EA042041B1 - REMNANT TUMOR-INFILTERING LYMPHOCYTES AND METHODS FOR THEIR PRODUCTION AND APPLICATION - Google Patents

Description

Cross-reference to related applications

This international application claims priority under U.S. Provisional Application No. 62/423,750, filed November 17, 2016, and U.S. Provisional Application No. 62/460,441, filed February 17, 2017, the entire contents of which are incorporated herein by reference. .

The field of technology to which the invention belongs

In some embodiments, methods and compositions are described for propagating tumor-infiltrating lymphocytes from tumor remnants.

Prerequisites for the creation of the invention

Treatment of generalized, refractory cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) is an effective approach to the treatment of patients with a poor prognosis. Gattinoni et al., Nat. Rev. Immunol. 2006, 6, 383-393. Adoptive T-cell therapy with autologous TIL provides a 55% objective response rate and long-term regression in >25% of patients with metastatic melanoma. Successful immunotherapy requires a large amount of TIL, and a functional and reliable method is needed to commercialize this approach. The development of such a method has been elusive due to the technical, logistical and regulatory challenges associated with cell propagation. Propagation of TILs using IL-2 followed by a rapid expansion process (RRP) has become the preferred method for propagating TILs due to the speed and efficiency of the process. Dudley et al., Science 2002, 298, 850-54; Dudley et al., J. Clin. oncol. 2005, 23, 2346-57; Dudley et al., J. Clin. oncol. 2008, 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J. Immunother. 2003, 26, 332-42. Methods for obtaining TIL from resected tumors include the step of grinding tumors into fragments of 1-3 mm ³ and propagation of TIL in the presence of interleukin 2 (IL-2) at the stage preceding the protocol of rapid propagation (the stage of prePBR or initiation). In the pre-PBR step, resident tumor immunocytes migrate out of the tumor and proliferate, and these TILs are subjected to a second PBR process with irradiated peripheral blood mononuclear cells (PBMC) as nurses, anti-CD3 antibody (OCT-3, muromonab) and IL-2 , which leads to a significant increase in their number. So far, in all TIL propagation methods, residual tumor fragments are discarded after the pre-PBR process.

Direct enzymatic digestion of resected tumors has previously been explored as an alternative to pre-PBR, however, fewer TIL cultures have been reported, making the TIL production process less efficient than pre-PBR IL-2 initiation processes. Dudley et al., J. Immunother. 2003, 26, 332-42. As a result, tumor splitting was no longer studied in the development of TIL as an anti-cancer therapy.

To date, the vast majority of clinical research on TILs has focused on pre-PBR and PBR-derived TILs, with very modest clinical results, and this area of research remains problematic, particularly in extending TIL therapy beyond melanoma treatment. for the treatment of other types of tumors. Goff et al., J. Clin. oncol. 2016, 34, 2389-97; Dudley et al., J. Clin. oncol. 2008, 26, 5233-39; Rosenberg et al., Clin. Cancer Res. 2011, 17, 4550-57. Much attention has been given to selection of TILs during expansion, either to select specific cell subsets (such as CD8+ T cells) or to target driver mutations, such as the ERBB2IP mutant epitope or driver mutations in the KRAS oncogene. Tran et al., N. Engl. J. Med. 2016, 375, 2255-62; Tran et al., Science 2014, 344, 641-45. However, such selection approaches, even if they could be developed and demonstrated to be effective in large clinical trials, greatly increase the duration, complexity, and cost of TIL therapy and limit the widespread use of TIL therapy for different types of cancer. Thus, there is an urgent need to develop methods that allow the production of TIL with improved properties for use in anticancer therapy.

The invention is based on the surprising fact that TILs with improved properties can be obtained by methods based on the use of remnant tumor cells, and that such remnant TILs (rTIL) are phenotypically and functionally different from normal migrating TILs (mTIL). The use of rOIL and combinations of rOIL and mOIL in cancer immunotherapy provides significant advantages over previously used methods of treatment based on mOIL.

The essence of the invention

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic cleavage of tumor remnants into tumor remnant cells using

- 1 042041 splitting mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, the rILs express reduced amounts of the T-cell depletion marker compared to the mILs, and the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof, and at the same time the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, thrice negative breast tumor, cervical tumor tissue, ovarian tumor tissue, and bone marrow or tumor tissue in acute myeloid leukemia.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof, and at the same time irradiated feeder cells are irradiated allogeneic peripheral blood mononuclear cells.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding the tumor remnant cells using a second culture medium.

- 2 042041 cells containing cell culture medium, irradiated feeder cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain propagated remnant tumor-infiltrating lymphocytes (rTIL), while rTIL express reduced amounts of T-cell depletion marker compared to mOIL, wherein the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and wherein IL-2 is present in the second cell culture medium at an initial concentration of about 3000 IU/ml, and the OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng/ml.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, wherein the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the amount is at least one T-cell marker of depletion in CD8+ and CD4 ⁺ T cells in rROIL is reduced by at least 10% compared to mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the T-cell marker depletion is a LAG3 marker in CD8+ T cells, and the amount of LAG3 marker in rROIL is reduced by at least 2-fold compared to mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) propagating the remnant tumor cells using a second cell culture medium containing the cell culture medium, irradiated feeder cells, antibody

- 3 042041

OKT-3 and IL-2, in a gas-permeable container, to produce replicated remnant tumor-infiltrating lymphocytes (rTIL), wherein the rTIL express reduced amounts of T-cell depletion marker compared to mTIL, while the T-cell depletion marker is selected from the group consisting of from TIM3, LAG3, TIGIT, PD-1, as well as their combinations, and at the same time, the T-cell depletion marker is a TIM3 marker in CD8+ T cells, and the amount of the LAG3 marker in rOIL is reduced by at least 3 times in compared with MOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the T-cell marker depletion is a TIM3 marker in CD4+ T cells, and the amount of LAG3 marker in rROIL is reduced by at least 2-fold compared to mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the TIM3 marker and the marker LAG3 in rOIL are not detectable by flow cytometry.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor infiltrating lymphocytes (rTIL),

- 4 042041 wherein rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, wherein the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and at the same time expression of CD56+ in rOIL is reduced by at least 3 times in comparison with the expression of CD56+ in mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the expression of CD69+ in rOIL increased by at least 2 times in comparison with the expression of CD69+ in mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor infiltrating lymphocytes (rILL).

while rOIL express reduced amounts of T-cell depletion marker compared to mOIL, while T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the splitting mixture contains deoxyribonuclease, collagenase and hyaluronidase.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when in this case, rOIL express reduced amounts of the T cell depletion marker compared to mOIL, with the T cell depletion marker being selected from the group consisting of TIM3, LAG3, TIGIT,

- 5 042041

PD-1, as well as combinations thereof, wherein the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when in this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the second culture medium cells additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising administering a therapeutically effective amount of rROIL to the patient, wherein the rROIL is obtained by a method comprising the following steps:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when in this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the second culture medium cells additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of the T cell depletion marker compared to mOIL, with the T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and

- 6 042041 wherein the second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, (g) treating the patient using a non-myeloablative regimen lymphodepletion prior to the administration of rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL administration to the patient, wherein the cancer is selected from the group consisting of melanoma, double refractory melanoma, ovarian cancer, cervical cancer, lung cancer, cancer bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, sarcoma, non-small

- 7 042041 cell lung cancer (NSCLC) and triple negative breast cancer.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, lymphodepletion includes the stages of administration of cyclophosphamide at a dose of 60 mg/m ² /day for two days, followed by the administration of fludarabine at a dose of 25 mg/m ² /day for five days her;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient, wherein the high dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg of aldesleukin, or a biological analog or variant thereof, in as a 15-minute bolus intravenous infusion every eight hours until tolerance.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

- 8 042041 (b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, the rILs express reduced amounts of the T-cell depletion marker compared to the mILs, and the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof, and at the same time the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, thrice negative breast tumor, cervical tumor tissue, ovarian tumor tissue, and bone marrow or tumor tissue in acute myeloid leukemia.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof, and at the same time irradiated feeder cells are irradiated allogeneic peripheral blood mononuclear cells.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating the tumor tissue in a gas-permeable container with the first culture medium

- 9 042041 cells and interleukin 2 (IL-2), with the receipt of tumor remnants and migrating OIL (moIL);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and IL-2 is present in the second cell culture medium at an initial concentration of about 3000 IU/ml, and the OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng/ml.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when in this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, wherein the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the amount is at least one T-cell marker of depletion in CD8+ and CD4 ⁺ T cells in rROIL is reduced by at least 10% compared to mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the T-cell marker depletion is a LAG3 marker in CD8+ T cells, and the amount of LAG3 marker in rROIL is reduced by at least 2-fold compared to mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

- 10 042041 (d) removal of the MOIL;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the T-cell marker depletion is a TIM3 marker in CD8+ T cells, and the amount of LAG3 marker in rROIL is reduced by at least 3-fold compared to mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the T-cell marker depletion is a TIM3 marker in CD4+ T cells, and the amount of LAG3 marker in rROIL is reduced by at least 2-fold compared to mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the TIM3 marker and the marker LAG3 in rOIL are not detectable by flow cytometry.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic cleavage of tumor remnants into tumor remnant cells using

- 11 042041 splitting mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of T-cell depletion marker compared to mOIL, while T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the expression of CD56+ in rOIL reduced by at least 3 times in comparison with the expression of CD56+ in mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the expression of CD69+ in rOIL increased by at least 2 times in comparison with the expression of CD69+ in mOIL.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when In this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the digestion mixture contains deoxyribonuclease , collagenase and hyaluronidase.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) propagating the remnant tumor cells using a second cell culture medium containing the cell culture medium, irradiated feeder cells, antibody

- 12 042041

OKT-3 and IL-2, in a gas-permeable container, to produce replicated remnant tumor-infiltrating lymphocytes (rTIL), wherein the rTIL express reduced amounts of T-cell depletion marker compared to mTIL, while the T-cell depletion marker is selected from the group consisting of of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, wherein the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as their combinations.

In one of the embodiments, the invention relates to a method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when in this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the second culture medium cells additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising administering a therapeutically effective amount of rROIL to the patient, wherein the rROIL is obtained by a method comprising the following steps:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when in this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the second culture medium cells additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) propagating tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody and IL-2 in a gas-permeable container to obtain replicated remnant tumors

- 13 042041 infiltrating lymphocytes (rILD), wherein rILD express reduced amounts of T-cell depletion marker compared to mOIL, while T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and also combinations thereof, wherein the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, (g) treating the patient using the regimen non-myeloablative lymphodepletion prior to the administration of rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; And

- 14 042041 (i) treating a patient with a high-dose IL-2 regimen starting the day after the administration of rOIL to the patient, wherein the cancer is selected from the group consisting of melanoma, doubly refractory melanoma, ovarian cancer, cervical cancer, lung cancer , bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC), and triple-negative breast cancer.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, lymphodepletion includes the stages of administration of cyclophosphamide at a dose of 60 mg/m ² /day for two days, followed by the administration of fludarabine at a dose of 25 mg/m ² /day for five days her;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removal of MWF;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient, wherein the high dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg of aldesleukin, or a biological analog or variant thereof, in as a 15-minute bolus intravenous infusion each

- 15 042041 eight hours until tolerance.

In one embodiment, the invention relates to a method for obtaining expanded tumor remnant cells, including tumor-infiltrating lymphocytes (TIL) from a patient, for adoptive T-cell therapy. In some embodiments, the implementation of the method according to the invention may include the step of obtaining from the patient tumor tissue containing TIL. In some embodiments, the method of the invention may include the step of fragmenting tumor tissue. In some embodiments, the method of the invention may include the step of treating tumor tissue in a gas permeable container with cell culture medium and interleukin 2 (IL-2), as well as other T cell growth factors or agonist antibodies, to obtain tumor remnants and expanded TILs. In some embodiments, the implementation of the method according to the invention may include the step of removing propagated TIL. In some embodiments, the method of the invention may include the step of enzymatically cleaving tumor remnants into tumor remnant cells. In some embodiments, the method of the invention may include the step of treating tumor remnant cells with a cell culture medium containing irradiated nurse cells, an anti-CD3 monoclonal antibody (muromonab or OKT-3) and IL-2 to obtain expanded tumor remnant cells. In some embodiments, tumor remnant cells obtained by the methods of the invention may include TILs that express reduced amounts of at least one marker selected from the group consisting of TIM3, LAG3, PD-1, as well as combinations thereof. In some embodiments, the tumor tissue may be selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, lung tumor tissue, and colorectal tumor tissue.

In one embodiment, the invention may include a method of treating a tumor in a patient in need of such treatment. In some embodiments, treatment may include administering a therapeutically effective amount of expanded tumor remnant cells to a patient, where expanded tumor remnant cells can be obtained by any of the methods described herein.

Brief description of the figures

The foregoing summary of the invention, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings.

In FIG. 1 shows an illustrative scheme for preparing a tumor splitting solution;

in fig. 2 is an illustrative detailed diagram of a tumor splitting procedure. In this example, two cleavage methods are used simultaneously and cells are plated separately as part of two separate pre-PBR steps to compare the efficiency of these cleavage methods;

in fig. 3 shows differential phenotypic expression of key markers in mOIL and rOIL;

in fig. 4 shows the results of the study of mOIL and rOIL using 2-(N-(7nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (A) and Mitotracker (B) to assess metabolic capacity before rapid reproduction;

in fig. 5 - results of experiments in which mOIL and rOIL were stimulated with CD3/CD28/4-1BB and brefeldin A beads overnight for CD4+ and CD8+ T cells. PMA and ionomycin were added for 4-5 hours. The amount of interferon y was assessed in a flow cytometric analysis for intracellular proteins (n=3);

in fig. 6 - results demonstrating that (A) rOIL proliferate and (B) remain phenotypically different from mOIL during rapid multiplication;

in fig. 7 is a diagram of an exemplary method of treating a patient using the rPOIL of the invention;

in fig. 8 is an illustrative timeline for a method of treating a patient using a rPOIL according to the invention;

in fig. 9 shows the diversity of the TCRve repertoire (i.e., the diversity score) in the MOIL and ROIL;

in fig. 10 - the percentage of total CDR3 in the mDIL and rOIL;

in fig. 11 shows the results of cell proliferation assays from triple-negative breast carcinoma, colorectal carcinoma, lung carcinoma, renal carcinoma, and melanoma, demonstrating that either CD4+ or CD8+ mDIL populations in all five tumors exhibited an increased ability to proliferate when co-cultured with rOIL and anti-CD3 antibody, as evidenced by the shift (or dye dilution) observed for CellTrace™ dye compared to mOIL alone. Red color corresponds to mOIL and blue color corresponds to mOIL when co-cultivated with rOIL;

in fig. 12 is a heat map obtained from NanoString analysis showing that the gene expression profiles for mDIL and rTIL are significantly different;

- 16 042041 in FIG. 13 is a graph from NanoString analysis showing that several genes are significantly up-regulated or down-regulated in rTIL compared to mTIL;

in fig. 14 is a graph of clonotypes showing the major 50 CDR3s shared by MFIL and rROIL (for three pairs of MFIL/rROIL) from ovarian carcinoma;

in fig. 15 is a graph of clonotypes showing the major 50 CDR3s shared by MFIL and rROIL (for three pairs of MFIL/rROIL) from renal carcinoma;

in fig. 16 is a graph of clonotypes showing the major 50 CDR3s shared by mDIL and rOIL (for three mDIL/rOIL pairs) from a triple negative breast carcinoma.

Brief Description of the Sequence List

SEQ ID NO: 1 is the amino acid sequence of muromonab heavy chain.

SEQ ID NO: 2 is the amino acid sequence of the muromonab light chain.

SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

Detailed description of the invention

Unless otherwise indicated, all technical and scientific terms used in this document have the meaning that is usually given to them by specialists in the field to which the present invention relates. The entire contents of all patents and publications cited herein are incorporated by reference.

Definitions.

The terms depleted phenotype and depleted marker refer to cell surface markers characteristic of depletion of T cells in response to chronic stimulation of the T cell receptor (TCR) by antigen. Phenotype depleted T cells express inhibitory receptors such as T cell immunoglobulin and mucin domain protein 3 (TIM3 or TIM3), lymphocyte activation gene protein 3 (LAG3 or LAG-3), T cell immunoreceptor with immunoglobulin domains and ITIM (TIGIT) and programmed cell death protein 1 (PD-1), and lack the ability to produce effector cytokines and the ability to mount an effective immune response. Depletion in T cells is described in Yi et al., Immunology 2010, 129, 474-81, the contents of which are incorporated herein by reference.

As used herein, co-administration, co-administration, co-administration, co-administration, co-administration, and co-administration means the administration of two or more active pharmaceutical ingredients to a human subject such that both active pharmaceutical ingredients and/or their metabolites are present. in the human subject's body at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in compositions in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in compositions in which both agents are present are also contemplated in the methods of the invention.

The term in vivo refers to an event that takes place in a subject's body.

The term in vitro refers to an event that takes place outside the subject's body. In vitro assays include cellular assays that use live or dead cells and may also include cell-free assays that do not use any intact cells.

The term antigen means a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR if it is a major histocompatibility complex (MHC) molecule. As used herein, the term antigen also encompasses T-cell epitopes. In addition, the antigen can be recognized by the immune system. In some embodiments, the antigen may induce a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and/or T lymphocytes. In some cases, this may require that the antigen contain or be associated with a Th-cell epitope. An antigen may also have one or more epitopes (eg, B and T epitopes). In some embodiments, the antigen will preferably react in a generally highly specific and selective manner with its corresponding antibody or TCR, but not with a variety of other antibodies or TCRs that may be induced by other antigens.

- 17 042041

The term effective amount, or therapeutically effective amount, means the amount of a compound or combination of compounds described herein that is sufficient to effect the intended effect, including, but not limited to, treatment of a disease. The therapeutically effective amount may vary depending on the intended use (in vitro or in vivo) or on the human subject and disease state being treated (for example, body weight, age and sex of the subject), severity of the disease state, route of administration, etc. further, which can be easily determined by a person skilled in the art. The term is also applied to a dose that will induce a specific response in target cells (eg, reduced platelet adhesion and/or cell migration). The specific dosage will vary depending on the particular compound selected, the dosage regimen administered, whether the compound is administered in combination with other compounds, the time of administration, the tissue into which it is administered, and the physical delivery system in which the compound is located. The therapeutically effective amount may be an antitumor effective amount and/or a tumor inhibitory effective amount, which may be the precise amount of compositions of the present invention to be administered, as may be determined by the clinician, taking into account individual differences in age, body weight, tumor size, degree of infection, or metastasis, as well as the health status of the patient (subject). In general, it can be stated that a pharmaceutical composition containing cytotoxic lymphocytes or rOIL described herein can be administered at a dose of 104-1011 cells/kg body weight (for example, 105-10 ⁶ , 105-10 ¹⁰ , 105-1011, 10 ⁶ -10 ¹⁰ , 10 ⁶ 1011.107-1011, 107-10 ¹⁰ , 108-1011, 108-10 ¹⁰ , 109-10 ¹¹ or 10 ⁹ -10 ¹⁰ cells/kg of body weight), including all integer values in within these ranges. Cytotoxic lymphocyte or rOIL compositions can also be administered at these doses multiple times. Cytotoxic lymphocytes or rOIL can be administered by the infusion method often used in immunotherapy (see, for example, Rosenberg et al., N. Eng. J. Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one of ordinary skill in the art of medicine by monitoring the patient's signs of disease and adjusting treatment accordingly.

As used herein, the term therapeutic effect means a therapeutic benefit and/or a prophylactic benefit to a human subject. A prophylactic effect includes delaying or eliminating the manifestation of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, arresting or reversing the progression of a disease or condition, or any combination thereof.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" should include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers, or pharmaceutically acceptable excipients, for active pharmaceutical ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, may also be included in the described compositions and methods.

The terms therapy, treatment, treat, and the like, refer to the achievement of a desired pharmacological and/or physiological effect. The effect may be prophylactic, consisting in the complete or partial prevention of the disease or a symptom thereof, and/or may be therapeutic, consisting in the partial or complete cure of the disease and/or adverse effect associated with the disease. As used herein, the term treatment encompasses any treatment of a disease in a mammal, in particular a human, and includes:

(a) preventing the onset of a disease in a subject who may be predisposed to the disease but not yet diagnosed as having it;

(b) inhibiting the disease, that is, stopping its development or progression; and (c) alleviating the disease, that is, causing regression of the disease and/or alleviating one or more symptoms of the disease.

Treatment also includes delivering an agent to provide a pharmacological effect, even in the absence of a disease or condition. For example, treatment includes delivery of a composition that elicits an immune response or results in immunity in the absence of a disease state, such as in the case of a vaccine.

The term "heterologous" when applied to fragments of a nucleic acid or protein indicates that the nucleic acid or protein contains two or more subsequences that are not naturally in the same relationship with each other. For example, a nucleic acid is typically produced by recombinant techniques whereby two or more sequences from unrelated genes are combined to produce a new functional nucleic acid, such as a promoter from one source and a coding region from another source, or coding regions from different sources.

- 18 042041 nicknames. Similarly, the term heterologous protein indicates that a protein contains two or more subsequences that do not naturally occur in the same relationship to each other (eg, a fusion protein).

The term proliferation means an increase in the number of antigen-specific TILs by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) within a week, more preferably at least about 10-fold (or 20, 30 , 40, 50, 60, 70, 80 or 90 times) per week, or most preferably at least about 100 times per week. Several protocols for rapid propagation are described in this document.

As used herein, the term tumor-infiltrating lymphocytes, or TIL, means a population of cells, initially obtained as white blood cells, that leave the bloodstream in a subject's body and migrate to a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and Ml macrophages. OILs include both primary and secondary OILs. Primary TILs are cells that are derived from patient tissue samples as described herein (sometimes referred to herein as freshly harvested) and secondary TILs are any cell populations of TILs that have expanded or proliferated as described herein, including , but without limitation, total TIL and multiplied TIL (PBR TIL or post-PBR TIL). In specific embodiments, the term primary OIL may include rOIL and mixtures of mOIL and rOIL.

Used in this document, the term population of cells (including TIL) means a number of cells with common features. In general, populations typically contain cell numbers ranging from 1x106 to 1x10 ¹⁰ , with different TIL populations containing different cell numbers. For example, the initial growth of primary TILs in the presence of IL-2 results in a total TIL population of about 1x108 cells. Propagation in the PBR phase usually ends when populations containing 1.5x109 to 1.5x1010 cells for infusion are obtained.

The terms peripheral blood mononuclear cells and PBMCs refer to peripheral blood cells having a round nucleus, including lymphocytes (such as T cells, B cells, and NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen presenting cells.

Used in this document, the term cryopreserved TIL means that TIL, either primary, total, or propagated (PBR TIL), processed and stored in the temperature range from about -150 to -60°C. General methods of cryopreservation are also described in other sections of this document. To be clear, cryopreserved TILs are distinct from frozen tissue samples, which can be used as a source of primary TILs, including rTILs.

As used herein, the term defrosted cryopreserved TILs means a population of TILs (such as rTILs) that has previously been cryopreserved and then raised to room temperature or above, including, but not limited to, the cell culture temperature, or the temperature at which the TILs may be administered to the patient.

The terms sequence identity, percent identity, and percent sequence identity, in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned. (with spaces inserted as needed) for maximum matching, not counting any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison programs or algorithms, or by visual inspection. Various algorithms and programs are known in the art that can be used to align amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the US Government's National Center for Biotechnology Information at the BLAST web address. Comparisons between two sequences can be made using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleotide sequences while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign available from DNASTAR are additional public domain computer programs that can be used to align sequences. One of ordinary skill in the art can determine the appropriate parameters for maximum alignment using specific sequence alignment programs. In particular embodiments, the alignment programs use default parameters.

- 19 042041

The term conservative amino acid substitutions refers to amino acid sequence modifications that do not interfere with the binding of an antibody to an antigen. Conservative amino acid substitutions include the replacement of an amino acid from one class with an amino acid from the same class, the class being determined by the general physicochemical properties of the amino acid side chains and the high frequency of substitutions in homologous proteins found in nature, which is determined, for example, using a standard frequency matrix Deyhoff substitutions or the BLOSUM matrix. Six major classes of amino acid side chains have been characterized and they include: class I (Cys); class II (Ser, Thr, Pro, Ala, Gly); class III (Asn, Asp, Gln, Glu); class IV (His, Arg, Lys); class V (Ile, Leu, Val, Met) and class VI (Phe, Tyr, Trp). For example, replacing Asp with another class III residue such as Asn, Gln, or Glu is a conservative substitution. Thus, a predicted non-essential amino acid residue in a protein is preferably replaced with another amino acid residue from the same class. Methods for identifying conservative amino acid substitutions that do not interfere with antigen binding are well known in the art (see, for example, Brummell et al., Biochemistry 1993, 32, 1180-1187; Kobayashi et al., Protein Eng. 1999, 12, 879- 884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 1997, 94, 412-417).

PEGylation means the modification of an antibody or fusion protein, or fragment thereof, which typically reacts with a polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions whereby one or more PEG groups are attached to the antibody or fragment. antibodies. Pegylation can, for example, increase the biological (eg, serum) half-life of an antibody. Preferably, the pegylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol should encompass any form of PEG that has been used to derivatize other proteins, such as mono(C ₁ -C ₁₀ ) alkoxy or aryloxy polyethylene glycol or polyethylene glycol maleimide. The protein or antibody to be pegylated may be an aglycosylated protein or antibody. PEGylation techniques are known in the art and can be applied to antibodies of the invention as described, for example, in EP 0154316 and EP 0401384 and US Pat.

The term OKT-3 (another name used herein as OKT) refers to a monoclonal antibody, or biological analog or variant thereof, including human, humanized, chimeric, or murine antibodies that are directed against the CD3 receptor on the T cell antigen receptor of mature T cells, and encompasses commercially available forms such as OKT-3 (30 ng/ml, MACS GMP CD3 pure, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and muromonab, or conservative amino acid substitution variants thereof, glycoforms, or biological analogs. The amino acid sequences of the muromonab heavy and light chains are shown in Table 1. 1 (SEQ ID NO: 1 and SEQ ID NO: 2). The hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and has accession number ATCC CRL 8001. The hybridoma capable of producing OKT-3 is also deposited with the European Collection of Authenticated Cell Cultures (ECACC) and has catalog number 86022706. Ahtu-CD3 antibodies also include antibodies of the UHCT1 clone (commercially available from BioLegend, San Diego, CA, USA), also known as T3 and CD3ε.

- 20 042041

Table 1. Muromonab amino acid sequences

Name Sequence (single-letter amino acid designations) SEQID NO: 1 QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY 60 Muromonab, heavy NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120 chain KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL 180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG 240 PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPHEPQ VYTLPPSRDE 360 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQID NO: 2 QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH 60 Muromonab light chain FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120 SEQLTSGGAS WCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL 180 TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC 213

The term IL-2 (also referred to herein as IL2) means the T cell growth factor known as interleukin-2 and encompasses all forms of IL-2, including its human and mammalian forms with conservative amino acid substitutions, glycoforms, biological analogs, and options. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the contents of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is shown in Table 1. 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (a proleukin commercially available from many vendors at 22 million IU in single use vials) as well as a form of recombinant IL-2 commercially available from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (cat. no. CYT-209-b), and other commercial equivalents from other suppliers. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated form of human recombinant IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of an aldesleukin suitable for use in the invention is shown in Table 1. 2 (SEQ ID NO: 4). The term IL-2 also encompasses PEGylated forms of IL-2 as described herein, including PEGylated IL2 as a prodrug of NKTR-214 available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and PEGylated IL-2 suitable for use in the invention are described in US Patent Application Publication No. US 2014/0328791 A1 and International Patent Application Publication No. WO 2012/065086 A1, the contents of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the invention are described in US Pat. Nos. 4,766,106; 5,206,344; IL-2 formulations suitable for use in the invention are described in US Pat. No. 6,706,289, the contents of which are incorporated herein by reference.

Table 2. Amino acid sequences of interleukins

Name Sequence (single-letter amino acid designations) SEQ ID NO: 3 MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK recombinant ATELKHLQCL 60 human EEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD IL-2 (rhIL-2) ETATIVEFLN 120

-21 042041

RWITFCQSII STLT 134 SEQ ID NO: 4 Aldesleukin PTSSSTKKTQ ELKHLQCLEE ELKPLEEVLN ATIVEFLNRW ITFSQSIIST LQLEHLLDL 60 LAQSKNFHLR 120 LT 132 QMILNGINNY PRDLISNINV KNPKLTRMLT IVLELKGSET FKFYMPKKAT TFMCEYADET SEQ ID NO: 5 recombinant MHKCDITLQE TVLRQFYSHH IIKTLNSLTE 60 QKTLCTELTV TDIFAASKNT TEKETFCRAA human IL-4 (rhIL-4) EKDTRCLGAT ENFLERLKTI MREKYSKCSS AQQFHRHKQL 120 130 IRFLKRLDRN LWGLAGLNSC PVKEANQSTL SEQ ID NO: 6 recombinant MDCDIEGKDG NKEGMFLFRA KQYESVLMVS 60 IDQLLDSMKE IGSNCLNNEF NFFKRHICDA human IL-7 (rhIL-7) ARKLRQFLKM TKSLEENKSL KEQKKLNDLC NSTGDFDLHL LKVSEGTOHE 120 FLKRLLQEIK TCWNKILMGT KEH LNCTGQVKGR 153 KPAALGEAQP SEQ ID NO: 7 recombinant MNWVNVISDL ISLESGDASI KKIEDLIQSM 60 HIDATLYTES DVHPSCKVTA MKCFFLELQV human IL-15 (rhIL-15) HDTVENLIIL FINTS 115 ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM SEQ ID NO: 8 recombinant MQDRHMIRMR KAQLKSANTG QLIDIVDQLK 60 NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ human IL-21 (rhIL-21) NNERIINVSI KSLLQKMIHQ HLSSRTHGSE KKLKRKPPST 120 DS 132 NAGRRQKHRL TCPSCDSYEK KPPKEFLERF

The term IL-4 (also referred to herein as IL4) refers to a cytokine known as interleukin 4, which is produced by Th2 T cells as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation of IL-4, Th2 T cells then further produce IL-4 in a positive feedback mechanism. IL-4 also stimulates B cell proliferation and class II MHC expression, and also induces class switch to IgE and IgG ₁ expression by B cells. Recombinant human IL-4 suitable for use in the invention is commercially available from many vendors including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (recombinant human IL-4 protein, Gibco catalog number CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the invention is shown in Table 1. 2 (SEQ ID NO: 5).

The term IL-7 (also referred to herein as IL7) means a glycosylated, tissue-derived cytokine known as interleukin-7, which can be derived from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the alpha chain of the IL-7 receptor and the common gamma chain of the receptor, which are involved in a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the invention is commercially available from many vendors including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (recombinant human IL-7 protein, Gibco catalog number PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is shown in Table 1. 2 (SEQ ID NO: 6).

The term IL-15 (also referred to herein as IL15) means the T-cell growth factor known as interleukin-15 and encompasses all forms of IL-15, including its human and mammalian forms with conservative amino acid substitutions, glycoforms, biological analogs, and options. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the contents of which are incorporated herein by reference. IL-15 shares β and γ signal receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine residue) with a molecular weight of 12.8 kDa.

- 22 042041

Recombinant human IL-15 is commercially available from many vendors including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (recombinant human IL-15, cat. no. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the invention is shown in Table 1. 2 (SEQ ID NO: 7).

The term IL-21 (also referred to herein as IL21) means the pleiotropic cytokine protein known as interleukin-21 and encompasses all forms of IL-21, including its human and mammalian forms with conservative amino acid substitutions, glycoforms, biological analogs, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. drug. Disc. 2014, 13, 37995, the contents of which are incorporated herein by reference. IL-21 is mainly produced by natural killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single, non-glycosylated, 132 amino acid polypeptide chain with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from many vendors including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, uSa (catalog no. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (recombinant human IL-21, cat. no. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the invention is shown in Table 1. 2 (SEQ ID NO: 8).

The term biological analog means a biological agent, including a monoclonal antibody or fusion protein, that has a high degree of similarity to a U.S. licensed reference biological agent despite minor differences in clinically inactive components, and for which there are no clinically significant differences between the biological agent and the reference agent in safety, purity and efficacy of the product. In addition, a similar biological or biologically similar medicinal product is a biological medicinal product similar to another biological medicinal product that has already been approved for use by the European Medicines Agency. The term biological analogue is also used interchangeably by other national and regional regulatory authorities. Biological agents or biological drugs are drugs that have been formulated or obtained from a biological source such as bacteria or yeast. They can be composed of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the IL-2 reference protein is aldesleukin (proleukin), the protein approved for use by drug regulatory authorities with reference to aldesleukin is biologically similar to aldesleukin or is a biological analogue of aldesleukin. In Europe, a biological analogue or biologically analogous medicinal product is a biological medicinal product that is similar to another biological medicinal product that has already been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for the use of biological analogues in Europe is Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC, as last amended, and thus in Europe a biological analogue can be authorized, approved for authorization or submitted for examination for authorization in accordance with Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC. In Europe, an already authorized originator biological medicinal product can be called a reference medicinal product. Some of the requirements for a product to be considered as a biological analogue are set out in the CHMP guidelines for similar biological medicinal products. In addition, tool-specific guidelines, including those related to monoclonal antibody biological analogs, are provided by the EMA for each tool and are posted on the organization's website. A biological analogue described herein may be similar to a reference drug in terms of quality, biological activity, mechanism of action, safety and/or efficacy profiles. In addition, the biological analogue can be used, or be intended for use, in the treatment of the same pathological conditions as the reference drug. Thus, the biological analogue described herein can be considered to have qualitative characteristics similar, or highly similar, to those of the reference drug. Alternatively, or additionally, a biological analog described herein may be considered to have a biological activity similar, or highly similar, to that of a reference drug. Alternatively, or additionally, a biological analogue described herein can be considered to have a safety profile similar, or highly similar, to that of a reference drug. Alternatively, or additionally, a biological analog described herein can be considered to have an efficacy similar to, or highly similar to, that of a reference drug. As described in this document

- 23 042041 mente, a biological analogue in Europe is compared with a reference drug that has been authorized by the EMA for use. However, in some cases, a biological analogue may be compared with a biological medicinal product that has been authorized outside the European Economic Area (non-EEA authorized comparator) in certain studies. Such studies include, for example, certain clinical and in vivo preclinical studies. As used herein, the term biological analog also means a biological drug that has been, or can be, compared to a non-EEA authorized comparator. Some biological analogs are proteins such as antibodies, antibody fragments (eg, antigen binding fragments), and fusion proteins. A protein biological analog may have an amino acid sequence having minor modifications in amino acid structure (including, for example, deletions, additions, and/or substitutions of amino acids) that do not significantly affect the function of the polypeptide. The biological analogue may contain an amino acid sequence having a sequence identity of 97% or more, for example, 97%, 98%, 99% or 100%, with the amino acid sequence of its reference drug. A biological analogue may have one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and/or truncation, which/are/are different from the post-translational modifications of the reference drug, provided that the differences do not result in a change in safety and /or the effectiveness of the drug. The biological analog may have an identical or different glycosylation pattern compared to the reference drug. In particular, but not exclusively, a biological analog may have a different glycosylation pattern if the differences eliminate, or should eliminate, the safety problems associated with the reference drug. In addition, the biological analogue may differ from the reference drug, for example, in terms of efficacy, pharmaceutical form, formulation, excipients and/or formulation, provided that the safety and efficacy of the medicinal product does not change for the worse. A biological analogue may differ, for example in pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles, from the reference drug, but is still considered sufficiently similar to the reference drug to be authorized or considered eligible for authorization. In certain circumstances, the biological analogue exhibits different binding characteristics compared to the reference drug, and the different binding characteristics are not considered by regulatory authorities, such as EMA, to be an obstacle to authorization as a similar biological agent. The term biological analogue is also used interchangeably by other national and regional regulatory authorities.

As used herein, the term variant includes, but is not limited to, antibodies or fusion proteins that contain an amino acid sequence that differs from that of the reference antibody by one or more substitutions, deletions, and/or additions at certain positions within or at the edges of the amino acid sequence of the reference antibody. . A variant may have one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of a reference antibody. Conservative substitutions may include, for example, substitution with identically charged or uncharged amino acids. The variant retains the ability of the reference antibody to specifically bind the antigen. The term variant also encompasses pegylated antibodies or proteins.

PEGylation means the modification of an antibody, or fragment thereof, which is generally reacted with a polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody, antibody fragment, or squirrel. Pegylation can, for example, increase the biological (eg, serum) half-life of an antibody or protein. Preferably, the pegylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol should encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy or aryloxy polyethylene glycol or polyethylene glycol maleimide. The antibody or protein to be pegylated may be an aglycosylated antibody. PEGylation techniques are known in the art and can be applied to antibodies of the invention as described, for example, in European Patent Nos. EP 0154316 and EP 0401384.

The term hematological malignancy refers to cancers and tumors of mammalian hematopoietic and lymphoid tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic system. Hematologic malignancies are also called liquid tumors. Hematologic malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (MLL), acute myelogenous leukemia (AML), chronic

- 24 042041 myelogenous leukemia (CML), acute monocytic leukemia (AML), Hodgkin's lymphoma and non-Hodgkin's lymphomas. The term B-cell hematologic malignancy refers to hematologic malignancies involving B cells.

The term solid tumor means an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term solid malignant tumor refers to malignant, neoplastic or cancerous solid tumors. Malignant solid tumors include, but are not limited to, sarcomas, carcinomas, and lymphomas such as cancers of the lung, breast, prostate, colon, rectum, and bladder. The tissue structure of solid tumors includes interconnected tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, among which cancer cells are distributed and which may provide a supportive microenvironment.

The term liquid tumor refers to an abnormal mass of cells that is liquid in nature. Liquid malignancies include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematologic malignancies. TILs derived from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MBCL).

As used herein, the term microenvironment may refer to the microenvironment of a solid or hematological tumor as a whole, or to a particular subgroup of cells within that microenvironment. As used herein, the term tumor microenvironment refers to the complex mixture of cells, soluble factors, signaling molecules, extracellular matrices, and mechanical extrinsic stimuli that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from the host immune system, promote therapeutic resistance, and provide niches for development. major metastases, as described in Swartz et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that must be recognized by T cells, tumor eradication by the immune system is a rare event due to immune suppression by the microenvironment. tumors.

As used herein, the terms fragmentation, fragmentation, and fragmented describe methods for destroying a tumor, including mechanical fragmentation methods such as crushing, cutting, separating, and destroying tumor tissue, as well as any other method for destroying the physical structure of tumor tissue.

The terms approximately and approximately mean within a statistically significant range of magnitude. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of the specified value or range. The allowable variation encompassed by the terms approximately or approximately depends on the particular system under study, and can be readily determined by one of ordinary skill in the art. In addition, as used herein, approximately and approximately means that distances, sizes, compositions, parameters, shapes, and other quantities and characteristics are not and need not be exact, but may be approximate and/or greater or smaller, if necessary, reflecting tolerances, conversion factors, rounding of values, measurement errors, and the like, as well as other factors known to specialists in this field. As a rule, distances, dimensions, compositions, dimensions, shapes, and other quantities and characteristics are approximate or approximate, whether or not specifically indicated. It should be noted that for embodiments with different sizes, shapes and proportions, the described conditions can be used.

Transitional terms including, consisting essentially of, and consisting of when used in the appended claims, in original and modified form, define the scope of the claim in relation to which of the unlisted additional elements or steps of the claim, if any, are excluded from the scope of the claim (s) formulas. The term including should be inclusive, or open-ended, and should not exclude any additional, unlisted element, method, step, or material. The term consisting of excludes any element, step or material other than those specified in the claim and, in the latter case, impurities normally associated with said material(s). The term consisting essentially of limits the scope of the claim to the specified elements, steps, or material(s), as well as those that do not materially affect the basic and new(new) characteristics of the claimed invention. All compositions, methods, and kits described herein that are an integral part of the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms including, consisting essentially of, and consisting of.

For the avoidance of doubt, this document assumes that the specific features (e.g., integers, characteristics, values, uses, diseases, formulas, compounds, or groups) described in connection with a particular aspect, embodiment, or example of

- 25 042041 of the invention should be taken as applicable to any other aspect, embodiment or example described herein, except in cases of incompatibility. Thus, such features may be used, when appropriate, in conjunction with any of the definitions, claims, or embodiments described herein. All of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), and/or all of the steps of any disclosed method or process, may be combined in any combination, except for combinations in which at least some of the features and/or steps are mutually exclusive. The invention is not limited to any of the details of any of the disclosed embodiments. The invention extends to any new feature, or new combination of features, disclosed in this specification (including any accompanying claims, abstract, and drawings), or to any new step, or any new combination of steps, of any of the disclosed methods or processes.

Methods for reproduction of remnant tumor-infiltrating lymphocytes.

In one embodiment, the invention relates to a method for propagating remnant tumor-infiltrating lymphocytes (rILs) after tumor cleavage, as described herein.

In one embodiment, the invention relates to a method for propagating rPOIL, comprising contacting a population of rPOIL containing at least one rPOIL with IL-2, resulting in the propagation of rOIL.

In one embodiment, the invention relates to a method for propagating a rOIL population, comprising the steps described in Jin et al., J. Immunotherapy 2012, 35, 283-292, the contents of which are incorporated herein by reference. For example, a tumor can be placed in an enzyme medium and fragmented mechanically for about 1 minute. The mixture can then be incubated for 30 minutes at 37° C. in a 5% CO ₂ atmosphere and then mechanically fragmented again for about 1 minute. After incubation for 30 minutes at 37° C. in a 5% CO ₂ atmosphere, the tumor can be mechanically fragmented a third time in about 1 minute. If large pieces of tissue are still present after the third mechanical failure, the sample can be subjected to 1 or 2 additional mechanical failure steps, with or without an additional 30 min incubation at 37°C in an atmosphere with 5% CO ₂ . After completion of the last incubation, if the cell suspension contains a large number of erythrocytes or dead cells, ficoll density gradient separation can be performed to remove these cells. TIL culture can be started in 24-well plates (Costar, 24-well cell culture plates, flat-bottomed; Corning Incorporated, Corning, NY), 1x10 ⁶ cells from a split tumor or one tumor fragment of approximately 1-8 mm ³ in 2 ml complete medium (CM) with IL2 (6000 IU/ml; Chiron Corp., Emeryville, CA). SM consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes and 10 mg/ml gentamicin. Culture can be started in 40 ml gas permeable flasks with 10 cm ² gas permeable silicone bottom (G-Rex 10; Wilson Wolf Manufacturing, New Brighton), 10-40x10 ⁶ viable split tumor cells or 5-30 tumor cells can be placed in each flask. fragments in 10-40 ml SM with IL-2. G-Rex 10 and 24-well plates can be incubated in a humidified incubator at 37°C and 5% CO2, 5 days after the start of culture, half of the medium can be removed and replaced with fresh CM medium with IL-2, and after day 5, half media can be changed every 2-3 days. Rapid Propagation Protocol (RRP) RTL can be performed using T-175 vials and gas permeable bags or G-Rex gas permeable vials as described elsewhere in this document. For PBR in T-175 vials, 1x10 ⁶ rOIL can be suspended in 150 ml of medium in each vial. rOIL can be cultured in a 1:1 mixture of CM and AIM-V media (medium 50/50) supplemented with 3000 IU/ml IL-2 and 30 ng/ml anti-CD3 antibody (OCT-3). T-175 vials can be incubated at 37°C in a 5% CO2 atmosphere. Half of the medium can be changed on day 5 using 50/50 medium with 3000 IU/ml IL-2. On day 7, cells from 2 vials of T-175 can be pooled in a 3 L bag and 300 ml of AIM-V with 5% human AB serum and 3000 IU/ml of IL-2 can be added to 300 ml of TIL suspension. The number of cells in each bag can be counted every day or every two days, and fresh medium can be added to maintain the cell number at 0.5 to 2.0 x 10 ⁶ cells/ml. For PBR in 500 ml vials with a 100-cm ² gas-permeable silicone bottom (e.g. G-Rex 100, Wilson Wolf Manufacturing, described elsewhere in this document), 5x10 ⁶ or 10x10 ⁶ OIL can be cultured in 400 ml of 50/50 medium with the addition of 3000 IU/ml IL-2 and 30 ng/ml anti-CD3 antibody (OCT-3). Vials of G-Rex 100 can be incubated at 37°C in an atmosphere with 5% CO ₂ . On day five, 250 ml of the supernatant can be withdrawn, placed in centrifuge beakers and centrifuged at 1500 rpm (491 d) for 10 minutes. The resulting TIL pellets can be resuspended in 150 ml of fresh medium 50/50 with 3000 IU/ml IL-2 and reintroduced into G-Rex 100 vials. -Rex 100 can be suspended in the 300 ml of medium present in each vial and the cell suspension can be divided into three 100 ml aliquots which can be used for inoculation

- 26 042041 3 vials of G-Rex 100. Approximately 150 ml AIM-V with 5% human AB serum and 3000 IU/ml IL-2 can then be added to each vial. The G-Rex 100 vials can then be incubated at 37°C in a 5% CO ₂ atmosphere, and four days later 150 ml AIM-V with 3000 IU/ml IL-2 can be added to each G-Rex 100 vial. can be completed by harvesting cells on day 14 of culture.

In one embodiment, a method for cell expansion, or cancer treatment, comprises the step of obtaining TIL from a patient's tumor sample. A sample of a patient's tumor can be obtained by methods known in the art. For example, TIL can be cultured from tumor enzymatic digests and tumor fragments (about 1 to about 8 mm ³ in size) obtained during acute excision. Such tumor digests can be prepared by incubating samples in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer medium containing 2 mM glutamate, 10 μg/ml gentamicin, 30 units/ml DNase, and 1.0 mg/ml collagenase) , followed by mechanical destruction (for example, using a tissue homogenizer). Tumor digests can be prepared by placing the tumor in an enzyme medium and mechanically fragmenting the tumor for about 1 minute, followed by a 30 min incubation at 37°C in a 5% CO ₂ atmosphere, followed by repeated cycles of mechanical disruption and incubation in the above conditions until only small pieces of tissue are present. After completion of the process, if the cell suspension contains a large number of erythrocytes or dead cells, a density gradient separation of the branched hydrophilic ficoll polysaccharide can be performed to remove these cells. You can use alternative methods known in this field, such as those described in the publication of US patent application No. 2012/0244133 A1, the contents of which are incorporated herein by reference. Any of the above methods can be used in any of the embodiments described herein for TIL propagation methods or cancer treatment methods.

In one embodiment, the implementation of the PBR ROIL can be carried out in a gas-permeable container in any suitable way. For example, rOIL can be rapidly propagated using non-specific stimulation of the T-cell receptor in the presence of interleukin-2 (IL-2), interleukin-15 (IL-15) and/or interleukin-21 (IL-21), as described, for example, in International Patent Application Publication Nos. WO 2015/189356 A1 and WO 2015/189356 A1, the contents of each of which are incorporated herein by reference. A non-specific T cell receptor stimulus may include, for example, OKT-3 at about 30 ng/mL, an anti-CD3 monoclonal antibody (commercially available from Ortho-McNeil, Raritan, NJ, USA or Miltenyi Biotech, Auburn, CA, USA). TILs can be rapidly propagated by additional stimulation of TILs in vitro with one or more antigens (including antigenic fragments thereof, such as epitope(s)) of cancer cells, which may optionally be expressed from a vector, such as a human leukocyte antigen A2 (HLA) binding peptide -A2), e.g. 0.3 μM MART-1:26-35 (27 L), or gpl 00:209-217 (210 M), optionally in the presence of T cell growth factor, e.g. IL-2 or IL- 15, at a concentration of 300 IU/ml. Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic fragments thereof. TIL can also be rapidly propagated by re-stimulation with the same cancer antigen(s) on HLA-A2 expressing antigen-presenting cells. Alternatively, TIL can also be restimulated with, for example, irradiated, autologous lymphocytes or HLA-A2+ irradiated allogeneic lymphocytes and IL-2.

In one embodiment, the method for propagating TIL may include using from about 5000 ml to about 25000 ml of cell culture medium, from about 5000 ml to about 10000 ml of cell culture medium, or from about 5800 ml to about 8700 ml of cell culture medium. In one embodiment, the method for propagating TIL may include using from about 1000 ml to about 2000 ml of cell culture medium, from about 2000 ml to about 3000 ml of cell culture medium, from about 3000 ml to about 4000 ml of cell culture medium, about 4000 ml to about 5000 ml of cell culture medium, from about 5000 ml to about 6000 ml of cell culture medium, from about 6000 ml to about 7000 ml of cell culture medium, from about 7000 ml to about 8000 ml of culture medium cells, about 8000 ml to about 9000 ml of cell culture medium, from about 9000 ml to about 10000 ml of cell culture medium, from about 10000 ml to about 15000 ml of cell culture medium, from about 15000 ml to about 20000 ml of medium for cell culture or about 20,000 ml to about 25,000 ml of cell culture medium. In one embodiment of the TIL propagation method, no more than one type of cell culture medium is used. Any suitable cell culture medium can be used, for example AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, CA, USA). In this regard, patent-pending methods have the advantage of reducing the amount of media as well as the number of media types needed to propagate TIL. In one of the embodiments, the implementation of the reproduction of TIL may include cell feeding no more often than every third or

- 27 042041 the fourth day. Propagation of cells in a gas permeable container simplifies the procedures required for cell expansion by reducing the frequency of feeding required for cell expansion.

In one embodiment, rapid propagation is carried out using a gas permeable container. Such embodiments allow cell populations to proliferate from about 5x10 5 cells/cm ² to 10x10 ⁶ to 30x10 ⁶ cells/cm ² . In one embodiment, this propagation occurs without feeding. In one embodiment, this propagation occurs without feeding as long as the medium remains at a height of about 10 cm in a gas-permeable vial. In one of the embodiments, the implementation of the reproduction occurs without feeding, but with the addition of one or more cytokines. In one embodiment, the cytokine may be added as a bolus without the need to mix the cytokine with the medium. Such containers, devices, and methods are known in the art and have been used to propagate TILs, and include those described in US Patent Application Publication No. US 2014/0377739 A1, International Patent Application Publication No. WO 2014/210036 A1, US Patent Application Publication No. US 2013/0115617 A1, International Publication No. WO 2013/188427 A1, US Patent Application Publication No. US 2011/0136228 A1, US Patent Publication No. 8809050, International Patent Application Publication No. WO 2011/072088 A2, US Patent Application Publication No. US 2016 /0208216 A1, US Patent Application Publication No. US 2012/0244133 A1, International Patent Application Publication No. WO 2012/129201 A1, US Patent Application Publication No. US 2013/0102075 A1, US Patent Publication No. 8956860, International Patent Application Publication No. WO 2013/ 173835 A1 and US Patent Application Publication No. US 2015/0175966 A1, the contents of which are incorporated herein by reference. Such methods are also described in Jin et al., J. Immunotherapy 2012, 35, 283-292, the contents of which are incorporated herein by reference.

In one embodiment, the gas permeable container is a GRex 10 vial (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas permeable container has a 10 cm ² gas permeable culture surface. In one embodiment, the gas permeable container may contain 40 ml of cell culture medium. In one embodiment, 100,300 million oil is produced in a gas-permeable container after 2 media changes.

In one embodiment, the gas permeable container is a GRex 100 vial (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas permeable container has a 100 cm ² gas permeable culture surface. In one embodiment, the gas permeable container may contain 450 ml of cell culture medium. In one embodiment, 1-3 billion oil is produced in a gas-permeable container after 2 media changes.

In one embodiment, the gas permeable container is a GRex 100M vial (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas permeable container has a 100 cm ² gas permeable culture surface. In one embodiment, the gas permeable container may contain 1000 ml of cell culture medium. In one embodiment, 1-3 billion oil is produced in a gas-permeable container without changing the medium.

In one embodiment, the gas permeable container is a GRex 100L vial (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas permeable container has a 100 cm ² gas permeable culture surface. In one embodiment, the gas permeable container may contain 2000 ml of cell culture medium. In one embodiment, 1-3 billion oil is produced in a gas-permeable container without changing the medium.

In one embodiment, the gas-permeable container is a 24-well G-Rex plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas permeable container is a well plate, with each well having a 2 cm ² gas permeable culture surface. In one embodiment, the gas-permeable container is a well plate, where each well may contain 8 ml of cell culture medium. In one embodiment, 20-60 million cells per well are obtained in a gas permeable container after 2 medium changes.

In one embodiment, the gas permeable container is a 6-well G-Rex plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas permeable container is a well plate, with each well having 10 cm ² of gas permeable culture surface. In one embodiment, the gas-permeable container is a well plate, where each well may contain 40 ml of cell culture medium. In one embodiment, 100-300 million cells per well are obtained in a gas permeable container after 2 medium changes.

In one embodiment, the cell culture medium in the first and/or second gas permeable container is unfiltered. Using an unfiltered medium for

- 28 042041 cell culture can simplify the procedures required for cell propagation. In one embodiment, the cell culture medium in the first and/or second gas permeable container does not contain beta-mercaptoethanol (BME).

In one embodiment, the implementation of the period of time spent on the method, including obtaining a sample of tumor tissue from a mammal; culturing the tumor tissue sample in the first gas-permeable container containing the cell culture medium; obtaining TIL from a sample of tumor tissue; propagation of the TIL in the second gas-permeable container containing the cell culture medium is from about 14 to about 42 days, for example, about 28 days.

In one embodiment, the ratio of rOIL to PBMC during the rapid expansion phase is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 in 225, about 1 in 250, about 1 in 275, about 1 in 300, about 1 in 325, about 1 in 350, about 1 in 375, about 1 in 400, or about 1 in 500. In one embodiment, the ratio rIL and PBMC during the rapid expansion phase is between 1 in 50 and 1 in 300. In one embodiment, the ratio of rIL and PBMC in the rapid expansion phase is between 1 in 100 and 1 in 200.

In one embodiment, the ratio of rOIL to PBMC (rOIL:PBMC) is selected from the group consisting of 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1: 40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1: 165 1:170 1:175 1:180 1:185 1:190 1:195 1:200 1:225 1:250 1:275 1:300 1:350 1:400, 1:450 and 1:500. In a preferred embodiment, the ratio of rOIL to PBMC (rOIL:PBMC) is about 1:90. In a preferred embodiment, the ratio of rOIL to PBMC (rOIL:PBMC) is about 1:95. In a preferred embodiment, the ratio of rOIL to PBMC (OIL:PBMC) is about 1:100. In a preferred embodiment, the ratio of rOIL to PBMC (OIL:PBMC) is about 1:105. In a preferred embodiment, the ratio of rOIL to PBMC (OIL:PBMC) is about 1:110.

In one embodiment, the cell culture medium also contains IL-2. In a preferred embodiment, the cell culture medium contains about 3000 IU/ml IL-2. In one embodiment, the cell culture medium contains about 1000 IU/ml, about 1500 IU/ml, about 2000 IU/ml, about 2500 IU/ml, about 3000 IU/ml, about 3500 IU/ml, about 4000 IU/ml ml, about 4500 IU/ml, about 5000 IU/ml, about 5500 IU/ml, about 6000 IU/ml, about 6500 IU/ml, about 7000 IU/ml, about 7500 IU/ml or about 8000 IU/ml IL -2. In one embodiment, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/ml, 7000 to 8000 IU/ml or 8000 IU/ml IL-2.

In one embodiment, the cell culture medium contains the OKT-3 antibody. In a preferred embodiment, the cell culture medium contains about 30 ng/ml of OKT-3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/ml, about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 50 ng/ml ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 200 ng/ml, about 500 ng/ml, and about 1 μg/ml antibody OKT-3. In one embodiment, the cell culture medium contains 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL up to 20 ng/ml, 20 ng/ml to 30 ng/ml, 30 ng/ml to 40 ng/ml, 40 ng/ml to 50 ng/ml, and 50 ng/ml to 100 ng/ml antibodies OKT-3.

In one embodiment, the TIL rapid expansion method can be performed using T-175 vials and gas permeable bags as previously described (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother 2003, 26, 332-42), or gas-permeable culture containers (G-Rex vials, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). For rapid multiplication of TIL in T-175 flasks, 1x106 TIL suspended in 150 ml of medium can be added to each T-175 flask. TIL can be cultured in a 1:1 mixture of CM and AIM-V media supplemented with 3000 IU (international units) of IL-2 per ml and 30 ng of anti-CD3 antibody (eg OKT-3) per ml. Vials of T-175 can be incubated at 37°C in an atmosphere with 5% CO ₂ . Half of the medium can be changed on day 5 using 50/50 medium with 3000 IU/ml IL-2. On day 7, cells from two T-175 flasks can be pooled in a 3 liter bag and 300 ml AIM-V with 5% human AB serum and 3000 IU/ml IL-2 added to 300 ml TIL suspension. The number of cells in each bag can be counted every day or every other day, and fresh medium added to maintain the cell number at 0.5-2.0x106 cells/ml.

In one embodiment, for the rapid propagation of OIL in 500 ml gas permeable vials with a 100 cm ² gas permeable silicone bottom (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5x10 ⁶ or 10x10 ⁶ TIL can be cultured in 50/50 medium supplemented with 5% human AB serum, 3000 IU/ml IL-2 and 30 ng/ml anti-CD3 (OCT-3). Vials of G-Rex 100 can be incubated at 37°C in an atmosphere with 5% CO ₂ . On a day, 5250 ml of supernatant can be withdrawn, placed in centrifuge beakers and centrifuged at 1500 rpm (rpm; 491xg) for 10 minutes. TIL pellets can be resuspended in 150 ml of fresh medium with 5% human AB serum, 3000 IU/mL IL-2 and returned to the original G-Rex 100 vials. Each vial of GRex 100 can be suspended in 300 ml of the medium present in each vial and the cell suspension can be divided into 3 x 100 ml aliquots which can be used to inoculate 3 vials of G-Rex 100. Then 150 ml of AIM- can be added to each vial. V with 5% human AB serum and 3000 IU/ml IL-2. G-Rex 100 vials can be incubated at 37°C in a 5% CO ₂ atmosphere, and after 4 days 150 ml AIM-V with 3000 IU/ml IL-2 can be added to each G-Rex 100 vial. Cells can be harvested in day 14 cultivation.

In one of the embodiments, the implementation of the OIL can be obtained as follows. 2 mm ³ tumor fragments are cultured in complete medium (CM) consisting of AIM-V medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mM glutamine (Mediatech, Inc. Manassas, VA), 100 U/ml penicillin ( Invitrogen Life Technologies), 100 μg/ml streptomycin (Invitrogen Life Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc. Winchester, VA), and 600 IU/ml rhIL-2 (Chiron, Emeryville, CA). For enzymatic digestion of solid tumors, tumor samples are finely cut in RPMI-1640 medium, washed and centrifuged at 800 rpm for 5 min at 15-22°C, resuspended in enzymatic digestion buffer (0.2 mg/ml collagenase and 30 units/ml of DNase in RPMI-1640), followed by stirring overnight at room temperature. TIL derived from fragments can be grown for 3-4 weeks in SM and propagated fresh, or cryopreserved in heat-inactivated human AV serum supplemented with 10% dimethyl sulfoxide (DMSO) and stored at -180°C until use in the study. Tumor-associated lymphocytes (TAL) derived from harvested ascites are plated at a density of 3x10 ⁶ cells/well in a 24-well plate in CM. TIL growth is monitored approximately every other day using an inverted microscope at low magnification.

In one embodiment, the OIL is propagated in gas-permeable containers. Gas-permeable containers are used to propagate TIL in the presence of PBMC using methods, compositions, and devices known in the art, including those described in US Patent Application Publication No. 2005/0106717 A1, the contents of which are incorporated herein by reference. In one embodiment, the implementation of the TIL is propagated in gas-permeable bags. In one embodiment, TILs are propagated using a cell expansion system in which TILs are propagated in gas permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TIL is expanded using a cell expansion system in which TIL is expanded in gas permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system includes a gas-permeable cell bag having a volume selected from the group consisting of about 100 ml, about 200 ml, about 300 ml, about 400 ml, about 500 ml, about 600 ml, about 700 ml. ml, about 800 ml, about 900 ml, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about 16 L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L and about 30 L. In one embodiment, the cell propagation system includes a gas permeable cell bag having a volume in the range selected from the group consisting of a volume of 50 to 150 ml, 150 to 250 ml, 250 to 350 ml, 350 to 450 ml, 450 to 550 ml, 550 to 650 ml, 650 to 750 ml, 750 to 850 ml, 850 to 950 ml and 950 to 1050 ml. In one embodiment, the cell expansion system includes a gas-permeable cell bag having a volume in the range selected from the group consisting of a volume of 1 L to 2 L, 2 L to 3 L, 3 L to 4 L, 4 L up to 5 l, from 5 l to 6 l, from 6 l to 7 l, from 7 l to 8 l, from 8 l to 9 l, from 9 l to 10 l, from 10 l to 11 l, from 11 l to 12 L, 12 L to 13 L, 13 L to 14 L, 14 L to 15 L, 15 L to 16 L, 16 L to 17 L, 17 L to 18 L, 18 L to 19 l and from 19 l to 20 l. In one embodiment, the cell expansion system includes a gas-permeable cell bag having a volume in the range selected from the group consisting of a volume of 0.5 L to 5 L, 5 L to 10 L, 10 L to 15 L, from 15 l to 20 l, 20 l to 25 l and 25 l to 30 l. In one embodiment, the cell expansion system uses a swing time of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours. h, about 10 h, about 11 h, about 12 h, about 24 h, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days,

- 30 042041 approximately 14 days, approximately 15 days, approximately 16 days, approximately 17 days, approximately 18 days, approximately 19 days, approximately 20 days, approximately 21 days, approximately 22 days, approximately 23 days, approximately 24 days, approximately 25 days , approximately 26 days, approximately 27 days and approximately 28 days. In one embodiment, the cell propagation system uses a swing time of 30 minutes to 1 hour, 1 hour to 12 hours, 12 hours to 1 day, 1 day to 7 days, 7 days to 14 days, 14 days up to 21 days and from 21 days to 28 days. In one embodiment, the cell expansion system uses a pumping speed of about 2 pumps/minute, about 5 pumps/min, about 10 pumps/min, about 20 pumps/min, about 30 pumps/min, and about 40 pumps/min. In one embodiment, the cell propagation system uses a pumping speed of 2 pumps/min to 5 pumps/min, 5 pumps/min to 10 pumps/min, 10 pumps/min to 20 pumps/min, 20 pumps/min up to 30 strokes/min and from 30 strokes/min to 40 strokes/min. In one embodiment, the cell propagation system uses a rocking angle of about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about 11 ° and approximately 12°. In one embodiment, the cell propagation system uses a rocking angle of 2° to 3°, 3° to 4°, 4° to 5°, 5° to 6°, 6° to 7°, 7° up to 8°, 8° to 9°, 9° to 10°, 10° to 11° and 11° to 12°.

In one embodiment, the method for propagating rROIL further comprises selecting rROIL for superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in US Patent Application Publication No. 2016/0010058 A1, the contents of which are incorporated herein by reference, can be used to screen TIL for superior tumor reactivity.

Characteristics of ROIL.

In one embodiment, the rROILs of the invention have a depleted T cell phenotype characterized by the presence of one or more T cell depletion markers. In one embodiment, the rROILs of the invention have a depleted T cell phenotype characterized by the presence of one or more T cell depletion markers as determined by flow cytometric analysis. In one embodiment, the T cell depletion marker is PD-1. In one embodiment, the T cell depletion marker is LAG3. In one embodiment, the T cell depletion marker is TIM3.

In one embodiment, PD-1 expression in rOIL is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least at least 140% or at least 150% in comparison with the MOIL. In one embodiment, the expression of PD-1 in rOIL is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold in comparison with MOIL.

In one embodiment, LAG3 expression in rOIL is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% , or at least 150% in comparison with MOIL. In one embodiment, LAG3 expression in rOIL is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold times, at least 8 times, at least 9 times, or at least 10 times in comparison with MOIL. In one embodiment, LAG3 expression in rOIL is not detectable by flow cytometry.

In one embodiment, TIM3 expression in rOIL is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% in comparison with MOIL. In one embodiment, TIM3 expression in rOIL is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold times, at least 8 times, at least 9 times, or at least 10 times in comparison with MOIL. In one embodiment, TIM3 expression in rOIL is not detectable by flow cytometry.

In one embodiment, TIGIT expression in rOIL is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least

- 31 042041

130%, at least 140% or at least 150% compared to MWL. In one embodiment, the expression of TIGIT in rOIL is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold times, at least 8 times, at least 9 times, or at least 10 times in comparison with MOIL. In one embodiment, the expression of TIGIT in rOIL is not detectable by flow cytometry.

In one embodiment, CTLA-4 expression in rOIL is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% compared to MWL. In one embodiment, the expression of CTLA-4 in rOIL is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold in comparison with MOIL. In one embodiment, the expression of CTLA-4 in rROIL is not detectable by flow cytometry.

In one embodiment, CD69 expression in rOIL is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% compared to MOIL. In one embodiment, CD69 expression in rOIL is increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold times, at least 8 times, at least 9 times, or at least 10 times in comparison with MOIL.

In one embodiment, the expression of S1PR1 (sphingosine-1-phosphate receptor 1) in rROIL is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least at least 140% or at least 150% in comparison with the MOIL. In one embodiment, the expression of S1PR1 in rOIL is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold times, at least 8 times, at least 9 times, or at least 10 times in comparison with MOIL.

In one embodiment, telomere length in rOIL is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% compared to MOIL. In one embodiment, the telomere length in rOIL is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold times, at least 8 times, at least 9 times, or at least 10 times in comparison with MOIL.

In one embodiment, CD28 expression in rOIL is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% compared to MOIL. In one embodiment, CD28 expression in rOIL is increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold times, at least 8 times, at least 9 times, or at least 10 times in comparison with MOIL.

In one embodiment, CD27 expression in rTIL is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% compared to MOIL. In one embodiment, CD27 expression in rOIL is increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold times, at least 8 times, at least 9 times, or at least 10 times in comparison with MOIL.

In some embodiments, the methods described herein may optionally include cryopreservation of the mOIL and/or rOIL in a storage medium (e.g., a medium containing 5% DMSO) prior to the additional step described herein or after the completion of the RRP step. described herein prior to transport, thawing and/or administration to a patient. In some embodiments, the methods described in

- 32 042041 herein, may include the step of thawing cryopreserved ILs (for example, cryopreserved oILs, cryopreserved roILs, or a combination or mixture thereof) before carrying out the additional step described herein. In some embodiments, an additional step may be additional or re-propagation of mOIL and/or rOIL (e.g., rePBR), which can be performed on thawed cells using, for example, cell culture supplemented medium containing IL-2, OKT- 3 and/or nurse cells (e.g. antigen presenting cells) typically containing peripheral blood mononuclear cells (PBMCs; or alternatively antigen presenting cells), wherein the further expansion step can be carried out for at least 14 days. In some embodiments, such a medium may also contain combinations of IL-2, IL-15 and/or IL-23, and not just IL-2 alone.

As described herein, cryopreservation can be performed at many points in time during the propagation of TIL. In some embodiments, a population of total TILs (eg, mTIL, rTIL, or a combination or mixture thereof) may be cryopreserved after propagation. Cryopreservation, in general, can be performed by placing a population of TIL in a solution for freezing, for example, containing 85% AB serum with inactivated complement and 15% dimethyl sulfoxide (DMSO). Cells in solution are placed in cryogenic flasks and stored for 24 hours at -80° C., optionally transferred to nitrogen gas freezers for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, the implementation of the OIL described herein can be cryopreserved in 5% DMSO. In some embodiments, the TILs described herein can be cryopreserved in cell culture medium plus 5% DMSO.

As needed, cryopreserved cells as described herein, such as cryopreserved rPOIL, are removed from the freezer and thawed in a 37° C. water bath until about 4/5 of the solution is thawed. Cells are typically resuspended in complete medium and optionally washed one or more times. In some embodiments, the implementation of thawed TIL can be counted and assessed for their viability by a method known in this field.

Methods for Digesting Tumors to Produce rPOIL In one embodiment, the method for producing rPOIL comprises the step of digesting a tumor with one or more enzymes. Enzymes suitable for digesting tumors are described in Volvitz et al., BMC Neuroscience 2016, 17, 30, the contents of which are incorporated herein by reference.

In some embodiments, the invention may include methods for producing rOIL, including the step at which the tumor, which may be a tumor tissue or part of it, is cleaved using deoxyribonuclease, collagenase, hyaluronidase, or a combination thereof.

In one embodiment, the method for producing rOIL includes the step of digesting the tumor using any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, resulting in degradation of the DNA. In one embodiment, the method for producing rOIL includes the step of digesting the tumor using deoxyribonuclease (DNase). In one embodiment, the method for producing rOIL includes the step of digesting the tumor using deoxyribonuclease and at least one other enzyme. In one embodiment, the deoxyribonuclease is deoxyribonuclease I. In one embodiment, the deoxyribonuclease is deoxyribonuclease II. In one embodiment, the deoxyribonuclease is bovine pancreas deoxyribonuclease I (Sigma D5025 or equivalent). In one embodiment, the deoxyribonuclease is recombinant bovine deoxyribonuclease I expressed in Pichia pastoris (Sigma D2821 or equivalent). In one embodiment, the deoxyribonuclease is recombinant human deoxyribonuclease I (rhDNase I, also known as dornase alfa, commercially available under the name Pulmozyme from Genentech, Inc.). In one embodiment, the deoxyribonuclease is bovine spleen deoxyribonuclease II (Sigma D8764 or equivalent). In one embodiment, the deoxyribonuclease is porcine spleen deoxyribonuclease II (Sigma D4138 or equivalent). In one embodiment, any of the above deoxyribonucleases is present in the tumor digest. The preparation and properties of deoxyribonucleases suitable for use in the invention are described in US Pat.

In one embodiment, the method for producing rOIL includes the step of cleavage of the tumor using any enzyme that catalyzes the cleavage of peptide bonds in collagen, resulting in collagen degradation. In one of the embodiments, the implementation of the method of obtaining ROIL includes the step of splitting the tumor using collagenase. In one of the embodiments

- 33 042041 method for obtaining rOIL includes the step of splitting the tumor using collagenase and at least one other enzyme. In one embodiment, the collagenase is a collagenase from Clostridium histolyticum. In one embodiment, the collagenase is clostridiopeptidase A. In one embodiment, the collagenase is collagenase I. In one embodiment, the collagenase is collagenase II. In one embodiment, the collagenase is Clostridium histolyticum collagenase (Sigma C5138 or equivalent). The preparation and properties of collagenases suitable for use in the invention are described in US Pat. Nos. 3,201,325; 3,705,083; 3,821,364;

In one of the embodiments, the method of obtaining rOIL includes the step of splitting the tumor using any enzyme that catalyzes the degradation of hyaluronic acid. In one of the embodiments, the implementation of the method of obtaining ROIL includes the step of splitting the tumor using hyaluronidase. In one embodiment, the method for producing rOIL includes the step of splitting the tumor using hyaluronic glucosidase. In one embodiment, the hyaluronidase is bovine testis type I hyaluronidase (Sigma H3506 or equivalent). In one embodiment, the hyaluronidase is a type II hyaluronidase from sheep testis (Sigma H2126 or equivalent). In one embodiment, the hyaluronidase is a type III hyaluronidase. In one embodiment, the hyaluronidase is type IV (type IV-S) hyaluronidase from bovine testis (Sigma H3884 or equivalent). In one embodiment, the hyaluronidase is a type V hyaluronidase from sheep testis (Sigma H6254 or equivalent). In one embodiment, the hyaluronidase is type VIII hyaluronidase from bovine testes (Sigma H3757 or equivalent). In one embodiment, the hyaluronidase is recombinant human hyaluronidase (commercially available under the name Gilenex from Halozyme, Inc.). The preparation and properties of hyaluronidases suitable for use in the invention are described in US Pat. Nos. 4,820,516; 5,593,877; 6,057,110;

In one of the embodiments, the implementation of the method of obtaining ROIL includes the step of splitting the tumor using deoxyribonuclease and hyaluronidase. In one of the embodiments, the implementation of the method of obtaining ROIL includes the step of splitting the tumor using deoxyribonuclease and collagenase. In one of the embodiments, the implementation of the method of obtaining ROIL includes the step of splitting the tumor using hyaluronidase and collagenase. In one embodiment, the method for producing rOIL includes the step of digesting the tumor using deoxyribonuclease, hyaluronidase, and collagenase.

In one embodiment, the method for producing rOIL includes the step of digesting the tumor using deoxyribonuclease and hyaluronidase, as well as at least one additional enzyme. In one embodiment, the method for producing rOIL includes the step of digesting the tumor using deoxyribonuclease and collagenase, as well as at least one additional enzyme. In one of the embodiments, the method of obtaining rOIL includes the step of splitting the tumor using hyaluronidase and collagenase, as well as at least one additional enzyme. In one embodiment, the method for producing rOIL includes the step of digesting the tumor using deoxyribonuclease, hyaluronidase, and collagenase, as well as at least one additional enzyme. In any of the above embodiments, the additional enzyme is selected from the group consisting of caseinase, clostripain, trypsin, and combinations thereof.

In one embodiment, the method for producing rOIL includes the step of cleavage of the tumor using any of the enzymes described above, and further includes the step of mechanically disrupting or fragmenting the tumor before, during, or after cleavage.

In one embodiment, the method for producing rOIL comprises the step of digesting the tumor using any of the enzymes described above, wherein the digestion is carried out for a period of time selected from the group consisting of 15 minutes, 30 minutes, 45 minutes, 1 hour, 90 minutes, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 18h, 24h, 36h and 48h.

In one embodiment, the method for producing rOIL comprises the step of digesting the tumor using any of the enzymes described above, wherein the digestion is carried out for a period of time selected from the group consisting of about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour , about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18h, about 24h, about 36h and about 48h.

In one embodiment, the method for producing rOIL comprises the step of digesting the tumor using any of the enzymes described above, wherein the digestion is carried out for a period of time selected from the group consisting of less than 15 minutes, less than 30 minutes, less than 45 minutes, less than 1 hour. , less than 90 minutes, less than 2 hours, less than 3 hours, less than 4 hours, less than 5 hours, less than 6 hours, less than 7 hours, less than 8 hours, less than 9 hours, less than

- 34 042041 hours, less than 11 hours, less than 12 hours, less than 18 hours, less than 24 hours, less than 36 hours and less than 48 hours.

In one embodiment, the method for producing rOIL includes the step of digesting the tumor using any of the enzymes described above, wherein the digestion is carried out for a period of time selected from the group consisting of more than 15 minutes, more than 30 minutes, more than 45 minutes, more than 1 hour , more than 90 min, more than 2 h, more than 3 h, more than 4 h, more than 5 h, more than 6 h, more than 7 h, more than 8 h, more than 9 h, more than 10 h, more than 11 h, more than 12 h, more 18 hours, more than 24 hours, more than 36 hours and more than 48 hours.

In one embodiment, the method for producing rOIL includes the step of digesting the tumor using any enzyme described above, wherein the digestion is carried out for a period of time selected from the group consisting of a period of time from 30 minutes to 1 hour, from 1 hour to 2 hours , 2 to 3 hours, 3 to 4 hours, 4 to 5 hours, 5 to 6 hours, 6 to 12 hours, 12 to 18 hours, 18 to 24 hours, and from 24 h to 48 h.

In one embodiment, the method for producing rOIL comprises the step of digesting the tumor using any of the enzymes described above, wherein the digestion is carried out at a temperature selected from the group consisting of about 20°C, about 25°C, about 30°C, about 35 °C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C and about 80°C.

In one embodiment, the method for producing rOIL comprises the step of digesting the tumor using any of the enzymes described above, wherein the digestion is carried out at a temperature selected from the group consisting of 20°C to 25°C, 25°C to 30° C, 30°C to 35°C, 35°C to 40°C, 40°C to 45°C, 45°C to 50°C, 50°C to about 55°C, from 55 °C to 60°C, 60°C to 65°C, 65°C to 70°C, 70°C to 75°C and 75°C to 80°C.

In one embodiment, the method for producing rOIL comprises the step of digesting the tumor using any of the enzymes described above, wherein both the time and temperature of digestion are reduced in the case of digestion of tumor remnants (after pre-PBR). In one embodiment, the method for producing rOIL comprises the step of digesting the tumor using any of the enzymes described above, wherein both the time and temperature of digestion are increased in the case of digestion of whole tumor fragments (no pre-PBR).

Methods for modulating the ratio of rOIL and MOIL.

In one embodiment, the concentration of rOIL versus mOIL can be modulated or controlled using any of the multiplication and cleavage steps described herein (including pre-PBR) such that a therapeutic TIL for use in the treatment of cancers described herein may have the desired ratio of rPOIL to MOIL. In one embodiment, the invention relates to a method for removing mOIL from a mixture of mOIL and rOIL. In one embodiment, the invention relates to a process for removing rOIL from a mixture of rOIL and rOIL.

In some embodiments of the methods of the invention, mOIL and/or rOIL can be added to the culture prior to the initial propagation step, during the first propagation step (eg, pre-PBR) and/or at the second propagation step (eg, PBR). In some embodiments of the methods of the invention, mOIL may be cultured separately according to the culture or propagation steps described herein using one, two, three or more propagation cycles and added to a population of rOIL and mOIL at a selected ratio of rOIL and mOIL. In some embodiments of the methods of the invention, rOIL can be cultured separately in accordance with the cultivation or propagation steps described herein using one, two, three or more propagation cycles and added to the mOIL population, producing a mixture of rOIL and mOIL with a selected ratio of rOIL and MOIL.

In one embodiment, rPOIL produced by the methods described herein can be added to a population of rOIL to achieve a selected ratio of rOIL to MOIL in the resulting rOIL/moIL mixture. In one embodiment, rOIL produced by the methods described herein can be added to a population of rOIL to achieve a selected ratio of rOIL to MIL in the resulting rOIL/mOIL mixture.

In one embodiment, the invention relates to a method of treating cancer comprising administering a therapeutically effective amount of TIL to a patient, wherein the ratio of rROIL to moIL in the TIL (e.g., selected ratio of rROIL to moIL) is selected from the group consisting of about 0:100, about 1 :99, approximately 5:95, approximately 10:90, approximately 15:85, approximately 20:80, approximately 25:75, approximately 30:70, approximately 35:65, approximately 40:60, approximately 45:55, approximately 50 :50, approximately 55:45, approximately 60:40, approximately 65:35, approximately 70:30, approximately 75:25, approximately 80:20, approximately 85:15, approximately 90:10, approximately 95:5, approximately 99 :1 and about 100:0 ROIL to MOIL.

In one embodiment, the ratio of rROIL to mOIL is adjusted using a selection method that can be used to increase or decrease the amount of rOIL relative to mOIL, as is known to the skilled artisan. In one embodiment, the selection method is based on the absence of depletion markers, including TIM3, LAG3, TIGIT, PD-1, and CTLA-4. In one embodiment, the selection method is based on increased expression

- 35 042041

CD69. In one embodiment, the selection method is based on higher mitochondrial mass. In one embodiment, the selection method is based on a subset of cell surface proteins. In one embodiment, the selection method is based on phenotype. In one embodiment, the selection method is based on a function.

In one embodiment, the ratio of rOIL to mOIL is adjusted by co-culturing rOIL and mOIL in the same cell culture medium until the desired ratio is reached. In one embodiment, the ratio of rOIL to mOIL is adjusted by co-cultivating rOIL and mOIL in the same cell culture medium, including adding rOIL or mOIL to the cell culture medium at different times during propagation until the desired ratio is reached. In one embodiment, the growth of rOIL is preferably stimulated in the cell culture medium by the addition of cytokines other than IL-2, including IL-4, IL-7, IL-15 and/or IL-21.

In one embodiment, the ratio of rOIL to mOIL (e.g., the selected ratio of rOIL to mOIL) produced by the methods described herein may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23% , 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,

38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54% , 55%, 56%,

57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73% , 74%, 75%,

76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%,

95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% ROIL relative to MAIL.

In one embodiment, the ratio of rOIL to mOIL (e.g., the selected ratio of rOIL to mOIL) obtained by the methods described herein may be no more than 1%, 2%, 3%, 4%, 5%, 6%, 7 %, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,

39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55% , 56%, 57%,

58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% , 75%, 76%,

77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%,

96%, 97%, 98%, 99%, 99.5% or 99.9% ROIL relative to MWL.

In one embodiment, the ratio of rOIL to mOIL (e.g., the selected ratio of rOIL to mOIL) produced by the methods described herein may be about 1%, 2%, 3%, 4%, 5%, 6%, 7% , 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24 %, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,

39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55% , 56%, 57%,

58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% , 75%, 76%,

77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%,

96%, 97%, 98%, 99%, 99.5% or 99.9% ROIL relative to MWL.

Methods for the treatment of cancer and other diseases.

rOIL, as well as combinations of rOIL and moIL described herein, can be used in a method of treating diseases in humans. In one embodiment, they are for use in the treatment of a hyperproliferative disease. In some embodiments, the hyperproliferative disease is cancer. In some embodiments, the hyperproliferative disease is a solid cancer. In some embodiments, the solid cancer is selected from the group consisting of melanoma, doubly refractory melanoma (i.e., melanoma refractory to at least two prior therapies, including chemotherapy and immune checkpoint blockade), ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, human papillomavirus cancer, head and neck cancer, kidney cancer, renal cell carcinoma and sarcoma. In some embodiments, the hyperproliferative disease is a hematologic malignancy (or liquid cancer). In some embodiments, the hematologic malignancy is selected from the group consisting of acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, and mantle cell lymphoma. rOIL and the combinations of rOIL and moIL described herein may also be used in the treatment of other diseases described herein in the following paragraphs.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising administering a therapeutically effective amount of rOIL to the patient, wherein the rOIL is obtained by a method comprising the following steps:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating the tumor tissue in a gas-permeable container with the first culture medium

- 36 042041 cells and interleukin 2 (IL-2), with the receipt of tumor remnants and migrating OIL (mOIL);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody and IL-2, in a gas-permeable container, to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), when in this case, rOIL express reduced amounts of the T-cell depletion marker compared to mOIL, while the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and the second culture medium cells additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

In one embodiment, the invention relates to a method for treating cancer in a patient in need of such treatment, comprising the following steps:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient.

In one embodiment, the invention relates to a method for treating cancer in a patient in need of such treatment, comprising the following steps:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient has one

- 37 042041 temporarily enter a therapeutically effective amount of mOIL mixed with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient.

In one embodiment, the invention relates to a method for treating cancer in a patient in need of such treatment, comprising the following steps:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL administration to the patient, wherein the cancer is selected from the group consisting of melanoma, double refractory melanoma, ovarian cancer, cervical cancer, lung cancer, cancer bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC), and triple negative breast cancer.

In one embodiment, the invention relates to a method for treating cancer in a patient in need of such treatment, comprising the following steps:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, lymphodepletion includes the stages of administration of cyclophosphamide at a dose of 60 mg/m ² /day for two days, followed by the administration of fludarabine at a dose of 25 mg/m ² /day for five days her;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient.

In some embodiments of the methods described herein, the step of removing

- 38 042041 at least a plurality of mOIL includes the removal of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13 %, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,

46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% , 63%, 64%,

65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% , 82%, 83%,

84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99, 5%, 99.9% or

100% MOIL.

The efficacy of the compounds and combinations of compounds described herein for the treatment, prevention and/or control of these diseases or disorders can be tested using various models known in the art that allow the development of guidelines for the treatment of a human disease. For example, models for determining the effectiveness of ovarian cancer treatments are described in, for example, Mullany et al., Endocrinology 2012, 153, 1585-92; and Fong et al., J. Ovarian Res. 2009, 2, 12. Models for determining the effectiveness of pancreatic cancer treatments are described in Herreros-Villanueva et al., World J. Gastroenterol. 2012, 18, 1286-1294. Models for determining the effectiveness of breast cancer treatments are described, for example, in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models for determining the effectiveness of melanoma treatments are described in, for example, Damsky et al., Pigment Cell & Melanoma Res. 2010, 23, 853-859. Models for determining the effectiveness of lung cancer treatments are described in, for example, Meuwissen et al., Genes & Development, 2005, 19, 643-664. Models for determining the effectiveness of lung cancer treatments are described, for example, in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60; and Sano, Head Neck Oncol. 2009, 1, 32.

Co-administration of IL-2.

In one embodiment, the invention relates to a method for treating cancer in a patient in need of such treatment, comprising the following steps:

(a) obtaining rOIL from a tumor resected from a patient by the method described herein;

(b) treating the patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(c) administering a therapeutically effective amount of rOIL to the patient; and (d) treating the patient with an IL-2 administration regimen starting the day after administration of rOIL to the patient.

In one embodiment, the IL-2 administration regimen is a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen comprises intravenous administration of aldesleukin, or a biological analog or variant thereof, beginning the day following administration of a therapeutically effective amount of the third population. TIL, while aldesleukin, or its biological analogue or variant, is administered at a dose of 600,000 or 720,000 IU/kg (patient's body weight) as a 15-minute bolus intravenous infusion every eight hours until tolerance, a maximum of 14 doses. After 9 days of rest, this regimen may be repeated for a further 14 doses, for a maximum of 28 doses.

In one embodiment, the IL-2 administration regimen is a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen comprises intravenous administration of aldesleukin, or a biological analog or variant thereof, beginning the day following administration of a therapeutically effective amount of the third population. TIL, while aldesleukin, or its biological analogue or variant, is administered at a dose of 0.037 mg/kg or 0.044 mg/kg (patient's body weight) as a 15-minute bolus intravenous infusion every eight hours until tolerance, a maximum of 14 doses. After 9 days of rest, this regimen may be repeated for a further 14 doses, for a maximum of 28 doses.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of the T cell depletion marker compared to mOIL, with the T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and

- 39 042041 wherein the second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof, (g) treating the patient using a non-myeloablative regimen lymphodepletion prior to the administration of rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a high dose IL-2 regimen starting the day after rOIL is administered to the patient, wherein the high dose IL-2 regimen comprises administering 600,000 or 720,000 IU/kg of aldesleukin, or a biological analog or variant thereof, in as a 15-minute bolus intravenous infusion every eight hours until tolerance.

In one embodiment, the IL-2 administration regimen is a decrescendo IL-2 administration regimen. Decrescendo regimens for IL-2 administration are described in O'Day et al., J. Clin. oncol. 1999, 17, 2752-61 and Eton et al., Cancer 2000, 88, 1703-9, the contents of which are incorporated herein by reference. In one embodiment, the decrescendo regimen for IL-2 administration comprises 18x106 IU/m ² IV for 6 hours, followed by 18x106 IU/m ² IV for 12 hours, followed by 18x106 IU/m ² IV for 24 hours. h, followed by the introduction of 4.5x106 IU/m ² intravenously for 72 h. This cycle of treatment can be repeated every 28 days, using a maximum of four cycles. In one decrescendo embodiment, the IL-2 administration regimen comprises administering 18,000,000 IU/m ² on day 1, 9,000,000 IU/m ² on day 2, and 4,500,000 IU/m ² on days 3 and 4.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain tumor remnants and migrating TILs (moILs);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a decrescendo IL-2 regimen starting the day after rOIL administration to the patient, wherein the decrescendo IL-2 regimen comprises administering 18x106 IU/m ² intravenously over 6 hours followed by 18x106 IU /m ² IV for 12 hours, followed by 18x106 IU/m ² IV for 24 hours, followed by 4.5x106 IU/m ² IV for 72 hours, repeating the cycle every 28 days and using a maximum of four cycles .

In one embodiment, the implementation of the mode of administration of IL-2 includes the introduction of pegylated IL2, including pegylated aldesleukin. In one embodiment, the IL-2 administration regimen comprises administering pegylated IL-2 every 1, 2, 4, 6, 7, 14, or 21 days at a dose of 0.10 mg/day to 50 mg/day.

In one embodiment, the invention relates to a method of treating cancer in a patient in need of such treatment, comprising:

(a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL);

(b) fragmentation of tumor tissue;

(c) treating the tumor tissue in a gas-permeable container with the first culture medium

- 40 042041 cells and interleukin 2 (IL-2), with the receipt of tumor remnants and migrating OIL (moIL);

(d) removing at least a plurality of MOILs;

(e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture;

(f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated nurse cells, OKT-3 antibody, and IL-2 in a gas-permeable container to obtain expanded remnant tumor-infiltrating lymphocytes (rTIL), wherein rOIL express reduced amounts of T cell depletion marker compared to mOIL, with T cell depletion marker selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof, and a second cell culture medium additionally contains a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, as well as combinations thereof, (g) treating a patient with a non-myeloablative lymphodepletion regimen prior to administering rOIL to the patient;

(h) administering a therapeutically effective amount of rOIL to the patient, wherein the patient is simultaneously administered a therapeutically effective amount of rOIL in admixture with rOIL; and (i) treating the patient with a pegylated IL-2 administration regimen starting the day following rOIL administration to the patient, wherein the pegylated IL-2 administration regimen comprises administering pegylated IL-2 every

1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50 mg/day.

Non-myeloablative lymphodepletion with chemotherapy.

In one embodiment, the invention relates to a method for treating cancer with a rROIL population, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of rROIL according to the invention. In some embodiments, a rROIL population may be provided with a mOIL population, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of rOIL and mOIL of the invention. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (days 27 and 26 before rOIL infusion) and fludarabine 25 mg/m ² /day for 5 days (days 27 -23 before rOIL infusion). In one embodiment, following non-myeloablative chemotherapy and rOIL infusion (Day 0), in accordance with the present invention, the patient receives an intravenous infusion of IL-2 at 720,000 IU/kg every 8 hours until physiological tolerance.

Experimental results show that lymphodepletion before adoptive transfer of tumor-specific T-lymphocytes plays a key role in improving the effectiveness of treatment by eliminating regulatory T-cells and competing elements of the immune system (cytokine scavengers). Accordingly, in some embodiments, a lymphodepletion step (sometimes also referred to as immunosuppressive conditioning) is performed on the patient prior to administration of the rOIL of the invention.

As a rule, lymphodepletion is carried out by the administration of fludarabine or cyclophosphamide (the active form is called mafosfamide) and their combinations. Such methods are described in Gassner et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley et al., J. Clin. oncol. 2008, 26, 5233-5239, and Dudley et al., J. Clin. oncol. 2005, 23, 2346-2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/ml - 10 μg/ml. In some embodiments, fludarabine is administered at a concentration of 1 μg/ml. In some embodiments, fludarabine is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, fludarabine is administered at 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day or 45 mg/kg/day. In some embodiments, fludarabine is administered over 2-7 days at a dose of 35 mg/kg/day. In some embodiments, fludarabine is administered over 4-5 days at a dose of 35 mg/kg/day. In some embodiments, fludarabine is administered over 4-5 days at a dose of 25 mg/kg/day.

In some embodiments, mafosfamide, the active form of cyclophosphamide, reaches a concentration of 0.5 μg/ml - 10 μg/ml when cyclophosphamide is administered. In some embodiments, mafosfamide, the active form of cyclophosphamide, reaches a concentration of 1 μg/ml upon administration of cyclophosphamide. In some embodiments, cyclophosphamide is administered over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the implementation of cyclophosphamide is administered at a dose of 100 mg/m ² /day, 150 mg/m ² /day, 175 mg/m ² /day, 200 mg/m ² /day, 22 5 mg/m ² /day, 250 mg/ ^m2 /day, 275 mg/ ^m2 /day or 300 mg/ ^m2 /day. In some embodiments, cyclophosphamide is administered intravenously (IV). In some embodiments, cyclophosphamide is administered over 2-7 days at a dose of 35 mg/kg/day. In some embodiments, cyclophosphamide is administered to those

- 41 042041 for 4-5 days at a dose of 250 mg/m ² /day i.v. In some embodiments, cyclophosphamide is administered for 4 days at a dose of 250 mg/m ² /day IV.

In some embodiments, lymphodepletion is performed by co-administering fludarabine and cyclophosphamide to the patient. In some embodiments, fludarabine is administered at a dose of 25 mg/m ² /day IV and cyclophosphamide is administered at a dose of 250 mg/m ² /day IV for 4 days.

In one embodiment, lymphodepletion is achieved by administering cyclophosphamide at 60 mg/m ² /day for two days followed by fludarabine at 25 mg/m ² /day for five days.

Pharmaceutical compositions, doses and dosing regimens.

In one embodiment, rOIL propagated using the methods of the invention is administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of rOIL in sterile buffer. rOIL propagated using the methods of the invention may be administered by any suitable route of administration known in the art. Preferably, rOIL is administered as a single intra-arterial or intravenous infusion, which preferably lasts about 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic routes of administration.

In one embodiment, the implementation of rOIL and mOIL, propagated using the methods of the invention, is administered to the patient in the form of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of rOIL and moIL in sterile buffer. rOIL and moIL propagated using the methods of the invention may be administered by any suitable route of administration known in the art. Preferably, rOIL and moIL are administered as a single intra-arterial or intravenous infusion, which preferably lasts about 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic routes of administration.

Any suitable dose of rOIL may be administered. Preferably, from about 2.3x10 ¹⁰ to about 13.7x1010 rROIL is administered, averaging about 7.8x10 ¹⁰ rROIL, particularly if the cancer is melanoma. In one embodiment, about 1.2 x 10 ¹⁰ to about 4.3 x 10 ¹⁰ rOIL is administered.

You can enter any suitable dose of ROIL and MOIL. Preferably, about 2.3 x 10 ¹⁰ to about 13.7 x ^{10 10} rOIL and mOIL are administered, averaging about 7.8 x ^{10 10} rOIL and mOIL, particularly if the cancer is melanoma. In one embodiment, about 1.2 x 10 ¹⁰ to about 4.3 x 10 ¹⁰ rOIL and MOIL are administered.

In some embodiments, the number of rOIL in the pharmaceutical compositions of the invention is about 1x10 ⁶ , 2x10 ⁶ , 3x10 ⁶ , 4x10 ⁶ , 5x10 ⁶ , 6x106 , 7x10 ⁶ , 8x10 ⁶ , 9x10 ⁶ , 1x10 ⁷ , 2x10 ⁷ ,

3x10 ⁷ , 4x10 ⁷ , 5x10 ⁷ , 6x10 ⁷ , 7x10 ⁷ , 8x10 ⁷ , 9x10 ⁷ , 1x10 ⁸ , 2x10 ⁸ , 3x10 ⁸ , 4x10 ⁸ , 5x10 ⁸ , 6x10 ⁸ , 7x10 ^{8 , 8}

9x10 ⁸ , 1x10 ⁹ , 2x10 ⁹ , 3x10 ⁹ , 4x10 9, 5x10 ⁹ , 6x10 ⁹ , 7x10 9, 8x10 ⁹ , ^9x109 , 1x10 ¹⁰ , 2x10 ¹⁰ , 3x10 ¹⁰ , 4x10 ¹⁰ , 5x10 ¹⁰ , 6x10 ¹⁰ , ^{7x10 10} , 8x10 ¹⁰ , 9x10 ¹⁰ , 1x10 ¹¹ , 2x10 ^p , 3x1011, 4x1011, 5x10 ¹¹ , 6x10 ^p , 7x10 ¹¹ , 8x1011, 9x10 ^p , 1x10 ¹² ,

2x10 ¹² , 3x10 ¹² , 4x10 ¹² , 5x10 ¹² , 6x10 ¹² , 7x10 ¹² , 8x10 ¹² , 9x10 ¹² , 1x10 ¹³ , 2x10 ¹³ , 3x10 ¹³ , ^{4x10 13 ,} 5x10 ¹³

7x10 ¹³ , 8x10 ¹³ and 9x10 ¹³ . In one embodiment, the number of rOIL in the pharmaceutical compositions of the invention ranges from 1x10 ⁶ to 5x10 ⁶ , from 5x10 ⁶ to 1x10 ⁷ , from 1x10 ⁷ to 5x10 ⁷ , from 5x10 ⁷ to 1x10 ⁸ , from 1x10 ⁸ to 5x10 ⁸ , from 5x10 ⁸ to 1x10 ⁹ , from 1x10 ⁹ to 5x10 ⁹ , from 5x10 ⁹ to 1x10 ¹⁰ , from 1x10 ¹⁰ to 5x10 ¹⁰ , from 5x10 ¹⁰ to 1x1011, from 5x10 ¹¹ to 1x10 ¹² , from 1x10 ¹² to ² from 1 5x10 ¹² to 1x10 ¹³ .

In some embodiments, the number of rOIL and moIL in the pharmaceutical compositions of the invention is about 1x10 ⁶ , 2x10 ⁶ , 3x10 ⁶ , 4x10 ⁶ , 5x10 ⁶ , 6x106 , 7x10 ⁶ , 8x10 ⁶ , 9x10 ⁶ , 1x10 ⁷ ,

2x10 ⁷ , 3x10 ^{7, 4x10 7} , 5x10 ⁷ , 6x10 ⁷ , 7x10 ⁷ , 8x10 ⁷ , 9x10 ⁷ , 1x10 ⁸ , 2x10 ⁸ , 3x10 ^{8 ,} 4x10 ⁸ , 5x10 ⁸ , 6x10 ^{8 , 8}

8x10 ⁸ , 9x10 ⁸ , 1x10 ⁹ , 2x10 ⁹ , 3x10 9, 4x10 ⁹ , 5x10 ⁹ , 6x10 ⁹ , 7x10 ⁹ , 8x10 ⁹ , 9x10 ⁹ , 1x10 ¹⁰ , 2x10 ¹⁰ , 3x10 ¹⁰ , 4x10 ¹⁰ , 5x10 ¹⁰ , ^{6x10 10} 7x10 ¹⁰ , 8x10 ¹⁰ , 9x10 10, 1x10 ¹¹ , 2x1011, 3x1011, 4x1011, 5x10 ¹¹ , 6x10 ^p , 7x10 ¹¹ , 8x1011, 9x10 ¹¹ , 1x10 ¹² , 2x10 ¹² , 3x10 ¹² , 4x10 ¹² , 5x10 ^{12, 6x10 12 ,} 6x10 ¹² , 7x10 ¹² , 8x10 ¹² , 9x10 ¹² , 1x10 ¹³ , 2x10 ¹³ , 3x10 13, 4x10 ¹³ , 5x10 ¹³ , ^{6x10 13 ,} 7x10 ¹³ , 8x10 ¹³ and 9x10 ¹³ . In one embodiment, the number of rOIL and mOIL in the pharmaceutical compositions of the invention ranges from 1x10 ⁶ to 5x10 ⁶ , from 5x10 ⁶ to 1x10 ⁷ , from 1x10 ⁷ to 5x10 ⁷ , from 5x10 ⁷ to 1x10 ⁸ , from 1x10 ⁸ to 5x10 ⁸ , from 5x10 ⁸ to 1x10 ⁹ , from 1x10 ⁹ to 5x10 ⁹ , from 5x10 ⁹ to 1x10 ¹⁰ , from 1x10 ¹⁰ to 5x10 ¹⁰ , from 5x10 ¹⁰ to 1x1011, from 5x10 ^p to 1x10 ¹² , from 1x10 ¹⁰ 2 to ⁵ from 5x10 ¹² to 1x10 ¹³ .

In some embodiments, the concentration of rOIL in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17 %, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009 %, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w, w/ v or v/v of the pharmaceutical composition.

- 42 042041

In some embodiments, the concentration of rOIL and mOIL in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18% , 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05 %, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0 .0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w, wt ./v or v/v from the pharmaceutical composition.

In some embodiments, the concentration of rOIL in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25 %, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50% , 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%, 11%, 10, 75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8 %, 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5, 25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50 %, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% , 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008 %, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0, 0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the concentration of rOIL and mOIL in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19 .25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16, 50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75 %, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11% , 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25 %, 8%, 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50% , 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0 .1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009 %, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005% , 0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of rOIL in the pharmaceutical compositions of the invention ranges from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02 % to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24%, from about 0.08% to about 23%, from about 0.09% to about 22%, from about 0.1% to about 21%, from about 0.2% to about 20%, from about 0.3% to about 19%, from about 0.4% to about 18%, from about 0.5% to about 17%, from about 0.6% to about 16%, from about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v/v from the pharmaceutical composition.

In some embodiments, the concentration of rOIL and mOIL in pharmaceutical compositions of the invention ranges from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0 02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25 %, from about 0.07% to about 24%, from about 0.08% to about 23%, from about 0.09% to about 22%, from about 0.1% to about 21%, from about 0, 2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16% , from about 0.7% to about 15%, from about 0.8% to about 14%, from about 0.9% to about 12%, or from about 1% to about 10% wt./wt., wt. /about or about/about from the pharmaceutical composition.

In some embodiments, the concentration of rOIL in the pharmaceutical compositions of the invention ranges from about 0.001% to about 10%, from about 0.01% to about 5%, from about 0.02% to about 4.5%, from about 0 03% to about 4%, from about 0.04% to about 3.5%, from about 0.05% to about 3%, from about 0.06% to about 2.5%, from about 0.07 % to about 2%, from about 0.08% to about 1.5%, from about 0.09% to about 1%, from about 0.1% to about 0.9% wt./wt., wt. /about or about/about from the pharmaceutical composition.

In some embodiments, the concentration of rOIL and mOIL in the pharmaceutical compositions of the invention ranges from about 0.001% to about 10%, from about 0.01% to about 5%, from about 0.02% to about 4.5%, from about 0.03% to about 4%, from about 0.04% to about 3.5%, from about 0.05% to about 3%, from about 0.06% to about 2.5%, from about 0 07% to about 2%, from about 0.08% to about 1.5%, from about 0.09% to about 1%, from about 0.1% to about 0.9% w/w, wt./about or about/about from the pharmaceutical composition.

- 43 042041

In some embodiments, the amount of rOIL in the pharmaceutical compositions of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g 0.007 g g, 0.0003 g, 0.0002 g or 0.0001 g.

In some embodiments, the amount of rOIL and moIL in the pharmaceutical compositions of the invention is equal to, or less than, 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g 0.007 g 0.006 g 0.005 g 0.004 g 0.003 g 0.002 g 0.001 g 0.0009 g 0.0008 g 0.0007 g 0.0006 g 0.0005 g 0 .0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

In some embodiments, the amount of rOIL in the pharmaceutical compositions of the invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 0.0009 g 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g , 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g , 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8, 5 g, 9 g, 9.5 g or 10 g.

In some embodiments, the amount of rOIL and moIL in the pharmaceutical compositions of the invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0 .0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0, 55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g , 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.

rOIL in pharmaceutical compositions according to embodiments of the invention are effective over a wide dosage range. The exact dosage will depend on the route of administration, the form in which the compound is administered, the sex and age of the subject being treated, the body weight of the subject being treated, and the preference and experience of the attending physician. Clinically accepted doses of rOIL can also be used if needed.

The amounts of pharmaceutical compositions administered in the methods described herein, for example, doses of rOIL, will depend on the human or mammal being treated, the severity of the disease or condition, the route of administration, the nature of the active pharmaceutical ingredients, and the judgment of the attending physician.

rOIL and moIL in pharmaceutical compositions according to embodiments of the invention are effective over a wide range of doses. The exact dosage will depend on the route of administration, the form in which the compound is administered, the sex and age of the subject being treated, the body weight of the subject being treated, and the preference and experience of the attending physician. Clinically accepted doses of rOIL and mOIL can also be used if needed. The amounts of pharmaceutical compositions administered in the methods described herein, for example, doses of rOIL and moIL, will depend on the human or mammal being treated, the severity of the disease or condition, the route of administration, the nature of the active pharmaceutical ingredients, and the judgment of the attending physician.

In some embodiments, the implementation of ROIL can be administered in a single dose. Such administration may be by injection, eg intravenous injection. In some embodiments, the implementation of ROIL can be administered in multiple doses. Dosing can be performed once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be done once a month, once every two weeks, once a week, or every other day. The introduction of ROIL can be continued as long as necessary.

In some embodiments, the implementation of rPOIL and moIL can be administered in a single dose. Such administration may be by injection, eg intravenous injection. In some embodiments, the implementation of the rPOIL and moIL can be administered in multiple doses. Dosing can be performed once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be done once a month, once every two weeks, once a week, or every other day. The introduction of ROIL and MOIL can be continued as long as necessary.

In some embodiments, the effective dose of ROIL is about 1x10 ⁶ , 2x10 ⁶ , 3x10 ⁶ , 4x10 ⁶ , 5x10 ⁶ , 6x10 ⁶ , 7x10 ⁶ , 8x10 ⁶ , 9x10 ⁶ , 1x10 ⁷ , 2x10 ⁷ , 3x10 ⁷ , 5x10 ^{7 .} , 6x10 ⁷ , 7x10 ⁷ , 8x10 ⁷ ,

9x10 ^{7 ,} 1x10 ⁸ , 2x10 ⁸ , 3x10 ⁸ , 4x10 ⁸ , 5x10 8, 6x10 ⁸ , 7x10 ⁸ , 8x10 ⁸ , 9x10 ⁸ , 1x10 ⁹ , 2x10 ⁹ , 3x10 ⁹ , 4x10 ⁹ , ⁹

6x10 ⁹ , 7x10 ⁹ , 8x10 ⁹ , 9x10 ⁹ , 1x10 ¹⁰ , 2x10 10, 3x10 ¹⁰ , 4x10 ¹⁰ , 5x10 ¹⁰ , 6x10 ¹⁰ , 7x10 ¹⁰ , 8x10 ¹⁰ , 9x10 ¹⁰ , 1x1011, 2x10 ¹¹ , 3x10111, ^4x1011 , 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x10 ¹² , 2x10 ¹² , 3x10 ¹² , 4x10 ¹² , 5x10 ¹² , 6x10 ¹² ,

7x10 ¹² , 8x10 ¹² , 9x10 ¹² , 1x10 ¹³ , 2x10 ¹³ , 3x10 ¹³ , 4x10 ¹³ , 5x10 ¹³ , 6x10 ¹³ , 7x10 ¹³ , 8x10 ¹³ and 9x10 ¹³ . In some

- 44 042041 embodiments, the effective dose of rOIL is in the range from 1x10 ⁶ to 5x10 ⁶ , from 5x10 ⁶ to 1x10 ⁷ , from 1x10 ⁷ to 5x10 ⁷ , from 5x10 ⁷ to 1x10 ⁸ , from 1x10 ⁸ to 5x10 ⁸ , from 5x10 ⁸ to 1x10 ⁹ , from 1x10 ⁹ to 5x10 ⁹ , from 5x10 ⁹ to 1x10 ¹⁰ , from 1x10 ¹⁰ to 5x10 ¹⁰ , from 5x10 ¹⁰ to 1x1011, from 5x1011 to 1x10 12, from 1x10 12 to 5x10 ¹² and from 5x10 ¹² to 1x10 ¹² and from 5x10 ^{10 .}

In some embodiments, the effective dose of rROIL and moIL is about 1x10 ⁶ , 2x10 ⁶ , 3x10 ⁶ , 4x10 ⁶ , 5x10 ⁶ , 6x10 ⁶ , 7x10 ⁶ , 8x10 ⁶ , 9x10 ⁶ , 1x10 ⁷ , 2x10 ⁷ , 3x10 ^{7 .} 5x10 ⁷ , 6x10 ⁷ ,

7x10 ^{7 ,} 8x10 ^{7, 9x10 7} , 1x10 ⁸ , 2x10 ⁸ , 3x10 ⁸ , 4x10 ⁸ , 5x10 ⁸ , 6x10 ⁸ , 7x10 ⁸ , 8x10 ⁸ , 9x10 ⁸ , 1x10 ⁹ , 2x10 ⁹ , ⁹

4x10 ⁹ , 5x10 ⁹ , 6x10 ⁹ , 7x10 ⁹ , 8x10 ⁹ , 9x10 ⁹ , 1x10 ¹⁰ , 2x10 10, 3x10 ¹⁰ , 4x10 ¹⁰ , 5x10 ¹⁰ , 6x10 ¹⁰ , 7x10 ¹⁰ , 8x10 ¹⁰ , 9x10 ¹⁰ , 1x1011, 2x10 ^{p ,} 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x10 12, 2x10 ¹² , 3x10 ¹² , 4x10 ¹² , 5x10 ¹² , 6x10 ¹² , 7x10 ¹² , 8x10 ¹² , 9x10 ¹² , 1x10 13, 2x10 13, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, ^{3 , 3} , 310 ¹³ , 3, 3, 3, 3, 310 ¹³ 4x10 ¹³ , 5x10 ¹³ , 6x10 ¹³ , 7x10 ¹³ , 8x10 ¹³ and 9x10 ¹³ . In some embodiments, the effective dose of rOIL and moIL is in the range of 1x10 ⁶ to 5x10 ⁶ , 5x10 ⁶ to 1x10 ⁷ , 1x10 ⁷ to 5x10 ⁷ , 5x10 ⁷ to 1x10 ⁸ , 1x10 ⁸ to 5x10 ⁸ , 5x10 ⁸ up to 1x10 ⁹ , from 1x10 ⁹ to 5x10 ⁹ , from 5x10 ⁹ to 1x10 ¹⁰ , from 1x10 ¹⁰ to 5x10 ¹⁰ , from 5x10 ¹⁰ to 1x1011, from 5x1011 to 1x10 ¹² , from 1x10 ¹² to ^{1x10 12} and from ⁵ .

In some embodiments, an effective dose of rOIL is in the range of about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg /kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg /kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/ kg to about 0.8 mg/kg, from about 0.7 mg/kg to about 0.75 mg/kg, from about 0.7 mg/kg to about 2.15 mg/kg, from about 0.85 mg /kg to about 2 mg/kg, from about 1 mg/kg to about 1.85 mg/kg, from about 1.15 mg/kg to about 1.7 mg/kg, from about 1.3 mg/kg mg up to about 1.6 mg/kg, from about 1.35 mg/kg to about 1.5 mg/kg, from about 2.15 mg/kg to about 3.6 mg/kg, from about 2.3 mg/kg to about 3.4 mg/kg, from about 2.4 mg/kg to about 3.3 mg/kg, from about 2.6 mg/kg to about 3.15 mg/kg, from about 2.7 mg/kg to about 3 mg/kg, from about 2.8 mg/kg to about 3 mg/kg, or from about 2.8-5 mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dose of rOIL and moIL is in the range of from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0. 3 mg/kg to about 3.2 mg/kg, from about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0 .3 mg to about 2.15 mg/kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0, 3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to about 0.8 mg/kg, from about 0.7 mg/kg to about 0.75 mg/kg, from about 0.7 mg/kg to about 2.15 mg/kg, from about 0, 85 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 1.85 mg/kg, from about 1.15 mg/kg to about 1.7 mg/kg, from about 1.3 mg/kg kg mg to about 1.6 mg/kg, from about 1.35 mg/kg to about 1.5 mg/kg, from about 2.15 mg/kg to about 3.6 mg/kg , from about 2.3 mg/kg to about 3.4 mg/kg, from about 2.4 mg/kg to about 3.3 mg/kg, from about 2.6 mg/kg to about 3.15 mg/ kg, from about 2.7 mg/kg to about 3 mg/kg, from about 2.8 mg/kg to about 3 mg/kg, or from about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dose of rOIL is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg up to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, from about 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

In some embodiments, an effective dose of rOIL and moIL is in the range of from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg , from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 to

- 45 042041 approximately 207 mg.

An effective amount of rOIL and/or mOIL may be administered in either single or multiple doses by any of the accepted routes of administration for agents having similar properties, including bloodstream infusion, tumor infusion, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneously, topically, intranasally by administration, by transplantation or inhalation.

Examples

Further, embodiments of the present invention are described with reference to the following examples. These examples are provided for the purpose of illustration only, and the invention described herein should in no way be considered limited to these examples, but rather should be considered to cover any and all variations that become apparent based on the ideas set forth herein.

Example 1. Propagation of rOIL from tumor hydrolysates.

Tumor remnants were digested according to the following exemplary procedure.

This procedure describes the digestion of a fresh human tumor sample to a single viable cell suspension to obtain and isolate tumor-infiltrating lymphocytes, and DNase-collagenase-hyaluronidase (DHA) mixture methods (as described herein) or digestion protocols using Human Tumor Dissociation MACS Kit (TDK) (Miltenyi Biotech, Inc., San Diego, CA, USA).

The preparation of the working culture medium CM1+IL-2 is carried out as follows. Place 500 ml of RPMI 1640, 200 mM L-glutamine and 100 ml of human AB serum in a 37° C. water bath and equilibrate for at least 30 minutes. This mixture is transferred from the water bath to the biological safety cabinet, where the stock solution of βME and the stock solution of gentamicin at a concentration of 50 mg/ml are transferred from the refrigerator 1000x. Withdraw 50 ml from RPMI 1640 medium, add: 50 ml human AB serum, 5 ml 200 mM L-glutamine, 500 μl 1000X β-ME and 500 μl 50 mg/ml gentamicin. Complete the preparation of medium 1 by adding 500 μl of 6000 U/ml reconstituted human rhIL-2 (CellGenix, Inc., Portsmouth, NH, USA).

A 10x stock solution of DHA is prepared using the following procedure, shown schematically in FIG. 1. First, calculate the volume required to dilute each enzyme to the desired working concentration of the solution. For example, dilute 150,000 U (international units) of deoxyribonuclease in 15 ml to obtain a working solution with a concentration of 10,000 U / ml. The resulting working solution is divided into aliquots. Dilute the lyophilized enzymes in a pre-calculated amount of sterile Hank's Balanced Salt Solution (HBSS, Sigma H6648, Sigma-Aldrich Co., St. Louis, MO, USA, or equivalent) at room temperature. Collect any residual powder from the walls of the vials and from the protective foil. Mix by pipetting up and down several times and shake to ensure complete dissolution. To sterile HBSS add 100,000 U of DNase (deoxyribonuclease I from bovine pancreas, Sigma D5025 or equivalent), 1 g of collagenase (Sigma C5138 from Clostridium histolyticum or equivalent), and 100 mg of hyaluronidase (type V from sheep testis, Sigma H6254 or equivalent) to a final volume of 100 ml, yielding 10x a three-enzyme stock solution for digestion of human tumors. The resulting working solutions of enzymes are divided into aliquots containing 10,000 U/ml of DNase, 10 mg/ml of collagenase and 1 mg/ml of hyaluronidase. 10x stock solution of DHA in a final volume of 100 ml has the following concentrations: DNase I 1000 U/ml, collagenase 10 mg/ml and hyaluronidase 1 mg/ml. For tumor digestion, the 10x DHA stock solution is diluted to 1x DHA in HBSS.

In parallel, to compare DHA digestion with MACS TDK digestion, prepare the reagents included in the MACS TDK kit according to the manufacturer's instructions, if necessary. Aliquots that have been stored at -20° C. are thawed at room temperature.

The tumor for splitting can be prepared as follows. The tumor is removed from its primary and secondary packaging, the container is weighed, the mass is recorded and transferred to the biological safety cabinet. The tumor is cut into fragments, or the tumor is morcellated. Several fragments are selected for use in the digestion protocol, and additional fragments are saved for histological studies and DNA extraction, if necessary.

An exemplary procedure for splitting a tumor using DKG is shown schematically in FIG. 2 and includes the following steps. The 10x DHA stock solution should be diluted to 1x the working concentration for digestion. Calculate the total volume required to split the tumor, which is approximately 5 ml of solution per cm ² of the tumor. Dilute the DHA solution to 1x working solution by adding 1 part DHA to 9 parts HBSS. Transfer the tumor fragments to a 50 ml Falcon conical tube in a volume of HBSS calculated as described above. A calculated amount of 10x DHA is added, capped and optionally sealed. Transfer to a MACS tube rotator (Miltenyi Biotech, Inc., San Diego, CA, USA) in a 37° C. and humidified incubator.

- 46 042041 atmosphere with 5% CO2 for constant rotation for 1-2 hours. Alternatively, tumor fragments can be split at room temperature overnight, also with constant rotation. Attach a 0.70 µm strainer to a sterile Falcon conical tube. Remove the tumor hydrolyzate from the incubator and transfer the entire hydrolyzate to the filter using a pipette. Use the plunger end of a sterile syringe to push any solids through the filter. The tube is closed, it contains rOIL obtained by splitting DHA. Cells can be washed to remove the digestion cocktail, counted, and resuspended in PBR expansion medium as described elsewhere in this document.

If a pre-PBR step is desired in order to generate a mTIL for comparison with rTIL (as in the following examples), pre-PBR G-REX vials are seeded using the rTIL derived from DHA digestion. Label the correct number of G-REX 10 vials and add the hydrolysate. CM1+IL-2 is added until a final volume of 40 ml is reached. Place the vials in a 5% CO2 incubator in a humidified atmosphere at 37°C. Cell viability can be counted and determined on a Nexcelom Cellometer K2 instrument using 40 µl of sample and 40 µl of acridine orange and propidium iodide double stain solution (AOPI) and counted in duplicate for each hydrolysate or condition, diluting as needed . Samples should be mixed thoroughly to avoid clumping, and AOPI should be pipetted rapidly before each sample is analyzed to ensure viability results are not affected by the cytotoxic effect of propidium iodide.

The DHA procedure described above has been found to be surprisingly superior to the MACS TDK Enzyme Digestion Blend and the corresponding procedure for several reasons. Conducted three independent experiments using a mixture of MACS TDK. The first experiment was carried out using a melanoma tumor, while a significant decrease in the population of CD4+/CD8+ cells in rROIL was observed by flow cytometry in the case of using the MACS system. This effect was not observed for rOIL upon DHA cleavage, suggesting that the MACS cleavage procedure may adversely affect the expression of surface markers. In the second experiment, an estrogen receptor positive (ER ⁺ )/progesterone receptor positive (PR+) breast tumor was used, and digestion with MACS resulted in detritus in a 24-well G-REX plate, while digestion with DKG resulted in a clear material, indicating poor digestion with the MACS enzyme cocktail. Finally, a second digestion of another ER+/PR+ breast tumor using an enzymatic digestion mixture and the MACS TDK procedure resulted in poor yield and poor viability of rOIL.

Example 2 Phenotype characterization of rOIL from tumor hydrolysates.

At the pre-PBR stage, resident TILs migrate as mTILs and proliferate. The duration of the pre-PBR used to obtain mOIL for comparison with rOIL can vary between 11-21 days depending on cell growth. Residual tumor fragments (residues) are usually discarded, and expanded mTILs are subjected to PBR with irradiated PBMCs as feeders, anti-CD3 and IL-2. Viable TIL remaining in tumor remnants (rTIL) after pre-PBR was examined after the cleavage described in Example 1 above to evaluate their function and phenotype in comparison to mTIL.

Cell populations from tumor remnants and pre-PBR suspension (ie, expanded cell population) for melanoma, head and neck, breast, kidney, pancreas, lung, and colorectal tumors (n=17) were evaluated and compared. Interestingly, rROIL invariably differs phenotypically from mOIL as determined in this study and demonstrated based on the differential expression of various markers, including LAG3, TIM-3, PD-1, CD69, CD45RO, CD27, CD56, CD57, and HLA-DR. PBR of tumor remnants and pre-PBR populations resulted in comparable reproduction rates, however, similar to the results in the pre-PBR stage, the phenotypic profiles differed in the two populations for LAG3, TIM-3, HLA-DR, and CD28.

rOIL and mOIL derived from melanoma, breast, kidney, pancreas, lung and colorectal cancer tumors (n=9) were evaluated and compared. Tumor rOIL invariably differed phenotypically from mOIL as determined by differential expression of various markers (Table 3 and Fig. 3).

- 47 042041

Table 3 Summary of phenotype characterization results for nine tumors

Marker expression LAG3 (CD8 ⁺ /CD4 ⁺ ) CIF TIM3 (CD8 ⁺ /CD4 ⁺ ) CIF PD-1 CD8V CD4 ⁺ % CD69 CD8 ⁺ /CD4 ⁺ CIF CD154 (CD8 ⁺ /CD4 ⁺ ) CIF CD28 (CD8 ⁺ /CD4 ⁺ ) CIF CD57 (CD8 ⁺ / CD4 ⁺ ) % CD56 % MOIL 507/ 144 2832/ 1756 36.95/ 47 1320/ 1543 1498/ 3751 1163/ 5036 18.76/ 19.6 5.615 ROIL 209/ 106 877/ 742 42.8/48 3437/ 223.4 1034/ 1167 458.3/ 2795 9.16/ 8.5 1.027 *P-values (CD8/ CD4) 0.05/ 0.21 0.05/ 0.01 0.38/ 0.89 0.11/ 0.001 0.55/ 0.01 0.05/ 0.11 0.05/ 0.06 0.05

*P-values refer to the differences between rTIL and mTIL using an unpaired Student's t-test.

The fundamental differences between rOIL and mOIL were: increased expression of CD69 (7-fold difference in the mean fluorescence intensity (MIF) in CD4 ⁺ ) (p<0.0001), reduced expression of LAG3 (2-fold difference in MIF in CD8 ⁺ T cells ) (p<0.05) and TIM3 expression (3- and 2-fold difference in CIF in CD8 ⁺ and CD4 ⁺ T cells, respectively) (p<0.05/0.01), decreased expression of CD154 (3-fold difference SIF in CD4 ⁺ T cells) (p<0.01) and decreased expression of CD56 (5%) (p<0.05). Surprisingly, PBR rROIL and mOIL resulted in comparable reproduction rates, similar to those of pre-PBR, as described in Example 4. The phenotypic profile of rROIL was maintained after PBR, maintaining individual levels of LAG3, TIM3, and CD28 expression, as described in Example 4. More Moreover, since CD57 is a terminal differentiation-associated receptor, the results suggest that rTILs are less terminally differentiated than mTILs (ie, less likely to die).

The results shown in FIG. Ziv tab. 3 show that mOIL and rOIL have clear and consistent differences in the expression of phenotypic markers in histologically distinct tumors. Of particular note, there was a decrease in the expression of so-called depletion markers (LAG3 and TIM3) in rOIL. Interestingly, PD-1 expression was similar in mOIL and rOIL. In addition, there was an increase in CD69 expression in rOIL compared to mOIL, although KLRG1 expression was similar in the two populations (data not shown). These results provide additional evidence in favor of the fact that rROIL, apparently, are not terminally differentiated, but are phenotypically similar to tissue-resident effector memory T cells.

Taken together, these results revealed significant differences in the biology of cell populations that remain in the tumor or proliferate and migrate out of the tumor, as well as signals associated with migration and retention.

Example 3 Functional characterization of rOIL from tumor hydrolysates.

T-cell dysfunction is directly related to the loss of mitochondrial function. Sharping et al., Immunity 2016, 45, 374-88. In addition, reprogramming of T cells to stimulate mitochondrial biogenesis can lead to increased persistence and function of T cells within the tumor. Thus, T cells with more active metabolism are critical for developing an effective immune response to the tumor. To evaluate the functionality of mOIL and rOIL, we compared mOIL and rOIL in terms of metabolic capacity using the Mito tracker and 2-(N-(7nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose ( 2-NBDG). 2-NBDG can be used to measure glucose uptake, but it is not possible to accurately determine the underlying metabolic process; that is, complete oxidation in the mitochondria, or only glycolysis and lactate formation. Mitotracker dye (ThermoFisher Scientific, Inc., Waltham, MA, USA) can be used to measure mitochondrial mass. Comparison of mOIL and rOIL using this approach demonstrated an increase in glucose uptake in rOIL, as shown in FIG. 4. This result is unexpected since rOILs directly released from the tumor are expected to be more glycolytic; however, when the mitochondrial mass of rOIL was assessed, they had a slightly elevated level of Mitotracker compared to mOIL. These results indicate that rROILs were more metabolically active than mOILs, although they were expected to be less active, and suggest that rOILs may have a greater ability to mount an immune response to a tumor than mOILs.

To further evaluate their functional capacity, rOILs were stimulated overnight with brefeldin A and anti-COP, anti-CO28, and anti-CO137 antibody coated beads (DYNABEADS, cat. no. 11162D, commercially available from ThermoFisher Scientific, Inc., Waltham, MA, USA), and measured the level of IFN-γ. Cells were harvested, stained for intracellular IFN-γ after permea

-48042041 bilization and evaluated by flow cytometry. The results are shown in FIG. 5.

Cells were also stimulated with phorbol-12-myristate-13-acetate (PMA) and ionomycin to assess the ability of TIL to produce a cytokine. The results are also shown in Fig. 5. Granzyme B, TNF-α and IL-17A levels were also assessed. There was no significant difference between rROIL and mOIL for IL-17A and no difference for TNFa or granzyme B (data not shown). Surprisingly, under conditions of stimulation with anti-CD3/anti-CD28/anti-CD137 beads and PMA/ionomycin, a slightly elevated level of IFN-γ was observed in the subgroup of CD4+ cells (but not in CD8+ T cells) (n=3) in rOIL in compared with MOIL. These data indicate that rOIL are functionally competent cells and show signs of greater functional competence than mOIL.

Example 4 Comparison of PBR for rOIL and MOIL from tumor hydrolysates.

For mOIL and rOIL, a Rapid Propagation Protocol (RRP) was performed with irradiated PBMC as nurse cells, anti-CD3 antibody (OCTZ) and IL-2 for 14 days. Viability and cell count were evaluated in duplicate for 3 independent tumors (n=3). Phenotypic expression was assessed by flow cytometry. Successful initiation in mini-PBR experiments was observed for both rOIL and MOIL. In PBR, the results of expansion of TIL with anti-CD3 antibody and nurse cells were similar (Fig. 6A), although in the case of rTIL, unexpectedly, the number of cells was slightly increased (p<0.08) compared to mTIL. Similar to the observations before PBR (see example 2), mOIL and rROIL obtained after PBR were phenotypically different. Many of the phenotypic differences observed in the pre-PBR phase were maintained during the PBR process, such as decreased LAG3 and TIM3 expression in rROIL (Fig. 6B).

Additional properties of mOIL and rOIL can be compared based on the results of (1) deep TCR sequencing, (2) co-culture proliferation (co-culture of rOIL/mOIL with cytokine mixtures) and additional functional assays, (3) transcription profiling assays (e.g., with using the NCOUNTER system from NanoString Technologies). TCR sequencing allows assessment of the clonality and/or diversity of the TCR repertoire, including the Vb repertoire. Telomere length can also be assessed to compare rOIL with mOIL.

EXAMPLE 5 Treatment of human diseases with rOIL and combinations of rOIL and MOIL.

The rOILs of the invention may be used in the treatment of the cancers described herein. The general scheme of the technological process for the propagation of rOIL from the patient's tumor and the treatment of the patient is shown in Fig. 7. As shown, this process allows you to individually adjust the ratio of rOIL and mOIL in the TIL preparation administered by infusion to the patient. The ratio of rOIL to mOIL can be selected using an affinity assay or other cell sorting assay known to those skilled in the art based on the differential expression of CD69 and/or T-cell depletion markers in rOIL and mOIL.

In FIG. 8, a timeline of an exemplary process of obtaining rROIL from a patient's tumor, expanding rROIL from tumor remnants after pre-PBR using the PBR stage, performing lymphodepletion and infusion of rROIL to the patient is shown in combination with a parallel process for rTIL.

Example 6. Study to evaluate the repertoire of Ve in the MOIL and ROIL.

Differences in Ve T-cell receptor repertoires between mOIL and rOIL were assessed in terms of diversity and frequency of occurrence.

In this study, 6 pre-PBR pairs of mTIL/rTIL were collected for the following cancers: ovarian cancer; kidney cancer (n=2) and breast cancer (TNBC n=2, ER+PR+ n=1). Cell pellets were sent on dry ice to iRepertoire (Huntsville, AL, USA) for RNA extraction and Ve sequencing.

The results of this study are shown in Fig. 9-10 and 14-16. In particular, in FIG. 9 and 10 show the diversity score and % of total CDR3, respectively. In addition, three clonotype plots showing the major total 50 CDR3s are shown in FIG. 14-16 for ovarian carcinoma, renal carcinoma, and triple negative breast carcinoma, respectively.

Surprisingly, the diversity of the TCRve repertoire is more pronounced in rROIL than in mOIL (Fig. 9). Approximately 30-50% of all CDR3s in mTIL and rTIL are shared (FIG. 10), indicating that a large percentage of all CDR3s are differentially expressed in these two populations. However, of the total CDR3s, mainly the core 50 clones are shared between the two populations, indicating that mTIL and rTIL have clones with similar antigen specificity (see FIGS. 14-16). In addition, the frequency of occurrence of the main 50 clones varies, again suggesting that mTIL and rTIL are surprisingly different T-cell populations.

Example 7 Co-Cultivation Proliferation Assay.

The mDIL and rOIL were assessed to determine if rOIL could change the proliferation status of the mOIL (or vice versa) when co-cultivated.

In this study, 5 pre-PBR mOIL/rOIL pairs were collected for the following cancers: kidney cancer, triple-negative breast cancer (TNBC), melanoma, lung and colorectal cancer.

-

Claims (5)

cancer. rOIL was isolated from tumor remnants by 60-minute enzymatic digestion at 37°C. MILs were stained with CellTrace™ yellow and rILs were stained with CellTrace™ red to track two different populations independently. 1e6 mOIL, 5e5 mOIL+5e5 rOIL, and 1e6 rOIL were cultured for 4 days at 37°C with IL-2 ± and OCTH (anti-CD3 antibody) and proliferation was assessed by flow cytometry. The results of this study are shown in Fig. 11. In FIG. The 11 mOIL from either CD4+ or CD8+ populations in all five tumors showed an increase in proliferative capacity when co-cultured with rROIL and anti-CD3 antibody, as indicated by the shift (or dye dilution) observed for CellTrace™ dye compared to one MOIL. Red color corresponds to mOIL and blue color corresponds to mOIL when co-cultivated with rOIL. Example 8 Co-Cultivation Proliferation Assay. The mOIL and rOIL were evaluated to identify similarities and/or differences in the gene expression profile of rOIL and mOIL. This study used nCounter® NanoString technology, which uses a color barcode multiplexed onto mRNA to provide a numerical measure of gene expression. Purified RNA (RNeasy, Qiagen) was hybridized from six paired samples of mOIL and rOIL with the code set of the nCounter® Immunology V2 panel for 16 h in a thermal cycler. The code sets consist of a mixture of capture and reporter probes that form multiplexes with the target RNA through a 22-bp pathway. interactions during thermal cycling. Samples were applied to a 12-well SPRINT cartridge and analyzed in an nCounter® SPRINT device. Count data was exported in a custom RCC format and matched to an RLF file that maps gene names to probe IDs. Normalization and analysis were performed using the nSolver 3.0 program (NanoString Technologies, Inc.). The results of this study are shown in Fig. 12 and 13. As shown in FIG. 12 and 13, the gene expression profiles differ significantly when comparing mDIL and rROIL (see heat map in FIG. 12). There are several genes that are significantly upregulated or downregulated in rTIL compared to mTIL (FIG. 13). CLAIM 1. A method for obtaining remnant tumor-infiltrating lymphocytes (rTIL) for adoptive T-cell therapy, including: (a) obtaining from the patient tumor tissue containing tumor-infiltrating lymphocytes (TIL); (b) fragmenting tumor tissue to obtain fragmented tumor tissue; (c) treating the fragmented tumor tissue in a gas-permeable container with a first cell culture medium and interleukin 2 (IL-2) to obtain migrating TILs (mTILs) and tumor remnants containing remnant TILs (rTILs); (d) removing at least a plurality of MWLs; (e) enzymatic digestion of tumor remnants into tumor remnant cells using a digestion mixture, wherein the digestion mixture contains one or more of deoxyribonuclease, collagenase, and hyaluronidase; and (f) expanding tumor remnant cells using a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody and IL-2, in a gas-permeable container to obtain propagated rOILs, wherein rOILs express reduced amounts The T-cell depletion marker versus mOIL and the T-cell depletion marker is selected from the group consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations thereof. 2. The method of claim 1, wherein the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, and bone marrow or acute myeloid leukemia tumor tissue. 3. The method according to claim 1 or 2, wherein the irradiated nurse cells are irradiated allogeneic peripheral blood mononuclear cells. 4. The method according to any one of claims 1 to 3, characterized in that IL-2 is present in the second cell culture medium at an initial concentration of about 3000 IU/ml and the OKT-3 antibody is present in the second cell culture medium at an initial concentration of about 30 ng/ml. 5. The method according to any one of claims 1 to 4, wherein the amount of at least one T cell depletion marker in CD8+ and CD4+ T cells in rROIL is reduced by at least 10% compared to mOIL. -