Venetoclax increases intra-tumoral effector T cells and anti-tumor … · 2020. 9. 4. · FJH, DH,...
Transcript of Venetoclax increases intra-tumoral effector T cells and anti-tumor … · 2020. 9. 4. · FJH, DH,...
1
Venetoclax increases intra-tumoral effector T cells and anti-tumor efficacy in combination
with immune checkpoint blockade
Frederick J Kohlhapp1, Dipica Haribhai
2, Rebecca Mathew
1, Ryan Duggan
1, Paul A Ellis
1, Rui
Wang2, Elisabeth A Lasater
3, Yan Shi
1, Nimita Dave
4, Jacob J Riehm
2, Valerie A Robinson
2, An
D Do5, Yijin Li
5, Christine J Orr
3, Deepak Sampath
3, Aparna Raval
5, Mark Merchant
3, Anahita
Bhathena2, Ahmed Hamed Salem
4,6, Keith M Hamel
1, Joel D Leverson
7, Cherrie Donawho
1,
William N Pappano1 and Tamar Uziel
2
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:
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.
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
2
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.
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
3
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,
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
4
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).
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
5
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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
6
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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
7
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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
8
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.
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
9
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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
10
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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
11
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-2D
b) (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).
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
12
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 1x10
5 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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
13
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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
14
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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
15
(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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
16
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.
REFERENCES:
1. Marsden VS, Strasser A. Control of apoptosis in the immune system: Bcl-2, BH3-only
proteins and more. Annu Rev Immunol. 2003;21:71-105.
2. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM. Cloning of the chromosome
breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science.
1984;226(4678):1097-9.
3. Opferman JT, Kothari A. Anti-apoptotic BCL-2 family members in development. Cell
Death Differ. 2018;25(1):37-45.
4. Nakayama K, Nakayama K, Negishi I, Kuida K, Shinkai Y, Louie MC, et al.
Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science.
1993;261(5128):1584-8.
5. Veis DJ, Sorenson CM, Shutter JR, Korsmeyer SJ. Bcl-2-deficient mice demonstrate
fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell.
1993;75(2):229-40.
6. Grayson JM, Zajac AJ, Altman JD, Ahmed R. Cutting edge: increased expression of Bcl-
2 in antigen-specific memory CD8+ T cells. J Immunol. 2000;164(8):3950-4.
7. Grayson JM, Murali-Krishna K, Altman JD, Ahmed R. Gene expression in antigen-
specific CD8+ T cells during viral infection. J Immunol. 2001;166(2):795-9.
8. Boise LH, Minn AJ, Noel PJ, June CH, Accavitti MA, Lindsten T, et al. CD28
costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity.
1995;3(1):87-98.
9. Nakayama K, Nakayama K, Negishi I, Kuida K, Sawa H, Loh DY. Targeted disruption of
Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease, and
lymphocytopenia. Proc Natl Acad Sci U S A. 1994;91(9):3700-4.
10. Wojciechowski S, Tripathi P, Bourdeau T, Acero L, Grimes HL, Katz JD, et al. Bim/Bcl-
2 balance is critical for maintaining naive and memory T cell homeostasis. The Journal of
experimental medicine. 2007;204(7):1665-75.
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
17
11. Leverson JD, Sampath D, Souers AJ, Rosenberg SH, Fairbrother WJ, Amiot M, et al.
Found in Translation: How Preclinical Research Is Guiding the Clinical Development of the
BCL2-Selective Inhibitor Venetoclax. Cancer Discov. 2017;7(12):1376-93.
12. Tam CS, Anderson MA, Pott C, Agarwal R, Handunnetti S, Hicks RJ, et al. Ibrutinib plus
Venetoclax for the Treatment of Mantle-Cell Lymphoma. N Engl J Med. 2018;378(13):1211-23.
13. Kumar S, Kaufman JL, Gasparetto C, Mikhael J, Vij R, Pegourie B, et al. Efficacy of
venetoclax as targeted therapy for relapsed/refractory t(11;14) multiple myeloma. Blood.
2017;130(22):2401-9.
14. Moreau P, Chanan-Khan A, Roberts AW, Agarwal AB, Facon T, Kumar S, et al.
Promising efficacy and acceptable safety of venetoclax plus bortezomib and dexamethasone in
relapsed/refractory MM. Blood. 2017;130(22):2392-400.
15. Lok SW, Whittle JR, Vaillant F, Teh CE, Lo LL, et al. A phase 1b dose-escalation and
expansion study of the BCL-2 inhibitor venetoclax combined with tamoxifen in ER and BCL-2-
positive metastatic breast cancer. Cancer Discov. 2019; 9(3):354-369.
16. Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science.
2018;359(6382):1350-5.
17. Lu P, Fleischmann R, Curtis C, Ignatenko S, Clarke SH, Desai M, et al. Safety and
pharmacodynamics of venetoclax (ABT-199) in a randomized single and multiple ascending
dose study in women with systemic lupus erythematosus. Lupus. 2018;27(2):290-302.
18. Khaw SL, Merino D, Anderson MA, Glaser SP, Bouillet P, Roberts AW, et al. Both
leukaemic and normal peripheral B lymphoid cells are highly sensitive to the selective
pharmacological inhibition of prosurvival Bcl-2 with ABT-199. Leukemia. 2014;28(6):1207-15.
19. Salem AH, Dunbar M, Agarwal SK. Pharmacokinetics of venetoclax in patients with 17p
deletion chronic lymphocytic leukemia. Anticancer Drugs. 2017;28(8):911-4.
20. Ribas A, Shin DS, Zaretsky J, Frederiksen J, Cornish A, Avramis E, et al. PD-1 Blockade
Expands Intratumoral Memory T Cells. Cancer Immunol Res. 2016;4(3):194-203.
21. Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, et al.
Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma.
Cell. 2018;175(4):998-1013 e20.
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
18
22. Gros A, Robbins PF, Yao X, Li YF, Turcotte S, Tran E, et al. PD-1 identifies the patient-
specific CD8(+) tumor-reactive repertoire infiltrating human tumors. J Clin Invest.
2014;124(5):2246-59.
23. Inozume T, Hanada K, Wang QJ, Ahmadzadeh M, Wunderlich JR, Rosenberg SA, et al.
Selection of CD8+PD-1+ lymphocytes in fresh human melanomas enriches for tumor-reactive T
cells. J Immunother. 2010;33(9):956-64.
24. Kamphorst AO, Pillai RN, Yang S, Nasti TH, Akondy RS, Wieland A, et al. Proliferation
of PD-1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung cancer patients.
Proc Natl Acad Sci U S A. 2017;114(19):4993-8.
25. Haikala HM, Anttila JM, Marques E, Raatikainen T, Ilander M, Hakanen H, et al.
Pharmacological reactivation of MYC-dependent apoptosis induces susceptibility to anti-PD-1
immunotherapy. Nature communications. 2019;10(1):620.
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
19
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
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
20
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.
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
A
Figure 1
C
F
2
3
1 4 5
1. CD8+, Bcl-2+, CD62L-, CD44+, PD-1+ 2. CD8+, Bcl-2+, CD62L+, CD44-, PD-1-
3. B cells (CD19+) 4. CD4+ 5. CD4-, CD8-, Thy1.2+
B
Isotype + anti-PD-1 + anti-PD-L1
- ve
ne
tocl
ax
+ ve
ne
tocl
ax
PD-1
E CD8+ T cells
Days Post-Inoculation
Tum
or
Vo
lum
e (m
m3)
13 23 33 43 530
500
1000
1500
2000 Isotype
anti-PD-1
venetoclax
Combination
a-PD-1
venetoclaxDays Post-Inoculation
Per
cen
t to
10
00
mm
3
0 10 20 30 40 50 60 70 80 90 100 1100
10
20
30
40
50
60
70
80
90
100Isotype
Combination
venetoclax
a-PD-1
Isotype anti-PD-1 venetoclax anti-PD-1 + venetoclax
Isotype anti-PD-1 venetoclax anti-PD-1 + venetoclax
13 23 33 43 53 63 73 83 93
0
1000
2000
3000
Days Post-Inoculation
Tu
mo
r V
olu
me (
mm
3)
Isotype
13 23 33 43 53 63 73 83 93
0
1000
2000
3000
Days Post-Inoculation
Tu
mo
r V
olu
me (
mm
3)
anti-PD-1
13 23 33 43 53 63 73 83 93
0
1000
2000
3000
Days Post-Inoculation
Tu
mo
r V
olu
me (
mm
3)
venetoclax
13 23 33 43 53 63 73 83 93
0
1000
2000
3000
Days Post-Inoculation
Tu
mo
r V
olu
me (
mm
3)
a-PD-1venetoclax
Pe
rcen
t su
rviv
al (
%)
CD8+ T cells
D
Cell number per 50 mm3 of
tumor volume
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
A
B
DMSO venetoclax (1M )
DMSO
CD4+ T cells CD8+ T cells
CD8+ T cells CD4+ T cells
BCL-2 (MFI) BCL-2 (MFI)
T naive T central memory T effector memory T effector memory RA
E
CD8+ T cells CD4+ T cells
Unstimulated
Stimulated
B C L -2 B C L -X L
0
2
4
6
8
1 0
Re
lati
ve
ex
pre
ss
ion
to u
ns
tim
ula
ted
ce
lls
B C L -2 B C L -X L
0
2
4
6
8
1 0
Re
lati
ve
ex
pre
ss
ion
to u
ns
tim
ula
ted
ce
lls
Re
lati
ve e
xpre
ssio
n
Re
lati
ve e
xpre
ssio
n
BCL-2 BCL-XL BCL-2 BCL-XL
venetoclax (1M )
C
venetoclax (M) venetoclax (M)
CD4+ T cells CD8+ T cells
D
Figure 2
DMSO 0.1 0.3 10
2.010 5
4.010 5
6.010 5
8.010 5
1.010 6
Total CD4+ T cells
Cell
num
ber
DMSO 0.1 0.3 10
510 0 4
110 0 5
210 0 5
210 0 5
310 0 5
Total CD8+ T cells
Cell
num
ber
*** ***
***
*** ***
***
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
C
Figure 3
A B
PB
MC
s A
lon
e
0.1 0.3 1 0.1 0.3 1
venetoclax (M)
BCL-XL i (M)
PB
MC
s +
MA
RT1
PB
MC
s +
CM
V
0
5000
10000
15000
20000
IFN
(pg/
ml)
D
E
0
5.0 x 105
1.0 x 106
1.5 x 106
Via
ble
CD
8+
T ce
ll /m
l
PB
MC
s A
lon
e
0.1 0.3 1 0.1 0.3 1
venetoclax (M)
BCL-XL i (M)
Donor 1 Donor 2
Lum
ine
scen
ce u
nit
s DMSO
venetoclax
anti-PD-1
anti-PD-1 + venetoclax
Unstimulated T cells
No Treatment (MLR)
anti-PD-1 (MLR)
venetoclax (MLR)
anti-PD-1 + venetoclax (MLR)
BCL-2 (MFI) BCL-XL (MFI)
ns
ns **
ns
**
*
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
CD8+ T-cells CD4+ T-cells
Subject 1
Subject 2
Subject 3
Days
A
B
B cells CD4+ CD8+
Figure 4
Subject number: 1 2 3
Day -1 Day 7 Day -1 Day 7
Effector cells Non-effector cells
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759
Published OnlineFirst September 4, 2020.Cancer Discov Frederick J Kohlhapp, Dipica Haribhai, Rebecca Mathew, et al. blockadeanti-tumor efficacy in combination with immune checkpoint Venetoclax increases intra-tumoral effector T cells and
Updated version
10.1158/2159-8290.CD-19-0759doi:
Access the most recent version of this article at:
Material
Supplementary
http://cancerdiscovery.aacrjournals.org/content/suppl/2020/09/04/2159-8290.CD-19-0759.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerdiscovery.aacrjournals.org/content/early/2020/09/04/2159-8290.CD-19-0759To request permission to re-use all or part of this article, use this link
Research. on August 13, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 4, 2020; DOI: 10.1158/2159-8290.CD-19-0759