstitution of NK cells.
In a fourth aspect, the present invention provides an NK cell transfusion preparation comprising: a first preparation comprising ex vivo expanded NK cells, the content of the ex vivo expanded NK cells in the first preparation being not less than 1 x 10 7 /kg, the first formulation is for preventing cytomegalovirus infection, or for clearing cytomegalovirus infection in vivo, or for promoting immune reconstitution of NK cells;
optionally, the NK cell reinfusion formulation further comprises a second formulation; the second formulation comprises IL-2, the level of IL-2 in the second formulation being 40 ten thousand IU/M2, the second formulation being administered after administration of the first formulation.
In a fifth aspect, the invention provides a combined preparation for preventing cytomegalovirus infection after hematopoietic stem cell transplantation, which comprises an NK cell reinfusion preparation and IL-2, wherein the content of in-vitro amplified NK cells in the NK cell reinfusion preparation is not less than 1 x 10 7 The content of the IL-2 is 40 ten thousand IU/M2; the NK cell transfusion preparation was administered twice on 20 days and 27 days after hematopoietic stem cell transplantation, respectively, and IL-2 was administered three times per week after the first administration of the NK cell transfusion preparation.
In a sixth aspect, the invention provides a combined preparation for eliminating cytomegalovirus infection after hematopoietic stem cell transplantation, which comprises an NK cell return preparation and IL-2, wherein the content of in-vitro amplified NK cells in the NK cell return preparation is not less than 1 x 10 7 The content of the IL-2 is 40 ten thousand IU/M2; the NK cell transfusion preparation was administered twice on 20 days and 27 days after hematopoietic stem cell transplantation, respectively, and IL-2 was administered three times per week after the first administration of the NK cell transfusion preparation.
In a seventh aspect, the present invention provides a combined preparation for promoting immune reconstitution of NK cells after hematopoietic stem cell transplantation, the combined preparation comprising an NK cell reinfusion preparation in which the content of in vitro expanded NK cells is not less than 1X 10 and IL-2 7 The content of the IL-2 is 40 ten thousand IU/M2; the NK cell reinfusion formulation was administered twice on days 20 and 27 after hematopoietic stem cell transplantation, and IL-2 was administered three times per week after the first administration of the NK cell reinfusion formulation.
Compared with the prior antiviral drugs for preventing or treating CMV infection, the invention has the following advantages:
the NK cell feedback is safe and well-tolerated, no serious side effect related to infusion occurs, moreover, the adoption of the NK cell feedback can not cause the occurrence of virus-resistant strains, and can not cause a series of drug side effects such as obvious bone marrow suppression and renal function damage, and the NK cell feedback can prevent HCMV infection, promote HCMV elimination and promote NK cell immune reconstitution, and the NK cell phenotype in a patient body after the NK cell feedback is more mature and has stronger function, so that the immune function of the patient is improved, and the infection of other pathogens is prevented.
Current studies apply CMV-specific T cells for the prevention or treatment of CMV infection, but the application of T cells has the restriction of MHC sites, which presents difficulties for donor selection. Compared with T cells, NK cells do not need MHC mediation to generate cytotoxicity, so that the restriction of MHC is avoided, the function is rapidly exerted, and the source selection of donor cells is more extensive.
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FIG. 1 shows the proportion of NK cells in liver, spleen, lung, periphery after NK cell reinfusion for 14 d;
figure 2 shows HCMV clearance in liver, spleen, lung, periphery following NK cell reinfusion for 14 d;
figure 3 shows a comparison of NK reinfusion groups with control group HCMV infection and refractory HCMV infection;
figure 4 shows a comparison of the duration of HCMV infection in NK reinfusion groups versus control groups;
FIG. 5 shows a comparison of NK reconstitution of NK cells in NK reinfused group and control group;
FIG. 6 shows in vivo monitoring of NK cells after 20 patient NK cell reinfusion;
FIG. 7 shows a comparison of HCMV and refractory HCMV infection in IL-2-injected and non-injected IL-2 groups;
FIG. 8 shows the percentage and absolute number of NK cells for the IL-2-injected group and the IL-2-non-injected group;
FIG. 9 shows the absolute numbers of NKG2C + NK subpopulations and the expression of the activation receptor NKP30 in the IL-2-injected group and the IL-2-non-injected group compared to each other.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents and other instruments used are not indicated by manufacturers, and are conventional reagent products which can be purchased in the market.
NK cells are the earliest reconstituted immune cells after hematopoietic stem cell transplantation. It was found that reconstitution of HCMV-specific NK cells 180 days after transplantation was negatively correlated with the incidence of CMV infection. Our previous studies also showed that patients with rapid IFN-. Gamma. + NK cell reconstitution had a low HCMV infection rate 15 days after transplantation. Although there is much clinical evidence that there is a correlation between NK cell function and CMV infection, whether NK cells have anti-CMV activity in vivo to directly kill viruses or inhibit the spread of viruses has not been experimentally confirmed.
Adoptive reinfusion of NK cells holds great promise in the treatment of tumors. However, since the number of NK cells in human peripheral blood is limited, at present, in clinical studies, the in vitro amplified NK cells are mainly used for adoptive transfusion of NK, and the inventors have found through in vitro experiments that the NK cells amplified by mbIL21/4-1BBL have stronger expression of activating receptors, such as NKP30, NKG2D, DNAM-1 and the like. However, CMV has strict species specificity, and in humans, NK cell deficient patients are more likely to relapse after recovery from herpes virus infection, and it is unknown whether NK cells have anti-HCMV effects in clinical applications.
The inventor further explores and discovers that: the NK cell feedback can prevent cytomegalovirus infection, eliminate cytomegalovirus infection and promote the immune reconstitution of NK cells.
Based on the above, in a first aspect, the embodiments of the present invention provide a use of NK cells in the preparation of a preparation for preventing cytomegalovirus infection, wherein the preparation for preventing cytomegalovirus infection comprises a first preparation; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 7 /kg, the first formulation is for use in preventing cytomegalovirus infection.
Alternatively, the first formulation is administered two times each 20 days and 27 days after hematopoietic stem cell transplantation.
The inventors have also found that interleukin-2 (IL-2) can enhance NK cell proliferation, stimulate NK cell activation and cytotoxicity.
In an alternative embodiment of the invention, it is proposed that the agent for preventing cytomegalovirus infection further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M 2 The second formulation is administered after administration of the first formulation, the second formulation being used to assist the first formulation in preventing cytomegalovirus infection.
In the embodiment of the invention, the preparation for preventing cytomegalovirus infection can be applied after all operations (including hematopoietic stem cell transplantation and tumor transplantation) which can cause cytomegalovirus infection, so as to prevent the cytomegalovirus infection caused by the operations.
In the embodiment of the invention, the preparation for preventing cytomegalovirus infection can be applied to all people with low immunity (including HIV infected people, immunodeficiency patients and patients with rheumatic immunity) so as to prevent the people with low immunity from infecting cytomegalovirus.
Based on the same inventive concept, in a second aspect, the invention provides an application of NK cells in preparing a cytomegalovirus removing preparation, wherein the cytomegalovirus removing preparation comprises a first preparation; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 7 Kg, said first formulation is for use in clearing cytomegalovirus infection.
Alternatively, the first formulation is administered two times each 20 days and 27 days after hematopoietic stem cell transplantation.
Optionally, the agent for clearing cytomegalovirus infection further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M 2 The second agent is administered after administration of the first agent, the second agent being used to aid the first agent in clearing cytomegalovirus infections.
In the embodiment of the invention, the preparation for eliminating cytomegalovirus infection can be applied after all operations (including hematopoietic stem cell transplantation and tumor transplantation) which can cause cytomegalovirus infection, so as to eliminate the cytomegalovirus infection caused by the operations.
In the embodiment of the invention, the preparation for eliminating cytomegalovirus infection can be applied to all people with low immunity (including HIV infectors, immunodeficiency patients and rheumatoid immunity patients) so as to eliminate cytomegalovirus infected by people with low immunity.
Based on the same inventive concept, in a third aspect, the present invention provides a use of NK cells for preparing an immune reconstitution promoting agent, the immune reconstitution promoting agent comprising a first agent; the first preparation comprises in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 7 /kg, said first formulation is for promoting immune reconstitution of NK cells.
Alternatively, the first formulation is administered two times each 20 days and 27 days after hematopoietic stem cell transplantation.
Optionally, the agent that promotes immune reconstitution further comprises a second agent; the second preparation comprises IL-2, and the content of the IL-2 in the second preparation is 40 ten thousand IU/M 2 A second formulation for administration after administration of the first formulation, the second formulation for assisting the first formulation in promoting immune reconstitution of NK cells.
In embodiments of the invention, the agent for eliminating cytomegalovirus infection can be applied after all operations (including hematopoietic stem cell transplantation and tumor transplantation) which can cause cytomegalovirus infection, so as to promote the immune reconstitution of NK cells in patients after the operations.
In the embodiment of the invention, the preparation for eliminating cytomegalovirus infection can be applied to all people with low immunity (including HIV infected people, immunodeficiency patients and patients with rheumatic immunity) so as to promote the immune reconstruction of NK cells in people with low immunity.
Based on the same inventive concept, in a fourth aspect, the present invention provides an NK cell transfusion preparation, comprising: a first formulation comprising ex vivo expansion of NK cells, in which first formulation N is expanded in vitroThe content of K cells is not less than 1 × 10 7 /kg, the first formulation is for preventing cytomegalovirus infection, or for clearing cytomegalovirus infection in vivo, or for promoting immune reconstitution of NK cells.
In an alternative embodiment of the invention, the NK cell back-infusion formulation further comprises: a second formulation comprising IL-2, the IL-2 content of the second formulation being 40 ten thousand IU/M 2 And a second agent administered after administration of the first agent, the second agent being for aiding the first agent in preventing cytomegalovirus infection, or in clearing cytomegalovirus infection in vivo, or in promoting immune reconstitution of NK cells.
The NK cell reinfusion preparation provided by the embodiment of the invention can be applied after all operations (including hematopoietic stem cell transplantation and tumor transplantation) which can cause cytomegalovirus infection, so as to prevent the cytomegalovirus infection caused by the operations, or eliminate the cytomegalovirus infection caused by the operations, or promote the immune reconstruction of NK cells in a patient after the operations.
The NK cell back-transfusion preparation provided by the embodiment of the invention can also comprise: HIV infected person, immunodeficiency patient, and rheumatic immune disease patient) to prevent cytomegalovirus infection of immunocompromised people, eliminate cytomegalovirus infection of immunocompromised people, or promote NK cell immune reconstitution in immunocompromised people.
Specifically, when the NK cell reinfusion formulation of the present embodiment is used for a patient after hematopoietic stem cell transplantation, the administration method may specifically include: the first preparation is administered two times on 20 days and 27 days after hematopoietic stem cell transplantation, and the second preparation is administered three times per week after the first preparation to prevent cytomegalovirus infection after hematopoietic stem cell transplantation, or to eliminate cytomegalovirus infection after hematopoietic stem cell transplantation, or to promote immune reconstitution of NK cells after hematopoietic stem cell transplantation.
Based on the same inventive concept, in a fifth aspect, the invention provides a combined preparation for preventing cytomegalovirus infection after hematopoietic stem cell transplantation,the combined preparation comprises an NK cell return preparation and IL-2, and the content of in-vitro expanded NK cells in the NK cell return preparation is not less than 1 x 10 7 The content of the IL-2 is 40 ten thousand IU/M2; the NK cell reinfusion formulation was administered twice on days 20 and 27 after hematopoietic stem cell transplantation, and IL-2 was administered three times per week after the first administration of the NK cell reinfusion formulation.
Based on the same inventive concept, in a sixth aspect, the invention provides a combined preparation for eliminating cytomegalovirus infection after hematopoietic stem cell transplantation, which comprises an NK cell reinfusion preparation and IL-2, wherein the content of in-vitro amplified NK cells in the NK cell reinfusion preparation is not less than 1 x 10 7 The content of the IL-2 is 40 ten thousand IU/M2; the NK cell reinfusion formulation was administered twice on days 20 and 27 after hematopoietic stem cell transplantation, and IL-2 was administered three times per week after the first administration of the NK cell reinfusion formulation.
Based on the same inventive concept, in a seventh aspect, the present invention provides a combined preparation for promoting immune reconstitution of NK cells after hematopoietic stem cell transplantation, the combined preparation comprising an NK cell reinfusion preparation in which the content of in vitro expanded NK cells is not less than 1X 10 and IL-2 7 The content of the IL-2 is 40 ten thousand IU/M2; the NK cell reinfusion formulation was administered twice on days 20 and 27 after hematopoietic stem cell transplantation, and IL-2 was administered three times per week after the first administration of the NK cell reinfusion formulation.
In order to make the present invention better understood by those skilled in the art, the application of the NK cells, the NK cell transfusion preparation and the combination preparation provided by the present invention are illustrated by specific examples below.
Example 1: mouse model experiments:
6-8 week female NSG mice were irradiated with X-ray sublethal dose and HCMV-IGG seropositive G-CSF mobilized donor peripheral blood stem cells 1X 10 were returned via the tail vein 6 A/only; after 2 weeks, MRC-5 cells infected with HCMV strain AD169 were intraperitoneally injected; 4 weeks after transplantation, adoptive reinfusion of expanded NK cells or primary NK cells 1X 10 7 One, and 5 ten thousand of single were intraperitoneally injected every other day from the infusion of NK cellsPosition IL-2. Taking the liver, spleen and lung of the mouse 14 days after the NK cells are back-transfused, and detecting the proportion of the NK cells by using a flow cytometry; based on the in situ hybridization method, the number of HCMV positive cells was detected using an HCMV-RNA probe.
Fig. 1 shows the proportion of NK cells in liver, spleen, lung, periphery after NK cell reinfusion for 14 d.
As can be seen from fig. 1, after NK cell transfusion 14d, sustained presence of NK cells could be detected in the tissues, and the proportion of NK cells in the in vitro expanded NK transfusion group was significantly higher compared to the primary NK transfusion group in liver, spleen, lung, periphery.
Fig. 2 shows HCMV clearance in liver, spleen and lung after NK cell reinfusion 14d, where the dark spots in the histopathological map are HCMV infected cells.
As shown in FIG. 2, the HCMV-infected cells were identified by the dark spots indicated by black arrows in the in situ hybridization method of HCMV-RNA. The virus-cleared mice were counted for positive mouse histiocyte status and the HCMV clearance of the mice was calculated, and the results showed that the ex vivo expanded NK reinfused group had a stronger HCMV clearance than the primary NK reinfused group, in the liver (76.5% vs.33.3%, p = 0.032), in the lungs (82.4% vs.44.4%, p = 0.046).
Example 2: clinical study:
collecting peripheral blood 60-80ml (determined by WBC count of peripheral blood of donor) 10 days before transplantation of haplotype hematopoietic stem cells, or collecting peripheral blood stem cells (3-4 ml) after mobilization of donor, separating PBMC, freezing, and recovering PBMC2 x 10 of donor 10 days after transplantation 7 The donor-derived NK culture was initiated.
20 patients with haplotypic hematopoietic stem cell transplantation were selected for inclusion as reinfusion NK subjects under the following conditions: (1) diagnosing as follows: acute leukemia, or MDS, MM or lymphoma. (2) age 16-65 years; (3) seropositive for donor HCMV-IGG; (4) HCMV negative within 20 days after transplantation; (5) No 1-4 degrees aGVHD occurred within 20 days post-transplantation, or aGVHD had been relieved and glucocorticoid dose <0.5mg/kg/d within 72h before reinfusion of NK cells; (6) patient consent to group.
For 20 patients in transitDonor-derived in vitro expanded NK cells were returned 20 days (+ -3 d) and 27 days (+ -3 d) post-implantation (1X 10 each time) 7 In kg). Meanwhile, from the first reinfusion, IL-2 (40 ten thousand IU/M) is performed according to the wishes of the patients 2 ) Subcutaneous injections were administered 3 times a week, 3 weeks after the first reinfusion.
20 patients with reinfusion were compared to historical control patients without reinfusion. The cumulative incidence of HCMV infection, the cumulative incidence of refractory HCMV infection, and the duration of HCMV infection were compared.
The results of the study are as follows:
(1) First, from a safety point of view, no side effects were observed in patients after NK reinfusion, and there was no statistical difference between the reinfusion and control groups in death, disease recurrence, and aGVHD.
(2) Fig. 3 shows the comparison between the NK reinfusion group and the control group for HCMV infection and treatment-refractory HCMV infection, and it can be seen from fig. 3 that the cumulative incidence of HCMV infection and treatment-refractory HCMV infection is reduced in the NK reinfusion group compared to the control group, indicating that NK reinfusion can prevent HCMV infection after transplantation.
In patients following HSCT, HCMV infection develops primarily from primary infection, or in the early stages of immunodeficiency, HCMV is reactivated from pre-existing latency in the transplant or recipient. Chronic use of antiviral drugs results in increased mortality from refractory HCMV infections. The development of refractory HCMV infection means that HCMV infection persists for more than 2 weeks under standard antiviral treatment, reflecting the therapeutic effect of HCMV clearance. The results of the embodiment of the invention show that the mechanism of HCMV infection after NK cell infusion and HSCT resistance is to effectively promote HCMV clearance by remarkably reducing intractable HCMV infection, shortening HCMV duration and reducing HCMV-DNA copy number.
(3) Fig. 4 shows the comparison of the duration of HCMV infection in NK reinfusion group with the control group, and it can be seen from fig. 4 that the duration of HCMV infection is shortened in NK reinfusion group compared to the control group, indicating that NK reinfusion can promote clearance of HCMV infection after transplantation.
(4) Figure 5 shows the NK cell reconstitution of NK cells compared to control NK cells. As can be seen from fig. 5, NK cell reconstitution in patients was better in the NK reinfusion group than in the control group, as shown by: the number of NK is more, and the expression of activating receptors NKG2C, NKP30 and NKG2D is stronger, which indicates that NK feedback can promote the immune reconstitution of NK cells.
(5) Fig. 6 shows the monitoring of NK cells in vivo after NK cell reinfusion in 20 patients, and it can be seen from fig. 6 that both the percentage and absolute number of NK cells in vivo after NK cell reinfusion increased significantly and peaked and then declined around 2-3 weeks after NK cell reinfusion. In addition to NKP30, the expression of activation receptors NKG2C, NKG2D and DNAM-1 is increased. The first week after NK cell reinfusion, the proliferative capacity of NK cells in vivo increased, as indicated by increased expression of KI-67. In addition, increased secretion of NK cell granzyme B and perforin could be detected. The number of NKG2A + NK subgroups is reduced, and the KIR + NK subgroups are increased, which indicates that NK cell infusion can promote proliferation, activation and maturation of NK cells after allogeneic HSCT.
(6) FIG. 7 shows a comparison of HCMV and refractory HCMV infection in IL-2-injected and non-injected IL-2 groups; among them, cohort a-no IL-2 injection group (10 persons) and cohort B-IL-2 injection group (10 persons), it can be seen from the figure that there is no difference in the cumulative incidence of HCMV infection in the two groups, a trend towards less refractory HCMV infection is seen in the IL-2 group, a trend towards shorter duration is also seen, and no statistical difference is likely due to the limited number of patients in the group.
(7) FIG. 8 shows the percentage and absolute number of NK cells in the IL-2-injected and IL-2-non-injected groups, and FIG. 9 shows the absolute number of NKG2C + NK subpopulations in the IL-2-injected and IL-2-non-injected groups and the expression of the activating receptor NKP 30.
As can be seen in fig. 8 and 9, patients in cohort B exhibited a higher percentage and absolute number of NK cells, a higher absolute number of NKG2C + NK subpopulation and higher expression of the activation receptor NKP30 than patients in cohort a, indicating that NK cells in cohort B patients may have greater function under IL-2 stimulation. The percentage and absolute number of NK cells in patients in cohort B were higher than in patients in cohort a, and the expression of the activating receptors NKP30 and NKG2C on NK cells was also higher than in patients in cohort a. Therefore, IL-2 may reverse NK cell depletion, stimulate NK cell expansion, and enhance NK cell function in vivo.
The experimental results of the examples of the invention show that there was no significant difference in patient mortality, relapse and aGVHD between the NK cell infusion cohort and the control cohort. Therefore, ex vivo expanded NK cell infusion is safe and well tolerated without the occurrence of serious side effects associated with infusion. In addition, the NK cells expanded in vitro can prevent HCMV infection, promote HCMV clearance and promote NK cell immune reconstitution. Combination with IL-2 treatment can improve the efficacy of NK cell infusion.
The experimental result also shows that the number of NK cells in the returned NK cell group is obviously more than that of the control group, the expression of activating receptors NKP30 and NKG2D is higher than that of the control group, and the absolute number of NKG2C + NK subgroups is also higher than that of the control group. Further, it was shown that, in comparison with the control group, the reconstructed NK cell phenotype of the patients in the in vitro amplified NK cell infusion group was more mature and functional.
The application of the NK cells, the NK cell reinfusion formulation and the combined formulation provided by the present invention are described in detail above, and the principle and the embodiment of the present invention are explained in the present document by applying specific examples, and the description of the above examples is only used to help understanding the method and the core concept of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
Claims (10)
1. Use of NK cells for the preparation of a formulation for the prevention of cytomegalovirus infection, wherein the formulation for the prevention of cytomegalovirus infection comprises a first formulation; the first preparation comprises donor-derived in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 7 /kg, said in vitro expanded NK cells are used to reduce the refractory cytomegalovirus infection rate after surgery of said donor.
2. The use of claim 1, wherein the agent for preventing cytomegalovirus infection further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M 2 What is, what isThe second formulation is administered after the first formulation is administered;
the IL-2 is used for reversing NK cell exhaustion, stimulating NK cell amplification and enhancing NK cell function in vivo.
3. The use of claim 1, wherein the first formulation is administered two times each on 20 and 27 days after the hematopoietic stem cell transplantation surgery; subcutaneously injecting the second formulation 3 times a week from the first administration of the first formulation to 3 weeks after the first administration of the first formulation.
4. The use of claim 3, wherein said first formulation is obtained by harvesting peripheral blood from a donor prior to hematopoietic stem cell transplantation, or harvesting peripheral blood stem cells from said donor after mobilization, isolating PBMC for cryopreservation, resuscitating PBMC from said donor after hematopoietic stem cell transplantation, and initiating in vitro culture of NK cells derived from said donor.
5. Use of NK cells in the preparation of a cytomegalovirus-scavenging formulation, wherein the cytomegalovirus-scavenging formulation comprises a first formulation; the first preparation comprises donor-derived in vitro expanded NK cells, and the content of NK cells in the first preparation is not less than 1 x 10 7 /kg, said in vitro expanded NK cells are used to shorten the duration of cytomegalovirus infection after surgery of said donor, and to reduce cytomegalovirus DNA copy number.
6. The use of claim 5, wherein the agent that clears cytomegalovirus further comprises a second agent; the second preparation comprises IL-2, and the content of IL-2 in the second preparation is 40 ten thousand IU/M 2 Said second formulation being administered after administration of said first formulation;
the IL-2 is used for reversing NK cell exhaustion, stimulating NK cell amplification and enhancing NK cell function in vivo.
7. The use of claim 5, wherein the first formulation is administered two times on days 20 and 27 after hematopoietic stem cell transplantation, and the second formulation is subcutaneously injected 3 times a week from the first administration to 3 weeks after the first administration.
8. The use of claim 7, wherein said first formulation is obtained by harvesting peripheral blood from a donor prior to hematopoietic stem cell transplantation, or harvesting peripheral blood stem cells from said donor after mobilization, isolating PBMCs for cryopreservation, resuscitating PBMCs from said donor after hematopoietic stem cell transplantation, and initiating in vitro culture of NK cells from said donor.
9. An NK cell transfusion preparation, wherein said NK cell transfusion preparation comprises a first preparation; the first preparation comprises in vitro expanded NK cells, and the content of the in vitro expanded NK cells in the first preparation is not less than 1 x 10 7 /kg。
10. A combined preparation based on in-vitro expanded NK cells is characterized by comprising an NK cell return preparation and IL-2, wherein the content of the in-vitro expanded NK cells in the NK cell return preparation is not less than 1 x 10 7 Per kg, the content of the IL-2 is 40 ten thousand IU/M 2 (ii) a In vitro expanded NK cells in the NK cell reinfusion formulation are of donor origin, said NK cell reinfusion formulation is administered twice on 20 and 27 days after hematopoietic stem cell transplantation of said donor, respectively, and IL-2 is administered three times per week after the first administration of the NK cell reinfusion formulation.
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