DOI: 10.1002/JLB.5MA0420-375RRRRRMEETING ARTICLETNFR2 blockade alone or in combination with PD-1 blockadeshows therapeutic efficacy in murine cancer models
Katherine Case1

Lisa Tran1

Michael Yang1

Hui Zheng2

Willem M. Kuhtreiber1

Denise L. Faustman1
1 Immunobiology Laboratory, Massachusetts
General Hospital and Harvard Medical School,
Boston, Massachusetts, USA

Abstract
Immune checkpoint inhibitors are profoundly transforming cancer therapy, but response rates

2 Biostatistics Center, Massachusetts Generalvary widely. The efficacy of checkpoint inhibitors, such as anti-programmed death receptor-1

Hospital, Boston, Massachusetts, USA(anti-PD-1), might be increased by combination therapies. TNFR2 has emerged as a new target due

Correspondence
Denise L. Faustman, Massachusetts General
Hospital and Harvard Medical School, Building 149, 13th Street, Room 3602, Boston, MA
02129, USA.
Email: Faustman@helix.mgh.harvard.edu

to its massive expression on highly immunosuppressive regulatory T cells (Tregs) in the microenvironment and on certain tumor cells. In murine colon cancer models CT26 and MC38, we evaluated the efficacy of a new anti-TNFR2 antibody alone or in combination with anti-PD-1 therapy.
Tumor-bearing mice were treated with placebo, anti-PD-1 alone, anti-TNFR2 alone, or combination anti-PD-1 and anti-TNFR2. We found that combination therapy had the greatest efficacy bycomplete tumor regression and elimination (cure) in 65–70% of animals. The next most effective
therapy was anti-TNFR2 alone (20–50% cured), whereas the least effective was anti-PD-1 alone(10–25% cured). The mode of action, according to in vivo and in vitro methods including FACS
analysis, was by killing immunosuppressive Tregs in the tumor microenvironment and increasing
the ratio of CD8+ T effectors (Teffs) to Tregs. We also found that sequence of antibody delivery
altered outcome. The two most effective sequences were simultaneous delivery (70% cured) followed by anti-TNFR2 preceding anti-PD-1 (40% cured), and the least effective was by anti-PD-1
preceding anti-TNFR2 (10% cured). We conclude that anti-PD-1 is best enhanced by simultaneous administration with anti-TNFR2, and anti-TNFR2 alone may be potentially useful strategy for
those do not respond to, or cannot tolerate, anti-PD-1 or other checkpoint inhibitors.
KEYWORDS

ADCC, antibodies, cancer, checkpoint blockade, combination immunotherapy, immunotherapy,
oncology, PD-1, Teff, TNFR2, Tregs

1

INTRODUCTION

to reinvigorate so-called “exhausted” tumor-specific Teffs to enhancetheir cytotoxic function against the tumor.

Immunotherapy has strikingly improved cancer survival with the use

The TNFR2 receptor sets the balance between Tregs and Teffs (alsoof blocking antibodies to programmed death receptor-1 (PD-1) or pro-known as cytotoxic T cells); the receptor acts as a bidirectional switch

grammed death-ligand 1 (PD-L1), among others, but response rates dif-that can cause Treg cell expansion (with agonism) or Treg contrac-

fer, depending on cancer type.1-4 New immunotherapies are needed totion (with antagonism).6,7 TNFR2-expressing Tregs or myeloid sup-combine with checkpoint inhibitors to boost their efficacy, or to usepressor cells in the tumor infiltrate are the most potent immuno-

as alternative single agents. One targeted approach presented here issuppressive cells identified to date that dampen the host immune

designed to shift the tumor microenvironment toward tumor cell death

response.8,9 TNFR2 agonism is potentially therapeutic in diverseby depletion of suppressive regulatory T cells (Tregs), which, in turn,

human autoimmune diseases where there are too many Teffs andlifts suppression of tumor-specific T effectors (Teffs). The purpose is

too few highly expressing TNFR2-expressing Treg cells.10 By contrast,
TNFR2 antagonism is potentially therapeutic in many cancers where

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; PD-1, programmed death
receptor-1; Teff, T-effector cells; Tregs, regulatory T cells.

Received: 6 September 2019
J Leukoc Biol. 2020;1–11.

Revised: 1 April 2020

there are too many TNFR2-expressing Tregs in the tumor microenvironment and too few Teffs, often referred to as exhausted T cells. The

Accepted: 14 April 2020www.jleukbio.org

c 2020 Society for Leukocyte Biology
○

1

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CASE ET AL .

accumulation of TNFR2-expressing Tregs in the tumor microenviron-manufacturer’s instructions. Antibody protein samples were also run

ment or soluble TNFR2 in the serum carry poor prognosis in humanon NuPAGE 4–12% Tris/glycine gels with NuPAGE LDS sample buffer

and mice tumors.11-14 In the CT26 colon cancer model and the 4T1(Life Technologies), NuPAGE sample reducing agent (Life Technolo-

breast mouse model, antibodies that block TNFR2 signaling enhancegies), and Tris/glycine/SDS running buffer (Life Technologies) at 200 V

survival alone or in combination with other therapies such as CpG andfor 35 min. Gels were stained for 45 min with SimplyBlue SafeStain

anti-CD25 antibodies.15 Murine testing of TNFR2 combined with PD-(Invitrogen, Carlsbad, CA, USA) under reducing conditions.

1 antagonistic antibodies has not yet been reported.
TNFR2 is an attractive target for cancer immunotherapy16 TNFR2
in the human is an identified oncogene across diverse cancer
types.17,18 The exact genetic mutations of TNFR2 itself or the signaling
pathway it involves are not fully identified in every cancer type. We aredefining oncogene as mutations in the case of human lymphomas andhuman myelofibrosis and on tumor tissue as the aberrant expression
on a parenchymal cell or cell line when the normal cells do not express
this protein linked to a well-known growth pathway. TNFR2 overexpression is also observed on myeloid-derived monocyte suppressor
cells, another target in the tumor microenvironment.19 Human datasuggests that after checkpoint failures in metastatic melanoma, TNFR2
can be even more highly expressed in the tumor microenvironment on
Tregs, acting as a possible escape mechanism after failed therapy.20
Our previously identified human-directed TNFR2 antagonistic antibodies in culture kill tumor-infiltrating Tregs, proliferate tumor associated Teffs, and also directly kill human oncogene expressing tumor
cells, but with lessened activity against normal lymphoid cells outside the tumor microenvironment.21,22 Human-directed TNFR2 antagonistic antibodies can directly kill Tregs with the natural agonistic
ligand TNF present and do not require antibody-dependent cellularcytotoxicity (ADCC) or Fc receptor function, a feature that likely will
improve their toxicology profile, as will TNFR2’s limited body-wide
expression.21 Likewise human-directed TNFR2 antagonistic antibodies have specificity for only rapidly proliferating Tregs in the tumor
microenvironment with little or lessened activity against nontumor
associated circulating human Tregs.21,22
Here we identify and begin to characterize a new murine-directed
TNFR2 antibody (TY101; hereinafter referred to as anti-TNFR2) that

2.2 Strong cation exchange (SCX) fractionation
with HPLC
SCX fractionation was performed on an Agilent 1260 model HPLC. Key
components included Quat Pump model number: G1311B, Multiwavelength detector model number: G1365C, Fraction Collector model
number G1330B. Data analysis was performed on Agilent Chemstation. The column used was a Proteomix SCXNP5 4.6 × 250 mm
column from Sepax Technologies, Inc. (Newark, Delaware, USA; catalogue number 401NP5-4625PK). Running conditions were as follows: flow rate: 1 mL/min, detector: UV 280 nm, column temperature: 25◦ C. A total of 20 µL of the antibody was directly injected on
to the column in each run. The sample was kept at 4◦ C in a temperature controlled autosampler. The mobile phase was composed of
2-Morpholinoethanesulfonic acid„ 4-Morpholinepropanesulfonic acid,
TRIS-hydroxymethyl methylamino propanesulfonic acid, 3-cyclohexyl2-hydroxy-1-propanesulfonic acid, NaCl, and Milli Q water. Each run
had a gradient of mobile phase A of 5% mobile and this was comparedto phase B of 35% mobile during a 30 min period followed by 2 min of
isocratic flow. A 4.5 min wash in mobile phase C was performed at theend of each gradient to clean the column. Fractions were collected andsubsequently pooled. Purity of each fraction was verified by HPLC run
on the same column and same instrument. The recovery yield was calculated by comparing peak area between the crude and purified sample after normalizing for injection volume and concentration. Pooled
fractions were stored at 4◦ C.

2.3 Impact on Treg cells with TNFR2
antagonistic antibodies

possesses features of previously identified human-directed TNFR2
antagonistic antibodies. We evaluate its therapeutic activity alone

CD4+ cells were isolated using the EasySep Mouse T cell Isolation

or in combination with anti-PD-1 antibody in vitro and in vivo inKit (catalogue #19852) from normal nontumor-bearing mice. Isolated

two mouse models of colon cancer, CT26 and MC38. We also test

CD4+ cells were re-suspended in RPMI 1640; GlutaMAX (Life Tech-

the mode of action by evaluating the impact on Tregs and Teffs innologies) plus 10% FBS (Sigma-Aldrich, St louis, MO), murine IL2tumor cell infiltrates using FACS analysis. Finally, we evaluate the best(100 U/ml) (Sigma-Aldrich), and 1% penicillin streptomycin (Life Tech-

sequence of delivery of both antibody therapies to optimize combina-

nologies). Cells were seeded at a concentration of 0.2 × 106 cells pertion immunotherapy.

well in 96-well round-bottom plates, treated with the TNFR2 antagonist, and incubated at 37◦ C and 5% CO2 for 72 h. After incubation,
cells were collected by centrifugation, washed once with 1× HBSS

2MATERIALS AND METHODS

2.1

Protein gel electrophoresis(Invitrogen, Carlsbad, CA, USA), and stained for FACS analysis. FACS
data were processed using FlowJo software (version 10.1).

Monoclonal antibody protein samples were run on NativePAGE 4–

2.4

Reagents and flow cytometry

16% Bis-Tris gel with Tris-Glycine Native Running Buffer (Life Tech-

Cells were prepared for flow cytometry using mouse regulatory T

nologies, Carlsbad, CA, USA) at 225 V for 2.5 h. Gels were stainedcell staining kit #2 (catalogue #88-8118-40, Thermo Fisher Scien-for 30 min with Pierce Silver Stain (Thermo Fisher Scientific, Inc.,

tific, Inc., Waltham, MA, USA) according to the manufacturer’s instruc-

Waltham, MA, USA) under nonreducing conditions according to thetions. Fluorescently stained cells were resuspended in 1× HBSS3CASE ET AL .

(Invitrogen, Carlsbad, CA, USA) and analyzed using a BD FACS Cal-

Biotec, Inc., Somerville, MA, USA). After dissociation, 2 mL/sample

ibur Flow Cytometer machine (Becton Dickinson, Franklin Lakes, NJ,

of 1× lysing buffer was used to further lyse any contaminating

USA). Antibodies used for FACS analysis of Tregs included clone FJK-

RBCs. Cell were incubated for 20 min on a tube rocker at room

165 anti-mouse FOXP3 for intracellular staining of FOXP3 and clone

temperature. Cells were centrifuged at 300× g for 4◦ C for 5 min.

PC61.5 anti-mouse CD25 (Thermo Fisher Scientific, Inc., Waltham,

After the centrifugation 2 mL of FACS Buffer was added to the

MA, USA). Treg populations and Teff/Treg ratios were assessed bycells and centrifuged at 300× g for 4◦ C for 5 min. Cells were re-FACS. Antibodies for in vivo studies were anti-mouse PD-1 (CD279,

suspended in 0.5–1 mL FACS buffer and were counted and used forclone RMP-14) (BioXCell, West Lebanon, NH, USA) and the anti-mouse

FACS staining.

TNFR2 (TY101), which was isolated from a collection of antibodies
within the Harvard and MGH Antibody Cores (Boston, MA, USA).
This collection of research reagents were internally produced antibodies donated from various investigators and screened by our groupfor the desired activities. After confirming the intended immunogen2.6 In vitro killing assays of murine tumor cells
treated with TNFR2 antagonistic antibodies
CT26 and MC38 cells were purchased from ATCC and grown up in

was TNFR2 or a fragment of TNFR2, the initial screening on murine

medium of RPMI GlutaMAX (Life Technologies) plus 10% FBS (Sigma-

TNFR2 ELISA plates for TNFR2 binding was initiated. These screens

Aldrich, St louis, MO), IL2 (100 U/ml) (Sigma-Aldrich, St louis, MO), andalso included early confirmation of no TNFR1 binding. If sole TNFR2

1% penicillin streptomycin. CT26 and MC38 cells were cultured in 96-

binding was observed, all new antibodies to mouse TNFR2 continued

well round-bottom plates at a concentration of 1 × 104 cells per wellinto functional assays. Immunoprecipitation gels showed that the pro-in 200 µl of media. Cells were treated directly with TNFR2 antagonis-

tein brought down by TY101 was mouse TNFR2 by the use of poly-

tic antibodies and incubated for 7 d. This time period was chosen after

clonal anti-TNFR2 immunoblotting methods.

optimization experiments were first conducted. The goal was to con-

For functional assays, the Treg cells within isolated mouse CD4 cells

firm under extended incubation conditions such as 14 d, the cells were

were demonstrated to be killed after brief 48–72 h timing on murine

totally eliminated. We then chose a more moderate incubation time tosplenocytes in a dose-specific manner. Longer incubation times atmonitor dose responses of a test antibody. After incubation, cells were

the start were performed to confirm cell disappearance and also flow

detached from the plate and stained with trypan blue (Sigma-Aldrich,cytometry experiments included a total cell count. The moderate dura-

St louis, MO) to count viable cells.

tion of the incubation time was chosen so an antibody dose response
could be observed. We selected the best antibody with these traits.
Because in humans we were knowledgeable of the optimized TNFR2

2.7

In vivo efficacy analysis

receptor binding sites for dominant antagonism, defined as antagonism

Mice were tested and monitored for tumor growth by either Cham-that is not disrupted by the natural ligand TNF, we next characterized

pions Oncology (Hackensack, NJ, USA) or an independent third party.

the mouse-directed lead candidate with TNF in the media to confirm it

Animals were randomized to receive subcutaneous injection into theremained an antagonist and the used the Pepscan technology using theflank with two different mouse tumor cell lines (CT26 and MC38).

precision epitope mapping methods (Pepscan, Lelystad, The Nether-

Animals were dosed with antibody once tumors reached a volume

lands). Antibody TY101 was the lead candidate based on binding

between 100 and 200 mm3 . Animals were dosed biweekly i.p. with 100

to only mouse CRD3 and CRD4 TNFR2 receptor regions, similar toµg twice per week anti-PD-1 or/and anti-TNFR2 antibody. Unless oth-

our past screening of human-directed antagonistic antibodies. Thiserwise specified all antibodies administered in vivo had endotoxin <1

receptor site binding is believed to result in the formation of TNFR2

EU/mg. Tumor volumes were measured manually with calipers byanti-parallel dimers that block the TNF binding site.15,20 This is theblinded examiner over a 20 or 21 d course. Animals were euthanizednear identical binding region for the optimized human antibodies, only

if tumor sizes reached 1500 mm3 or the tumor ulcerated through thereactive to the human TNFR2 receptor with strong antagonism. TY101

skin. The flow gating methods for the tumor into single cell suspensions

was then moved forward into additional characterization studies out-

and then gating on select populations is shown in Supporting Informa-

lined in the paper. TY101 was purified using affinity chromatographytion Figure S1.

followed by PBS solution. The purity of the antibody was >95%. Bacterial endotoxin levels were determined using the Endosafe-PTS system
and Endosafe PTS cartridges (Charles River Laboratories, Wilming-

2.8

Pepscan mapping studies

tom, MA). Antibody concentration was determined by measuring

The 3D peptide mapping of anti-TNFR2 binding to TNFR2 wasabsorbance at 280 nm and the results were calculated using the stan-

performed using chemically linked peptides on scaffolds (CLIPS)

dard extinction coefficient 205, 500 M−1 cm−1 for 1.0 mg/ml = A280.

technology (Pepscan). Briefly, the target protein was converted
into a library of up to 10,000 overlapping linear peptides, which

2.5

Isolation of tumor infiltrating lymphocytes

were then bound to a solid support and shaped into a matrixof CLIPS constructs. The affinity of the antibody to the vari-

Tumors were surgically dissected from mice and then dissociated using

ous peptide conformations were used to determine the precise

Miltenyi Dissociation kit protocol (catalogue #130-096-730) (Miltenyi

discontinuous epitopes.

4

CASE ET AL .

F I G U R E 1 Murine-directed TNFR2 antagonistic antibody exhibits mild in vitro cytotoxic activity against regulatory T cells (Tregs) and tumor
cell lines. (A) Low expression of TNFR2 on murine colon cancer cell lines. The mean fluorescent intensity (MFI) of TNFR2 was measured onmouse colon cancer cell lines CT26 (left) and MC38 (right). CT26 tumor cells showed lower expression of the aberrantly expressed TNFR2 protein (MFI = 65); MC38 tumor cells also had low expression but slightly higher than CT26 (MFI = 109). Pink represents TNFR2 staining; blue isbackground staining with an isotype control. This is a representative experiment but the experiment performed >10 times. (B) A nonreducing gelof the mouse-directed IgG1 TNFR2 antagonist (TY101) showed two distinct bands (left). These isoforms were separated by HPLC (right) into two
fractions (F1 and F2). (C) The parent anti-TNFR2 antibody (TY101) and its two HPLC separated band fractions (F1 and F2) were tested for cytotoxic
function against three potential targets: Tregs (CD4+ cells) from normal mice and TNFR2-low expressing tumor cells (CT26 and MC38). Freshly
isolated CD4+ cells were treated with the parent antibody, HPLC fraction F1, and fraction F2 (0 to 25 µg/mL) for 72 h, whereas CT26 and MC38
tumor cells were treated for 7 d. The dose–response for the parent TNFR2 antagonist (TY101) (column 1) as well as each fraction (columns 2 and 3),
showed mild Treg, MC38, and CT26 cell killing. Data are expressed as mean ± SEM for at least n = 6–7 experiments/group for the MC38 and CT26
killing. For the splenic Treg killing by the three antibody fractions, a characteristic Treg experiment was presented (the experiment was performed
two times). The mixed effects statistical model showed for both TY101 and fraction F1 and F2 a downward slope. All killing slopes against the tumor
cell lines showed a change from baseline that was statistically significant (P < 0.05)

5CASE ET AL .

2.9

Statistical analysis

Data analysis was performed by Student’s t-test using Excel(Microsoft) or GraphPad Prism 5 software (GraphPad Software).
Significance was determined by P < 0.05.

Enzymatic removal of the Fc region of this antibody did not affect invitro killing of Tregs or of TNFR2-expressing CT26 or MC38 cells (datanot shown); therefore, this antibody did not require ADCC mechanism
for activity. Detailed peptide mapping studies of the anti-TNFR2 binding region with Pepscan mapping technology confirmed binding to the
CRD3 and CRD4 regions of the mouse TNFR2 receptor. These binding

3

RESULTSregions are conducive to antagonistic activity.21

3.1 CT26 and MC38 cell lines express low levels of
TNFR2, making them susceptible to modest
anti-TNFR2-induced cell death in vitro

3.2 Anti-TNFR2 therapy alone or in combination
with anti-PD-1 is efficacious in vivo in CT26-tumor
implanted animals

We profiled CT26 and MC38 murine tumor cells for aberrant TNFR2

To profile the efficacy of each antibody therapy in vivo, immunocom-

surface expression using flow cytometry. Figure 1A shows that bothpetent mice were first subcutaneously implanted into the flank with

CT26 and MC38 tumor cells had low yet stable aberrant TNFR2

CT26 colorectal murine tumor cells. When tumors reached a thresholdexpression. Using nonreducing SDS gels we next observed that thevolume of 100–200 mm3 , we administered four different therapies atmouse-directed IgG1 TNFR2 antibody (TY101) expressed two anti-doses of 100 µg of sterile antibody twice per week for 20–21 d: placebo

body bands (Fig. 1B, left). The bands were separable by HPLC and had

control, anti-PD-1 alone, anti-TNFR2 alone, and combination anti-PD-

identical sequences. Multiple antibody bands on nonreducing gels are

1+anti-TNFR2 (Fig. 2A, right). We assessed the daily impact on tumor

often due to the amino acid sequences of the hinge region forming dif-volume by group (Fig. 2A, left), by individual animals in spider plots

ferent disulfide linkages. On reducing gels, anti-TNFR2 showed only

(Fig. 2B), by percentage of animals achieving cure (cure rate) as mea-

one heavy chain and one light chain, confirming the suspected inter-

sured by complete tumor regression (Fig. 2C), and, in the few animals

chain disulfide hinge variations previously described (Supporting Infor-

who failed to be cured, the percentage of Tregs remaining (Fig. 2D, leftmation Fig. S2).
Next we examined the functional capacity in vitro of the parent

panel) and the ratio of CD8+ cells/Tregs in the tumor infiltrate (Fig. 2D,
right panel), measured by FACS analysis.

antibody (anti-TNFR2 antibody, TY101) and each of the two HPLC iso-Mice receiving the three active therapies displayed significantly

lated antibody fractions, F1 and F2 (Fig. 1B, right, Fig. 1C). The desired

smaller tumor volumes than controls (n = 8) (Fig. 2A). The display oftrend line is downward Treg cells as the dose of antibody is increased,

the individual tumor implanted mice in Figure 2B helped to define dif-even when tested on nontumor microenvironment CD4 T cells isolated

ferences between the treated mice. Anti-TNFR2 antibody (n = 18) wasfrom normal mice (Fig. 1C, top row). Anti-TNFR2 antibody displayed

the best solo therapy based on cure rate at days 20–21 of 10/18 mice,modest killing of normal mouse CD4 Tregs cells in a dose–responseor 55% (Fig. 2B, C). Anti-PD-1 antibody (n = 8) performed relatively

manner (Fig. 1C, second row, left most panel). The CD4 Tregs werepoorly, with a cure rate of only 25% (Fig. 2B, C). Combination therapy

isolated from normal spleen, because of insufficient numbers of T-with anti-TNFR2 and anti-PD-1 (n = 8) performed better than any solo

lymphocytes to harvest from the tumor microenvironment. The tumor

therapy, with a cure rate of 62% (Fig. 2B, C).

microenvironment shows more pronounced Treg killing with TNFR2

The few surviving mice with visible but greatly reduced residual

antagonistic antibodies but in mice this experiment is not feasible duetumors at days 20–21 (n = 2 per study group) were studied for con-to small tumor volumes; therefore, normal splenocytes were used

stituents of the tumor microenvironment, specifically the number offor screening antibodies. The tumor microenvironment shows more

Tregs and the ratio of Teffs:Tregs. FACS analysis of the tumor infiltratepronounced Treg kills of TNFR2 antagonistic antibodies but in mice

revealed that mice receiving either anti-TNFR2 alone or combination

this experiment is not feasible. Furthermore, both F1 and F2 HPLC-

therapy had Treg depletion and corresponding increases in the ratio ofseparated antibody fractions retained this modest dose–response

Teffs to Tregs (Fig. 2D; Supporting Information Fig. S1). Mice treatedkilling of murine Tregs (Fig. 1C, top row, two right-most panels). Char-with anti-PD-1 alone had no changes in Tregs or Teffs relative to con-acteristic flow cytometry histograms are represented in Supporting

trols as would be expected.

Information Figure S3. Similarly, anti-TNFR2 antibody modestly killed
CT26 or MC38 mouse tumor cells within 7 d of exposure, and again
this activity was observed in the both the F1 and F2 fractions (Fig. 1C,
two bottom rows). It is important to keep in mind that, in the human,
optimized antagonistic antibodies preferentially bind only newly

3.3 Anti-TNFR2 therapy alone or in combination
with anti-PD-1 is the most efficacious in vivo inMC38-tumor implanted animals as it relates to curessynthesized TNFR2 proteins on rapidly proliferating tumor-infiltrating

MC38 tumor implanted mice receiving each of the active therapies had

Tregs because a new inhibitory dimeric surface structure is formed

smaller tumor volumes than controls (Fig. 3A, left). Anti-PD-1 aloneor stabilized. Therefore we were not too surprised that in vitro thisresulted in only 10% of mice being cured but still with reduced tumor

new antibody might not be very potent, so in vivo experiments werevolumes compared to controls (P = 0.0002; Fig. 3B, top right; Fig. 3C).

required scientifically.

Anti-TNFR2 alone cured 20% of the mice and again all mice had

6

CASE ET AL .

F I G U R E 2 Single and combination therapy reduced tumor volume in CT26-implanted animals. (A) Schematic of therapy administration (right)
with biweekly dosing with 100 µg/mouse antibody or placebo over a 20–21 d course. All three active therapy groups demonstrated a significant
reduction in mean tumor volume relative to the control group. Anti-programmed death receptor-1 (anti-PD-1; 156 ± 118.72 mm3 , P = 0.002, n = 8);
anti-TNFR2 (146 ± 65.95 mm3 , P < 0.0001, n = 18); and combination anti-PD-1+anti-TNFR2 (147 ± 124.33 mm3 ; P = 0.0004, n = 8) tumors were
all smaller than controls (798 ± 493.026 mm3 ; n = 8). (B) Tumor volumes in individual animals over time using spider plots (control in top left; antiPD-1in top right; anti-TNFR2 in bottom left; and combination therapy in bottom right). (C) Cure rates (complete tumor elimination) at days 20–21
were best for combination therapy (62% of animals cured) and anti-TNFR2 therapy (55% cured) and worst for programmed death receptor-1 (PD1; 25% cured) and control (0% cured) (D) Flow cytometry of tumor infiltrate in the only animals with visible but shrunken tumors at day 21 showed
depletion of regulatory T cells (Tregs; CD4+ T cells) and increased ratio of Teffs (CD8+ T cells) to Tregs only for anti-TNFR2 alone and combination
therapy, but not for anti-PD-1 alone. Data are expressed as mean ± SEM. All statistics are the treatment group compared to the control group. Using
the CT26 murine model this experiment was performed at two separate times with similar data

CASE ET AL .

7

F I G U R E 3 Single and combination anti-programmed death receptor-1 (anti-PD-1) and anti-TNFR2 therapy reduce tumor volume in MC38implanted mice. (A) Schematic of therapy administration, right panel, in which mice were dosed biweekly with 0.1 mg/kg of anti-PD-1, anti-TNFR2,
or combination anti-TNFR2 + anti-PD-1 therapy, and mean tumor volumes were measured over a 20–21 d course (left panel). All three active treatment groups demonstrated a significant reduction in tumor growth relative to control. Anti-TNFR2 alone and the combination anti-TNFR2+antiPD-1 therapy showed greater improvement (vs. control) than did anti-PD-1 alone (left). Control group (2064 ± 210.15 mm3 ; n = 10), anti-PD-1 (680
±165.00 mm3 ; P = 0.0002, n = 10), anti-TNFR2 (336 ± 122.94 mm3 ; P < 0.0001, n = 10), and combination therapy (245 ± 168.71 mm3 ; P < 0.0001,
n = 10) (B) Individual tumor volumes are shown (control in top left; anti-PD-1in top right; anti-TNFR2 in bottom left; and combination therapy
in bottom right). (C) Cure rates (complete tumor elimination) at days 20–21 were best for combination therapy (70% of animals cured) and antiTNFR2 therapy (20% cured) and worst for programmed death receptor-1 (PD-1; 10% cured) and control (0% cured). (D) Flow cytometry of tumor
infiltrate at day 8 (n = 8) and day 15 (n = 8) were performed for each treatment condition to capture the evolution of tumor infiltrates and prevent
bias at days 20–21, a time point where only failed animals could be samples. Anti-TNFR2 and anti-TNFR2 therapy with anti-PD1 were the therapies to most dramatically to deplete regulatory T cells (Tregs; CD4+ T cells) (P = 0.0142, P = 0.0179) and increase CD8+/Treg ratio (P = 0.0044,
P = 0.0072) at day 15. Data are expressed as mean ± SEM. Using the MC38 murine model this experiment was performed at two separate times
with similar data
significantly reduced tumor volumes (P < 0.0001; Fig. 2B, lower left;

evolution of the Tregs and CD8/Treg tumor cellular changes. The

Fig. 3C). The combination of anti-PD-1 and anti-TNFR2 was highly effi-

flow cytometry gating patterns are shown in Supplementary Figure

cacious, with 70% cured (P < 0.0001; Fig. 3B lower right; Fig. 3C).

S1. This was important to perform because sampling only at days

A time course of flow cytometry was performed to understand the

20–21, the experimental end point, leaves only a few failure mice

8

CASE ET AL .

and many mice with no tumors so the flow cytometry picture bias isapy also had the highest ratio of CD8/Tregs, but it is important to keep

toward nonresponding mice. Both TNFR2 therapy as a sole therapy

in mind that the flow cytometry at day 20 could only be performed on

and in combination with anti-PD1 changed the tumor microenviron-the two mice with tumor remaining; thus the flow cytometry at ear-ment with the targeted depletion of Tregs and augmented Teffs mea-lier time points is likely more reflective of the tumor microenvironment

sured as the CD8/Treg ratio both at day 8 (Treg: anti-TNFR2 alonechanges shown in Figure 3D. Characteristic flow cytometry pictures of

P = 0.0045, anti-PD1 alone P = 0.1716; anti-TNFR2 plus anti-PD1

the tumor cell infiltrates for a day 8 and for day 20 harvested MC 38

P = 0.0046; CD8/Treg ratio: anti-TNFR2 alone P = 0.0046, anti-PD1

tumors are shown (Supporting Information Figs. S4 and S5). The tumor

alone P = 0.0413, combination therapy P = 0.0045) and at day 15 (Treg:

infiltrates were evaluated to the number of Tregs and the umber ofsole therapy P = 0.0142, combination therapy = 0.0179; CD8/Treg

Teffs in mice receiving different treatments.

ratio: sole therapy P = 0.0044, anti-TNFR2 plus anti-PD1 P = 0.0045)(Fig. 3D, right panel).

3.5 Lowering endotoxin contamination of therapy
improves in vivo efficacy

3.4 The sequence of antibody therapy
administration alters efficacy(<1 endotoxin units, EU, per mg) of each of the three active therapies

Two previous reports in mice given combination antibody therapy with

was associated with lower tumor volumes than the same therapy with

anti-PD1 therapy suggest that the order of antibody administration

higher endotoxin administration (<2 EU/mg) (Supporting Information

significantly affects outcomes.23,24 The impact of therapeutic sequenc-

Fig. S6, left vs. right columns). As a result of this experiment we used

ing in MC38-implanted mice is depicted in Figure 4. We evaluated four

the higher purification on all other experiments reported in this study.

In MC38-implanted animals (n = 90), less endotoxin contaminationdifferent antibody administration sequences (Fig. 4A, right panel): (i)control (placebo for 20 d); (ii) anti-PD-1 for 7 d followed by anti-TNFR2
until day 20; (iii) anti-TNFR2 for 7 d followed by anti-PD-1 until day

4

DISCUSSION

20; and (iv) the co-administration of anti-PD-1 and anti-TNFR2 for
20 d. Although each of the three active therapies is superior to con-

Our study finds that (i) anti-TNFR2 immunotherapy alone or in com-trol in terms of reduced tumor volume (Fig. 4A, left panel) and curebination with anti-PD-1 is a powerful new therapeutic approach inrate (Fig. 4C), the data also clearly show that antibody ordering affects

a commonly used mouse models of cancer; (ii) the mode of action

efficacy. When anti-PD-1 preceded anti-TNFR2 therapy, efficacy wasof anti-TNFR2 appears to be by dramatically changing the tumor

weakest (Fig. 4B, upper right). Efficacy was improved when anti-TNFR2

microenvironment with tumor-specific Treg elimination and Teff

preceded anti-PD-1 (Fig. 4A, left panel, Fig. 4B lower left, Fig. 4C). Thisaugmentation to allow the immune system of the host to become

ordering of antibodies resulted in 40% cures, (P = 0.0001) (Fig. 4C).

active again; and (iii) delivering anti-TNFR2 at the same time as, or

The best efficacy was achieved by co-administration of anti-PD-1 andbefore, anti-PD-1 carries the greatest benefit. Anti-PD-1 precedinganti-TNFR2, with 70% of mice cured (P < 0.0001) (Fig. 4B, lower right,

TNFR2 is to be avoided.

Fig. 4C). This high efficacy rates suggest synergy compared to the otherWe were surprised by the potent in vivo efficacy of anti-TNFR2

two active treatment groups. Flow cytometry was performed at boththerapy compared to its lackluster in vitro killing of Tregs, CT26, andday 8, when all mice had tumors, and also at day 20, on the few remain-

MC38 cell lines. One explanation may be that the Tregs in the ining animals where a tumor could be found. The flow cytometry of thevitro experiments were from normal spleen rather than the tumor

tumor infiltrates was studied as it is related to the order the admin-

microenvironment (because of too few T cells to harvest for anal-

istered antibodies. The tumor infiltrates in the anti-PD1 then anti-

ysis in mouse tumors). Normal Tregs have less TNFR2 expression

TNFR2 antibody combination (with low cure rates) was compared toand less newly made TNFR2 than that on the highly suppressive

the anti-TNFR2 followed by anti-PD1 antibody (with high cure rates).

Tregs of the tumor microenvironment, suggesting insufficient antag-

To further define the time course an early and late time points wasonism to be cytotoxic. TNFR2 antagonistic antibodies do not dis-used to better understand the tumor infiltrate evolution (Fig. 4D). An

rupt already stabilized resting TNFR2 trimers with TNF ligand; they

early treatment of time point of day 8 allowed sufficient tumor mate-

only stabilize or make new TNFR2 dimers on rapidly dividing cells

rial prior to a cure with all mice having tumors (n = 8). The data showin vivo.

that the combination therapy of anti-PD1 first followed by anti-TNFR2

Based on our findings, we believe anti-tumor efficacy of TNFR2

allowed persistent high Tregs and lower Teff compared to anti-TNFR2

blockade is by selective killing of high TNFR2-expressing tumor-

first followed by anti-PD1 or simultaneous treatment where the Tregs

residing Tregs that confer maximal suppression of the “exhausted”

were lowered. This more successful combination, or avoidance of anti-

Teffs. TNFR2 antagonism kills tumor-associated Tregs by preventing

PD1 first, shows at day 8 the Treg cells decreasing and Teff increasing

signaling through NF-kB, which is necessary for Treg survival.5 Deplet-compared to the reverse treatment or to controls. This same compar-

ing the suppressive Tregs, in turn, allows Teff awakening. Anotherison at day 20 demonstrated the same trend (Fig. 4D) and shows thatmechanism of efficacy may be by direct killing of tumor cells, which

only one of the active treatments reduced the number of Tregs: anti-

has been found with other tumor cell lines.21,22 It is also known that

TNFR2 preceding anti-PD-1 therapy or the combo together This ther-myeloid-derived suppressor cells (MDSC) express TNFR2 and thisCASE ET AL .

9

F I G U R E 4 Sequential administration of anti-programmed death receptor-1 (anti-PD-1) and anti-TNFR2 has differential effects on tumor volume in MC38-implanted mice. (A) Schematic of therapy administration (Fig 4A, right). MC38-implanted mice were dosed biweekly with 0.1 mg/kg
of sequential or concurrent administration of anti-PD-1 and anti-TNFR2 antibodies, and mean tumor volumes were measured over a 20–21 dcourse (Fig. 4A. left). Administering anti-TNFR2 before anti-PD-1 showed a significantly greater reduction in tumor volume than administeringanti-PD-1 before anti-TNFR2, and this reduction was similar to the results in the simultaneous anti-TNFR2+ anti-PD-1 treatment group (n = 10,n = 10, n = 10). (B) Individual tumor volumes for control (2064 ± 210.159; n = 10), anti-TNFR2/ anti-PD1 (387 ± 269.563; P = 0.0001, n = 10),anti-PD1/ anti-TNFR2 (999 ±226.17; P = 0.0003, n = 10), and concurrent anti-PD1 and anti-TNFR2 treatment (245 ± 168.717; P < 0.0001, n = 10)
are shown. (C) Cure rate (complete tumor regression) was highest for simultaneous combination therapy (70% of animals cured), followed by antiTNFR2 first (40% cured), and last by anti-PD-1 first (10% cured) and control (0%). (D) Flow cytometry data of tumor infiltrate in the animals thatfailed to be cured revealed that anti-TNFR2 first depleted regulatory T cells (Tregs) and enhanced Teff/Treg ratio, and this effect was not observedin either the simultaneous treatment group or the group where anti-PD-1 was presented first at least for the two nonresponsive mice. Data areexpressed as mean ± SEM. Using the MC38 murine model this experiment was performed one time

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CASE ET AL .

may also contribute to their immunosuppressive effects in the tumor
microenvironment.23
Unlike the Tregs of the tumor environment in mice and humans,
which have massive TNFR2 expression, the Teffs of the tumor microenvironment have null to suboptimal TNFR2 expression. Normal Teffs are

AUTHORSHIP
K.C., L.T., and M.Y. performed experiments; W.M.K. analyzed theresults; H.Z. performed the statistics; and D.L.F. interpreted data,
supervised experiments, and wrote the manuscript.

known to have low but detectable TNFR2. PD-1 blockade is known torestore activation marker expression on Teffs to normal and to restore

DISCLOSURES

their proliferative abilities.24 This may explain the significance of the

The authors declare no conflicts of interest on the data generated insequence of delivery. We found the relatively weakest efficacy withthese mice experiments. Also all mouse experiments were performed

PD-1 blockade preceding TNFR2 blockade. This sequence may inad-by third parties blinded to the reagents to ensure data integrity andvertently inactivate or kill the newly invigorated Teffs (in addition tono bias.

the Tregs). In other words, PD-1 blockade restores TNFR2 expression to Teffs, leaving them vulnerable to inactivation or death by the
TNFR2 blockade.
We are not the first group to show that combination cancer

ORCID
Denise L. Faustman

https://orcid.org/0000-0002-0871-4812

immunotherapies can often improve with selection of the proper
sequencing of immunotherapy administrations. Two groups haveshown that concurrent PD-1 blockade negates the effects of OX40
agonistic antibody in murine tumor models.25,26 We now show similar
but not identical data with TNFR2 antagonism; TNFR2 antagonism
works best if administered first followed by PD-1 blockade or concurrently with PD-1 blockade, but loses its added efficacy if administeredafter PD-1 blockade. Checkpoint inhibitors nonspecifically activate
the immune system and may revive the Teff response. Teff augmentation in murine models with a TNFR2 agonistic approach, especially if
the tumor is deficient in Tregs, could be an alternative.27 If pretreatment with PD-1 blockade unleashes the immune system, the benefitof a targeted immunotherapy like TNFR2 blockade is likely diminishedbecause of greater the Teff inactivation.
This study also addresses the technical issue of endotoxin contamination of antibodies used in murine studies. Most commonly it isacceptable to use in vivo mouse antibodies with endotoxin levels of <2
EU/mg as reported broadly in the literature. What we observe here isthat when mouse-directed immunotherapeutic antibodies are purified
to a higher standard, such as endotoxin to levels of <1 EU/mg, the efficacy of active therapies is enhanced. This finding may be explained byendotoxin’s nonspecific immune activation.
We conclude that TNFR2 blockade is a promising immunotherapy
alone or, better yet, when combined with PD-1 blockade, based on
results in two murine cancer models. Anti-TNFR2 alone performed better than anti-PD alone. TNFR2 blockade lacks potential for autoimmunity because only a minor subset of peripheral CD4 T cells express
TNFR2, and it is likely to have a favorable toxicology profile due to its
selective expression on a subpopulation of potent Tregs, myeloid suppressor cells. TNFR2 ligands, agonists, and antagonists have minimalin vivo toxicity in primates.26 Our data also reinforce the importanceof studying mechanism of action, and potency of TNFR2 antagonism
alone, as well as sequence of delivery.

ACKNOWLEDGMENT
We thank Dr. Miriam Davis for the critical read and editing of
the manuscript.

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SUPPORTING INFORMATION
Additional information may be found online in the Supporting Information section at the end of the article.

How to cite this article: Case K, Tran L, Yang M, Zheng
H, Kuhtreiber WM, Faustman DL. TNFR2 blockade alone
or in combination with PD-1 blockade shows therapeutic
efficacy in murine cancer models. J Leukoc Biol. 2020;1–11.
https://doi.org/10.1002/JLB.5MA0420-375RRRRRSupplementary Figure 1

Cells

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