New Treatment for Sepsis

Abstract

The present invention relates to a method for the treatment of sepsis in a subject, comprising administering to said subject a pharmaceutical composition comprising an effective amount of allogeneic natural killer cells, and to a method for predicting a subject's susceptibility to such a treatment method.

Description

[Technical field]

[0001] The present invention relates to the field of therapeutic therapy of human and animal subjects, and in particular to methods of using allogeneic cells in the treatment of sepsis. [Background technology]

[0002] Sepsis is a very dangerous condition caused by the body's response to infection or trauma. Blockade of inflammatory mediators or pathogen recognition signaling pathways often fails, and immune-modulating therapy may be a new approach to be used in sepsis.

[0003] During sepsis, the immune system becomes hyperactivated and produces excessive levels of cytokines, signaling molecules that attract immune cells. Elevated levels of these cells secrete more cytokines, and this “cytokine storm” recruits even more immune cells, intensifying the vicious cycle. Instead of stopping the initial infection, immune factors attack the body’s tissues and organs, causing organ failure and death. Clinical trials of interventions targeting cytokine production or effects have so far been unsuccessful (Chousterman et al., 2017). Even if patients survive the hyperinflammatory phase, the subsequent immunosuppressive phase remains life-threatening.

[0004] Allogeneic natural killer ("NK") cells have been proposed for use in adoptive immunotherapy for the treatment of cancer (Geller et al., 2011). Infusion of allogeneic NK cells after ex vivo expansion has been reported to be largely safe (Lim et al., 2015). Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a new therapeutic method for treating sepsis. [Means for solving the problem]

[0006] In a first aspect, the present invention relates to a method for the treatment of sepsis in a subject, comprising administering to the subject a pharmaceutical composition comprising an effective amount of allogeneic natural killer cells.

[0007] In one embodiment, the administration of allogeneic natural killer cells is performed in multiple administrations.

[0008] In one embodiment, the allogeneic natural killer cells are administered by at least one intravenous infusion.

[0009] In one embodiment, at least 10 per administration 9 In some embodiments, up to 10 allogeneic natural killer cells are administered per administration. 10 pcs or up to 10 11 Allogeneic natural killer cells are administered.

[0010] In one embodiment, the subject is immunocompromised, hi some embodiments, the subject is immunocompromised as a result of ongoing drug treatment for cancer.

[0011] In one embodiment, the subject is suffering from sepsis caused by a microbial infection.

[0012] In one embodiment, the allogeneic natural killer cells are administered simultaneously, separately, or sequentially with an anti-tumor therapy or an anti-bacterial infection therapy. In another embodiment, the anti-tumor therapy is any one of, or a combination of, radiation therapy, chemotherapy, and immunotherapy.

[0013] In one embodiment, at least 70% of the cells in the pharmaceutical composition are CD3 - and CD56 + It is.

[0014] In one embodiment, at least 70% of the cells in the pharmaceutical composition are CD56 + and CD16 + It is.

[0015] In one embodiment, the circulating sepsis biomarker is decreased by administration of allogeneic natural killer cells. In some embodiments, the circulating sepsis biomarker is any one of IL6, procalcitonin, C-reactive protein, D-dimer, or a combination thereof.

[0016] In one embodiment, serum levels of inflammatory cytokines are reduced by administration of allogeneic natural killer cells. In some embodiments, the inflammatory cytokines are any one of IL6, IL10, IL8, IL-1RA, or a combination thereof.

[0017] In one embodiment, the percentage of NK cell subsets associated with killing function (CD56+CD16+) is increased. In another embodiment, the percentage of NK cell subsets associated with killing function (CD56+CD16+) is increased from about 8.0% to 18.0%.

[0018] In some embodiments, the allogeneic natural killer cells are derived from peripheral blood or umbilical cord blood.

[0019] In one aspect, the present invention relates to allogeneic natural killer cells for use in the methods according to the above aspects of the invention.

[0020] In one aspect, the invention relates to the use of allogeneic natural killer cells in the manufacture of a pharmaceutical composition for use in a method according to the above aspects of the invention.

[0021] In one aspect, the invention relates to a method for predicting the susceptibility of a subject to a treatment method according to the above aspects of the invention, comprising co-incubating serum from the subject with allogeneic natural killer cells and monitoring the level of interleukin-6 in the serum over time, wherein a decrease in the level of interleukin-6 over time is indicative of the subject's susceptibility to the treatment method.

[0022] In one aspect, the invention relates to a method for predicting the susceptibility of a subject to a treatment method according to the above aspects of the invention, comprising co-incubating serum from the subject with allogeneic natural killer cells and measuring the proportional expression of at least one cell surface marker protein on the cell surface of the allogeneic natural killer cells before and after co-incubation with serum, wherein a reduced proportional expression of at least one cell surface marker protein on the cell surface of the allogeneic natural killer cells is indicative of the subject's susceptibility to the treatment method, wherein the at least one cell surface marker protein is selected from the group consisting of CD56, CD16, DNAM-1, NKG2D, and NKP30 / NKP46.

[0023] In one aspect, the present invention provides a method for predicting the susceptibility of a subject to a treatment method according to the above aspect of the invention, comprising the steps of: - co-incubating the subject's serum with allogeneic natural killer cells and monitoring the level of interleukin-6 in the serum over time; and - co-incubating the subject's serum with allogeneic natural killer cells and measuring the proportional expression of at least one cell surface marker protein on the cell surface of the allogeneic natural killer cells before and after co-incubation with serum, wherein the reduced proportional expression of at least one cell surface marker protein on the cell surface of the allogeneic natural killer cells, the at least one cell surface marker protein being selected from the group consisting of CD56, CD16, DNAM-1, NKG2D, and NKP30 / NKP46; The method relates to a method in which a decrease in the level of interleukin-6 over time combined with a reduced proportional expression of at least one cell surface marker protein on the cell surface of the allogeneic natural killer cells is indicative of the subject's susceptibility to a therapeutic method.

[0024] In one embodiment of this aspect, the serum and allogeneic natural killer cells are co-incubated for 16-24 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] definition For purposes of this disclosure, "natural killer cells" or "NK cells" are defined as a subset of lymphocytes that can specifically destroy tumor cells or pathogens without prior exposure to the target cell or presentation with histocompatibility antigens. Characteristic surface markers for NK cells are CD45+, CD3-, and CD56+.

[0026] Detailed Description The present invention is based on the discovery that adoptive transfusion of allogeneic NK cells can be used to treat patients with sepsis. NK cell therapy can reduce the levels of pro-inflammatory cytokines in patients and simultaneously increase anti-inflammatory cytokines. This therapy may help improve the patient's ability to fight infection.

[0027] In a first aspect, the present invention relates to a method for the treatment of sepsis in a subject, comprising administering to said subject a pharmaceutical composition comprising an effective amount of allogeneic natural killer cells.

[0028] The purification and expansion of natural killer cells is generally known in the art and can be carried out according to established methods. Exemplary procedures for the purification and expansion of NK cells are provided in the Examples.

[0029] The pharmaceutical composition is preferably suitable for intravenous infusion into a subject of interest, such as a human subject. A suitable pharmaceutical composition includes water for injection, and optionally includes human albumin and other pharma- ceutically acceptable excipients. A composition for intravenous injection must be pyrogen-free and have the appropriate pH, isotonicity, and stability. Suitable compositions may utilize isotonic vehicles, such as, for example, sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. Additionally, the formulation of suitable pharmaceutical compositions is known in the art, for example, by Remington, The Science and Practice of Pharmacy, 23rd Edition (Elsevier, Inc. 2020, ISBN 978-0-12-820007-0).

[0030] Administration of the composition comprising allogeneic natural killer cells is generally performed by single or multiple administrations, such as at least one intravenous infusion. In one embodiment, administration is performed by at least two administrations, at least three administrations, at least four administrations, at least five administrations, at least six administrations, at least seven administrations, at least eight administrations, at least nine administrations, or at least ten administrations. In one embodiment, at least 10 administrations are performed per administration. 9 Allogeneic natural killer cells are administered.

[0031] In some embodiments, the subject is immunocompromised. In some embodiments, the subject is immunocompromised due to ongoing drug treatment for other conditions, such as ongoing treatment for cancer or autoimmune disease, or due to organ transplantation. In some embodiments, the subject is immunocompromised due to disease or dysfunction caused by or associated with disease. As disclosed in the examples of the present disclosure, immunocompromised patients may not respond adequately to standard treatment for sepsis. Therefore, the method according to the present invention may be highly beneficial for this patient population.

[0032] In some embodiments, the subject is suffering from sepsis caused by a microbial infection, such as a bacterial infection, a viral infection, a fungal infection, or any combination thereof.

[0033] In some embodiments, at least 70% of the cells in the pharmaceutical composition are CD3 - and CD56 + In some embodiments, at least 70% of the cells in the pharmaceutical composition are CD56 + and CD16 + It is.

[0034] In one aspect, the present invention relates to allogeneic natural killer cells for use in the methods according to the above aspects of the invention.

[0035] In one aspect, the present invention relates to a pharmaceutical composition comprising allogeneic natural killer cells as described above, for use in a method according to the above aspect of the invention.

[0036] In one aspect, the present invention relates to the use of allogeneic natural killer cells in the manufacture of a pharmaceutical composition for use in the method according to the above aspect of the present invention. A suitable pharmaceutical composition comprises water for injection, and optionally human albumin and other pharma- ceutically acceptable excipients. A composition for intravenous injection must be pyrogen-free and have the appropriate pH, isotonicity, and stability. A suitable composition may utilize an isotonic vehicle, such as, for example, sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary. Additionally, the formulation of suitable pharmaceutical compositions is known in the art, for example, by Remington, The Science and Practice of Pharmacy, 23rd Edition (Elsevier, Inc. 2020, ISBN 978-0-12-820007-0).

[0037] Furthermore, a biomarker useful for predicting therapeutic efficacy is provided.The plasma of prospective patients is co-incubated with NK cells.If NK cells reduce the IL6 level in plasma and / or reduce the marker of NK cell killing function, this indicates that NK cell therapy may be effective in treating patients.

[0038] Thus, in one aspect, the present invention relates to a method for predicting the susceptibility of a subject to a treatment method according to the above aspect of the invention, comprising co-incubating serum from the subject with allogeneic natural killer cells and monitoring the level of interleukin-6 in the serum over time, wherein a decrease in the level of interleukin-6 over time is indicative of the subject's susceptibility to the treatment method.

[0039] In one aspect, the invention relates to a method for predicting the susceptibility of a subject to a treatment method according to the above aspects of the invention, comprising co-incubating serum from the subject with allogeneic natural killer cells and measuring the proportional expression of at least one cell surface marker protein on the cell surface of the allogeneic natural killer cells before and after co-incubation with serum, wherein the at least one cell surface marker protein is selected from the group consisting of CD56, CD16, DNAM-1, NKG2D and NKP30 / NKP46, and wherein a reduced proportional expression of the at least one cell surface marker protein on the cell surface of the allogeneic natural killer cells is indicative of the subject's susceptibility to the treatment method.

[0040] In one aspect, the present invention provides a method for predicting the susceptibility of a subject to a treatment method according to the above aspect of the invention, comprising the steps of: - co-incubating the subject's serum with allogeneic natural killer cells and monitoring the level of interleukin-6 in the serum over time; and - co-incubating the subject's serum with allogeneic natural killer cells and measuring the proportional expression of at least one cell surface marker protein on the cell surface of the allogeneic natural killer cells before and after co-incubation with the serum, wherein the at least one cell surface marker protein is selected from the group consisting of CD56, CD16, DNAM-1, NKG2D, and NKP30 / NKP46; The method relates to a method in which a decrease in the level of interleukin-6 over time combined with a reduced proportional expression of at least one cell surface marker protein on the cell surface of the allogeneic natural killer cells is indicative of the subject's susceptibility to a therapeutic method.

[0041] In one embodiment of this aspect, the serum and allogeneic natural killer cells are co-incubated for 16-24 hours.

[0042] The following examples are included as an illustration of the invention and should not be construed as limiting the scope of the invention, as defined in the appended claims. EXAMPLES

[0043] Example 1 A 58-year-old male patient was admitted to the inventor's clinic with advanced lung cancer. He underwent various antitumor treatments, including radiation, chemotherapy, and immunotherapy, but the tumor continued to progress rapidly. Several treatments caused severe liver and kidney dysfunction, immunosuppression, and fungal and bacterial co-infections. These complications prevented the patient from continuing antitumor therapy. The patient required plasma exchange and hemodialysis and was in intensive care. Antibiotic treatment failed to control the infection.

[0044] Biomarkers of sepsis, including circulating C-reactive protein (CRP), procalcitonin (PCT), and serum IL-6 concentrations, were elevated. The patient was delirious.

[0045] After agreement with the patient and family, the patient received an infusion of expanded allogeneic natural killer ("NK") cells obtained from the hospital's laboratory. Flow cytometry was used to identify NK cells by their characteristic cell surface markers CD45+, CD3-, and CD56+. NK cells were cultured from peripheral blood mononuclear cells of healthy donors. After stimulation with cytokines such as IL2, IL7, and IL15, the NK cells were polarized and proliferated 300-500-fold. The purity of NK cells (CD3-CD56+) in the product was 78.3%, and the activation rate (CD56+CD16+) was 74.2% of NK cells.

[0046] Patients were randomly assigned to receive 10 9 ~2×10 9 The patients received seven doses of intravenous infusion of allogeneic NK cells. After NK cell infusion, circulating sepsis biomarkers were significantly reduced (Table 1), and the patients' body temperature decreased, their subjective sensation improved, and their consciousness improved.

[0047] [Table 1]

[0048] Serum cytokine concentrations were measured before and after NK cell therapy. The data showed that treatment reduced serum levels of proinflammatory cytokines (IL6, IL10, IL8, and IL-1RA) and increased levels of anti-inflammatory cytokines (IFN-γ and TNF-α) (Table 2).

[0049] [Table 2]

[0050] After three NK cell infusions over a 24-hour period, the percentages of NK cell subsets in the patient's blood were analyzed. When the patients' peripheral blood NK cell subsets were subdivided, the data showed that the NK cell subset associated with killing function (CD56 + CD16 +) increased from 8.1% to 16.8%. Furthermore, NK cell treatment also caused increased expression of cell surface markers associated with NK cell killing: DNAM-1, NKG2D, and NKP30 / NKP46 (Table 3).

[0051] [Table 3]

[0052] Example 2 To demonstrate the therapeutic effect of NK cells in vitro, they were co-incubated with patient serum for 16–24 h. These cells were seeded in 96-well U-bottom plates and maintained in a humidified atmosphere containing 5% CO2 at 37 °C. NK cells 1 × 10 5 50 μL of NK cells and patient serum were added together to one well of a 96-well plate. Serum was used as a control. Changes in IL-6 concentration in the medium were evaluated. NK cells reduced IL-6 levels in serum (Table 4).

[0053] [Table 4]

[0054] This study was performed on three different batches of NK cell products (NK1, NK2, and NK3) expanded from three different healthy donors.

[0055] Changes in cell surface markers expressed on NK cells in the coculture system were examined using the same panel of NK cell surface markers CD56, CD16, DNAM-1, NKG2D, and NKP30 / NKP46.

[0056] Interestingly, the changes in the expression of cell surface markers differ between therapeutic use in vivo and co-culture with serum in vitro. After treatment with patient plasma, the killing function of NK cells was impaired, as represented by a decrease in the percentage of cytotoxic subsets from 78.4% to 59.1%, as well as a decrease in the expression of NKG2D, DNAM-1, and NKP30 / NKP46 (Table 5). Without being bound to a particular theory, NK cells may transition from an active state to an exhausted or resting state after the changes in the cytokine profile in plasma. This indicates that the batch of allogeneic NK cells is functional.

[0057] [Table 5]

[0058] Example 3 Exemplary procedures for collection, expansion, and preparation of NK cells. 50 ml of peripheral blood from healthy donors is collected in sterile vacutainer tubes containing anticoagulant (Na heparin). Plasma is collected for subsequent culture. Peripheral blood mononuclear cells (PBMCs) are isolated using Ficoll-Paque™ (Cytiva).

[0059] NK proliferation was performed using anti-CD16, 10% autologous plasma, IL-15, and IL-2, but without feeder cells at 1.5–4 × 10 6 Start with PBMCs. The cell culture density is 1.5 x 10 in X-VIVO15 medium in a T75 flask. 6 After 3 days of culture, X-VIVO15 medium, autologous plasma, and IL-2 are added.

[0060] On day 5, IL2 and X-VIVO15 medium (Lonza) were added and the cell density was adjusted to 1.0 × 10 6 Adjust to / ml.

[0061] NK cells (CD3 - CD56 +The percentage of NK cell subsets (NK cells) is examined by flow cytometry on day 6. If the percentage of NK cell subsets is greater than 10% on day 6, the culture is continued; otherwise, the culture is considered unsuccessful and discarded.

[0062] IL2 and RPMI1640 medium are added on day 7. The cells are transferred to a culture bag and the cell density is adjusted to 0.7×10 6 / ml. IL2 and RPMI 1640 medium are added continuously from day 9 to day 12. Cell clumping is avoided.

[0063] NK cells (CD3 - CD56 + The percentage of NK cells and the expression level of CD16 on NK cells are evaluated on day 13. If the percentage of NK cells is greater than 70% and the CD16 positivity rate of NK cells is greater than 80%, the cells will be tested for sterility and harvested.

[0064] The cells are washed twice with saline and then transferred to a saline bag containing 5% human serum albumin.

[0065] References Bjorkstrom et al. (2010). CD56 negative NK cells: origin, function, and role in chronic viral disease. Trends in Immunology, 31(11), 401-406. Chousterman et al. (2017). Cytokine storm and sepsis disease pathogenesis. Semin Immunopathol, 39, 517-528. doi:https:d future of allogeneic natural kill Geller et al. (2011). Use of allogeneic NK cells for cancer immunotherapy. Immunotherapy, 3(12). Lim et al. (2015, June 3). Present and future of allogeneic natural killer cell therapy. Frontiers in Immunology, 6(Article 286).

Claims

1. 1. A pharmaceutical composition for administration to a subject for the treatment of sepsis, the pharmaceutical composition comprising an effective amount of allogeneic natural killer cells.

2. The pharmaceutical composition of claim 1, wherein the allogeneic natural killer cells are administered in multiple doses.

3. The pharmaceutical composition of claim 1 or 2, wherein the allogeneic natural killer cells are administered by at least one intravenous infusion.

4. At least 10 per dose ９ The pharmaceutical composition according to any one of claims 1 to 3, wherein said allogeneic natural killer cells are administered.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject is immunocompromised.

6. The pharmaceutical composition of claim 5, wherein the subject is immunocompromised as a result of ongoing drug treatment for cancer.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the subject is suffering from sepsis caused by a microbial infection.

8. At least 70% of the cells in the pharmaceutical composition are CD3 － and CD56 ＋ The pharmaceutical composition according to any one of claims 1 to 7,

9. At least 70% of the cells in the pharmaceutical composition are CD56 ＋ and CD16 ＋ The pharmaceutical composition according to any one of claims 1 to 8,

10. Allogeneic natural killer cells for use in a pharmaceutical composition according to any one of claims 1 to 9.

11. Use of allogeneic natural killer cells in the manufacture of a pharmaceutical composition according to any one of claims 1 to 9.

12. A method for predicting a subject's susceptibility to treatment for sepsis, comprising: co-incubating the subject's serum with allogeneic natural killer cells; and monitoring the level of interleukin-6 in the serum over time; a decrease in the level of interleukin-6 over time indicates the subject's sensitivity to said treatment; the treatment comprising administering to the subject a pharmaceutical composition comprising an effective amount of the allogeneic natural killer cells; method.

13. A method for predicting a subject's susceptibility to treatment for sepsis, comprising: co-incubating the subject's serum with allogeneic natural killer cells; and measuring the proportional expression of at least one cell surface marker protein on the cell surface of said allogeneic natural killer cells before and after co-incubation with serum; at least one cell surface marker protein is selected from the group consisting of CD56, CD16, DNAM-1, NKG2D, and NKP30 / NKP46; a decreased proportional expression of at least one cell surface marker protein on the cell surface of said allogeneic natural killer cells is indicative of the subject's susceptibility to said treatment method; the treatment comprising administering to the subject a pharmaceutical composition comprising an effective amount of the allogeneic natural killer cells; method.

14. The method of claim 12 further comprising the steps of the method of claim 13.

15. The method of any one of claims 12 to 14, wherein the serum and the allogeneic natural killer cells are co-incubated for 16 to 24 hours.