WO2024123175A1 - Compositions for treating immune checkpoint blockade therapy resistant cancers - Google Patents

Abstract

The present invention provides a pharmaceutical combination for use in treating cancer comprising an ITK inhibitor and an immune checkpoint inhibitor, wherein the ITK inhibitor is formulated for intermittent administration or for administration at a dose that partially inhibits ITK enzymatic activity in T cells. The present invention further provides a method of reversing T cell exhaustion resulting from persistent TCR stimulation comprising exposing an exhausted T cell to an ITK inhibitor, wherein said T cell may be a CD8+ T cell or a CAR T cell.

Description

Title: Compositions for treating immune checkpoint blockade therapy resistant cancers

FIELD OF THE INVENTION

The invention is in the field of cancer treatment, in particular treatment of immune checkpoint blockade therapy resistant cancers. The invention provides pharmaceutical combinations for use in such treatment, and methods of reversing T cell exhaustion resulting from persistent TCR stimulation.

BACKGROUND OF THE INVENTION

The programmed cell death protein 1 (PD-1) pathway as a target for cancer treatment through immunotherapy has become a focal point in modern cancer treatments. PD-1 is a T cell co-inhibitory receptor with two ligands, PD-L1 and PD-L2. Binding of PD-1 to either PD-L1 or PD-L2 interferes with early T-cell receptor (TCR) and CD28 signaling and inhibits T cell proliferation, T cell effector functions, and T cell survival. Tumor cells frequently up-regulate PD-L1, allowing them to suppress anti-tumor T cell responses and evade immune system destruction through PD-1. Similar effects occur via CTLA-4, LAG3 (CD223), TIGIT, TIM-3, PSGL-1, and CD200. Thus, PD-1, CD200, LAG3 (CD223), TIGIT, TIM-3, PSGL-1 and CTLA-4 serve as immune checkpoints for inhibition of the immune response. Blocking the functions of these immune checkpoints with antagonistic monoclonal antibodies has been shown to boost the anti-tumor immune response in many different types of human cancer. Immune checkpoint blockade (ICB) therapy, involving administration of an immune checkpoint inhibitor (ICI), such as, for instance, an anti-PD-l/PD-Ll antibody to block checkpoint inhibition over the PD-1/PD-L1 axis, is one of the most promising therapeutic approaches in treating solid tumors. However, not all patients show complete responsiveness to ICB therapy. Patients who do not respond to initial PD-1/PD-L1 blockade are referred to as having “primary resistance”. Other patients develop “acquired resistance” to immunotherapy, which is a clinical scenario whereby a cancer initially responds to immunotherapy, but after a period of time, relapses and progresses. Hence, despite ICB’s success, there still remains a large proportion of patients that do not benefit from this therapy. A better understanding of PD-1 pathway-mediated immunosuppression and its blockage is therefore needed in order to treat patients with ICB resistant cancers.

Cytotoxic T lymphocytes (CTLs), also known as CD8+ T cells, are a critical component of the adaptive immune system and play a pivotal role in immune defense against cancer cells. Through their expression of PD-1 they also play a key role in the response to ICB therapy. Tumor-specific CD8+ T cells are exposed to persistent antigenic stimulation which induces a dysfunctional state called “exhaustion” and ICB therapy should effectively reverse such exhaustion. In case of ICB resistance, this is clearly not the case, and there may be further functional impairments of CTL other than through cancer-controlled checkpoint inhibition that plays a role in resistance to ICB therapy. Alternatively, ICB may not be sufficient to fully reactivate exhausted cells because ICB efficacy depends on the presence or magnitude of subsets of exhausted cells that retain a level of proliferative capacity.

It is an aim of the present invention to provide novel treatments and treatment strategies that improve the efficiency of ICB therapy, particularly in treatment of ICB resistant cancers.

SUMMARY OF THE INVENTION

The present inventors earlier investigated the functional decline in CTL in relation to chronic antigen stimulation-induced T cell exhaustion, and found that the exhaustion was caused by chronic T cell receptor (TCR) signaling. The inventors have now found that dampening this persistent TCR stimulation ameliorates CTL exhaustion. In fact, the inventors found methods to reinvigorate exhausted CTL, found that such reinvigorated CTLs display a stem cell-like transcription factor T cell factor 1 positive (TCF1+) phenotype, and that such TCF1+ CD8+ T cells restore efficacy of ICB therapy in ICB resistant tumors.

The present invention now provides a pharmaceutical combination for use in treating cancer comprising an ITK inhibitor and an immune checkpoint inhibitor, wherein the ITK inhibitor is administered (or formulated for administration) using an intermittent dosing regimen or wherein the ITK inhibitor is administered at a dose that partially inhibits ITK enzymatic activity in T cells, preferably CD8+ T cells.

In a preferred embodiment of a pharmaceutical combination for use according to the present invention, the cancer is an ICB resistant cancer, preferably an ICB resistant solid tumor.

In another preferred embodiment of a pharmaceutical combination for use according to the present invention, the ITK inhibitor is selected from BMS-431051, BMS-488516, BMS-509744, PF 06465469, HY- 11066, CPI-818, ibrutinib, bosutinib, CTA056, GSK-2250665A, and combinations thereof.

In yet another preferred embodiment of a pharmaceutical combination for use according to the present invention, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, an anti-Tim-3 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, an anti-PSGL-1 antibody, an anti-CD200 antibodyand combinations thereof, more preferably selected from nivolumab, camrelizumab, cemiplimab, dostarlimab, MEDI0680, pembrolizumab, prolgolimab, retifanlimab, sasanlimab, spartalizumab, STI- All 10, tislezlizumab, toripalimab, atezolizumab, avelumab, durvalumab, KD033, and STI-A1014, ipilimumab, tremelimumab, botensilimab, cobolimab sabatolimab, surzebiclimab, Sym023, R07121661, LY3321367, ICAGN02390, BMS-986258, tiragolumab, domvanalimab, vibostolimab, ociperlimab, BMS-986207, COM-902, AGEN-1307, ieramilimab, relatlimab, samalizumab, VTX-0811, SelK2, and combinations thereof.

In yet another preferred embodiment of a pharmaceutical combination for use according to the present invention, the intermittent dosing regimen of the ITK inhibitor comprises a repeated treatment cycle comprising a treatment interval (a period of time) of about 1-7 days, preferably about 3 days, of administration of the ITK inhibitor, and wherein said treatment cycle further comprises a non-treatment (or off treatment) interval of about 1-7 days, preferably about 3 days, of non-administration of the ITK inhibitor. This cycle of administration and non-administration of the ITK inhibitor may, in aspects of this invention, be repeated until progression or unacceptable toxicity. In the intermittent dosing regimen in aspects of this invention the treatment interval and the non-treatment interval in a treatment cycle may be the same or different. In aspects of this invention the intermittent dosing regimen of the ITK inhibitor comprises at least 2 treatment cycles, more preferably at least 5, 10, 20, or 50 cycles. During the treatment interval of the cycle of intermittent dosing, the dosing preferably comprises an administered dose of about 1-10 mg/kg body weight per day of the ITK inhibitor. The intermittent dosing regimen of the ITK inhibitor in aspects of this invention preferably reduces ITK enzymatic activity sufficient to reverse T cell exhaustion. The intermittent dosing regimen of the ITK inhibitor in aspects of this invention preferably does not result in a steady-state plasma concentration of the ITK inhibitor.

In yet another preferred embodiment of a pharmaceutical combination for use according to the present invention, the ITK inhibitor dose that is effective for partial inhibition of ITK enzymatic activity is 0.01-5 mg/kg body weight per day, such as 0.01-2.5 mg/kg per day. Partial inhibition of ITK enzymatic activity preferably comprises continuous dosing, preferably whereby steady- state plasma levels are attained.

In yet another preferred embodiment of a pharmaceutical combination for use according to the present invention or a method of the present invention, the an immune checkpoint inhibitor is formulated for administration or is administered at a dose of 100-1000 mg Q2W, Q3W or Q4W, such as 240-480 mg every 2-4 weeks, preferably by IV administration (e.g., at about 2-4, such as 3 mg/kg body weight).

In another aspect, the present invention provides a method of treating a subject suffering from an ICB resistant tumor, comprising administering to the subject an ITK inhibitor, and further comprising administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor (ICI), wherein the ITK inhibitor is administered using an intermittent dosing regimen or wherein the ITK inhibitor is administered at a dose that partially inhibits ITK enzymatic activity in T cells, preferably CD8+ T cells, of said subject. In a preferred embodiment the administration of the ICI is at a dosage that is in accordance with normal or conventional ICB therapy.

The intermittent dosing regimen of the ITK inhibitor preferably comprises a repeated treatment cycle comprising a treatment interval of about 1-7 days, preferably about 3 days, of administration of the ITK inhibitor, and wherein said treatment cycle further comprises a nontreatment interval of about 1-7 days, preferably about 3 days, of nonadministration of the ITK inhibitor. During the treatment interval of the cycle of intermittent dosing, the dosing preferably comprises an administered dose of about 1-10 mg/kg per day of the ITK inhibitor.

Preferably, the subject is aspects of this invention is a mammal, more preferably a human.

In a preferred embodiment of a method of treating a subject suffering from an ICB resistant tumor, the ITK inhibitor is selected from BMS-431051, BMS-488516, BMS-509744, PF 06465469, HY-11066, CPI- 818, ibrutinib, bosutinib, CTA056, GSK-2250665A, and combinations thereof.

In another preferred embodiment of a method of treating a subject suffering from an ICB resistant tumor, the intermittent administration of the ITK inhibitor comprises administration at a dose of 1-10 mg/kg body weight per day and the intermittent treatment regimen comprises a repeated interval of 1-7 days of continuous treatment, followed by 1-7 days off treatment.

In another preferred embodiment of a method of treating a subject suffering from an ICB resistant tumor, the ITK inhibitor dose that is effective for partial inhibition of ITK enzymatic activity is 0.01-5 mg/kg body weight per day.

In another preferred embodiment of a method of treating a subject suffering from an ICB resistant tumor, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti- CTLA-4 antibody, an anti-Tim-3 antibody, an anti-TIGIT antibody, an anti- LAG3 antibody, an anti-PSGL-1 antibody, an anti CD200 antibody, and combinations thereof.

In another preferred embodiment of a method of treating a subject suffering from an ICB resistant tumor, the ICI is selected from nivolumab, camrelizumab, cemiplimab, dostarlimab, MEDI0680, pembrolizumab, prolgolimab, retifanlimab, sasanlimab, spartalizumab, STI-A1110, tislezlizumab, toripalimab, atezolizumab, avelumab, durvalumab, KD033, and STI-A1014, ipilimumab, tremelimumab, botensilimab, cobolimab sabatolimab, surzebiclimab, Sym023, R07121661, LY3321367, ICAGN02390, BMS-986258, tiragolumab, domvanalimab, vibostolimab, ociperlimab, BMS-986207, COM-902, AGEN-1307, ieramilimab, relatlimab, samalizumab, VTX-0811, SelK2, and combinations thereof. In another aspect, the present invention provides a method of reversing T cell exhaustion resulting from persistent TCR stimulation comprising exposing said T cell to an effective amount of an ITK inhibitor. Preferably in a method of reversing T cell exhaustion according to the invention, said T cell is a CD8+ T cell or a CAR T cell, more preferably a CD8+ T cell. Preferably in a method of reversing CD8+ T cell exhaustion according to the invention, the exposure to an effective amount of an ITK inhibitor effects in said T cell one or more of: increasing TCF1 expression; decreasing TOX expression; decreasing surface expression of inhibitory receptors PD-1, CTLA-4, TIGIT, Tim-3, Lag-3, CD 160, PSGL-1 or CD200; increasing production of cytokines IFNy, TNFct or IL-2; increasing cytotoxicity; and increasing proliferative capacity.

In a preferred embodiment of a method of reversing T cell exhaustion resulting from persistent TCR stimulation according to the present invention, the exposure to an ITK inhibitor is intermittent or at a concentration that partially inhibits ITK enzymatic activity in said T cell. In preferred embodiments thereof, intermittent exposure to the ITK inhibitor comprises exposure to a dose of O.lnM to about 5gM and preferably the intermittent exposure regimen comprises a repeated interval of 1-7 days, preferably about 3 days, of continuous exposure, followed by 1-7 days, preferably about 3 days, of non-exposure. In other preferred embodiments thereof, said ITK inhibitor dose that is effective for partial inhibition of ITK enzymatic activity is about O.lnM to about lgM.

The present invention further provides a method of reversing CD8+ T cell exhaustion or CAR T cell exhaustion comprising exposing an exhausted CD8+ T cell or CAR T cell to an interleukin-2 inducible T-cell kinase (ITK) inhibitor, wherein the inhibition of ITK in said exhausted CD8+ T cell or CAR T cell is partial or intermittent. A method of reversing T cell exhaustion in accordance with the invention, is followed by a step wherein said T-cell is exposed to an antigen capable of TCR signaling to thereby induce activation of said T-cell. Preferably, said antigen is a tumor antigen. The method of reversing CD8+ T cell exhaustion or CAR T cell exhaustion in aspects of this invention is preferably performed in a cyclic manner, whereby after the step of exposing said T-cell to an antigen capable of TCR signaling to thereby induce activation of said T-cell, said T-cell activation is continued until said activated T cell is exhausted or shows an exhaustion-related transcriptional profile, whereaftrer the method is repeated. The method may be performed in vitro, ex vivo or in vivo. Preferably the method is performed in vivo.

Partial or intermittent inhibition of ITK in aspects of this invention is preferably effected by administrating the ITK inhibitor at a dose and/or for a duration that reduced but does not block TCR signaling. The level of TCR signaling should ensure T-cell activation. The proposed method preserves CTL function and prevents the excessive and persistent activation that results in CTL exhaustion. That CTL function is preserved while excessive CTL activation is prevented can, for instance, be observed through cell markers, such as transcription factor T cell factor 1 (TCF1) expression and TOX expression. Preferably the dose and/or duration of the exposure of the exhausted CD8+T cell to an ITK inhibitor results in the CTL having a stem cell-like transcription factor T cell factor 1 (TCF1) positive phenotype. Preferably the dose and/or duration of the exposure of the exhausted CD8+T cell to an ITK inhibitor results in the CTL wherein TOX expression is downregulated.

As ITK is a critical tyrosine kinase that regulates TCR signaling and T cell activation, blocking of ITK inhibits T cell activation. In aspects of this invention, complete and continuous ITK inhibition is considered detrimental to T cell immunity. Partial inhibition, in contrast to complete inhibition, allows sufficient activation to ensure that function and expansion is preserved while preventing excessive activation that leads to CTL exhaustion. Partial ITK inhibition may in one embodiment be attained by intermittent exposure to an ITK inhibitor, wherein a period of exposure to an ITK inhibitor is followed by a period without exposure to an ITK inhibitor, for instance in a cycled manner, preferably through periodic exposure. Also, partial inhibition of activation may be attained by be continuous but a submaximal level of exposure, such as exposing the exhausted CTL to a dose of the ITK inhibitor that results in partial inhibition of activation, e.g., exposing the exhausted CTL in vitro or in vivo to a concentration of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the half-maximal inhibitory concentration (IC50) of the ITK inhibitor. Intermittent exposure in aspects of this invention may temporarily comprise a full inhibition of ITK, such as attainable at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the half-maximal inhibitory concentration (IC50) of the ITK inhibitor. The in vivo inhibitory concentrations are preferably attained in blood plasma, more preferably in the tumor microenvironment.

DESCRIPTION OF THE DRAWINGS

Figure 1. ITK inhibitor enhances the anti-tumor effects of anti- PD-1 in the murine B16-F10-OVA model. (A) Scheme of in vivo experimental set up shown. (B) Individual tumor growth curves of B16-F10- OVA tumor experiments are shown. (C) Group based tumor volume curve (left) and Kaplan-Meier survival curve (right) are depicted. (D) Absolute numbers of donor CD 8+ T cells in the draining lymph nodes shown. Tumor growth curve is presented as mean values ± SE. Two-way ANOVA with Tukey's multiple comparison test was used to compare tumor growth between groups. P values are shown for individual days (Isotype vs. ITK inh+aPD-l:#P <0.05, ## P<0.01, ### P<0.001, #### PO.OOOl; ITK inh vs. ITK inh+aPD-1: &P <0.05,&&P<0.01,&&&P<0.001,&&&&P<0.0001; ctPD-1 vs. ITK inh:§P<0.05,§§P<0.01; Isotype vs. ITK inhifP

P<0.001, tttt P<0.0001;) Log-rank- tests was performed to compare the survival curves of groups. Mann Whitney test was used to test significant differences between treatment groups. Each symbol represents one animal (n=9-15), 4 independent experiments performed. Lines depict mean ± SE. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2. ITK inhibitor augments the antitumor effect of ICB in ICB-unresponsive tumors. (A) Scheme of in vivo experimental set up of AE17 tumor experiments is shown. (B) Individual tumor growth curves of AE17 tumor experiments are shown. (C) Group based tumor growth curve (left) and Kaplan-Meier survival curve (right) of AE17 tumor experiments are depicted. n=12, 3 independent experiments performed. (D) Scheme of in vivo experimental set up of 4662 tumor experiments. (E) Individual tumor growth curves of 4662 tumor experiments are shown. (F) Group based tumor growth curve (left) and Kaplan-Meier survival curve (right) of 4662 tumor experiments are depicted. n=17-18, 4 independent experiments performed. For all group-based tumor growth curves (C and F), data are presented as mean values ± SE. For tumor growth, statistical analyses were performed using two-way ANOVA followed by Tukey's multiple comparison test. P values are shown for individual days (ctPD-1 vs. ITK inh+ctPD-1: *P<0.05, **P<0.01. ***P<0.001, ****P<0.0001; Isotype vs. ITK inh+ctPD-l:#P <0.05,##P<0.01, ### PC0.001, #### PC0.0001; ITK inh vs. ITK inh+aPD-1: &P <0.05, && P<0.01, &&& PO.OOl, &&&& PO.OOOl; ctPD-1 vs. ITK inh:§P<0.05 ; Isotype vs. ITK inhifP <0.05,ttP<0.01;) Log-rank-tests was performed to compare the survival curves of groups. *P<0.05, **P<0.01, ***p<0.001, ****P<0.0001.

Figure 3. Ibrutinib rejuvenates in vitro exhausted CD8+ T cell functions. (A) Scheme of testing ibrutinib effects on in vitro CTL exhaustion. By repeatedly stimulating the purified OT-I T cells with OVA(257-264) peptide for 5 days, in vitro exhausted CTLs were induced. From day 5, the cells were treated with DMSO or lpM ibrutinib. On day 8, function and phenotype of the cells were determined. (B) Representative histograms depicting the expression of inhibitory receptors on DMSO or ibrutinib treated exhausted cells on day 8 of the in vitro exhaustion culture. (C) Pooled data showing the median fluorescence intensity (MFI) of the inhibitory receptors expressed on DMSO or ibrutinib treated cells. (D) Bar chart depicting frequency of cells expressing either one, two, three or four of the inhibitory receptors (IRs) PD-1, Lag3, Tim-3 and Tigit. (E) Representative FACS plots illustrating percentage of cytokine-producing CD8+ T cells upon OVA(257-264) peptide re- stimulation. Exhausted OT-I CD8+ T cells were re-stimulated on day 8 for 6 hours with OVA(257-264) peptide and intracellular cytokines were measured by flow cytometry. (F) Pooled data showing the frequency of cytokine producing CD8+ T cells. (G) Bar graph depicting the frequency of cells producing either one, two or three of the cytokines (IL-2, TNF-ct and IFN-y) simultaneously. Each symbol represents one animal (n=6), 4 independent experiments performed. Lines depict mean ± SE. Between the groups, paired-t test was performed, except for MFI of PD-1, CD 160 and Tim3, where the Wilcoxon matched-pairs signed rank test was used. *P<0.05, **P<0.01.

Figure 4. The T cell exhaustion decreasing effect of Ibrutinib is independent of Btk expression. Purified OT-I cells from wild type (WT) or btk deficient (btk-/-) mice were stimulated once with OVA (257-264) peptide or repeatedly stimulated for 5 days, and then treated with DMSO or lgM ibrutinib till day 8, when phenotype and function of cells was determined. (A) Pooled data depicting the expression of inhibitory receptors. (B) Pooled data showing the frequency of cytokine producing cells re-stimulated on day 8 with OVA(257-264) peptide for 6h. (C) Representative flow cytometry plots (left) and pooled data (middle) showing the frequency of TOX+ CD44+ CD8+ T cells from either WT or btk-/- mice after DMSO or lgM ibrutinib treatment. Bar graph (right) demonstrates the fold change of %TOX+ in exhausted T cells from either WT or btk-/- mice after treatment with DMSO or lgM ibrutinib. (D) Representative flow cytometry plots (left)and pooled data (middle) illustrating the frequency of TCF1+ CD44+ CD8+ T cells from either WT or btk-/- mice after treatment with DMSO or lgM ibrutinib. Bar graph (right) demonstrates the fold change of %TCF1+ in exhausted T cells from either WT or btk-/- mice after treatment with DMSO or lgM ibrutinib. Each symbol represents one animal (n=5), 5 independent experiments performed. Lines depict mean ± SE. To compare different groups, paired-t test was performed with exemption of (B) % of IL-2+ cells and C) % of TOX+ cells, where Wilcoxon matched-pairs signed rank test was used . *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 5. Inhibiting ITK reverses CTL exhaustion. Purified OT-I cells from wild type (WT) or btk deficient (btk-/-) mice were stimulated one time with OVA (257-264) peptide or repeatedly stimulated with the peptide for 5 days. For each of the Single peptide and Repeat peptide experiments, DMSO controls are depicted left, and ITK inhibitor tests are depicted right. (A) Pooled data showing the MFI of inhibitory receptors on cells. (B) Pooled data showing the frequency of cytokine producing cells. Cells were harvested on day 8 and re-stimulated for 6 hours with OVA(257-264) peptide and intracellular cytokines were measured by flow cytometry. (C) Pooled data depicting the MFI of transcription factors TOX (left) and the fold change of TOX MFI (right). (D) Pooled data depicting the MFI of transcription factors TCF1 (left) and the fold change of TCF1 MFI (right). (E) Representative histograms and pooled data showing ITK (Tyrl80) and PLC-yl (Tyr783) phosphorylation in exhausted T cells that were treated on day 5 with ibrutinib or DMSO and harvested on day 6. (F) Representative histograms and pooled data depicting ITK (Tyrl80) and PLC-yl (Tyr783) phosphorylation in exhausted T cells that were treated on day 5 with ITK inhibitor (BMS- 509744) or DMSO and stained on day 6. Each symbol represents one animal (n=7-10), 7-9 independent experiments performed. Lines depict mean ± SE. Between the groups, paired-t test was performed to test for statistical significance, except for A) Lag3 MFI, Tim3 MFI, B) frequency of TNF-ct+ and IFN-Y+ where Wilcoxon matched-pairs signed rank test was used. *P<0.05, **P<0.01, ***P<0.001.

Figure 6. Ibrutinib reduces the exhaustion-related transcriptional signature of in vitro exhausted cells. RNAseq was performed with the single peptide stimulated and repeat peptide stimulated cells after treatment of cells with ibrutinib or DMSO from day 5 to day 8. (A) Principle Component Analysis (PCA) plot of RNAseq results. (B) Heatmap depicting the significantly differentially expressed genes between ibrutinib-treated cells (right) and DMSO-treated exhausted T cells (left). Lower tree of DMSO- treated is primarily downregulated, while upper tree is primarily upregulated. The situation in ibrutinib-treated cells is vice versa. (C) Heatmap showing a specific subset of exhaustion-related genes (inhibitory receptors and Transcription factors). The color key in heat maps of B and C indicates transcript-based z-score expression values. (D) Differentially expressed genes expression changes induced by ibrutinib in exhausted T cells were analyzed using Gene set enrichment analysis (GSEA). The gene set downregulated in exhausted T cell is enriched in genes upregulated by ibrutinib (left) while the gene set upregulated in exhausted T cell is enriched in genes downregulated by ibrutinib (right).

Figure 7. BTK is not expressed in exhausted T cells. Western blot examining the expression of BTK in in vitro exhausted OT-I cells (day 5 of repeat peptide stimulation) or OT-I cells stimulated once with peptide. WT and btk+/- OT-I cells shown. WT and btk-/- splenocytes shown as positive and negative controls.

DETAILED DISCRIPTION OF THE INVENTION Definitions

The term “Interleukin-2-inducible T-cell kinase”, abbreviated as ITK herein, refers to the protein encoded by the ITK gene. In embodiments, the Interleukin-2-inducible T-cell kinase is a human Interleukin-2-inducible T-cell kinase. In embodiments, the Interleukin-2-inducible T-cell kinase ITK protein has the amino acid sequence set forth in or corresponding to Entrez 3702, UniProt Q08881, or RefSeq (protein) NP_005537, in particular the amino acid sequence as set forth in NP_005537.3. In embodiments, the Interleukin-2-inducible T-cell kinase ITK gene has the nucleic acid sequence set forth in RefSeq (mRNA) NM_005546.4.

The term “inhibitor”, as used herein, refers to a molecule that decreases, blocks, inhibits, abrogates the activity or activation of a protein, such as a kinase, or interferes with signal transduction resulting from the interaction of at least two proteins, preferably a receptor and its ligand, such as a T-cell receptor and its ligand. Preferably an inhibitor reduces activity of a protein or blocks binding between receptor and ligand by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

The term “ITK inhibitor”, as used herein, refers to a compound that inhibits interleukin-2 inducible T cell kinase (ITK) activity. In embodiments, an ITK inhibitor is a selective ITK inhibitor. An ITK inhibitor may result in a decrease in the phosphorylation and activation of ITK by the membrane resident Src family kinase Lek, a decrease in the phosphorylation of Y783 and activation of phospholipase- Cy 1 (PLCyl) by ITK, or a decrease in the interaction between ITK and adaptor proteins LAT and SLP-76. An ITK inhibitor may for instance be a small molecule compound specifically binding to the ATP binding pocket in the kinase domain of ITK thereby preventing the initiation of kinase activity. An inhibitor of ITK may for instance also be a small molecule compound specifically binding to the PH domain of ITK, since preventing the recruitment of ITK to the plasma membrane can prevent its activation. An inhibitor of ITK may for instance also be a small molecule compound specifically binding to the SH3 or SH2 domain of ITK thereby preventing interactions with potential substrates. All molecules that prevent the activation or activity of ITK may be used in aspects of this invention as ITK inhibitor.

The inhibition of ITK activation or activity may be determined using an enzyme assay or cellular assay. As an enzyme assay, a kinase activity assay, such as the LanthaScreen™ Eu Kinase Binding Assay for ITK (ThermoFisher Scientific) or ITK Kinase Enzyme System (Promega Cat no. V3191)) may suitably be used.

As used herein the term “selective interleukin-2 inducible T-cell kinase (ITK) inhibitor” means a compound with an IC50 of at least 2-fold greater for ITK than representative members of the kinase enzyme family. More preferably, the selective inhibitor has an IC50 of 5-fold, 10-fold, 20- fold, 100-fold, 500-fold greater than other representative members of the kinase enzyme family. The compound may be an ATP competitive ITK inhibitor. The compounds may be a covalent or irreversible ITK inhibitor. Examples of selective ITK inhibitors may be found in the scientific and patent literature. Charrier et al. 2013 Exp Opin Drug Disc 8(4) 369-381; Charrier et al. 2011 J Med Chem 54 2341-2350, e.g., 3-aminopyrid-2-ones; Das et al. 2006 Bioorg Med Chem Lett 16 2411-2415 and Das et al. 2006 Bioorg Med Chem Lett 16 3706-3712, e.g., (2-amino-5- [(thiomethyl)aryl] thiazoles); Dubovsky et al., 2013 Blood 122 2539-2549 (ibrutinib); Guo et al. 2012 Mol Pharm 82(5):938-47 (e.g., tricyclic imidazo quinoxalins such as CTA056); Harling et al., 2013 J Biol Chem 288:28195- 28206, e.g., acrylamide-based irreversible ITK inhibitors; Herdemann et al.

2011 Bioorg Med Chem Lett 21 1852-1856; Kaur et al. 2012 Eur J Pharm Sci 47 574-588; Lin et al. 2004 Biochem 43 11056-11062; Lo 2010; Maxwell et al. 2011 Am Chem Soc MEDL8 (e.g., aminobenzothiazoles); Meganathan et al. 2012 J Mol Model 2012 Sep. 27.; Sahu and August 2009 Curr Top Med Chem 690-703; Riether et al. 2009; Vargas et al. 2013 Scand J Immunol 78 130-139; Velankar et al. 2010 Bioorg Med Chem 18 4547-4559; Zapf et al.

2012 J Med Chem 2012 Nov. 12. (e.g., pyrazolopyrimidines); Dong et al. 2015 J Biol Chem. 6 290(10):5960-78 (PRN694); W02002/050071;

W02004/016609 (e.g., substituted pyrrolopyri dines); W02005/026175 (e.g., thienopyrazoles); WO 2007/058832 (e.g., benzimidazoles); W02007/076228 (e.g., 2-(ih-thieno [3,2-c] pyrazol-3yl)-ih-indole derivatives); WO 2011/110575 (e.g., aminobenzothiazoles); US2007/0293499 (e.g., aryl ketones), W02014/082085 (e.g., 2-amino-5-(thioaryl)thiazole, 2-amino-5- [(thiomethyl)aryl] thiazole, 4-arylpyrazolyl indole, 5-arylpyrazolyl indole, benzimidazole, imidazo quinoxalin, or pyrazolopyrimidine); and WO2022/130175 (e.g., imidazopyridine derivatives), the contents of which are hereby incorporated by reference in their entireties.

Suitable ITK inhibitors further include BMS-431051 (Won&Lee, 2008. Int. Rev. Immunol. 27:19—41), BMS-488516 (PubChem CID 11527400) and BMS-509744 (CAS No. 439575-02-7), PF 06465469 (CAS No. 1407966- 77-1), HY-11066 (CAS No. 439574-61-5), CPI-818 (Corvus Pharmaceuticals, Inc. Burlingame, CA, USA; ClinicalTrials.gov identifier NCT03952078; Khodadoust et al. 2020, Blood (2020) 136 (Supplement 1): 19—20, https://doi.org/10.1182/blood-2020-137782), ibrutinib (CAS No. 936563-96-1), bosutinib (CAS. No. 380843-75-4), CTA056 (CAS No: 1265822-30-7), GSK- 2250665A (CAS No. 1246030-96-5), and combinations thereof. Preferred ITK inhibitors are BMS-509744, CPI-818 and ibrutinib.

BMS-509744 (CAS# 439575-02-7) is a cell-permeable aminothioaryl-thiazolo compound that potently inhibits ITK kinase activity in an ATP-competitive manner by stabilizing ITK activation loop in a substrate-blocking, inactive conformation, inhibiting Fyn, IR, Lek, Btk only at high concentrations and exhibiting little or no activity against 14 other kinases.

Ibrutinib, molecular formula C25H24N6O2; chemical name 1- [(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-l- yl]piperidin-l-yl]prop-2-en-l-one; molecular weight-440.50 Da (formerly PCI-32765) is a potent, covalent inhibitor of Bruton's tyrosine kinase (BTK). In clinical studies, ibrutinib has been well-tolerated and has demonstrated profound anti-tumor activity in a variety of hematologic malignancies, most notably chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), leading to US FDA approval for relapsed CLL and MCL.

The ITK inhibitor can be administered to the subject, e.g., a mammal, preferably a human, orally, intravenously, or by injection.

In embodiments of aspects of this invention, the ITK inhibitor is administered intermittent or at a dose that is effective for partial inhibition of ITK enzymatic activity at a dosage of about 1 mg/day to about 1000 mg/day, preferably at a dosage of about 10 mg/day to about 100 mg/day.

The terms “intermittent exposure,” and “intermittent administration” in the context of administration of an ITK inhibitor, may be used interchangeably herein, and refer to a repeated, discontinuous, application of the ITK inhibitor compound to a subject, e.g., human subject, or cells, wherein a desired period of time lapses between applications or administrations, and wherein the therapeutic effect of the compound is discontinuous. Preferably, in said period between applications or administrations, the exposure to the ITK inhibitor compound and therefore the therapeutic effect is absent or at a sub-inhibitory or non-inhibitory level, e.g. the plasma concentrations in said subject are at a sub -inhibitory or non- inhibitory concentration. This may e.g. be achieved by low dosages.

In an embodiment of the invention, the ITK inhibitor is used in aspects of this invention at a (plasma) concentration of from about O.lnM to about 5gM. In an embodiment, the ITK inhibitor is used in the method at a (plasma) concentration of about O.lnM, 0.5nM, InM, 5nM, lOnM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, lOOnM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, lpM, 2gM, 3gM, 4gM, or 5gM The term "exhausted T cell", as used herein, refers to a CD8+ T cell having decreased levels of T-cell-specific transcription factor 1 (TCF1), increased levels of Thymocyte selection-associated high mobility group box protein TOX (TOX), increased surface expression of inhibitory receptors (e.g. PD-1, CTLA-4, TIGIT, Tim-3, Lag-3, CD160, PSGL-1, CD200), reduced capacity to produce cytokine (e.g. IFNy, TNFct and IL-2), reduced cytotoxicity/killing ability, and reduced proliferative capacity, as compared to a CD8+ T cell with normal T cell function that supports a protective immune response. When exhausted CD8+ T cells are reinvigorated these cells express higher levels of TCF1 or a higher percentage of CD8+ T cells are TCF1 positive, they express lower levels of TOX, are TOX negative, or fewer of these cells express TOX. Reinvigorated CD8+ T cells express fewer inhibitory receptors on their surface or express lower levels of these inhibitory receptors, they produce a wider variety and/or higher levels of cytokines, exhibit improved cytotoxic capacity (are more efficient at killing targets) and exhibit improved proliferative capacity.

The abbreviation “TOX”, as used herein, refers to Thymocyte selection-associated high mobility group box protein TOX (Uniprot 094900), which is a protein that in humans is encoded by the TOX gene (Gene ID: 9760).

The abbreviation “TCF1”, as used herein, refers to transcription factor T cell factor 1 (TCF1) (Uniprot P20823) and isoforms thereof, encoded by the Tcf7 gene (Gene ID: 6932).

The abbreviation “CD160”, as used herein, refers to a protein (Uniprot 095971) that in humans is encoded by the CD160 gene (Gene ID: 11126).

The term "resistant cancer" is used interchangeable with the term "cancer resistant to therapy" and refers to both (i) a cancer that is resistant to at least one therapeutic agent, wherein the resistance is acquired after treatment with said at least one therapeutic agent, i.e. a resistance-acquired cancer and (ii) a cancer that is resistant to at least one therapeutic agent wherein the resistance is de novo, i.e. a de novo resistant cancer wherein the resistance is present prior to treatment with said at least one therapeutic agent. The term "resistant cancer" refers to cancer cells that are able to survive in a subject in the presence of at least one therapeutic agent whereas a normal, non-resistant cancer cell would either show signs of cell toxicity, cell death or cellular senescence. The skilled person can easily assess whether a cancer is a resistant cancer, namely by assessing cell viability or apoptosis-inducing activity after bringing a suitable therapeutic agent in contact with a cancer originating from a subject. The skilled person is aware of the existence procedures to determine if a cancer is a resistant cancer. The resistant cancer may be a resistant adrenocortical carcinoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, carcinoid tumor, cardiac (heart) tumors, central nervous system tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, glioblastoma, eye cancer, fallopian tube cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor, head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, lung cancer, lip and oral cavity cancer, male breast cancer, metastatic squamous neck cancer, mouth cancer, nasal cavity and paranasal sinus cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, stomach (gastric) cancer, thyroid cancer, urethral cancer, vaginal cancer, and/or vulvar cancer. The resistant cancer is preferably not a lymphoma, chronic lymphocytic leukemia (CLL) or other B-cell malignancy, including but not limited to small lymphocytic lymphoma (SLL), high risk CLL, non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. The resistant cancer is preferably a melanoma, mesothelioma or pancreatic cancer.

The term “immune checkpoint” refers to molecules that control T- cell immune responses and that are physiologically expressed to downregulate T-cell activation and prevent immune-mediated damage to self. Immune checkpoints (and their human homologue UniprotID) in aspects of this invention include Programmed cell death protein 1 (PD-1) (Uniprot Q15116), Cytotoxic T-lymphocyte antigen 4 (CTLA-4) (Uniprot P16410), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) (Uniprot Q8TDQ0), T cell immunoreceptor with Ig and ITIM domains (TIGIT) (Uniprot Q495A1), Lymphocyte Activation Gene-3 (LAG-3) (Uniprot P18627), and P-selectin glycoprotein ligand 1 (PSGL-1) (Uniprot Q14242). These molecules may be targeted as part of ICB therapy. Further immune checkpoint molecules that may be targeted as part of ICB therapy include inhibitory checkpoint molecules A2AR & A2BR (Adenosine A2a and A2b receptors) (resp. Uniprot P29274 and P29275), B7-H3 (CD276, with checkpoint inhibitor enoblituzumab) (Uniprot Q5ZPR3), B7-H4 (VTCN1) (Uniprot Q7Z7D3), BTLA (B and T Lymphocyte Attenuator or CD272) (Uniprot Q7Z6A9), IDO (Indoleamine 2,3-dioxygenase) (Uniprot P14902 or Q6ZQW0), KIR (Killer-cell Immunoglobulin-like Receptor, with checkpoint inhibitor lirilumab) (e.g. Uniprot Q8N743), NOX2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 ) (Uniprot P04839), VISTA (V-domain Ig suppressor of T cell activation) (Uniprot Q9H7M9), and SIGLEC7/9 (Sialic acid-binding immunoglobulin-type lectin 7 or 9, resp. CD328 or CD329) (Uniprot Q9Y286 and Q9Y336, resp.).

The term "immune checkpoint inhibitor", or ICI, means a compound that inhibits immune checkpoint molecules, including checkpoint molecules, as well as ligands of these molecules, such as PD-L1 or PD-L2. ICIs are used as therapeutic compounds in ICB therapy to decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of the immune checkpoint molecules (such as PD-1) with one or more of its binding partners (such as PD-L1). An ICI reduces the negative co- stimulatory signal mediated by or through immune checkpoint proteins expressed on T lymphocytes so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In aspects of this invention, the ICI may include antibodies against immune checkpoint molecules, antigen binding fragments of such antibodies, immunoadhesins, fusion proteins, such as fusion proteins comprising (parts) of immune checkpoint molecules or binding partners thereof, oligopeptides, or antibodies against binding partners of immune checkpoint molecules and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of the immune checkpoint molecule with its binding partner.

In some preferred embodiments, the ICI is an anti-PD-1 antibody. Examples of preferred anti-PD-1 antibodies for use in aspects of this invention include nivolumab, camrelizumab, cemiplimab, dostarlimab, MEDI0680, pembrolizumab, prolgolimab, retifanlimab, sasanlimab, spartalizumab, STI -All 10, tislezlizumab, and toripalimab. In some preferred embodiments, the ICI is an anti-PD-Ll antibody. Examples of preferred anti-PD-Ll antibodies for use in aspects of this invention include atezolizumab, avelumab, durvalumab, KD033, and STI-A1014. In some preferred embodiments, the ICI is an anti-CTLA-4 antibody. Examples of preferred anti-CTLA-4 antibodies include ipilimumab, tremelimumab, and botensilimab. In some preferred embodiments, the ICI is an anti-Tim-3 antibody. Examples of preferred anti-Tim-3 antibodies for use in aspects of this invention include cobolimab and sabatolimab, surzebiclimab, Sym023 (Symphogen A/S), R07121661 (Hoffmann-La Roche), LY3321367 (Eli Lilly and Company), ICAGN02390 (Incyte Corporation), and BMS-986258 (Bristol-Myers Squibb). In some preferred embodiments, the ICI is an anti- TIGIT antibody. Examples of preferred anti-TIGIT antibodies for use in aspects of this invention include tiragolumab, domvanalimab, vibostolimab, ociperlimab, BMS-986207, COM-902 (Compugen Ltd), AGEN-1307 (Agenus Inc.). In some preferred embodiments, the ICI is an anti-LAG3 antibody. Examples of preferred anti-LAG3 antibodies for use in aspects of this invention include ieramilimab and relatlimab. In some preferred embodiments, the ICI is an anti-CD200 antibody. Examples of preferred anti-CD200 antibodies for use in aspects of this invention include samalizumab. In some preferred embodiments, the ICI is an anti-PSGL-1 antibody. Examples of preferred anti-PSGL-1 antibodies include VTX-0811 (Verseau Therapeutics Inc.) and SelK2 (Selexys Pharmaceuticals),

In preferred aspects of this invention, the ICB therapy comprises the use of ICI targeting different immune checkpoints simultaneously, such as PD-1 and TIMIT, PD-1 and LAG-3, PD-1 and Tim-3, PD-1 and PSGL-1, CTLA-4 and PD-1, etc.. For such combined ICB therapies, bispecific antibodies may also be used. The functional impairment that accompanies CTL exhaustion represents a significant barrier for efficient immunity in chronic infections and cancer. Immunotherapies that aim to reinvigorate exhausted CTL can contribute to tumor control, especially in melanoma, but also in other cancers. Up until now, the most clinically successful immunotherapy for solid tumors has been the blocking of the PD-1/PD-L1 inhibitory receptor pathway. Progenitor exhausted CTLs are critical for this ICB efficacy, and are defined as transcription factor T cell factor 1 (TCF1) positive. Despite ICB’s success, there still remains a large proportion of patients that do not benefit from ICB therapy. Therefore, developing novel treatments and treatment strategies that could improve the efficiency of ICB in ICB resistant cancers is greatly needed.

Chronic antigen-mediated TCR stimulation is a driving force of T cell exhaustion. Therefore, any strategy that dampens this persistent TCR stimulation would be expected to ameliorate CTL exhaustion. Therefore, targeting components of the TCR signaling pathway can potentially reduce such exhaustion. ITK is a critical tyrosine kinase that regulates TCR signaling and blocking it inhibits T cell activation. Although continuous ITK inhibition may be detrimental to T cell immunity, intermittent inhibition may allow enough activation to ensure function is preserved while preventing excessive activation that leads to CTL exhaustion. There is potentially indirect evidence that ITK inhibition is beneficial for ICB therapy. This arises from ibrutinib, a potent inhibitor of BTK, which is a critical kinase for normal B cell development and function, but also malignant B cell survival. Ibrutinib is an efficient therapy in chronic lymphocytic leukemia (CLL) and other B-cell malignancies. The anti-tumor activity of ibrutinib has been attributed to its direct effect on malignant B cells, but there is cumulative evidence suggesting that it also affects T cell immunity. Ibrutinib treatment reduces chronic activation markers, like CD39 and HLA-DR, on T cells in leukemia patients. Furthermore, Ibrutinib improves CD 19 chimeric antigen receptor T-cell (CAR T cell) viability and expansion, decreases PD-1 expression on CAR T cells, and this is accompanied by improved clinical responses. T cells in ibrutinib treated CLL patients have reduced PD-1 expression and improved cytokine production, indicating possible effects on T cell exhaustion. Finally, in mouse lymphoma and CLL models, ibrutinib can synergize with anti-PD-Ll treatment. However, the mechanism by which ibrutinib affects T cells in hematopoietic tumors remains obscure. This effect could be either direct or indirect. As mentioned, chronic antigen stimulation is a driving force of T cell exhaustion, therefore by acting on malignant B cells and reducing tumor load, ibrutinib could be reducing chronic antigen stimulation and thus indirectly affecting T cell exhaustion. On the other hand, ibrutinib can also indirectly affect T cell exhaustion by reducing PD-L1 expression in CLL or other immune cells, such as myeloid cells that also express BTK, and thus indirectly mitigate T cell exhaustion. The question remains, however, whether in addition to indirect effects on T cells, ibrutinib can deliver direct effects on T cells despite BTK not being expressed in T cells. Besides BTK, ibrutinib also targets other TEC-family tyrosine kinases, such as the bone marrow-expressed kinase (BMX) and ITK. By acting on ITK, ibrutinib could be tempering TCR signaling in the face of chronic antigen stimulation, thus mitigating T cell exhaustion.

In the Example described below, the present inventors investigated whether ITK inhibition can improve ICB therapy in ICB resistant tumors. It was found that in vivo intermittent ITK inhibitor treatment, as described herein, synergizes with ICB in different resistant solid tumors. Furthermore, inhibiting ITK with the selective ITK inhibitor BMS-509744 or with the non-selective ITK inhibitor ibrutinib can directly improve key functional and molecular aspects of T cell exhaustion in an in vitro CTL exhaustion model. The present invention shows that intermittent inhibition of ITK could be a strategy to reinvigorate exhausted CTLs and improve anti-tumor immunotherapies in resistant cancers.

The present invention now provides a pharmaceutical combination. The term “pharmaceutical combination”, as used herein, defines either a fixed combination in one dosage unit form or a kit of parts for the combined administration where the ITK inhibitor and the immune checkpoint inhibitor may be administered independently at the same time or separately within time intervals that allow that the combination partners show a cooperative, e.g., synergistic, effect.

The term “a combined administration” is defined herein to include reference to a “kit of parts” in the sense that the combination of active compounds can be dosed independently or by use of different fixed combinations with distinguished amounts and distinguished administration regimes of the combination partners, i.e., simultaneously or at different time points. The parts of the kit of parts can then e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partner (ITK inhibitor) to the combination partner (ICI) to be administered in the combined preparation can be varied, e.g., in order to cope with the needs of a patient sub-population to be treated or the needs of the single patient.

The term “combined administration” as used herein further includes the administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

The term “treating” or “treatment” as used herein comprises a treatment relieving, reducing or alleviating at least one symptom in a subject or effecting a delay of progression of a disease. For example, treatment can be the diminishment of one or several symptoms of a disorder or complete eradication of a disorder, such as cancer. Within the meaning of the present invention, the term “treat” also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.

In a pharmaceutical combination for use in treating cancer according to the present invention, the ITK inhibitor may be formulated for intermittent administration. In an intermittent administration, the ITK inhibitor may be administered at a dose of about 1-10 mg/kg body weight per day. In embodiments of intermittent administration aspects of this invention, each treatment interval may for instance comprise 1-7 days, such as 3 days, of continuous treatment followed by 1-7 days, such as 3 days, of non-treatment (off treatment). Such a treatment interval may be repeated, for instance 3 times over a period of 18 days. During or after a treatment period with an ITK inhibitor, the reversal of exhaustion of T cells is preferably assessed, as the intermittent administration is aimed to reverse exhaustion, but thereafter allowing (re-)activation of T cells, as continuous or unhalted ITK inhibition would inhibit such activation. The inhibition of activation in an intermittent administration regimen of an ITK inhibitor may therefor only be temporary, in order to allow the exhausted T cells to revert to a non-exhausted state. Administration of an ITK inhibitor should be halted once the T cells reach a non-exhausted state. This may be determined by the use of exhaustion markers as described herein. During the period that administration is halted, the T cells may be activated whereby the immune response to cancer is restored.

In a pharmaceutical combination for use in treating cancer according to the present invention, the ITK inhibitor may also be formulated for administration at a dose that partially inhibits ITK enzymatic activity in T cells. Partially inhibition of ITK enzymatic activity could be continuous, at a low dose, and may be simultaneous, separate or sequential to the administration of the ICI inhibitor as part of ICB therapy. Partially inhibition of ITK enzymatic activity is aimed at balancing the population of T cells in allowing some T cells to revert from exhausted state, while at the same time allowing other (exhaustion reverted) T cells to be activated.

A pharmaceutical combination in aspects of this invention may comprise the active compounds, e.g., in the form of a free base or a pharmaceutically acceptable salt thereof.

A “pharmaceutical combination” in aspects of this invention includes reference to two separate “pharmaceutical compositions”, whereby the term “pharmaceutical composition” refers to a mixture of one or more of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, such as pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

“Pharmaceutically acceptable excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

“Pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the parent compound. Such salts may include: (1) acid addition salt which is obtained by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D)- or (L)-malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like, preferably hydrochloric acid or (L) -malic acid; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

The compounds used in aspects of this invention may also be used in the form, or be designed in the form, of a prodrug. A “prodrug” refers to an agent, which is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. An example, without limitation, of a prodrug would be a compound, which is, administered as an ester (the “prodrug”), phosphate, amide, carbamate, or urea.

“Therapeutically effective amount” refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated. In reference to the treatment of cancer, a therapeutically effective amount refers to that amount which has the effect of: (1) reducing the size of the tumor; (2) inhibiting tumor metastasis; (3) inhibiting tumor growth; and/or (4) relieving one or more symptoms associated with the cancer.

As used herein, “administer” or “administration” refers to the delivery of an inventive compound or of a pharmaceutically acceptable salt thereof or of a pharmaceutical composition containing an inventive compound or a pharmaceutically acceptable salt thereof of this invention to an organism for the purpose of prevention or treatment of a protein kinase- related disorder.

Suitable routes of administration may include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injections. In certain embodiments, the preferred routes of administration are oral and intravenous. Alternatively, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor- specific antibody. In this way, the liposomes may be targeted to and taken up selectively by the tumor.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in any conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the compounds used in the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient. Pharmaceutical preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding other suitable auxiliaries if desired, to obtain tablets or dragee cores. Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, maize starch, wheat starch, rice starch and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers may be added in these formulations, also. Pharmaceutical compositions which may also be used include hard gelatin capsules. The capsules or pills may be packaged into brown glass or plastic bottles to protect the active compound from light. The containers containing the active compound capsule formulation are preferably stored at controlled room temperature (15-30° C.). The compounds may also be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating materials such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of a water soluble form, such as, without limitation, a salt, of the active compound. Additionally, suspensions of the active compounds may be prepared in a lipophilic vehicle. Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers and/or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. A compound of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materials (for instance, in an emulsion with a pharmacologically acceptable oil), with ion exchange resins, or as a sparingly soluble derivative such as, without limitation, a sparingly soluble salt.

A non-limiting example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer and an aqueous phase such as the VPD cosolvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD cosolvent system (VPD: D5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This cosolvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of such a cosolvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the cosolvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80, the fraction size of polyethylene glycol may be varied, other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone, and other sugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. In addition, certain organic solvents such as dimethylsulfoxide also may be employed, although often at the cost of greater toxicity.

Additionally, the compounds may be delivered using a sustained- release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The pharmaceutical compositions herein also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Many of the ITK inhibitory compounds may be provided or administered in aspects of this invention as physiologically acceptable salts wherein the compound may form the negatively or the positively charged species. Examples of salts in which the compound forms the positively charged moiety include, without limitation, quaternary ammonium, salts such as the hydrochloride, sulfate, carbonate, lactate, tartrate, malate, maleate, succinate wherein the nitrogen atom of the quaternary ammonium group is a nitrogen of the selected compound of this invention which has reacted with the appropriate acid. Salts in which a compound of this invention forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the compound with an appropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH)₂ ), etc.).

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose, e.g., the modulation of protein kinase activity and the treatment or prevention of the malignant disorder.

More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any compound used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of ITK, or surrogate marker activity). Such information can then be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein is within the realm of common general knowledge, e.g., both the IC50 and the LD50 for the ITK inhibitors or ICIs is readily available from public data sources. These data may be used to optimize the efficacy of the pharmaceutical combination or combination therapy as disclosed herein, and combination therapy in cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS , Ch. 3, 9th ed„ Ed. by Hardman, J„ and Limbard, L., McGraw-Hill, New York City, 1996, p. 46.)

Dosage amount and interval may be adjusted individually to provide plasma levels of the active species which are sufficient to maintain the kinase modulating and immune checkpoint blocking effects. These plasma levels are generally referred to as minimal effective concentrations (MECs). The MEC will vary for each compound but can be estimated from in vitro data, e.g., the concentration necessary to achieve 50-90% inhibition of ITK may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.

Suitable therapeutically effective amounts of ITK inhibitors during the course of intermittent administration wherein ITK is inhibited, may range from approximately 2.5 mg/m² to 1500 mg/m² of subject body surface per day. Additional illustrative amounts range from 0.2-1000 mg/day, 2-500 mg/day, and 20-500 mg/day, such as 140-420 mg/day, or about 250 mg/day, preferably per os (PO).

In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration, and other procedures known in the art may be employed to determine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

In a method of treating a subject suffering from an ICB resistant tumor, a therapeutically effective amount of an ITK inhibitor, and a therapeutically effective amount of an immune checkpoint inhibitor, are administering to the subject. These compounds can be administered separately, sequential or simultaneaously. The ITK inhibitor treatment may be started prior to ICI treatment, or may be provided as a supplement to an ongoing treatment with an ICI inhibitor. Preferably the ICI treatment is started after the ITK inhibitor treatment, preferably about 1-7 days thererafter. As explained herein, the ITK inhibitor is administered intermittently to said subject, or at a dose that partially inhibits ITK enzymatic activity in CD 8+ T cells of said subject. The compounds are preferably administered in the form of a pharmaceutical combination according to the present invention. Preferably the administration of the pharmaceutical combination comprises the separate administration of an ITK inhibitor and an ICI, as the dosing regimes of these compounds will differ.

In a method of reversing T cell exhaustion resulting from persistent TCR stimulation, in accordance with the present invention, an exhausted T cell, as defined herein, is exposed to an effective amount of an ITK inhibitor. The effective amount in this context may be determined by experimentation, wherein, for instance, the expression of TCF1, TOX, PD-1, CTLA-4, TIGIT, Tim-3, Lag-3, CD160, CD200, IFNy, TNFct and/or IL-2 is measured on T-cells. One of skill in the art is aware of how such expression is to be determined, for instance by using anti-CD8, anti-Lag3, anti-PD-1, anti-Tim3, anti-TIGIT, anti-IFN-y, anti-TNF-a, anti-IL-2, anti-Tox, and anti-TCFl antibodies are used in combination with, for instance, flow cytometric evaluation as described in the Examples below. Alternatively, or in addition, RNA-seq may be used to determine the transcriptional profile of T-cells.

The above uses and methods can also be carried out in combination with radiation therapy or chemotherapy, wherein the amount of an inventive compound in combination with the radiation therapy or chemotherapy is effective in treating the cancer. Techniques for administering radiation therapy or chemotherapy are known in the art, and these techniques can be used in the combination therapy described herein. The administration of the compound of the invention in this combination therapy can be determined as described herein. EXAMPLES

Example 1: Overcoming immune checkpoint blockade resistance in solid tumors with ITK inhibition

In this Example, the effect of ITK inhibitor BMS- 509744 is tested in three ICB-resistant solid tumor (melanoma, mesothelioma or pancreatic cancer) C57BL/6 mouse models. To mechanistically understand how BMS- 509744 and ibrutinib, another inhibitor that can target ITK, can enhance immunotherapies, we used an in vitro exhaustion model to examine whether they directly act on T cells and reinvigorate exhausted CTL in vitro. Inhibitory receptor expression, transcription factors, cytokine production and the transcriptional profile of in vitro exhausted CTLs were analyzed by flow cytometry and RNAseq.

Methods

Animals

C57BL6/J mice were purchased from Charles River, France. Inhouse-bred OT-I CD45.1+ mice on the C57BL6/J background were generated by backcrossing C57BL/6 Tg (TcraTcrb) 1 lOOMjb/J (OT-I) with B6.SJL-Ptprca Pepcb/BoyJ (CD45.1+) mice (both from Charles River France). Btk deficient (btk-/-) OT-I mice were generated by cross breeding OT-I CD45.1+ mice with btk deficient C57BL/6J (Hendriks et al., 1996. EMBO J. 15(18):4862-72). Btk-/- OT-I mice were housed in a certified barrier facility at Erasmus University Medical Center. OT-I CD45.1 expression was confirmed using PCR and flow cytometry analysis. Btk-/- deficiency was confirmed by PCR. All experiments in this study were carried out under ethical approval by the Instantie voor Dierenwelzijn (IvD). The Project Proposal (AVD101002015179) was approved by CCD (Centrale Commissie Dierproeven). In vitro CTL exhaustion induction and inhibitor treatment

We have previously described in detail the induction of in vitro exhausted murine CD8+ T cells (Zhao et al., 2020. PLoS Pathog. 16(6):el008555). In brief, CD8+ T cells were purified from splenocytes of an OT-I mouse based on negative selection (EasySep, Stemcell Technologies). Repeat peptide stimulated cells were induced by adding OVA peptide (2 ST- 264) (Anaspec) daily for five days with cells being split on day 4. For single peptide stimulated cells, OVA peptide (257-264) was added once to the cells for 48 hours and washed away, followed by resting for 3 days. After a total of 5 days, cells were harvested and phenotypically and functionally characterized to analyze their exhaustion status. From day 5, single peptide and repeat peptide stimulated cells were treated with different concentrations, as indicated in the figures, of ibrutinib (MedChemExpress, No. HY10997) or BMS-509744 (ITK inhibitor) (MedChemExpress, No. HY11092). On day 8, cells were harvested and either stained or reactivated with peptide. Concentrations of ibrutinib used in this study were based on results from previous clinical studies, which indicate a peak plasma concentration of ibrutinib between 340 nmol/L and 450 nmol/L after oral administration (Byrd et al., 2016. N Engl J Med. 374(4):323-32. Byrd et al., 2013. N Engl J Med. 369(l):32-42). In addition, our preliminary experiments indicate that lpM of ibrutinib does not induce cell death.

Tumor cell culture and tumor models

The B16-F10-OVA melanoma cell line (Sanquin, Amsterdam) was cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco), 2 mM glutamine (Life Technologies), lOOU/ml penicillin (Gibco), lOOgg/ml Streptomycin- sulfate (Gibco), and 50 pM B-mercaptoethanol (Sigma). In the T cell adoptive transfer experiments, 6-weeks-old C57BL/6J mice received 0.5*10⁶ B16-F10-OVA melanoma cells per mouse subcutaneously in the shaved right flank. Adoptive transfer of 0.5*10⁶ CD8+ T cells from naive OT-I CD45.1+ mice was performed on day 7 when the tumors reached an average size of 60-80 mm³. All of the treatments started from day 12 post tumor injection.

AE-17 cells (Dr. Delia Nelson, Curtin University, Perth, Australia) were maintained in RPMI 1640 supplemented with 10% FBS, 48 mg/L Gentamicin, 60 mg/L benzylpenicillin, 2 mM L-glutamine and 0.05 mM 2-mercaptoethanol (Sigma). Murine Pancreatic tumor cell line 4662 (Pancreatic Ductal Adenocarcinoma, PDA) (kind gift from Prof. Robert H. Vonderheide, University of Pennsylvania, PA, USA) was cultured in DMEM supplemented with 10% FBS, 100 units/mL Penicillin/Streptomycin, 2 mM Glutamine, and 100 mg/L Gentamicin (Sigma). 6-7 weeks old C57BL/6 mice received 0.5*10⁶ of either AE-17 or PDA 4662 cells subcutaneously in the flank. Due to the variability in tumor growth between cell lines, treatment was started for the two different tumor cell lines at different time points when the tumor reached an average size of 60-100 mm³. The 4662 cell line grew faster in vivo, and therefore the treatment was started on day 5 - 8 post tumor injection. For the AE-17 cell line, the treatment for the mice started from day 20 post tumor cell injection. Size of the tumors were measured every other day using a digital vernier caliper. The volume of tumor was calculated using the formula: V = L*W*H, where V is tumor volume, L is the length of the tumor (longer diameter), W is the width of the tumor (shorter diameter) and H is the height of the tumor. Mice were monitored for tumor growth and survival. Mice were euthanized and organs were harvested when the tumor volume reached 1500mm³, or when they met the humane endpoint criteria defined in the project proposal such as necrosis.

In vivo treatment of mice with inhibitors

The ITK inhibitor BMS-509744 (MedChemExpress, cat. No. HY11092) was injected intraperitoneally (i.p.) at a dose of 5mg/kg per day. Each treatment interval consisted of 3 days continuous treatment followed by 3 days off treatment, with this interval repeated 3 times over a period of 18 days. Anti-PD-1 antibodies (RMP1-14, cat: 114119, Biolegend) or isotype control antibodies (RTK2758, cat: 400565, Biolegend) were i.p. injected at a dose of lOOpg/mouse, twice per week. Treatment of mice was started when tumors reached an average size of 60- 100mm³.

Tissue collection and sample preparation

When the tumor volumes reached 1500mm³ or on the end date of the experiments, tumor draining lymph nodes were harvested. Single cell suspensions were obtained after processing these tissues. As described previously (Hope et al., 2019. Front Immunol. 10:3074), lymph nodes were mechanically dissociated and filtered through a 40-pm cell strainer (Falcon). After washing two times with medium (RPMI medium containing 5% heat- inactivated FBS, and 2 mM L-glutamine), single cell suspensions were obtained. Cells were counted using Trypan blue on an automated cell counter (Countess, Life Technologies).

Flow cytometry

To exclude apoptotic and dead cells, Annexin V conjugated with either APC, Cy5.5 or PerCP-Cy5.5 (BD Biosciences) was included in all stains and 2.5 mM calcium chloride (CaC12) was added to all solutions and washes. The following fluorochrome-conjugated monoclonal antibody combinations against surface and intracellular antigens were used to stain the in vitro or ex vivo harvested cells: anti-CD8a-eFluor 450 (53-6.7, eBioscience), anti-CD4-BV510 (RM4-5, Biolegend), anti-CD160-PE-CF594 (CNX46-3, BD Biosciences), anti-Lag3-APC (C9B7W, BD Biosciences), anti- CD244-PE (2B4, BD Biosciences; eBio244F4, eBioscience), anti-PD-l-APC- Cy7 (19F.1A12, Biolegend), anti-Tim3-PE-Cy7 (RMT3-23, Invitrogen), anti- TIGIT-FITC (GIGD7, eBioscience); anti-CD44-BV786 (IM7, BD Biosciences), anti-CD45.1-FITC (A20, BD Biosciences), anti-CD45.1-PE-CF594 (A20, BD Biosciences), anti-IFN-y-APC (XMG1.2, eBioscience), anti-TNF-ct-AF488 (MP6-XT22, eBioscience), anti-IL-2-PE (JES6-5H4, eBioscience), anti-Tox- PE (TXRX10, eBioscience), anti-TCFl-A647 (C63D9, Cell Signaling). PE- conjugated tetramers of H-2Kb major histocompatibility complex class I loaded with OVA(257— 264) were used to identified the endogenous antigenspecific CTLs.

For surface staining, cells were washed with FACS wash (HBSS containing 3% FBS and 0.02% sodium azide) and incubated with Fc receptor blocking antibody (2.4G2, BD Biosciences) for 10 minutes on ice, followed by pre- determined optimal concentrations of the fluorochrome-conjugated monoclonal antibodies at 4°C in the dark for 20 minutes. The cells were then washed one time with FACS wash and fixed with 1% PFA. For the intranuclear staining of Ki-67 and transcription factors, cells were first stained for surface antigens as described above. After washing, cells were fixed with FoxP3 Fixation Buffer (005523, eBioscience) for 60 minutes in the dark at 4°C, washed with Perm/Wash buffer (008333, eBioscience) and stained with a mix of antibodies against transcription factors and Ki-67 for 45 minutes at 4°C in the dark. Cells were then washed twice with Perm/Wash buffer and fixed with 1% PFA. Appropriate isotype controls were included for staining of transcription factors.

To analyze phosphorylation of the in vitro exhausted CTLs, cells were first induced to be exhausted by in vitro CTL exhaustion induction method. On day 5, exhausted CTLs were treated with ibrutinib or BMS- 509744 or left without treatment after washing away OVA peptide. On day 6, cells were harvested and fixed immediately with pre-warmed Fixation buffer (420801, Biolegend) for 20 minutes at 37°C. After washing with Cell Staining buffer (420201, Biolegend), cells were permeabilized by adding prechilled True-Phos™ Perm Buffer (425401, Biolegend) overnight at -20°C. The next day, cells were stained with Anti-BTK Phospho (Tyr223)/ITK Phospho (Tyrl80) antibody (A16128C, Biolegend) , anti-PLCyl Phospho (Tyr783) antibody (A17025A, Biolegend) or isotype antibody (MOPC-21, Biolegend) for 40 minutes at room temperature in the dark. After staining, cells were washed twice with Cell Staining buffer and fixed with 1% PFA.

In order to detect cytokine production, in vitro cultured cells or the ex vivo samples were re-stimulated with lOgg/ml OVA(257-264) SIINFEKL peptide for 6 hours at 37°C, 5% CO2 in the presence of GolgiPlug (BD Biosciences). Cells were then stained with surface antibodies as described above. After washing with FACS wash, cells were fixed with IC Fixation Buffer (88-8824, eBioscience) at 4°C overnight, washed with Perm/Wash buffer and stained for intracellular cytokines for 45 minutes in the dark at 4°C. After staining, cells were washed twice with Perm/Wash buffer and fixed with 1% PFA. All the samples were measured within 48 hours after fixation.

Samples were measured on a LSRFortessa (BD Biosciences) using application settings and at least 200,000 targeted events for in vitro samples and 500,000 targeted events from ex vivo samples were collected. Data was then analyzed with FlowJo software (Version 9.9.4, Treestar, Ashland, OR, USA).

Western blot assay

Btk+/- and WT OT-I cells were harvested from in vitro exhaustion inducing cultures on day 5. Btk-/- and WT splenocytes were used as control. Western blot was performed as previously described (Hope et al., 2017 Frontiers in Immunology 8(1696)). In brief, cell pellets were washed twice with PBS buffer and then lysed in RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0). The total protein concentration was determined by using the Pierce BCA Protein Assay Kit (Thermo Scientific, 23227). The same amount of proteins were loaded into a 4-15% Mini-PROTEAN TGX Gel (Bio-Rad, 456-1084) and transferred to PVDF membrane (Merck Millipore, IPVH00010). Membranes were blocked with 5% BSA in Tris-buffered saline with tween (TBST), primary antibodies were incubated overnight at 4°C with in TBST as follows: anti-BTK (Cell Signaling, D3H5), anti-B-Actin (Cell signaling, 8H10D10). Membranes were washed and incubated with secondary antibodies as follows: Goat anti-Mouse IgG (LI-COR Biosciences, 926-32210) and Goat anti-Rabbit IgG (LI-COR Biosciences, 926-68071) in 3% milk/TBST for 1 h at room temperature, and imaged using an Odyssey imaging system.

RNA sequencing

Using in vitro exhaustion model, single peptide stimulated and repeat peptide stimulated cells were treated from day 5 to day 8, with DMSO, Ibrutinib or BMS-509744 as described above. On day 8, 0.5-l*10^A6 live CD8+ T cells were washed with PBS and immediately lysed with TRIzol LS reagent (Life Technologies) and stored at -80°C. RNA was extracted according to manufacturer’s instructions and a bioanalyzer (Agilent) was used to determine the quality and quantity of the extracted RNA. Barcoded sequencing libraries were generated using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760S/L). mRNA was isolated from total RNA using the oligo-dT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplification of the resulting product. The quality and yield after sample preparation was measured with the Fragment Analyzer. The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Paired-end sequencing was performed on a Hiseq2500 machine (Illumina) for 150 cycles.

Paired-end raw FASTQ files were analyzed with the nf-core/RNA- seq pipeline (v3.0) using Nextflow (v20.11.0-edge) and its default settings (Ewels et al., 2020 Nat Biotechnol. 38(3):276-78; Di Tommaso et al., 2017. Nat Biotechnol. 35(4):316-19). Quality of the sequencing was reported with FastQC (vll.9). Subsequently, bases with low Phred scores (<= 30) were trimmed and the reads with a low mean Phred score were removed using Trim Galore! (v6.6). Trimmed FASTQ reads were mapped to the mouse reference genome version GRCm38 and Ensembl GRCm38.81.gtf gene annotation file using RSEM (vl.3.1), which umbrellas STAR (v2.7.6a) as read aligner. Next, SAMtools (v 1.10) processed the alignment files and extracted mapping statistics for post-alignment stats (Li & Dewey, 2011. BMC Bioinformatics 12:323; Dobin et al., 2013. Bioinformatics 29(1):15-21; Li et al., 2009. Bioinformatics 25(16):2078-9). Quality of each sample alignment was visually inspected using reports derived from RSeQC (v3.0.1), Qualimap (v2.2.2-dev) and Preseq (v2.0.3), including read inner distance plots, splice junction annotations, the genomic origin of the mapped reads, and the estimated complexity of the sequencing library (Wang et al., 2012. Bioinformatics 28(16):2184-5; Okonechnikov et al., 2016.

Bioinformatics 32(2):292-4; Daley & Smith, 2013. Nat Methods 10(4):325-7). RSEM estimated transcript counts were imported into R (v4.0.3), transformed to gene counts using tximport (vl.18) and analyzed with DESeq2 (vl.30.0) (Soneson et al., 2015. FlOOORes. 4:1521; Love et al., 2014 Genome Biol. 15(12): 550). Only “protein_coding” and “IncRNA” genes were kept using biomaRt (v2.46.0) annotation (Durinck et al., 2009. Nat Protoc. 4(8): 1184-91). Gene counts were transformed using the “rlog” function of DESeq2 for visual inspection and for Principal Component Analysis (PCA) utilizing its “plotPCA” function.

Differential expression analysis

Differentially expressed genes were calculated using DESeq2. p- values were calculated using Wald statistical tests, and either the single peptide experiments or DMSO control were defined as the reference. Fold Changes (FC) were shrunk with the DESeq2 function “Ifcshrink” using method “apeglm” [Zhu A, et al. 2019. Bioinformatics 35(12):2084-92],

Genes were indicated as differential expressed with an adjusted p-value < 0.05, calculated with the Benjamini- Hochberg multiple hypothesis testing method, a BaseMean > 50 counts, and an absolute log2FC > 0.2. Heatmaps were made using the R package pheatmap (vl.0.12), Z-scores were calculated per gene on the rlog counts. Hierarchical clustering was performed using complete clustering and Euclidean distances for both the samples and genes in heatmaps with all statistical significant differentially expressed genes. In the heatmap with a subset of exhaustion-related genes the samples were clustered using the Ward algorithm with a Pearson's correlation coefficient matrix.

Gene Set Enrichment Analysis (GSEA) was performed with the complete gene- set after shrinkage of the fold changes (Subramanian et al., 2005. PNAS 102(43):15545-50]. Genes were ranked on the FC and compared with CD8+ T cell specific exhaustion gene-sets. The CD8+ T cell exhaustion gene-sets were based on the publication of Bengsch B, et al. and downloaded from PubMed Central (Immunity. 2018;48(5): 1029-45 e5). Enrichment plots were made using R package fgsea (vl.16.0) with 100.000 permutations. The GSEA was calculated separately for the upregulated and downregulated genes. Ingenuity Pathway Analysis (IPA, Qiagen) was performed on all significant differentially expressed genes using IPAs default parameters.

Statistics

Statistical analysis was performed using Prism software (GraphPad Prism 9, Version 9.0.0). Normal distribution of the data was evaluated by Shapiro- Wilk normality test. For the paired samples, two- tailed, paired-t test was used for normally distributed data and Wilcoxon matched-pairs signed rank test for not normally distributed data. As the in vivo data was normally distributed, one way ANOVA with Tukey's post-test was used, for the non-normally distributed data, two-tailed Mann Whitney test was utilized as mentioned in the figure legends. Tumor growth curves were compared using two-way ANOVA with Tukey's multiple comparison test. Kaplan-Meier survival analysis was used to determine the survival of treated tumor bearing mice. P values lower than 0.05 were considered statistically significant with the numbers of stars in the figures indicating the p value: * P<0.05, ** P<0.01 and *** P<0.001 and **** P<0.0001.

Results

In vivo treatment of three ICB-resistant solid tumors with BMS- 509744 significantly improved ICB immunotherapy. This was accompanied by increased draining lymph node TCF1+ anti-tumor CTL, the cells that respond to ICB. Treatment of in vitro exhausted T cells with BMS-509744 and ibrutinib enhanced cytokine production, decreased inhibitory receptor expression, and downregulated the transcription factor TOX while upregulating TCF1. RNA-seq revealed that inhibitors directly reduced the exhaustion related transcriptional profile of these cells.

ITK inhibition enhances the anti-tumor effect of checkpoint blockade therapy Since ICB mediates its therapeutic effect by reinvigorating exhausted CTL and chronic TCR signaling is a major driver of such exhaustion (Bucks et al. 2009. J Immunol. 182(ll):6697-708; Utzschneider et al., 2016. J Exp Med. 213(9):1819-34), we investigated whether inhibiting ITK, an important kinase that regulates TCR signaling, would enhance the anti-tumor effect of ICB in ICB-resistant C57BL/6 mouse tumor models. For this we used BMS-509744, a selective ITK inhibitor. To avoid continuous ITK inhibition from blocking T cell activation and impairing T cells responses, we administrated the inhibitor intermittently using a 3-day cycle. We first tested animals injected with the incompletely ICB refractory B16-F10-OVA melanoma tumor that expresses SIINFEKL peptide of ovalbumin (Sanchez-Paulete et al., 2016. Cancer Discov . 6(l):71-9), which 7 days later received adoptive transfers of OT-I T cells that recognize the SIINFEKL peptide of ovalbumin (Fig 1A). We treated animals with established tumors with BMS-509744 after day 12 of tumor injection to avoid inhibition of naive T cell priming. As expected, B16-F10-OVA melanoma was only partially sensitive to anti-PD-1 therapy, moreover, the addition of ITK inhibitor treatment further improved tumor growth inhibition (Fig IB and 1C left) and survival (Fig 1C right). At the end of the experiment, 0% of mice that received isotype control treatment and 15.4% of mice with ITK inhibitor treatment alone survived. Treatment with anti-PD- 1 therapy alone increased survival to 38.5% and this was even further improved by combining ITK inhibitor with anti-PD-1 therapy to 72.7% survival on day 35 (Fig 1C right). When CD8+ T cells in draining lymph nodes were assessed at the time that animals reached the predefined endpoint, more donor OT-I cells could be found in the tumor draining lymph node of animals treated with ITK inhibitor and nearly all of these cells maintained TCF1 expression(Fig ID). Taken together, these data illustrated that ITK inhibitor improves the antitumor effects of ICB, by increasing pre- terminally exhausted TCF1+ tumor- specific cells, cells known to be required for the protective effect of ICB, in the draining lymph nodes.

ITK inhibition enhances anti-tumor effects of ICB in resistant tumors We next evaluated the anti-tumor effects of ITK inhibitor in combination with ICB, in two tumors insensitive to ICB therapy, the AE17 mesothelioma and 4662 pancreatic tumor cell lines (Morrison et al., 2018. Trends Cancer. 4(6):418-28; De La Maza et al., 2017. Clin Cancer Res. 23(18):5502-13). With the AE17 tumor, the treatment started 20 days after the inoculation of tumor (Fig 2A) and only ITK inhibitor combined with anti- PD-1 had an effect on tumor growth (Fig 2B and C). The beneficial effects of the combined therapy were also demonstrable in terms of survival of these tumor-bearing animals (Fig 2C). On day 46, 36.4% of mice that received isotype control antibody and 22.2% mice with ITK inhibitor treatment alone survived. Anti-PD-1 treatment alone did not increase survival as 25% survived on day 46. In contrast, combining ITK inhibitor with anti-PD-1 therapy significantly increased survival to 90%.

In the 4662 pancreatic tumor model, treatments started when the tumor was established (Days 5-8) (Fig 2D). Similar to the AE17 tumor model, combined ITK inhibitor and anti-PD-1 treatment decreased tumor growth and improved the survival of mice (Fig 2E and F). Experiment was ended due to tumor ulcerations in animals on day 29. By day 29, 5.9% of mice that received isotype control antibody and 0% of mice with ITK inhibitor treatment alone survived. Anti-PD-1 treatment alone increased survival to 22.2% and this was significantly improved to 52.9% survival when combining ITK inhibitor with anti-PD-1 treatment. Overall, these results from ICB insensitive tumor models demonstrate that ICB and ITK inhibitor treatment synergize and potently suppress tumor growth.

ITK inhibitors reverse the exhaustion-related phenotype of in vitro exhausted CTLs

We next examined whether ITK inhibition can directly ameliorate T cell exhaustion as improving CTL exhaustion by ITK inhibition could explain the synergy with ICB. We therefore examined whether the selective ITK inhibitor BMS-509744 could affect T cell exhaustion in an in vitro exhaustion model we established (Zhao et al., 2020. PLoS Pathog. 16(6):el008555). This model uses purified OVA-specific OT-I transgenic CD8+ T cells that are driven to exhaustion by repeated stimulation with OVA(257-264) peptides for 5 days (Zhao et al., 2020. Supra). Using this model, in vitro exhausted cells were treated with lgM BMS-509744 for an additional 3 days, and then cells were analyzed for exhaustion characteristics (Fig 3A). ITK inhibition was found to downregulate surface expression of multiple inhibitory receptors including PD-1, Lag3, Tigit, Tim3 and CD160 (Fig 3B). Furthermore, the frequency of IL-2 and TNF-ct producing cells increased after ITK inhibitor treatment (Fig 3C). Finally, ITK inhibition downregulated TOX expression although the expression of TCF1 remained unchanged (Fig 3D and 3E).

ITK is an important kinase that regulates TCR signaling in CD4+ and CD8+ T cells by phosphorylating downstream Phospholipase C- yl (PLC-yl) in T cells. Since chronic TCR signaling can lead to exhaustion, we examined whether ITK inhibitor BMS- 509744 or ibrutinib, which is also known to inhibit ITK, affected ITK activity in in vitro exhausted CTLs. We found that the phosphorylation of both ITK (Tyrl80) and its downstream target, PLC-yl (Tyr783) were both decreased in exhausted T cells by ITK inhibitor BMS-509744 or ibrutinib treatment (Fig 3F and 3G).

CD8+ T cell function is improved in CLL patients who receive long term ibrutinib treatment (Parry et al., 2019. Front Immunol. 10:2832), however, it is unknown whether this is an indirect effect or whether ibrutinib can directly reduce CTL exhaustion. We therefore examined if ibrutinib would directly affect exhausted CTL in the in vitro exhaustion model (Zhao et al., 2020. Supra). For this, in vitro exhausted cells were treated with lgM ibrutinib for an additional 3 days after the 5 day exhaustion induction, and then cells were analyzed for exhaustion characteristics (Fig 4A). We found that ibrutinib treatment lowered the expression of inhibitory receptors (Fig 4B and 4C, 5A). Cells expressing multiple inhibitory receptors were also decreased with ibrutinib treatment (Fig 4D). Cytokine production after peptide re- stimulation improved with ibrutinib treatment, and there were more cells producing IL-2, TNF-ct and IFN-y (Fig 4E and 4F, 4B). When the simultaneous production of these three cytokines was analyzed, the frequency of double and triple cytokine producing cells was significantly increased upon ibrutinib treatment (Fig 4G), which indicates that ibrutinib treatment partially rescues the polyfunctionality of exhausted T cells. Ibrutinib treatment also improved the exhaustion-related transcription factor profile of the exhausted cells as it decreased TOX expression while increasing TCF1 expression in wild type cells (Fig 5C and 5D). Overall, these results indicated that ibrutinib directly acts on T cells to ameliorate key features of CTL exhaustion by downregulating inhibitory receptors, improving cytokine production and polyfunctionality while downregulating TOX and increasing TCF1 transcription factors.

Ibrutinib improves CD8+T cell exhaustion independent of BTK

In order to understand how ibrutinib is acting in a BTK independent manner to directly reverse exhaustion, we first performed a Western blot analysis to exclude that BTK is expressed in in vitro exhausted T cells (Fig 7). To further exclude a role for BTK in ibrutinib’s effect, we submitted btk-/- OT-I cells to our in vitro exhaustion protocol and found that btk-/- OT-I cells, similar to wild type OT-I cells, were readily exhausted in vitro. Furthermore, when btk-/- OT-I cells were in vitro exhausted and treated with ibrutinib, ibrutinib retained its ability to ameliorate exhaustion-related features of these cells. Ibrutinib could decrease inhibitory receptor expression (Fig 5 A) as well as improve cytokine production in btk-/- OT-I cells (Fig 5B). Ibrutinib also prevented the upregulation of TOX in in vitro exhausted btk-/- OT-I cells (Fig 5C). Additionally, a higher frequency of in vitro exhausted btk-/- T cells maintained TCF1 expression after ibrutinib treatment (Fig 5D).

From the above, it is clear that dampening ITK signaling activity with pharmacological inhibitors, such as BMS-509744 and ibrutinib, can directly reverse key features of CTL exhaustion in vitro. Ibrutinib improves the transcriptional profile of in vitro exhausted CD8+ T cells

To further confirm that ibrutinib treatment of in vitro exhausted CD8+ T cells could directly change the transcriptional profile of these cells, we performed RNA-seq. For this we first exhausted OT-I cells for 5 days and then treated cells with ibrutinib or left them without treatment till day 8. Cells were then analyzed by RNA-seq. By visualizing the RNA-seq results with Principal Component Analysis (PCA), we found that single peptide stimulated cells and repeat peptide stimulated cells were distinctly separated (Fig 6A). However, ibrutinib treatment drove the repeat peptide stimulated cells to cluster more closely with single peptide stimulated cells. There were 1745 significant differentially expressed genes between the repeat peptide stimulated exhausted cells with or without ibrutinib treatment (Fig 6B). Specifically, ibrutinib downregulated the expression of genes encoding for the inhibitory receptors Pdcdl (1.8 fold), Lag3 (1.9 fold) and Tigit (2.1 fold), but did not affect Havcr2/Tim3a, Cdl60 and Cd244a significantly (Fig 6C). Ibrutinib also corrected some of the transcription factor changes that are induced by T cell exhaustion. Tox and Irf4 were downregulated by 1.6 and 2 fold, respectively. Tcf7 and Eomes were upregulated (1.8 fold and 2.2 fold, respectively) while Tbx21, Batf and Nfatcl were not affected (Fig 6C). We next used gene set enrichment analysis (GSEA) to confirm whether ibrutinib reduced the exhausted related transcriptional profile of in vitro exhausted CD8+ T cells. We found that ibrutinib treated cells downregulated genes that are upregulated in T cell exhaustion while upregulating genes that are downregulated in exhaustion (Fig 6D), thus mitigating the gene expression changes induced by exhaustion. Performing ingenuity pathway analysis (IPA) on the differentially expressed genes between ibrutinib treated and untreated in vitro exhausted CD8+ T cells, revealed activation of interferon signaling and inflammation related signaling (Table 1). At the same time, the exhaustion-associated metabolic pathways, check-point blockade pathway and cell death were all inhibited after ibrutinib treatment (Table 2). Table 1. IPA analysis depicting the canonical pathways activated by treating with ibrutinib in vitro exhausted CTL. The percentage of molecules regulated in the pathway, the z-score and the p-value are shown.

Table 2. IPA analysis depicting the canonical pathways activated inhibited by treating with ibrutinib in vitro exhausted CTL. The percentage of molecules regulated in the pathway, the z-score and the p-value are shown.

Overall, these results indicated that ibrutinib acts directly on exhausted CD8+ T cells and decreases their exhaustion-associated gene transcriptional signature.

We report here that dampening TCR signaling with an ITK inhibitor can confer ICB sensitivity to ICB resistant solid tumors. Our data suggest that the prevention of CTL exhaustion may be a major factor in this ICB treatment improvement. We found that ITK inhibition in vivo results in more TCF1+ cells in the draining lymph nodes of tumor bearing mice. The efficacy of ICB therapy depends on stem cell-like TCF1+ T cells (Siddiqui et al. 2019, Immunity 50(l):195-211 elO; Kurtulus et al., 2019, Immunity 50(1): 181-94 e6), which maintain the potential to proliferate, therefore the accumulation of such cells when ITK was inhibited could explain the increased efficacy of ICB in combination treatment. Our studies were performed using animals with established tumors. Because ITK plays a critical role in CD8+ T cell activation (Grasis et al., 2011, J Signal Transduct. 2011:297868; Broussard et al., 2006, Immunity. 25(l):93-104), treatments were started a few days after tumor growth to avoid effects on antigen-specific T cell priming. We applied intermittently ITK inhibitor in vivo, namely, in a series of three- day treatments followed by resting for another three-days, to avoid a continuous blocking of T cell activation, which would be detrimental to the anti-tumor response. Further optimization of this ITK inhibition scheme could potentially improve the effect of ICB therapy combination.

Our studies provide an explanation for ibrutinib’s effects on T cell immunity in B cell malignancies that have been described in recent years. It remains unclear if these effects are the result of reduced antigenic load as a consequence of BTK inhibition of malignant B cells or a potential indirect effect on other immune cell populations because of on-target or off-target inhibitory effects. In a previous clinical study, it was found that the CLL patients who received ibrutinib treatment maintained increased CD4+ and CD8+ T cell counts while it markedly diminished PD-1 and CTLA-4 expression on T cells (Long et al., 2017, J Clin Invest. 127(8):3052-64). Since chronic antigen stimulation plays a critical role in T cell exhaustion (Bucks et al., 2009. J Immunol. 182(ll):6697-708; Utzschneider et al., 2016, J Exp Med. 213(9):1819-34) reducing B cell tumor load with ibrutinib, could indirectly affect T cell exhaustion. Indeed, these T cell effects were suggested to be due to a BTK independent mechanism, because the highly specific BTK inhibitor, zanubrutinib or acalabrutinib, could not induce these T cell changes (Davis et al., 2021, Journal of translational medicine 19(1):1- 13; Patel et al., 2017, Clin Cancer Res. 23(14):3734-43). When CD19- targeted CAR T cell therapy was combined with ibrutinib, it was found that ibrutinib enhanced the function and engraftment of CAR T cells (Fraietta et al., 2016. Blood. 127(9): 1117-27). Although the effects of enhancing CAR-T cell expansion could also be caused by the reduced B cell tumor burden, improved CAR T cell expansion was also observed in patients who had failed ibrutinib treatment alone (Gauthier et al., 2020. Blood. 135(19):1650-60). Therefore, the greater expansion of polyfunctional CAR-T cells could be due to ibrutinib acting on CAR T cells or other immune cell populations. In our study, we demonstrated that ibrutinib can directly act on T cells and mitigate T cell exhaustion. We showed this by using an in vitro CTL exhaustion induction method where only CD8+ T cells are present (Zhao et al., 2020. PLoS Pathog. 16(6):el008555]. In this system, ibrutinib was found to ameliorate many of the functional, transcription factor and transcriptional changes of T cell exhaustion. Ibrutinib not only downregulated inhibitory receptors, but also improved these cells functionally shown by increased cytokine production. Ibrutinib resulted in a less exhausted transcriptional signature with changes in key exhaustion- related transcription factors. Thus ibrutinib can act directly on T cells and reverse or prevent T cell exhaustion, and this could improve ICB and CAR T cell therapy in solid tumors.

By far, the most promising tumor immunotherapy to date, ICB, exhibits only limited efficacy in many solid tumors. Therefore, identifying treatments that enhance ICB effects is critical for expanding its efficacy. Ibrutinib has been shown to augment ICB in tumor models in Balb/c mice but the mechanism of action remains unknown (Sagiv-Barfi et al., 2015. Proc Natl Acad Sci U S A. 112(9):E966-72). We provide evidence that this is a direct effect of ibrutinib on T cell exhaustion and, at least for a major part, through its known ITK inhibitory capacity. We have shown that selectively inhibiting ITK with BMS-509744 can improve the anti-tumor effect of ICB in three solid tumor models. BMS-509744 is a selective and potent ITK kinase inhibitor that can block ITK phosphorylation and downstream PLC-y phosphorylation in both human and mouse cells (Lin et al., 2004. Biochemistry. 43(34): 11056-62; Mamand et al., 2018. Sci Rep. 2018;8(l):14216). Ibrutinib is actually a more potent inhibitor of ITK compared to BMS-509744 as determined by ITK phosphorylation (Mamand et al., 2018. Sci Rep. 2018;8(l):14216) and this may explain why in vitro ibrutinib had a stronger effect restoring in vitro exhausted CTL. However, the higher potency of ibrutinib may also be due to its ability to inhibit additional targets, such as MEK1/2 (Mamand et al., 2018. Sci Rep. 2018;8(l):14216), which could also contribute to the T cell exhaustion improvement seen in the in vitro experiments. Indeed, an inhibitor of MEK, which suppresses MAPK-PI3K-mT0R pathways activity, has been shown to improve the anti-tumor effects of CD8+ T cells and ICB (Liu et al., 2015. Clin Cancer Res. 21(7):1639-51; Ebert et al., 2016. Immunity. 2016;44(3):609-21; Hu-Lieskovan et al., 2015. Sci Transl Med. 7(279):279ra41; Verma et al., 2021. Nat Immunol. 22(l):53-66.). This could indicate that using ibrutinib to enhance immunotherapies may have additional benefits compared to exclusively targeting ITK. Our study demonstrates that intermittent ITK inhibition can dampen TCR signaling thereby mitigating T cell exhaustion and augmenting ICB therapy in ICB-resistant cancers. We provide evidence and a rationale for ITK inhibitors to be tested together with ICB for the treatment of patients with cancer that have been excluded from ICB immunotherapy.

Claims

Claims

1. A pharmaceutical combination for use in treating cancer comprising an ITK inhibitor and an immune checkpoint inhibitor, wherein the ITK inhibitor is administered using an intermittent dosing regimen or wherein the ITK inhibitor is administered at a dose that partially inhibits ITK enzymatic activity in T cells, preferably CD8+ T cells.

2. Pharmaceutical combination according to claim 1, wherein the cancer is an ICB resistant cancer, preferably an ICB resistant solid tumor.

3. Pharmaceutical combination according to claim 1 or 2, wherein the ITK inhibitor is selected from BMS-431051, BMS-488516, BMS-509744, PF 06465469, HY- 11066, CPI-818, ibrutinib, bosutinib, CTA056, GSK- 2250665A, and combinations thereof.

4. Pharmaceutical combination according to any one of the preceding claims, wherein the immune checkpoint inhibitor is selected from an anti- PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, an anti- Tim-3 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, an anti- PSGL-1 antibody, and combinations thereof, more preferably selected from nivolumab, camrelizumab, cemiplimab, dostarlimab, MEDI0680, pembrolizumab, prolgolimab, retifanlimab, sasanlimab, spartalizumab, STI- All 10, tislezlizumab, toripalimab, atezolizumab, avelumab, durvalumab, KD033, and STI-A1014, ipilimumab, tremelimumab, botensilimab, cobolimab sabatolimab, surzebiclimab, Sym023, R07121661, LY3321367, ICAGN02390, BMS-986258, tiragolumab, domvanalimab, vibostolimab, ociperlimab, BMS-986207, COM-902, AGEN-1307, ieramilimab, relatlimab, samalizumab, VTX-0811, SelK2, and combinations thereof.

5. Pharmaceutical combination according to any one of the preceding claims, wherein the intermittent dosing regimen comprises a repeated treatment cycle comprising a treatment interval of about 1-7 days of administration of the ITK inhibitor, wherein said treatment cycle further comprises a non-treatment interval of about 1-7 days days of nonadministration of the ITK inhibitor, and the intermittent dosing regimen comprises at least 2 treatment cycles, preferably wherein during the treatment interval the dosing comprises an administered dose of about 1-10 mg/kg body weight per day of the ITK inhibitor, and preferably wherein the administration is per os (PO).

6. Pharmaceutical combination according to any one of claims 1-4, wherein said ITK inhibitor dose that is effective for partial inhibition of ITK enzymatic activity is 0.01-5 mg/kg body weight per day.

7. Pharmaceutical combination according to any one of the preceding claims, wherein said an immune checkpoint inhibitor is formulated for administration at a dose of 100-1000 mg Q2W, Q3W or Q4W, such as 240- 480 mg every 2-4 weeks, preferably by IV administration.

8. A method of treating a subject suffering from an ICB resistant tumor, comprising administering to the subject an ITK inhibitor, and further comprising administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor, wherein the ITK inhibitor is administered using an intermittent dosing regimen or wherein the ITK inhibitor is administered at a dose that partially inhibits ITK enzymatic activity in T cells, preferably CD8+ T cells of said subject.

9. Method according to claim 8, wherein the ITK inhibitor is selected from BMS-431051, BMS-488516, BMS-509744, PF 06465469, HY-11066, CPI-818, ibrutinib, bosutinib, CTA056, GSK-2250665A, and combinations thereof.

10. Method according to claim 8 or 9, wherein the intermittent dosing regimen comprises a repeated treatment cycle comprising a treatment interval of about 1-7 days of administration of the ITK inhibitor, wherein said treatment cycle further comprises a non-treatment interval of about 1-7 days of non-administration of the ITK inhibitor, and wherein the intermittent dosing regimen comprises at least 2 treatment cycles, preferably wherein during the treatment interval the dosing comprises an administered dose of about 1-10 mg/kg body weight per day of the ITK inhibitor, and preferably wherein the administration is per os (PO); preferably wherein said ITK inhibitor dose that is effective for partial inhibition of ITK enzymatic activity is 0.01-5 mg/kg per day.

11. Method according to any one of claims 8-10, wherein the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, an anti-Tim-3 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, an anti-PSGL-1 antibody, and combinations thereof, preferably wherein the immune checkpoint inhibitor is selected nivolumab, camrelizumab, cemiplimab, dostarlimab, MEDI0680, pembrolizumab, prolgolimab, retifanlimab, sasanlimab, spartalizumab, STI-A1110, tislezlizumab, toripalimab, atezolizumab, avelumab, durvalumab, KD033, and STI-A1014, ipilimumab, tremelimumab, botensilimab, cobolimab sabatolimab, surzebiclimab, Sym023, R07121661, LY3321367, ICAGN02390, BMS-986258, tiragolumab, domvanalimab, vibostolimab, ociperlimab, BMS-986207, COM-902, AGEN-1307, ieramilimab, relatlimab, samalizumab, VTX-0811, SelK2, and combinations thereof.

12. A method of reversing T cell exhaustion resulting from persistent TCR stimulation, comprising exposing an exhausted T cell to an effective amount of an ITK inhibitor, preferably wherein said T cell is a CD8+ T cell or a CAR T cell, more preferably a CD8+ T cell, wherein after the method of reversing T cell exhaustion, said T-cell is exposed to an antigen capable of TCR signaling to thereby induce T-cell activation, preferably a tumor antigen.

13. The method according to claim 12, wherein said exposure to an effective amount of an ITK inhibitor effects in said T cell one or more of: increasing TCF1 expression; decreasing TOX expression; decreasing surface expression of inhibitory receptors PD-1, CTLA-4, TIGIT, Tim-3, Lag-3, CD 160, or CD200; increasing production of cytokines IFNy, TNFct or IL-2; increasing cytotoxicity; and increasing proliferative capacity.

14. The method according to any one of claims 12-13, wherein said exposure to an ITK inhibitor is intermittent or at a concentration that partially inhibits ITK enzymatic activity in said T cell.

15. The method according to any one of claims 12-14, wherein the intermittent exposure to the ITK inhibitor comprises an exposure period of about 1-7 days followed by a non-exposure period of about 1-7 days, optionally followed by a further exposure period to the ITK inhibitor of about 1-7 days, preferably wherein said exposure and non-exposure periods are repeated in at least 2 cycles of exposure and non-exposure to the ITK inhibitor, and wherein during said non-exposure said T cell is activated by exposure to an antigen capable of TCR signaling in said T-cell, preferably a tumor antigen.