Venetoclax Increases Intra-Tumoral Effector T Cells and Anti-Tumor Efficacy in Combination with Immune Checkpoint Blockade

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Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Venetoclax increases intra-tumoral effector T cells and anti-tumor efficacy in combination with immune checkpoint blockade

Frederick J Kohlhapp1, Dipica Haribhai2, Rebecca Mathew1, Ryan Duggan1, Paul A Ellis1, Rui Wang2, Elisabeth A Lasater3, Yan Shi1, Nimita Dave4, Jacob J Riehm2, Valerie A Robinson2, An D Do5, Yijin Li5, Christine J Orr3, Deepak Sampath3, Aparna Raval5, Mark Merchant3, Anahita Bhathena2, Ahmed Hamed Salem4,6, Keith M Hamel1, Joel D Leverson7, Cherrie Donawho1, William N Pappano1 and Tamar Uziel2

1Oncology Discovery, AbbVie Inc. North Chicago, Illinois. 2Translational Oncology, AbbVie Inc. North Chicago, Illinois. 3Translational Oncology, Genentech, Inc. South San Francisco, California. 4Clinical Pharmacology and Pharmacometrics, AbbVie Inc. North Chicago, Illinois. 5Oncology Biomarker Development, Genentech, Inc. South San Francisco, California. 6Faculty of Pharmacy, Ain Shams University, Egypt. 7Oncology Development, AbbVie Inc. North Chicago, Illinois.

Note: F. Kohlhapp, D. Haribhai and R. Mathew contributed equally to this article. C. Donawho, W. N. Pappano and T. Uziel contributed equally to this article.

Corresponding Authors: Tamar Uziel, Translational Oncology, AbbVie Inc. 1 North Waukegan Road, North Chicago, IL 60064. Phone: 847-938 0502; Fax: 847-935 5165; Email: [email protected] ; and William N Pappano, Oncology Discovery, AbbVie Inc. 1 North Waukegan Road, North Chicago, IL 60064. Phone: 847-935-3321; Fax: 847-935 5165; Email: [email protected]

Running Title: venetoclax effect on T cells

Keywords: venetoclax, anti-PD-1, anti-PD-L1, T cells, immunotherapy.

Conflict of Interest and Funding:

FJH, DH, RM, RD, PAE, YS, JJR, VAR, AB, AHS, KMH, JDL, WNP, and TU are employees of AbbVie. EAL, ADD, AR, YL, CJO and MM are employees of Genentech. RW, ND, and CD were employees of AbbVie at the time of the study. DS was an employee of Genentech at the time of the study. The design, study conduct, and financial support for this research were provided by AbbVie and Genentech. AbbVie and Genentech participated in the interpretation of data, review, and approval of the publication.

Downloaded from cancerdiscovery.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT:

The anti-apoptotic protein BCL-2 plays critical roles in regulating lymphocyte development, immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL-2 is critical for anti-tumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL-2, would influence the anti-tumor activity of immune checkpoint inhibitors (ICIs). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the anti-tumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T cell function in response to antigen stimuli in vitro and did not antagonize T cell activation induced by anti-PD-1. Further,

we demonstrate that the anti-apoptotic family member BCL-XL provides a survival advantage in effector T cells following inhibition of BCL-2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy.

SIGNIFICANCE

The anti-apoptotic oncoprotein BCL-2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development and immune system regulation. Here we demonstrate that venetoclax, the first FDA/EMA approved BCL-2 inhibitor, unexpectedly, can be combined preclinically with immune checkpoint inhibitors to enhance anti-cancer immunotherapy warranting clinical evaluation of these combinations.

INTRODUCTION

The regulation of programmed cell death is crucial for orchestrating the development of the immune system and the complex cellular dynamics of immune responses. B cell lymphoma protein 2 (BCL-2) family proteins, which regulate the intrinsic apoptosis pathway, play a key role in immune cell development, response and homeostasis (1). The BCL-2 family can be sub- divided into anti-apoptotic (pro-survival) and pro-apoptotic (pro-death) proteins, whose expression levels and dynamic interactions dictate whether a cell lives or dies. Cloned from the breakpoint of the t(14;18) translocation in human B cell lymphoma, BCL-2 was the first member

of the family to be identified (2). Like its closely related family members BCL-XL and MCL-1, BCL-2 is a pro-survival protein and plays crucial roles during embryogenesis, hematopoiesis, and development of the immune system (3). Bcl-2 deficient mice undergo normal embryonic development of the hematopoietic system, but develop lymphocytopenia by 3-4 weeks after birth as a result of cells of the thymus and spleen that undergo massive apoptosis (4, 5). Models of murine infection demonstrate that memory CD8+ T cells express higher levels of Bcl-2 than naïve T cells, and that Bcl-2 is upregulated during antigen-induced stimulation (6, 7). In addition

to BCL-2, BCL-XL is induced in anti-CD3/CD28-activated T cells, which has been shown to enhance their survival in response to apoptosis-inducing agents (8). Interestingly, even though Bcl2-deficient mice developed lymphocytopenia, lymphocytes that survive in these mice responded normally to numerous stimuli, such as anti-CD3, phorbol 12-myristate 13-acetate plus ionomycin, lipopolysaccharide, and anti-IgM antibody (9). Further, Bcl-2 was not required to maintain memory T cells (10).

BCL-2 has also been implicated in oncogenesis and maintaining the survival of numerous tumor types, making it an attractive therapeutic target (11). Venetoclax (ABT-199, Venclexta, Venclyxto) is a selective small-molecule inhibitor of BCL-2 that can induce tumor cell apoptosis. Venetoclax is approved as a single agent and in combination with rituximab for patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), with or without 17p deletion, who have received at least one prior therapy, as well as in combination with obinutuzumab for previously untreated patients with CLL or SLL. It also received accelerated approval from the FDA in combination with azacitidine or decitabine or low-dose cytarabine for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adults who are age 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy. Signs of clinical activity have also been observed in a variety of other malignancies including mantle cell lymphoma, multiple myeloma and breast cancer (12-15).

Tumor immunotherapy, especially targeting the immune checkpoint proteins programmed cell death protein-1 (PD-1) and its ligand programmed death ligand-1 (PD-L1), has shown clinical promise in reinvigorating the immune system against cancer (16). Nivolumab/ pembrolizumab / cemiplimab (anti-PD-1) and atezolizumab/ durvalumab/ avelumab (anti-PD-L1) are now approved for the treatment of various tumor types, including melanoma, Merkel cell carcinoma,

lung cancer, renal cell carcinoma, head and neck cancer, triple-negative breast cancer, urothelial cancer and unresectable metastatic solid tumors with microsatellite instability or mismatch repair deficiencies (16). While anti-PD-1/PD-L1 therapies are promising, only subsets of patients in certain types of tumors respond. Thus, combining anti-PD-1/PD-L1 with other therapeutic modalities is of interest to improve the overall response rate and the durability of remission.

Although the importance of Bcl-2 has been established for both effector and memory T cell responses during homeostasis or following infection, (9, 10), little is known about the role of Bcl-2 during anti-tumor immune responses. Because venetoclax can cause reductions in T-cell numbers (17, 18), we hypothesized that combining it with immune checkpoint inhibitors would antagonize their anti-tumor activity.

The studies presented herein refute our hypothesis indicating instead, that the combinations of venetoclax with anti-PD-1 or anti-PD-L1 enhance tumor growth inhibition in syngeneic mouse models via an immune dependent mechanism. Although venetoclax reduced lymphocytes, including T cell numbers, it neither inhibited T cell function in response to antigen nor antagonized the activity of immune checkpoint inhibitor to antigen stimuli. The reduction in T cell numbers was attributed primarily to the sensitivity of naïve T cells to venetoclax, whereas T

effector cells were largely insensitive, likely due to increased reliance on BCL-XL. Tumor- bearing mice treated with venetoclax demonstrated an increase in CD8+PD-1+ T effector memory (TEM) cells within the tumors. Furthermore, venetoclax augmented anti-PD-1/PD-L1 activity in immunocompetent mouse tumor models without compromising the memory T cell response. Moreover, administration of venetoclax in healthy human subjects showed that the proportion of effector cells increased following treatment, confirming our findings in murine models and human cells cultured ex vivo. Together, these data indicate that T cells critical for an anti-tumor immune response are unaffected by venetoclax and that combination of venetoclax with immune checkpoint blockade improves anti-tumor activity in preclinical models.

RESULTS

Venetoclax augments the anti-tumor activity of anti-PD-1 and anti-PD-L1 antibodies in vivo

To investigate the effects of venetoclax on the anti-tumor activity of anti-PD-1/PD-L1 treatment, we performed tumor efficacy studies in immunocompetent C57BL/6 mice bearing MC38 tumors. Analysis from eight independent studies indicated that, as expected, anti-PD-1 led to significant tumor growth inhibition compared to isotype control (p-value = 0.0001) and this was, unexpectedly, further enhanced by co-treatment with venetoclax (venetoclax / anti-PD-1 co- treatment vs. anti-PD-1 alone, p-value = 0.005) (Fig. 1A, Supplementary Fig. 1A-I). Venetoclax also increased tumor growth inhibition when combined with anti-PD-L1 (Supplementary Fig. 2).

We also observed increased tumor growth inhibition with venetoclax plus anti-PD-1 or anti-PD- L1 compared to anti-PD-1 or anti-PD-L1 alone in immunocompetent BALB/c mice bearing ant- PD-1/PD-L1-resistant CT26 tumors (Supplementary Fig. 3A, 3B).

These findings warranted exploration of whether the augmented checkpoint inhibitor activity in vivo may be the result of venetoclax-dependent tumor cell-intrinsic effects. When cultured in vitro, the MC38 colon cancer cell line was resistant to venetoclax-mediated killing up to concentrations as high as 3 µM (Supplementary Fig. 4). We observed no decreases in viability or proliferation, and no signs of increased apoptosis over 3 days of treatment (Supplementary Fig. 4A - 4C). Additionally, no increases in immunomodulatory markers including secreted cytokines and cell surface MHC I or PD-L1 were observed following venetoclax treatment (Supplementary Fig. 4D and 4E). At the transcriptional level, we did not observe modulation of gene expression in MC38 cells following venetoclax treatment in vitro (data not shown). Treatment of MC38 tumor-bearing severe combined immune-deficient (SCID) mice with venetoclax showed minimal changes in gene expression compared to the vehicle-treated mice (only 62 genes significantly modulated (p-value < 0.05, and Log2 fold-change > 0.5), with no enrichment of distinct pathways; data not shown). To determine whether the anti-tumor activity of venetoclax with anti- PD-1 or anti-PD-L1 was immune dependent, we next tested the efficacy of these combinations in SCID mice transplanted with MC38 cells. As expected, treatments with either anti-PD-1 or anti- PD-L1 were not effective in this mouse strain. Consistent with the lack of in vitro activity against MC38 cells, venetoclax showed no anti-tumor activity in SCID mice, either as a single agent or in combination with the checkpoint inhibitors (Supplementary Fig. 5A and 5B). Further, CD8 T cell depletion was sufficient to abrogate the combination effect of venetoclax with anti-PD-1 (Supplementary Fig. 5C). These data suggest that cancer cell-intrinsic inhibition of Bcl-2 in vitro or in vivo does not result in activation of cell death pathways and does not intrinsically enhance immunogenicity of MC38 tumor cells.

Following in vivo treatment in immunocompetent mice bearing MC38 or CT26 tumors, all mice achieving complete regression (CR) remained tumor-free for over three months without evidence of tumor regrowth. Surviving CR mice were challenged by re-inoculation with MC38 or CT26 tumor cells, respectively, to evaluate retention of tumor-specific memory T cells. Following re- challenge, none of the CR mice grew tumors, demonstrating that these mice had developed and retained immunological memory. In addition, splenocytes isolated from these mice secreted IFNγ when co-cultured with irradiated MC38 or CT26 cells, respectively, ex vivo (Supplementary Fig. 6), demonstrating tumor-specific immune memory. In total, these data demonstrate that the augmented efficacy of the combination treatment is T cell-driven and indicate that venetoclax improves anti-PD-1/PD-L1-driven anti-tumor immune responses.

Venetoclax increases the number of intra-tumoral CD8+ T effector memory cells

To examine the effect of venetoclax on tumor-infiltrating lymphocytes (TILs), we performed flow cytometry analysis of excised MC38 tumors on day 14 following tumor inoculation (7 days

after initial dosing). At day 14, the tumors across all treatment groups were of similar size (Supplementary Fig. 7A). We observed that venetoclax decreased the total number of B and T cells in the peripheral blood, as previously reported (18) and in the tumors of these mice (Supplementary Fig. 7B). t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis of tumor infiltrating immune cells revealed two discrete populations of CD8+ Bcl-2+ T cells: (1) a population of activated and effector-like cells that are CD62L-, CD44+ and PD-1+, and (2) a population of naïve-like cells that are CD62L+, CD44- and PD-1- (Fig. 1B). In response to venetoclax treatment, the B cell population was decreased (population 3; Fig. 1C) and no change was observed in the CD11b+, F4/80+ macrophage population (Supplementary Fig. 7C). Interestingly, venetoclax treatment led to a decrease of the naïve-like CD8+ T cells (population 2, PD-1-) and an enrichment of the activated, T effector memory (TEM)-like cells (population 1, PD-1+) (Fig. 1C, 1D). This increase in TEM-like cells was further enhanced with the combination of anti-PD-1 and venetoclax (Fig. 1D). To depict the data highlighted from t-SNE analysis, we show, using bivariate scatter plots, the significant reduction in the naïve-like T cells (CD62L+, BCL2+, PD-1-) with venetoclax while enriching for activated effector-like T cells (Fig. 1E and Supplementary Fig. 7D). This phenomenon is the same regardless of combination treatment with anti-PD-L1 or anti-PD-1. The data presented show that venetoclax specifically targets naïve-like T cells. Previous studies have shown that activated T cells upregulate

additional anti-apoptotic molecules, including BCL-XL (8). Thus, we hypothesized that TEM- like cells infiltrating the tumor microenvironment (TME) might upregulate Bcl-xL, which could render these cells insensitive to Bcl-2 inhibition. Indeed, we found that the CD8+ T cells

remaining within the tumor after venetoclax treatment were enriched for Bcl-xL expression (Fig. 1F and Supplementary Fig. 7E), providing a possible explanation for their resistance to venetoclax.

These data demonstrate that venetoclax treatment can augment the anti-tumor activity of anti- PD-1 or anti-PD-L1 antibodies. Moreover, venetoclax treatment not only spares TEM-like cells but also increases the absolute number of these effector T cells in the tumors of the MC38 syngeneic model. The number of TEM cells can be further increased by combining venetoclax with checkpoint inhibitors. Collectively, these data show that venetoclax and checkpoint inhibitors can work in concert to increase effector T cells in the TME and reduce tumor growth.

Venetoclax treatment differentially affects human T cell subsets in vitro

We next explored the potential effects of venetoclax on lymphocyte subsets in cultured human peripheral blood mononuclear cells (PBMCs). Treated samples exhibited a concentration- dependent decrease in the number of B cells and T cells (CD4+ and CD8+ T cells; Supplementary Fig. 8A). While B lymphocytes were the most sensitive to venetoclax , CD8+ T cells were more sensitive than CD4+ T cells (Fig. 2A and Supplementary Fig. 8A), confirming data previously reported (18). Natural Killer cells and T regulatory cells (Tregs) were also less sensitive to venetoclax than CD8+ T cells (Supplementary Fig. 8A). With further

characterization of the T cells subsets we found that naïve T cells (TN; CD62L+CD45RA+) were the most sensitive T cell subset, and both their number and proportion decreased with increasing venetoclax concentrations. In contrast, even though the total number of memory T cells decreased with venetoclax treatment, their relative proportion, specifically CD8+ TEM cells (CD62L-CD45RA-) increased (Fig. 2B, 2C, Supplementary Fig. 8B). CD8+ central memory T cells (TCM; CD62L+CD45RA-) and terminally differentiated effector T cells / T effector memory RA cells (TEMRA; CD62L-CD45RA+) were also sensitive to venetoclax, but to a lesser extent than CD8+ naïve T cells, and their relative proportion increased with increasing venetoclax concentrations. Interestingly, anti-CD3/CD28-activated T cells were resistant to venetoclax treatment, and cytokine production by CD8+ T cell subsets was not affected (Supplementary Fig. 8C, 8D). In resting human PBMCs, BCL-2 expression was similar across all T cell subsets (Fig. 2D). Upon anti-CD3/CD28 T cell activation, a two-fold increase in BCL-2

expression and a nine-fold increase in BCL-XL expression were observed in all CD4+ and CD8+ subsets (Fig. 2E). These data suggest that BCL-XL likely accounts for the survival of activated T cells during BCL-2 inhibition. Consistent with the results from the MC38 syngeneic mouse model, the in vitro data indicate that venetoclax spares TEM cells and activated T cells.

Venetoclax treatment does not impair human T cell function and anti-PD-1 activity in vitro

We next examined whether venetoclax would affect antigen stimuli of T cells, in vitro. We tested antigen-specific recall response of cytomegalovirus (CMV) CD8+ T cells from human CMV+ PBMCs. T2 cells loaded with CMV peptide were incubated with CMV+ PBMCs in the presence

of increasing concentrations of venetoclax or the BCL-XL inhibitor A-1331852. CMV+ human PBMCs were also incubated with T2 cells without any peptide or loaded with MART1 peptide, serving as controls to assess antigen-specific response. Venetoclax treatment reduced overall

CD8+ T cells viability, whereas the BCL-XL inhibitor did not (Fig. 3A). Despite the reduction in cell number following venetoclax treatment, similar amounts of interferon  (IFN) were

secreted compared to the control (Fig. 3B). In contrast, BCL-XL inhibitor treatment reduced the production of IFN. This suggests that venetoclax treatment does not restrict the response of antigen-specific memory T cells, which may mimic anti-tumor recall activity in cancer patients.

We further evaluated the effect of venetoclax on human T cell function in response to antigen stimulation with or without anti-PD-1 co-treatment in an allogenic mixed lymphocyte reaction (MLR). In this assay, we observed that venetoclax reduced total CD4+ T cell viability in a concentration-dependent manner but did not limit the proliferation of the surviving T cells (Supplementary Fig. 9A). Although the overall number of CD4+ T cells was reduced, there was no decrease in the amount of secreted IFNγ with or without PD-1 blockade (Fig. 3C). We hypothesized that the decrease in CD4+ T cell number was the result of selective killing of non- activated T cells and that the responding T cells remain unaffected by venetoclax treatment. Therefore, we next measured the proportion of T cells that produce IFN on the final day of the MLR. Venetoclax treatment resulted in a higher percentage of CD4+ T cells producing IFN

regardless of PD-1 blockade (Fig. 3D and Supplementary Fig. 9B). Thus, venetoclax does not antagonize functional T cell response and does not abrogate anti-PD-1 activity in the MLR. Next,

we asked whether the activated T cells from the MLR upregulate BCL-XL and thus might be more resistant to venetoclax-induced apoptosis. Indeed, both BCL-2 and BCL-XL were upregulated as CD4+ T cells were activated in the MLR (Fig. 3E and Supplementary Fig. 9C).

While BCL-2 upregulation was uniform, BCL-XL was bi-modal, with one population showing expression similar to baseline and another with increased expression. However, when treated

with venetoclax, the remaining CD4+ T cells in the MLR exhibited only high BCL-XL expression (Fig. 3E and Supplementary Fig. 9C). In contrast to venetoclax, the BCL-XL inhibitor did not affect CD4+ T cell viability (Supplementary Fig. 9A), but reduced IFN production (reminiscent of the CD8+ CMV-recall assay) and diminished the effect of anti-PD-1 (Fig. 3D). Together with Figure 2E, these data demonstrate that activated T cells express higher levels of

BCL-XL than resting T cells, which may render them insensitive to venetoclax. In conclusion, our data demonstrate that human naïve T cells are more sensitive to venetoclax than activated and memory T cells when treated in vitro. Venetoclax did not impair antigen- recall or alloantigen-specific T cell activation or function and did not antagonize the response of T cells to anti-PD-1.

Collectively these data show that antigen-specific functional human T cell responses are unaffected by venetoclax. Importantly for the potential clinical combination of venetoclax with immune checkpoint inhibitors, functional T cell responses to anti-PD-1 are not inhibited by venetoclax.

Effect of venetoclax on T cell subsets in human subjects

To confirm that effector T cells are more resistant to venetoclax than non-effector T cells, we analyzed PBMC samples from three healthy volunteers who received a single 100 mg dose of venetoclax under fasting conditions. T cell subsets in peripheral blood were measured by flow cytometry one day before and seven days after venetoclax administration. Peak venetoclax concentrations in these subjects ranged between 0.08 and 0.34 µg/mL, which is approximately 10% of the mean peak concentration at the approved venetoclax dose of 400 mg in CLL (19). Minimal changes were observed in the number of B cells, CD4+ and CD8+ T cells (Fig. 4A), as expected for a single dose of 100 mg. However, we did observe differences in the proportion of T cell subsets, with the fraction of CD4+ and CD8+ effector memory cells (TEMs and TEMRA) increased, and the proportion of non-effector cells (TN and TCM) decreased (Fig. 4B). Although preliminary and limited by the number of subjects, these data are consistent with our observations in syngeneic mice (Fig. 1C, 1D), as well as human PBMCs treated in vitro (Fig. 2C), and show that venetoclax primarily affects naïve T cells, leading to an increased proportion of effector memory cells.

DISCUSSION

To date, clinical development of venetoclax has proceeded exclusively in hematologic malignancies and estrogen-positive (ER+) breast cancer, where its ability to directly induce tumor cell apoptosis is the primary driver of efficacy. The data presented herein suggest that venetoclax may also have utility in additional solid tumors, through a previously unappreciated ability to augment anti-tumor T cell responses mediated by immune checkpoint inhibitors.

Like tumor cells, immune cells depend on members of the BCL-2 family for their development and survival. Mouse studies have shown the importance of Bcl-2 in T cell homeostasis and in the contraction phase of the effector T cell response (1). Although Bcl2 knock-out mice are lymphopenic, the remaining effector lymphocytes can proliferate in response to immune-stimuli (9), suggesting that Bcl-2 deletion does not impair their activation. Additional studies demonstrated that while Bcl-2 was required for cytokine-driven T cell survival, it was dispensable for the survival of memory T cells (10). Several studies have shown a role for Bcl-2 during homeostasis and during acute infections. Whether or not tumor-infiltrating T cells rely on BCL-2 for survival or require BCL-2 for reinvigoration in response to checkpoint inhibitors was an open question. Here we used venetoclax to study the effect of BCL-2 inhibition on T cell subsets and evaluated whether venetoclax could be combined with anti-PD-1 or anti-PD-L1 antibodies for cancer immunotherapy. Clinically, venetoclax is viewed as a lymphodepleting agent, and thus our initial working hypothesis was that venetoclax-induced apoptosis of T cells and TILs would reduce the adaptive immune system’s ability to attack tumors and impair immune checkpoint inhibitors’ activity. Surprisingly, we found that even though venetoclax treatment led to a decrease in T cells, it was able to augment the activity of immune checkpoint inhibitors and did not interfere with the establishment of immune memory.

Though we observed improved efficacy for the combination of venetoclax and anti-PD-1/PD-L1, we recognize that venetoclax does reduce overall T cell number. Subsequent assessment of TILs revealed a depletion of naïve-like T cells but an increase in the number and proportion of CD8+ PD-1+ TEM cells. Importantly, reinvigoration of anti-tumor immune responses with checkpoint inhibitors in patients’ malignancies is associated with intra-tumoral expansion of CD8+ memory T cells (20). Clinical responses have been correlated with increases in memory T cells (21), specifically, CD8+ TEMs (20). PD-1 expression on CD8+ TILs has also been identified as a biomarker for the enrichment of tumor-specific T cells (22, 23). Moreover, reinvigoration of T cells was found to be accompanied by proliferation of CD8+ PD-1+ T cells in the peripheral blood that also express low levels of BCL-2 (24). In fact, all responding CD8+ Ki67+ PD-1+ T cells had low levels of BCL-2 and high levels of T cell activation and effector markers, suggesting that this population might be spared by venetoclax. The CD8+ T cell subset reported to be responsible for the anti-tumor activity of immune checkpoint inhibitors, was increased by venetoclax in our studies. Although venetoclax-mediated depletion of naïve T cells did not

antagonize the activity of checkpoint inhibitors in our syngeneic tumor models, it is unclear what impact this might have on the depth and durability of clinical responses. Of potential relevance, the frequency of CD8+ naïve T cells in the peripheral blood of lung cancer patients undergoing anti-PD-1 therapy was much lower than healthy subjects (24), implying that this population of T cells may be less involved in anti-tumor immune response.

Based on the data presented here, a simple mechanistic hypothesis could focus on the ability of venetoclax to induce apoptosis of certain immune cell subsets dictated by the BCL-2 family dependence profiles of the various immune cell populations. Venetoclax treatment may merely select for the most active, tumor-directed effector T cells and enable them to accumulate at their intended site of action. In support of this, we show that activated T effector cells and TEMs

upregulate BCL-XL both in vitro and in vivo and are resistant to venetoclax, whereas naïve T cells expressing low levels of BCL-XL are depleted. This was observed in human PBMCs cultured ex vivo and confirmed in the MC38 syngeneic mouse model where PD-1+ CD8+ TILs

expressed high levels of Bcl-xL and were enriched in tumors after venetoclax treatment. Any of these cells that are inhibited through PD-1-PD-L1 interactions could then be unleashed through the action of immune checkpoint inhibitors. Notably, Tregs and macrophages are resistant to venetoclax indicating that the increased efficacy is not the result of depleting these cells in the tumor microenvironment. Of course, it is likely that the mechanism is more complex and could also involve venetoclax rendering cancer cells more susceptible to T cell driven cytotoxicity. In a study using a Myc-dependent breast cancer model (WapMyc mouse), treatment with venetoclax and the diabetes drug metformin inhibited tumor growth and increased the intra-tumoral infiltration of PD-1-positive T-cells, indicative of an initial immune-mediated response followed by immune exhaustion (25). Subsequent experiments showed that adding neoadjuvant anti-PD-1 to this regimen led to significant improvements in durability of the anti-tumor response. Further understanding of the mechanism of venetoclax mediated immune modulation and anti-tumor response remains an area of active investigation for our laboratories.

We have provided the first data to suggest that venetoclax could contribute to anti-tumor activity through a distinct mechanism which is essentially immuno-modulatory in nature. By enriching the number (and potentially the quality) of PD-1+ effector T cells within tumors, venetoclax may have the potential to augment the efficacy of immune checkpoint inhibitors. Further, based on the absence of direct cancer cell intrinsic effects of venetoclax in the syngeneic models presented here, our data support the notion that venetoclax requires an active anti-tumor immune response as is the case with anti-PD-1 reinvigoration of the T cell. These hypotheses are under active clinical investigation (NCT04274907, NCT03000257) and may inform the combination of venetoclax and immunotherapy. Of course, it may be attractive to leverage both the apoptosis- inducing and immune-related effects of venetoclax simultaneously for cancer therapy. Indeed, clinical studies combining it with the anti-PD-L1 antibody atezolizumab were initiated in lymphoma, CLL, SLL and small cell lung cancer (NCT03276468, NCT02846623, NCT04422210), where venetoclax has already demonstrated signs of clinical activity. The

results of Haikala et al., (25) and those presented here suggest that venetoclax-checkpoint inhibitor combinations merit exploration in cancers not previously expected to respond to venetoclax alone, including an array of solid tumor malignancies.

METHODS

Reagents and cell lines

Venetoclax, BCL-XL inhibitor (A-1331852), anti-human PD-1(MDX-1106) AB426 [hu IgG1/k], anti-mouse PD-1 antibody (17D2[mu IgG2a/k] DANA), anti-PD-L1 antibody (YW243.55.S70 [hu/muIgG2a/k], and anti-mouse CD8 antibody used for depletion of CD8 T cells in vivo (PR- 1928513) were synthesized at AbbVie. Antibodies used for flow cytometry are listed in Supplementary Table 1. MC38 (mouse colon 38) cell lines were obtained from the National Cancer Institute (NIH; Rockville, MD) or from Kerafast (Boston, MA). CT26 was obtained from ATCC (Manassas, VA). The cells were tested regularly for Mycoplasma using MycoAlert Detection Kit (Lonza; Basel, Switzerland), and authenticated via PCR using nine short tandem repeat (STR) markers (IDEXX BioResearch; Columbia, MO).

Mouse MC38 cell line in vitro studies

Cells were plated in 384-well plates in the presence of increasing doses of venetoclax or DMSO control. Viability was measured via CellTiter-Glo® according to the manufacturer's protocol (Promega; Madison, WI). Confluency and apoptosis were assessed via IncuCyte® live-cell analysis and IncuCyte® Caspase-3/7 Green Apoptosis Assay Reagent, respectively (Essen BioScience, Sartorius; Ann Arbor, MI). For assessment of immune stimulation cells were plated in 96-well plate treated in the presence or absence of interferon gamma (IFN) for 3 days. Secretion of immune-stimulatory cytokines was measured by mouse IFN-α/IFN- 2 plex ELISA ProcartaPlex kit according to the manufacturer's protocol (Thermo Fisher Scientific). Cells were stained with anti-PD-L1 and anti- MHC class I (H-2Kb/H-2Db) (both from BioLegend, San Diego, CA) and evaluated by flow cytometry. Gene expression was determined by Clariom™ S Assay (Affymetrix, Thermo Fisher Scientific).

Mouse in vivo studies

All experiments were conducted in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals guidelines in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). C57BL/6, BALB/c and C.B-17 SCID mice were obtained from Charles River (Wilmington, MA).

Venetoclax was formulated in 10% ethanol + 30% PEG 400 + 60% Phosal® 50PG. Venetoclax was administered orally once a day for 14 days at 50 mg/kg/day. Anti-PD-1 antibody (17D2[mu IgG2a/k] DANA) and anti-PD-L1 antibody (YW243.55.S70) were formulated in 1X phosphate buffered saline and were administered by intraperitoneal (IP) injection 3 times every 4 days at 10 mg/kg. MC38 cells (5x104 for the Kerafast line and 1x105 for the NIH line) in a 0.1 ml of a 1:1 mixture of cells in culture media and Matrigel (BD Biosciences, Bedford, MA) were inoculated subcutaneously into the lower right flank of the mice and 7, 11 or 15 days later treatment was initiated (8-10 mice per group). Tumor volume was determined via measurements of the length (L) and width (W) of the tumor with electronic calipers and the volume was calculated according to the following equation: V = (L x W2)/2 using Study Director Version 2.1.11 (Studylog Systems Inc., South San Francisco). % tumor growth inhibition (TGI) was calculated as follow: 1 – (mean tumor volume of treatment group/ mean tumor volume of treatment control group) x 100. All %TGI comparisons were based on data collected at the same study time point. Study log stats (AbbVie Inc.) was used for the statistical analysis and P values are derived from Student’s T test comparison (one-sided two-sample) of log transformed data of treatment group vs. control group. Due to substantial variability in treatment responses caused by alternate sources of the MC38 cell line (NIH or Kerafast) and amongst studies, the anti-tumor growth efficacy data obtained from eight separate studies was evaluated using mixed effect modeling applied with the R software. The analysis aggregated information from all studies to evaluate the time trend of tumor growth and took into consideration the correlation between time points within each mouse and the correlation between mice within the same arm of the same study. The dependent variable of the model is the log of tumor fold change from the first time point (taken at baseline). The fixed effect includes time, treatment by time interaction, source by time interaction, and treatment by source by time interaction, where time is treated as a continuous variable. The random effect includes the time effect for each mouse and for each arm in each study. The fixed effect of treatment by source by time interaction could be removed from the model if it is discovered to be not significant.

Mouse tumor digestion and flow cytometry

Tumors were dissociated using a mouse tumor dissociation kit following the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). In brief, tumors were cut into 2-4 mm pieces and incubated with enzyme mix for 30 minutes in a 37 oC shaker (200 rpm). Cells were strained with 100 µm and 70 µm strainers (Falcon / Corning) before washing twice with Roswell Park Memorial Institute (RPMI1640) media containing 10% fetal bovine serum (FBS), GlutaMax™, penicillin, streptomycin and gentamicin. Single cell suspensions from tumors were counted and up to 2x106 live cells per tissue were stained with antibodies as indicated in Supplementary Table 1 adding Zombie UV reagent (BioLegend, San Diego, CA) to assess live

vs. dead cells and analyzed by flow cytometry using a LSRFortessa™ X-20 instrument (BD Biosciences, San Jose, CA). FCS 3.1 data files were exported from FACSDiVa and analyzed using FlowJo v10.4.1. Briefly, compensation was performed using single-stained bead controls and applied to all samples. Instrument acquisition anomalies were removed using a time-based histogram gate. Dead-cells, cell aggregates, and debris were removed from analysis utilizing Zombie UV intensity, pulse processing channels, and scatter, respectively. Lymphocyte subsets were gated according to a hierarchy to identify various T cell subsets as well as characterize their phenotype. An example layout of the gating hierarchy is found in the Supplement Fig. 10. Absolute cell counts were normalized to tumor volume using the following method: digested whole tumors were counted by flow cytometry. Analyses of nucleated and live/dead cell yielded the initial count. Up to 2x106 live cells were stained per sample, and the number of live cells detected during final acquisition was tabulated as the final count. The ratio of the initial count to final count provided the cell number normalization factor to calculate the total number of cells per tumor in any given gated population. A tumor size normalization factor was created by dividing all tumor sizes by 50 mm3. A normalized cell count could finally be plotted for any given population by first multiplying by the cell number normalization factor and then dividing by the tumor size factor to yield the cell number per 50 mm3 tumor volume metric for each sample. For t-SNE analysis, data files were passed through a preprocessing pipeline that included cleanup for viability, cell aggregates, and instrument acquisition anomalies using a combination of manual gating and the flowAI plug-in (FlowJo Exchange). Files were down sampled to a fixed number of lymphocytes after gating for Live/Singlet/CD45pos/CD3pos or CD19pos events per sample. Down sampled events were concatenated into a single file and the t-SNE algorithm was applied using all antibodies in the panel as parameter input values. t-SNE X and Y parameters were plotted for the fully concatenated file (Fig. 1B) or the deconvolved, individual treatment group files (Fig. 1C) to assess global changes in population frequencies. A third parameter was displayed by marker heatmap that revealed major immune cell subsets as well as marker expression within these subsets (Fig. 1E, 1F). Visible populations based on the tSNE plot were manually gated to explore relevant subsets and expression of additional markers on these subsets.

Human peripheral blood mononuclear cells (PBMCs) in vitro studies

Frozen viable human PBMCs were thawed and cultured overnight in 30 U/mL of interleukin-2 (IL-2, BD Biosciences), then washed once with RPMI1640 complete media supplemented with 10% FBS, GlutaMax™, and penicillin/ streptomycin (Gibco / ThermoFisher Scientific). Cells were treated with increasing concentrations of venetoclax for 24 hours and then harvested, counted and stained with antibody cocktail containing anti-CD19, anti-CD3, anti-CD4, anti-CD8, anti-CD45RA and anti-CD62L (Supplementary Table 1) and viability dye and analyzed by flow cytometry using a BD LSRII instrument (BD Biosciences). FCS 3.0 data files were exported from FACSDiVa and analyzed using FlowJo v10.4.1. Each subset of cell numbers was

calculated taking in account the viable cells followed by the percentage of CD19+ B cells and CD3+ positive T cells. T cells were further analyzed to denominate each immune cell subset (TN, TCM, TEM, TEMRA) and plotted using GraphPad Prism software. For 48-hour viability experiments, fresh PBMCs from healthy donors were plated at 2x105 cell per well in 96 well plate. Venetoclax was added at indicated concentrations. After 2 days, cells were collected and stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD127, anti- CD56, anti-CD19, and 7-AAD (BD Biosciences). Live CD3+CD4+ T, CD3+CD8+ T, CD19+ B, CD56+ NK, and CD3+CD4+CD25+CD127low Treg cell numbers were quantified by flow cytometry LSRFortessa™ X-20 instrument (BD Biosciences, San Jose, CA). For activation of T cells, cells were plated in anti-CD3-coated wells (2.5 µg/mL; ThermoFisher Scientific, clone OKT3) and soluble anti-CD28 was added (1 µg/mL; ThermoFisher Scientific, clone CD28.2).

Cytokine analysis in CD8 T cell subsets

CD8+ T cells were enriched from PBMCs utilizing RosetteSepTM Human CD8+ T Cell Enrichment Cocktail (Stemcell Technologies; Vancouver, Canada). CD8+ T cells were stained for CCR7 and CD45RA and naïve (CD45RA-/CCR7++), effector (CD45RA+/CCR7-), effector memory (CD45RA-/CCR7-) and central memory (CD45RA+/CCR7+) subsets were sorted on a BD FACSAriaTM Fusion (BD Biosciences, San Jose, CA). Cells were resuspended in RPMI 1640 media supplemented with 10% FBS, L-glutamine, and penicillin/streptomycin with or without 400 nM venetoclax, activated with Dynabeads Human T-Activator CD3/CD28 (Life Technologies; 1 bead:2 cells) and incubated at 37 oC for 18 hours. Supernatant was harvested and analyzed by Luminex. Cytokines and chemokines were quantified using a Milliplex human multiplex-bead–based 30-plex Luminex assay (Millipore; Burlington, MA) according to the manufacturer’s protocol. Data were acquired on a verified and calibrated FlexMap3D system (Luminex, Inc; Austin, TX) and analyzed with Bio-Plex Manager 6.0 software (Bio-Rad Labs; Hercules, CA).

Cytomegalovirus (CMV) Recall Assay

HLA-A*0201-restricted cytotoxic T cell peptide from the CMV protein pp65 (NLVPMVATV) was used to stimulate CMV+ CD8+ T cells from donor PBMCs (both from Astarte Biologics, Bothell, WA). CMV pp65 peptide (2 g/ml) was loaded on T2 cells (ATCC, Manassas, VA) in RPMI1640 media supplemented with 10% FBS, 1% antibiotics and Brefeldin-A (BD Biosciences). After 3 hours loading, T2 cells were irradiated (30 Gy) and washed with AIM V media (Invitrogen, Carlsbad, CA). Irradiated T2 cells loaded with CMV peptide were then incubated with CMV+ donor PBMCs (n=3 separate donors) in AIM V media at 37 °C in a 5%

CO2 incubator. These cells were treated with venetoclax or the BCL-XL inhibitor for the duration of the 4 days incubation period. Cells were stained with CD8 antibody and Zombie green fixable viability dye (BioLegend, San Diego, CA) and analyzed using a LSRFortessa™ X-20 instrument

(BD Biosciences, San Jose, CA). Secreted IFN was measured by MSD ELISA (Meso Scale Diagnostics; Rockville, MD).

Mixed Lymphocyte Reaction (MLR)

Monocyte-derived dendritic cells (MoDCs) were generated from fresh human blood. Briefly, human PBMCs were isolated using a Ficoll gradient and allowed to adhere to the plate for 2 hours, after which cells in suspension were removed. Fresh AIM V™ medium (ThermoFisher) supplemented with 80 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF, PeproTech, Rocky Hill, NJ) and 50 ng/mL IL-4 (R&D Systems, Minneapolis, MN) were added to the culture. After 5 days, the MoDCs were stimulated with IL-1α and TNF-α (0.2 ng/mL each, PeproTech, Rocky Hill, NJ) for 48 hours to increase expression of major histocompatibility complex class II molecules (MHCII). Activated MoDCs were then co-cultured with viably thawed CD4 T cells (Biological Specialty Corporation, Colmar, PA) at a ratio of 10:1 (T cells:MoDCs) in a mixed lymphocyte reaction (MLR). The cells were treated with control IgG (Isotype) or anti-PD-1 antibody (10 µg/mL) along with venetoclax. The MLR was cultured for 5 days, after which the cells were analyzed by flow cytometry using an LSRFortessa™ X-20 instrument (BD Biosciences) to determine cell number and functional cytokine (IFNγ) responses. Secreted IFN was analyzed using a human IFN AlphaLISA Detection Kit per manufacturer recommendation (Perkin Elmer).

Venetoclax study in healthy subjects

Three female volunteers, 20, 47 and 58 years old received one 100 mg commercial venetoclax tablet orally. Venetoclax plasma concentrations were evaluated prior to oral dosing and at 1, 2, 4, 6, 8, 10, 12, 24, 48 and 72 hours postdose. A liquid–liquid extraction and liquid chromatography with tandem mass spectrometric detection method was used to determine venetoclax plasma concentrations. The effect of venetoclax on T cells was assessed as described above for in vitro experiments.

Statistical Analyses

Unless otherwise specified, GraphPad Prism was used for statistical analyses using t-test statistical calculations.

Acknowledgments The authors would like to thank AbbVie colleagues Dr. Alexander Shoemaker, Dr. Doug Kline and Dr. Fiona Harding for critically reading the manuscript, Dr. Claudie Hecquet for analysis of

MC38 in vivo studies, Dr. Yan Sun for statistical analysis of MC38 in vivo studies, and Drs. Xin Lu and Weiguo Feng for gene expression analyses.

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FIGURE LEGENDS

Figure 1. Venetoclax enhances antitumor response of anti–PD-1 antibody in mouse MC38 tumor model. A. Tumor outgrowth in mm3, and Kaplan-Meier analysis / survival curve comparing venetoclax, anti-PD-1 and the combination of venetoclax with anti-PD-1 treatments to isotype / vehicle control in MC38 tumor bearing C57BL/6 mice. B. t-SNE maps representing lymphocyte populations within the tumors. Major infiltrating lymphocyte populations in the tumors were quantified 7 days after initiation of treatment by surface expression of total lymphocytes (CD45+), CD4+ and CD8+ T cells, B cells (CD19+), and pan-T cells (Thy1.2). Additional markers were included to further characterize T cell subsets (CD62L, CD44 and PD-1) as well as

measurement of Bcl-2 and Bcl-xL expression. t-SNE analysis of tumor lymphocytes revealed two CD8+ Bcl-2+ T cells: a population of activated and effector-like cells that are CD62L-, CD44+ and PD-1+ (marked as population #1), and a population of naïve-like cells that are CD62L+, CD44-, PD-1- (marked as population #2). C. Venetoclax effect, with or without anti-PD-1/PD- L1, on tumor infiltrating lymphocytes depicted by PD-1 expression. Following venetoclax treatment an increase in PD1+CD44+ CD8+ T cells (population #1) and a decrease in PD-1- CD44-CD8+ T cells (population #2) is observed. D. The effect of anti-PD-1, venetoclax and the combination on intra-tumoral CD8+ T cell subset numbers (naïve-like T (TN) cells: CD62L+ CD44-; central memory T (TCM) cells: CD62L+ CD44+, and effector memory T (TEM) cells: CD62L-- CD44+). (Unpaired t test: ns -not significant (p > 0.05), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to isotype). E and F. Representative frequencies and scatter plots of CD8+ T

cells in control and treated mice correlating PD-1 and Bcl-2 (E) or Bcl-xL (F) expression and overlaid with CD62L using a third parameter heat map.

Figure 2. Venetoclax treatment differentially affects human T cell subsets in vitro. A. Total CD8+ and CD4+ T cell numbers following 24 hours treatment with increasing concentrations of venetoclax. B. Total cell numbers from CD8+ and CD4+ T cell subsets following 24 hours treatment with venetoclax (TN - Naïve T Cells, TCM – Central Memory T cells; TEM – Effector Memory T cells; TEMRA – Effector Memory T cells expressing CD45RA, also known as terminally differentiated effector memory T cells). C. Venetoclax effect on the proportion of T cell subsets (average of samples from nine donors examined in B). D. BCL-2 expression in CD8+ and CD4+ T cell subsets (fluorescent intensity determined by flow cytometry). E. BCL-2 and BCL-XL relative protein expression in resting and CD3/CD28 activated T cells (fluorescent intensity determined by flow cytometry). Paired t test was used for statistical analysis: ns -not significant (p > 0.05), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to DMSO control. (MFI- mean fluorescence intensity).

Figure 3. Venetoclax treatment does not impair human T cell function in response to antigen stimuli. A. CMV+ PBMCs were stimulated by CMV peptide loaded on T2 cells in the presence of increasing concentrations of venetoclax or BCL-XL inhibitor (A-1331852) for 4 days and viability of CD8+ T cells was assessed. As controls, CMV+ PBMCs were incubated with T2

cells without any peptide. B. Concentration of IFN secretion measured with the assay described in A. MART1 peptide was used as a control. Representative viability (A) and cytokine secretion (B) data shown from one donor with treatments in duplicate ± standard deviation. C. IFN secretion in mixed lymphocyte reaction (MLR) with anti-PD-1 (10 μg/mL) and/or venetoclax (1 M) treatment for 5 days. Paired t test: ns -not significant (p > 0.05), * p ≤ 0.05, ** p ≤ 0.01 compared to DMSO control. D. Percentage of CD3+T cells producing IFN as measured by intracellular flow cytometry on the final day of the MLR in the presence of increasing

concentrations of venetoclax or the BCL-XL inhibitor (A-1331852). E. BCL-2 and BCL-XL protein expression as determined by flow cytometry prior to and after the MLR (MFI - mean fluorescence intensity).

Figure 4. Venetoclax increases the proportion of effector memory cells in the blood of human subjects. A. Three healthy volunteers were administered a single 100 mg dose of venetoclax. The fold change of B cells, CD4+ and CD8+ T cells in the peripheral blood was determined by flow cytometry one day before (Day -1) and seven days after (Day 7) exposure to the drug. B. Assessment of the fraction of CD4+ and CD8+ T effector cells (TEM and TEMRA) in the blood of the human subjects following venetoclax administration.

Figure 1

A 2000 IsotypeIsotype 100 IsotypeIsotype anti-anti-PPDD-1-1 90 anti-a-PD-PD1 -1 venetoclaxvenetoclax

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Unstimulated Downloaded from cancerdiscovery.aacrjournals.org Stimulated on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Venetoclax increases intra-tumoral effector T cells and anti-tumor efficacy in combination with immune checkpoint blockade

Frederick J Kohlhapp, Dipica Haribhai, Rebecca Mathew, et al.

Cancer Discov Published OnlineFirst September 4, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-19-0759

Supplementary Access the most recent supplemental material at: Material http://cancerdiscovery.aacrjournals.org/content/suppl/2020/09/04/2159-8290.CD-19-0759.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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