METHOD FOR EXPANSION AND DIFFERENTIATION OF T LYMPHOCYTES AND NK CELLS FOR ADOPTIVE TRANSFER THERAPIES

Abstract

The present invention describes a method for obtaining lymphoid cells having a desired phenotype for adoptive transfer therapies useful for the treatment of cancer. Especially, this invention is related to strategies for inducing preferential signaling through the intermediate affinity IL-2 receptor in order to expand the cells with a desired central memory phenotype. The method of the present invention is useful for obtaining tumor-infiltrating lymphocytes, TCR or chimeric antigen receptor engineered T cells for the treatment of cancer.

Claims

1. A method for the differentiation and expansion of T lymphocytes and NK cells with a central memory phenotype useful in adoptive cell transfer therapies comprising three stages: i) extraction of lymphoid cells from a subject, ii) in vitro expansion and differentiation of T lymphocytes and NK cells, which have been further genetically engineered, while inducing a preferential signaling through the intermediate affinity IL-2 receptor (IL2Rβγ), and iii) transference of the activated cells to the subject with cancer.

2. The method according to claim 1 wherein the strategy for inducing preferential signaling through the intermediate affinity IL2 receptor (IL2Rβγ) while expanding the lymphoid cells is selected from the group comprising of: culturing the lymphoid cells with a soluble IL-2 mutein, which preferentially interact and signal though the intermediate affinity IL2R (IL2Rβγ), genetically engineering of the lymphoid cells to secrete IL2 muteins, which preferentially interact and signal though the intermediate affinity IL2R, culturing the T lymphocytes and the NK cells in the presence of native IL-2 and a pharmacologic agent which block IL2-IL2Rα interaction, culturing the T lymphocytes and the NK cells in the presence of native IL-2 and genetically engineering of such cells to secrete a soluble protein, which block the IL2-IL2Rα interaction, culturing the T lymphocytes and the NK cells in the presence of native IL-2 and genetically engineering of such cells to decrease the expression of the IL2Rα receptor subunit at the cell surface, culturing the T lymphocytes and the NK cells in the presence of native IL-2 and genetically engineering of such cells to increase the expression of the intermediate affinity IL-2 receptor at the cell surface.

3. The method according to claim 2 wherein the IL-2 mutein is selected from the group comprising of: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7.

4. The method according to claim 2 wherein the pharmacologic agent, which block the IL2-IL2Rα interaction, is selected from the group comprising of: an antibody or antibody fragment, which bind either to IL2Rα or to IL2 a peptide or a chemically defined small molecule, which bind either to IL2Rα or to IL2. a soluble form of the IL2Rα or a modified variant of it.

5. The method according to claim 1 wherein the stage (iii) could be done in the same donor or in a different one.

6. The method according to claim 2 wherein the signaling by the intermediate affinity IL-2 receptor in stage ii could occur in different moments of cells culturing or said signaling could be maintained in vitro and in vivo.

7. The method according to claim 1 to prolong the persistence of the transferred cells and a higher in vitro and in vivo antitumor effect.

8. The method according to claim 1 in combination with other cytokines selected from the group comprising: IL12, IL17, IL15 and IL21.

9. The method according to claim 1 wherein lymphoid cells subject to the method could be any of the followings: a mixture of T cells; purified CD4 or CD8 T cells; NK cells; NKT cells.

10. The method according to claim 1 wherein the T lymphocytes and NK cells could be obtained in stage i either from peripheral blood mononuclear cells, tumor draining lymph nodes or tumor infiltrates.

11. The method according to claim 1 wherein lymphoid cells in the methods could have been further engineered to: express a CAR; express a TCR of desired specificity; express other receptors of interest in the cell membrane, secrete different cytokines or soluble proteins of interest.

12.-15. (canceled)

16. A method for enrichment and expansion ex vivo of lymphocytes with a central memory phenotype comprising: a. Obtaining a population of lymphocytes from a subject. b. Expanding the lymphocytes culturing the cells in conditions that activate the β/γ dimeric IL-2 receptor.

17. The method of claim 16, wherein the step (b) is performed by using at least one IL2 mutein.

18. The method of claim 16, wherein the step (b) is performed by regulating the expression of at least one subunit of the IL-2 receptor.

19. The method of claim 18, wherein the subunit of the IL-2 receptor is the alpha (CD25), beta, or gamma subunit.

20. The method of claim 16, wherein the step (b) is performed by inhibiting the interaction of IL-2 receptor with its cognate protein.

21. The method of claim 20, wherein the inhibition of the interaction of IL-2 receptor with its cognate protein occurs by downregulating the alpha subunit of IL-2 receptor, or by upregulating the beta and gamma subunits.

22. The method of claim 20, wherein the inhibition of the interaction of IL-2 receptor with its cognate protein occurs by incubating the cells with an anti-CD25 antibody.

23. The method of claim 16, wherein the step (b) is performed by transducing lymphocytes with a nucleic acid coding for the expression and secretion of mutant IL-2 sequence.

24. (canceled)

25. A method of treating a cancer in a subject in need therefore, the method comprising administering lymphocytes with a central memory phenotype comprising: a. Obtaining a population of lymphocytes from a subject; b. Expand the lymphocytes population by activating in said lymphocytes the β/γ dimeric IL-2 receptor; and c. Administering a therapeutically effective dosage of lymphocytes from step (b) to the subject.

26. The method of claim 25, wherein activating the β/γ dimeric IL-2 receptor comprises incubating said lymphocytes with mutant IL-2.

27. The method of claim 25, wherein activating the β/γ dimeric IL-2 receptor comprises introducing in said lymphocytes a nucleotide sequence encoding mutant IL-2.

28. The method of claim 25, wherein activating the β/γ dimeric IL-2 receptor comprises introducing a nucleotide sequence to upregulate the expression of the subunits β/γ of the IL-2 receptor in said lymphocytes and incubating said lymphocytes with wild-type IL-2.

29. The method of claim 25, wherein activating the β/γ dimeric IL-2 receptor comprises introducing a nucleotide sequence to downregulate the expression of the alpha subunit of the IL-2 receptor in said lymphocytes and incubating said lymphocytes with wild-type IL-2.

30. The method of claim 25, wherein activating the β/γ dimeric IL-2 receptor comprises incubating said lymphocytes with wild-type IL-2 and anti-CD25 antibodies.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0086] FIG. 1. Comparison of T cells phenotype obtaining when cultured with native IL2 or with the IL2 mutein that preferential signaling through the IL2Rβγ. A) Culture survival, B) Percentage of memory and effector cells.

[0087] FIG. 2. Comparison of the phenotypes recovered on T cells when cultured with native IL2 or IL2-mutein that preferentially signals through the intermediate affinity receptor IL2Rβγ in a broad range of doses. A) Culture survival, B) Percentage of PD1 positive cells and C) Percentage of cells with Central memory-like phenotype.

[0088] FIG. 3. Comparison of the phenotypes recovered on T cells when cultured with native IL2 or IL2-mutein that preferentially signals through the intermediate affinity receptor IL2Rβγ combined with IL7 and IL15 cytokines A) Culture survival, B) Percentage of memory and effector cells.

[0089] FIG. 4. Comparison of the phenotype recovered on T cells when cultured with native IL2 or IL2-mutein that preferentially signals through the intermediate affinity receptor IL2Rβγ, in a broad range of doses, combined with IL7 and IL15. A) Culture survival, B) Percentage of PD1 positive cells and C) Percentage of cells with central memory-like phenotype.

[0090] FIG. 5. Comparison of the phenotypes recovered on T cells when stimulated with IL2 or IL2-mutein only or with IL2 or IL2-mutein combined with IL7 and ID 5.

[0091] FIG. 6A. Histograms showing the percentage of eGFP positive cells, which measure the efficiency of transduction for IL2 or IL2 mutein in T cells.

[0092] FIG. 6B. Comparison of the percentage of cells with central memory and effector memory phenotype recovered in cultures when T lymphocytes were transduced to produce native IL2 or IL2-mutein.

[0093] FIG. 7A. Histograms showing the efficiency of CD25 downregulation after transduction with different shRNA constructs.

[0094] FIG. 7B. Percentage of cells with central memory and effector memory phenotype when T lymphocytes have reduced expression of CD25.

[0095] FIG. 8. Comparison of the effect of provide an IL2Rβγ-biased-IL2 signaling when employed an anti-CD25 antibody on CD8+ T cells A) Culture survival and B) Percentage of memory and effector cells.

[0096] FIG. 9. Functional capacity of CD8+ T cells with IL2 alone, IL2+IL15+IL7, IL2-mutein alone or IL2-mutein+IL15+IL7. A) Granzyme β production by OTI CD8+ T cells re-stimulated with the peptide SIINFEKL, B) Tumor killing capacity against B16-OVA cells of OT1 cells cultured with IL2, IL2-mutein alone or combined with IL7 and IL15.

[0097] FIG. 10. Measuring of tumor volume in MB16OVA tumor bearing animals that received OT1 cells cultured with native IL2 or IL2 mutein.

[0098] FIG. 11. Comparison of antitumor in vivo effect of OT1 T cells transduced to produce native IL2 or IL2-mutein that preferentially signals through the intermediate affinity receptor IL2Rβγ. A) Tumor volume, B) Survival over time.

[0099] FIG. 12. Characterization of CD8+ TILs before and after culturing with IL2 or IL2 mutein: A) Expansion level, B) Ki-67 expression percentage by TILs and C) Distribution of naïve T cells (CD62L+CD44-), central memory (CD62L+CD44+) and (CD62L-CD44+).

[0100] FIG. 13. Characterization of CD8+ TILs before and after culturing with IL2 or IL2 mutein: A) Percentage of expression of inhibitory markers and B) IFNγ, TNFα and Granzyme β.

[0101] FIG. 14. Expansion of NK cells in TILs cultured with IL2 or IL2 mutein: A) Percentage of NK cells and B) NK cells number.

EXAMPLES

Example 1. Substitution of Native IL2 by IL2-Mutein, which Preferentially Signals Through the IL2Rβγ, Preserves Lymphocyte Viability and Confers Central Memory-Like Phenotype

[0102] CD8+ T cells are isolated from naïve OT1 mouse spleen. AntiCD3/antiCD28 coated beads are used as TCR stimulation for seven days, together with native IL2 or the IL2 mutein for 10 days, the last one obtained in the Center of Molecular Immunology (batch 1201602) which and divulged in SEQ ID NO 6 of U.S. Pat. No. 9,206,243. Every two days culture is expanded to keep the density at 1×10.sup.6 and the cytokines are added with fresh media. The successful expansion of a large numbers of T cells is achieved in both culture conditions and similar fold-increase in cell number is observed. FIG. 1A shows that the viability in culture is improved when using the IL2 mutein in comparison to the native IL2 (p<0.0001). Additionally, the quantity of memory cells and the expression of inhibitory markers was measured by flow cytometry. A significantly high percentage of cells with central memory-like phenotype (defined by CD62L+/CD44+) was observed in cells in when using IL2-mutein in comparison to native IL2 (FIG. 1B). Phenotypic analysis of PD-1, Lag-3, Tim-3, and Tigit markers showed that IL2-mutein activated cells repressed the expression of the immune checkpoint ligands (PD-1, Lag-3, Tim-3, Tigit) (Table 1).

TABLE-US-00001 TABLE 1 Exhaustion markers expression (percent of positive cells) when native IL2 or IL2-mutein is used for in vitro T cells activation/expansion. Marker native IL-2 IL-2 mutein PD1 2.61 0.38 TIM3 17.0 0.42 Lag3 79.9 15.9 Klrg1 12.4 0.06

[0103] The described effect in terms of viability (FIG. 2A), PD1 expression (FIG. 2B) and CD62L expression (FIG. 2C) is consistent in a broad range of doses for both the native IL2 and the IL2-mutein.

[0104] Taken together these results indicate that the substitution of native IL2 by IL2-mutein with preferential IL2Rβγ stimulation, promotes a central memory phenotype and not the well-described effector phenotype induced by native IL2. IL2-mutein more than native IL2 confers potential for lymphoid recirculation (more CD62L expression) and resistance to exhaustion (less PD1 expression), a more desired phenotype for in vivo transference.

Example 2. Substitution of Native IL2 by IL2-Mutein, which Preferentially Signal Through the IL2Rβγ, Favors Central Memory Phenotype in Combined Culture Protocols with IL7 and IL15

[0105] As previously described by other authors, the combination of IL7 and IL15 with IL2 improves T cells fitness when compared with IL2 alone (Redeker and Arens, 2016, Front Immunol. 6(7): 345). In our experiment, native IL2 or IL2-mutein were combined in culture with IL7 and 11_15 for OT-1T cells activation/expansion protocol.

[0106] In this strategy, the same TCR stimulation described in Example 1 was used. Native IL2 or IL2-mutein is added at the beginning of the culture until day 5th, then the cells are kept in IL7/IL15 for 10 days. In this scenario, both culture conditions give similar cell density in culture, high viability (FIG. 3A), and favors the prevalence of memory rather than effector cells. However, the use of IL2-mutein showed higher proportion of central memory cells in culture compared to native IL2 (p<0.0015) as shown in FIG. 3B.

[0107] At the same time, the cells cultured with the mutein showed a lower expression of exhaustion markers as shown in Table 2.

TABLE-US-00002 TABLE 2 Exhaustion markers expression (percent of positive cells) when native IL2 or IL2-mutein is used for in vitro T cells activation/expansion combined with IL7 and IL15. Marker native IL-2 IL-2 mutein PD1 1.69 0.48 TIM3 10.3 0.18 Lag3 84.3 21.1 Klrg1 6.07 0.05

[0108] The evaluation of different concentrations in culture, for both the native IL2 and the IL2-mutein combined with IL7/IL15, demonstrates that the described effect is consistent in a broad range of doses, in terms of viability (FIG. 4A), PD1 expression (FIG. 4B) and central memory-like phenotype (FIG. 4C).

[0109] Although the combination of native IL2 with IL7 and 11_15 improves culture conditions when compared with native IL2 alone, no improvement is observed for the IL2-mutein when using combined protocols, since IL2-mutein alone is highly effective in preserving T cells viability and inducing central memory phenotype. Additionally, IL2-mutein alone is as effective as the combination of IL2, IL7 and IL15 in inducing central memory phenotype-like on activated T cells (FIG. 5).

Example 3. Genetically Engineered T Cells that Produce IL2-Mutein, which Preferentially Signal Through the IL2Rβγ, Acquire a Central Memory Phenotype

[0110] In order to generate a continued cytokine support source both in vitro and in vivo, we genetically engineered T cells to produce the IL2-mutein that preferentially activate the intermediate affinity IL2 receptor. Retroviral constructs encoding the enhanced green fluorescent protein (eGFP) and the native IL2 or IL2-mutein are used. The transduction procedure is initiated by stimulating freshly isolated naïve OT-I T cells with anti-CD3/anti-CD28 coated beads and native IL2 or IL2-mutein. Concentrated retroviral vector supernatant is added at 24 and 48 hours to a retronectin coated-plate with the cells. After transduction, TCR stimulation with anti-CD3/anti-CD28 coated-beads is maintained for seven days together with IL7 and IL15 for 10 days. The transduction efficiency was confirmed by eGFP expression and was higher than 80% for both constructs (FIG. 6A). The secreted molecule was detected by ELISA (native IL2 or IL2-mutein).

[0111] In a similar manner to that observed when OT1 T cells were cultured with the soluble IL2 mutein, the cells engineered to produce IL2-mutein had the memory phenotype in a higher percentage when compared to the cells engineered to produce the native IL2 which were mainly effector cells (p<0.0001) (FIG. 6B). Also, the cells engineered to produce IL2-mutein had less exhaustion markers expression compared to the cells engineered to produce the native IL2 (Table 3).

TABLE-US-00003 TABLE 3 Exhaustion markers expression (percent of positive cells) when T cells are engineered to produce native IL2 or IL2-mutein Marker native IL-2 IL-2 mutein PD1 3.02 0.67 TIM3 17.0 0.35 Lag3 80.1 36.2

[0112] Taken together these results confirms the hypothesis that stimulation through IL2Rβγ with the IL2-mutein, favors central memory differentiation with this new culture method when the IL2-like signaling is constant and sustained.

Example 4. Genetically Engineering to Downregulate CD25 Expression, Favors a Central Memory Phenotype on T Cells Expanded with Native IL2

[0113] Vectors encoding for eGFP and a small hairpin RNA (shRNA) targeting il2ra were used, with the aim of blocking the expression of IL2 alpha chain gen (CD25). The transduction procedure is initiated by stimulating freshly isolated naïve OT1 T cells with anti-CD3/anti-CD28 coated beads and native IL2. Three different constructions for shRNA were used, obtaining variable knockdown efficiencies based on surface expression of CD25. Transduction with lentivirus containing scramble shRNA is used as control. After two rounds of transduction, cells are maintained in culture for 5 days with native IL2 (50 U/ml). The reduction on CD25 surface expression after transduction with the different constructs was evaluated by Flow Cytometry (FIG. 7A). The percentage of central memory cells after five days of native IL2 stimulation is superior when better CD25 downregulation is achieved (FIG. 7B).

[0114] Downregulation of CD25 on T cells provides a scenario for preferentially stimulation of the intermediate affinity receptor IL2Rβγ using in culture the native IL2. Under these conditions, the differentiation of CD8+ T to the central memory-like phenotype is favored.

Example 5. Central Memory Phenotype is Obtained when Cells are Stimulated with Native IL2 but CD25 Interaction is Blocked with a Pharmaceutical Agent Simultaneously Added on the Culture

[0115] In another attempt to preferentially direct native IL2 signaling through intermediate affinity receptor IL2Rβγ, a pharmaceutical agent is added on culture together with native IL2 during in vitro T cells activation. The pharmacological agent is added at high concentration to guarantee the blocking effect. OT-I T cells are cultured with anti-CD3/anti-CD28 coated beads, 50 IU/ml of IL2 and 10 μg/ml of anti-CD25 monoclonal antibody (BioLegend clone 3C7). The anti-CD25 is added since day 0 and is renewed when adding fresh medium with native IL2 every two days. An antibody with irrelevant specificity is used as isotype control. Phenotyping of the cells after 10 days of culture revealed that the addition of anti-CD25 during native IL2 activation, increases the cell viability (FIG. 8A) and the central memory-like population (FIG. 8B) (p<0.0001) when compared with the isotype control. Additionally, when native IL2 interaction with CD25 is disrupted with the antibody, less expression of exhaustion markers is obtained (Table 4).

TABLE-US-00004 TABLE 4 Exhaustion markers expression (percent of positive cells) when T cells are stimulated with native IL2 but interaction with CD25 is disrupted with an antiCD25 antibody Marker Isotype Control Anti-CD25 PD1 4.12 3.33 TIM3 18.5 0.54 Lag3 80.2 21.3

[0116] Together, these results suggest that directing native IL2 signaling through IL2Rβγ by impairment of the interaction with CD25, the cells are directed to a central memory phenotype, which is more suitable for adoptive cell therapies.

Example 6. OT-I T Cells which Receive Preferential Activation Through Dimeric Receptor IL2Rβγ, are Polyfunctional and Show Efficient T Cell Antigen-Specific Cytotoxicity In Vitro

[0117] In order to assess the functional capacity of the central memory T cells, activated through the IL2Rβγ, a cytotoxicity assay was performed. OT1 cells activated as described in Examples 1, 3 were stimulated for 16 hours with the cognate peptide SIINFEKL, and flow cytometry analysis was performed to determine the production of Granzyme β. In order to evaluate the killing capacity of the OT-I T cells, the cells were co-cultured with B16 melanoma cells transduced to express the OVA nominal antigen in the surface (B16-OVA) at the 1:1 proportion. Non transduced B16 melanoma cells were used as control. IncuCyteCytotox reagent was added and the killing capacity was evaluated each two hours during 60 hours. The lysis of the target cells was antigen specific, due to no lysis was observed in the controls cells.

[0118] OT1 cells activated with the IL2 mutein that preferentially signaling through IL2Rβγ showed an ex vivo effective direct lysis activity, showing when signal is given through the IL2Rβγ the cells are differentiated into potent killing cells. (FIGS. 9A and B)

Example 7. T Cells, which Receive Preferential Activation Through Dimeric Receptor IL2Rβγ, Show Better Antitumor Activity when Used for ACT in a Melanoma Model

[0119] The OT1 model together with B16/OVA tumor cell line is one of the most used models and represents a relevant preclinical approximation of ACT. C57BL/6 recipient mice received a subcutaneous injection of 1×10.sup.6 OVA-expressing melanoma cells on day 0 and ten days after tumor cells inoculation; mice were sub-lethally irradiated (5 Gy).

[0120] Group 1: received no treatment (control group).

[0121] Group 2: received OT1 T cells activated with native IL2 (as in Example 1).

[0122] Group 3: received OT1 T cells activated with IL2-mutein (as in Example 1).

[0123] Group 4: received OT1 T cells activated with native IL2+IL7+IL15 (as in Example 2).

[0124] As shown in FIG. 10, ACT with cells primed with IL2-mutein better controlled tumor growth compared to native IL2-activated cells. Together these results indicates that the initially induced memory phenotype with low exhaustion markers expression obtained with IL2-mutein activation was an effective combination for tumor control in vivo.

Example 8. T Cells Engineered to Secrete IL2-Mutein, which Preferentially Signal Through the IL2Rβγ, Showed Robust Antitumor Activity when Used for ACT

[0125] In an effort to improve the effectiveness of ACT, we genetically engineered T cells to produce the IL2-mutein. The insertion of the gen allows for continued and sustained IL2-mutein signal on T cells, not just in vitro but in vivo as well. C57BL/6 recipient mice received a subcutaneous injection of 1×10.sup.6 OVA-expressing melanoma cells on day 0 and ten days after tumor cells inoculation; mice were sub-lethally irradiated (5 Gy). The animals were divided in three groups of treatment and on days 11 and 15 they received an intravenous transference of 1×10.sup.6T cells from OT1 mice as follow:

[0126] Group 1: received no treatment (control).

[0127] Group 2: received native IL2_engineered OT1 T cells (as the transduction protocol described in Example 3)

[0128] Group 3: received IL2-mutein_engineered OT1 T cells (as the transduction protocol described in Example 3).

[0129] As shown in FIG. 11, the mice receiving OT1 T cells engineered for IL2-mutein production, experienced a good control of the stablished tumors (FIG. 11A) and a higher survival (FIG. 11B).

Example 9 Generation of TILs Using IL2-Mutein

[0130] Since ACT therapies can be performed using TILs, we evaluated the effects of IL2-mutein on the TILs expansion. To study the role of the activation of the β/γ dimeric IL2 receptor in tumor-infiltrating lymphocytes, MC38 tumors were isolated from C57BL/6 mice between days 19-21 following tumor cell inoculation. Tumors were mechanically dissociated and were cultured in presence of IL2 or IL2-mutein for 14-16 days. We found that TILs expansion was higher when IL2-mutein was used in culturing conditions. Consistently, IL2-mutein increased the proliferation rate of TILs in terms of ki67 marker (FIGS. 12A and B). Furthermore, TILs cultured ex vivo with IL2-mutein showed a significant expansion of central memory-like T cells as compared to the controls (FIG. 12C). Overall, these data show that IL2-mutein induces CD8+ TILs expansion favoring central memory differentiation phenotype.

Example 10. TILs Expanded with Mutant IL-2 are Polyfunctional and Show Reduction of Activation and Exhaustion Markers

[0131] TILs obtained with the methodology of Example 9 were characterized functional and phenotypically before the expansion, they were stimulated during 4 hours in presence of anti-CD3 and anti-CD2 antibodies.

[0132] We found that inhibitory receptors PD-1, Lag-3, and Tim-3 were drastically reduced in TILs expanded with IL2-mutein compared with TILs expanded with native IL2 (FIG. 13A). TILs were also analyzed for their functionality. (FIG. 13B). Collectively, these data show the ability of IL2-mutein to expand TILs and enrich them for central memory-like phenotype without compromising their functionality.

Example 11. Generation of NK Cells from TILs Using Mutant IL-2

[0133] To study the role of the activation of the β/γ dimeric IL2 receptor in the proportion of NK cells among the total expanded TILs, MC38 tumors were isolated from C57BL/6 mice between days 19-21 following tumor cell inoculation. Tumors were mechanically dissociated and were cultured in presence of native IL2 or IL2-mutein for 14-16 days. TILs cultured ex vivo with IL2-mutein showed a significant expansion of NK cells as compared to the native IL2, in terms of proportion form the total (FIG. 14A) and in terms of absolute numbers (FIG. 14B).