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Purpose: To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL). Patients and Methods: In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients. Results: In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4–mediated inside–out activation. Transcriptomes of CD49d+ and CD49d− cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK–STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d− CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d− CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d− were estimated progression-free at 8 years (P = 0.0004). Conclusions: CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression.

CD19-redirected chimeric antigen receptor (CAR) T cells have shown remarkable activity against B-cell cancers. While second-generation CARs induce complete remission in >80% of patients with acute lymphoblastic leukemia, similar monotherapy induces long-term remissions in only 26% of patients with chronic lymphocytic leukemia (CLL). This disparity is attributed to cell-intrinsic effector defects in autologous CLL-derived T cells. However, the mechanisms by which leukemic cells impact CAR T-cell potency are poorly understood. Herein we describe an in vitro assay that recapitulates endogenous CLL-mediated T-cell defects in healthy donor CAR T cells. Contact with CLL cells insufficiently activates, but does not irreversibly impair, CAR T-cell function. This state is rescuable by strong antigenic stimulation or IL2, and is not driven by immune suppression. Rather, this activation defect is attributable to low levels of costimulatory molecules on CLL cells, and exogenous costimulation enhanced CAR T-cell activation. We next assessed the stimulatory phenotype of CLL cells derived from different niches within the same patient. Lymph node (LN)-derived CLL cells had a strong costimulatory phenotype and promoted better CAR T-cell degranulation and cytokine production than matched peripheral blood CLL cells. Finally, in vitro CD40L-activated CLL cells acquired a costimulatory phenotype similar to the LN-derived tumor and stimulated improved CAR T-cell proliferation, cytokine production, and cytotoxicity. Together, these data identify insufficient activation as a driver of poor CAR T-cell responses in CLL. The costimulatory phenotype of CLL cells drives differential CAR T-cell responses, and can be augmented by improving costimulatory signaling. Significance: CLL cells insufficiently activate CAR T cells, driven by low levels of costimulatory molecules on the tumor. LN-derived CLL cells are more costimulatory and mediate enhanced CAR T-cell killing. This costimulatory phenotype can be modeled via CD40 L activation, and the activated tumor promotes stronger CAR T-cell responses.

Supplementary Figure S4. Clinical impact of CD49d bimodal expression on durability of BTKi therapy.

Supplementary Table S6. DEGs for Baseline Comparison of CD49d+ and CD49d

Supplementary Methods

Purpose:To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL).Patients and Methods:In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients.Results:In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4–mediated inside–out activation. Transcriptomes of CD49d+ and CD49d− cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK–STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d− CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d− CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d− were estimated progression-free at 8 years (P = 0.0004).Conclusions:CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression.

Chronic lymphocytic leukemia (CLL) is an immunosuppressive disease characterized by increased infectious morbidity and inferior anti-tumor activity of immunotherapies. Targeted therapy with Bruton's tyrosine kinase inhibitors (BTKis), or the Bcl-2 inhibitor venetoclax, have profoundly improved treatment outcomes in CLL. To overcome or prevent drug resistance and extend duration of response after time-limited therapy, combination regimens are tested. Anti-CD20 antibodies that recruit cell and complement mediated effector functions are commonly used. Epcoritamab (GEN3013), an anti-CD3xCD20 bispecific antibody (bsAb) that recruits T cell effector functions, has demonstrated potent clinical activity in patients with relapsed CD20+ B-cell non-Hodgkin's lymphoma. Development in CLL is ongoing. To characterize epcoritamab mediated cytotoxicity against primary CLL cells, peripheral blood mononuclear cells (PBMCs) from treatment-naïve and BTKi-treated patients, including patients progressing on therapy were cultured with epcoritamab alone or in combination with venetoclax. Ongoing treatment with a BTKi and high effector:target ratios were associated with superior in vitro cytotoxicity. Cytotoxic activity was independent of CD20 expression on CLL cells, and observed in samples from patients progressing on a BTKi. Epcoritamab induced significant T-cell expansion, activation, and differentiation into Th1 and effector memory cells, in all patient samples. In patient-derived xenografts, epcoritamab reduced blood and spleen disease burden compared to mice receiving a non-targeting control. In vitro, the combination of venetoclax with epcoritamab induced superior killing of CLL cells than either agent alone. These data support the investigation of epcoritamab in combination with BTKis or venetoclax to consolidate responses and target emergent drug resistant subclones.

Supplemental Table 7. Targeting epigenetic resistance to BTK inhibitor treatment in ABC DLBCL, relates to Fig7

Supplemental Table 1. Epigenetic Ibrutinib Resistance in ABC DLBCL lines, , relates to Fig. 1

CD19-redirected chimeric antigen receptor (CAR) T cells have shown remarkable activity against B-cell cancers. While second-generation CARs induce complete remission in >80% of patients with acute lymphoblastic leukemia, similar monotherapy induces long-term remissions in only 26% of patients with chronic lymphocytic leukemia (CLL). This disparity is attributed to cell-intrinsic effector defects in autologous CLL-derived T cells. However, the mechanisms by which leukemic cells impact CAR T-cell potency are poorly understood. Herein we describe an in vitro assay that recapitulates endogenous CLL-mediated T-cell defects in healthy donor CAR T cells. Contact with CLL cells insufficiently activates, but does not irreversibly impair, CAR T-cell function. This state is rescuable by strong antigenic stimulation or IL2, and is not driven by immune suppression. Rather, this activation defect is attributable to low levels of costimulatory molecules on CLL cells, and exogenous costimulation enhanced CAR T-cell activation. We next assessed the stimulatory phenotype of CLL cells derived from different niches within the same patient. Lymph node (LN)-derived CLL cells had a strong costimulatory phenotype and promoted better CAR T-cell degranulation and cytokine production than matched peripheral blood CLL cells. Finally, in vitro CD40L-activated CLL cells acquired a costimulatory phenotype similar to the LN-derived tumor and stimulated improved CAR T-cell proliferation, cytokine production, and cytotoxicity. Together, these data identify insufficient activation as a driver of poor CAR T-cell responses in CLL. The costimulatory phenotype of CLL cells drives differential CAR T-cell responses, and can be augmented by improving costimulatory signaling.Significance:CLL cells insufficiently activate CAR T cells, driven by low levels of costimulatory molecules on the tumor. LN-derived CLL cells are more costimulatory and mediate enhanced CAR T-cell killing. This costimulatory phenotype can be modeled via CD40 L activation, and the activated tumor promotes stronger CAR T-cell responses.

Supplemental Information- Figures & Legends (Supplemental Tables loaded separately)

Supplemental Table 6. RAC2 protein interactions are a marker of epigenetic ibrutinib resistance

Supplemental Table 4. Altered dependencies in ibrutinib resistant ABC DLBCL, relates to Fig. 4

Supplementary Methods

Supplemental Figure 1: An overview of study design choices. Supplemental Figure 2: IL-2 Supplementation Enhances the APC phenotype of CLL cells. Supplemental Figure 3: Exogenous Co-stimulation Improves Second-generation CAR T cell Activation. Supplemental Figure 4: aCLL Stimulation Shows Inconsistent Cytokine Production After a 6hr Incubation. Supplemental Table 1: Full Antibody List. Supplemental Table 2: CLL Donor List. Supplemental Table 3: Full list of adjusted P-values from Holm-Sidak multiple comparisons.

Supplemental Table2. Tracking the evolution of ibrutinib resistance phenotypes, relates to Fig. 2

Oligo and Primer Tables

Supplemental Table 5. RAC2 as a mediator of epigenetic ibrutinib resistance, , relates to Fig5

The use of Bruton tyrosine kinase (BTK) inhibitors to block B-cell receptor (BCR)–dependent NF-κB activation in lymphoid malignancies has been a major clinical advance, yet acquired therapeutic resistance is a recurring problem. We modeled the development of resistance to the BTK inhibitor ibrutinib in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma, which relies on chronic active BCR signaling for survival. The primary mode of resistance was epigenetic, driven in part by the transcription factor TCF4. The resultant phenotypic shift altered BCR signaling such that the GTPase RAC2 substituted for BTK in the activation of phospholipase Cγ2, thereby sustaining NF-κB activity. The interaction of RAC2 with phospholipase Cγ2 was also increased in chronic lymphocytic leukemia cells from patients with persistent or progressive disease on BTK inhibitor treatment. We identified clinically available drugs that can treat epigenetic ibrutinib resistance, suggesting combination therapeutic strategies.Significance:In diffuse large B-cell lymphoma, we show that primary resistance to BTK inhibitors is due to epigenetic rather than genetic changes that circumvent the BTK blockade. We also observed this resistance mechanism in chronic lymphocytic leukemia, suggesting that epigenetic alterations may contribute more to BTK inhibitor resistance than currently thought.See related commentary by Pasqualucci, p. 555.This article is highlighted in the In This Issue feature, p. 549

Supplemental Table 3. Epigenetic retuning of oncogenic signaling in ibrutinib resistance, relates to Fig. 3

Purpose:In chronic lymphocytic leukemia (CLL), the T-cell receptor (TCR) repertoire is skewed and tumor-derived antigens are hypothesized as drivers of oligoclonal expansion. Ibrutinib, a standard treatment for CLL, inhibits not only Bruton tyrosine kinase of the B-cell receptor signaling pathway, but also IL2-inducible kinase of the TCR signaling pathway. T-cell polarization and activation are affected by ibrutinib, but it is unknown whether T cells contribute to clinical response.Experimental Design:High-throughput TCRβ sequencing was performed in 77 longitudinal samples from 26 patients with CLL treated with ibrutinib. TCRβ usage in CD4+ and CD8+ T cells and granzyme B expression were assessed by flow cytometric analysis. Antitumor cytotoxicity of T cells expanded with autologous CLL cells or with antigen-independent anti-CD3/CD28/CD137 beads was tested.Results:The clonality of the TCR repertoire increased at the time of response. With extended treatment, TCR clonality remained stable in patients with sustained remission and decreased in patients with disease progression. Expanded clonotypes were rarely shared between patients, indicating specificity for private antigens. Flow cytometry demonstrated a predominance of CD8+ cells among expanded clonotypes. Importantly, bulk T cells from responding patients were cytotoxic against autologous CLL cells in vitro and selective depletion of major expanded clonotypes reduced CLL cell killing.Conclusions:In patients with CLL, established T-cell responses directed against tumor are suppressed by disease and reactivated by ibrutinib.See related commentary by Zent, p. 4465

Supplementary Figure S1. Ibrutinib alters the absolute expression of inflammatory cytokines and chemokines in serum from patients with CLL. Supplementary Figure S2. Ibrutinib treatment reduces tumor burden. Supplementary Figure S3. Both CD4+ and CD8+ T cell subsets are decreased on ibrutinib. Supplementary Figure S4. Grading of macrophage interaction with CLL cells in bone marrow specimens. Supplementary Figure S5. Ibrutinib alters the absolute expression of chemokines in bone marrow supernatant from patients with CLL.

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc–expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling.Significance:Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.

Purpose: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B-cell receptor (BCR) signaling. Ibrutinib, a Bruton tyrosine kinase inhibitor, disrupts BCR signaling and leads to the egress of tumor cells from the microenvironment. Although the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the in vivo effects of ibrutinib on the tumor microenvironment.Experimental Design: Patients received single-agent ibrutinib on an investigator-initiated phase II trial. Serial blood and tissue samples were collected pretreatment and during treatment. Changes in cytokine levels, cellular subsets, and microenvironmental interactions were assessed.Results: Serum levels of key chemokines and inflammatory cytokines decreased significantly in patients on ibrutinib. Furthermore, ibrutinib treatment decreased circulating tumor cells and overall T-cell numbers. Most notably, a reduced frequency of the Th17 subset of CD4+ T cells was observed concurrent with reduced expression of activation markers and PD-1 on T cells. Consistent with direct inhibition of T cells, ibrutinib inhibited Th17 differentiation of murine CD4+ T cells in vitro. Finally, in the bone marrow microenvironment, we found that ibrutinib disaggregated the interactions of macrophages and CLL cells, inhibited secretion of CXCL13, and decreased the chemoattraction of CLL cells.Conclusions: In conjunction with inhibition of BCR signaling, these changes in the tumor microenvironment likely contribute to the antitumor activity of ibrutinib and may impact the efficacy of immunotherapeutic strategies in patients with CLL. Clin Cancer Res; 22(7); 1572–82. ©2015 AACR.See related commentary by Bachireddy and Wu, p. 1547

Supplementary Table S1: Evaluation of serum cytokine levels on ibrutinib Supplementary Table S2: Evaluation of chemoattractant levels in bone marrow supernatant on ibrutinib supplementary figure legends for supplementary figures S1-S5

Conflict of Interest Form from Disruption of in vivo Chronic Lymphocytic Leukemia Tumor–Microenvironment Interactions by Ibrutinib – Findings from an Investigator-Initiated Phase II Study

(A) Frequency of predominant clonotypes as detected by NGS and of the corresponding CD8+ TRBV families as measured by flow cytometry. Grey bars indicate TRBV families that did not have commercially available fluorescent-labelled antibodies. (B) Pie chart of the most expanded clonotypes and expression of granzyme B (GrB). CD8+ GrB+ clonotypes comprising >2% of the repertoire at the time of response are shown in color.

Scatter plot of the TRBV frequency as detected by next generation sequencing (NGS) and flow cytometry. Spearman rho correlation coefficient and two tail p-value are shown.

Histogram of PD-1 expression in total CD3+, total CD8+, and the most abundant CD8+ clonotype in each patient after expansion with beads or autologous CLL cells.

T cell counts at baseline, time of ibrutinib response, and progressive disease (PD). Individual values, median and interquartile range are shown. Dashed lines indicate the lower and upper limits of the normal reference range.

Expansion of T cells under different conditions. Total CD3+, CD8+, CD4+ counts at Day 0 and Day 7 of expansion in the presence of anti-CD3/CD28/CD137 beads or autologous CLL cells.

Supplementary Tables

The management of chronic lymphocytic leukemia (CLL) has undergone unprecedented changes over the last decade. Modern targeted therapies are incorporated into clinical practice. Unfortunately, patients have begun to develop resistance or intolerance to multiple classes. Symptomatic patients previously treated with a BTK inhibitor (BTKi) and venetoclax represent a new and rapidly growing unmet need in CLL. Here, we define unmet needs in a modern treatment context. We also critically review the literature for PI3K inhibitors and chemoimmunotherapy and lack of data to support their utility following BTKis and venetoclax. Finally, we suggest opportunities to ensure the continued innovation for patients with CLL.

Bruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).

Reversing or preventing immunodeficiency in patients with chronic lymphocytic leukemia (CLL) is of the highest priority. The past decade of research has met the challenge of treating CLL for most patients. Patients continue to struggle, however, with infections and second primary malignancies related to immunodeficiency. Strategies addressing this need currently are limited to vaccinations, with suboptimal efficacy, and immunoglobulin replacement. Correlative studies have provided insights into immunologic alterations on treatment. Understanding vulnerabilities in the immune system may help identify potential interventions to boost immunity. An emphasis on systematically testing such interventions is required to restore immunocompetence in patients with CLL.

Recent advances in cancer characterization have consistently revealed marked heterogeneity, impeding the completion of integrated molecular and clinical maps for each malignancy. Here, we focus on chronic lymphocytic leukemia (CLL), a B cell neoplasm with variable natural history which is conventionally categorized into two subtypes distinguished by extent of somatic mutations in the heavy chain variable region of immunoglobulin genes (IGHV). To build the ‘CLL map,’ we integrated genomic, transcriptomic, and epigenomic data from 1148 patients. We identified 202 candidate genetic drivers of CLL (109 novel) and refined the characterization of IGHV subtypes, which revealed distinct genomic landscapes and leukemogenic trajectories. Discovery of new gene expression subtypes further subcategorized this neoplasm and proved to be independent prognostic factors. Clinical outcomes were associated with a combination of genetic, epigenetic, and gene expression features, further advancing our prognostic paradigm. Overall, this work reveals fresh insights into CLL oncogenesis and prognostication.

Chimeric antigen receptor (CAR) T cell therapy has been promising in chronic lymphocytic leukemia (CLL) treatment showing durable remission. However, only a third of CLL patients can achieve complete remission with persistent effect featured by early memory T cells. This disparity is attributed to T cell-intrinsic defects or tumor-mediated immunosuppression. The mechanism how CLL cells impact CAR T cell potency still remains poorly understood. Here we used second-generation CD19- and ROR1-directed CAR T cells with a 4-1BB intracellular signaling domain. To recapitulate CLL-derived T cell defects, we developed an in vitro model of chronic antigen stimulation using CAR T cells recursively exposed to primary CLL cells. Upon each round of CLL stimulation, both types of CAR T cells were lack of proliferation and differentiation, accompanied with cytokine production failure in IL-2, suggesting hypofunction of CAR T cell induced by CLL. We next found that this lack of CAR T cell response is not permanent but can be rescued by strong antigen stimulus or IL-2 administration. On the other hand, IL-2 affects phenotype of CLL cells by enhancing expression of co-stimulatory molecules CD80 and CD86, suggesting that IL-2 supplementation or auxiliary co-stimulation via CD80/CD86 can rescue CAR T cell reactivity against these tumor cells. To test this hypothesis, we employed the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide that have previously been demonstrated to upregulate co-stimulatory molecules on CLL and restore T-cell function via IL-2 upregulation. We sought to test if IMiDs can improve CAR T cell response to CLL. CAR T cells repeatedly exposed to primary CLL cells were co-treated with IMiDs at each round, showing significantly increased proliferation compared to the group without drug treatment. Furthermore, IMiDs can enhance the functions of these CLL stimulated CAR T cells, including differentiation (higher frequency of effector memory T cells), activation (HLA-DR, CTLA-4), proliferation (Ki67), and effector function (Granzyme B). In addition, IMiDs can increase cytotoxic efficacy of CD8+ CAR19 T cells on CLL cells. Overall, our study unveiled that in CLL, activation defect of 4-1BB, CD3-signaling anti-CD19 CAR T cells was attributable to low levels of co-stimulatory molecules on CLL cells and can be effectively overcome via upregulation of co-stimulation. Our data suggested that CLL resistance to CAR T cell can be successfully rescued by concurrent treatment with IMiDs. This study provides a potential translational approach to overcome the bottleneck of CLL treatment by CAR T cells. Citation Format: Ziran Zhao, McKensie Collins, Clare Sun, Adrian Wiestner, J. Joseph Melenhorst. Co-stimulatory signaling boosts CAR T cell efficacy against chronic lymphocytic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4100.

Purpose: In chronic lymphocytic leukemia (CLL), the T-cell receptor (TCR) repertoire is skewed and tumor-derived antigens are hypothesized as drivers of oligoclonal expansion. Ibrutinib, a standard treatment for CLL, inhibits not only Bruton tyrosine kinase of the B-cell receptor signaling pathway, but also IL2-inducible kinase of the TCR signaling pathway. T-cell polarization and activation are affected by ibrutinib, but it is unknown whether T cells contribute to clinical response. Experimental Design: High-throughput TCRβ sequencing was performed in 77 longitudinal samples from 26 patients with CLL treated with ibrutinib. TCRβ usage in CD4+ and CD8+ T cells and granzyme B expression were assessed by flow cytometric analysis. Antitumor cytotoxicity of T cells expanded with autologous CLL cells or with antigen-independent anti-CD3/CD28/CD137 beads was tested. Results: The clonality of the TCR repertoire increased at the time of response. With extended treatment, TCR clonality remained stable in patients with sustained remission and decreased in patients with disease progression. Expanded clonotypes were rarely shared between patients, indicating specificity for private antigens. Flow cytometry demonstrated a predominance of CD8+ cells among expanded clonotypes. Importantly, bulk T cells from responding patients were cytotoxic against autologous CLL cells in vitro and selective depletion of major expanded clonotypes reduced CLL cell killing. Conclusions: In patients with CLL, established T-cell responses directed against tumor are suppressed by disease and reactivated by ibrutinib. See related commentary by Zent, p. 4465