DOI: 10.1002/JLB.5AB0320-415RRRRR

BRIEF CONCLUSIVE REPORT

Optimizing TNFR2 antagonism for immunotherapy with tumor
microenvironment specificity
Michael Yang1

Lisa Tran1

Katherine Case1

Heather Torrey1

Hui Zheng2

Yaerin Song1

Hiroyuki Takahashi1

Haley Perkins1

Willem M. Kuhtreiber1

Denise L. Faustman1
1 Immunobiology Laboratories, Massachusetts

General Hospital and Harvard Medical School,
Boston, Massachusetts, USA

Abstract
Most approved cancer immunotherapies lack T-regulatory (Treg) or tumor specificity. TNF recep-

2 Biostatistics Center, Massachusetts General

tor 2 (TNFR2) antibody antagonism is emerging as an attractive immunotherapy due to its tumor

Hospital, Boston, Massachusetts, USA

microenvironment (TME) specificity. Here we show that the human TNFR2 receptor is overex-

Correspondence
Denise L. Faustman, Massachusetts General
Hospital and Harvard Medical School, 13thStreet, Building 149, Rm 3602, Boston MA
02193, USA.
Email: faustman@helix.mgh.harvard.edu

pressed on both human tumor cells and on human tumor-residing Tregs, but negligibly expressedon beneficial T effectors (Teffs). Further, we found widespread, if variable, TNFR2 expression on
788 human tumor cell lines from diverse cancer tissues. These findings provided strong rationale for developing a targeted immunotherapy using a TNFR2 antibody antagonist. We designed
a novel, human-directed TNFR2 antibody antagonist and tested it for function using three cellbased TME assays. The antagonist showed TME specificity by killing of TNFR2-expressing tumor
cells and Tregs, but sparing Teffs, which proliferated. However, the antagonist shuffled between
five isoforms, only one of which showed the desirable function. We designed and tested several
new chimeric human versions of the antagonist, finding that the IgG2 isotype functioned better
than the IgG1 isotype. To further improve function, we introduced targeted mutations to its aminoacid sequence to stabilize the natural variability of the IgG2 isotype’s hinge. Altogether, our findings suggest that optimal TNFR2 antagonists are of the human IgG2 isotype, have hinge stabilization, and have wide separation of antibody arms to bind to newly synthesized TNFR2 on rapidly
growing tumor cells. Antagonistic antibodies with these characteristics, when bound to TNFR2,
can form a nonsignaling cell surface dimer that functions with high TME specificity.
KEYWORDS

3 cell-based assays, hinge stabilization, IgG2 isoform

1

environment immunosuppression.4–6 TNFR2 is also overexpressed

INTRODUCTION

in the tumor microenvironment (TME), which consists of infiltrating
The TNFR2 receptor on T regulatory (Treg) cells is a master control

TNFR2+ Treg cells, myeloid suppressor cells, and tumor cells.7,8

switch with broad therapeutic implications for diseases of immune

Agonism of TNFR2 sets off downstream proliferation through tran-

dysregulation.1 Agonism of TNFR2 is advantageous for treating

scription factor NF-kB for preferential survival.1 Antagonism of

autoimmunity because it stimulates adult Treg expansion into potent

TNFR2, by contrast, kills target cells through inactivation of NF-kB

immunosuppressor cells and reciprocally kills CD8 cytotoxic T cells.2,3

signaling and the stabilization of strong hexagonal networks of recep-

Antagonism of TNFR2, on the other hand, is advantageous for treating

tors with poor signaling activity.4,9,10 In most human tumors, every

cancer because it inactivates or kills Tregs, thereby removing tumor

Treg in the TME expresses newly made and high-density TNFR2.7
Human cancers with high TNFR2 expression in the TME have poor
prognosis and secrete into the serum soluble TNF (sTNFR2), a prog-

Abbreviations: HPLC, high-pressure liquid chromatography; MFI, mean fluorescent intensity;
SS, Sezary syndrome; sTNFR2, soluble TNFR2 receptor; Treg cells, T-regulatory cells; Teff,
T-effector cells or cytotoxic T cells; TME, tumor microenvironment.

Received: 15 September 2019
J Leukoc Biol. 2020;1–10.

Revised: 5 March 2020

nostic marker.11–15 The most suppressive subset of Tregs highly
express TNFR2 in the TME of both humans and rodents.5–7 Therefore,

Accepted: 7 March 2020www.jleukbio.org

c 2020 Society for Leukocyte Biology
○

1

2

YANG ET AL .

the central role of TNFR2 in diseases of immune dysregulation, such

T cell lymphoma (Sézary syndrome; SS) patients (n = 28 new sam-as autoimmunity and cancer, is established.1–5,10

ples from 10 subjects) and healthy controls (n = 11 samples; 11 sub-

The most suppressive human-directed TNFR2 antagonistic anti-

jects) according to a human studies protocol approved by both thebodies appear to have specificity for the TME due in part to differ-

Massachusetts General Hospital (MGH) Human Studies Committee

ential expression of TNFR2 on key target cells. In peripheral blood,(MGH-2001P001379) and Stanford University Human Studies Com-

only a subpopulation of Tregs expresses TNFR2 as a stable trimer withmittee (study# 2001P001379 IRB 5538 and IRB 13844). For additionalbound TNF for effective agonistic signaling and most lymphoid cell

human Treg and Teff experiments, blood samples were collected from

populations are negative for TNFR2.4 Antagonist antibodies preferen-

23 non cancer subjects according to the same MGH human studies

tially kill human tumor cells expressing the TNFR2 oncogene, and they

protocol (MGH-2001P001379). All donors provided written informed

kill infiltrating TNFR2-expressing Tregs, which, in turn, lifts suppres-consent. For SS lymphoma subjects, almost all were in stage IV dis-sion of tumor-fighting T effectors (Teffs).4,10 Human TNFR2 antago-ease and by the requirements of this study were not taking antimitotic

nistic antibodies have little activity against normal circulating human

drugs; this drug class interferes with effectiveness of TNFR2 antago-

T cells.4 Antagonistic antibodies cannot disrupt preformed trimers,

nism, which requires rapidly proliferating cells.4 Blood was collectedbut can bind to newly synthesized TNFR2 and stabilize it by form-

into BD Vacutainer EDTA Tubes (BD Diagnostics, Franklin Lakes, NJ,

ing an anti-parallel dimer that creates a nonsignaling complex.4,9 The

USA) and processed within 24 h of phlebotomy.

antiparallel dimer may be a preferred structure for the TNF superfamily receptor antagonism; recent mechanistic and structural data findthat the CD40 receptor can also be inhibited by antibodies that stabi-

2.2

Isolation and culture of cells

lize surface receptor dimers.16 The stabilization of TNFR2 as antiparal-

Fresh human blood was processed within 24 h of venipuncture. Bloodlel dimers prior to trimerization with TNF may allow an open stabilized

was washed twice with 1× HBSS (Invitrogen, Carlsbad, CA, USA) plus

hexagonal complex to form on the cell surface for inhibited signaling

2% FBS (Sigma-Aldrich, St. Louis, MO, USA), and CD4+ cells were(Graphical Abstract).4,9

isolated using Direct Human CD4+TCell Isolation Kit (Stem Cell

Our previous experience with a TNFR2 antagonist similar to the one

Technologies, Vancouver, Canada). Isolated CD4+ cells were resus-

studied here found that (i) it was not dependent on Fc receptor bind-pended in Roswell Park Memorial Institute (RPMI) GlutaMAX (Lifeing or antibody-dependent cellular cytotoxicity (ADCC) mechanism, (ii)Technologies, Carlsbad, CA, USA) plus 10% FBS (Sigma-Aldrich Corpo-

it maintained its antagonism in the presence of natural TNF, and (iii)

ration, Natick, MA) and 1% penicillin-streptomycin (Life Technologies).

it did not require cross-linking for activity.4,10 These features suggest

Because isolated and cultured human T cells die without IL-2 in thethat antibody regions other than the Fc region are crucial for functional

media, all culture conditions in all experiments contained a low amount

impact, and they suggest lower toxicity when translated into humanof IL-2 (200 U/ml) to prevent IL-2 withdrawal from influencing the data.

clinical trials.

CD4+ cell cultures were incubated at 37◦ C with 5% CO2 . Colorectal

Here we first examine differential TNFR2 antigen density on nearly

adenocarcinoma cell line SW480 [SW-480] (ATCC CCL-228) was

800 tumor cell lines followed by a study of the impact of density on

cultured in Leibovitz’s L-15 Medium (ATCC 30-2008) supplemented

TNFR2 antagonistic antibody killing in dose–response experiments.

with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Life

Our additional focus using these refined cell-based killing assays is theTechnologies). SW480 cell cultures were incubated at 37◦ C with notesting and refinement of a new set of human-directed TNFR2 antag-

CO2 exchange. Both the T cell leukemia cell line MOTN-1 (DSMZ ACC

onistic antibodies to optimize activity. Using three cell-based TME559) and T cell lymphoma JEKO-1 (ATCC CRL-3006) were cultured

assays (tumor cells, Treg cells, Teff cells), we test the antibody’s capac-

in RPMI GlutaMAX (Life Technologies), 20% FBS (Sigma-Aldrich), andity to kill TNFR2-expressing tumor cells and Tregs, and spare Teffs,

1% penicillin-streptomycin (Life Technologies) plus additional IL-2

which can proliferate because of removal of Tregs. Teff expansion is(100U/ml) for MOTN-1.

crucial for immune system destruction of tumor cells. We test several

We tested 788 different human tumor cells lines, and cultured the

IgG2 human chimeric isoforms of our antagonistic antibody for theircells according to the vendor’s instructions (ATCC Manassas, VA, USA

potency and TME specificity. Finally, we introduce targeted mutations

and DSMZ, Braunschweig, Germany). From approximately 5 × 106

to the antibody antagonist’s amino acid structure to determine whatcells, we isolated RNA using an mRNA isolation kit (Sigma-Aldrich).

factors optimize its TME specificity.

After isolation the RNA was stored in the freezer. Quantitative PCR(qPCR) was applied using PCR technology (Bio-Rad, Hercules, CA,
USA) and commercial primers to the human TNFR2 gene (TNFRSF1B).

2

MATERIALS AND METHODS

2.1

Human samples

The known amplification primer was forward CGTTCTCCAACACGACTTCATCC and the reverse primer was ACGTGCAGACTGCATCCATGCT. qPCR also used SYBR Green ER Super Mix (Invitrogen),200 nM primers, and 200 ng cDNA followed the recommended

This study was conducted with full local institutional review board

testing cycling conditions. The Invitrogen Superscript IV kit and theapproval and involved written and informed consent of all humanProtoScript kits were used for the cDNA synthesis with oligo(dT)subjects. Human blood samples were collected from cutaneous(deoxythymine) primers. PCR primers were designed as constructs

3

YANG ET AL .

with the assistance of either GeneFisher or Primer3.16,17 Real-time

intracellular staining of FOXP3 and human CD25 (clone BC96;

quantitative PCR (qPCR) was performed using the Bio-Rad PCR

BioLegend) for cell surface staining of CD25. This is a standard kit

machinery, and all reactions were performed according to the manu-

and method for identifying the Treg population within isolated CD4facturers’ instructions. For these quantitative experiments of relative

T cells. Treg populations, which were assessed by FACS with FL2mRNA expression between different cell lines, our endogenous control(red) versus FL1 (green); Tregs were defined as CD4+ CD25hiFoxP3-

gene was 18S ribosomal RNA (Amplifluro Human 18S rRNA Primer

positive, whereas Teff populations were defined as CD4+ CD25low

Set, Sigma-Aldrich).

and FoxP3-negative. Antibody MAB2261 (R&D Systems, Minneapolis,
MN, USA) was used for measuring TNFR2 cell surface expression.

2.3

Treg assays

All unlabeled antibodies at our lab are labeled with Allophycocyanin(APC) using the Lightning Link APC Conjugation Kit from Novus

For all Treg assays, freshly isolated CD4+ cells were seeded in 96-wellround-bottom plates at a concentration of 0.2 × 106 to 1 × 106 cells per

Biologicals (Littleton, CO, USA). FACS data were processed using
FlowJo software (version 10.1).

well, treated with TNFR2 antagonists and various reagents, and incubated for 48 to 72 h. After incubation, cells were collected and stained

2.6

Construction of human chimeric variants

for FACS analysis.
We designed several new human chimeric constructs to improve

2.4

Direct cancer-killing assays

TNFR2 antibody efficacy. These constructs were based on the literature or from direct protein sequencing of the antibody from the Kabat

SW480 colon cancer cells were cultured in 96-well flat-bottom plates

database of antibody heavy and light chain amino acid sequences.19

at a concentration of 1 × 104 cells per well in 200 µl of media. Cells

The asterisk (*) is the continuation of the conserved region of thewere treated directly with TNFR2 antagonistic antibodies and incu-

sequence. The cDNAs of the light and heavy chains of the antibodies

bated for various time points but optimally for 7 d. MOTN-1 cells were

were chemically synthesized with optimization for mammalian expres-cultured in 96-well U-bottom plates at a concentration of 1 × 105 cells

sion. The cDNA were cloned into expression vector pATX2. The heavy

per well in 200 µl of media. Cells were treated directly with TNFR2

and light chains were expressed in 500 µl in cultures of 293F cells.

antagonistic antibodies and incubated for 72 h or longer but optimally

Purification was done with affinity chromatography on protein G resin.

for 72 h. JEKO-1 cells were also cultured in 96-well U-bottom plates

The media was TBS with pH 7.5/8.0 buffer and elution with 0.1 M

at a concentration of 1 × 105 cells per well in 200 µl of media. After

glycine at pH 3.0. Neutralization was with 1 M Tris-HCL, pH 8.5. All

incubation, cells were detached from the plate and stained with trypan

purified antibody variants were run on SDS-PAGE and all endotoxin

blue (Sigma-Aldrich) to count viable cells. The past, full dose–response

was removed so final levels were <1EU/ml. Bacterial endotoxin levels

curves were performed too see if killing increased with the dose orwere determined using the Endosafe-PTS system and Endosafe PTS

if killing reach a plateau followed by a decline, a trait of many anti-

cartridges (Charles River Laboratories, Willington, MA). For removal

bodies dependent of Fc receptor binding or antibodies that agonize

the Toxin Eraser Endotoxin Removal System was used (Genescript).

death receptors.18

The regions of the humanized variants for the chimeric antibodies areindicated below. All other antibody regions were of mouse origins.

2.5

Reagents and flow cytometry

Murine monoclonal antibody (mAb) directed against human TNFR2
was produced, as previously described.4,10 In this paper, we moved
forward with the murine TNFR2 antibody presented in our mostrecent cancer paper.10 Recombinant human TNF was purchased from
Sigma-Aldrich, and recombinant human IL-2 was purchased from
Sigma (Aldrich Corporation, Natick, MA). Cells were prepared forflow cytometry using Human Treg Flow Kit (BioLegend, San Diego,
CA, USA) according to the manufacturer’s instructions. Fluorescently
stained cells were resuspended in 1 × HBSS (Invitrogen) with 2% heat
inactivated human serum and analyzed using a BD FACS Calibur flow
cytometer machine (Becton Dickinson, Franklin Lakes, NJ). Prior tostarting any new cell line flow analysis, isotype control antibodies tothe staining antibody were compared to 2% human serum or Fc blocking reagent (Bio-Rad) for any possible nonspecific background staining
and the blocking of that background staining. In almost all cases, theaddition of 2% human serum eliminated even low background nonspecific staining. Antibodies used for FACS analysis of Tregs included
Alexa Fluor 488 anti-human FOXP3 (clone 259D; BioLegend) for

IgG2 consensus
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS
C232S, C233SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSC232SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSC233SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSC127S
ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS
IgG2 Consensus
NTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
C232S, C233S4

YANG ET AL .

NTKVDKTVERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE

crude and purified sample after normalizing for injection volume and

VTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN

concentration. Pooled fractions were stored at 4◦ C.

C232S
NTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE

2.9

Statistical analysis

VTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFN
Data analysis was performed by Student’s t-test using ExcelC233S
NTKVDKTVERKCSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE(Microsoft) or SPSS (IBM). Significance was determined by P < 0.05.

VTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNC127S
NTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE

3

RESULTS AND DISCUSSIONVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNThe Kabat numbering system was used for all antibody sequences.
The Kabat numbering scheme is the numbering of amino acid residues
in antibodies based upon variable regions.

3.1 TNFR2 is differentially expressed on cells of the
TME; TNFR2 oncogene expression on tumor cell lines
is widespread: TNFR2 density on tumor cells
determines rate of killing
Log mean fluorescent intensity (MFI) by FACS analysis of TNFR2

2.7

Protein gel electrophoresis

in lymphoid cell populations of humans with end-stage cutaneous T cell lymphoma (SS) demonstrates the following: (i) Treg

The mAb protein samples were run on NuPAGE 3–8% Tris-Acetate gelswith Tris-Glycine Native Running Buffer (Life Technologies) at 225V
for 2.5 h. Gels were stained for 45 min with SimplyBlue SafeStain (Invitrogen) under nonreducing conditions.

cells (CD4+ CD25hi Foxp3hi ) from SS samples had a significantly
higher TNFR2 MFI level than that of Treg cells from control lymphocytes (from normal blood donors) (P = 0.04); (ii) Teff cells(CD4+ CD25lo Foxp3− ) from SS subjects had a TNFR2 MFI level similar
to control, both of which were close to background level of fluores-

2.8 SCX fractionation with high-pressure liquid
chromatography (HPLC)
SCX fractionation (strong cation-exchange chromatography) 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(catalogue number 401NP5-4625PK). Running conditions were as
follows: flow rate: 1 mL/min; detector: UV 280 nm; and 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 (MES), MOPS,
N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 3Cyclohexylamino 2-hydroxy-1-propanesulfonic acid (CAPSO), NaCl,and HPLC-grade water was used throughout. Mobile phase A was composed of 16 mM MES, 10 mM MOPS, 10 mM TAPS, and 10 mM CAPSO
pH 4.5. Mobile phase B was composed of10 mM MES, 12 mM MOPS,
14 mM TAPS, 16 mM CAPSO, and 30 mM NaCl pH. Mobile phase C
was composed of 10 mM MES, 12 mM MOPS, 14 mM TAPS, 16 mM
CAPSO, and 30 mM NaCl pH 10.2 + 1 M NaCl. Each run had a gradient of mobile phase A of 5% mobile and this was compared to phasecence (dashed line); and (iii) the CD4+ lymphoma cells within the
CD26− cellular subset had a higher TNFR2 MFI level than controls,that is, donors or noncancer cells from the same patient. MFI, wasbelow background (P = 0.03) (Supporting Information Fig. S1). Similarfindings were reported previously with a similar TNFR2 antibody
antagonist, but with fewer samples.10
A survey of 788 commercially available human cancer cell lines by
qPCR showed widespread, if variable, expression of the TNFR2 oncogene. The greatest expression was on hematopoietic and lymphoid celllines, as well as lung and breast tumor lines (Fig. 1A). The SW480 coloncancer cell line has low TNFR2 oncogene expression, with an MFI of
44; TNFR2 antagonistic antibody killing of this tumor cell line wasslow, taking 7 d (Fig. 1B). In contrast, MOTN-1 (leukemia cell line) and
JEKO-1 (lymphoma cell line) had high TNFR2 density and rapid TNFR2
antagonistic killing—within only 48–72 h of incubation time. These
data suggest that the antagonist worked most rapidly against tumor
cell lines with the highest density of TNFR2. The total elimination oftumor cells with either TNFR2 antagonistic antibody at high doses orwith extended culture was consistent with antibody killing not cell conversions or loss of phenotype.

3.2 IgG2b TNFR2 antagonist shuffles between
five isoforms

B of 35% mobile during a 30 min period followed by 2 min of isocratic

For years, the important antibody region for target specificity was

flow. A 4.5 min wash in mobile phase C was performed at the end of

the variable region, but here the focus is on antibody isotypes

each gradient to clean the column. Fractions were collected and pooled

and isoforms. Antibody isotypes are known to have an impact on

together after all the fractions were collected. Purity of each fraction

function.20–23 Antibody shuffling between isoforms is defined as thewas verified by HPLC, run on the same column and same instrument.

process of creating variants through covalent intrachain interactions

The recovery yield was calculated by comparing peak area between thein the hinge made naturally in vivo and influenced by manufacturing

5

YANG ET AL .

AB

F I G U R E 1 Widespread aberrant TNFR2 expression on tumor
cell lines; TNFR2 antagonist appears to kill faster-growing
tumors more rapidly. (A) Analysis of TNFR2 mRNA expression
in 788 human tumor cell lines shows widespread, if variable,
expression across body and tissue sites of the cancer. Background
expression of negative cells with control primers from normal tissue donors was set at 4. (B) Left panels show histogram of TNFR2
expression. Right panels indicate in vitro culture of the cell linewith TNFR2 antagonistic antibodies. Colorectal adenocarcinoma
cell line, SW480, for 24–48 h or for 7 d with various doses of the
TNFR2 antagonist antibody (0 to 50 µg/ml) demonstrated killingby 7 d. Data are means ± SEM from n = 23 independent experiments; all Ps < 0.05 at 0.5, 2.5, 5, 12.5, 25, and 50 µg/ml; paired
t-test. A significant linear downward trend in SW480 viability
occurred with increasing dose, P < 0.0001 with extended 7 d culture on this low expressing cell line (MRI = 42.2). In contrast, killingof the leukemia cell line MOTN-1 occurred rapidly (within 48–
72 h) in a dose–response manner with TNFR2 antagonist antibody(0 to 50 µg/ml) (all Ps < 0.05), as did killing of the lymphoma cellline JEKO-1(all Ps < 0.05). Both MOTN-1 and JEKO-1 have high
TNFR2 surface density of the TNFR2 growth receptor (MFI = 246;
MFI = 775). The pink histogram shows tumor cells; the blue background histogram shows isotype controls. The full TNFR2 antagonist antibody dosing scale is: 0, 0.05, 0.1, 0.5, 2.5, 5, 25, 50 but due
to a limitation of space, not all doses are represented

conditions.22–29 Our murine IgG2b anti-human TNFR2 antagonistic

Teff proliferation, and SW480 colon cancer cell killing (Fig. 2B). Using

antibody, using standard production, showed on nonreducing gels,

HPLC, the single mAb separated into 5 discrete bands, labeled F1-F5,

4–5 IgG2 isoforms attributable to various types of hinge region

for testing in functional assays (Fig. 2C).

disulfide bonds (Fig. 2A). Reducing conditions always generated ongels one heavy and one light chain band (Supporting Information
Fig. S2), confirming that the nonreducing gel band variability was due
to antibody hinge sulfhydryl bonds. Despite consisting of various hinge

3.3 Only one of the five IgG2 isoforms has desired
antitumor activity on cell-based TME assays

isoforms, the parent monoclonal displayed the desired antitumor

Of the five visible IgG2b bands on HPLC, only fraction F1 had thefunctional activity in our three cell-based TME assays: Treg killing,

desired functional activity compared to the other hinge isoforms of the6

YANG ET AL .

F I G U R E 2 Native mouse IgG2b anti-human TNFR2 has desired TNFR2 antagonistic “shuffled” isoforms. (A) The parent mouse antibody, an
IgG2b isoform, has the five bands expected on nonreducing gels. (B) When this mixture of IgG2b isoforms of this single monoclonal TNFR2 antagonistic antibody is tested in fresh peripheral blood, the mixture performs as desired: it kills T-regulatory cells (Tregs), induces proliferation of T
effectors (Teffs), and kills SW480 tumor cells. For all three test parameters, the P-value was <0.05 using the mixed effects model. This experimenton fresh CD4 T cells is a representative sample but has been performed over 100× on normal blood donors. The p value is from a mixed effects
model of antibody dose compared to baseline on 100 donor samples; the experiment on SW480 tumor cells was performed >10 times with identical results and the figure shown is pooled data from 10 separate experiments (C). Strong cation-exchange chromatography (SCX) fractionation was
performed on an Agilent 1260 model HPLC to separate the various IgG2b isoforms for further functional testing on an individual basis. Using thismethod of charge separation, at least 5 antibody peaks could be physically separated for further studies

same mAb (Figs. 2 and 3). For these screening assays, noncancerous

tested the functional impact of chimeric antibody structure (Fig. 4).

blood samples were utilized due to lack of availability of fresh periph-

We created two chimeric antibodies by replacing the mouse IgG2beral blood from SS donors on a daily basis, so the magnitude of theheavy chain constant region with that of either human IgG1 or IgG2.

effect is not as great as that obtained with TME infiltrates. Peripheral

The human IgG1 native framework hinge yielded, as expected, one

blood from a liquid tumor, such as SS, constitutes the TME.

band on nonreducing gel; the human IgG2 native framework yielded,
as expected, two bands on nonreducing gels (Fig. 4C, right panels).

3.4 Human chimeric IgG2 is functionally superior tothe human chimeric IgG1 antibody variant

The human chimeric IgG1 antibody had no or almost undetectablefunctional activity in relation to Treg killing and Teff proliferation(Fig. 4B). It had minimal activity against SW480 direct tumor cell killing.

Having established on the murine IgG2b antibody framework that the

The human chimeric IgG2 antibody showed a paradoxical inverted V-function of the antagonistic antibody varies with isoform shuffling, weshaped Treg killing curve with a downward V-shaped Teff proliferation

7

YANG ET AL .

F I G U R E 3 HPLC separations of the mouse anti-human IgG2b antibody shows that only one isoform fraction has the desired antitumor activity. HPLC separation of the mouse IgG2b antibody into the various isoforms revealed 5 physical protein peaks (Fig. 2) that were tested separately
in functional assays in this experiment, HPLC fractions F1-F5. Each fraction was tested in functional assays of the rate of T-regulatory (Treg) killing(left panels), T effectors (Teff) expansion (middle panels), and killing of the SW480 tumor cell line (right panel). Data involve the culture of fresh
peripheral human CD4+ cells in response to the TNFR2 antagonist (0-50 µg/ml) for 48–72 h. The functional assay was the killing of SW480 colorectal adenocarcinoma cultured for 7 d with the TNFR2 antagonist (0–50 µg/ml). The percentage Tregs or Teff is in comparison to total CD4 T cells.
Data are mean ± SEM. Statistical analysis by 1-way ANOVA, Tukey’s multiple comparison test, and for the slope compared to baseline the mixed
effects model **P < 0.01. All fractions were tested on fresh CD4 cells for the Tregs and Teff or on SW480 tumor cells at least 5 times; a representative experiment is shown for the Tregs and Teff experiments. For the SW480 cells, the error bars represent 8 separate experiments. Data aremean ± SEM
curve (Fig. 4C). These bi-model shaped curves maybe driven by bivalent antibody binding versus monovalent target protein binding withhigh concentrations. Also it could be that one isoform of the native

3.5 A double IgG2 mutation stabilizes the antibody
hinge, enlarges the arms, and has desired functional
activity

IgG2 antibody had a very different function from the other isoform
driving this “mixed” response. It did show direct and strong SW480

For the IgG2 isotype, known mutations in the hinge region of cys-tumor cell killing (Fig. 4C). This suggests that the ratios of IgG2 shuf-

teine (C) to serine (S) prevent shuffling, and past reports confirm

fled forms may vary in these preparations and could account for thethat these stabilized mutants have distinct functions.22–29 The hingelate TNFR2 antagonism at high concentrations on Tregs and the lateforms are shown in Supporting Information Figure S4. We mutated theproliferation of Teffs. The antibody heterogeneity conferring antibody

human IgG2 chimeric antibody into three variants to test their func-

functional variability might be the result of the manufacturing condi-

tional impact in the TME assays: double mutant C232S C233S, sin-tions creating a varying ratio of IgG2 isoforms.

gle mutant C232S, and single mutant C233S. Figure 5 shows that the

As an additional experiment to verify that the 4–5 bands of the

double mutant was the only one to successfully reproduce the func-

murine IgG2b were due to the murine hinge region shuffling, thetion of the parent murine IgG2a antibody (Treg killing, Teff prolifera-

murine hinge of the native IgG2b antibody was placed onto the humantion, and SW480 tumor killing in a dose-dependent manner). The datachimeric IgG2 construct (Supporting Information Fig. S3). As expected,

also shows the single hinge mutants were only partially successful. The

the human chimeric IgG2 with the murine hinge once again on nonre-

enlarged arm width of the double mutant is likely necessary to bind toducing gels had 4–5 bands. The functional consequences were mixed.

the wide size of TNFR2 on rapidly growing tumor cells. The nonreduc-This isoform construction, like the parent human IgG2, had an inverted

ing gel confirmed the presence of the desired single IgG2 antibody iso-V pattern of killing on Tregs and a V curve on Teffs, with modest

form (Fig. 5, left).

SW480 tumor cell killing. This suggested that antibody hinge drives
antitumor function.

The importance of the IgG2 hinge on function has been recently
confirmed by a study of the anti-CD40 antibody agonist for cancer

8

YANG ET AL .

F I G U R E 4 Native IgG2 shows superior performance than IgG1 TNFR2 antagonist. Chimeric anti-human TNFR2 antibodies were constructedusing a human IgG1heavy chain (A) Antibody activity assays with their indicated desired properties for tumor microenvironment specificity. (B)
Human chimeric IgG1 showed a single band on a nonreducing gel, but no activity in the T-regulatory (Treg) or T effector (Teff) assay and minimal
activity in the SW480 tumor cell killing assay. (C) Human chimeric IgG2 showed two bands on a nonreducing gel with bi-modal Treg, Teff activity
but excellent SW480 tumor killing. All fractions were tested on fresh CD4 cells for the Tregs and Teff or on SW480 tumor cells. A representative
experiment is shown for the Tregs and Teff experiments but the experiment was performed on >5 normal donors. The percentage Tregs or Teff isin comparison to total CD4 T cells. For the SW480 cells, the error bars represent 8 separate experiments. Data are mean ± SEM. Statistical analysis
by 1-way ANOVA and Tukey’s multiple comparison test, **P < 0.01

F I G U R E 5 Mutated hinge variants of IgG2 produce single and stable isoforms showing different functional activity in cell-based tumor
microenvironment (TME) assays. Chimeric human IgG2 antibodies were constructed with hinge mutations at C232S and C233S (A), C232C only(B), and C233S only (C). (A) Human IgG2 isoform with the hinge stabilization mutants C232S, C233S showed a single band on a nonreducing gel andrestored T-regulatory (Treg) killing, T effector (Teff) proliferation and SW480 tumor cell killing. (B-C) Human IgG2 isoform with the hinge stabilization mutant C232S or C233S showed a single band on a nonreducing gel, but no Treg killing, good Teff activity, and sluggish SW480 tumor killing.
A representative experiment is shown for the Tregs and Teff experiments, performed >5 times on different normal blood donors. For the SW480
cells, the error bars represent 8 separate experiments. Data are mean ± SEM. Statistical analysis by 1-way ANOVA and Tukey’s multiple comparison
test, **P < 0.01

9

YANG ET AL .

F I G U R E 6 Adverse functional consequences of narrowing a human chimeric IgG2 antagonist’s antibody arms by alternative hinge point
mutation. Another point mutation was made in the amino acid sequence at residue C127S to bring the arms of the antibody closer together. We
hypothesized that this would convert the antibody from an antagonist into an agonist as past data has shown. This hypothesis did not hold true, as
there was some weak killing of the T-regulatory (Treg) cells, slight proliferation of the T effector (Teff) cells, but no distinct pattern for the SW480
cells. A representative experiment is shown for the Tregs and Teff experiments; overall the experiment was performed 5 times on different normal
blood donors. For the SW480 cells, the error bars represent 7 separate experiments. Data are mean ± SEM. Statistical analysis by 1-way ANOVA
and Tukey’s multiple comparison test, **P < 0.01
immunotherapy.20,22,24 The structurally rigid IgG2 isoforms with theantagonist with potential as a targeted cancer immunotherapy. The

C127S mutation optimized agonism. The CD40 receptor is expressed

IgG2 isotype of the antibody antagonist functioned best against tar-on the surface of APCs greatly increases their antigen presentation

get cells in the TME. It is known to have hinge variants that confer

and costimulatory capacity, resulting in a more effective activation of

widely spaced and symmetrical arms for antigen binding. Stabilization

cytotoxic T cells if this receptor was agonized. The natural ligand forof those open IgG2 structures with hinge disulfide double mutations

the CD40 receptor is CD154; this interaction also facilitates effector

at C232S and C233S optimized the antibody’s antagonism. It has been

T cell dependent B cell activation.30

proposed by us that the antibody locks in the nonsignaling antiparallel dimer state to inhibit NF-kB signaling, leading to cell death. This

3.6 Another mutation that brings the IgG2
antibody arms closer together reduces its desired
functional activity
Unmodified IgG2 isotype, compared to other isotypes, has a considerably broader arm span.27,28,30,31 It is known that if the heavy chain
C127S mutation is introduced, it will stabilize the human IgG2 hinge,

finding, combined with the knowledge that this form of antagonism isindependent of the Fc region of the antibody, should encourage development of therapeutic TNFR2 antagonistic antibodies with greater
TME specificity and improved safety.
ACKNOWLEDGMENTS

prevent disulfide shuffling, and tighten the arm distance. This muta-

We thank Dr. Miriam Davis for her critical read and edits of thetion (C127S) succeeded in preventing the shuffling in the hinge, accord-manuscript. We also thank Dr. Youn Kim and her staff for her collab-

ing to the single band on nonreducing gel (Fig. 6, right). Compared tooration on the SS patients.

the parent IgG2b monoclonal, its Treg killing, Teff proliferation, andSW480 tumor killing was modest to nonexistent (Fig. 6). This muta-

AUTHORSHIP

tion, based on previous reports, converts antibodies into agonists. We
did not observe this, but we did observe poor recovery of the desired

D.L.F. conceived the experiments. M.Y. and H. Takahashi performed

antagonistic response.

the tumor cell line experiments; M.Y. and K.C. organized the Sepax
experiments, prepared antibody, and performed gels; L.T., H. Torrey,
and Y.S. performed the fresh lymphocyte experiments; H.P., M.Y., and4

CONCLUSION

K.C. recruited subjects for the fresh blood studies; W.M.K. analyzed
the results; M.Y. and K.C. prepared figures with subsequent writing

This study took advantage of the differential expression of TNFR2 on

and figure preparation undertaken by D.L.F.; and H.Z. performed thelymphoid cells of the TME to develop a novel, human-directed TNFR2

statistics on the represented data.

10

YANG ET AL .

DISCLOSURES
None of the authors consult nor have board seats on the commercialization of this technology. All technology is owned by the
Massachusetts General Hospital.

ORCID
Denise L. Faustman

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

REFERENCES
1. Okubo Y, Mera T, Wang L, Faustman DL. Homogeneous expansion ofhuman T-regulatory cells via tumor necrosis factor receptor 2. Sci Rep.
2013;3:3153-3160.
2. Chen X, Baumel M, Mannel DN, Howard OMZ, Oppenheim JJ. Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells. J Immunol. 2007;179:
154-161.
3. Ban L, Zhang J, Wang L, Kuhtreiber W, Burger D, Faustman DL. Selective death of autoreactive T cells in human diabetes by TNF or TNF
receptor 2 agonism. Proc Natl Acad Sci U S A. 2008;105:13644-13649.
4. Torrey H, Butterworth J, Mera T, et al. Targeting TNFR2 withantagonistic antibodies inhibits proliferation of ovarian cancer cells
and tumor-associated Tregs. Sci Signaling. 2017. https://doi.org/
10.1126/scisignal.aaf8608.eaaf8608.
5. Chen X, Subleski JJ, Kopf H, Howard OMZ, Mannel DN, Oppenheim
JJ. Cutting edge: expression of TNFR2 defines a maximally suppressive subset of mouse CD4+CD25+FoxP3+ T regulatory cells: applicability to tumor-infiltrating T regulatory cells. J Immunol. 2008;180:6467-6471.
6. Govindaraj C, Scalzo-Inguanti K, Madondo M, et al. Impaired Th1
immunity in ovarian cancer patients is mediated by TNFR2+ Tregs
within the tumor microenvironment. Clin Immunol. 2013;149:97-110.
7. Williams GS, Mistry B, Guillard S, et al. Phenotypic screening reveals
TNFR2 as a promising target for cancer immunotherapy. Oncotarget.
2016;7:68278-68291.
8. Ungewickell A, Bhaduri A, Rios E, et al. Genomic analysis of mycosis fungoides and Sezary syndrome identifies recurrent alterations in
TNFR2. Nat Genet. 2015;47:1056-1060.
9. Vanamee E, Faustman DL. TNFR2: a novel target for cancer
immunotherapy. Trends Mol Med. 2017;23:1037-1046.
10. Torrey H, Khodadoust M, Tran L, et al. Targeted killing of TNFR2expressing tumor cells and Tregs by TNFR2 antagonistic antibodies inadvanced Sezary syndrome. Leukemia. 2019;33:1206-1218.
11. Tarhini AA, Lin Y, Yeku O, et al. A four-marker signature of TNF-RII,
TGF-alpha, TIMP-1 and CRP is prognostic of worse survival in high-risk
surgically resected melanoma. J Transl Med. 2014;12:19.
12. Heemann C, Kreuz M, Stoller I, et al. Circulating levels of TNF receptor
II are prognostic for patients with peripheral T-cell non-Hodgkin lymphoma. Clin Cancer Res. 2012;18:3637-3647.
13. Babic A, Shah SM, Song M, et al. Soluble tumour necrosis factor receptor type II and survival in colorectal cancer. Br J Cancer. 2016;14:995-1002.
14. Warzocha K, Bienvenu J, Ribeiro P, et al. Plasma levels of tumour
necrosis factor and its soluble receptors correlate with clinicalfeatures and outcome of Hodgkin’s disease patients. Br J Cancer.
2998;77:2357-2362.
15. Dobrzycka B, Terlikowski SJ, Kowalczuk O, Kinalski M. Circulating levels of TNF38 alpha and its soluble receptors in the plasma of patientswith epithelial ovarian cancer. Eur Cytokine Netw. 2009;20:131-134.

16. Giegerich R, Meyer F, Schleiermacher C. GeneFisher - software support for the detection of postulated genes. Proc Int Conf Intell Syst Mol
Biol. 1996;4:67-77.
17. Untergasser A, Cutcutache I, Koressaar T, et al. Primer3 - new capabilities and interfaces. Nucleic Acids Res. 2012;40(15):e115.
18. Mayes P, Hance K, Hoos A. The promise and challenges of immune
agonist antibody development in cancer. Nat Rev Drug Discov. 2018.
https://doi.org/10.1038/nrd.2018.75.
19. Kabat EA, Wu TT, Perry H, Gottesman K, Foeller C. Sequences ofProteins of Immunological Interest, Fifth Edition. NIH Publication No.
1991:91-3242.
20. Argiriadi MA, Menatuil L, Dubrovska I, et al. CD40/anti-CD40 antibody complexes which illustrate agonist and antagonistic structuralswitches. BMS Mol Cell Biol. 2019;20:29-42.
21. Vanamee ES, Faustman DL. Structural principles of tumor necrosis factor superfamily signaling. Sci Signal. 2018;11:eaao4910.
22. Li F, Ravetch JV. Inhibitory Fc𝛾 receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies. Science.
2011;333:1030-1034.
23. Adame-Gallegos JR, Shi J, McIntosh RS, Pleass RJ. The generationand evaluation of two panels of epitope-matched mouse IgG1, IgG2a,
IgG2b and IgG3 antibodies specific for Plasmodium falciparum and Plasmodium yoelii merozoite surface protein 1–19 (MSP119). Exp Parasitol.
2012;130:384-393.
24. White AL, Chan HTC, French RR, Willoughby J, Mockridge CI, Roghanina A. Conformation of the human immunoglobulin G2 hinge imparts
superagonistic properties to immunostimulatory anticancer antibodies. Cancer Cell. 2015;27:138-148.
25. Yoo EM, Wims LA, Chan LA, Morrison SL. Human IgG2 can form covalent dimers. J Immunol. 2003;170:3134-3138.
26. Martinez T, Guo A, Allen MJ, et al. Disulfide connectivity ofhuman immunoglobulin G2 structural isoforms. Biochemistry. 2008;47:7496-7508.
27. Wypych J, Li M, Guo A, et al. Human IgG2 antibodies display disulfidemediated structural isoforms. J Biol Chem. 2008;283:16194-16205.
28. Guo A, Han M, Martinez T, et al. Electrophoretic evidence for the presence of structural isoforms specific for the IgG2 isotype. Electrophoresis. 2008;29:2550-2560.
29. Liu YD, Chen x, Zhang-van Enk J, Plant M, Dillon TM, Flynn GC.
Human IgG2 antibody disulfide rearrangement in vivo. J Biol Chem.
2008;283:29266-29272.
30. Bishop GA, Hostager BS. The CD40-CD154 interaction in B cell-T cell
liaisons. Cytokine Growth Factor Rev. 2003;14:297-309.
31. Sheriff S, Jeffrey PD, Bajorath J. Comparison of Ch1 domains in different classes of murine antibodies. J Mol Biology. 1996;263:385-389.
32. Canfield SH, Morrison SL. The binding affinity of human IgG for its
high affinity Fc receptor is determined by multiple amino acids in
the Ch2 domain and is modulated by the hinge region. J Exp Med.
1991;173:1483-1491.

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

How to cite this article: Yang M, Tran L, Torrey H, et al.
Optimizing TNFR2 antagonism for immunotherapy withtumor microenvironment specificity. J Leukoc Biol. 2020;1–10.
https://doi.org/10.1002/JLB.5AB0320-415RRRRR