Ibrutinib for improved chimeric antigen receptor T‐cell production for chronic lymphocytic leukemia patients

Chimeric antigen receptor T (CART) cells targeting CD19 have shown promising results in the treatment of chronic lymphocytic leukemia (CLL). However, efficacy seems to be inferior compared to diffuse large B‐cell lymphoma or acute lymphoblastic leukemia. Impaired T‐cell fitness of CLL patients may be involved in treatment failure. Less‐differentiated naïve‐like T cells play an important role in CART expansion and long‐term persistence in vivo. These cells are sparse in CLL patients. Therefore, optimization of CART cell production protocols enriching less differentiated T cell subsets may overcome treatment resistance. The B‐cell receptor inhibitor ibrutinib targeting Bruton's tyrosine kinase (BTK) is approved for the treatment of CLL. Besides BTK, ibrutinib additionally inhibits interleukin‐2‐inducible T‐cell kinase (ITK) which is involved in T‐cell differentiation. To evaluate the effect of ibrutinib on CART cell production, peripheral blood mononuclear cells from nine healthy donors and eight CLL patients were used to generate CART cells. T‐cell expansion and phenotype, expression of homing and exhaustion makers as well as functionality of CART cells were evaluated. CART cell generation in the presence of ibrutinib resulted in increased cell viability and expansion of CLL patient‐derived CART cells. Furthermore, ibrutinib enriched CART cells with less‐differentiated naïve‐like phenotype and decreased expression of exhaustion markers including PD‐1, TIM‐3 and LAG‐3. In addition, ibrutinib increased the cytokine release capacity of CLL patient‐derived CART cells. In summary, BTK/ITK inhibition with ibrutinib during CART cell culture can improve yield and function of CLL patient‐derived CART cell products.

or diffuse large B-cell lymphoma (DLBCL) 5 and Yescarta (Axicabtagene Ciloleucel) for the treatment of DLBCL. 6 In comparison with ALL and DLBCL, the efficacy of CD19-specific CART cells in CLL seems to be lower with overall response rates (ORRs) of 50% to 70% and complete response (CR) rates of only 20% to 30% 7,8 compared to DLBCL with ORRs and CRs of 82% and 54%. 6 Intrinsic T-cell defects may contribute to these differences in therapeutic efficacy of CART cells. 9 The differentiation status of the transfused CART cells can have a major impact on in vivo proliferative capacity and therapeutic efficacy. [10][11][12][13][14] Less-differentiated CART cells, particularly naïve-like T cells (T N ), and stem cell memory-like T cells (T SCM ) have a high capacity for engraftment and long-term persistence in vivo. [15][16][17] Optimized CART cell production protocols enriching for these T cell subsets may improve therapeutic outcome. 18 Several specific pathway inhibitors can interfere with the differentiation of T cells. For example, mTOR inhibition not only enhances the formation of CD8+ memory T cells but also augments their antitumor functions. 19 Phosphatidylinositol 3-kinase (PI3K) inhibition with idelalisib can significantly improve CART cell products, particularly enrich for less-differentiated CART cells and decrease the expression of the exhaustion markers programmed cell death protein 1 (PD-1) and T-cell immunoglobulin mucin-3 (TIM-3). 18 It was also reported that addition of interleukin (IL)-21 and the GSK3β inhibitor SW119 to the culture medium can lead to an enrichment of CD19-CAR-modified CD8+ T SCM with enhanced metabolic fitness and robust, long-lasting antitumor responses. 16,20,21 AKT inhibitors as well as adenosine monophosphate-activated protein kinase agonists can have similar effects on CART cell production. 22,23 Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, showed encouraging clinical activity in patients with B-cell malignancies, particularly in CLL patients. [24][25][26] Ibrutinib interferes with BCR signaling by inhibiting multiple targets, including BTK as well as B-lymphocyte kinase and impairs phosphorylation of downstream effectors. 27,28 Ibrutinib also targets the interleukin-2-inducible T-cell kinase (ITK), can thereby deplete T helper 2 (Th2) cells and induce a shift toward Th1 cells 29 or increase Th17 cell subsets. 30 It was reported that CLL patients after treatment with ibrutinib can reverse the exhausted T-cell phenotype by reducing the expression of PD-1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). 30 Furthermore, pretreatment with ibrutinib before T cell collection for CART cell therapy can reverse the dysfunction of T cells and induce the variation of CART cell phenotypes. 9 In addition, ibrutinib combined with CART therapy in preclinical in vivo models can result in high tumor killing and sustained long-term remissions. 31 However, treatment of patients with ibrutinib is not always possible and other strategies for exploiting the beneficial effects on CART cell therapy may be desirable.
In our study, we investigated the impact of ibrutinib supplemented to the CART cell culture medium for enhanced CART cell production and improved treatment of CLL patients.

| Flow cytometry
Phenotypic analysis was performed by flow cytometry as described previously. 18,32,33,35 Viability was determined by near-IR (Thermo

| Cytotoxicity assay
The cytotoxic activity of transduced effector cells was evaluated using a 4-hour chromium-51 ( 51 Cr; Hartmann Analytic, Braunschweig, Germany) release assay as described previously. 34

| Statistical analysis
Statistical analysis was performed using Excel (Microsoft, Redmond, WA). P values were calculated either using the two-way t test or the one-way analysis of variance (ANOVA). P values <.05 were considered statistically significant. Graphs and tables were designed using Excel or Prism 6 (GraphPad Software, Inc., La Jolla, CA). If not otherwise mentioned, results are presented as mean ± SD.

| Distribution of CD4+ and CD8+ T cells
Higher numbers of CD4+ T cells and lower numbers of CD8+ T cells were observed in CLL patient-derived CART cells compared to HDs (Figure 2A,B). Cultivation of CLL and HD-derived CART cells in presence of ibrutinib did not have any influence on the distribution of CD4+ and CD8+ T cells (Figures 2A,B and S3).

| Analysis of homing and exhaustion markers
Expression of CXCR3 and CD62L was determined to evaluate T-cell homing capacities of CART cells generated in presence of ibrutinib.

| Evaluation of antigen-specific cytotoxicity
Functional analysis for CART cell-mediated cytotoxicity was performed by 51 Cr release assay. Ibrutinib for CART cell generation did not have significant effects on in vitro cytotoxic capacities of HD and CLL patient-derived CART cells against CD19-positive Daudi cells (10:1 ratio without vs with ibrutinib: 52% ± 15% vs 42% ± 12%, P = .3 and 35% ± 18% vs 40% ± 13%, P = .5; Figure 5D,E). No relevant unspecific cytotoxicity against CD19-negative K-562 cells was observed ( Figure S5). A significant difference in cytotoxic capacity was seen between HD and CLL patient-derived CART cells generated in absence of ibrutinib (3:1 ratio HD vs CLL: 42% ± 11% vs 20% ± 12%, P = .0421, Figure S6). This may reflect the T-cell defect and the reduced effector function of CLL-derived T cells.

| DISCUSSION
CD19-CART cell therapy is a promising treatment approach for patients with CD19-positive B-cell malignancies. However, efficacy is clearly lower for CLL patients compared to ALL and DLBCL. Therefore, novel strategies are needed to improve the clinical benefit using CART cells for CLL patients. Optimization of the CART production process may be a simple but effective approach to improve efficacy of CART cells in CLL patients. In our study, we evaluated the effects of F I G U R E 5 Functional characterization of CD19-specific CART cells generated with or without ibrutinib. Production of TNF-α (A) and IFN-γ (B) was measured on Day 15 of CART cell production in HD-derived (n = 4) and CLL patient-derived (n = 8) CART cells. CART cells were stimulated with CD19+ Daudi cells for 6 hours. C, Multifunctional CART cells producing both, TNF-α and IFN-γ. In vitro cytotoxic capacity of cryopreserved CD19-specific CART cells was determined by 51 Cr release assay after coculture with CD19+ Daudi target cells for 4 hours. The cytotoxic potential was assessed for CART cells derived from HDs (n = 4; D) and CLL patients (n = 8; E). Average lysis was determined in different effector (CART cells) to target (Daudi cells) ratios (30:1, 10:1, 3:1, 1:1). Mean values were calculated for each group; error bars indicate SD. Significance was calculated using the two-way t test and is represented as * for P values <.05. CART, chimeric antigen receptor T cells; CLL, chronic lymphocytic leukemia; HDs, healthy donors; IFN-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha ibrutinib-supplemented CART cell production with a particular focus on CLL patient-derived CART cells.
Intrinsic defects of CLL patient-derived T cells impair both, the feasibility of CART cell generation as well as in vivo efficacy. 7,8,18,33 It was reported that CLL patients pretreated with >5 cycles of ibrutinib therapy can recover the T-cell defects leading to effective ex vivo expansion of their CART cells with potentially enhanced in vivo functionality. 9 However, pretreatment of CLL patients with ibrutinib is not always possible: side effects including cardiotoxicity and bleedings as well as treatment resistance may prohibit prolonged application. In the current study, ibrutinib supplemented to the CART cell production process could not only achieve significantly higher expansion of T cells but also lead to increased viability as well as transduction efficiency and thereby achieve significantly higher CART cell yields from CLL patients.
CLL patients tend to have higher CD4:CD8 T cell ratios when compared to HDs. A more balanced CD4:CD8 ratio might be beneficial for efficient CART cell therapy. 17 In our study, ibrutinib supplemented to CART cell culture medium could not overcome this limitation in CLL patients. This stands in contrast to our previous findings that PI3K inhibition with idelalisib can lead to potentially more favorable CD4:CD8 ratios in CLL patients. 18 This indicates that different pathways may be targeted by ibrutinib and idelalisib for optimized CART cell production.
Less-differentiated T cells, particularly T N and T SCM , are important for CART cell engraftment and in vivo persistence as well as efficient tumor eradication. 14-16 Differentiation of primed T N cells can be suppressed by certain cytokines, or by small molecules targeting key metabolic and developmental pathways. 11 For example, we previously reported inhibition of the PI3K/AKT/mTOR pathway with the clinically approved PI3Kδ inhibitor idelalisib can mediate CART cells with a T N cell phenotype. 18 In the current study, we focused on the BTK inhibitor ibrutinib that is also in clinical use for the treatment of CLL.
Ex vivo supplementation of ibrutinib to the culture medium significantly enriched less-differentiated CART cells with up to four times more CART cells with T N cell phenotype in the final cell product. This effect was not only seen in CART cells generated from CLL patientderived PBMCs but also in CART cells from HDs. This indicates that ex vivo CLL cell elimination alone cannot explain the beneficial effects of ibrutinib for the CART cell production process.

More differentiated T cells such as T EM and T Eff cells rather prefer
homing into the peripheral tissue. 37,38 In contrast, less-differentiated T cells, including T N , T SCM and T CM cells, tend to migrate more into the lymphoid tissue. 37,39 In the current study, CART cell generation in presence of ibrutinib did not influence the expression of CXCR3 that is mediating trafficking into the peripheral tissue. 40 In contrast, significantly higher expression of the lymphoid homing marker CD62L was observed in CART cells generated from CLL patients in presence of ibrutinib. CD62L expression was associated with enhanced antitumor activity in preclinical models of adoptive cell therapy 14,17 and ibrutinib-based CART cell production may be beneficial for lymphoid malignancies with higher migration directly to the tumor site. Considering the high amounts of CXCR3 and CD62L positive cells, CART cells coexpressing both markers may have the ability to reach the lymphoid tissue as well as extranodal sites. 41 PD-1 is a key immune-checkpoint receptor expressed on activated T cells. It is a negative regulator of T-cell immune response, inhibits T-cell proliferation as well as cytokine production and reduces T-cell survival. 42,43 Importantly, CLL patient-derived T cells were reported to be impaired with high PD-1 expression. 44,45 LAG-3 and TIM-3 can also negatively impact T-cell function and induce cell death. 46,47 Reduced expression of these negative regulators of T cells may increase immune activation and decrease CART cell exhaustion. 48 We observed that ibrutinib-supplemented CART cell generation significantly reduced the expression of these exhaustion markers, especially on CLL patient-derived CART cells. LAG-3 and PD-1 coexpression was reported to characterize highly dysfunctional and exhausted T cells. 49 inhibitor leading to an enrichment of less-differentiated CART cells with potentially increased antitumor activity. 51 The current study provides, to our knowledge, the first proof of concept that ibrutinib-based CART cell production may be beneficial for efficient CART cell products in CLL patients. Different pathways are probably involved in ibrutinib and PI3K-based optimization of CART cell production. Additional improvements may be achieved by the combination of both strategies with the benefits of a more balanced CD4:CD8 distribution from PI3K inhibition and the higher expansion benefits of less-differentiated phenotypes as well as increased CD62L expression achieved with ibrutinib in CLL patient-derived CART cells. However, the proportions of CLLderived CART cells with less-differentiated phenotypes are in the onedigit percentage ranges and there is still room for improvement. For example, AKT inhibition may be a promising option to further enrich for CART cells with less-differentiated phenotypes. 52,53 Previous reports by Fraietta et al demonstrated improved ex vivo expansion, decreased PD1 expression as well as improved IFN-γ production of T cells from CLL patients who received >5 cycles of ibrutinib in vivo. 9 In addition, they reported that ex vivo ibrutinib exposure did neither alter T-cell proliferation nor transduction efficiency. 9 Our current study confirms these observations in our setup with ibrutinib supplemented ex vivo to the CART cell culture medium: similar effects including higher CART cell numbers, reduced PD1 expression as well as improved IFN-γ production of CART cells were achieved. Furthermore, we identified that ex vivo ibrutinib can even increase transduction efficiency as well as CART cell yields.
In conclusion, our study provides evidence that BTK/ITK inhibition with ibrutinib during CART cell generation may improve CLL patient-derived CART cell products and may have the potential to enhance CART cell function. Ibrutinib-supplemented CART cell pro-

ETHICS STATEMENT
Sample collection and analysis were approved by the Ethics Committee of the University of Heidelberg (S-254/2016) and written informed consent was obtained from all patients.

DATA AVAILABILITY STATEMENT
Data can be made available upon reasonable request.