阅读说明：本技术 用于癌症诊断的肿瘤来源的外泌体的免疫应答谱分析 (Immune response profiling of tumor-derived exosomes for cancer diagnosis ) 是由 应仪如 郑毅然 李怡霏 于 2018-12-06 设计创作，主要内容包括：本发明总体涉及产生肿瘤来源的外泌体诱导的免疫应答或癌症特异性应答谱,其通过从患有特定癌症类型的患者的血液样品分离肿瘤来源的外泌体,并体外测定肿瘤来源的外泌体对免疫细胞尤其是T细胞的免疫学影响来进行。所述肿瘤来源的外泌体诱导的免疫应答或产生的癌症特异性应答谱可用于检测或诊断受试者中的癌症或癌症类型以及鉴定受试者患有的癌症类型是否响应于该癌症类型的治疗的方法中。(The present invention relates generally to generating tumor-derived exosome-induced immune responses or cancer-specific response profiles by isolating tumor-derived exosomes from blood samples of patients with specific cancer types and determining in vitro the immunological impact of the tumor-derived exosomes on immune cells, particularly T cells. The tumor-derived exosomes induce immune responses or generate cancer-specific response profiles that are useful in methods of detecting or diagnosing cancer or a type of cancer in a subject and identifying whether a subject has a type of cancer responsive to treatment for that type of cancer.)

A method of (i) detecting cancer or a type of cancer in a subject, or (ii) simultaneously testing or differentiating between multiple types of cancer in a subject, the method comprising the steps of: identifying a cancer or cancer type in the subject using a cancer-specific response profile created based on in vitro measurements of the functional impact of tumor-derived exosomes on immune cells, wherein the tumor-derived exosomes are isolated from the subject, and wherein the immune cells are preferably T-cells.

2. A method of screening a subject for prevalence of one or more cancer types, the method comprising the steps of: using a cancer-specific response profile created for each subject, said cancer-specific response profile created based on an in vitro measurement of the functional effect of tumor-derived exosomes on immune cells, to identify cancer or a cancer type in each said subject, wherein said tumor-derived exosomes are isolated from said subject, and wherein said immune cells are preferably T-cells.

3. A method of managing a subject having a type of cancer, the method comprising the steps of:

(1) identifying a type of cancer in the subject using a cancer-specific response profile created based on an in vitro measurement of the functional impact of tumor-derived exosomes on immune cells, wherein the tumor-derived exosomes are isolated from the subject; and

(2) administering the subject if the subject is found to have the cancer type,

wherein the immune cell is preferably a T cell.

4. A method of identifying whether a subject having a type of cancer is responsive to management of the type of cancer, the method comprising the steps of:

(1) identifying a type of cancer in the subject using a cancer-specific response profile created based on an in vitro measurement of the functional impact of tumor-derived exosomes on immune cells, wherein the tumor-derived exosomes are isolated from the subject; and

(2) comparing the respective cancer-specific response profiles created before and during and/or after management of the cancer type, wherein a change in the cancer-specific response profile identifies the subject as responsive to management of the cancer type,

wherein the immune cell is preferably a T cell.

5. Tumor-derived exosomes-induced immune responses or cancer-specific response profiles created based on in vitro measurements of the functional effects of tumor-derived exosomes on immune cells, preferably T-cells, for use in or in the detection or diagnosis of cancer or cancer types in a subject.

6. Tumor-derived exosomes induce immune responses useful or useable for generating a cancer-specific response profile that measures in vitro the functional effects of tumor-derived exosomes on immune cells, preferably T-cells.

7. Use of tumor-derived exosomes to induce an immune response in generating a cancer-specific response profile that measures in vitro the functional impact of tumor-derived exosomes on immune cells, wherein the tumor-derived exosomes are isolated from a subject with cancer and the cancer-specific response profile is indicative of the type of cancer in the subject, and wherein the immune cells are preferably T-cells.

8. A method of generating a response profile specific to a cancer type, the method comprising the steps of:

(1) measuring in vitro the functional effect of tumor-derived exosomes on immune cells, wherein the tumor-derived exosomes are isolated from a subject having a specific cancer type; and

(2) creating a cancer-specific response profile based on functional impact specific to the type of cancer,

wherein the immune cell is preferably a T cell.

9. The method of any one of claims 1-4 and 8, the response of claim 5 or 6, the profile of claim 5, or the use of claim 7, wherein said immune cells comprise one or more of the group consisting of T cells, natural killer (NK cells), and B cells.

10. The method, response, profile or use of claim 9, wherein said immune cell is a T cell.

11. The method, response, profile or use of claim 10, wherein said T cells are selected from the group consisting of CD8T cells and CD4T cells.

12. The method, response, profile or use of claim 11, wherein said T cells are selected from naive CD8⁺T cells, naive CD4⁺T cell, activated (Act) CD8⁺T cells and Act CD4⁺T cells.

13. The method of any one of claims 1-4 and 8-12, the response of any one of claims 5,6 and 9-12, the profile of any one of claims 5 and 9-12, or the use of any one of claims 7 and 9-12, comprising the step of measuring in vitro the functional effect of tumor-derived exosomes on immune cells to generate said cancer-specific response profile.

14. The method of any one of claims 1-4 and 8-13, the response of any one of claims 5,6 and 9-13, the profile of any one of claims 5 and 9-13, or the use of any one of claims 7 and 9-13, wherein creating a cancer specific response profile comprises measuring one or more of: inhibiting immune cell function; attenuating an immune cell response to the stimulus; promoting the expansion of regulatory immune cells; inducing apoptosis of cytotoxic immune cells; or immunostimulation.

15. The method, response, profile or use of claim 14, wherein creating a cancer specific response profile comprises measuring immunosuppression due to one or more of I L-10, TGF- β, PD-1, PD L-1, TRAI L, Fas L, CD39 and CD 73.

16. The method, response, profile or use of claim 14, wherein creating a cancer specific response profile comprises measuring the immunostimulatory effect due to one or more of the following molecules: tumor antigens and heat shock proteins.

17. The method of any one of claims 1-4 and 8-16, the response of any one of claims 5,6 and 9-16, the profile of any one of claims 5 and 9-16 or the use of any one of claims 7 and 9-16, wherein creating a cancer specific response profile comprises measuring at least one expression level of a marker on and/or in an immune cell.

18. The method, response, profile or use of claim 17, wherein said marker is selected from the group consisting of an immune cell activation marker, an immune cell proliferation marker, an immune cell depletion marker, an immune cell cytotoxicity and apoptosis marker, and an immune cell suppression marker.

19. The method, response, profile or use of claim 18, wherein said activation marker is CD69, CD25 or pSTAT 5.

20. The method, response, profile or use of claim 18, wherein said proliferation marker is ki 67.

21. The method, response, profile or use of claim 18, wherein the wasting marker is Tim 3.

22. The method, response, profile or use of claim 18, wherein said cytotoxic marker is granzyme B or IFN γ.

23. The method, response, profile or use of claim 18, wherein said cytotoxic and apoptotic marker is Fas L.

24. The method, response, profile or use of claim 18, wherein said inhibitory marker is PD-1 or CT L a 4.

25. The method of any one of claims 1-4 and 8-24, the response of any one of claims 5,6 and 9-24, the profile of any one of claims 5 and 9-24 or the use of any one of claims 7 and 9-24, wherein the cancer is selected from the group consisting of: renal cancer, colorectal cancer (colon and/or rectal cancer), skin cancer (including basal cell carcinoma, squamous cell carcinoma, and melanoma), leukemia, lymphoma, central nervous system tumor, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, mesothelioma, gastric cancer, liver cancer, renal cancer including wilms, bladder cancer, esophageal cancer, laryngeal cancer, parotid cancer, cholangiocarcinoma, endometrial cancer, adenocarcinoma, small cell carcinoma, neuroblastoma, adrenocortical cancer, epithelial cancer, desmoid, fibroproliferative small round cell tumor, endocrine tumor, ewing's sarcoma family tumor, germ cell tumor, hepatoblastoma, hepatocellular carcinoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, peripheral primitive neuroectodermal tumor, peripheral carcinoma, neuroblastoma, melanoma, bladder cancer, bladder, Retinoblastoma and rhabdomyosarcoma.

26. The method of any one of claims 1-4 and 8-25, the response of any one of claims 5 and 9-25, or the profile of any one of claims 5 and 9-25, comprising the step of comparing the created cancer specific response profile of the subject with one or more previously created reference cancer specific response profiles, wherein each of said reference profiles is created based on the subject diagnosed with a particular type of cancer.

27. The method of any one of claims 1-4 and 8-26, the response of any one of claims 5 and 9-26, or the profile of any one of claims 5 and 9-26, comprising the step of comparing the created cancer specific response profile of the subject to a pre-established reference cancer specific response profile database, wherein a match or close match of the subject profile and the reference profile indicates the type of cancer from which the subject suffers.

28. The method of any one of claims 1-4 and 8-27, the response of any one of claims 5,6 and 9-27, the profile of any one of claims 5 and 9-27, or the use of any one of claims 7 and 9-27, wherein a plurality of different functional impact types are used to create the cancer specific response profile.

29. The method of any one of claims 1-4 and 8-28, the response of any one of claims 5,6 and 9-28, the profile of any one of claims 5 and 9-28, or the use of any one of claims 7 and 9-28, wherein measuring the functional impact of tumor-derived exosomes on immune cells to create a cancer-specific response profile (or reference profile) comprises the steps of: calculating a first "parameter" score based on the functional effect, normalizing with respect to a control, wherein the first parameter score is calculated by dividing the geometric mean of the functional effect by the average geometric mean of the control, and then performing a log-2 transformation to obtain the first parameter score for the functional effect.

30. The method, response, spectrum or use of claim 29, wherein said first parameter score is calculated according to the formula:

whereinIs the geometric mean of the functional impact.

31. The method, response, profile or use of claim 30, wherein measuring the functional impact of tumor-derived exosomes on immune cells to create a cancer-specific response profile (or reference profile) comprises the step of calculating a second ('Exo') score based on the mean absolute value of the first parameter score.

32. The method, response, spectrum or use of claim 31, wherein said second Exo score is calculated according to the formula:

where Mi is the first parameter score and n is the number of parameter scores.

33. The method, response, profile or use of claim 32, wherein said creating a cancer specific response profile (or reference profile) based on the functional impact of tumor-derived exosomes on immune cells comprises the step of calculating a third "bias" score based on the average of the absolute values of the mean normalized bias of the first parameter score.

34. The method, response, spectrum or use of claim 33, wherein said third deviation score is calculated according to the formula:

wherein the NPD includes a normalized parameter deviation calculated according to:

where x is the parameter score of the test sample for functional impact i, and M is the identification parameter score for that functional impact.

35. The method, response, profile or use of claim 34, wherein the functional impact included in the response profile is selected after linear regression and spearman rank order correlation tests on the first parameter score data.

36. The method, response, profile, or use of claim 35, wherein the first parameter score, second Exo score, and third deviation score are based on fluorescence intensity of marker expression levels on immune cells.

37. The method, response, profile or use of claim 36, wherein said first parameter score, second Exo score and/or third deviation score is used to generate a cancer specific response profile.

38. The method, response, profile or use of claim 37, wherein said first parameter score, second Exo score and/or third deviation score is used to create a reference profile from a subject known to have cancer.

39. The method, response, profile or use of claim 38, wherein said first parameter score, second Exo score and/or third deviation score is used when comparing a cancer specific response profile of a subject to a reference profile.

40. The method, response, profile or use of claim 39, wherein said second Exo score is used to give an overall "yes" or "no" answer as to whether cancer is present in the subject.

41. The method, response, profile or use of claim 40, wherein said third deviation score is used to determine the type of cancer present in said subject.

42. The method, response, profile or use of claim 41, wherein the cancer specific response profile and the reference profile of the subject are each in the form of an immune response signature barcode.

43. The method, response, profile or use of claim 42, wherein with respect to a marker expressed on or in an immune cell, the method comprises:

(a) calculating a first parameter score based on the expression level of the marker on the immune cells, wherein the expression level of the marker on the immune cells is normalized to the expression level of the marker on control immune cells;

(b) calculating a second Exo score based on the mean absolute value of the first parameter score;

(c) calculating a third deviation score based on an average of the absolute values of the average normalized deviations of the first parameter scores; and

(d) comparing the first parameter score, the second Exo score, and/or the third deviation score of the subject response profile to a set of reference profiles prepared from subjects known to have cancer.

44. The method of any one of claims 1-4 and 8-43, the response of any one of claims 5,6 and 9-43, the profile of any one of claims 5 and 9-43, or the use of any one of claims 7 and 9-43, wherein the subject is a human.

45. A test, assay, kit, device or apparatus for or useful in: the method of any one of claims 1-4 and 8-44, the response of any one of claims 5,6 and 9-44, the spectrum of any one of claims 5 and 9-44, or the use of any one of claims 7 and 9-44.

46. The method of any one of claims 1-4 and 8-44, the response of any one of claims 5,6 and 9-44, the profile of any one of claims 5 and 9-44, or the use of any one of claims 7 and 9-44, wherein said tumor-derived exosomes are isolated from a liquid biopsy obtained from said subject.