IMPROVED PROCESS FOR CULTURING TUMOR-INFILTRATING LYMPHOCYTES FOR THERAPEUTIC USE
20230018646 · 2023-01-19
Inventors
- Ulrik Cordes (Charlottenlund, DK)
- Christina Friese (Copenhagen Ø, DK)
- Nikolaj Kirketerp-Møller (Copenhagen N, DK)
- Christina Heeke (Copenhagen NV, DK)
Cpc classification
A61K35/17
HUMAN NECESSITIES
C07K16/2809
CHEMISTRY; METALLURGY
C12N5/0638
CHEMISTRY; METALLURGY
C07K2317/70
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention is targeted towards reinvigorating exhausted Tumor Infiltrating Lymphocytes (TILs) in vitro by co-culturing excised TIL containing tumor fragments with checkpoint inhibitors, stimulating the TILs with other interleukins known to revert T cell exhaustion), and/or inhibiting the effect of regulatory T cells secreted factors (such as IL-10) thereby creating a favorable tumor microenvironment (TME) where exhausted T-cells can expand faster and to higher numbers than currently established TIL expansion protocols.
Claims
1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) culturing autologous T cells by obtaining a first population of tumor infiltrating lymphocytes (TILs) from a tumor resected from a mammal; (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more Tumor Microenvironment (TME) stimulators to produce a second population of TILs, wherein the one or more TME stimulators are selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, urelumab and utomilumab; and (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, an OKT3 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is a therapeutic population.
2-20. (canceled)
21. The method of claim 1, further comprising administering to the mammal the therapeutic population of T cells.
22. The method of claim 21, wherein the therapeutic population of T cells is administered to the mammal after a nonmyeloablative lymphodepleting chemotherapy is administered to said mammal.
23. The method of claim 1, wherein the one or more TME stimulators comprise pembrolizumab.
24. The method of claim 1, wherein the one or more TME stimulators comprise ipilimumab.
25. The method of claim 1, wherein the one or more TME stimulators comprise urelumab.
26. The method of claim 1, wherein the concentration of the TME stimulators is 0.1 μg/mL to 300 μg/mL.
27. The method of claim 1, wherein steps (a) through (b) are performed within a period of about 7 days to about 28 days.
28. The method of claim 1, wherein step (c) is performed within a period of about 7 days to about 21 days.
29. The method of claim 22, wherein the mammal has breast cancer, renal cell cancer, bladder cancer, melanoma, cervical cancer, gastric cancer, colorectal cancer, lung cancer, head and neck cancer, ovarian cancer, Hodgkin lymphoma, pancreatic cancer, liver cancer, or a sarcoma.
30. The method of claim 1, wherein step (c) produces 1×10.sup.7 to 1×10.sup.12 cells.
31. The method of claim 1, wherein the TME stimulators are added together or 1, 2, 3, 4, 5, 6 or 7 days apart.
32. The method of claim 1, wherein the antigen-presenting cells (APCs) are selected from the group consisting of allogeneic feeder cells, PBMCs, and artificial antigen-presenting feeder cells.
33. The method of claim 1, further comprising processing of the resected tumor into multiple tumor fragments.
34. The method of claim 33, wherein the fragments have a size of 1 to 10 mm.sup.3.
35. The method of claim 1, further comprising formulating a composition to include at least 1×10.sup.8 to 5×10.sup.11 cells from the therapeutic population.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1—One or More Immune Modulators Reinvigorate Exhausted T-Cells Ex Vivo
[0172] Step 1.1: Effect of single immune modulators
[0173] a. Resected TIL-containing tumor tissue from various patients is dissected 30-50 tumor fragments per cm.sup.3 tissue and transferred into 24-well cell culture plates. 2 mL of cell culture medium containing 6000 IU/mL IL-2 and either none (baseline) or a low, mid, or high concentration of each of the immune-modulators listed in Table 1.
[0174] b. The cell culture plates are incubated at 37° C., 5% CO.sub.2 where cell culture medium is changed frequently. Cell cultures should not increase 1.5×10.sup.6 cells per well and should be split into new wells.
[0175] c. After a number of days, cells are harvested, cells are counted to determine amount, and analyzed by flowcytometry viability and phenotype
[0176] Step 1.2 Effect of PD1 co-blockade and/or blockade/stimulation
[0177] a. As PD1 blockade is clearly identified as key pathway to reinvigorate exhausted T-cells, a new experiment including IL-2, optimal concentration of PD1 and the remaining immune modulators listed in Table 1, and the specific combinations with IL-2 listed in Tables 2-21 is setup and performed as above.
[0178] Step 1.3 Effect of CTLA4 co-blockade and/or blockade/stimulation
[0179] a. As CTLA4 blockade is clearly identified as key pathways to reinvigorate exhausted T-cells, a new experiment including IL-2, optimal concentration of PD1 and the remaining immune modulators listed in Table 1, and the specific combinations with IL-2 listed in Tables 22-41 is setup and performed as above.
[0180] Step 2: possibly further fine tune concentration of immune modulators
[0181] Step 3: Understanding of combinatorial effects
[0182] a. A new experiment is setup in a similar way using the best performing immune modulators at the optimal concentration from the first experiment in a combinatorial approach to determine possible synergistic effects by adding several immune modulators simultaneously with the same readout as described above.
[0183] b. The above is run in several iterations eventually revealing combinations with a shortened time, a higher expansion rate and/or improved phenotype
[0184] Step 4: validation of optimal combination in patients versus standard TIL manufacturing protocol
[0185] a. Initial TIL culture expansion is run in parallel in a number of TIL therapy eligible patients to validate the effects on a real patient setting
Example 2—“Young” Tumor-Infiltrating Lymphocytes (TILs) with TME Stimulators
[0186] This example demonstrates the manufacturing process for generation of “young” tumor-infiltrating lymphocytes (TILs) with TME stimulators.
[0187] Tumor material of various histologies was obtained from commercial sources. Fourteen independent patient tumors or tumor digests were obtained (3 ovarian cancer, 3 metastatic melanoma, 3 head and neck cancer, 2 lung cancer, 2 colorectal cancer, 1 cervical cancer; Table 42). Cryopreserved or fresh tumor material was shipped to Cbio A/S in sterile freezing or transport medium. The tumor material was handled in a laminar flow hood to maintain sterile conditions.
[0188] TILs were prepared as previously described in detail in the standard TIL manufacturing protocol (Friese, C. et al., CTLA-4 blockade boosts the expansion of tumor-reactive CD8+ tumor-infiltrating lymphocytes in ovarian cancer. Sci Rep 10, 3914 (2020); Jin, J. et al., Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes in Gas-permeable Flasks to Numbers Needed for Patient Treatment, Journal of Immunotherapy, 35—Issue 3 (2012)). Briefly, TIL cultures were set up using tumor fragments or tumor digest. The tumors were divided into 1-3 mm.sup.3 fragments and placed into a G-Rex 6-well plate (WilsonWolf; 5 fragments per well) with 10 ml complete medium (CM) including 6000 IU/mL IL-2 (6000 IU/ml, Clinigen) only (baseline) or in combination with TME stimulators in low, medium, high, or very high concentrations of each of the PD-1/PD-L1 antagonists (group A), CTLA-4 antagonist (group B), LAG-3 antagonist (group C), TIGIT antagonist (group D), 4-1BB agonist together with anti-CD3 (group J) and CD28 agonist (group K) listed in Table 43, in a humidified 37° C. incubator with 5% CO.sub.2. CM was added every 4-5 days until a total volume of 40 ml was reached. Subsequently, half of the medium was removed and replaced with CM and IL-2 every 4-5 days. TIL cultures from tumor digest were initiated by culturing single-cell suspensions (5×10.sup.5/ml) obtained by overnight enzymatic digestion in flat-bottom 96-well plates in 250 μL CM and IL-2 (6000 IU/ml, Clinigen) in a humidified 37° C. incubator with 5% CO.sub.2. Half of the medium was removed and replaced with CM every 2-3 days.
[0189] CM consisted of RPMI1640 with GlutaMAX, 25 mM HEPES pH 7.2 (Gibco), 10% heat-inactivated human AB serum (Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 1.25 μg/ml Fungizone (Bristol-Myers Squibb).
[0190] This example demonstrates the generation of “young” tumor-infiltrating lymphocytes (TILs) with TME stimulators having an age of 10-28 days.
Example 3—Phenotype Analysis of “Young” TIL Cultures with TME Stimulators
[0191] This example demonstrates the phenotype analysis of “young” TIL cultures with TME stimulators performed as described in example 2.
[0192] When cultures designated for young TIL generation were harvested, their phenotype was assessed by flow cytometry.
[0193] TIL phenotype was determined by assessment of the viability and the CD3+ subset, the CD3+CD8+ subset, the CD3+CD4+ subset and the NK subset in both frequency and absolute cell count. Additionally, differentiation status, activation status, the expression of exhaustion markers and senescence of TILs were assessed. Flow cytometry was conducted using the following markers:
TIL Panel 1: CD3, CD4, CD8, CD45RA, CD56, CCR7, FVS780, BTLA, LAG-3, PD-1, TIM-3
TIL Panel 2: CD3, CD4, CD8, CD45RA, CD56, CCR7, FVS780, CD-27, CD28, CD57, CD69
[0194] Briefly, about 0.5×10.sup.6 young TILs per panel were washed and then incubated with titrated antibodies (BD Biosciences, Table 45) and Brilliant Stain Buffer (BD Biosciences) for 30 min at 4° C. Cells were washed twice with PBS and directly analyzed by flow cytometry (CytoFLEX, Beckman Coulter).
[0195] This example demonstrates the phenotype analysis of “young” TIL cultures with TME stimulators.
Example 4—TME-Stimulators Increased the Success Rate of TIL Expansion Ex Vivo
[0196] This example demonstrated that the success rate of TIL expansion ex vivo was increased, when TME stimulators were added to the culture medium when TIL cultures were initiated performed as described in example 2.
[0197] The success rate of TIL expansion was investigated by determining cell number per tumor fragment when harvesting TIL cultures. 5×10.sup.4 TILs/fragment was set as a threshold for successful TIL culture.
[0198] Determining the success rate of TIL expansion demonstrated that the success rates of TIL cultures were increased when TME stimulators were added to the “young” TIL cultures (avelumab 68%, relatlimab 70%, tiragolumab 76.5%, pembrolizumab 82.1%, ipilimumab 88.5%, theralizumab 90.9%, nivolumab 92.3%, and urelumab/OKT3 100%) compared to baseline cultures 61.5%, illustrated in
[0199] Grouping the TME stimulators according to their targets, the example also demonstrated that adding inhibitors from group C (70%, LAG-3 inhibitors), group A (76.3%, including inhibitors of PD1 and its ligand PD-L1), group D (76.5%, TIGIT inhibitors), group B (88.5%, inhibitors of CTLA-4 and ligand), group K (90.9%, CD28 agonist) and group J (96.3%, 4-1BB agonist together with anti-CD3) also increased the success rate of TIL cultures compared to baseline cultures 61.5%, illustrated in
[0200] This example demonstrates that the success rate of TIL expansion ex vivo was increased, when TME stimulators were added to the culture medium when TIL cultures were initiated as compared to the standard TIL manufacturing protocol.
Example 5—Checkpoint Blockade or Co-Stimulation Increased the TIL Yield and Reduced Culture Time of TILs
[0201] This example demonstrated that the TIL yield was increased and the culture time of TILs was reduced, when TME stimulators were added to the culture medium when TIL cultures were initiated, performed as described in example 2.
[0202] The TIL yield and the culture time of TILs were investigated when harvesting TIL cultures. This analysis demonstrated that the TIL yield increased, and the culture time decreased, when TME stimulators were added to the culture medium when TIL cultures were initiated compared to TILs cultured in IL-2 alone (
[0203] This example demonstrated that the TIL yield was increased and the culture time of TILs was reduced, when TME stimulators were added to the culture medium, when TIL cultures were initiated as compared to the standard TIL manufacturing protocol.
Example 6—Different Concentrations of TME Stimulators Induced TIL Expansion Ex Vivo
[0204] This example performed as described in example 2 demonstrated that the TIL yield was increased, when TME stimulators were added to the culture medium in different concentrations, when TIL cultures from various tumor types were initiated.
[0205] The TIL yield expansion was investigated when harvesting TIL cultures. The first analysis in
[0206] In
[0207] In
[0208] This example 6 demonstrates how different concentrations of TME stimulators influenced TIL growth in a dose dependent manner.
Example 7—TME-Stimulators as a Whole and from Different Groupings Enhances TIL Growth
[0209] Example 7 illustrated in
[0210] Breaking the TME stimulators up into the underlying subgroupings, the example also demonstrated that adding inhibitors from group A (including inhibitors of PD1 and its ligand PD-L1), group B (inhibitors of CTLA-4), group J (4-1BB agonist together with anti-CD3) and group K (CD28 agonist) also significantly increased TIL growth. Although not significant in this example there was a tendency that adding TME stimulators from groups C (LAG-3 inhibitors) and D (TIGIT inhibitors) also improved TIL growth.
[0211] In
[0212] Summing up this example, adding TME stimulators to the young TIL processing step provided a novel improvement over the existing standard TIL protocol that allowed for a faster TIL therapy manufacturing protocol.
Example 8: TIL Stimulator Agonists, Antagonists, T-Cell Depleting, T-Cell Reinvigorating, and Stimulators of CD28 Family Origin Significantly Increased TIL Growth Rates
[0213] Example 8 illustrated in
[0214] Breaking the TME stimulators up into the subgroupings according to their functionality, the example also demonstrated that both T-cell antagonists, agonists, reinvigorating, depleting and members of the CD28 family of receptors all had a significant effect on TIL growth. Whereas a representative amount of different TME antagonists exemplified here including 2 different PD-1 inhibitors (pembrolizumab and nivolumab), 2 different PD-L1 inhibitors (avelumab and durvalumab), a CTLA-4 inhibitor (ipilimumab), a TIGIT inhibitor (tiragolumab), showed a 3-5-fold increase over the standard young TIL process, TME agonists here exemplified by stimulators targeting 4-1BB (urelumab together with anti-CD3 (OKT3)) and CD28 (theralizumab) seemed to further speed up growth.
[0215] Further dividing the TME antagonists into whether they allow for depletion of regulatory T cells through antibody-dependent cellular toxicity (ADCC) such as ipilimumab and tiragolumab or only allow for T-cell reinvigoration through checkpoint inhibition also both demonstrated a significant improvement in TIL growth rates over standard young TIL protocol conditions as illustrated in
[0216] Looking specifically on TME stimulators originating from the CD28 family of proteins exemplified here by inhibitors of PD-1, CTLA-4 and CD28 or their ligands originating from the B7-family of proteins exemplified here by two different inhibitors of PD-L1, it was demonstrated that they also significantly enhanced TIL growth as compared to the standard young TIL protocol. Although not shown here, other receptors expressed on T cells originating from the CD28 protein family such as BTLA and ICOS could have a similar growth stimulating effect for young TIL cultures.
[0217] In
[0218] Summing up this example, adding TME stimulators that were either antagonizing receptors expressed on T cells (or their ligands), agonizing receptors expressed on T-cells, reinvigorating exhausted T-cells (or their ligands), depleting regulatory T-cells and/or targeting receptors expressed on T cells originating from the CD28 family (or their ligands originating from the B7 family of proteins) to the young TIL processing step provided a novel improvement over the existing standard TIL protocol that allowed for a faster TIL therapy manufacturing protocol.
Example 9—TME Stimulator Antagonists Targeting Receptors Expressed on T Cells or their Ligands Demonstrated a Similar TIL Growth Stimulating Effect
[0219] Example 9 illustrated in
[0220] In
Example 10—TME Stimulators from Different Manufacturers Demonstrated a Similar TIL Growth Stimulating Effect
[0221] Example 10 illustrated in
[0222] Two PD-1 inhibitors (pembrolizumab, Merck Sharp Dome and nivolumab, Bristol Myers Squibb) and two PD-L1 inhibitors (avelumab, Merck KgaA and durvalumab, AstraZeneca) were tested in this example. All the different TME stimulators showed significant improvement over the standard young TIL protocol in the ability to accelerate TIL growth. There was a tendency that the four different antibodies showed similar effects as compared to group A as well as between the individual inhibitors.
[0223] This was an example of how TME stimulators from various manufacturers in general were interchangeable and could be used to optimize the young TIL manufacturing process.
[0224] In
Example 11—Combinations of TME Stimulators Further Enhanced Young TIL Growth
[0225] This example performed as described in example 2 demonstrated that the TIL yield was increased compared to the standard TIL manufacturing protocol, when TME stimulators in various combinations were added to the culture medium, when TIL cultures from various tumor types were initiated.
[0226] The TIL yield was investigated when harvesting TIL cultures. The first analysis illustrated in
[0227] In another analysis illustrated in
[0228] In
[0229] In
Example 12—TME-Stimulators Alone or in Combination Increased the Success Rate of TIL Expansion Ex Vivo
[0230] This example demonstrated that the success rate of TIL expansion ex vivo was increased, when TME stimulators were added to the culture medium during TIL culture initiation performed as described in example 2.
[0231] The success rate of TIL expansion was investigated by determining cell number per tumor fragment when harvesting TIL cultures. 5×10.sup.4 TILs/fragment was set as a threshold for successful TIL culture.
[0232] Determining the success rate of TIL expansion demonstrated that the success rates of TIL cultures were increased when TME stimulators from different groups were added to the “young” TIL cultures either alone or in combinations (group A 76.3%, group B 88.5%, group J 100.0%, group A+B 83.3%, group B+J 100.0%, group A+J 100.0%, and group A+B+J triple combo 96.0%) compared to baseline cultures 61.5%, illustrated in
[0233] This example demonstrated that the success rate of TIL expansion ex vivo was increased, when TME stimulators alone or in combinations were added to the culture medium when TIL cultures were initiated compared to the standard TIL manufacturing protocol.
Example 13—TME-Stimulators as a Whole, from Different Groupings and in Combinations Enhance the Frequency and the Number of T Cells
[0234] Example 13 illustrated in
[0235] Breaking the TME stimulators up into the underlying subgroupings, the example also demonstrated that adding inhibitors from group A (including inhibitors of PD1 and its ligand PD-L1) or group B (inhibitors of CTLA-4 and ligand), also significantly increased the frequency of T cells compared to IL-2 alone (
[0236] Breaking the TME stimulators up into the underlying subgroupings, the example also demonstrated that adding inhibitors from group A (including inhibitors of PD1 and its ligand PD-L1), group B (inhibitors of CTLA-4 and ligand), group K (CD28 agonists and group J (4-1 BB agonist together with anti-CD3) also significantly increased the number of viable T cells per tumor fragment compared to IL-2 alone (
[0237] Summing up this example, adding TME stimulators to the young TIL manufacturing step provided a novel improvement over the existing standard TIL protocol that allowed for generation of a TIL product containing an increased frequency of T cells and, an increased number of viable T cells.
Example 14—TME-Stimulators as a Whole, from Different Groupings and in Combinations Maintain the Frequency of Effector-Memory T Cells
[0238] Example 14 illustrated in
Example 15—TME-Stimulators in Combination Enhance the Frequency and Number of CD8+ T Cells
[0239] Example 15 illustrated in
[0240] The example also demonstrated that adding a combination of TME stimulators from group J (4-1BB agonist together with anti-CD3), group A (including inhibitors of PD1 and its ligand PD-L1) and group B (inhibitors of CTLA-4) to the standard young TIL showed a tendency to enhance CD8+ T cells growth compared to adding TME stimulators from group A, group B or group J alone (
[0241] An increased frequency of CD8+ T cells in the TIL infusion product has previously been associated with beneficial clinical outcome of TIL therapy in patients with metastatic melanoma (Radvanyi, L. G. et al., Specific lymphocyte subsets predict response to adoptive cell therapy using expanded autologous tumor-infiltrating lymphocytes in metastatic melanoma patients. Clin. Cancer Res. 18, 6758-6770 (2012)). Thus, methods increasing CD8+ T-cell frequency could induce clinical responses in cancer patients that do not respond to TILs manufactured using the standard TIL protocol.
[0242] Summing up this example, adding TME stimulators alone and in combinations to the young TIL processing step provided a novel improvement over the existing standard TIL manufacturing protocol that allowed for generation of a TIL product containing an increased frequency of CD8+ T cells.
Example 16—TME-Stimulators in Combination Reduce the Frequency of CD4+ T Cells
[0243] Example 16 illustrated in
[0244] Summing up this example, adding TME stimulators to the young TIL processing step provided a novel improvement over the existing standard TIL manufacturing protocol that allowed for generation of a TIL product containing a reduced frequency of CD4+ T cells.
Example 17—TME-Stimulators from Different Groups Reduce the Frequency of NK Cells
[0245] Example 17 illustrated in
[0246] Furthermore, the example demonstrated that adding TME stimulators from group B showed a tendency to a reduced NK cell frequency compared to group J (4-1BB agonist together with anti-CD3) and group K (CD28 agonists).
[0247] Summing up this example, adding TME stimulators to the young TIL processing step provided a novel improvement over the existing standard TIL manufacturing protocol that allowed for generation of a TIL product containing a reduced frequency of NK cells.
Example 18—TME-Stimulators from Different Groups or in Combination Affect the Frequency of NK and T Cells in Total and CD8+ T Cells Specifically
[0248] Example 18 illustrated in
[0249] Furthermore, the example demonstrated that adding urelumab/OKT3 (group J) and ipilimumab (group B) reduced the CD8+ T cell frequency compared to urelumab/OKT3 alone, urelumab/OKT3 and pembrolizumab (group A) and urelumab/OKT3, ipilimumab and pembrolizumab (
[0250] Therefore, the example demonstrated that adding urelumab/OKT3 (group J), ipilimumab (group B) and pembrolizumab (group A) could be favorable compared to urelumab/OKT3 and ipilimumab only.
[0251] Summing up this example, adding TME stimulators to the young TIL processing step provided a novel improvement over the existing standard TIL manufacturing protocol that allowed for generation of a TIL product containing a reduced frequency of NK cells but an increased frequency of CD8+ T cells.
Example 19—TME-Stimulators in Combination Added with Time Delay Enhance the Frequency of CD3+ and CD8+ T Cells and Reduce the Frequency of NK Cells and CD4+ T Cells
[0252] Example 19 illustrated in
[0253] Summing up this example, adding TME stimulators with a time delay to the young TIL processing step provided a novel improvement over the existing standard TIL manufacturing protocol that allowed for generation of a TIL product containing an increased frequency of T cells in total, CD8+ T cells and a reduced frequency of NK cells and CD4+ T cells.
Example 20—TME-Stimulators Alone or in Combination Enhance the Frequency of LAG3+ T Cells
[0254] Example 20 illustrated in
[0255] Furthermore, the example demonstrated that adding a combination of TME stimulators in group A (including inhibitors of PD1 and its ligand PD-L1), group B (inhibitors of CTLA-4), or group J (4-1BB agonist together with anti-CD3) in a time delay as described in example 19 compared to adding TME stimulators in combination from group A, B and J showed a tendency to increased reinvigoration of tumor-specific CD8+ T cells resulting in an increased frequency of CD8+ LAG-3+ T cells (
[0256] Summing up this example, adding TME stimulators to the young TIL processing step provided a novel improvement over the existing standard TIL manufacturing protocol that allowed for generation of a TIL product containing an increased frequency of tumor-specific LAG-3+ T cells. As LAG-3 is known to be a marker for T-cell exhaustion and that T cells that have a higher affinity to tumor antigens generally have an increased tendency to get exhausted, expansion of CD8+ LAG-3+ T cell clones can lead to a higher proportion of tumor-reactive T-cells possibly leading to an improved clinical outcome of this novel approach to TIL therapy.
Example 21—TME-Stimulators Increased the Frequency of CD8 T-Cells with a Younger Phenotype being CD28+
[0257] Example 21 illustrated in
[0258] Furthermore, the example demonstrated that adding a combination of TME stimulators from group A (including inhibitors of PD1 and its ligand PD-L1), group B (inhibitors of CTLA-4), or group J (4-1BB agonist together with anti-CD3) compared to adding TME stimulators from group A or group B alone showed a tendency to increased expansion of T cells with a younger phenotype resulting in an increased frequency of CD8+CD28+ T cells (
[0259] Furthermore, the example demonstrates that adding a combination of TME stimulators from group A (including inhibitors of PD1 and its ligand PD-L1), group B (inhibitors of CTLA-4) and group J (4-1BB agonist together with anti-CD3) with time delay as described in example 19 compared to adding TME stimulators from group A or group B alone or a combination of TME stimulators from group A, group B and group J without time delay showed a tendency to increased expansion of T cells with a younger phenotype resulting in an increased frequency of CD8+CD28+ T cells (
[0260] Summing up this example, adding TME stimulators to the young TIL processing step provided a novel improvement over the existing standard TIL manufacturing protocol that allowed for generation of a TIL product containing an increased frequency of CD8+ T cells with a younger phenotype expressing CD28.
Items
[0261] 1. A method for promoting regression of a cancer in a mammal by expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: [0262] (a) culturing autologous T cells by obtaining a first population of TILs from a tumor resected from a mammal, [0263] (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulators to produce a second population of TILs; [0264] (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is a therapeutic population; and [0265] (d) after administering nonmyeloablative lymphodepleting chemotherapy, administering to the mammal the therapeutic population of T cells, wherein the T cells administered to the mammal, whereupon the regression of the cancer in the mammal is promoted.
[0266] 2. A method for treating a subject with cancer comprising administering expanded tumor infiltrating lymphocytes (TILs) comprising: [0267] (a) culturing autologous T cells by obtaining a first population of TILs from a tumor resected from a mammal, [0268] (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulators to produce a second population of TILs; [0269] (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is a therapeutic population; and [0270] (d) after administering nonmyeloablative lymphodepleting chemotherapy, administering to the mammal the therapeutic population of T cells, wherein the T cells administered to the mammal, whereupon the regression of the cancer in the mammal is promoted.
[0271] 3. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: [0272] (a) culturing autologous T cells by obtaining a first population of TILs from a tumor resected from a mammal [0273] (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulators to produce a second population of TILs; and [0274] (c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is a therapeutic population.
[0275] 4. The method of any of the preceding items, wherein the one or more TME stimulators are selected from the groups consisting of: [0276] (x) one or more substances that are capable of antagonizing and/or inhibiting receptors expressed on T-cells (or their ligands) known to cause T-cell downregulation, deactivation and/or exhaustion, [0277] (y) one or more substances that are capable of agonizing and/or stimulating receptors expressed on T-cells known to cause T-cell upregulation, activation, and/or reinvigoration, [0278] (z) one or more substances that are capable of antagonizing and/or inhibiting soluble molecules and cytokines and their receptors known to cause T-cell downregulation, deactivation, and/or exhaustion, and [0279] (v) one or more substances that are capable of downregulating and/or depleting regulatory T-cells thereby favoring ex-vivo effector T-cell expansion, and [0280] (w) specific combinations of one or more substances from the groups (x), (y), (z) and/or (v) as listed in Tables 2-41.
[0281] 5. The method of any of the preceding items, wherein the one or more TME stimulators is/are one or more checkpoint inhibitors or inhibitors of their ligands such as anti-PD1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-LAG3, anti-AZAR, anti-B7-H3, anti B7-H4, anti-BTLA, anti-IDO, anti-HVEM, anti-IDO, anti-TDO, anti-KIR, anti-NOX2, anti-TIM3, anti-galectin-9, anti-VISTA, anti-SIGLEC7/9, and wherein the one or more checkpoint inhibitors or inhibitors of their ligands optionally also are added to the second expansion.
[0282] 6. The method of any of the preceding items, wherein the substances that are capable of antagonizing and/or inhibiting receptors expressed on T-cells (or their ligands) known to cause T-cell downregulation, deactivation and/or exhaustion are selected from the groups consisting of: [0283] A: substances that act through the PD-1 receptor on T-cells, [0284] B: substances that act through the CTLA-4 receptor on T-cells, [0285] C: substances that act through the LAG-3 receptor on T-cells, [0286] D: substances that act through the TIGIT/CD226 receptor on T-cells, [0287] E: substances that act through the KIR receptor on T-cells, [0288] F: substances that act through the TIM-3 receptor on T-cells, [0289] G: substances that act through the BTLA receptor on T-cells, and [0290] H: substances that act through the A2aR receptor on T-cells.
[0291] 7. The method of item 6, wherein the substance of group A is selected from one or more from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, and durvalumab.
[0292] 8. The method of item 6-7, wherein the substance of group B is selected from one or more from the group consisting of ipilimumab and tremelimumab.
[0293] 9. The method of item 6-8, wherein the substance of group C is selected from one or more from the group consisting of relatlimab, eftilagimo alpha, and sym022.
[0294] 10. The method of item 6-9, wherein the substance of group D is tiragolumab.
[0295] 11. The method of item 6-10, wherein the substance of group E is lirilumab.
[0296] 12. The method of item 6-11, wherein the substance of group F is sym023.
[0297] 13. The method of item 6-12, wherein the substance of group G is 40E4 and PJ196.
[0298] 14. The method of any of the preceding items, wherein the substances that are capable of agonizing and/or stimulating receptors expressed on T-cells known to cause T-cell upregulation, activation, and/or reinvigoration are selected from the groups consisting of: [0299] I: substances that act through the OX40/CD134 receptor on T-cells, [0300] J: substances that act through the 4-1BB/CD137 receptor on T-cells, [0301] K: substances that act through the CD28 receptor on T-cells, [0302] L: substances that act through the ICOS receptor on T-cells, [0303] M: substances that act through the GITR receptor on T-cells, [0304] N: substances that act through the CD40L receptor on T-cells, and [0305] 0: substances that act through the CD27 receptor on T-cells.
[0306] 15. The method of item 14, wherein the substance of group J is selected from one or more from the group consisting of urelumab and utomilumab.
[0307] 16. The method of item 14, wherein the substance of group K is theraluzimab.
[0308] 17. The method of item 14, wherein the substance of group O is valilumab.
[0309] 18. The method of any of the preceding items, wherein the substances that are capable of antagonizing and/or inhibiting soluble molecules and cytokines and their receptors known to cause T-cell downregulation, deactivation, and/or exhaustion are selected from the groups consisting of: [0310] P: substances that act through the IDO1/2 receptor on T-cells, [0311] Q: substances that act through the TGFβ receptor on T-cells, [0312] R: substances that act through the IL-10 receptor on T-cells, and [0313] S: substances that act through the IL-35 receptor on T-cells.
[0314] 19. The method of item 14, wherein the substance of group P is epacedostat.
[0315] 20. The method of item 14, wherein the substance of group Q is linrodostat.
[0316] 21. The method of item 14, wherein the substance of group R is galunisertib.
[0317] 22. The method of any of the preceding items, wherein the substances that are capable of downregulating and/or depleting regulatory T-cells thereby favoring ex-vivo effector T-cell expansion are selected from the groups consisting of: [0318] T: cyclophosphamides, [0319] U: TKIs, [0320] V: substances that act through aCD25, and [0321] X: IL2/Diphteria toxin fusions.
[0322] 23. The method of item 20, wherein the substance of group U is sunitinib.
[0323] 24. The method of item 20, wherein the substance of group V is selected from one or more from the group consisting of sorafenib, imatinib and daclizumab.
[0324] 25. The method of item 20, wherein the substance of group X is dinileukin diftitox.
[0325] 26. The method of any of the preceding items, wherein the concentration of substance in is 0.1 μg/mL to 300 μg/mL, such as 1 μg/mL to 100 μg/mL, such as 10 μg/mL to 100 μg/mL, such as 1 μg/mL to 10 μg/mL.
[0326] 27. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat a cancer type selected from the groups consisting of: [0327] 1: solid tumors, [0328] 2: ICI naïve tumors, [0329] 3: MSI-H tumors, [0330] 4: Hematological tumors, and [0331] 5: Hyper-mutated tumors.
[0332] 28. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat a cancer type selected from the groups consisting of breast cancer, renal cell cancer, bladder cancer, melanoma, cervical cancer, gastric cancer, colorectal cancer, lung cancer, head and neck cancer, ovarian cancer, Hodgkin lymphoma, pancreatic cancer, liver cancer, and sarcomas.
[0333] 29. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat a breast cancer.
[0334] 30. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat renal cell cancer.
[0335] 31. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat bladder cancer.
[0336] 32. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat melanoma.
[0337] 33. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat cervical cancer.
[0338] 34. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat gastric cancer.
[0339] 35. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat colorectal cancer.
[0340] 36. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat lung cancer.
[0341] 37. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat head and neck cancer.
[0342] 38. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat ovarian cancer.
[0343] 39. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat Hodgkin lymphoma.
[0344] 40. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat pancreatic cancer.
[0345] 41. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat liver cancer.
[0346] 42. The method of any of the preceding items, wherein the therapeutic population of T cells is used to treat sarcomas.
[0347] 43. The method according to any of the preceding items, wherein steps (a) through (c) or (d) are performed within a period of about 20 days to about 45 days.
[0348] 44. The method according to any of the preceding items, wherein steps (a) through (c) or (d) are performed within a period of about 20 days to about 40 days.
[0349] 45. The method according to any of the preceding items, wherein steps (a) through (c) or (d) are performed within a period of about 25 days to about 40 days.
[0350] 46. The method according to any of the preceding items, wherein steps (a) through (c) or (d) are performed within a period of about 30 days to about 40 days.
[0351] 47. The method according to any of the preceding items, wherein steps (a) through (b) are performed within a period of about 10 days to about 28 days.
[0352] 48. The method according to any of the preceding items, wherein steps (a) through (b) are performed within a period of about 10 days to about 20 days.
[0353] 49. The method according to any of the preceding items, wherein step (c) is performed within a period of about 12 days to about 18 days.
[0354] 50. The method according to any of the preceding items, wherein step (c) is performed within a period of about 10 days to about 28 days.
[0355] 51. The method according to any of the preceding items, wherein step (c) is performed within a period of about 10 days to about 20 days.
[0356] 52. The method according to any of the preceding items, wherein step (c) is performed within a period of about 12 days to about 18 days.
[0357] 53. The method according to any of the preceding items, wherein step (b) results in 1×10.sup.6 to 1×10.sup.7 cells, such as 2×10.sup.6 to 5×10.sup.6 cells.
[0358] 54. The method according to any of the preceding items, wherein step (c) results in 1×10.sup.7 to 1×10.sup.12 cells, such as 1×10.sup.8 to 5×10.sup.9 cells, such as 1×10.sup.9 to 5×10.sup.9 cells, such as 1×10.sup.8 to 5×10.sup.10 cells, such as 1×10.sup.9 to 5×10.sup.11 cells.
[0359] 55. The method according to any of the preceding items, wherein the APCs are artificial APCs (aAPCs) or allogeneic feeder cells.
[0360] 56. The method according to any of the preceding items, wherein the therapeutic population of TILs are infused into a patient.
[0361] 57. The method according to any of the preceding items, wherein the cells are removed from the cell culture and cryopreserved in a storage medium prior to performing step (c).
[0362] 58. The method according to any of the preceding items, further comprising the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable fragment antibody fused with at least one endodomain of a T-cell signaling molecule.
[0363] 59. The method according to any of the preceding items, wherein step (c) further comprises a step of removing the cells from the cell culture medium.
[0364] 60. The method according to any of the preceding items, wherein step (a) further comprises processing of the resected tumor into multiple tumor fragments, such as 4 to 50 fragments, such as 20 to 30 fragments.
[0365] 61. The method according to item 60, wherein the fragments have a size of about 5 to 50 mm.sup.3, 20 to 50 mm.sup.3.
[0366] 62. The method according to any of the preceding items, wherein the mammal is a human.
[0367] 63. The method according to any of the preceding items, wherein the cell culture medium is provided in a container selected from the group consisting of a G-Rex container and a Xuri cellbag.
[0368] 64. The method according to any of the preceding items, wherein the anti-CD3 antibody is OKT3.
[0369] 65. A population of tumor infiltrating lymphocytes (TILs) obtainable by a method of any of the previous items.
[0370] 66. Expanded tumor infiltrating lymphocytes (TILs) for use in treating a subject with cancer, the treatment comprising the steps of: [0371] culturing autologous T cells by obtaining a first population of TILs from a tumor resected from a mammal [0372] performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulators to produce a second population of TILs; [0373] performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is a therapeutic population; and [0374] after administering nonmyeloablative lymphodepleting chemotherapy, administering to the mammal the therapeutic population of T cells, wherein the T cells administered to the mammal, whereupon the regression of the cancer in the mammal is promoted.
[0375] 67. A population of tumor infiltrating lymphocytes (TILs) obtainable by a method comprising:
culturing autologous T cells by obtaining a first population of TILs from a tumor resected from a mammal
performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more TME stimulators to produce a second population of TILs; and
performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is a therapeutic population.
[0376] 68. A therapeutic population of TILs comprising IL-2 and one or more TME stimulators.
[0377] 69. A therapeutic population of TILs comprising IL-2, one or more TME stimulators, IL-2, anti-CD3 antibody, and antigen presenting cells (APCs).