MOLECULAR CANCER THERAPEUTICS | LARGE MOLECULE THERAPEUTICS
Brentuximab Vedotin–Driven Microtubule Disruption
Results in Endoplasmic Reticulum Stress Leading to
Immunogenic Cell Death and Antitumor Immunity
Ryan A. Heiser, Anthony T. Cao, Weiping Zeng, Michelle Ulrich, Patrick Younan, Martha E. Anderson,
Esther S. Trueblood, Mechthild Jonas, Robert Thurman, Che-Leung Law, and Shyra J. Gardai
ABSTRACT
◥
Brentuximab vedotin, a CD30-directed antibody–drug conju-
gate (ADC), is approved for clinical use in multiple CD30-expres-
sing lymphomas. The cytotoxic payload component of brentux-
imab vedotin is monomethyl auristatin E (MMAE), a highly potent
microtubule-disrupting agent. Preclinical results provided here
demonstrate that treatment of cancer cells with brentuximab
vedotin or free MMAE leads to a catastrophic disruption of the
microtubule network eliciting a robust endoplasmic reticulum
(ER) stress response that culminates in the induction of the classic
hallmarks of immunogenic cell death (ICD). In accordance with
the induction of ICD, brentuximab vedotin–killed lymphoma cells
drove innate immune cell activation in vitro and in vivo. In the
“gold-standard” test of ICD, vaccination of mice with brentuximab
vedotin or free MMAE-killed tumor cells protected animals
Introduction
Tumors evade immune surveillance through tolerance of tumor-
associated neoantigens or by evolving immunosuppressive tumor
microenvironments (TME; refs. 1, 2). Therapeutics that kill tumor
cells directly while also promoting tumor-specific T-cell responses may
potentiate checkpoint inhibitor therapies, and therefore promote
durable clinical responses (3).
Apoptotic cell death is a normal aspect of cellular turnover and is
associated with silent or tolerogenic immune responses (4). In
contrast, immunogenic cell death (ICD) is an inflammatory cell
death process that promotes the activation and recruitment of innate
immune cells (1). During the pre-apoptotic stage of ICD, cells
present a defined combination of hallmark signals that provoke a
localized immune reaction to the dying cells (5). Cardinal hallmarks
of ICD include heightened translocation of calreticulin (CRT) from
the endoplasmic reticulum (ER) to the cell surface, active secretion of
ATP, and passive release of non-histone chromatin-binding protein
high-mobility group box 1 (HMGB1; Supplementary Fig. S1; ref. 6).
CRT exposure promotes phagocytosis of dying cells by antigen-
presenting cells (APC) via interactions with low-density lipoprotein
receptor-related protein 1. In addition, ATP release into the extra-
Seagen Inc., Bothell, Washington.
Corresponding Author: Ryan A. Heiser, Seagen Inc., 21823 30th Drive S.E,
Bothell, WA, 98021. E-mail: rheiser@seagen.com
Mol Cancer Ther 2024;23:68–83
doi: 10.1158/1535-7163.MCT-23-0118
This open access article is distributed under the Creative Commons Attribution-
NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.

2023 The Authors; Published by the American Association for Cancer Research
from tumor rechallenge; in addition, T cells transferred from
previously vaccinated animals slowed tumor growth in immu-
nodeficient mice. Immunity acquired from killed tumor cell
vaccination was further amplified by the addition of PD-1
blockade. In a humanized model of CD30þ B-cell tumors,
treatment with brentuximab vedotin drove the expansion and
Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024
recruitment of autologous Epstein-Barr virus–reactive CD8þ T
cells potentiating the activity of anti–PD-1 therapy. Together,
these data support the ability of brentuximab vedotin and
MMAE to drive ICD in tumor cells resulting in the activation
of antigen-presenting cells and augmented T-cell immunity.
These data provide a strong rationale for the clinical combina-
tion of brentuximab vedotin and other MMAE-based ADCs with
checkpoint inhibitors.
cellular environment promotes APC recruitment via purinergic
receptors and activation via the nucleotide-binding domain, leu-
cine-rich–containing family, pyrin domain–containing-3 (NLRP3)
inflammasome. Finally, HMGB1 release directly activates APCs
via Toll-like receptor 4 (TLR4; refs. 6–11). Together, the spatio-
temporal presentation of these damage-associated molecular pat-
terns (DAMP), concomitant with tumor antigen exposure from
dying tumor cells, can result in the development of antigen-specific
antitumor immunity (5).
Although a variety of anticancer therapeutic approaches with
different molecular mechanisms of action have been described pre-
viously to elicit ICD (radiotherapy, photodynamic therapy, anthra-
cyclines, and oxaliplatin; refs. 5, 12, 13), a common factor is the
dysregulation of ER homeostasis (1, 14–18). Overloading the ER’s
capacity for unfolded polypeptides, or other disruptions to the protein-
folding environment, initiates a canonical ER stress response known as
the unfolded protein response (UPR). A strong UPR, coupled with the
associated release of ER Ca2þ stores, is required to trigger ICD-related
hallmark DAMPs (7, 16, 17, 19). Translocation of CRT to the cell
surface and active secretion of ATP resulting from cellular autophagy
follow severe ER stress (7, 20–22), whereas the release of nuclear
HMGB1 is thought to occur as the dying cell loses nuclear and
plasma membrane integrity (22–24). Synthetic knockdown of the
ER stress response during treatment with ICD-inducing therapeu-
tics abrogates ICD (21, 25). Conversely, combining pharmacolog-
ical agents that perturb ER function with certain non–ICD-inducing
compounds have been shown to increase the immunogenicity of
dying tumor cells (15, 26–28).
Although some systemic chemotherapies elicit ICD, they can also be
associated with lymphopenia and systemic immune alterations often
requiring months for T cells to return to pre-treatment levels (29–32).
Antibody–drug conjugates (ADC) are a clinically validated strategy for
the selected delivery of cytotoxic molecules to tumors that largely avoid
AACRJournals.org | 68

persistent depletion of peripheral lymphocytes (33). The combination
of ICD-inducing ADCs with checkpoint inhibitors may therefore drive
complementary immune mechanisms. Multiple combinations of
ADCs with checkpoint inhibitors are being actively evaluated in the
clinic with promising early results (34, 35).
Multiple preclinical studies have described successful combina-
tions of ADCs with checkpoint inhibitors and other immunothera-
pies. M€ uller and colleagues (36) were the first to show that the
HER2-directed ADC trastuzumab emtansine (T-DM1) drove
immune infiltration in breast cancer lesions in the clinic. In
addition, T-DM1 combined with cytotoxic T-lymphocyte antigen 4
(CTLA-4) and programmed cell death protein 1 (PD-1) blockade
amplified antitumor responses in preclinical models (36). Data from
this group supported the direct activation of APCs by dolastatins,
from which monomethyl auristatin E (MMAE) was synthetically
derived, and also MMAE itself (37, 38). Rios-Doria and collea-
gues (39) showed that tubulysin and pyrrolobenzodiazepine dimer
(PBD)-based ADCs successfully combined with immunotherapies
in syngeneic tumor models, supportive of ICD induction. Finally,
monomethyl auristatin F, a derivative of MMAE with greatly
reduced membrane permeability, was shown to drive ICD with the
B-cell maturation antigen-directed ADC belantamab mafodotin
and combine with a checkpoint modulator (40). Together, these
studies highlight the potential immunomodulatory abilities of ADC
payloads, particularly those causing microtubule destabilization.
Indeed, results supporting ICD induction from vinca domain-
binding microtubule-destabilizing agents were predicted by early
ICD-screening platforms (41, 42).
Brentuximab vedotin is a CD30-directed vedotin ADC, current-
ly approved for use in the US for multiple CD30-expressing
lymphomas (43). Brentuximab vedotin comprises a CD30-directed
monoclonal antibody (mAb) conjugated to a protease cleavable
maleimidocaproyl–valine-citrulline (mc-vc) linker and the micro-
tubule-disrupting agent (MMAE; refs. 44, 45). Upon internalization,
the protease-cleavable linker enables the preferential release of
MMAE within CD30-expressing target cells, resulting in microtu-
bule disruption and cell death (45–48). We previously reported on
the ability of brentuximab vedotin and MMAE to induce ICD in
target cells via microtubule disruption and ER stress in a series of
in vitro and in vivo experiments (49, 50). Furthermore, the potential
for brentuximab vedotin or MMAE treatment to activate antitumor
immunity in combination with PD-1 inhibition was previously
evaluated (51). Here, we expand upon our initial findings of
brentuximab vedotin and MMAE-induced ICD and the enhanced
immune response observed in combination with PD-1 inhibition.
Materials and Methods
Animals and animal care
All animal studies were executed in compliance with institutional
guidelines and regulations and approved by an Institutional Animal
Care and Use Committee (IACUC) and internal review committee
under protocols SGE-024 and SGE-029. Mice were age- and sex-
matched and randomly assigned to treatment groups in all experi-
ments with females being predominantly used for studies. The number
of mice used in each experiment was determined on the basis of
previous experience with variability of model performance. Mice were
housed in an IACUC-certified housing facility under the supervision of
a licensed veterinarian. All animal studies were designed to minimize
animal usage and distress.
AACRJournals.org
Brentuximab Vedotin and Immunogenic Cell Death
Cell lines
Cell lines L540 (DSMZ, ACC72; RRID:CVCL_1362; human
female), KARPAS 299 (DSMZ, ACC31, RRID:CVCL_1324; human
male), HDLM-2 (DSMZ, ACC17; RRID:CVCL_0009; human male),
and A20 (ATCC, TIB-208; RRID:CVCL_1940; mouse) were originally
purchased from the vendors listed and since maintained by the Seagen
cell bank (2016–2023). For each study, cell lines were requested from
the cell bank and aliquots were thawed and cultured for periods
between 1 and 3 weeks. Cell bank cell lines are routinely checked
for contamination, including Mycoplasma, and cell line identity
authentication by an STR-based DNA profiling and multiplex PCR,
CellCheck 16–Human, or CellCheck 19 Mouse Plus, IDEXX Labora-
tories, Inc. Following any cell line modifications or cloning, expanded
lines were resubmitted for pathogen burden and cell line identity
validation before experimental use.
ICD hallmark assessment
CRT exposure
Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024
The lymphoma cell line L540cy was grown in RPMI and 20% FBS
(Gibco). Cells were treated with IC50 or IC90 concentrations of
brentuximab vedotin, doxorubicin, etoposide, or oxaliplatin for 0,
18, 24, or 48 hours. Cells were also treated with controls of a non-
binding ADC (IgG-MMAE), chimeric anti-CD30 antibody (cAC10)
naked mAb at the IC50 or IC90 concentrations of brentuximab vedotin.
Cells were harvested and stained for cell-surface CRT (FMC75, Enzo,
RRID:AB_11180758). Staining was concomitant with propidium
iodide (PI; Sigma) and all staining was performed as per the manu-
facturer’s recommendations. Surface levels of CRT on PI-negative cells
were analyzed by flow cytometry.
ATP and HMGB1 release assays
L540cy cells were plated at 200,000–3,000,000 cells/well on a 24-well
dish and allowed to reach 50% confluence. Cells were then treated with
IC90 (1 mg/mL) of brentuximab vedotin or a non-binding ADC (IgG-
MMAE), 100 nmol/L (IC90) MMAE, 1 mg/mL oxaliplatin, or 30 mmol/L
etoposide for 18 hours. For additional cell lines shown in the Sup-
plementary Figures, approximately 50,000 cells were incubated with
IC20–IC50 concentrations of test articles for 48–72 hours. After treat-
ment of cells, 250 mL of media were harvested from each well by tilting
the plate and transferred to a 1.5-mL Eppendorf tube. Cells were spun
at 1,600 rpm (200 rcf) for 1 minute in a microcentrifuge. Subsequently,
50 mL of media were transferred to wells in a 96-well clear-bottom plate
and 50 mL of CellTiter Glo (Promega) was added to each well and
shielded from light. Plates were shaken for 1 minute on an orbital
shaker, incubated for an additional 10 minutes at room temperature to
stabilize luminescent signal, and immediately read on an Envision
Plate Reader. ATP levels were normalized to levels from untreated cells.
HMGB1 release was assessed in a similar manner to ATP release.
L540cy cells were plated and treated for 18–72 hours. Extracellular
HMBG1 was measured in supernatants using an HMGB1 ELISA
(Tecan, IBL International).
Fluorescent staining
An Enzo Life Sciences’ ER-ID kit was used for fluorescent labeling of
the ER in L540cy cells. Antibodies against phosphorylated inositol–
requiring enzyme 1 (pIRE1; NB100–2323, Novus Biologicals, RRID:
AB_0145203) and anti-tubulin (YOL1/34, Abcam, RRID:AB_305329),
were used for fluorescent imaging. L540cy cell nuclei were stained using
Diamond Antifade Mountant with 40 ,6-diamidino-2-phenylindole
(Thermo Fisher Scientific).
Mol Cancer Ther; 23(1) January 2024 69
 Heiser et al.
ER stress determination by western blot analysis
L540cy cells were plated at 500,000 cells/well in a 12-well plate
overnight. Cells were then treated with IC90 (1 mg/mL) of brentuximab
vedotin or a non-binding ADC (IgG-MMAE), 100 nmol/L (IC90)
MMAE, vincristine, or paclitaxel for 18 hours. Treated L540cy cells
were collected, lysed in RIPA buffer (Cell Signaling Technology) and
centrifuged at 14,000–16,000 rpm for 10 minutes and stored at 20 C.
Cell pellets were resuspended in 4X BOLT LDS sample buffer (Thermo
Fisher Scientific) and lysed by heating at 95 C for 5–10 minutes to
produce cell lysates. Sample lysates were run on a Bis-Tris 4%–12%
gradient gel at 140 V for 1 hour 40 minutes in MOPS buffer. The Bis-
Tris gel was then transferred onto a nitrocellulose membrane using an
iBlot2. Membranes were washed once in 1X TBS and incubated
overnight at 4 C with antibodies against total and pIRE1, total and
phosphorylated c-Jun N-terminal kinase (pJNK), and activating tran-
scription factor 4 (ATF4) (Cell Signaling Technology and Novus;
RRID:AB_10145203, RRID:AB_2058748). Equal loading was con-
firmed with total actin or tubulin assessment (Cell Signaling Tech-
nology, RRID:AB_330288, RRID:AB_2210548). Membranes were
washed four times in 1X TBS-T for 5–10 minutes each. Signals were
normalized against actin.
C/EBP homologous protein (CHOP) luciferase assay
Induction of CHOP was measured using a reporter system for
CHOP activity according to the manufacturer’s instructions (Bright-
Glo Luciferase Assay System, Promega). In brief, 100,000 Mia-PaCa-2
cells/well (Signosis; SL-0025-FP) were plated in a 96-well, flat-bottom
clear plate (aliquot 150 mL/well). At 24, 48, and 72 hours, plates were
removed from the incubator and allowed to come to room temper-
ature. 100 mL of media were removed from the wells and 100 mL of
Bright-Glo reagent was added to each well. Plates were shaken for at
least 2 minutes before measurement on an Envision plate reader
(PerkinElmer).
Immune cell activation
Cancer cell/peripheral blood mononuclear cell (PBMC) co-cultures:
induction of innate and secondary T-cell responses following
co-culture of human PBMCs with cancer cells induced to undergo
ICD following exposure to brentuximab vedotin or MMAE
L540cy cancer cells were plated in 12-well plates and treated with
1 mg/mL of brentuximab vedotin, IgG-MMAE, or 0.1 mmol/L of
MMAE for 16 hours. Cells were harvested by moving samples to a
15-mL conical tube and centrifuging for 5 minutes at 400  g. Tissue
culture supernatants were removed and cells resuspended in com-
plete RPMI (10% FCS, 1 sodium pyruvate, 1 MEM NEAA,
1 GlutaMax; Gibco) at a total of 1107 cells/mL and plated at
250,000 cells/well in a 24-well plate. Human PBMC were thawed and
washed three times in complete RPMI and resuspended at a total of
1107 cells/mL. PBMCs were mixed with treated tumor cells 1:1 at
500,000 cells/well and incubated at 37 C, 5% CO2, for 24 or 48 hours.
Tissue culture supernatants were harvested for analysis of innate and
adaptive cell activation using the MILLIPLEX MAP Human Cyto-
kine/Chemokine Magnetic Bead Panel (Sigma).
L540cy xenograft
Five million L-540cy cells were implanted subcutaneously in
25% Matrigel (Corning) in female (RRID:IMSR_TAC:CB17SC) severe
combined immune deficient (SCID) mice (Taconic). When tumors
reached 200 mm3, mice (n ¼ 5) were randomized into treatment
groups and treated intraperitoneally (i.p.) with 3 mg/kg brentuximab
vedotin, IgG-MMAE, or cAC10 antibody. Seventy-two hours after
70 Mol Cancer Ther; 23(1) January 2024
treatment, tumors were harvested, weighed, and manually dissoci-
ated through a 70-mm cell strainer to generate single-cell suspen-
sions for flow cytometry. Tumor cell suspensions were stained with
Live/Dead Viability Dye (Thermo Fisher Scientific) and fluorescent
antibodies targeting murine CD45 (RRID:AB_2563542), CD11c
(RRID:AB_313779), CD11b (RRID:AB_312791), Ly-6G (RRID:
AB_10643269), Siglec-F (RRID:AB_2904295), and MHC II
(RRID:AB_493147; BioLegend). Alternatively, portions of each
tumor were weighed and lysed in RIPA buffer (Cell Signaling
Technology) for cytokine analysis using the MILLIPLEX MAP
Human Cytokine/Chemokine Magnetic Bead Panel (Sigma).
In vivo autologous lymphoblastoid cell line (LCL) PBMC
tumor model
A total of 2.5106 Epstein-Barr virus (EBV)-transformed CD30þ
LCLs, derived from a healthy donor, were implanted subcutaneously
into groups of eight nonobese diabetic (NOD)/SCID/gamma-chain
mice (NSG) mice (RRID:IMSR_JAX:005557). For assessing PBMC-
Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024
mediated rejection of LCL tumors, 0, 2.5, 5, or 10 million thawed
autologous donor PBMCs were given to mice intravenously 5 days
after tumor implant following which tumor volumes were measured
periodically.
For experiments testing the effect of brentuximab vedotin on
responding immune cells, mice received a single subtherapeutic dose
of brentuximab vedotin or a non-binding ADC (human IgG-MMAE;
1 mg/kg, i.p.) when tumor volumes averaged 250 mm3, then received
2.0106 autologous PBMCs 3 days post-dose. Four days following the
transfer of PBMCs, tumors were harvested from six mice. Tumor
RNA was purified using the RNeasy Plus Kit (Qiagen, Germany),
and RNA evaluated using the NanoString nCounter Human Immu-
nology panel (NanoString Technologies). Data were processed and
analyzed using nSolver software (NanoString Technologies). Eleven
days following transfer of PBMCs, tumors were harvested from
five mice, weighed, and manually dissociated through a 70-mm cell
strainer for FACS analysis. Tumor cell suspensions were stained
with Zombie Aqua Viability Dye (BioLegend) followed by incubation
with fluorescently labeled antibodies targeting human CD19 (RRID:
AB_2564143), CD2 (RRID:AB_2572066), CD8 (RRID:AB_2562790),
CD4 (RRID:AB_2572097), CD56 (RRID:AB_2563915), CD45
(RRID:AB_2562057), programmed death-ligand 1 (PD-L1; RRID:
AB_2563852), PD-1 (RRID:AB_2922606), and murine CD45.1
(RRID:AB_2562565; BioLegend) in staining buffer (PBS, 2% FCS,
1% normal rabbit serum, 0.05% NaN3) at 4 C for 30 minutes. Cells
were washed and resuspended in 120 mL of staining buffer for plate-
based flow cytometry using an Attune NXT flow cytometer. Total
events were collected from 80 mL of sample, and flow cytometry-
measured cell concentrations were used to calculate numbers of
infiltrating immune cells. CD8þ T cells were identified as viability
dye negative, hCD45þ, mCD45.1, CD2þ, and CD8þ cells. Natural
killer (NK) cells were identified as viability dye negative, hCD45þ,
mCD45.1, CD2þ, and CD56þ cells. Experiments combining bren-
tuximab vedotin with nivolumab treatments were conducted as
described above, with mice receiving two doses of nivolumab
(10 mg/kg, i.p.) 2 and 7 days after adoptive transfer of PBMCs.
Immunohistochemistry
The detection of infiltrating cells in the A20 syngeneic model was
performed as follows: mouse tissues were dissected and embedded in
OCT (TissueTek; #62550–01) and frozen in isopentane cooled in
liquid nitrogen. Sections were cut at 4 mm and stored frozen at 80 C.
Slides with frozen tissue sections were thawed, fixed in acetone for
MOLECULAR CANCER THERAPEUTICS
 Brentuximab Vedotin and Immunogenic Cell Death

A Surface calreticulin B Surface calreticulin+/PI–

100 Untreated
Oxaliplatin
80 Etoposide
BV

Positive (%)
Untreated cAC10
60
Oxaliplatin
40
Etoposide

cAC10 20

BV
0
100 101 102 103 104 6 24 48
Time (h)

C ATP secretion D HMGB1 release

Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

ns
*
* ns
* ns
8 *** * 4
* *
6 3
Fold change

Fold change

4 2

2 1

0 0
NT BV lgG-MMAE MMAE Etop Oxal NT BV lgG-MMAE MMAE Etop Oxal

Figure 1.
Brentuximab vedotin–treated Hodgkin lymphoma cells express ICD hallmarks. A, MFI histograms of surface CRT staining on live L540cy cells measured by flow
cytometry following treatment for 18 hours with the IC50 or IC90 concentrations of BV, oxaliplatin, etoposide, and cAC10. B, L540cy cell-surface expression of
CRT measured by flow cytometry following treatment over a 48-hour time course with BV, cAC10, oxaliplatin, or etoposide. C, Extracellular ATP measured from
L540cy cell-free supernatant following treatment for 18 hours with BV, IgG-MMAE, MMAE, oxaliplatin, or etoposide. D, Extracellular HMGB1 measured from L540cy
cell-free supernatant following treatment for 18 hours with BV, IgG-MMAE, MMAE, etoposide, or oxaliplatin. Representative data from three independent experiments.
Symbols represent biological replicates.  P < 0.05;   P < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparisons test) compared with BV. Bars at the
mean. ANOVA, analysis of variance; ATP, adenosine trisphosphate; BV, brentuximab vedotin; cAC10, chimeric anti-CD30 antibody; CRT, calreticulin; Dox, doxorubicin;
Etop, etoposide; h, hour; HMGB1, non-histone chromatin-binding protein high-mobility group box 1; IC, inhibitory concentration; ICD, immunogenic cell death; IgG,
immunoglobulin G; MFI, mean fluorescence intensity; MMAE, monomethyl auristatin E; PI, propidium iodide; ns, not significant; NT, not treated.

10 minutes at 20 C, and then air-dried for 10 minutes. Immunos-
taining was performed on the Bond-Max and BondIII autostainers
(Leica Biosystems, Inc.) using Bond Polymer Refine Detection (DAB)
Kits (Leica; #DS9800). Endogenous peroxidase activity was blocked
using PeroxAbolish (BioCare Medical; #PXA969). Protein Block
(Dako; #X0909) was used to block non-specific staining. Sections
were then incubated with Biotinylated Rat anti-Mouse CD8 clone
4SM15 (eBioscience, RRID:AB_2572771) or Biotinylated Rat IgG2a
clone eBR2a (eBioscience, RRID:AB_470084) as isotope control at
3 mg/mL. Bound antibody was detected using the Vectastain Elite ABC
HRP Kit (Vector Laboratories) and developed with DAB, from the
Bond Polymer Refine Staining Kit (Leica).
Formalin-fixed, paraffin-embedded LCL tumor tissues were sec-
tioned onto adhesive slides, deparaffinized, and rehydrated to dis-
tilled water. Slides were steamed for antigen retrieval in Diva solution
(Biocare). Sections were quenched with 3% H2O2 and blocked with
0.5% Casein and 5% Goat Serum in TBS. Mouse anti-human CD8
(Abcam RRID:AB_443686) was applied for 1 hour, washed, and
detected with Biotinylated Goat anti-Mouse IgG (The Jackson
Laboratory, RRID:AB_2338569). Sections were then developed with
Vectastain Elite ABC HRP Kit (#PK-6100) and Vector ImmPact
DAB (#SK-4103).
A20hCD30 cell line vaccination experiments
To generate human CD30-expressing A20 mouse lymphoma cells,
cells were transfected with plasmid constructs encoding human
TNFRSF8 (NM_001243.4) and sgRNA/Cas9 for control under the
endogenous Rosa26 site for constitutive expression. FACS yielded a
clonal population of A20 cells that stably expressed human CD30,
called A20hCD30.

AACRJournals.org Mol Cancer Ther; 23(1) January 2024 71

 Heiser et al.

lgG-
A B NT BV MMAE MMAE

hlgG- pIRE1
MMAE
IRE1

pJNK
MMAE

JNK

ATF4
BV
Actin
Nuclei Tubulin ER Merged L540cy

C D
NT BV
NT

pIRE

MMAE IRE
L540cy
Nuclei ER pIRE1 Merged

Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

E L540cy XBP1 HDLM-2 XBP1 KARPAS 299 XBP1 A20hCD30 XBP1
30 50 **** ns 80 100 ***
**** **** ***
Positive (%)
Positive (%)

80

Positive (%)
40

Positive (%)
*** ns ** 60 *
20 **** **
30 60
40
10 20 40
10 20 20
0 0 0 0
NT IgG- BV MMAE NT IgG- BV MMAE NT IgG- BV MMAE NT IgG- BV MMAE
MMAE MMAE MMAE MMAE

L540cy ATF6 HDLM-2 ATF6 KARPAS 299 ATF6 A20hCD30 ATF6

50 **** 50 80 100 ***
*** ***
****
Positive (%)

**** *

Positive (%)
Positive (%)

40 40 80
Positive (%)

** 60 **** *
30 **** ns 60
30
40
20 20 40
10 10 20 20
0 0 0 0
NT IgG- BV MMAE NT IgG- BV MMAE NT IgG- BV MMAE NT IgG- BV MMAE
MMAE MMAE MMAE MMAE

L540cy pPERK HDLM-2 pPERK KARPAS 299 pPERK A20hCD30 pPERK

20 * 60 80 ****
ns ns * 30 ****
Positive (%)
Positive (%)

ns ****
Positive (%)

ns 60
**** ns
Positive (%)

15
40 ***
10 20 40
5 20 10 20
0 0 0 0
NT IgG- BV MMAE NT IgG- BV MMAE NT IgG- BV MMAE NT IgG- BV MMAE
MMAE MMAE MMAE MMAE

F NT MMAE Paclitaxel Vincristine NT MMAE Paclitaxel Vincristine

pIRE1 pJNK

IRE1 JNK

G Dose-CHOP-luciferase induction H CHOP-luciferase activity

4 15
MMAE NT
Paclitaxel MMAE
Fold over pre-dose

3 Vincristine Paclitaxel
Fold change

10 Vincristine

2
5
1

0 0
0.1 1 10 100 1,000 0 24 48 72 96
nmol/L Hours post-dose

72 Mol Cancer Ther; 23(1) January 2024 MOLECULAR CANCER THERAPEUTICS

 Brentuximab Vedotin and Immunogenic Cell Death

A20hCD30 cells were cultured in RPMI-1640 with 10% FBS,
10 mmol/L HEPES, 1 mmol/L sodium pyruvate, penicillin (100 U/mL),
and streptomycin (100 mg/mL). A20hCD30 cells were treated with
1 mg/mL brentuximab vedotin or 100 nmol/L mc–vc–MMAE for
4 days. To prepare dying cells for immunization, treated A20hCD30
cells were overlaid atop Histopaque and centrifuged at 2,000  g for
30 minutes. Dead and dying cells were pelleted underneath the
Histopaque layer and viability was assessed to be <20% live cells by
trypan blue exclusion. Dead and dying A20hCD30 cells were resus-
pended in PBS and a total of 2  106 cells were injected into the
peritoneum of immune-competent BALB/c mice (n ¼ 8). Flash-frozen
A20hCD30 cells were prepared by submerging cells in liquid nitrogen for
10 seconds, followed by immersion in 37 C water until completely
thawed. The liquid nitrogen freeze-thaw process was repeated five
times. Fourteen days later, mice received a second immunization with
dead and dying cells prepared in the same manner.
Twenty-one days after initial immunization, mice were subcuta-
neously implanted with a total of 5  106 wild-type (WT) A20 cells and
ciences). Cells were then washed with FACS buffer þ Annexin V
binding buffer before fixation and permeabilization using the True
Nuclear Kit, 96-well plate protocol (BioLegend). Overnight intracel-
lular staining at 4 C was then performed upon the addition of 100 mL
of permeabilization buffer containing antibodies to each well. A list of
antibodies with corresponding RRIDs used for intracellular staining is
provided in Supplementary Table S1. Cells were then washed three
times in permeabilization buffer, resuspended in PBS, and analyzed on
an Attune Flow Cytometer.
Statistical analysis
Descriptive statistics were used throughout. Where applicable,
significance was determined by an ANOVA with Sidak or Dunnett’s
multiple comparisons tests, calculated using GraphPad Prism 8.1
(RRID:SCR_000306).
Data and materials availability
All available data are presented.

Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

monitored for tumor growth. In some experiments, mice were treated
with four administrations of anti–PD-1 over 2 weeks (1 mg/kg;
BioLegend, RRID:AB_2783090).
Immune-competent BALB/c mice (n ¼ 4) were immunized with
brentuximab vedotin-killed or flash-frozen–killed A20hCD30 cells as
previously described. Sixteen weeks after initial immunization, spleens
were harvested from immunized mice or naive BALB/c mice and
manually homogenized. Splenic homogenates were combined from
four mice per immunization group and CD3þ T cells were isolated
using the EasySep Mouse T-cell Enrichment Kit (Stem Cell Technol-
ogies). One million CD3þ T cells were administered intravenously into
WT A20 tumor-bearing NSG mice when tumors averaged 100 mm3
(n ¼ 5–8). Tumors were subsequently monitored for growth and at
study takedown for CD8þ T-cell content by flow cytometry.
Intracellular staining of UPR markers by flow cytometry
Cells were harvested 48 hours following treatment with test articles.
Supernatants were transferred to round-bottom plates to collect non-
adherent cells. Versene (200 mL; Thermo Fisher Scientific) was then
added to each well, and upon detachment, cells were transferred to the
corresponding round-bottom plates used to collect non-adherent cells.
Cells were pelleted by spinning at 1,500 rpm for 5 minutes. Plates were
decanted, washed with PBS and, following an additional spin, cells
were stained with Live/Dead Yellow cell viability dye (Thermo Fisher
Scientific) per the manufacturer’s protocol. Surface staining for calre-
ticulin and Annexin V was then performed in FACS buffer (2%
FBS/PBS) with the addition of Annexin V binding buffer (BD Bios-
Results
Brentuximab vedotin–treated Hodgkin lymphoma (HL) cells
express ICD hallmarks
Cell surface CRT expression, the first hallmark of ICD, was eval-
uated on CD30þ L540cy cells to determine whether cell death induced
by brentuximab vedotin is consistent with ICD induction. The L540cy
cell line is a derivative of the CD30þ HL L540 line adapted for growth
as a xenograft. L540cy cells were treated with brentuximab vedotin,
IgG-MMAE, oxaliplatin (a known ICD inducer; ref. 12), or etoposide
(which does not induce ICD; ref. 27). After 18 hours of treatment, cells
were harvested, stained for cell-surface CRT expression, and levels
were analyzed by flow cytometry. The mean fluorescence intensity of
surface CRT at 18 hours was increased in live cells (Annexin V/PI)
after brentuximab vedotin treatment, whereas IgG-MMAE or etopo-
side-treated cells exhibited minimal changes in surface CRT (Fig. 1A).
Furthermore, the proportion of brentuximab vedotin–treated cells
expressing surface CRT was increased at 48 hours (Fig. 1B). Impor-
tantly, similar increases in expression of cell surface CRT were
observed in three separate human lymphoma cell lines and the murine
A20 tumor cell line engineered to express human CD30 (A20hCD30;
Supplementary Fig. S2A), demonstrating a common effect across
multiple target cell lines.
The second hallmark signature of ICD induction is active secretion
of ATP through pannexin channels. Release of ATP serves as a potent
chemotactic signal recruiting immune cells to the inflamed site (52).

Figure 2.
Brentuximab vedotin–induced ICD is associated with severe ER stress. A, Fluorescent staining of tubulin, ER, and nuclei in L540cy cells treated for 16 hours with
human IgG-MMAE, MMAE, or BV. B, Western blot analysis of ER stress markers in L540cy cells treated for 18 hours with BV, IgG-MMAE, and MMAE. C, Spatial
relationship of ER stress markers with the ER network in L540cy cells treated for 18 hours with MMAE. D, Western blot analysis of pIRE1 and IRE1 expressions in L540cy
cells treated for 18 hours with a titration of BV. E, Intracellular detection of the proportion of viable lymphoma cells (L540cy, HDLM-2, KARPAS 299, and A20hCD30)
upregulating UPR-associated proteins XBP1, ATF6, and pPERK by flow cytometry 48 hours after treatment with IC20–IC50 doses of the indicated agents.
F, Representative western blot analysis of pIRE1, IRE1, pJNK, and JNK expression in L540cy cells treated for 18 hours with MMAE, vincristine, or paclitaxel. G, Dose–
CHOP luciferase induction in Mia-PaCa-2 cells treated with MMAE, vincristine, or paclitaxel in vitro. H, CHOP-luciferase activity in Mia-PaCa-2 cells treated
intratumorally with MMAE, vincristine, or paclitaxel in vivo. Western blot analysis band intensity quantification provided in Supplementary Fig. S3. Symbols
represent biological replicates.  P < 0.05;   P < 0.01;    P < 0.001;     P < 0.0001, (one-way ANOVA followed by Dunnett’s multiple comparisons test)
compared with BV (for human lines), or MMAE (for A20hCD30). Bars at the median. ANOVA, analysis of variance; ATF4, activating transcription factor 4; ATF6,
activating transcription factor 6, BV, brentuximab vedotin; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; hIgG, human immunoglobulin G; IC,
inhibitory concentration; IgG, immunoglobulin G; IRE1, inositol-requiring enzyme 1; JNK, c-Jun N-terminal kinase; MMAE, monomethyl auristatin E; ns, not
significant; NT, not treated; pIRE1, phosphorylated IRE1; pJNK, phosphorylated JNK; pPERK, phosphorylated protein kinase RNA-activated–like endoplasmic
reticulum kinase; UPR, unfolded protein response; XBP1, X-box binding protein 1.

AACRJournals.org Mol Cancer Ther; 23(1) January 2024 73

 Heiser et al.

A Human dendritic cells Human T cells

MIP-1α IP10/CXCL10 IFNγ TNFα
* ns ns
ns ns ns
ns
** *
* *
ns ns
3,000 1,200 1,500 * 500
ns
ns 400
900
2,000 1,000
pg/mL

pg/mL

pg/mL

pg/mL
300
600
200
1,000 500
300
100

0 0 0 0

Ig M BV
-M AE

O E
Et l
op
Ig M B V
-M AE

O E
Et l
op

Ig M BV
-M AE

O E
Et l
op

Ig M BV
-M AE

O E
Et l
op
xa
xa

xa

xa
A
A

A
G M
M
G M
M

G M
M

G M
M

Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

B Dendritic cells Dendritic cells/tumor Neutrophils/immune cells
ns
***
Percentage of CD11c/total cells
Percentage of CD11c of CD45+

*** ns

Percentage of Ly6g/CD45+
25 5 15
***
20 4
*
10
15 3

10 2
5
5 1

0 0 0
cAC10 IgG-MMAE BV cAC10 IgG-MMAE BV cAC10 IgG-MMAE BV

Intratumoral IP10/CXCL10 Intratumoral MIP-1α Intratumoral TNFα

C
**
* ns *
5,000 1,000 250 ns
ns
4,000 800 200
pg/mL

pg/mL

pg/mL

3,000 600 150

2,000 400 100

1,000 200 50

0 0 0
cAC10 IgG-MMAE BV cAC10 IgG-MMAE BV cAC10 IgG-MMAE BV

D Serum IP10/CXCL10 Serum MIP-1α Serum TNFα

ns ns
ns
600 * 80 ns 15 *
60
400 10
pg/mL

pg/mL

pg/mL

40
200 5
20

0 0 0
cAC10 IgG-MMAE BV cAC10 IgG-MMAE BV cAC10 IgG-MMAE BV

74 Mol Cancer Ther; 23(1) January 2024 MOLECULAR CANCER THERAPEUTICS

 Brentuximab Vedotin and Immunogenic Cell Death

L540cy cells were treated with brentuximab vedotin, a non-binding
ADC, free MMAE, oxaliplatin, or etoposide for 18 hours. Cell-free
supernatant was collected and analyzed for ATP levels that were
normalized to levels from untreated cells. Following treatment with
brentuximab vedotin or free MMAE, extracellular ATP levels approx-
imated or were greater than those obtained following treatment
with oxaliplatin (Fig. 1C). Treatment with etoposide, a cytotoxic
agent that does not induce ICD, did not induce robust ATP secretion
from dying L504cy cells. Heightened ATP secretion following bren-
tuximab vedotin and MMAE treatment was also observed with
additional lymphoma lines KARPAS 299 and the murine A20hCD30
(Supplementary Fig. S2A).
Finally, extracellular HMGB1 elicits inflammatory responses as a
ligand for TLR4, TLR2, and advanced glycosylation end product-
specific receptor (AGER, RAGE), triggering potent NF-kB responses
in immune cells (1, 6, 10). After 18 hours of treatment with brentux-
imab vedotin, free MMAE, etoposide, or oxaliplatin, HMGB1 was
elevated in the cell-free supernatant of L540cy cells (Fig. 1D).
and pJNK were minimally expressed with controls (no treatment and
IgG-MMAE; Fig. 2B and D). Furthermore, microscopy showed pIRE1
to be spatially localized with the structurally disrupted ER in MMAE-
treated L540cy cells compared with control, corroborating a potent
UPR response (Fig. 2C). Further evidence for activation of the three
arms of the UPR was provided by staining of intracellular XBP1, ATF6,
and phosphorylated protein kinase RNA-activated-like ER kinase by
flow cytometry for CD30-expressing lymphoma lines 48 hours after
treatment with brentuximab vedotin or MMAE (Fig. 2E). Significant
increases in UPR stress markers were observed for each cell line treated
with brentuximab vedotin compared with untreated controls. Com-
pared with human lymphoma lines, the engineered A20hCD30 synge-
neic lymphoma expressed much lower levels of CD30 (Supplementary
Fig. S2B) and showed stronger UPR induction from free MMAE
treatment compared with brentuximab vedotin.
The effects of using microtubule-destabilizing agents (MMAE
and vincristine) versus a stabilizing agent (paclitaxel) were inves-
tigated to determine whether the underlying mechanism behind ER

Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

Increased release of HMGB1 was also observed for KARPAS 299,
HDLM-2, and A20hCD30 cell lines following brentuximab vedotin or
MMAE treatment at 72 hours (Supplementary Fig. S2A). Elevation of
HMGB1, a late-stage marker of ICD, was seen upon etoposide
treatment, but occurred in the absence of robust CRT exposure and
ATP release, indicating that molecules like MMAE are unique in their
ability to drive all three markers of ICD. Overall, these results
demonstrate that treatment of CD30þ lymphoma cell lines with
brentuximab vedotin or MMAE was associated with the presentation
of three hallmarks of ICD.
MMAE-induced microtubule disruption by brentuximab vedotin
leads to severe ER stress
The ER is a dynamic cellular organelle that expands and contracts
depending on the functional needs of the cell (53, 54). Expansion and
contraction occur along the tubulin highway (55) and catastrophic
disruption of that network can inhibit normal ER function (56). As
such, the relationship between MMAE-mediated disruption of the
microtubule network and the resultant effects on ER structure and
stress response in treated cells was investigated. Compared with
control (IgG-MMAE), treatment of L540cy cells with brentuximab
vedotin or free MMAE led to a striking qualitative disorganization of
the perinuclear ER network (Fig. 2A). Disruption of protein folding in
the ER results in a well-defined stress response, referred to as the UPR,
purposed for re-establishing ER homeostasis. To determine whether
the observed disorganization of the ER network occurs concomitant
with the UPR response, L540cy cells were treated for 18 hours with
brentuximab vedotin or free MMAE. After 18 hours, there was a rapid
induction and activation of multiple arms of the UPR, including
increased pIRE1, downstream pJNK, and ATF4 expression; pIRE1
stress induction was associated with collapse of the microtubule
network. Although IRE1 phosphorylation occurred after treatment
with all agents, indicating acute ER stress and initial activation
of stress responses, only the microtubule-destabilizing agents
MMAE and vincristine elicited downstream JNK phosphorylation
(Fig. 2F). Pixel densities for all western blot analysis images are
provided in Supplementary Fig. S3A–S3D. Robust pIRE1 and pJNK
upregulation was also observed following treatment with brentux-
imab vedotin or MMAE in additional lymphoma lines by intracel-
lular flow cytometry (Supplementary Fig. S4A). Sustained activation
of the UPR response ultimately leads to the initiation of ICD
through the induction of CHOP. To assess CHOP induction from
microtubule-stabilizing versus -destabilizing compounds, an ER
stress reporter system of Mia-PaCa-2 cells transduced with a
CHOP-driven luciferase was employed. In this system, paclitaxel-
driven microtubule stabilization did not drive equivalent CHOP
induction as the microtubule-destabilizing compounds MMAE and
vincristine, despite visible cell death (Fig. 2G). CHOP induction
was confirmed in three additional lymphoma lines treated with
brentuximab vedotin or free MMAE by intracellular flow cytometry
(Supplementary Fig. S4B).
Induction of ER stress with microtubule-disrupting agents, includ-
ing MMAE, was validated in vivo by engrafting CHOP-luciferase Mia-
PaCa-2 cells into NSG. Xenografts were treated intratumorally with
MMAE, vincristine, or paclitaxel and monitored daily for luciferase
activity. Soon after administration, CHOP induction, as monitored by
luciferase activity, was observed from tumors treated with MMAE or
vincristine, peaking at 72 to 96 hours after treatment (Fig. 2H).
Notably, luciferase was detectable 24 hours after treatment, indicating
that the response happened quickly and increased over time.

Figure 3.
Proinflammatory chemokine and cytokine production in human dendritic and T cells and BV-induced ICD in L540cy xenografts. A, Chemokine and cytokine
measurements by immunoassay (Luminex) following washing out of treatment agents. B, Recruitment of CD11cþ DCs compared with Ly6gþ cells into a L540cy
xenograft 72 hours after administration of a single dose of BV, IgG-MMAE, or cAC10 (murine model; n ¼ 5); levels of immune cells were determined by flow cytometry.
C, Intratumoral murine cytokine activity in mice harboring L540cy xenografts 72 hours after administration of a single dose of BV, IgG-MMAE, or cAC10 (n ¼ 5);
cytokines were measured by immunoassay (Luminex). D, Upregulation of murine serum chemokines and cytokines in mice harboring L540cy xenografts 72 hours
after administration of a single dose of BV, IgG-MMAE, or cAC10 (n ¼ 5); cytokines were measured by immunoassay (Luminex). Representative data from two
independent experiments, each symbol represents individual mice (n ¼ 4–5 per group).  P < 0.05;   P < 0.01;   P < 0.001, (one-way ANOVA followed by Dunnett’s
multiple comparisons test) compared with BV treatment. Bars represent the mean and SE. ANOVA, analysis of variance; BV, brentuximab vedotin; cAC10, chimeric
anti-CD30 antibody; CCL2, chemokine (C-C motif) ligand 2; CXCL10, C-X-C motif chemokine ligand 10; Etop, etoposide; ICD, immunogenic cell death; IFN, interferon;
IgG, immunoglobulin G; IP10, interferon gamma-induced protein 10; MIP-1a, macrophage inflammatory protein-1a; MMAE, monomethyl auristatin E; ns, not
significant; NT, not treated; Oxal, oxaliplatin; SE, standard error; TNF, tumor necrosis factor.

AACRJournals.org Mol Cancer Ther; 23(1) January 2024 75

 Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

MOLECULAR CANCER THERAPEUTICS

76 Mol Cancer Ther; 23(1) January 2024
Heiser et al.
 Brentuximab Vedotin and Immunogenic Cell Death

Treatment with microtubule-stabilizing paclitaxel did not elicit lucif-
erase activity, but inhibited tumor growth, indicating that antitumor
activity of paclitaxel did not rely on ER stress induction.
Immune cell activation by cells undergoing ICD in response to
brentuximab vedotin
After confirming that cells treated with brentuximab vedotin exhibit
signs of severe ER stress and express the key ICD hallmarks, we
questioned whether the dying cells were able to activate innate and
adaptive immune responses. To this end, brentuximab vedotin–killed
L540cy cells were fed to human monocyte-derived CD11cþ dendritic
cells (DC) in vitro and proinflammatory chemokine and cytokine
production were measured in supernatants. Notably, macrophage
inflammatory protein-1a (MIP-1a) and IFN gamma–induced protein
10/C-X-C motif chemokine ligand 10 (IP10/CXCL10) were increased
in supernatants from DCs that were fed cells treated with brentuximab
vedotin, MMAE, etoposide, or oxaliplatin compared with IgG-MMAE
(Fig. 3A). Treatment with the non-targeting ADC did not significantly
significant or trending increases in intratumoral and serum murine
proinflammatory cytokines TNFa, IP10, and MIP-1a (Fig. 3C
and D). Furthermore, tumor-derived cytokine concentrations were
notably higher than those detected in circulation, consistent with
tumor-localized production. In vitro activation of human immune
cells and recruitment of murine DCs in the L540Cy xenograft
model following treatment with brentuximab vedotin are consis-
tent with ICD induction and highlight the potential for driving
focused immune activation in the TME following treatment with
MMAE-based ADCs.
Brentuximab vedotin–induced ICD promotes CD8þ T-cell and
NK-cell recruitment to human LCL tumors
The ability of brentuximab vedotin treatment to promote proin-
flammatory cytokine production in xenografts is consistent with ICD-
driven activation of innate immunity. To further investigate the impact
of this inflammatory response on the recruitment and activation of
antigen-specific cytotoxic T cells to the TME, we used a humanized
LCL/PBMC model. This model pairs EBV-transformed CD30þ LCL

Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

promote proinflammatory mediators, showing that delivery of MMAE
to tumor cells was required for driving proinflammatory cytokines
from human DCs. Oxaliplatin and etoposide, which were both shown
to induce HMGB1 release from L540Cy cells (Fig. 1D), also drove
proinflammatory cytokine secretion consistent with this component of
ICD. In addition, when brentuximab vedotin- and MMAE-treated
cells were co-cultured with DCs and autologous T cells, increased
production of T-cell cytokines IFNg and TNFa was observed, com-
pared with IgG-MMAE and a trending increase compared with
etoposide-treated cells (Fig. 3A). Together, the increase in DC- and
T-cell–derived proinflammatory cytokines demonstrates the ability of
MMAE- and brentuximab vedotin–killed tumor cells to promote APC
activity.
The host innate immune response to brentuximab vedotin–
treated tumors was investigated in vivo in a human L540cy xeno-
graft model in SCID mice. SCID mice lack T and B cells but retain
innate immune cells enabling evaluation of APC activation and
recruitment following ADC killing of xenograft tumors. Although
brentuximab vedotin treatment did not significantly influence the
scale of immune infiltration into the tumor by 72 hours, it did begin
to reshape the immune landscape as shown by increased propor-
tions of antigen-presenting CD11cþ DCs in tumors relative to
Ly6gþ neutrophils (Fig. 3B). Skewing of the available innate
immune compartment toward mature DCs (CD11cþ DCs) may
reflect recruitment of DCs into the tumor or differentiation of
infiltrating monocytes following tumor cell killing by brentuximab
vedotin. Furthermore, consistent with innate immune cell activa-
tion and recruitment following ICD induction, brentuximab vedo-
tin treatment, compared with IgG-MMAE and cAC10, led to
tumors with adoptively transferred EBV-reactive autologous PBMCs to
evaluate antigen-specific T-cell responses in vivo. In this model, intra-
venous transfer of autologous PBMCs results in a titratable, immune-
mediated elimination of the tumor as measured by tumor volume
reduction (Fig. 4A). To evaluate whether treatment of LCL tumors
with brentuximab vedotin can influence the innate and adaptive arms of
the immune system, mice with established LCL tumors (200 mm3) were
given a subtherapeutic dose of brentuximab vedotin (the highest dose
without consistent tumor reduction; 1 mg/kg) followed by adoptive
transfer of autologous PBMCs 3 days later. Tumors from brentuximab
vedotin–treated and control groups were harvested for analysis 4 and
11 days after PBMC transfer (Fig. 4B). NanoString analysis of brentux-
imab vedotin-treated LCL tumors harvested 4 days after PBMC transfer
showed increased lysosome-associated membrane glycoprotein 3 tran-
script consistent with UPR-induced autophagy in tumor cells (Fig. 4C;
ref. 57). UPR-induced autophagy in tumor cells undergoing ICD drives
the hallmark release of extracellular ATP that signals via purinergic
receptors to activate the NLRP3 inflammasome in neighboring macro-
phages (22, 58, 59). Consistent with increased extracellular ATP, LCL
tumors treated with brentuximab vedotin showed increased NLRP3
transcript (Fig. 4C). Finally, signatures reflecting early innate (CD11b,
CXCL9) and cytotoxic immune cell (IFNg, Granzyme B) activity and
recruitment were strongly upregulated in brentuximab vedotin-treated
samples compared with non-targeted ADC and antibody alone controls
(Fig. 4C). Eleven days after PBMC transfer, flow cytometry of processed
tumors showed significantly increased CD8þ T cells and CD56þ NK
cells in brentuximab vedotin–treated mice, demonstrating enhanced
recruitment, and/or proliferation of innate and adaptive cytotoxic

Figure 4.
Brentuximab vedotin treatment of humanized mice bearing LCL tumors enhances intratumoral immune activation. NSG mice (n ¼ 5) harboring CD30þ LCL tumors
were injected intravenously with autologous donor PBMCs 5 days after tumor implantation. A, Tumor volume was measured over time in NSG mice (n ¼ 5 per group)
receiving no PBMCs and 2.5, 5, and 10 million PBMCs. B, Mice were treated with BV, cAC10, or a non-binding control (human IgG-MMAE) 3 days before receiving a total
of 2.0  106 PBMCs (n ¼ 10 per group). Tumors were collected for analysis of immune activity 4 and 11 days after transfer of PBMCs (n ¼ 5 per time-point). C, Immune-
related transcripts (NanoString) were measured 4 days following PBMC transfer in NSG mice treated with BV, cAC10, or a non-binding control (IgG-MMAE) 3 days
before PBMC transfer. D, Tumors taken 11 days after PBMC transfer in NSG mice treated with BV or a non-binding control (IgG-MMAE) 3 days before PBMC transfer
were processed and evaluated by flow cytometry for immune cell infiltration. Each symbol represents NanoString analysis or flow cytometry of immune cells from
individual mice (n ¼ 4–7 per group).  P < 0.05;   P < 0.01;    P < 0.001;     P < 0.0001 (one-way ANOVA followed by Dunnett’s multiple comparisons test)
compared with BV treatment. Bars represent the mean and SE. ANOVA, analysis of variance; BV, brentuximab vedotin; cAC10, chimeric anti-CD30 antibody; CXCL9,
C-X-C motif chemokine ligand 9; GZMB, granzyme B; IFNg, interferon gamma; hIgG, human immunoglobulin G; IgG, immunoglobin G; ITGAM, integrin alpha M;
LAMP3, lysosomal-associated membrane glycoprotein 3; LCL, lymphoblastoid cell line; MMAE, monomethyl auristatin E; NK, natural killer, NLRP3, nucleotide-binding
domain, leucine-rich–containing family pyrin domain–containing-3; NOD, nonobese diabetic; NSG, NOD SCID gamma; PBMC, peripheral blood mononuclear cells;
SCID, severe combined immune deficient; SE, standard error; TIL, tumor-infiltrating lymphocyte; TNF, tumor necrosis factor.

AACRJournals.org Mol Cancer Ther; 23(1) January 2024 77

 Heiser et al.

A 1,250
Untreated
1,250
MMAE-killed A20
1,250
BV-killed A20
1,250
Freeze-thawed A20

Tumor volume (mm3)

Tumor volume (mm3)

Tumor volume (mm3)

Tumor volume (mm3)

1,000 1,000 1,000 1,000
1/5 CR 6/8 CR 5/8 CR 3/8 CR
750 750 750 750
500 500 500 500
250 250 250 250
0 0 0 0
0 7 14 21 28 35 42 0 7 14 21 28 35 42 0 7 14 21 28 35 42 0 7 14 21 28 35 42
Days post-tumor implant Days post-tumor implant Days post-tumor implant Days post-tumor implant

B Non-immunized
Immunized with
MMAE-killed A20 cells
Immunized with
BV-killed A20 cells

200 µm

C Untreated MMAE-killed A20 BV-killed A20

Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

Tumor volume (mm3)

Tumor volume (mm3)

Tumor volume (mm3)

1,000 1,000 1,000
0/9 CR 5/9 CR 4/9 CR
750 750 750
500 500 500
250 250 250
0 0 0
0 7 14 21 28 35 0 7 14 21 28 35 0 7 14 21 28 35
Days post-tumor implant Days post-tumor implant Days post-tumor implant

Anti–PD-1 MMAE-killed A20 + anti–PD-1 BV-killed A20 + anti–PD-1

Tumor volume (mm3)

Tumor volume (mm3)

Tumor volume (mm3)

1,000 1,000 1,000
750 2/9 CR 750 9/9 CR 750 8/9 CR

500 500 500

250 250 250
0 0 0
0 7 14 21 28 35 0 7 14 21 28 35 0 7 14 21 28 35
Days post-tumor implant Days post-tumor implant Days post-tumor implant

D No T Cells T cells from naïve BALB/c T cells from BV-killed A20 T cells from freeze-thawed A20
Tumor volume (mm3)

Tumor volume (mm3)

Tumor volume (mm3)

Tumor volume (mm3)

1,250 1,250 1,250 1,250

1,000 1,000 1,000 1,000
750 750 750 750
500 500 500 500
250 250 250 250
0 0 0 0
0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
Days post-tumor implant Days post-tumor implant Days post-tumor implant Days post-tumor implant

Tumor AUC from

E T-cell adoptive transfer F Intratumoral CD8
ns
***
ns
15,000 8
total tumor cells (CD45+)

**
AUC to day 19

Percentage of

* 6

10,000 4

2
5,000
0
aw e-
ve

SG
aw e-

ve
Fr d

ed
ed

BV
ille

th ez
th z

aï

aï
ee

N
N

e
-k

N
Fr
BV

78 Mol Cancer Ther; 23(1) January 2024 MOLECULAR CANCER THERAPEUTICS

 Brentuximab Vedotin and Immunogenic Cell Death

cells (Fig. 4D; Supplementary Fig. S5). Results from the LCL/PBMC
xenograft model are consistent with upregulation of inflammatory
murine chemokines in L540cy xenografts treated with brentuximab
vedotin (Fig. 3) and substantiate enhancement of both innate cell and
antigen-specific T-cell responses following brentuximab vedotin–driven
ICD induction.
Brentuximab vedotin–mediated ICD drives T-cell memory and
protects mice from subsequent tumor challenge
The “gold-standard” experiment for assessing a compound’s ability
to elicit functional ICD involves vaccination of mice with treated
syngeneic tumor cells followed later by tumor rechallenge. Bona fide
ICD-inducing compounds drive lasting antigen-specific memory
responses that prevent tumor growth upon subsequent implant with
the same cell line (6, 12, 60). To validate ICD induction and tumor-
specific adaptive immune responses following brentuximab vedotin
treatment, the human CD30-engineered syngeneic A20 B-cell lym-
phoma model (A20hCD30) was used. A20hCD30 cells were killed in vitro
To further demonstrate the impact of MMAE-induced ICD on the
long-lived cytotoxic T-cell response, CD3þ T cells were isolated from
the spleens of tumor cell-immunized BALB/c mice 5 months after
vaccination and adoptively transferred to NSG mice harboring existing
A20 tumors (approximately 100 mm3). Use of NSG mice precludes
host antitumor immunity, enabling the assessment of adoptively
transferred T-cell activity. NSG mice receiving no T cells, T cells
transferred from naive mice, or T cells from mice immunized with
control freeze-thawed–killed tumor cells showed minimal tumor
control and were sacrificed at study take down (day 19), when all
control tumors exceeded 1,000 mm3. In contrast, mice receiving T cells
from mice immunized with brentuximab vedotin–killed A20hCD30
tumor cells showed significantly slowed tumor growth up to day 19 as
determined by the tumor volume area under the curve (Fig. 5D and E).
Furthermore, mice receiving T cells from brentuximab vedotin–
immunized mice showed a trend toward increased CD8þ T-cell
infiltration in A20 tumors at take down (Fig. 5F). Together, these
results are consistent with enhanced T-cell immunity elicited by

Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

with brentuximab vedotin or MMAE and injected into WT BALB/c
mice. As a negative control, A20hCD30 cells were flash-frozen in liquid
nitrogen providing tumor-associated antigens in the absence of
ICD induction. Mice were immunized two times, and 21 days after
the initial immunization mice were implanted with WT A20 lympho-
ma cells and monitored for tumor growth. The majority of mice
immunized with brentuximab vedotin- or MMAE-killed cells began
to reject the tumors around 7 days after implant, resulting in 5/8 and
6/8 complete responses (CR)s, respectively, consistent with a
primed adaptive immune response. In contrast, unimmunized naive
mice and mice immunized with control freeze-thawed cells showed
accelerated tumor growth and fewer CRs, 1/5 and 3/8 CRs, respec-
tively (Fig. 5A). Consistent with memory T-cell generation fol-
lowing vaccination, mice that had previously received brentuximab
vedotin- or MMAE-killed cells showed rapid infiltration and
increased murine CD8þ T cells within A20 tumors shortly after
implant (Fig. 5B).
Clinical response rates to PD-1/PD-L1 therapies correlate with the
presence of CD8þ T cells in the TME (61). Given that T cells from mice
immunized with brentuximab vedotin–killed tumor cells could confer
anamnestic antitumor protection, the combination with PD-1 inhi-
bition was investigated. BALB/c mice were immunized with brentux-
imab vedotin- or MMAE-killed A20hCD30 cells, implanted with WT
A20 tumor cells 14 days later, and then given anti–PD-1 7 days after
tumor implantation. Although immunization with brentuximab vedo-
tin- or MMAE-killed A20 cells alone again provided antitumor
immunity, the addition of anti–PD-1 robustly increased the propor-
tion of CRs (Fig. 5C). These results demonstrate that MMAE-induced
ICD can be combined with PD-1 blockade to improve antitumor
immunity.
known ICD inducers in similar models alone and in combination
with anti-PD-1 (21, 27, 39) and provide evidence that MMAE is an
ICD-inducing agent capable of promoting long-lived T-cell responses.
Combination of brentuximab vedotin treatment and PD-1
inhibition improves antitumor immunity in the autologous
LCL/PBMC xenograft model
The combination of brentuximab vedotin and PD-1 inhibition was
also evaluated in the humanized autologous LCL/PBMC xenograft
model. In this model, LCL tumor clearance is mediated by autologous
EBV-specific cytotoxic T-cell responses. As previously described,
treatment of LCL tumors with a subtherapeutic dose of brentuximab
vedotin augmented infiltration of cytotoxic T and NK cells and
increased production of IFNg (Fig. 4C and D). Inhibitory PD-L1
expression can be driven though IFNg and the JAK/STAT pathway
following activation of cytotoxic T cells and may limit antitumor
immunity (62). Flow cytometric evaluation of on-study LCL tumor
cells and intra-tumoral CD8þ T cells in this model showed high
expression of PD-L1 and PD-1, respectively, supporting the presence
of this inhibitory axis in LCL tumors (Fig. 6A).
PBMC-humanized mice receiving a subtherapeutic dose of bren-
tuximab vedotin showed accelerated LCL tumor rejection relative to
untreated humanized mice, consistent with enhanced T-cell activity
(Fig. 6B and C). Although blocking of human PD-1 with nivolumab
did not significantly accelerate tumor rejection as a single agent, the
combination of brentuximab vedotin and nivolumab showed superior
tumor rejection compared with either treatment alone (Fig. 6B and C).
Results from the humanized LCL tumor model are consistent with the
murine A20 vaccination model and further substantiate the comple-
mentarity of MMAE-driven ICD and PD-1 inhibition. In addition to

Figure 5.
Brentuximab vedotin–induced ICD protects against subsequent tumor challenges. A20 lymphoma cells expressing human CD30 (A20hCD30) were treated with BV or
MMAE in vitro, and dying cells were used to immunize wild-type BALB/c mice. Cells flash-frozen in liquid nitrogen were injected as a non-ICD control. A, Mice (n ¼ 8
per treatment group) immunized with BV-killed, MMAE-killed, or flash-frozen A20hCD30 cells were challenged with wild-type A20 cells on day 14 and monitored for
tumor growth. B, Detection of CD8þ T cells within the tumor by IHC after 9 days. C, BALB/c mice (n ¼ 9 per group) were immunized with BV- or MMAE-killed A20 cells
and rechallenged 14 days later. Anti–PD-1 (1 mg/kg) was administered on days 6, 9, 14, and 17 after tumor challenge and tumor growth was monitored. Numbers in
figures reflect surviving mice out of total at day 29. D, A20 tumor-bearing NSG mice (n ¼ 5–8 per treatment group) received CD3þ T cells from mice immunized with
BV-killed or flash-frozen–killed A20 cells, or T cells from unimmunized mice (naive) and monitored for tumor growth. E, Tumor volume AUC for mice receiving T cells
from mice immunized with brentuximab vedotin–killed A20hCD30 tumor cells. F, CD8þ T-cell infiltration. Representative data from two independent experiments,
each symbol represents an individual mouse.  P < 0.05;   P < 0.01;    P < 0.001, (one-way ANOVA followed by Dunnett’s multiple comparisons test) compared
with BV treatment. Bars represent the mean. ANOVA, analysis of variance; AUC, area under the curve; BV, brentuximab vedotin; CR, complete response; ICD,
immunogenic cell death; IHC, immunohistochemistry; MMAE, monomethyl auristatin E; NOD, nonobese diabetic; ns, not significant; NSG, NOD SCID gamma; NT, not
treated; PD-1, programmed cell death protein 1; SCID, severe combined immune deficiency.

AACRJournals.org Mol Cancer Ther; 23(1) January 2024 79

 Heiser et al.

Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024

Figure 6.
Protective immunity from brentuximab vedotin- or MMAE-mediated immunization is enhanced by anti–PD-1 treatment. A, Representative expression of PD-1 and
PD-L1 on T cells and LCL tumor cells, respectively, on day 35 by flow cytometry in the autologous PBMC/LCL tumor model. Staining demonstrates the presence
of an active PD-1/PD-L1 axis between T cells and LCL tumor cells. B, LCL tumor volumes were analyzed for treatment conditions relative to mice that received
only PBMCs. Tumor-bearing mice that received PBMCs and a subtherapeutic dose of BV (1 mg/kg, i.p.) with or without anti–PD-1 antibody (10 mg/kg, i.p. 2 and
7 days after PBMCs), showed enhanced immune-mediated tumor regression. C, Tumor volumes (AUC). Error bars indicate SEM.  P < 0.05;     P < 0.001
(one-way ANOVA followed by Dunnett’s multiple comparisons test). ANOVA, analysis of variance; AUC, area under the curve; BV, brentuximab vedotin; IgG,
immunoglobulin G; i.p., intraperitoneal injection; LCL, lymphoblastoid cell line; nivo, nivolumab; NT, not treated; PBMC, peripheral blood mononuclear cells;
PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; SEM, standard error of the mean; TIL, tumor-infiltrating lymphocyte.

direct tumor cell killing by brentuximab vedotin, priming and recruit-
ment of T cells following ICD induction may help potentiate thera-
peutic blockade of PD-1/PD-L1 in CD30-expressing lymphomas.
Discussion
Failures in immunosurveillance can lead to cancer progression (1, 2).
Specifically, immunological ignorance of tumor-associated antigens,
immune exclusion, or active immunosuppression within the TME are
key barriers to protective cancer immunity (1). Overcoming these
barriers may be possible by eliciting tumor cell death and providing
tumor-associated antigens in an inflammatory context (6). In contrast
with normal cellular apoptosis, ICD elicits the release of inflammatory
DAMPs that promote APC activation and productive T-cell priming
against tumor antigens (6). Therefore, induction of ICD by anticancer
treatments is highly desirable to improve patient immune responses
to cancer.
Brentuximab vedotin is an ADC that selectively delivers the highly
potent microtubule inhibitor MMAE to CD30-expressing target
cells (44). MMAE is a synthetic derivative of the dolostatin 10 family
of microtubule inhibitors with mechanistic similarities to vinca alkaloid
antineoplastic agents, vincristine and vinorelbine (45). Here, treatment
of CD30þ lymphoma cells with brentuximab vedotin or MMAE was
associated with induction of the three hallmarks of ICD: expression of
CRT on the cell surface, secretion of ATP, and extracellular release of
HMGB1 to a similar degree as the known ICD-inducing agent oxali-
platin. Etoposide, a compound described not to induce ICD, was unable
to fulfill all three of these characteristics (a schematic of the proposed
mechanism is shown in Supplementary Fig. S6C). Unlike other known
ICD-inducing agents that affect DNA replication/integrity (18, 12),
these results demonstrate that MMAE-mediated ICD is induced
through microtubule disruption and subsequent disorganization of the
ER, leading to a potent ER stress response. Phagocytic uptake of
brentuximab vedotin–killed cells by APCs led to the production of
proinflammatory cytokines and efficiently primed the host immune
system to mount a long-lived antitumor T-cell response. Overall, our
results expand upon our understanding of brentuximab vedotin’s
multifaceted anticancer mechanism of action (Supplementary Fig. S6).

80 Mol Cancer Ther; 23(1) January 2024 MOLECULAR CANCER THERAPEUTICS


Brentuximab vedotin–induced innate cell activation was observed
in an in vivo murine model by inducing high expression of hallmark
ICD cytokines. Blood biomarker analyses from a phase I/II study in
patients with relapsed or refractory HL demonstrated a marked
increase in similar proinflammatory cytokines and chemokines fol-
lowing treatment with brentuximab vedotin (33). These in-human
results validate the findings from the preclinical experiments inves-
tigating the potential for brentuximab vedotin to induce ICD.
Antitumor immunity imparted by brentuximab vedotin–killed
cells led to increased CD8þ T-cell infiltration and improved tumor
clearance in both murine and humanized tumor models. Given
that clinical response rates to checkpoint inhibitors correlate with
the presence of CD8þ T cells in the TME (61), it was hypothesized
that brentuximab vedotin–mediated ICD and the downstream
effects on T cells could be augmented by checkpoint inhibition.
Our models confirmed that brentuximab vedotin–mediated ICD
and resultant immune-mediated tumor rejection were enhanced by
PD-1 inhibition.
These results provide a mechanistic rationale for investigating
the antitumor response of brentuximab vedotin and checkpoint
inhibition as a novel regimen in CD30þ lymphomas. Previously
published results of treatment with brentuximab vedotin with a
PD-1 inhibitor (nivolumab) in patients have demonstrated prom-
ising antitumor responses. In a phase I/II study in which 91 patients
with relapsed or refractory HL were treated with brentuximab
vedotin plus nivolumab, the CR rate was 67% and the objective
response rate was 85%. Both the CR and objective response
rates with brentuximab vedotin plus nivolumab were higher than
previously observed with brentuximab vedotin or nivolumab
alone (63). A similar study of brentuximab vedotin plus nivolumab
in patients with relapsed or refractory non-Hodgkin lymphomas
demonstrated an objective response rate of 70% and a 50% CR rate
in patients with mediastinal gray zone lymphoma (CHECKMATE
436; ref. 64). Ongoing studies are investigating the combination in
different populations; interim analyses of these studies have dem-
onstrated durable response rates in patients aged ≥60-years with
classical HL (65) and high progression-free survival rates in patients
aged 9–30 years with relapsed or refractory classical HL (66).
The results of this study further demonstrate that the small-
molecule MMAE itself is a potent driver of ICD irrespective of the
anti-CD30 antibody backbone. Therefore, the ability to induce tar-
geted immune activation is likely intrinsic to other vedotin ADCs.
Indeed, preclinical studies with other vedotin ADCs (tisotumab
vedotin and enfortumab vedotin) have demonstrated the presence of
References
1. Zhou J, Wang G, Chen Y, Wang H, Hua Y, Cai Z. Immunogenic cell death in
cancer therapy: present and emerging inducers. J Cell Mol Med 2019;23:4854–65.
2. de Souza AP, Bonorino C. Tumor immunosuppressive environment: effects on
tumor-specific and nontumor antigen immune responses. Expert Rev Antican-
cer Ther 2009;9:1317–32.
3. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle.
Immunity 2013;39:1–10.
4. Kazama H, Ricci JE, Herndon JM, Hoppe G, Green DR, Ferguson TA.
Induction of immunological tolerance by apoptotic cells requires caspase-
dependent oxidation of high-mobility group box-1 protein. Immunity 2008;
29:21–32.
5. Kroemer G, Galassi C, Zitvogel L, Galluzzi L. Immunogenic cell stress and death.
Nat Immunol 2022;23:487–500.
6. Galluzzi L, Vitale I, Warren S, Adjemian S, Agostinis P, Martinez AB, et al.
Consensus guidelines for the definition, detection and interpretation of immu-
nogenic cell death. J Immunother Cancer 2020;8:e000337.
AACRJournals.org
Brentuximab Vedotin and Immunogenic Cell Death
ICD hallmarks in ADC-treated cell lines (67, 68). Collectively, these
findings support the hypothesis that induction of ICD is a vedotin
ADC class effect and provide rationale for the clinical investigation of
vedotin ADCs in combination with checkpoint inhibitors and other
immunotherapies.
Authors’ Disclosures
These studies were funded by Seagen Inc. R.A. Heiser, W. Zeng, M. Ulrich,
P. Younan, and R. Thurman are employees of Seagen and hold stocks and shares in
Seagen. A.T. Cao, M.E. Anderson, M. Jonas, C.-L. Law, E.S. Trueblood, and S.J. Gardai
were employees of Seagen at the time of the studies. R.A. Heiser reports a patent
for US11299543B2 issued; as well as employment and being a stockholder of
Seagen Inc. A.T. Cao reports personal fees from Seagen during the conduct of the
study. P. Younan reports employment with Seagen. S.J. Gardai reports other support
from Seagen during the conduct of the study; as well as a patent for U.S. 201862668104
pending and U.S. 20200239585 pending. No other disclosures were reported by
the authors.
Authors’ Contributions
Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024
R.A. Heiser: Conceptualization, investigation, writing–original draft, writing–
review and editing. A.T. Cao: Conceptualization, data curation, formal analysis,
investigation. W. Zeng: Investigation. M. Ulrich: Investigation. P. Younan: Inves-
tigation, methodology. M.E. Anderson: Investigation. E.S. Trueblood: Investigation.
M. Jonas: Validation. R. Thurman: Writing–review and editing. C.-L. Law: Writing–
review and editing. S.J. Gardai: Conceptualization, investigation, writing–original
draft, writing–review and editing.
Acknowledgments
Medical writing support was provided by Moamen Hammad, and editorial
support, including formatting, proofreading, and submission, was provided by
George Chappell and Travis Taylor, all of Scion, London, UK, supported by
Seagen according to Good Publication Practice guidelines (https://www.acpjour
nals.org/doi/10.7326/M22-1460). Some of these results were presented at the
2015, 2016, and 2017 American Association for Cancer Research (AACR) annual
meetings (49–51).
The publication costs of this article were defrayed in part by the payment of
publication fees. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
Note
Supplementary data for this article are available at Molecular Cancer Therapeutics
Online (http://mct.aacrjournals.org/).
Received March 3, 2023; revised August 7, 2023; accepted September 26, 2023;
published first September 29, 2023.
7. Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer G. Immunogenic cell death in
cancer and infectious disease. Nat Rev Immunol 2017;17:97–111.
8. Iyer SS, Pulskens WP, Sadler JJ, Butter LM, Teske GJ, Ulland TK, et al. Necrotic
cells trigger a sterile inflammatory response through the Nlrp3 inflammasome.
Proc Natl Acad Sci USA 2009;106:20388–93.
9. Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-
Ullrich JE, et al. Cell-surface calreticulin initiates clearance of viable or
apoptotic cells through trans-activation of LRP on the phagocyte. Cell 2005;
123:321–34.
10. Yu M, Wang H, Ding A, Golenbock DT, Latz E, Czura CJ, et al. HMGB1 signals
through toll-like receptor (TLR) 4 and TLR2. Shock 2006;26:174–9.
11. Elliott MR, Chekeni FB, Trampont PC, Lazarowski ER, Kadl A, Walk SF, et al.
Nucleotides released by apoptotic cells act as a find-me signal to promote
phagocytic clearance. Nature 2009;461:282–6.
12. Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancer
therapy. Annu Rev Immunol 2013;31:51–72.
Mol Cancer Ther; 23(1) January 2024 81
 Heiser et al.
13. Pitt JM, Kroemer G, Zitvogel L. Immunogenic and non-immunogenic cell
death in the tumor microenvironment. Adv Exp Med Biol 2017;1036:65–79.
14. Tufi R, Panaretakis T, Bianchi K, Criollo A, Fazi B, Di Sano F, et al. Reduction of
endoplasmic reticulum Ca2þ levels favors plasma membrane surface exposure of
calreticulin. Cell Death Differ 2008;15:274–82.
15. Panaretakis T, Kepp O, Brockmeier U, Tesniere A, Bjorklund AC, Chapman DC,
et al. Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell
death. EMBO J 2009;28:578–90.
16. Kepp O, Menger L, Vacchelli E, Locher C, Adjemian S, Yamazaki T, et al.
Crosstalk between ER stress and immunogenic cell death. Cytokine Growth
Factor Rev 2013;24:311–8.
17. Rufo N, Yang Y, De Vleeschouwer S, Agostinis P. The “Yin and Yang” of
unfolded protein response in cancer and immunogenic cell death. Cells 2022;11:
2899.
18. Showalter A, Limaye A, Oyer JL, Igarashi R, Kittipatarin C, Copik AJ, et al.
Cytokines in immunogenic cell death: applications for cancer immunotherapy.
Cytokine 2017;97:123–32.
19. Lebeau PF, Platko K, Byun JH, Austin RC. Calcium as a reliable marker for the
quantitative assessment of endoplasmic reticulum stress in live cells. J Biol Chem
2021;296:100779.
20. Chaput N, De Botton S, Obeid M, Apetoh L, Ghiringhelli F, Panaretakis T, et al.
Molecular determinants of immunogenic cell death: surface exposure of calre-
ticulin makes the difference. J Mol Med 2007;85:1069–76.
21. Obeid M, Panaretakis T, Joza N, Tufi R, Tesniere A, van Endert P, et al.
Calreticulin exposure is required for the immunogenicity of gamma-
irradiation and UVC light-induced apoptosis. Cell Death Differ 2007;14:
1848–50.
22. Martins I, Wang Y, Michaud M, Ma Y, Sukkurwala AQ, Shen S, et al. Molecular
mechanisms of ATP secretion during immunogenic cell death. Cell Death Differ
2014;21:79–91.
23. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll-like
receptor 4-dependent contribution of the immune system to anticancer che-
motherapy and radiotherapy. Nat Med 2007;13:1050–9.
24. Tesniere A, Schlemmer F, Boige V, Kepp O, Martins I, Ghiringhelli F, et al.
Immunogenic death of colon cancer cells treated with oxaliplatin. Oncogene
2010;29:482–91.
25. Garg AD, Krysko DV, Verfaillie T, Kaczmarek A, Ferreira GB, Marysael T, et al.
A novel pathway combining calreticulin exposure and ATP secretion in immu-
nogenic cancer cell death. Embo j 2012;31:1062–79.
26. Michaud M, Sukkurwala AQ, Di Sano F, Zitvogel L, Kepp O, Kroemer G.
Synthetic induction of immunogenic cell death by genetic stimulation of
endoplasmic reticulum stress. Oncoimmunology 2014;3:e28276.
27. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al.
Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med
2007;13:54–61.
28. Martins I, Kepp O, Schlemmer F, Adjemian S, Tailler M, Shen S, et al. Restoration
of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic
reticulum stress. Oncogene 2011;30:1147–58.
29. Verma R, Foster RE, Horgan K, Mounsey K, Nixon H, Smalle N, et al.
Lymphocyte depletion and repopulation after chemotherapy for primary breast
cancer. Breast Cancer Res 2016;18:10.
30. Mackall CL, Fleisher TA, Brown MR, Andrich MP, Chen CC, Feuerstein IM, et al.
Distinctions between CD8þ and CD4þ T-cell regenerative pathways result in
prolonged T-cell subset imbalance after intensive chemotherapy. Blood 1997;89:
3700–7.
31. Kotsakis A, Sarra E, Peraki M, Koukourakis M, Apostolaki S, Souglakos J, et al.
Docetaxel-induced lymphopenia in patients with solid tumors: a prospective
phenotypic analysis. Cancer 2000;89:1380–6.
32. Fagnoni FF, Lozza L, Zibera C, Zambelli A, Ponchio L, Gibelli N, et al. T-
cell dynamics after high-dose chemotherapy in adults: elucidation of the
elusive CD8þ subset reveals multiple homeostatic T-cell compartments
with distinct implications for immune competence. Immunology 2002;106:
27–37.
33. Herrera AF, Moskowitz AJ, Bartlett NL, Vose JM, Ramchandren R, Feldman TA,
et al. Interim results of brentuximab vedotin in combination with nivolumab
in patients with relapsed or refractory Hodgkin lymphoma. Blood 2018;131:
1183–94.
34. Nicol o E, Giugliano F, Ascione L, Tarantino P, Corti C, Tolaney SM, et al.
Combining antibody–drug conjugates with immunotherapy in solid tumors:
current landscape and future perspectives. Cancer Treat Rev 2022;106:
102395.
82 Mol Cancer Ther; 23(1) January 2024
35. Hoimes CJ, Flaig TW, Milowsky MI, Friedlander TW, Bilen MA, Gupta S, et al.
Enfortumab vedotin plus pembrolizumab in previously untreated advanced
urothelial cancer. J Clin Oncol 2023;41:22–31.
36. M€ uller P, Kreuzaler M, Khan T, Thommen DS, Martin K, Glatz K, et al.
Trastuzumab emtansine (T-DM1) renders HER2þ breast cancer highly suscep-
tible to CTLA-4/PD-1 blockade. Sci Transl Med 2015;7:315ra188.
37. M€ uller P, Martin K, Theurich S, Schreiner J, Savic S, Terszowski G, et al.
Microtubule-depolymerizing agents used in antibody–drug conjugates induce
antitumor immunity by stimulation of dendritic cells. Cancer Immunol Res
2014;2:741–55.
38. Martin K, M€ uller P, Schreiner J, Prince SS, Lardinois D, Heinzelmann-Schwarz
VA, et al. The microtubule-depolymerizing agent ansamitocin P3 programs
dendritic cells toward enhanced antitumor immunity. Cancer Immunol Immun-
other 2014;63:925–38.
39. Rios-Doria J, Harper J, Rothstein R, Wetzel L, Chesebrough J, Marrero A, et al.
Antibody–drug conjugates bearing pyrrolobenzodiazepine or tubulysin pay-
loads are immunomodulatory and synergize with multiple immunotherapies.
Cancer Res 2017;77:2686–98.
40. Montes de Oca R, Alavi AS, Vitali N, Bhattacharya S, Blackwell C, Patel K, et al.
Belantamab mafodotin (GSK2857916) drives immunogenic cell death and
immune-mediated antitumor responses in vivo. Mol Cancer Ther 2021;20:
Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024
1941–55.
41. Menger L, Vacchelli E, Adjemian S, Martins I, Ma Y, Shen S, et al. Cardiac
glycosides exert anticancer effects by inducing immunogenic cell death.
Sci Transl Med 2012;4:143ra99.
42. Sukkurwala AQ, Adjemian S, Senovilla L, Michaud M, Spaggiari S, Vacchelli E,
et al. Screening of novel immunogenic cell death inducers within the NCI
mechanistic diversity set. Oncoimmunology 2014;3:e28473.
43. Seagen Inc. ADCETRIS US Full Prescribing Information. Updated November
2022. [Accessed October 9, 2023]. Available from: https://www.accessdata.fda.
gov/drugsatfda_docs/label/2022/125388s106lbl.pdf.
44. Yi JH, Kim SJ, Kim WS. Brentuximab vedotin: clinical updates and practical
guidance. Blood research 2017;52:243–53.
45. Gauzy-Lazo L, Sassoon I, Brun MP. Advances in antibody–drug conjugate
design: current clinical landscape and future innovations. SLAS Discov 2020;
25:843–68.
46. Jain N, Smith SW, Ghone S, Tomczuk B. Current ADC linker chemistry.
Pharm Res 2015;32:3526–40.
47. Sutherland MS, Sanderson RJ, Gordon KA, Andreyka J, Cerveny CG, Yu C, et al.
Lysosomal trafficking and cysteine protease metabolism confer target-specific
cytotoxicity by peptide-linked anti-CD30–Auristatin conjugates. J Biol Chem
2006;281:10540–7.
48. Barreca M, Stathis A, Barraja P, Bertoni F. An overview on anti-tubulin
agents for the treatment of lymphoma patients. Pharmacol Ther 2020;211:
107552.
49. Gardai SJ, Epp A, Law C-L. Abstract 2469: Brentuximab vedotin-mediated
immunogenic cell death. Cancer Res 2015;75:2469.
50. Cao A, Heiser R, Law C-L, Gardai SJ. Abstract 4914: Auristatin-based antibody–
drug conjugates activate multiple ER stress response pathways resulting in
immunogenic cell death and amplified T-cell responses. Cancer Res 2016;76:
4914.
51. Cao AT, Law C-L, Gardai SJ, Heiser RA. Abstract 5588: Brentuximabvedotin-
driven immunogenic cell death enhances antitumor immune responses, and is
potentiated by PD-1 inhibition in vivo. Cancer Res 2017;77:5588.
52. Ma Y, Adjemian S, Yang H, Catani JP, Hannani D, Martins I, et al. ATP-
dependent recruitment, survival and differentiation of dendritic cell precursors
in the tumor bed after anticancer chemotherapy. Oncoimmunology 2013;2:
e24568.
53. Terasaki M, Chen LB, Fujiwara K. Microtubules and the endoplasmic reticulum
are highly interdependent structures. J Cell Biol 1986;103:1557–68.
54. Schwarz DS, Blower MD. The endoplasmic reticulum: structure, function, and
response to cellular signaling. Cell Mol Life Sci 2016;73:79–94.
55. Klopfenstein DR, Kappeler F, Hauri HP. A novel direct interaction of endo-
plasmic reticulum with microtubules. Embo j 1998;17:6168–77.
56. Ko P, Choi JH, Song S, Keum S, Jeong J, Hwang YE, et al. Microtubule acetylation
controls MDA-MB-231 breast cancer cell invasion through the modulation of
endoplasmic reticulum stress. Int J Mol Sci 2021;22:6018.
57. Dominguez-Bautista JA, Klinkenberg M, Brehm N, Subramaniam M, Kern B,
Roeper J, et al. Loss of lysosome-associated membrane protein 3 (LAMP3)
enhances cellular vulnerability against proteasomal inhibition. Eur J Cell Biol
2015;94:148–61.
MOLECULAR CANCER THERAPEUTICS

58. Gombault A, Baron L, Couillin I. ATP release and purinergic signaling in NLRP3
inflammasome activation. Front Immunol 2012;3:414.
59. Wang Y, Martins I, Ma Y, Kepp O, Galluzzi L, Kroemer G. Autophagy-
dependent ATP release from dying cells via lysosomal exocytosis. Autophagy
2013;9:1624–5.
60. Kepp O, Senovilla L, Vitale I, Vacchelli E, Adjemian S, Agostinis P, et al.
Consensus guidelines for the detection of immunogenic cell death. Oncoimmu-
nology 2014;3:e955691.
61. Khagi Y, Kurzrock R, Patel SP. Next-generation predictive biomarkers for
immune checkpoint inhibition. Cancer Metastasis Rev 2017;36:179–90.
62. Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, Rodriguez GA,
et al. Interferon receptor signaling pathways regulating PD-L1 and PD-L2
expression. Cell Rep 2017;19:1189–201.
63. Advani R, Moskowitz AJ, Bartlett NL, Vose J, Ramchandren R, Feldman T, et al.
Brentuximab vedotin in combination with nivolumab in relapsed or refractory
Hodgkin lymphoma: 3-year study results. Blood 2021;138:427–38.
64. Santoro A, Moskowitz AJ, Ferrari S, Carlo-Stella C, Fanale MA, Francis S,
et al. Nivolumab combined with brentuximab vedotin for relapsed/refractory
AACRJournals.org
Brentuximab Vedotin and Immunogenic Cell Death
mediastinal gray zone lymphoma: primary efficacy and safety analysis of the
phase 2 CheckMate 436 study. Blood 2020;136:44–5.
65. Yasenchak CA, Bordoni R, Patel-Donnelly D, Larson T, Goldschmidt J,
Boccia RV, et al. Frontline brentuximab vedotin as monotherapy or
in combination for older Hodgkin lymphoma patients. Blood 2020;136:
18–9.
66. Cole P, Mauz-K€orholz C, Mascarin M, Michel G, Cooper S, Beishuizen A, et al.
Nivolumab and brentuximab vedotin (BV)-based, response-adapted treatment
in children, adolescents, and young adults (CAYA) with standard-risk relapsed/
refractory classical Hodgkin lymphoma (R/R cHL): primary analysis. J Clin
Oncol 2020;38:8013.
67. Gray E, Hensley K, Allred S, Trueblood E, Gosink J, Thurman R, et al. 617
Tisotumab vedotin shows immunomodulatory activity through induction of
immunogenic cell death. J Immunother Cancer 2020;8:A653.
68. Liu BA, Olson D, Snead K, Gosink J, Tenn E-M, Zaval M, et al. Abstract 5581:
enfortumab vedotin, an anti–Nectin-4 ADC demonstrates bystander cell killing
and immunogenic cell death antitumor activity mechanisms of action in
urothelial cancers. Cancer Res 2020;80:5581.
Downloaded from http://aacrjournals.org/mct/article-pdf/23/1/68/3397457/68.pdf by guest on 13 February 2024
Mol Cancer Ther; 23(1) January 2024 83