stm.sciencemag.org/cgi/content/full/12/549/eaba2325/DC1

Supplementary Materials for

Tumor-targeted CD28 bispecific antibodies enhance the antitumor efficacy of PD-1

immunotherapy

Janelle C. Waite, Bei Wang, Lauric Haber, Aynur Hermann, Erica Ullman, Xuan Ye, Drew Dudgeon, Rabih Slim, Dharani K. Ajithdoss, Stephen J. Godin, Ilyssa Ramos, Qi Wu, Erin Oswald, Patrick Poon, Jacquelynn Golubov,

Devon Grote, Jennifer Stella, Arpita Pawashe, Jennifer Finney, Evan Herlihy, Hassan Ahmed, Vishal Kamat, Amanda Dorvilliers, Elizabeth Navarro, Jenny Xiao, Julie Kim, Shao Ning Yang, Jacqueline Warsaw, Clarissa Lett, Lauren Canova, Teresa Schulenburg, Randi Foster, Pamela Krueger, Elena Garnova, Ashique Rafique, Robert Babb, Gang Chen, Nicole Stokes Oristian, Chia-Jen Siao, Christopher Daly, Cagan Gurer, Joel Martin, Lynn Macdonald, Douglas MacDonald, William Poueymirou, Eric Smith, Israel Lowy, Gavin Thurston, William Olson, John C. Lin,

Matthew A. Sleeman, George D. Yancopoulos, Andrew J. Murphy*, Dimitris Skokos*

*Corresponding author. Email: dimitris.skokos@regeneron.com (D.S.); andrew.murphy@regeneron.com (A.J.M.)

Published 24 June 2020, Sci. Transl. Med. 12, eaba2325 (2020)

DOI: 10.1126/scitranslmed.aba2325

The PDF file includes:

Materials and Methods Fig. S1. MC38/CD86 tumor growth inhibition is immune cell dependent and potentiated by therapeutic anti–PD-1. Fig. S2. Localization of PD-1/PD-L1 and CD28 at the immunological synapse in the presence of PD-1/PD-L1 mAbs. Fig. S3. PSMAxCD28 bispecific and PD-1 or PD-L1 blockade promote T cell activation in vitro. Fig. S4. MUC16xCD28, but not a MUC16xCD27 T cell binding control, promotes T cell activation. Fig. S5. SPR-Biacore sensorgrams for binding of human and murine CD28 to human and murine CD80 and CD86. Fig. S6. PSMAxCD28 and PD-1 mAb combination increases the frequency of tumor-specific T cells. Fig. S7. PSMAxCD28 cooperates with anti–PD-1 to induce intratumoral but not splenic or systemic cytokines. Fig. S8. Expression of T cell activation markers used to determine CITRUS clusters shown in Fig. 3. Fig. S9. Tumor antigen-specific (p15E+) CD8 T cell CITRUS analysis. Fig. S10. EGFRxCD28 bispecific potentiates T cell activation only in the presence of TCR stimulation. Fig. S11. A431 human xenograft tumor model. Fig. S12. MFI data for CITRUS clusters shown in Fig. 4.

Fig. S13. PSMAxCD28 alone or in combination with PD-1 mAb does not induce cytokine in non–tumor-bearing mice, in contrast to CD28 superagonist. Fig. S14. EGFRxCD28 alone or in combination with PD-1 mAb does not induce cytokines in human immune cell-engrafted STRG mice, in contrast to CD28 superagonist. Table S1. SPR-Biacore kinetics. Table S2. Measuring binding kinetics of human and mouse CD28/CD80/CD86 interactions using SPR. Table S3. Histopathology, organ weights, body weight, and clinical pathology findings in cynomolgus monkeys. References (75–77)

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/549/eaba2325/DC1)

Data file S1 (Microsoft Excel format). Primary data.

Materials and Methods

Cell lines

To generate MC38/CD86 and MC38/EV engineered tumor cell lines, the pLVX lentiviral

plasmid with EF1 promoter encoding mouse CD86 or empty vector and a puromycin resistance

gene (pLVX.EF1a.CD86-puro and pLVX.EF1a.EV-puro, respectively) was used to transfect

HEK293T (ATCC, CRL-11268) cells, facilitating the production of viral particles, which were

subsequently used to infect MC38 cells (National Cancer Institute, Laboratory of Tumor

Immunology & Biology). Engineered cell lines expressing CD86 were isolated by flow

cytometry. Cells were maintained under conditions recommended by ATCC in the presence of

0.5 µg/ml puromycin (Sigma).

Jurkat Clone E6-1 (ATCC, TIB-152) and Raji (ATCC, CCL-86) were cultured according

to ATCC recommended protocol. To generate hPD-L1 expressing cells, a lentiviral plasmid

encoding human PD-L1 (290 aa long; accession NM_14143.4) and a puromycin resistance gene

was used to transfect HEK293T cells, facilitating the production of viral particles, which were

subsequently used to infect Raji cells. Human PD-L1 positive cells were isolated by flow

cytometry. Jurkat cells were transduced with NFB-Luc using a lentivirus (Qiagen, CLS-013L)

and a lentiviral plasmid encoding human PD-1 and a puromycin resistance gene as previously

described (28). All generated cell lines were maintained in DMEM medium (Irvine Scientific)

supplemented with 10 % Fetal Bovine Serum (FBS, Seradigm), Penicillin-Streptomycin-

Glutamine (P/S/Q, Thermo Fisher Scientific), and Non-Essential Amino Acids (NEAA, Irvine

Scientific) and 500 µg/mL Geneticin A (G418, Thermo Fisher Scientific) or 0.5 µg/ml

puromycin (Sigma).

The DU145/hPSMA cell line was generated by transducing DU145 cells (ATCC, HTB-

81) with viral particles that were produced by HEK293T cells transfected with a lentiviral

plasmid encoding human PSMA (amino acids M1 to A750 of accession number Q04609) and a

neomycin resistance gene. After infection, cells were cultured in 500 g/ml of G418 (Thermo

Fisher Scientific) to select for cells stably expressing PSMA. The generated cell line,

DU145/PSMA, was maintained in MEM medium (Irvine Scientific) supplemented with 10 %

FBS (Seradigm), P/S/Q (Thermo Fisher Scientific), and 500 g/mL G418 (Thermo Fisher

Scientific).

For generation of MC38/hPSMA cells, a lentiviral plasmid encoding human PSMA

(amino acids M1 to A750 of accession number Q04609) and a neomycin resistance gene was

used to transfect HEK293T cells, facilitating the production of viral particles, which were

subsequently used to infect MC38 parental cells. Human PSMA-positive cells were isolated by

flow cytometry. MC38/hPSMA were maintained under conditions recommended by ATCC in

the presence 500 g/mL G418 (Thermo Fisher Scientific).

To generate Raji/DKO/hPD-L1 cells, CRISPR/Cas9 technology was used on a stable Raji

cell line (ATCC, CCL-86) to eliminate the expression of CD80 and CD86 to generate

Raji/CD80negative/CD86negative cells (Raji/DKO). For generation of Raji/DKO/hPD-L1 and

Raji/DKO/hPSMA/hPD-L1 cells, a lentiviral plasmid encoding human PDL1 (amino acids M1-

T290 of accession number NP_054862.1) and a puromycin resistance gene was used to transfect

HEK293T cells, facilitating the production of viral particles which were subsequently used to

transduce Raji/DKO cells. Stable PD-L1 expressing cell lines were established by selection with

1 g/ml puromycin (Sigma). In a similar manner, Raji/DKO/hPD-L1 cells were transduced with

lentiviral particles generated by HEK293T cells transfected with a lentiviral plasmid encoding a

gene for human PSMA (amino acids M1 to A750 of accession number Uniprot Q04609) and a

neomycin resistance gene. Stable PSMA expressing cell lines were established by selection with

1250 g/ml G418 (Thermo Fisher Scientific). Generated cell lines were maintained in RPMI

(Irvine Scientific) supplemented with 10% FBS (Seradigm), P/S/Q (Thermo Fisher Scientific),

Sodium Pyruvate (Millipore), HEPES (Irvine Scientific), 1 g/ml Puromycin (Sigma) for PD-L1

expressing cells, and 1250 g/ml G418 (Thermo Fisher Scientific) for PSMA expressing cells.

To generate HEK293/hCD20/hMUC16 cells, HEK293 cells (ATCC, CRL-1573) were

maintained in DME (Irvine Scientific), supplemented with 10% FBS (Seradigm) and P/S/Q

(Thermo Fisher Scientific). HEK293/hCD20 cell generation was previously described (20).

HEK293/hCD20 were maintained in HEK293 medium supplemented with 500 g/ml G418

(Gibco). To generate HEK293/hCD20/hMUC16, a lentiviral vector encoding human MUC16

(short form- amino acids P13810 to Q14507 of accession number NP_078966.2) and a

hygromycin resistance gene was cloned. HEK293T cells were co-transfected with the human

MUC16 encoding lentiviral vector and the lentiviral packaging vector Lenti-X VSV-G single

shot (Takara). The resulting lentivirus was used to transduce HEK293/hCD20, and transduced

cells were sorted by FACS for MUC16 expressing cells. HEK293/hCD20/hMUC16 were

maintained in HEK293/hCD20 medium supplemented with 100 g/ml Hygromycin

(InVivoGen).

Amnis image stream

Jurkat/hPD-1 T cells and Raji-WT or Raji/hPD-L1 target cells were incubated with

CD20xCD3-Alexa488 (Regeneron, 0.5 µg/ml) alone or together with anti-PD-1-Alexa647

(Regeneron, anti-PD-1 blocker or anti-PD-1 non blocker, 1 µg/ml) for 1 hour at 37 °C. Cells

were gently washed with flow cytometry (FACS) buffer consisting of 3 % FBS and 2 mM

ethylenediaminetetraacetic acid (EDTA) in Dulbecco’s Phosphate-Buffered Saline Solution (D-

PBS, Irvine Scientific) twice and stained with CD28-PE (BD, 2 µg/ml) and Hoechst 33342

(Thermo Fisher H3570, 1 µM) for 15 min at 4 °C. Cells were washed with FACS buffer and

stored in BD stabilizing fixative (BD 338036). Images of cells were collected on Amnis Imaging

Flow Cytometer and analyzed by IDEAS software. Cells were gated on doublet bright-field,

doublet nucleus, nucleus focus, single spot count, singlet CD28. Synapse area was defined by

valley mask based on nucleus staining. The ratio of PD-1 or CD28 in/out of synapse was

calculated by the following formula: intensity in synapse/(total intensity - intensity in

synapse)*100%.

Human primary CD3+ T cell isolation

Human peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor

leukocyte pack. PBMC isolation was accomplished by density gradient centrifugation using

50 mL SepMate tubes following the manufacturer’s recommended protocol. Briefly, 15 mL of

FicollPaque PLUS was layered into 50 mL SepMate tubes, followed by addition of 30 mL of

leukocytes diluted 1:2 with D-PBS. Subsequent steps were followed according to SepMate

manufacturer’s protocol. CD3+ T cells were subsequently isolated from PBMCs using an

EasySep Human T Cell Isolation Kit from StemCell Technologies and following manufacturer’s

recommended instructions. Isolated CD3+ T cells were frozen in FBS containing 10 % DMSO at

a concentration of 50 × 106 cells per vial.

IL-2 release from primary CD3+ T cells in an MLR reaction with DU145/PSMA cells

Previously isolated and frozen human CD3+ T cells were thawed the day of the assay in

stimulation medium consisting of X-VIVO 15 cell culture medium (Lonza) supplemented with

10 % FBS, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Irvine Scientific),

sodium pyruvate (NaPyr, Invitrogen), NEAA (Irvine Scientific), and 0.01 mM beta-

mercaptoethanol (BME, Sigma-Aldrich) containing 50 U/ml benzonase nuclease (EMD

Millipore). Cells were centrifuged at 1200 rpm for 10 minutes, resuspended in stimulation

medium, and plated out into 96-well round bottom plates at a concentration of 1 x 105 cells/well.

DU145/hPSMA were treated with 25 g/mL of Mitomycin C in primary stimulation medium at a

concentration of 10 × 106 cells/mL. After incubation for 1 hour at 37 °C, 5% CO2, mitomycin C-

treated cells were washed 3 times with D-PBS containing 2% FBS and added to the wells

containing CD3+ T cells at a final concentration of 5 × 104 cells per well. To prevent possible

CD28 agonistic activity through Fc-anchoring of CD28 antibody to Fc-receptors from occurring,

a saturating amount of non-specific human IgG antibody (100 nM of each: hIgG1, hIgG4, and

hIgG4s) was included in each assay well. Subsequently, PSMAxCD28 or hIgG4s isotype control

antibodies were titrated from 30 pM to 200 nM in a 1:3 dilution and added to wells. The final

point of the 10-point dilution contained no titrated antibody. Because DU145 cells endogenously

express PD-L1, the impact of PD-1 suppression of T cell activity was evaluated by adding a

constant 20 nM of the anti-PD-1 blocker or hIgG4 isotype control to wells. Plates were incubated

for 72 hours at 37 °C, 5 % CO2 and subsequently centrifuged to pellet the cells. 50 µL of

medium supernatant was collected and from this, 5 µL was tested in a human IL-2 AlphaLISA

(Perkin Elmer) assay according to the manufacturer’s protocol. The measurements were acquired

on Perkin Elmer’s multilabel plate reader Envision (Perkin Elmer). A standard curve of known

IL-2 concentrations was generated in order to extrapolate the concentration of IL-2 generated in

assay wells. All serial dilutions were tested in duplicates. The EC50 values of the antibodies were

determined from a four-parameter logistic equation over a 10-point dose-response curve using

GraphPad Prism software.

PD-L1 and PD-1 competition binding ELISA

PD-L1 or an isotype control antibody was incubated with human PD-L1-mFc proteins for

1 hour at room temperature and then transferred to 96-well plates coated with human PD-1-hFc.

After 1 hour, plate-captured hPD-L1-mFc was detected with horseradish peroxidase (HRP)–

conjugated goat anti-mouse Fcγ-specific polyclonal antibody (Jackson ImmunoResearch) and

developed with TMB colorimetric substrates (BD Biosciences). Absorbance at 450 nm was

detected on a Victor multilabel plate reader (PerkinElmer) and plotted as a function of the anti–

PD-L1 antibody concentrations.

PD-1 competition binding ELISA was performed by following the method previously described

(28) and using hPD-1-hFc, hPD-L1-mFc, and a horseradish peroxidase (HRP)–conjugated goat

anti-human IgG Fcγ-specific polyclonal antibody (Jackson ImmunoResearch) for detection.

Allogeneic response assay with Raji cells

Previously isolated and frozen human CD3+ T cells were thawed the day of the assay in

stimulation medium consisting of X-VIVO 15 cell culture medium (Lonza) supplemented with

10 % FBS, HEPES (Irvine Scientific), NaPyr (Invitrogen), NEAA (Irvine Scientific), and 0.01

mM BME (Sigma-Aldrich) containing 50 U/ml benzonase nuclease (EMD Millipore). Cells were

centrifuged at 1200 rpm for 10 minutes, resuspended in stimulation medium, and plated into 96-

well round bottom plates (Costar) at a concentration of 1 x 105 cells per well. Raji/DKO/hPD-

L1/PSMA were treated with 20 µg/ml of Mitomycin C (Sigma-Aldrich) in stimulation medium

at a concentration of 10 × 106 cells/ml. After incubation for 1 hour at 37 °C and 5% CO2, cells

were washed 3 times with D-PBS containing 2% FBS and added to the wells containing CD3+ T

cells at a final concentration of 2.5 × 104 cells per well. To all wells, irrelevant hIgG1 mAb was

added (100 nM/well) to block Fc receptors on Raji cells. A dose titration of PSMAxCD28

serially diluted 1:3 from 200 nM to 30 pM, with the final point of the 10-point dilution

containing no antibody, was added to wells. A constant amount of 20 nM anti-PD1, anti-PD-L1,

or isotype control antibody was then added to wells. Plates were incubated for 72 hours at 37 °C

and 5 % CO2, at which time 50 µl of culture supernatant was collected. 5 µl of this collected

supernatant was tested according to the manufacturer’s protocol in each of three AlphaLISA

assays (Perkin Elmer) assay according to the manufacturer’s protocol.: human IL-2 (AL221F),

human TNF(AL208F), and human IFN (AL217F). The measurements were acquired on an

Envision multilabel plate reader (Perkin Elmer). A standard curve of known IL-2, TNF or

IFN concentrations was generated to extrapolate the concentration of IL-2, TNF or IFN

generated in assay wells. All serial dilutions were tested in triplicate. The EC50 values of the

antibodies were determined from a four-parameter logistic equation over a 10-point dose-

response curve using GraphPad Prism software.

Allogeneic response assay with HEK293 Cells

Previously isolated human CD3+ T cells were thawed in stimulation medium. Cells were

centrifuged at 1200 rpm for 10 minutes, resuspended in stimulation medium, and 1 x 105 cells

per well was plated into 96-well round bottom plates. HEK293/hCD20/hMUC16 cells were

treated with 15 µg/ml Mitomycin C in stimulation medium at a concentration of 10 ×

106 cells/ml for 1 hour at 37 °C, 5% CO2. Cells were washed 3 times in D-PBS containing 2%

FBS, resuspended in stimulation medium and added to the wells containing CD3+ T cells at a

final concentration of 1 × 104 cells per well. MUC16xCD28 and MUC16xCD27 were generated

by pairing an anti-MUC16 with an anti-CD28 or anti-CD27 arm as previously described (20).

The CD28 arm of the MUC16xCD28 bispecific, when formatted as a bivalent hIgG1 or hIgG4,

exhibits ligand binding-antagonist activity and anchoring-dependent and soluble format agonism.

The CD27 arm of the MUC16xCD27 bispecific, when formatted as a bivalent hIgG1 or hIgG4,

exhibits slight ligand binding-antagonist activity and anchoring-dependent agonism but are not

agonistic in a soluble format. MUC16xCD28, MUC16xCD27, or isotype control antibodies were

diluted following a 9-point 1:4 serial dilution from 500 nM to 7 pM, with the 10th point

containing no antibody, and added to wells. Plates were incubated for 72 hours at 37 °C, 5%

CO2. 50 µl cell culture supernatant was collected, 5 L of which was used for IL-2 quantitation

by AlphaLISA assays (Perkin Elmer) according to the manufacturer’s protocol, and acquired on

an Envision multilabel plate reader (Perkin Elmer). A standard curve of known IL-2

concentrations was used to extrapolate the concentration of IL-2 generated in assay wells. To

assess T cell proliferation, medium was supplemented with a final concentration of 1.25 µM

[3H]thymidine (Perkin Elmer) and the cells were incubated at 37 °C, 5 % CO2 for 16 hours.

Plates were harvested using Microbeta Filermat-96 Cell Harvester (Perkin Elmer), 30 L

MicroScint-20 (Perkin Elmer) was added, and [3H]thymidine incorporation was measured using

Microplate Scintillation Counter TopCount NXT (Perkin Elmer). All serial dilutions were tested

in triplicate. The EC50 values of the antibodies were determined from a four-parameter logistic

equation over a 10-point dose-response curve using GraphPad Prism software.

Flow cytometric staining

For antibody binding assessment by flow cytometry, HEK293,

HEK293/hCD20/hMUC16, or primary human T cells from 2 donors (donor 129 and 130) were

plated at 5 x 105/well in V bottom 96 well plate and pre-incubated for 15 minutes with Fc

receptor binding inhibitor polyclonal antibody (Ebioscience) at 4 °C in FACS buffer (D-PBS + 2

% FBS). Cells were then incubated with 500 nM or 20 nM of MUC16xCD28, MUC16xCD27,

or hIgG4s isotype control antibodies in FACS buffer for 30 min at 4 °C. Cells were washed

twice in FACS buffer then stained with 100 nM AffiniPure Fab Goat anti-human IgG Alexa

Fluor 488 conjugated (Jackson Immunoresearch) for 30 min at 4 °C. Cells were washed once in

FACS buffer, once in D-PBS, then stained with fixable far red dead cell stain kit (Invitrogen) in

D-PBS for 30 min at 4 °C. Cells were washed twice in FACS buffer, fixed in Cytofix (BD) for

30 min at 4 °C. Cells were washed once in FACS buffer and acquired in FACS buffer on a

Cytoflex (Beckman Coulter). For Raji/DKO/hPSMA/hPD-L1 staining, cells were plated at 2 x

105/well in V bottom 96 well plate and pre-incubated for 15 minutes with ChromPure Human

IgG, whole molecule (Jackson Immunoresearch) at room temperature in FACS buffer. Cells

were then stained with either phycoerythrin (PE) labeled anti-human CD80 (BD Pharmingen),

allophycocyanin (APC) labeled anti-human CD86 (Biolegend), PE labeled anti-human PD-L1

(Biolegend), or APC anti-human PSMA (Biolegend) in FACS buffer for 30 min at 4 °C. Cells

were washed, stained with fixable violet dead cell stain kit, fixed, and acquired as described

above. Data were analyzed and geometric MFI quantified using FlowJo (Treestar) and plotted

using Prism (GraphPad).

Biacore

All binding kinetics and affinities were assessed using surface plasmon resonance

technology on a Biacore T200 or Biacore 8K instrument (GE Healthcare) using a Series S CM5

sensor chip in filtered and degassed HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 3

mM EDTA, 0.05% (v/v) polysorbate 20, pH 7.4). Different capture sensor surfaces were

prepared by covalently immobilizing with a mouse anti-human Fc mAb (Regeneron), rabbit anti-

mouse Fc polyclonal Ab (GE Healthcare), or anti-His mAb (GE Healthcare) onto the chip

surface using standard amine coupling chemistry, reported previously (75). After surface

activation, different capture reagents prepared in coupling buffer (10 mM sodium acetate buffer,

pH 5.0) were injected for 6-10 minutes, and the remaining active carboxyl groups on the CM5

chip surface were later blocked by injecting 1 M ethanolamine, pH 8.0 for 7 minutes. A typical

resonance unit (RU) signal of about ~5000-15000 RU was achieved after the immobilization

procedure.

SPR binding analysis of EGFRxCD28 bispecific antibody, PD-1 mAb, and PD-L1 mAb

The binding of EGFRxCD28 bispecific antibody, PD-1 mAb, and PD-L1 mAb to their

respective targets was performed on a CM5 sensor surface immobilized with either an anti-

human Fc mAb or an anti-mouse Fc polyclonal antibody at 37 ºC. At the end of each cycle, the

anti-human Fc and the anti-mouse Fc capture surfaces were regenerated using a 10-12 second

injection of 20 mM phosphoric acid and a 40 second injection of 10 mM glycine, pH 1.5,

respectively.

The binding of human EGFR ectodomain expressed with a C-terminal myc-myc-

hexahistidine tag (hEGFR.mmh) to EGFRxCD28 bispecific antibody was performed on a

Biacore T200 instrument. The EGFRxCD28 bispecific antibody was first captured on the anti-

human Fc mAb immobilized surface followed by the injection of different concentrations of

hEGFR.mmh (123 pM – 30 nM, three-fold serial dilution) at a flow rate of 50 L/min for 5

minutes. The dissociation of hEGFR.mmh from EGFRxCD28 bispecific antibody was monitored

for 10 min in the running buffer.

The binding of human CD28 to EGFRxCD28 bispecific antibody was performed on a

Biacore T200 instrument. The human CD28 ectodomain expressed with a C-terminal mouse

IgG2a Fc tag (hCD28.mFc) was captured on an anti-mouse Fc immobilized surface and different

concentrations of EGFRxCD28 bispecific antibody (0.37 nM – 90 nM, three-fold serial dilution)

at a flow rate of 50 µL/min for 4 minutes with a 5-minute dissociation phase in the running

buffer.

The binding of human PD-1 ectodomain expressed with a C-terminal myc-myc-

hexahistidine tag (hPD-1.mmh) and human PD-1 ectodomain expressed with a C-terminal mouse

IgG2a Fc tag (hPD-1.mFc) to the PD-1 mAb was performed on a Biacore 8K instrument. After

the capture of the PD-1 mAb on the anti-human Fc mAb immobilized surface, different

concentrations of hPD-1.mmh (18.75 nM – 1200 nM, two-fold serial dilution) or hPD-1.mFc

(9.38 nM – 600 nM, two-fold serial dilution) were injected for 3 minutes at a flow rate of 30

L/min with a 20-minute dissociation phase in the running buffer.

The binding of human PD-L1 ectodomain expressed with a C-terminal myc-myc-

hexahistidine tag (hPD-L1.mmh) or human PD-L1 ectodomain expressed with a C-terminal

mouse IgG2a Fc tag (hPD-L1.mFc) to PD-L1 mAbs was performed on a Biacore 8K instrument.

The PD-L1 mAbs were first captured on the on the anti-human Fc mAb immobilized surface

followed by the injection of different concentrations of hPD-L1.mmh or hPD-L1.mFc (1.25 nM

– 40 nM, two-fold serial dilution) for 3 minutes at a flow rate of 30 L/min. The dissociation of

the bound PD-L1 reagents from the PD-L1 mAb was monitored for 20 minutes in the running

buffer.

SPR binding analysis of human and murine CD28 to human and murine CD80 and CD86

Due to the weak monomeric binding affinities of CD28 for its ligands (76) and the

practical limitations thereof, we opted for an avidity format steady-state SPR analysis to

determine the relative affinities between dimeric human and murine CD28 to human and murine

CD80 and CD86 ligands. Binding studies were performed on a Biacore T200 instrument at 25

°C using a CM5 sensor surface. The hCD28.mFc and the murine CD28 ectodomain expressed

with a C-terminal human IgG1 Fc and hexahistidine tag (mCD28.hFc_6xHis, R&D Systems)

were captured on the anti-mouse Fc antibody and anti-His mAb immobilized surfaces,

respectively. Different concentrations of the ectodomain of the human and murine CD80 or

CD86 expressed with the C-terminal human IgG1 Fc tag (0.5 nM – 500 nM, two-fold serial

dilution, Sino Biologicals) were individually injected over the CD28 capture surfaces at a flow

rate of 50 μL/min for 90 seconds followed by a 3-minute dissociation in the running buffer.

SPR data analysis

All of the specific SPR binding sensorgrams were double-reference subtracted as

reported previously (77), and the kinetic parameters were obtained by globally fitting the double-

reference subtracted data to a 1:1 binding model with mass transport limitation using Scrubber

software (version 2.0c, BioLogic Software), Biacore T200 Evaluation software v 3.1 (GE

Healthcare), or Biacore Insight Evaluation software (GE Healthcare). The dissociation rate

constant (kd) was determined by fitting the change in the binding response during the dissociation

phase, and the association rate constant (ka) was determined by globally fitting analyte binding at

different concentrations. The equilibrium dissociation constant (KD) was calculated from the

ratio of the kd and ka. The dissociative half-life (t½) in minutes was calculated as ln2/(kd*60). The

steady state analysis was performed using the Scrubber software and the KD value was

determined.

Flow cytometry-based cytotoxicity assay

Cell lines endogenously expressing TSA (PEO1, EGFR+) were labeled with 1 µM of

Violet Cell Tracker and plated overnight at 37 ° C. Separately, human PBMCs (New York Blood

Center) or cynomolgus monkey PBMCs (Covance) were plated in RPMI medium (Irvine

Scientific) supplemented with 10 % FBS (Seradigm) and P/S/Q (Thermo Fisher Scientific) at 1 x

106 cells/mL and incubated overnight at 37 ° C in order to enrich for lymphocytes by depleting

adherent macrophages, dendritic cells, and some monocytes. The next day, the target cells were

co-incubated with adherent cell-depleted naïve human PBMCs (Effector/Target cell 4:1 ratio)

and a serial dilution of either TSAxCD3 or non-targeting CD3-based bispecific, alone or in

combination with a fixed concentration of indicated antibodies for 96 hours at 37 ° C. After

incubation, the cells were removed from the cell culture plates using an enzyme-free cell

dissociation buffer and analyzed by flow cytometry.

For flow cytometry analysis, cells were stained with a viability far red cell tracker

(Invitrogen) and directly conjugated antibodies to CD2, CD4, CD8, and CD25 (BD). Samples

were run with calibration beads for cell counting. For the assessment of specificity of killing,

target cells were gated as Violet cell tracker positive populations. Percent of live target cells was

calculated as follows: % viable cells=(R1/R2)*100, where R1= % live target cells in the presence

of antibody, and R2= % live target cells in the absence of test antibody. T cell activation was

measured by the percent of activated (CD25+) T cells out of CD2+/CD4+ or CD2+/CD8+ T cells.

T cell count was measured by calculating the number of live CD4+ or CD8+ cells per calibration

bead.

The concentrations of cytokines accumulated in the medium were analyzed using the BD

cytometric Bead Array (CBA) human Th1/Th2/Th17 Cytokine kit, following the manufacturer’s

protocol.

Supplementary Figures:

Fig. S1. MC38/CD86 tumor growth inhibition is immune cell dependent and potentiated by therapeutic anti–

PD-1. A. Evaluation of CD86 expression on MC38/CD86 and MC38/EV by flow cytometry. B. MC38/EV and

MC38/CD86 tumor growth in WT and Rag2KO mice. 1 x 106 MC38/EV or MC38/CD86 cells were implanted

subcutaneously on the right flank of either WT or Rag2 KO mice. Tumor volume over time. Data represented as

means ± SEM. Statistical significance was determined with 2-way ANOVA and Sidak’s multiple comparisons tests.

****, p<0.0001, WT-MC38/CD86 vs. WT-MC38/EV. n=5-6 mice per group. Data represent 2 experiments. C-D.

MC38/EV and MC38/CD86 tumor growth in WT mice with therapeutic anti-PD-1 treatment. 1 x 106 MC38/EV or

MC38/CD86 tumor cells were implanted subcutaneously on the right flank of WT mice. Isotype control (Iso Ctrl) or

PD-1 mAb were administered at 5 mg/kg on days 7, 10, 14, 17, and 21 after implant. n=10 mice per group. Data

represent 1 experiment. C. Tumor volume over time. Data represented as means ± SEM. Statistical significance was

determined using 2-way ANOVA and Tukey’s multiple comparisons test. **, p<0.01, MC38/EV + Iso Ctrl vs.

MC38/CD86 + Iso Ctrl or MC38/CD86 + PD-1 mAb. #, p<0.05, MC38/EV + PD-1 mAb vs MC38/CD86 + Iso Ctrl

or MC38/CD86 + PD-1 mAb. D. Survival over time. Data represented as means. Statistical significance was

determined using Log-rank (Mantel-Cox) curve comparison test. ***, p<0.001, MC38/EV + Iso Ctrl vs.

MC38/CD86 + Iso Ctrl or MC38/CD86 + PD-1 mAb. ###, p<0.001, MC38/EV + PD-1 mAb vs. MC38/CD86 + Iso

Ctrl or MC38/CD86 + PD-1 mAb.

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1000

1500

Day Post Implant

Tu

mo

r V

olu

me (

mm

3)

Rag2KO-MC38/EV

Rag2KO-MC38/CD86

WT-MC38/EV

WT-MC38/CD86

****

MC38/CD86 Unstained

MC38/EV + CD86-PEMC38/CD86 + Iso-PE

MC38/CD86 + CD86-PE

A B

0 5 10 15 20

0

500

1000

1500

Therapeutic PD-1 (RMP1-14)

Day Post Implant

Tu

mo

r V

olu

me (

mm

3)

MC38/EV + Iso Ctrl

MC38/CD86 + Iso Ctrl

MC38/EV + PD-1 mAb

MC38/CD86 + PD-1 mAb

B6-MC38/EV-Iso vs. B6-MC38/CD86-Iso

B6-MC38/EV-Iso vs. B6-MC38/EV-PD-1 (RMP1-14)

B6-MC38/EV-Iso vs. B6-MC38/CD86-PD-1 (RMP1-14)

B6-MC38/CD86-Iso vs. B6-MC38/EV-PD-1 (RMP1-14)

B6-MC38/CD86-Iso vs. B6-MC38/CD86-PD-1 (RMP1-14)

B6-MC38/EV-PD-1 (RMP1-14) vs. B6-MC38/CD86-PD-1 (RMP1-14)

**

ns

**

*

ns

*

** #0 5 10 15 20

0

500

1000

1500

Therapeutic PD-1 (RMP1-14)

Day Post Implant

Tu

mo

r V

olu

me (

mm

3)

MC38/EV + Iso Ctrl

MC38/CD86 + Iso Ctrl

MC38/EV + PD-1 mAb

MC38/CD86 + PD-1 mAb

B6-MC38/EV-Iso vs. B6-MC38/CD86-Iso

B6-MC38/EV-Iso vs. B6-MC38/EV-PD-1 (RMP1-14)

B6-MC38/EV-Iso vs. B6-MC38/CD86-PD-1 (RMP1-14)

B6-MC38/CD86-Iso vs. B6-MC38/EV-PD-1 (RMP1-14)

B6-MC38/CD86-Iso vs. B6-MC38/CD86-PD-1 (RMP1-14)

B6-MC38/EV-PD-1 (RMP1-14) vs. B6-MC38/CD86-PD-1 (RMP1-14)

**

ns

**

*

ns

*

** #

C D

0 10 20 30 40 50

0

20

40

60

80

100

Day Post ImplantS

urv

ival (%

)

***##

***##

Fig. S2. Localization of PD-1/PD-L1 and CD28 at the immunological synapse in the presence of PD-1/PD-L1

mAbs. A. PD-1 mAb binding on Jurkat/PD-1 by FACS. Data represented as means. B. PD-1 mAb competition

binding ELISA. Data represented as means. C-D. Images of T cell (Jurkat/PD-1) and target cell (Raji WT)

conjugates in the presence of a PD-1 non-blocker (C) or PD-1 blocker (D) and CD20xCD3 bispecific. Dotted lines

are outlines of cells based on the brightfield image. Scale bar is 7 m. E. PD-1 and CD28 localization in the

synapse. Data represented as means ± SEM. Statistical significance was calculated with an unpaired t test (ns, not

significant). PD-1 non-blocker, n=140 and PD-1 blocker, n=289. F. PD-L1 mAb binding on Raji/PD-L1 by FACS.

Data represented as means. G. PD-L1 mAb competition binding ELISA. Data represented as means. H-K. Images of

T cell (Jurkat/PD-1, H-I, or Jurkat WT, J-K) and target cell (Raji/PD-L1) conjugates in the presence of a PD-L1

non-blocker (H, J) or PD-L1 blocker (I, K) and CD20xCD3 bispecific. Dotted lines are outlines of cells based on

the brightfield image. Scale bar is 7 m. L. PD-L1 and CD28 localization in the synapse. Data represented as means

± SEM. Statistical significance was calculated with 1-way ANOVA (****, p < 0.0001). Jurkat/PD-1 and PD-L1

non-blocker, n=70, or PD-L1 blocker, n=73. Jurkat WT and PD-L1 non-blocker, n=143, or PD-L1 blocker, n=116.

Data in A-L are representative of at least 2 independent experiments.

0.0

0.2

0.4

0.6

0.8

Rati

o P

D-L

1 in

th

e s

yn

ap

se

****

PD-L1 Non-blocker, Jurkat/hPD-1

PD-L1 Blocker, Jurkat/PD-1

PD-L1 Non-blocker, Jurkat WT

PD-L1 Blocker, Jurkat WT

E

C

Bright field Nuclei CD20xCD3 CD28 mAb PD-1 mAb Merge

D

T cell

B cell

T cell

B cell

A B

J

H

I

F G

Bright field Nuclei CD20xCD3 CD28 mAb MergePD-L1 mAb

K

L

0.0

0.2

0.4

0.6

0.8

1.0

Rati

o C

D28 in

th

e s

yn

ap

se ****

-13 -12 -11 -10 -9 -8 -7 -60

5000

10000

15000

20000

25000

Ab [M]

Geo

me

tric

Mean

[M

FI]

PD-1 Blocker

hIgG4 isotype control

mIgG2a isotype control

PD-1 Non-blocker

-13 -12 -11 -10 -9 -8 -7 -60

100000

200000

300000

400000

Ab [M]

Geo

metr

ic M

ean

[M

FI]

PD-L1 Non-blocker

PD-L1 Blocker

Isotype control

-12 -11 -10 -9 -8 -7 -60.0

0.5

1.0

1.5

2.0

Ab [M]

Ab

so

rban

ce

, O

D 4

50n

m

PD-1 Non-blocker

PD-1 Blocker

mIgG2 Isotype

hPD-1

-12 -11 -10 -9 -8 -7 -60.0

0.5

1.0

1.5

2.0

2.5

Ab [M]

Ab

so

rban

ce, O

D 4

50n

m

PD-L1-hIgG1 Non-blocker

PD-L1-mIgG Blocker

hIgG1 Isotype

mIgGa isotype

hPD-L1 ecto.mFc

0.0

0.2

0.4

0.6

Rati

o P

D-1

in

th

e s

yn

ap

se

ns

PD-1 Non-blocker, Raji WT

PD-1 blocker, Raji WT

0.0

0.2

0.4

0.6

0.8

Rati

o C

D28 in

th

e s

yn

ap

se

ns

+ ++T cell

Raji/WT

PD-1

blocker

Schematic KeyPDL1 CD80TCR/CD3 CD28 PD1CD20CD20xCD3

PDL1 CD80TCR/CD3 CD28 PD1CD20CD20xCD3

++ +T cell

Raji/WT

PD-1 Non -blocker

+ ++Jurkat/PD-1

Raji/PD-L1

PD-L1Blocker

+ -Jurkat/PD-1

Raji/PD-L1

PD-L1

Non-blocker

+ ++Jurkat WT

Raji/PD-L1

PD-L1Blocker

+ ++

Jurkat WT

Raji/PD-L1

PD-L1 Non-

blocker

Fig. S3. PSMAxCD28 bispecific and PD-1 or PD-L1 blockade promote T cell activation in vitro. A. Additional

human T cell donors in cytokine release assay with DU-145/hPSMA cells co-cultured with the indicated antibodies

as described in Figure 2. IL-2 release at 96 hours. Data represented as the means ± SEM. Data shown from 2 of 3

human T cell donors tested and are representative of 2 independent experiments. B-C. Human T cell cytokine

release assay with Raji CD80 CD86 Double Negative/hPD-L1/hPSMA cells. B. Raji CD80 CD86 Double

Negative/hPD-L1/hPSMA cell-surface binding of anti-hPD-L1 (blue), anti-hPSMA (red), anti-hCD80 (purple), anti-

hCD86 (green), and their isotype control antibodies (black). APC, Allophycocyanin; PE, Phycoerythrin; h, human;

MFI, mean fluorescence intensity. C. Human T cells and Raji CD80 CD86 Double Negative/hPD-L1/hPSMA cells

were co-cultured with a dose titration of PSMAxCD28 and 20 nM of the indicated antibody. TNF, IFN and IL-2

release at 96 hours. Data represented as the means ± SEM. Data shown from 1 of 3 human T cell donors tested in 1

experiment.

-13 -12 -11 -10 -9 -8 -7 -60

2×102

4×102

6×102

Concentration of Antibody, Log10[M]

TN

Fa

pg

/ml

PD-L1 Blocker

hIgG4 Isotype

PD-1 Blocker

PD-1 Non-Blocker

-12 -11 -10 -9 -8 -7 -60

1×103

2×103

3×103

4×103

5×103

Concentration of Antibody, Log10[M]

IFN

g p

g/m

L

-12 -11 -10 -9 -8 -7 -60.0

5.0×102

1.0×103

1.5×103

2.0×103

2.5×103

Concentration of Antibody, Log10[M]

IL-2

pg

/ml

C

TNFa IFNg IL-2

B

hIgG4s Isotype

PSMAxCD28

hIgG4s Isotype

PSMAxCD28

+ hIgG4 Isotype(20nM)

+ PD-1 mAb(20nM)

Dose titration

Legend

Legend

Legend

Legend

Legend

Legend

Legend

Legend

Legend

Legend

Legend

Legend

Legend

Legend

Legend

Legend

-12 -11 -10 -9 -8 -7 -60

200

400

600

Concentration of antibody Log10[M]

IL-2

(p

g/m

L)

A

-12 -11 -10 -9 -8 -7 -60

500

1000

1500

Concentration of antibody Log10[M]

IL-2

(p

g/m

L)

Donor 2 Donor 3

Fig. S4. MUC16xCD28, but not a MUC16xCD27 T cell binding control, promotes T cell activation. A-B.

Primary T cells from donors 129 (left) and 130 (right) were incubated with HEK293 or HEK293/hCD20/hMUC16

and a titration of MUC16xCD28 (black), MUC16xCD27 (blue), or an isotype control (gray). 72 hours later, T cell

proliferation was assessed by [3H]thymidine incorporation (A) and IL-2 release (B). Data represented as the means ±

SEM. C. MUC16xCD27 and MUC16xCD28 binding to primary human T cells (left) or HEK293 and

HEK293/hCD20/hMUC16 (right) was assessed by flow cytometry of cells stained with with 20 nM or 500 nM anti-

MUC16xCD28 (blue), anti-MUC16xCD27 (black), or isotype control (gray) and secondary Alexa fluor 488 labeled

F(ab). Geometric mean fluorescence intensity ratio over the isotype staining is shown. Data shown from 2 human T

cell donors tested in 1 experiment.

-12 -11 -10 -9 -8 -7 -60

1×103

2×103

3×103

4×103

Antibody Concentration (log M)IL

-2 r

ele

ase (

pg

/ml) MUC16xCD28

MUC16xCD27

IgG4P-PVA

-12 -11 -10 -9 -8 -7 -60

1×103

2×103

3×103

4×103

5×103

Concentration of Antibody, Log10[M]

IL-2

rele

ase (

pg

/ml) MUC16xCD28

MUC16xCD27

IgG4P-PVA

-12 -11 -10 -9 -8 -7 -60

1×104

2×104

3×104

4×104

Antibody Concentration (log M)

3H

-Th

ym

idin

e In

co

rpo

rati

on

(CP

M)

MUC16xCD28

MUC16xCD27

IgG4P-PVA

-12 -11 -10 -9 -8 -7 -60

2×104

4×104

6×104

Concentration of Antibody, Log10[M]

3H

-Th

ym

idin

e In

co

rpo

rati

on

(CP

M)

MUC16xCD28

MUC16xCD27

IgG4P-PVA

C

ADonor 129 Donor 130

Donor 129 Donor 130B

50

0n

M

20

nM

50

0n

M

20

nM

50

0n

M

20

nM

50

0n

M

20

nM

50

0n

M

20

nM

50

0n

M

20

nM

0

2

4

6

Binding to T-Cell Target

Concentration of Antibody [nM]

Fo

ld B

ind

ing

ove

r Is

o

Donor 129 Donor 130

50

0n

M

20

nM

50

0n

M

20

nM

50

0n

M

20

nM

50

0n

M

20

nM

50

0n

M

20

nM

50

0n

M

20

nM

0

5

10

50

100

150

Binding to HEK293 Cells

Concentration of Antibody [nM]

Fo

ld B

ind

ing

over

Iso

MUC16xCD28

MUC16xCD27

IgG4P-PVA

HEK293 HEK293/hCD20/hMUC16

Fig. S5. SPR-Biacore sensorgrams for binding of human and murine CD28 to human and murine CD80 and

CD86. A. SPR-Biacore sensorgrams for binding of human and murine CD80 and CD86 to surface capture human

and murine CD28 at 25C and pH 7.4 on a Biacore T-200. Titration of CD28 ligands ranging from 1 nM to 500 nM

were injected in duplicate over an anti-mFc capture human CD28-mFc (429 RU) or anti-His capture murine CD28-

hFc.6xhis (290 RU) surfaces. B. Calculations of KD equilibrium fits for human CD28 (left) and murine CD28

(right) were plotted from the mean binding signal from 84 to 87 seconds as the steady-state binding response. The

steady state equilibrium dissociation constant (KD) was determined using Scrubber 2.0c, which fit the dose-

dependent binding signal to a 1:1 model using a floating RMax. Data represent at least 2 independent experiments.

Hu

man

CD

80

Mu

rin

e C

D80

Hu

man

CD

86

Mu

rin

e C

D86

Human CD28 Murine CD28

B

A

Fig. S6. PSMAxCD28 and PD-1 mAb combination increases the frequency of tumor-specific T cells. A.

MC38/PSMA tumor cells implanted in CD3/CD28/PSMA humanized mice and treated with isotype control,

PSMAxCD28, PD-1 mAb, or combination at 5 mg/kg on days 10 and 14 after implant. Spleens were harvested on

day 17. Splenocytes were cultured overnight in T cell medium with 10 g/ml peptide (p15E or OVA) and 2 g/ml

anti-CD28. After overnight incubation, intracellular cytokine staining was performed following standard procedures.

Data represented as the means ± SEM. Data are from 1 experiment. B-C. PSMAxCD28 and PSMAxCD3 induce

primary tumor clearance but not immunity to secondary tumor challenge. B. Schematic of experimental design. C.

Tumor volume over time from individual mice for indicated treatment groups. Number of tumor-free mice out of

total is indicated in the upper left corner for each data set. Data in B-C are representative of 2 independent

experiments.

Iso C

trl

PSMA

xCD28

PD-1

mAb

Combo

0.0

0.2

0.4

0.6

IFNg+ (% of CD8)

IFN

g+ (

% o

f C

D8)

No peptide

OVA

p15E

****

0 10 20 300

500

1000

1500

2000

days post implant

Tu

mo

r V

olu

me (

mm

3)

Naive

0 10 20 300

1000

2000

3000

days post implant

Tu

mo

r V

olu

me (

mm

3)

Re-Challenge

(V/T)

hCD28xhPSMA (5)

Combo (5+5)

Naive

0 10 20 300

500

1000

1500

2000

days post implantTu

mo

r V

olu

me (

mm

3)

Combo

(# tumor free/total)

0 10 20 30 400

500

1000

1500

day post implant

Tu

mo

r V

olu

me (

mm

3)

hIgG4s

0 10 20 30 400

500

1000

1500

days post implant

Tu

mo

r V

olu

me (

mm

3)

Combo(1/7) (4/7)

56%

(0/10) (0/4)

Isotype

PSMAxCD28

+ PSMAxCD3 Naive

Re-challenge

Tumor Free

Secondary

tumor

challenge

PSMAxCD28 (5mg/kg)

+ PSMAxCD3 (5mg/kg)

0 10 20

Dosing start day 0

2x/week (0, 3, 7)

Primary Tumor challenge:

MC38/hPSMA

60+

Second Tumor challenge:

MC38/hPSMA (same as primary)

B

C

A

Iso C

trl

PSMA

xCD28

PD-1

mAb

Combo

0.0

0.2

0.4

0.6

IFNg+ (% of CD8)

IFN

g+ (

% o

f C

D8)

No peptide

OVA

p15E

****

Fig. S7. PSMAxCD28 cooperates with anti–PD-1 to induce intratumoral but not splenic or systemic

cytokines. A. Ex vivo splenic and intratumoral cytokines. Points represent data from individual mice. Bar is the

average ±SEM. Data are representative of 2 independent experiments. B. CD3/CD28/PSMA triple humanized mice

were implanted with MC38/hPSMA and treated with the indicated antibody at 5 mg/kg on day 0. Blood was

collected from the submandibular vein at 4 hours after dosing for plasma cytokine analysis. Points represent data

from individual mice. Line is the average ±SEM. Statistical significance was calculated with 1-way ANOVA and

Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ****p<0.0001. Data are representative of at least 3

independent experiments.

Fig. S8. Expression of T cell activation markers used to determine CITRUS clusters shown in Fig. 3. A. Mean

fluorescence intensity (MFI) of the indicated markers on tumor CD8+ T cell clusters. Data represent the means ±

SEM. B. Scatter dot plot of CD28 and PD-1 expression on tumor CD8+ T cell clusters C1 (pink) and C2 (blue). Data

are representative of 2 independent experiments.

C1 C20

500

1000

1500

2000

2500

MF

I

PD1

C1 C2-500

0

500

1000

1500

MF

I

LAG3

C1 C20

100

200

300

400

MF

I

TIM3

C1 C20

200

400

600

800

MF

I

CD38

C1 C2-1000

-800

-600

-400

-200

0

MF

I

CD101

C1 C2-200

0

200

400

600

800

MF

I

Ki67

C1 C20

200

400

600

800

MF

I

ICOS

C1 C20

500

1000

1500

MF

I

Sca1

C1 C2-80

-60

-40

-20

0

20

MF

I

KLRG1

C1 C20

100

200

300

400

MF

I

CD122

C1 C20

100

200

300

MF

I

CD28

C1 C2-200

0

200

400

600

MF

I

CD44

C1 C20

500

1000

1500

2000

MF

I

CD62L

C1 C20

100

200

300

400

500

MF

I

TCF1

C1 C20

500

1000

1500

2000

MF

IEOMES

C1 C20

5000

10000

15000

MF

I

CD5

C1 C20

50

100

150

MF

I

CD127

A

BCD28

PD-1

C2

C1

Fig. S9. Tumor antigen-specific (p15E+) CD8 T cell CITRUS analysis. A. Histogram showing fluorescence

intensity of p15E pentamer staining on p15E+ or p15E- CD8+ T cells (concatenated data from all samples). B.

Frequency of p15E+ pentamer-positive CD8+ T cells in tumor. Data represented as the means ± SEM. C.

Frequencies of Cluster 6 (higher expression of exhaustion markers PD-1, LAG3) and Cluster 13 (higher expression

of memory-like markers Tcf-1, Eomes, CD62L). Data represented as the means ± SEM. D. tSNE and FlowSOM

clustering analysis of p15E+ CD8+ T cells overlayed with all identified clusters (top) and overlay of clusters 6 and 13

on total p15E+ cells (bottom). E. Expression of selected T cell markers differentiatly expressed on T cells from

clusters 6 and 13. F. FACS dot plot of overlayed clusters 6 and 13 showing CD28 and PD-1 expression. Data

represent 1 experiment.

0

5

10

15

% o

f cells

6

**

*

0

2

4

6

8

10

% o

f cells

13

***

*

CD28

PD-1

c6

c13

0

2

4

6

% o

f C

ells

Isotype

PD-1

EGFRxCD28

EGFRxCD28 + PD-1

% of p15E+

p15E Pentamer

A B

0

2

4

6

% o

f C

ells

Isotype

PD-1

EGFRxCD28

EGFRxCD28 + PD-1

PSMAxCD28

PSMAxCD28 + PD-1

CCluster 6 Cluster 13

D E

F

Fig. S10. EGFRxCD28 bispecific potentiates T cell activation only in the presence of TCR stimulation. A-B. Flow cytometry analysis shows that EGFRxCD28 bispecific antibody binds to CD28+ and EGFR+ cells. A. Jurkat

cells (CD28+ cells). B. PEO1 cells (EGFR+ cells). C-F. Human T cells were cultured with cancer target cells

expressing endogenous MUC16 and EGFR (ovarian cancer cell line PEO1) and the indicated bispecifics for 96

hours. C. Schematic of assay setup. D. Tumor cells, % viable cells. E. Frequency of CD25+ T cells (% of CD2+). F.

Supernatants from cytotoxicity assay were analyzed using a Cytometric Bead Array (CBA) kit. IFN release plotted

as pg/ml. Data represent 2 independent experiments.

A CD28+ cells EGFR+ cells

-13 -12 -11 -10 -9 -8 -70

20

40

60

80

100

Antibody Concentration Log10

(M)

Targ

et cells

% v

iabili

ty

MUC16xCD3 + EGFRxCD28 (2.5ug/ml)

CD3-binding control + EGFRxCD28 (2.5ug/ml)

MUC16xCD3

CD3-binding control

C

B

CD

3

CD28

EG

FR

MU

C1

6

Targetcell

T cell

Titration

MUC16xCD3 alone

MUC16xCD3+ EGFRxCD28 (2.5µg/ml)

CD

3

CD

28

EG

FR

MU

C1

6

Targetcell

T cell

Titration

Fixed 2.5µg/ml

D

-13 -12 -11 -10 -9 -8 -70

20

40

60

80

100

Antibody Concentration Log10

(M)

% C

D25 in C

D2+

MUC16xCD3 + EGFRxCD28 (2.5ug/ml)

CD3-binding control + EGFRxCD28 (2.5ug/ml)

MUC16xCD3

CD3-binding control

E

JUR

KA

T

-13 -12 -11 -10 -9 -8 -7 -60

2500

5000

7500

10000

12500

15000

Log10 [Antibody] (M)

MF

I_P

E

EGFRxCD28

Isotype ControlSecondary

Only

EGFRxCD28

Isotype Ctl

PEO

1

-13 -12 -11 -10 -9 -8 -7 -60

5000

10000

15000

20000

25000

Log10 [Antibody] (M)

MF

I_P

E

EGFRxCD28

Isotype ControlSecondary

Only

EGFRxCD28

Isotype Ctl

MUC16

xCD3

+ EG

FRxC

D28

(2.5

ug/m

l )MU

C16

xCD3

CD3-

binding

cont

rol +

EG

FRxC

D28

(2.5

ug/m

l )

CD3-

b inding

cont

rol

EGFR

xCD28

(2.5

ug/m

l )Cells

only

0

20

40

60

80

100

500100015002000

pg/m

L

IFNgF

Fig. S11. A431 human xenograft tumor model. A. Human CD45+ cell engraftment in peripheral blood before

treatment. B. A431 FACS analysis. Marker of interest is indicated for each plot. C. PSMA expression on MC38-

hPSMA (left panel) or A431 tumor cells (right panel). Data represent 2 independent experiments.

PD-L1EGFRxCD28 CD80

CD86 HLA-A,B,C HLA-DR

Isotype Control

Ab staining

B

A

C MC38-hPSMA A431

Isotype Control

Ab staining

0

20

40

60

80

100

% o

f C

ell

% of human CD45+ cells in peripheral blood

0

20

40

60

80

100

% o

f C

ell

Isotype

PD1

EGFRxCD28

EGFRxCD28 + PD1

PD-1 mAb

Isotype

PD1

PSMAxCD28

PSMAxCD28 + PD1

PD-1 mAb

PD-1 mAbPD-1 mAb

Fig. S12. MFI data for CITRUS clusters shown in Fig. 4. A. Gating strategy for tumor-infiltrating T cells. B. MFI

data for CD8+ T cell clusters. C. CD4+ T cell clusters. Data in B-C represented as the means + SEM. Data represent

2 independent experiments.

C

C1 C20

500

1000

1500

2000

CD45RA

C1 C20

1000

2000

3000

CCR7

C1 C20

2000

4000

6000

8000

PDL1

C1 C20

500

1000

1500

KI67

C1 C20

5000

10000

15000

20000

CD2

C1 C20

500

1000

1500

CD86

C1 C20

2000

4000

6000

TIGIT

C1 C20

500

1000

1500

2000

GITR

C1 C20

2000

4000

6000

ICOS

C1 C20

500

1000

1500

CD38

MF

I

B CD8+ T Cell

CD4+ T Cell

Live human CD45+ Single cells Size Mouse CD45neg

CD4+, CD8+ T CD3+ T CD14negCD19negCD4+ T

CD86

FMO

CD86

CD8+ T

CD86

FMO

CD86

C1 C2 C3 C4 C5-1000

0

1000

2000

3000

4000

CD45RA

C1 C2 C3 C4 C50

2000

4000

6000

8000

CCR7

C1 C2 C3 C4 C50

2000

4000

6000

8000

PD-L1

C1 C2 C3 C4 C50

100

200

300

400

500

Ki67

C1 C2 C3 C4 C50

5000

10000

15000

CD2

C1 C2 C3 C4 C50

200

400

600

CD86

C1 C2 C3 C4 C50

1000

2000

3000

TIGIT

C1 C2 C3 C4 C50

500

1000

1500

2000

GITR

C1 C2 C3 C4 C50

2000

4000

6000

8000

ICOS

C1 C2 C3 C4 C50

500

1000

1500

CD38

C1 C2 C3 C4 C50

500

1000

1500

2000

CD39

MF

I

A

C1 C2 C3 C4 C50

500

1000

1500

CD28 MFI

C1 C20

100

200

300

400

500

CD28 MFI

CD28

CD28

Fig. S13. PSMAxCD28 alone or in combination with PD-1 mAb does not induce cytokine in non–tumor-

bearing mice, in contrast to CD28 superagonist. CD3/CD28/PSMA triple humanized mice were treated with a

single dose of 2.5 mg/kg each antibody as indicated. Blood was collected from the submandibular vein for plasma

cytokine analysis at 4 hours (day 0) (A) and 72 hours (day 3) (B) post-dose. Symbols represents individual mice. n =

5 mice per group. Lines represent the means ± SEM. Statistical significance was calculated with 1-way ANOVA and

Holm-Sidak’s multiple comparisons test. *p<0.05, **p<0.01, ****p<0.0001. Data represent at least 3 experiments.

Cy

tok

ine

(p

g/m

l)C

yto

kin

e (

pg

/ml)

Cy

tok

ine

(p

g/m

l)

4h (day 0)A

72h (day 3)B

0

20

40

60

IL-10

0

2

4

6

IL-1b

0

500

1000

1500

2000

2500

KC/GRO

0

200

400

600

IL-5

*

0

5

10

15

IL-4

*

0

1

2

3

4

IFNg

0

20

40

60

80

100

IL-10*

0

2

4

6

IL-1b

0

2

4

6

8

10

IL-2

*

0

2

4

6

8

10

IL-4

*

0

500

1000

1500

IL-5

*

0

10

20

30

40

IL-6

*

0

100

200

300

KC/GRO

0

5

10

15

20

25

TNFa

*

Iso Ctrl

PD-1 mAb

PSMAxCD28

PSMAxCD28 + PD-1 mAb

CD28 mAb

CD28 SA

Iso Ctrl

PD-1 mAb

PSMAxCD28

PSMAxCD28 + PD-1 mAb

CD28 mAb

CD28 SA

Fig. S14. EGFRxCD28 alone or in combination with PD-1 mAb does not induce cytokines in human immune

cell-engrafted STRG mice, in contrast to CD28 superagonist. A-B. Human immune cell-engfrafted STRG mice

were dosed with the indicated antibodies and blood was collected from the submandibular vein at 4 hours post-dose

for plasma cytokine analysis. A. Data correspond with mice implanted with A431 as shown in Fig. 4A. Symbols

represent individual mice. n = 10 mice per group. Lines represent the means ± SEM. Data represent 2

experiments.B. Non-tumor-bearing mice. Symbols represent individual mice. n = 5 mice per group. Lines represent

the means ± SEM. Statistical significance was calculated with an unpaired t test (ns, not significant; *p<0.05,

**p<0.01). Data represent 3 experiments.

Iso Ctrl CD28 Superagonist0

10

20

30

40

Cyto

kin

e (

pg

/ml)

TNF-α

**

Iso Ctrl CD28 Superagonist0

5

10

15

Cyto

kin

e (

pg

/ml)

IL-8

ns

Iso Ctrl CD28 Superagonist0

5

10

15

Cyto

kin

e (

pg

/ml)

IL-6

ns

Iso Ctrl CD28 Superagonist0.0

0.1

0.2

0.3

Cyto

kin

e (

pg

/ml)

IL-4

*

Iso Ctrl CD28 Superagonist0

50

100

150

Cy

tok

ine

(p

g/m

l)

IL-2

**

Iso Ctrl CD28 Superagonist0

10

20

30

40

Cyto

kin

e (

pg

/ml)

IL-10

ns

Iso Ctrl CD28 Superagonist0

500

1000

1500

2000

2500

Cy

tok

ine

(p

g/m

l)

IFNg

*

0

10

20

30

40

pg

/m

TNF-a

0

50

100

150

pg

/m

IL-2

0.0

0.1

0.2

0.3

pg

/m

IL-4

0

100

200

300

pg

/m

IL-8

0

20

40

60

pg

/m

IL-6

Isotype

PD-1

EGFRxCD28

EGFRxCD28 + PD-1

0

500

1000

1500

2000

2500

pg

/mL

IFN-g

0

10

20

30

40

pg

/m

IL-10

A

B

Table S1. SPR-Biacore kinetics.

Binding kinetic parameters for antigen binding to their specific antibody

at 37oC

Capture Surface Test ligand Surface density of antibody captured

(RU) Antigen

bound (RU) ka (M-1

s-1

) kd (s-1

) KD (M) t½ (min) EGFRxCD28 bispecific

antibodya hEGFR.mmh 246 ± 1.0 73 4.41x 10

5 2.72x 10-3 9.59x 10

-9 4.2

PD-1 Non blocker (hIgG4)a hPD-1.mmh 343 ± 2.0 9 3.41 x 10

3 3.03 x 10-2 8.86 x 10

-6 0.4 hPD-1.mFc 346 ± 6.7 10 1.91 x 10

4 1.30 x 10-3 6.81 x 10

-8 8.9

PD-L1 Blocker (hIgG1)a hPD-L1.mmh 186 ± 2.2 62 1.05 x 10

6 2.48 x 10-4 2.37 x 10

-10 46.5 hPD-L1.mFc 174 ± 2.8 116 2.36 x 10

6 4.21 x 10-5 1.78 x 10

-11 274.6 PD-L1 Blocker (hIgG4)

a hPD-L1.mmh 176 ± 1.6 62 1.22 x 106 2.62 x 10

-4 2.14 x 10-10 44.2

hPD-L1.mFc 186 ± 1.2 130 2.86 x 106 3.94 x 10

-5 1.38 x 10-11 293.2

PD-L1 Non blocker (hIgG1)a hPD-L1.mmh 172 ± 1.7 36 2.37 x 10

5 2.28 x 10-4 9.63 x 10

-10 50.6 hPD-L1.mFc 193 ± 2.6 67 3.68 x 10

5 1.57 x 10-5 4.27 x 10

-11 734.6

Binding kinetic parameters for EGFRxCD28 bispecific antibody binding to

captured hCD28.mFc at 37oC

Capture Surface Test ligand Surface density of antigen captured

(RU) Antibody

bound (RU) ka (M-1

s-1

) kd (s-1

) KD (M) t½ (min)

hCD28.mFcb EGFRxCD28 bispecific

antibody 40 ± 0.2 25 2.54x 105 1.32x 10

-2 5.17x 10-10 0.9

aAntibody was captured on a anti-human Fc mAb-coupled sensor surface and different concentrations of specific antigen were injected.

bhCD28.mFc was captured surface on a anti-mouse Fc antibody-coupled sensor surface and different concentrations of EGFRxCD28 bispecific antibody were injected.

Table S2. Measuring binding kinetics of human and mouse CD28/CD80/CD86 interactions

using SPR. Equilibrium binding constant (KD) values at 25° C

Captured Surface

Human CD80-hFc

Human CD86-hFc

Mouse CD80-hFc

Mouse CD86-hFc

hCD28-mFc

990 ± 30 nM

3900 ± 300 nM

51 ± 1 nM

470 ± 10 nM

mCD28-hFc.6xhis

143 ± 3 nM

298 ± 6 nM

24 ± 0.5 nM

99 ± 2 nM

Table S3. Histopathology, organ weights, body weight, and clinical pathology findings in

cynomolgus monkeys. Endpoints Evaluated CD28 SA

(fold change)

PSMAxCD28 PSMAxCD28

+

PD-1 mAb

EGFRxCD28 EGFRxCD28

+

PD-1 mAb

Histopatholgy:

Tissue examined: Aorta, Bone

marrow (femur and sternum),

Brain, Epididymis, Esophagus,

Eye, Gallbladder, Glands

(adrenal, mammary,

parathyroid, pituitary, prostate,

salivary, seminal vesicle, and

thyroid), GALT, Heart, Kidney,

Large intestines (cecum, colon,

rectum), Liver, Lung, Lymph

nodes (mandibular, and

mesenteric), Muscle, Nerves

(optic and sciatic), Pancreas,

Site of injection, Skin, Small

intestines (duodenum, ileum,

jejunum), Spinal cord, Spleen,

Stomach, Testis, Thymus,

Tongue, Trachea and Urinary

bladder

Mononuclear

cell

infiltration

(perivascular,

minimal to

mild)

observed in

the following

tissues: Brain,

Gall bladder,

Liver, Kidney,

Sciatic nerve

and Seminal

vesicle

No significant

lesions

No

significant

lesions

No significant

lesions

No significant

lesions

Body Weight No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

Organ Weights

Adrenal, Brain, Heart, Kidneys,

Liver, Ovariesa, Spleen,

Thymus, Thyroid, Uterusa,

Pituitaryb, Prostateb,

Epididymisb, Testisb, Lung b

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

Urinalysis

Bilirubin, Clarity, Color,

Glucose, Ketones, Leukocytesa,

Nitritesa, Occult blood, pH,

Protein, Specific gravity,

Urobilinogen, and Volume

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

Coagulation

Activated partial

thromboplastin time (APTT),

Fibrinogen, and Prothrombin

time

↑APTT (+2

Seconds)d

↑ Fibrinogen

(1.6)d

No treatment

related

changes

observed

↑Fibrinogene No treatment

related

changes

observed

No treatment

related

changes

observed

Hematology

Hematocrit, Hemoglobin, Mean

corpuscular hemoglobin, Mean

corpuscular hemoglobin

concentration, Mean

corpuscular volume, Mean

platelet volume, Platelets, Red

blood cells, Red cell

distribution width (RDW),

Reticulocyte absolute count,

Reticulocyte percent, White

blood cells, and Differential

leukocyte absolute count:

Basophils, Eosinophils,

Lymphocytes, Monocytes, and

Neutrophils, Large unstained

cells (LAC)

↑lymphocytes

(1.7)c

↑ Basophils

(2.9)c

↑ LAC (4.1)c

↑ RDW (1.2) c

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

Serum Chemistry

Alanine Aminotransferase,

Albumin, Alkaline

Phosphatase, Aspartate

Aminotransferase, Blood Urea

Nitrogen, Calcium, Chloride,

Creatine Kinase, Creatinine,

Gamma Glutamyltransferase,

Globulin, Glucose, Inorganic

Phosphorus, Potassium,

Sodium, Total Bilirubin, Total

Cholesterol, Total Protein,

Triglyceride, C-Reactive

Protein (CRP), and Lactate

Dehydrogenase

↓albumin

(0.9) c

↑Globulin

(1.2) c

↑ CRP (11.4) d

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

No treatment

related

changes

observed

a Evaluated only in groups administered EGFRxCD28 and EGFRxCD28 + PD-1 mAb b Organ weight evaluated only in groups administered CD28 SA, PSMAxCD28 and PSMAxCD28 + PD-1 mAb

Fold change was determined by comparing the group mean value to the respective pretreatment value.

Change in APTT represents the actual difference from the pretreatment value in seconds c Change noted on day 15 d Change noted on day 2 e Change noted in one animal