**IMMUNOLOGY**
# High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics
1† 1† 2 1 2 3 4,5
**Yuan Wang, Ruina Jin, Bingqing Shen, Na Li, He Zhou, Wei Wang, Yingjie Zhao,**
**Mengshi Huang², Pan Fang², Shanshan Wang², Pascaline Mary², Ruikun Wang¹, Peixiang Ma³,**
4,5 1,3
**Ruonan Li¹, Yujie Tian¹, Youjia Cao¹, Fubin Li, Liang Schweizer², Hongkai Zhang ***
**Currently, high-throughput approaches are lacking in the isolation of antibodies with functional readouts beyond**
**simple binding. This situation has impeded the next generation of cancer immunotherapeutics, such as bispecific**
**T cell engager (BiTE) antibodies or agonist antibodies against costimulatory receptors, from reaching their full**
**potential. Here, we developed a highly efficient droplet-based microfluidic platform combining a lentivirus trans-**
**duction system that enables functional screening of millions of antibodies to identify potential hits with**
**desired functionalities. To showcase the capacity of this system, functional antibodies for CD40 agonism**
with low frequency (<0.02%) were identified with two rounds of screening. Furthermore, the versatility of
**the system was demonstrated by combining an anti-Her2 × anti-CD3 BiTE antibody library with functional**
**screening, which enabled efficient identification of active anti-Her2 × anti-CD3 BiTE antibodies. The plat-**
**form could revolutionize next-generation cancer immunotherapy drug development and advance medi-**
**cal research.**
Copyright © 2021
The Authors, some
rights reserved;
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American Association
for the Advancement
of Science. No claim to
original U.S. Government
Works. Distributed
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License 4.0 (CC BY-NC).
## INTRODUCTION
Cancer immunotherapies that harness the power of the immune
system to treat tumors have brought great progress and driven a
major paradigm shift in cancer treatment. The first generations of
cancer immunotherapy agents consist primarily of antagonist antibodies that block negative immune checkpoints, such as programmed
cell death protein 1 (PD-1) (*1*–*3*). Recently, next-generation cancer
immunotherapies, including bi/multispecific antibodies, such as
bispecific T cell engager (BiTE) antibodies and agonist antibodies
of costimulatory receptors, have been claiming the spotlight, showing
promise in triggering anticancer immunity (*4*–*7*).
In vitro display technology, such as phage display, allows the
selection of antibody binders from large combinatorial libraries
11
with a capacity of 10 diversities (*8*–*13*). This has been one of
the major technologies in conventional antibody drug development through the use of high- or low-throughput simple binding assays.
$$
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$$
Next-generation cancer immunotherapies, including agonist
or bi/multispecific antibodies, have yet to fulfill their full potential. This may be, at least in part, due to the low throughput of
functional screens (maximally, a few thousand antibodies can be
tested), representing a bottleneck that requires prescreens to narrow down potential candidates and may cause loss of certain target profiles, depending on the criteria set for the prescreens. In
addition, bi/multispecific antibody development may also suffer
from a lack of diversity driven by limited candidate generations
using validated monospecific antibodies with known bispecific
scaffolds.
Droplet microfluidic technology allows analysis and screening
of cells at the single-cell level with unprecedented throughput,
something that cannot be obtained using bulk population-based
assays. The microfluidic droplet system can encapsulate single
cells in water-in-oil droplets at a rate of thousands of droplets per
second (*14*). Antibodies generated by the cells are contained in the
droplet, enabling the maintenance of phenotype and genotype linkage within the droplet (*15*–*17*). Last, droplets containing desirable
cells are sorted by fluorescence-activated droplet sorting (FADS).
El Debs *et al*. and Shembekar *et al*. (*18*, *19*) described application
of the microfluidic droplet system for screening hundreds of thousands
of hybridoma cells for antibodies that inhibit enzyme angiotensin-converting enzyme 1 (ACE-1) or bind to target cells (*18*, *19*).
With the sophistication of the microfluidic droplet system, simultaneous analyses of millions of individual antibody-secreting
plasma cells for their antibody secretion rate and affinity can be
achieved (*20*). Millions of plasma cells can be screened for antibodies bound to vaccines or cancer targets (*21*). However, improvement is urgently needed to screen for functional antibodies.
Here, we developed an efficient technology platform to simultaneously screen for the binding and agonistic activity of antibodies
or the functions of bispecific antibodies. This approach combines
the strength of an autocrine-based lentiviral transduction system
with the microfluidic droplet system (*22*–*26*). The technical capabilities of the platform were demonstrated by the successful identification of rare potent costimulatory receptor CD40 agonist antibodies
and active anti-Her2 × anti-CD3 bispecific antibodies from a combinatorial antibody library. This streamlined technology enables
efficient and unbiased discovery of active antibodies with markedly
increased throughput and accuracy to serve as the next generation
of cancer immunotherapy.
1
State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences,
2
Nankai University, Tianjin, China. HiFiBiO (Shanghai) Co. Ltd., Shanghai, China.
3
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech Univer-
4
sity, Shanghai, China. Shanghai Institute of Immunology, Faculty of Basic Medicine,
5
Shanghai Jiao Tong University School of Medicine, Shanghai, China. Key Laboratory
of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai
Jiao Tong University School of Medicine, Shanghai, China.
*Corresponding author. Email: hongkai@nankai.edu.cn
†These authors contributed equally to this work.
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## RESULTS
## Outline of function-based antibody selection using droplet-based microfluidics
We designed the functional antibody selection platform as depicted
in Fig. 1. Cells were infected by a lentiviral antibody-expressing
library at a multiplicity of infections (MOI) equal to 1 so that each cell
expressed and secreted only one type of antibody. For microfluidicbased screening, individual antibody-secreting cells were coencapsulated with a reporter cell into a droplet by a microfluidic drop
maker. The resulting emulsion was incubated off-chip overnight
and injected into the sorting chip. Droplets containing activated
reporter cells were sorted by FADS. Subsequently, cells were recovered from the droplets, and functional antibodies were identified by
sequencing the antibody genes in the sorted cells.
## Development and optimization of droplet-based assay
Two microfluidic devices were used (i) to compartmentalize the
lentivirus-infected cells with reporter cells and detection reagents
(Fig. 2, A and B) and (ii) to sort droplets based on reporter cell activation and receptor binding signals using a surface acoustic wave–
based sorter (Fig. 2C) (*27*). The throughput of the microfluidic
device for droplet formation and sorting is on the order of millions
of droplets within hours per instrument.
Droplets generated by the microfluidic chip were incubated for
24 hours at 37°C. No merging of droplets was observed, indicating
that the droplets were stable (Fig. 2B). Next, we used an anti-
Her2 × anti-CD3 BiTE antibody as an example for the development and
optimization of the droplet-based assay. The anti-Her2 × anti-CD3
BiTE positive control was engineered to recognize Her2 on tumor
cells and CD3 on T cells using the variable domain sequences of trastuzumab and blinatumomab, respectively (*28*). Her2-overexpressing
$$
37^{\circ}\mathrm{C}
$$
K562-Her2 cells were infected with the anti-Her2 × anti-CD3 BiTE
positive control lentivirus at a low MOI, resulting in less than 10%
of cells being infected. K562-Her2 played a dual role in expressing
antibodies and providing Her2-mediated cross-linking of the secreted antibodies. Each infected K562-Her2 cell was encapsulated
with a Jurkat/pIL2–enhanced green fluorescent protein (eGFP) reporter cell (fig. S1, A and B) with 0.5 antibody-secreting cells and 1
reporter cell per droplet on average. The number of cells per droplet
was analyzed on the basis of the fluorescent signal from the
antibody-secreting cell labeled with CellTrace Violet dye and the
reporter cell labeled with CellTrace Yellow dye. The results were in
good agreement with the theoretical double Poisson distribution,
with 4524 droplets analyzed (fig. S2). After 16 hours of incubation,
the droplets were reinjected into the sort chip to analyze the activation of reporter cells in the droplet, and approximately 9.5% of the
reporter cells were activated. In contrast, when the anti-Her2 × anti-CD3
lentivirus-infected K562-Her2 cells were cocultured with reporter cells
in the plate well, 72.7% of the reporter cells were activated after 16 hours
of incubation (Fig. 3A). The divergence of reporter cell activation
in the plate well and droplets demonstrates that antibodies secreted by
cells in the droplet cannot diffuse between different droplets; thus, the
system is suitable for screening a library. In addition, NucGreen
Dead 488 was used to track the cell viability in droplets. Both K562-
Her2 and Jurkat/pIL2-eGFP cells in the droplets exhibited a high viability of approximately 90% after 16 hours of incubation (fig. S3).
## Screening anti-Her2 × anti-CD3 bispecific antibody using the function-based screening method
Because phage display technology can be used to screen an antibody
library with much larger diversity than is possible in eukaryotic systems, we first selected Her2-binding antibodies from an antibody
**Fig. 1. Schematic overview of functional antibody screening using a droplet-based microfluidic system.** The antibody genes were cloned into lentiviral vectors.
Eukaryotic cells were infected with the lentiviral antibody library, and individual transduced cells were coencapsulated with the reporter cell into droplets using a microfluidic system. The resulting emulsion was incubated off-chip overnight and injected into the sorting chip. Droplets containing antibody-secreting cells and activated
reporter cells were sorted. The sorted cells were cultured for the next round of selection. After multiple rounds of iteration, antibody genes were amplified from the
sorted cells and analyzed by Sanger sequencing or next-generation sequencing. The enriched antibodies were synthesized, and recombinant antibodies were expressed
and tested for function. PMTs, photomultiplier tubes. IDT, interdigital transducer.
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**A**
**Fig. 2. Microfluidic chips.** (**A**) The droplet generation chip is used to generate droplets to coencapsulate antibody-secreting cells with reporter cells. (**B**) Image of generated droplets. (**C**) The sorting chip is used to collect droplets based on the intensity of fluorescence. The functions of various inlets are indicated, and images of outlets for
droplet generation and sorting are shown.
phage display library. We panned a naïve human single-chain Fv
10
(scFv) library with a diversity of 10 members against the Her2
protein. One round of panning was performed, and the scFv genes
in phagemid were subcloned into a lentiviral vector that contained
a fixed blinatumomab-derived anti-CD3 scFv gene to express anti-
Her2 × antib-CD3 BiTE antibodies. The size of the combinatorial
5
bispecific antibody library was approximately 10 members.
$$
10^{10}
$$
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10^{5}
$$
K562-Her2 cells were infected with the BiTE antibody lentivirus
library at low MOI to ensure that most cells were infected with only
one virus and thus produced a single type of bispecific antibody.
Before cell encapsulation, K562-Her2 cells and Jurkat/pIL2-eGFP
cells were stained with CellTrace Violet and Yellow dyes, respectively.
The infected K562-Her2 cells were individually coencapsulated
with Jurkat/pIL2-eGFP reporter cells into droplets, which were
sorted after 16 hours of incubation (fig. S4). We first selected droplets for the presence of K562-Her2 and Jurkat/pIL2-eGFP cells based
on the cell staining fluorescence signals. Then, we selected droplets
if the reporter cell inside was activated on the basis of the GFP signal. Last, we selected droplets if the GFP signal was colocalized with
the reporter cell staining signal. Eleven million droplets were imaged,
approximately 0.26% of the droplets were sorted, and an image of
representative sorted droplets is shown in Fig. 3B. The anti-Her2
scFv genes were amplified from the sorted cells and cloned into a
mammalian expression plasmid. Twenty clones were selected for
Sanger sequencing, and five bispecific antibodies appeared more
than once. The purified bispecific antibodies were added to Jurkat/
pIL2-eGFP reporter cells in coculture with K562-Her2. Three of the
five antibodies (BiTE1, BiTE2, and BiTE3) activated the reporter
cell in the presence of K562-Her2 (Fig. 3C). To assess the antitumor
activity of BiTE1, we performed in vitro cytotoxicity assays by
coculturing peripheral blood mononuclear cells (PBMCs) and
HER2-expressing SK-BR-3 breast cancer cells. The results showed
that lysis of SK-BR-3 cells only occurred when they were incubated
with BiTE1, not with the control antibody anti-CD3 × anti-HEL
(hen egg lysozyme) (Fig. 3D). Flow cytometry analysis of T cells in
the coculture system showed that BiTE1 stimulated the expression
of the activation marker CD69 on T cells (Fig. 3E). Moreover, BiTE1
induced dose-dependent increases in interferon-g (IFN-g) and
interleukin-2 (IL-2) in the supernatants (Fig. 3, F and G). Overall,
the results demonstrate that this technology allows combinatorial
screening and profiling of large numbers of bispecific antibodies.
## Discrimination of the strong and weak hits by the method
To validate the potential of the method to discriminate between
strong and weak hits, trastuzumab-derived anti-Her2 × anti-CD3
positive control, BiTE1, and BiTE3 in descending order of potency
were each cloned into lentiviruses. Cells infected with each of these
bispecific antibodies encoding lentivirus were coencapsulated with the
reporter cell, and droplets were analyzed for the activation of the reporter cell. The order of droplet intensity was as follows: trastuzumabderived anti-Her2 × anti-CD3 positive control > BiTE1 ≈ BiTE3
(fig. S5). This work demonstrates the capability of the technology to
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**A**
**B**
**CD**
**F**
**EG**
**Fig. 3. Screening anti-Her2 × anti-CD3 BiTE antibody from a bispecific antibody library.** (**A**) Activation of reporter cells in plate well–based or droplet-based cocultures. K562-Her2 cells expressing the anti-Her2 × anti-CD3 BiTE positive control were cocultured with Jurkat/pIL2-eGFP reporter cells in plate wells or individually coencapsulated with reporter cells. The activation of reporter cells under both conditions was compared. (**B**) Representative images of a sorted droplet. Antibody-secreting
K562-Her2 cells and reporter cells were stained with CellTrace Violet and CellTrace Yellow dyes, respectively. K562-Her2 cells were individually coencapsulated with the
reporter cell, and droplets containing activated reporter cells were sorted. (**C**) Activation of reporter cells by the identified antibodies. K562-Her2 cells were cocultured
with reporter cells in the presence of the identified antibodies. Expression of GFP by the reporter cell was analyzed. (**D** to **G**) PBMC and tumor cell coculture assay. PBMCs
and SK-BR-3 cells were cocultured in the presence of BiTE1 or control antibody. Tumor cell lysis was determined by measuring the release of lactic acid dehydrogenase
(LDH) from tumor cells. (D) Expression of the activator marker CD69 on T cells was investigated (E), and levels of cell-secreted IFN-g (F) and IL-2 (G) in the culture supernatant were measured by ELISA.
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discriminate between weak and strong hits, which enables fast enrichment of high-potency hits using a more stringent gating strategy.
## Proof-of-concept screening of CD40 agonists from a spike-in library
To further validate the utility of the function-based screening method,
we sought to identify CD40 agonistic antibodies. CD40 is a promising drug target for cancer immune therapy (*5*). Activation of
CD40 on antigen-presenting cells (APCs) results in improved antigen processing and presentation and cytokine release, which enhances the T cell response.
Human Jurkat T cells were engineered to express human CD40
and GFP controlled by nuclear factor kB (NF-kB) response elements. The reporter cell line expressed GFP when CD40 was activated (fig. S6). Human embryonic kidney (HEK) 293FT cells
coexpressing red fluorescence protein (RFP) and the hexameric form
of CD40L were used as positive controls (CD40L cells). HEK293FT
cells coexpressing blue fluorescence protein (BFP) and HEL antibodies were used as negative controls (HEL cells). CD40L or HEL
cells were coencapsulated with Jurkat/NF-kB-GFP-hCD40 reporter
cells into droplets. After 16 hours of incubation, 24% of the droplets
containing the CD40L cell and reporter cell exhibited a GFP fluorescence signal, while the HEL cell and reporter cell coencapsulating
droplets exhibited a clean background of activation of the reporter
cell (0.5%) (fig. S7).
Next, CD40L cells were spiked into a 10-fold excess of HEL cells.
The mixture of CD40L cells and HEL cells was coencapsulated with
Jurkat/NF-kB-GFP-hCD40 reporter cells with 0.5 protein-secreting
cells per droplet. The droplets that contained activated reporter
cells were sorted on the basis of the green fluorescence. Before sorting, only 1.24% of the mixed cell population were RFP positive,
16.79% of cells were BFP positive, and the remaining droplets were
empty or contained only reporter cells; whereas after sorting, the
percentage of droplets containing RFP-positive CD40L cells increased
Downloaded from https://www.science.org on February 01, 2023 to 51.94%, where 49.24% were RFP positive and 2.70% were RFP
and BFP double positive (Fig. 4A). We also observed that most
droplets contained both RFP-positive CD40L cells and GFP-positive
reporter cells after sorting (Fig. 4B).
## Screening CD40 agonist antibody using the function-based screening method
We first selected CD40-binding antibodies from an antibody library
using phage display technology. The scFv genes in phagemid were
subcloned into a lentiviral vector that contained the immunoglobulin G1 (IgG1) Fc gene to express scFv Fc fusion proteins. The li-
5
brary size was approximately 10. HEK293FT cells were infected
with a lentivirus library at low MOI to ensure that most cells were
infected with only one virus and produced one type of monoclonal
antibody.
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$$
To screen CD40 agonist antibodies, antibody-producing cells were
coencapsulated with CellTrace Yellow–prestained Jurkat/NF-kB-
GFP-hCD40 reporter cells and DyLight 650–conjugated secondary
antibodies in droplets. The reporter cells were also coencapsulated
with soluble hexameric CD40L protein and anti-HEL antibody as
positive and negative controls, respectively. Droplets of different
populations (positive control, negative control, and the screening
population) were coded by adding different concentrations of
DY405. After 16 hours of incubation, the droplets were sorted on
the basis of the following criteria (fig. S8). The droplets containing
the screening population were gated on the basis of the intensity of
DY405. The CellTrace Yellow fluorescence signal showed the
presence of reporter cells in the droplet. The DyLight 650 fluorescence signal indicated binding of the secreting antibodies to CD40
on the surface of the reporter cell, and the GFP fluorescence signal
indicated activation of the reporter cell (fig. S8). The droplets displayed different patterns of fluorescence signals (Fig. 5A and fig.
S9). Thirteen million and 4.8 million droplets were imaged for the
first and the second round of screening, respectively. Whereas only
0.29% of the droplets were DyLight 650 and GFP double positive
for the first round of screening (among the droplets containing reporter cell signal), this value increased to 12.5% for the second
round of screening (Fig. 5B). Droplets were imaged after sorting,
and the number of DyLight 650 and GFP double-positive droplets was markedly increased (Fig. 5C).
The scFv genes were amplified from the cells after each round of
sorting and subjected to third-generation sequencing. Circular
consensus sequences (CCSs) from these reads were generated and
filtered by quality, and full-length scFvs were identified in 44,084
reads. Considering the errors introduced by polymerase chain reaction (PCR) or sequencing, similar (at 95% similarity) scFv sequences
were grouped into 561 scFv clusters, and consensus scFv sequences
of each cluster were created. Comparison of the sequence frequency
in each round revealed that some scFvs were enriched, while some
scFvs were eliminated during selection (Fig. 5D). The frequency of
scFv clusters C01, C03, C04, C05, and C06 was low before sorting
and showed a roundwise increase during the selection process. In
contrast, scFv cluster C02 was highly abundant before sorting, while
its frequency was substantially reduced after selection (Fig. 5E).
**A**
**Fig. 4. Screening CD40 agonist from a spike-in library using a microfluidic system.** (**A**) RFP-positive hexameric CD40L protein–secreting cells were spiked into a
10-fold excess of BFP-positive anti-HEL antibody–secreting cells, and the mixture of cells was coencapsulated with the reporter cells. After incubation, droplets containing
activated reporter cells were sorted. The proportion of droplets containing RFP- or BFP-positive cells before and after sorting was analyzed. (**B**) Bright-field and fluorescence images of droplets before and after sorting.
Downloaded from https://www.science.org on February 01, 2023
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**A**
Secreted antibody by infected HEK293FT
Rabbit anti-human IgG Fc DL650 Infected HEK293FT
Jurkat/NF-κB-GFP-hCD40 Drop code: DY405
With reporter cell
and
binding and
agonistic antibody
**B**
**C**
**D**
## Round Round Round E F
**E**
**Fig. 5. Screening CD40 agonist antibody from a monoclonal antibody library.** HEK293FT cells were infected with a lentivirus antibody library and individually coencapsulated with Jurkat/NF-kB-GFP-hCD40 reporter cells and fluorescence-labeled secondary antibodies in droplets. Droplets containing reporter cells activated by antibodies secreted by the coencapsulated antibody–expressing cells were sorted. The sorted cells were expanded for the second round of selection, and enriched
antibodies were identified by next-generation sequencing. (**A**) Schematic of possible time traces. (**B**) Proportions of different types of droplets for each round of selection
were analyzed. (**C**) Bright-field and fluorescence images of the sorted droplets after the second round of selection. (**D**) Bar plot for the top 20 scFv clusters and their frequencies during the selection process. (**E**) The change in frequencies of the selected antibodies during the selection process. (**F**) Agonist activity of the selected antibodies
was determined using the CD40 reporter cell line in the presence or absence of the cross-linking secondary antibody.
The full-length IgG C01, C02, C03, C04, C05, and C06 genes
were synthesized, and recombinant antibodies were expressed and
purified. The activity of these antibodies was tested using the
Jurkat/NF-kB-GFP-hCD40 reporter cell line. Reporter cells were
stimulated with different concentrations of antibody in the presence
or absence of the cross-linking secondary antibody. Antibodies C01,
C03, C04, C05, and C06 activated the reporter cell line. The activity
of C01, C03, and C05 was independent of cross-linking, while C04
and C06 activated the reporter cells in a cross-linking–dependent
manner (Fig. 5F). We compared the signal intensities between Fc-dependent C04 and Fc-independent C01 in the droplet-based assay,
and the results supported the differential Fc dependency of these hits
(fig. S10). The activity of CD40 agonist antibodies independent
of Fc receptor–mediated cross-linking might be disconcerting
due to the potential for adverse events (*29*, *30*). Therefore, the
crosslinking–dependent C04 antibody was chosen for further
## Jurkat/NF-kB-GFP-hCD40 reporter cell line. Reporter cells were cross-
characterization.
Flow cytometry and surface plasmon resonance (SPR) results
revealed that the C04 antibody bound to human, rhesus macaque,
and cynomolgus monkey CD40 with similar affinity (fig. S11, A and
B). In addition, enzyme-linked immunosorbent assay (ELISA)
results confirmed that antibody C04 specifically bound to CD40
rather than other tumor necrosis factor (TNF) receptor superfamily
members, such as TNF receptor superfamily member 18 (GITR), TNF
Downloaded from https://www.science.org on February 01, 2023 receptor superfamily member 4 (OX40), or TNF receptor superfamily
member 9 (4-1BB) (fig. S11C).
Given that FcgRIIB expression on tumor-infiltrating myeloid
cells is required for the agonistic activity of the cross-linking–
dependent CD40 agonist antibodies, the FcgRIIB dependency of
antibody C04 was confirmed. The Jurkat/NF-kB-GFP-hCD40 reporter
can be stimulated by C04 in the presence of FcgRIIB-overexpressing
cells. Results showed that the agonism of C04 was FcgRIIb dependent (Fig. 6A).
To investigate whether C04 promotes the activation of APCs, we
stimulated human dendritic cells (DCs) and B cells isolated from
PBMCs with C04 in the presence or absence of the cross-linking
antibody. Flow cytometry analysis showed that C04 up-regulated
the expression of the activation marker CD86 on DCs and B cells
and that anti–Fc-mediated cross-linking further enhanced the maximum response of the antibody (Fig. 6B).
The immunostimulatory activity of antibody C04 was further
+
assessed by the ovalbumin (OVA)–specific CD8 T cell response model
in FcgR/CD40-humanized mice. Mice treated with the CD40 agonist
antibody exhibited an increased number and percentage of OT-I cells
+
among CD8 T cells compared to mice treated with the isotype antibody, demonstrating that C04 had a robust adjuvant effect (Fig. 6C).
$$
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$$
In addition, the antitumor efficacy of C04 was assessed in syngeneic tumor models. When MC38 tumors were established (~100 mm³),
mice were treated with C04, CP-870,893 (Pfizer), or anti-HEL antibodies. C04 displayed comparable antitumor activity to CP-870,893,
which is the most potent CD40 agonistic antibody among those
studied in clinical trials. In addition, treatment with C04 caused
**A**
**C**
**B**
**D**
**Fig. 6. Characterization of the identified CD40 agonist antibody.** (**A**) The FcgRIIB dependency of antibody C04. Jurkat/NF-kB-GFP-hCD40 reporter cells were stimulated with C04 antibody or anti-HEL antibody in coculture with FcgRIIB-overexpressing HEK293FT cells. Activation of the reporter cell line was analyzed by flow cytometry.
(**B**) Activation of DCs or B cells by C04. DCs or B cells isolated from a donor were stimulated by C04 in the presence (left) or absence (right) of the cross-linking secondary
+
antibody. Expression of CD86 was analyzed by flow cytometry. (**C**) OVA-specific CD8 T cell response induced by C04 in FcgR/CD40-humanized mice. Transgenic mice
were adoptively transferred with OVA-specific OT-I cells and treated with DEC-OVA, together with C04 or isotype control antibody. Mice were euthanized for the analysis
of T cells. Each circle represents an individual mouse. (**D**) Antitumor effect of C04 in the syngeneic mouse model. FcgR/CD40-humanized mice were subcutaneously engrafted with MC38 tumor cells. When MC38 tumors were established (~100 mm³), mice were treated with C04, CP-870,893, or isotype control antibody. Tumor volume
and body weight were measured every 3 days until the end of the experiment. Data are represented as the means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001.
$$
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Downloaded from https://www.science.org on February 01, 2023 relatively less body weight reduction than CP-870,893 treatment,
suggesting that C04 has a favorable toxicity profile (Fig. 6D).
## DISCUSSION
Here, we describe a droplet-based microfluidic platform for the
functional screening of millions of antibodies. The platform shares
some key features with the most efficient selection methods, such as
phage display (*8*–*13*). First, the genotype and phenotype linkage
was maintained throughout the whole process. Second, the product
from one round can be directly amplified and used as the input in
the next round of selection. Thus, multiple rounds of iteration allow
the enrichment of rare hits. Compared to the conventional method
of individually expressing and assaying thousands of antibodies, the
throughput of this platform increased that limit to 10 million. This
is especially useful for the development of next-generation cancer
immunotherapies, such as agonist antibodies or bispecific antibodies, when simple binding assays may be inadequate. To demonstrate
the usefulness of this platform, we first applied it to discover
bispecific antibodies and agonist antibodies, whose development
was limited by low-diversity and/or low-throughput and potentially
biased screening.
Bispecific T or natural killer (NK) cell engagers (BiTEs or BiKEs)
hold great promise for cancer treatment, and a growing number of
BiTEs and BiKEs are making their way through various stages of
development (*6*). To identify the optimal BiTE or BiKE, we constructed a bispecific antibody library to address the complexity of
the array of tumor antigen–targeting antibodies. The large number
of bispecific antibodies in a given library can exceed the throughput
of existing methods. The described approach provides significant
opportunities to screen unprecedented numbers of molecules of
different formats and compositions, such as antibodies and nonantibody protein scaffolds (*31*). However, the method by itself cannot
guarantee the generation of functional hits of high potency, and the
successful isolation of potent antibodies also depends on the existence of such hits in the library. Similar to other in vitro display
methods, the efficiency of drug discovery will scale with library
size (*32*).
We performed a single round of screening to identify anti-Her2 ×
anti-CD3 antibodies. Such a single round could be potentially sufficient for some targets, but the false-positive rate could be high in
certain screenings. A few reasons could contribute to the falsepositive rate. First, the local concentration of antibody in the droplets is very high and may result in an increased tendency to form
high–molecular weight aggregates and nonspecific activation of the
reporter cell. Second, the gating strategy may not be stringent enough
to exclude all false-positive events. As used in well-established
methods, such as phage display, iterative rounds of screening may
help to enrich the true hits and eliminate false-positive hits. In addition, more stringent gating strategies can be applied in screening.
Research into costimulatory receptor agonists has been reignited
over the past decade due to substantial advances in the field of immunoncology. Costimulatory receptors are expressed on a number
of immune cell types, including T cells, B cells, and NK cells as well
as APCs, and engagement of these receptors promotes immune cell
function, proliferation, and survival. Nevertheless, there are no general rules to guide the screening of agonist antibodies. For example,
a panel of antibodies binds to the same or similar epitopes of the Fas
receptor but results in different biological effects, with some acting
as agonists and others as antagonists (*4*, *33*). The intrinsic complexity of agonist antibodies requires screening as many antibodies as
possible. We used our unique platform to screen for CD40 agonist
antibody and found a few potent CD40 agonist antibodies, most of
which were too rare (<0.02% frequency) to be found using a conventional screening platform.
Our method can also be applied to high-throughput analysis of
cell-cell interactions. For example, we can envision a scenario where
DCs infected with a lentivirus library encoding neoantigens are coencapsulated with tumor-infiltrating T cells to map the pairs of cognate antigens and T cell receptors (TCRs) (*34*). This method can
also be adapted to screen different types of molecules, such as cytokines (*35*–*37*). In addition to application in drug discovery, identification of intercellular signaling pathways has become an actively
growing field. Combining our technology with the power of CRISPR-
Cas9 library screening could enable the deciphering of cell-cell communications at scale (*38*–*40*). The innovative applications of this
activity-based selection method have been limited only by the imagination of the users.
In summary, we developed a unique high-throughput platform
for function-based screening of up to millions of antibodies. With
the capability to screen millions of antibody-producing cells without any presumptions other than the key function used for screening, this may revolutionize next-generation cancer immunotherapy
drug discovery and development as well as advance basic research
involving cell-to-cell interactions.
## MATERIALS AND METHODS Cell culture
HEK293FT cells (Thermo Fisher Scientific, R70007) were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher
Scientific). Jurkat cells [American Type Culture Collection (ATCC),
TIB-152] were cultured in RPMI 1640 medium (Thermo Fisher
Scientific). SK-BR-3 cells (ATCC, HTB-30) were cultured in McCoy’s
5A (modified) medium (Biological Industries). All the culture
medium was supplemented with 10% fetal bovine serum (FBS; Biological Industries), 1× nonessential amino acids, penicillin (100 U/ml),
streptomycin (100 mg/ml), and 12.5 mM Hepes (Thermo Fisher Scientific).
HEK293F cells (Thermo Fisher Scientific, R79007) were suspended
and cultured in FreeStyle 293 Expression Medium (Thermo Fisher
Scientific). All cells were maintained in a CO₂ incubator at 37°C.
$$
\mathrm{C O_{2}}
$$
$$
37^{\circ}\mathrm{C}.
$$
## IL2 reporter cell line
Jurkat/pIL2-eGFP was developed to monitor the activation of T cells.
The Jurkat cell line was transfected with a vector carrying the eGFP
reporter gene under the control of the full-length IL-2 promoter
(the region from −648 to −1 upstream of the translation initiation
codon of IL2). After stimulation with anti-CD3 and anti-CD28
antibodies, cells expressing high levels of GFP were sorted into wells
of a 96-well plate using fluorescence-activated cell sorter (FACS),
and individual clones of the Jurkat/pIL2-eGFP cell line were
characterized.
## CD40 reporter cell line
Jurkat/NF-kB-GFP-hCD40 reporter cells were developed to monitor
the activation of CD40. Jurkat cells were transfected with a vector
carrying the eGFP reporter gene under the control of the NF-kB
response element. After stimulation with TNFa (10 ng/ml; Sino
Downloaded from https://www.science.org on February 01, 2023
---
Biological), cells expressing a high level of eGFP were sorted by
FACS, and the Jurkat/NF-kB-eGFP cell line was characterized. The
resulting Jurkat/NF-kB-eGFP cell line was infected with lentivirus
expressing full-length human CD40. After stimulation with 100 nM
hexameric CD40L Fc fusion protein, individual cells with a high GFP
signal level were sorted into wells of a 96-well plate by FACS, and
individual clones of Jurkat/NF-kB-GFP-hCD40 cells were characterized.
## Microfluidic chip fabrication
All microfluidic chips were fabricated in polydimethylsiloxane (PDMS)
polymers (Sylgard 184 Elastomer Kit; Dow Corning Corp.) using
standard soft lithography as previously described (*41*). Masters
were made using one layer of SU-8 photoresist (MicroChem). The
depth of the two devices is 40 ± 1 mm to allow the droplet to generate or flow in a monolayer format. Device i is composed of one oil
inlet and two aqueous phase inlets where reporter cells, infected
cells, and bioassay reagents are added. For device ii, PDMS is bound
to a piezoelectric substrate (Y128-cut lithium niobate wafer), where
a gold interdigital electrode is patterned with standard lift-off
technology and aligned with the fluidic channel above. Microfluidic
devices were treated before use with 1% (v/v) 1*H*,1*H*,2*H*,2*H*
perfluorodecyltrichlorosilane (Alfa Aesar) in Novec HFE7500 fluorinated oil (3M) to prevent droplets from wetting the channel walls.
$$
40\pm1
$$
## Droplet production, collection, and incubation
Aqueous phases containing infected cells and reporter cells were
coflowed and partitioned into droplets with hydrodynamic flow
focusing in dripping mode on a microfluidic chip (Fig. 2A). The
nozzle is 15 mm wide, 40 mm deep, and 10 mm long. The continuous
phase was Novec HFE7500 fluorinated oil (3M) containing 2%
(w/w) 008-FluoroSurfactant (RAN Biotechnologies). Pressure pumps
(Fluigent) were used to generate monodispersed droplets of 100 ±
10 pl at 5000 Hz. The droplets were collected into a 10-ml tube and
incubated at 37°C in 5% CO₂ to allow antibody secretion and
subsequent activity to occur within each droplet before screening.
$$
37^{\circ}\mathrm{C}
$$
$$
\mathrm{C O_{2}}
$$
## Microfluidic droplet screening and recovery
Droplet fluorescence analysis and sorting operations were performed on a dedicated droplet microfluidic station, similar to that
described by Mazutis *et al.* (*15*). Pressure pumps (Fluigent) were
used to inject the collected droplets into the sorter device (Fig. 2C)
at a frequency of 1000 to 3000 Hz. The sorter device was mounted
on an inverted microscope (Applied Scientific Instrumentation
Microscope) equipped with a 940-nm light-emitting diode illumination source (Thorlabs, M940L3) and a fixed focus laser line (solidstate laser with a wavelength of 405, 488, 561, or 635 nm; Omicron)
with photomultiplier tube bandpass filters of 440/40 to 25, 525/40
to 25, 593/46 to 25, and 708/75 to 25 nm (Hamamatsu).
The fluorescence of each droplet was measured as the droplet
flowed past an observation constraint in the microfluidic channel
where the laser line was positioned. The emitted fluorescence was
detected with photomultiplier tubes, converted into corresponding
signal output voltages, and recorded by the data acquisition card
(FPGA PCIe-7842R). These voltages were then processed by the
card and custom LabVIEW software to identify droplets according
to their fluorescence intensity and size. These characteristics were
used to determine whether each droplet should be sorted.
Droplets were sorted on the basis of the surface acoustic wave
deflection as described by Franke *et al.* (*21*, *42*) with a gigahertz signal
generator (Wavetek, model 3010). Sorted droplets were collected in a
1.5-ml Eppendorf tube. Cells were recovered by adding 100 ml of
DMEM culture medium, followed by 100 ml of 1*H*,1*H*,2*H*,2*H*-
perfluoro-1-octanol (Sigma-Aldrich, 37053), and then cells were
pooled and centrifuged at 400*g* for 5 min at 4°C for subsequent
steps, such as subculture or DNA sequencing.
$$
4^{\circ}\mathrm{C}
$$
## Phage display
A human naïve scFv library was constructed using PBMCs from 30
healthy donors with standard protocols. The phage library was
incubated with biotinylated CD40-Fc fusion protein or Her2 recombinant protein (ACROBiosystems) for 2 hours at room temperature, and the phage-antigen complex was captured by Dynabeads
M280 (Life Technologies). Bound phages were eluted using glycine-
HCl (pH 2.2) for 10 min at room temperature and neutralized with
tris-HCl (pH 8.0) to adjust the pH to 7.5. The phagemid DNA was
isolated using a plasmid miniprep kit (QIAGEN).
## Lentivirus library construction
Both phagemids and the lentiviral vector pLV-ef1a-scFv-Fc were
-digested with the Sfi I enzyme. The lentiviral vector and scFv genes
were isolated after electrophoresis. ScFv genes were ligated into a
lentiviral vector. The product of the ligation reaction was transformed
into XL1-Blue competent cells by the electroporation transformation
technique, and most of the transformed bacteria were plated on LB
agar plates. The remaining bacteria were serially diluted and plated to
estimate the size of the library. The lentiviral plasmid was prepared
using a plasmid midiprep kit (QIAGEN) for lentivirus preparation.
## Lentivirus preparation
When cell confluence reached 80%, HEK293FT cells were transfected
with the lentiviral backbone plasmid and packaging plasmids using
polyethylenimine (PEI; Polysciences) transfection reagent. The medium was changed to fresh complete culture medium 6 hours after
transfection. The supernatant containing lentivirus was harvested
after 48 hours, centrifuged at 300*g* for 5 min at 4°C, and filtered
through a 0.45-mm filter to remove cell debris. Viral titer was measured using a P24 ELISA kit (Clontech). The virus was aliquoted
and stored at −80°C.
$$
4^{\circ}\mathrm{C}.
$$
$$
-80^{\circ}\mathrm{C}
$$
## Function-based screening of anti-Her2 × anti-CD3 bispecific antibody using microfluidics
For aqueous phase 1, Jurkat/pIL2-eGFP reporter cells were washed
with phosphate-buffered saline (PBS) and stained with 1 mM Cell-
Trace Yellow dye (Thermo Fisher Scientific, C34573) for 10 min at
37°C. The stained Jurkat/pIL2-eGFP reporter cells were washed two
times with RPMI 1640 and resuspended in cell culture medium
(RPMI 1640, 5% FBS, 25 mM Hepes, and 0.1% Pluronic F-68) containing anti-CD28 antibody (1 mg/ml; Invitrogen), which was added
here to enhance T cell activation (fig. S1).
$$
37^{\circ}\mathrm{C}.
$$
For aqueous phase 2, stable Her2-expressing K562 cells were infected with a lentiviral antibody library. The resulting antibodysecreting K562-Her2 cells were washed with PBS and stained with
1 mM CellTrace Violet dye (Thermo Fisher Scientific, C34571) for
10 min at 37°C. The stained cells were washed twice with RPMI
1640 and resuspended in cell culture medium containing 200 nM
DY647 (Dyomics, 647-00). K562-Her2 cells infected with positive
control lentivirus were resuspended in cell culture medium containing 1500 nM DY647.
$$
37^{\circ}\mathrm{C}
$$
Downloaded from https://www.science.org on February 01, 2023
---
Aqueous phases 1 and 2 were injected into the droplet generation chip from different inlets and used as the dispersion phase.
Novec HFE7500 fluorinated oil (3M) containing 2% (w/w) fluorosurfactant (RAN Biotechnologies) was used as the continuous phase
to produce droplets with an average size of 100 pl. The flow rates of
aqueous phase 1, aqueous phase 2, and oil phase were adjusted so
that 1 reporter cell and 0.5 antibody-secreting cells were coencapsulated per droplet on average. During droplet production, the cell
suspension was cooled using ice water to inhibit antibody secretion.
The droplets were collected and incubated at 37°C for 16 hours.
$$
37^{\circ}\mathrm{C}
$$
The droplets were first gated to eliminate coalesced droplets and
retain only droplets of the desired size. The positive control and the
screening population droplets were distinguished on the basis of the
different intensities of fluorescence of DY647. For the screening population, droplets were selected for the presence of Jurkat/pIL2-eGFP
reporter cells based on CellTrace Yellow signal and K562-Her2 cells
based on CellTrace Violet signal. FADS was performed to sort the
droplets containing Jurkat/pIL2-eGFP emitting GFP fluorescence.
Last, we gated and sorted the droplets with GFP signal colocalized
with reporter cell staining signal but not with K562-Her2 signal.
The cells were recovered from the sorted droplets by adding 200 ml
of RPMI 1640 medium supplemented with 10% FBS and 24%
Nycodenz (Serva, 31000.01), followed by the addition of 50 ml of
1*H*,1*H*,2*H*,2*H*-perfluoro-1-octanol (Sigma-Aldrich, 370533). After
mixing thoroughly and centrifuging at 300*g* for 5 min at 4°C, the
aqueous layer was completely separated and washed with RPMI
1640 medium. The recovered cells were lysed, and antibody genes
were amplified from the cells.
$$
4^{\circ}\mathrm{C}.
$$
## Anti-Her2 × anti-CD3 bispecific antibody in vitro experiment Jurkat/pIL2-eGFP reporter cell assay
To determine the activity of anti-Her2 × anti-CD3 antibody candidates, Jurkat/pIL2-eGFP reporter cells were stimulated with different
concentrations of anti-Her2/anti-CD3 antibodies and anti-CD28
antibody (1 mg/ml) in the presence of K562 cells or K562-Her2 cells.
After 16 hours of incubation, GFP expression in reporter cells was
detected by flow cytometry.
## Anti-Her2 × anti-CD3 bispecific antibody ex vivo assay
5
A total of 1 × 10 PBMC cells were cocultured with SK-BR-3 cells at
a 1:1 ratio. Different concentrations of anti-Her2 × anti-CD3 antibody or control antibody together with anti-CD28 antibody (1 mg/ml)
were added. After 48 hours of incubation, cells were collected and
stained with anti–CD3-FITC (BioLegend, 300406) and anti–CD69-
APC (BioLegend, 310910) for 30 min at 4°C. T cell activation was
determined by flow cytometry. Flow cytometry results were analyzed with FlowJo X software.
$$
4^{\circ}\mathrm{C}
$$
The cell supernatant was collected to quantify cytokine release
and cytotoxicity. IL-2 (BD, 550611) and IFN-g (R&D Systems,
VAL104) were measured with ELISA kits according to the manufacturer’s instructions. Cytotoxicity was analyzed by measuring the
levels of released lactate dehydrogenase (LDH) using the CytoTox
96 Non-radioactive Cytotoxicity Assay protocol (Promega).
## Function-based screening of CD40 agonist antibodies using microfluidics
For aqueous phase 1, Jurkat/NF-kB-GFP-hCD40 reporter cells
were washed with PBS and stained with 1 mM CellTrace Yellow dye
for 10 min at 37°C. The stained Jurkat/NF-kB-GFP-hCD40
$$
37^{\circ}\mathrm{C}
$$
reporter cells were washed two times with DMEM and resuspended at 20 million cells/ml in cell culture medium (DMEM, 5%
FBS, 25 mM Hepes, and 0.1% Pluronic F-68) containing 16.67 nM
DyLight 650–conjugated goat anti-human Fc IgG (Abcam,
ab98622) and 24% Nycodenz. The secondary antibody DyLight
650–conjugated goat anti-human Fc IgG was used to mimic the
cross-linking action by the Fc receptor (*43*).
For aqueous phase 2, for the screening population, HEK293FT
cells were infected with a lentiviral antibody library and resuspended
in cell culture medium containing 500 nM DY405 (Dyomics, 405-00).
For positive control droplets, HEK293FT cells were resuspended in
cell culture medium containing soluble hexameric CD40L protein
and 1500 nM DY405. For negative control droplets, HEK293FT
cells were resuspended in cell culture medium containing anti-HEL
antibody and 2500 nM DY405.
Aqueous phases 1 and 2 were injected into the droplet generation chip from different inlets and used as the dispersion phase.
Novec HFE7500 fluorinated oil (3M) containing 2% (w/w) fluorosurfactant (RAN Biotechnologies, 008-FluoroSurfactant) was used
as the continuous phase to produce droplets with an average size of
100 pl. The flow rates of aqueous phase 1, aqueous phase 2, and oil
phase were adjusted so, on average, 1 reporter cell and 0.5 antibodysecreting cell were coencapsulated per droplet. During droplet production, the cell suspension was cooled using ice water to inhibit
antibody secretion. The droplets were collected and incubated at
37°C for 16 hours.
The droplets were first gated to eliminate coalesced droplets and
retain only droplets of the desired size. Negative control droplets,
positive control droplets, and screening droplets were distinguished
on the basis of the different intensities of blue fluorescence of
DY405. For the screening population, droplets were selected for the
presence of Jurkat/NF-kB-GFP-hCD40 reporter cells in the droplet
based on the yellow fluorescence of CellTrace Yellow dye. Last,
FADS was performed to sort droplets containing Jurkat/NF-kB-
GFP-hCD40 emitting DyLight 650 and GFP fluorescence. The cells
were recovered from the sorted droplets by adding 200 ml of DMEM
supplemented with 10% FBS and 24% Nycodenz, followed by adding
50 ml of 1*H*,1*H*,2*H*,2*H*-perfluoro-1-octanol (Sigma-Aldrich, 370533).
After mixing thoroughly and centrifuging at 300*g* for 5 min at 4°C,
the aqueous layer was completely separated and washed with
DMEM. The cells were resuspended in DMEM containing 10% FBS
and 1% penicillin and streptomycin and then cultured for 1 to 2 weeks.
## Bioinformatic analysis of the PacBio sequencing results
A single-molecule real-time (SMRT) sequencing platform (Pacific
Biosciences) generated long sequencing reads with an average read
length of ~20 kb, which could sequence a scFv (subreads) more
than 10 times. The circular consensus sequencing program from
PacBio SMRT portal software (version 4.1.0) can take multiple subreads of the same SMRTbell sequence and combine them, using a
statistical model, to produce one high-quality CCS. After CCS from
long sequencing reads was generated, scFv flanking sequences were
trimmed, scFv DNA sequences were translated into proteins using
the CLC Genomics Workbench (version 11.0.1), and CDR1 to CDR3
regions for heavy and light chains were identified using IgBLAST
(version 1.15.0). CD-hit (version 4.8.1) was used to group scFvs
with a protein similarity of 95%. The frequency of scFvs for each
round was calculated in the Java program. ScFvs that appeared
in only one sample were removed because they were likely PCR
Downloaded from https://www.science.org on February 01, 2023 artifact products. The bar plot was drawn using the R package ggplot2 (version 3.2.1).
## Protein expression and purification of full-length IgG
Equal amounts of the heavy chain and light chain expression plasmids were cotransfected into HEK293F cells. Five days after transfection, cells were centrifuged at 3000 rpm for 10 min at 4°C, and
supernatants were harvested and passed through a 0.45-mm filter.
Antibodies were purified with a HiTrap Protein A column (GE)
using an ÄKTA purifier chromatography system.
$$
4^{\circ}\mathrm{C},
$$
## CD40 in vitro experiment
## Species cross-reactivity of antibody
Cross-reactivity of C04 was assessed by flow cytometry analysis.
Briefly, HEK293FT cells were transiently transfected with human CD40
(hCD40)– or rhesus macaque CD40 (rCD40)–expressing vector
using PEI. After 48 hours, HEK293FT-hCD40 or HEK293FTrCD40 cells
were incubated with different concentrations of antibody for 30 min
at room temperature. Then, cells were stained with Alexa Fluor 488–
conjugated goat anti-human Fc (Life Technologies) at room temperature
for 30 min and analyzed by flow cytometry. The fluorescence intensity
is equal to the percentage of GFP-positive cells multiplied by the median
fluorescence intensity (MFI). The fluorescence intensity was plotted
against the antibody concentrations using GraphPad Prism software.
***SPR analysis***
SPR experiments were performed with a Biacore T200 SPR system
+
(GE Healthcare). In brief, experiments were performed at 20°C in buffer [0.01 M Hepes, 0.15 M NaCl, and 0.05% (v/v) Surfactant P20]. Anti-his antibody was immobilized on a Series S CM5 HBS-P
chip by amine coupling, and his-tagged cynomolgus monkey CD40
was captured by immobilized anti-his antibody at a flow rate of
10 ml/min for 60 s. Twofold serially diluted CD40 antibodies were
injected through flow cells for 120 s followed by a 130-s dissociation
phase at a flow rate of 30 ml/min. Before the next assay cycle, the
sensor surface was regenerated with glycine-HCl (pH 1.5) for 30 s at
a flow rate of 30 ml/min. Background binding to blank immobilized
flow cells was subtracted, and equilibrium dissociation constant
(*K*D) values were calculated using the 1:1 binding kinetics model
built in Biacore T200 Evaluation Software (version 3.2).
$$
\ \mathrm{H B S\ }{\scriptstyle P^{+}}
$$
$$
20^{\circ}\mathrm{C}
$$
$$
(K_{\mathrm{D}})
$$
## CD40 antibody selectivity
Human CD40 (ACROBiosystems), GITR (ACROBiosystems), OX40
(ACROBiosystems), 4-1BB (ACROBiosystems), or bovine serum
albumin (BSA) (Solarbio) was plated onto a microtiter plate at 4°C
overnight. The coated wells were blocked with 0.5% BSA in PBS at
37°C for 1 hour. Serially diluted antibodies were added and incubated
at 37°C for 1 hour and washed eight times, followed by the addition
of goat anti-human IgG–horseradish peroxidase (SouthernBiotech)
and incubation at 37°C for 30 min. After eight washes, 2,2’-azinobis
[3-ethylbenzthiazoline-6-sulfonic acid] diammonium salt (ABTS)
substrate solution (Thermo Fisher Scientific) was added, and the
optical density at 405 nm was measured with a plate reader.
$$
4^{\circ}\mathrm{C}
$$
$$
37^{\circ}\mathrm{C}
$$
$$
37^{\circ}\mathrm{C}
$$
## Jurkat/NF-kB-GFP-hCD40 reporter cell assays
For CD40 agonist activity detection, Jurkat/NF-kB-GFP-hCD40 reporter cells were incubated with different concentrations of CD40
agonist antibodies with or without goat anti-human Fc antibody
(SouthernBiotech) for 24 hours. GFP expression was detected by
flow cytometry.
For the FcgRIIB dependency experiment, HEK293FT cells were
transiently transfected with the FcgRIIB-expressing vector using
PEI. After 36 hours, HEK293FT-FcgRIIB cells were plated in 48-well
plates and cultured overnight at 37°C. Then, Jurkat/NF-kB-GFPhCD40 reporter cells and different concentrations of C04 or anti-HEL
were cocultured with HEK293FT-FcgRIIB cells for 24 hours. GFP
expression was detected by flow cytometry.
$$
37^{\circ}\mathrm{C}
$$
For data analysis of Jurkat/NF-kB-GFP-hCD40 reporter cell assays, flow cytometry results were analyzed with FlowJo X software,
and the fluorescence intensity was equal to the percentage of
GFP-positive cells multiplied by MFI. The fluorescence intensity of
cells was plotted against the antibody concentrations using GraphPad
Prism software.
## CD40 ex vivo experiment
Following thawing and recovery of human PBMCs, monocytes
were selected by adhering to plastic and then cultured for 8 days in
RPMI containing 10% FBS (Gibco), granulocyte-macrophage colonystimulating factor (100 ng/ml; R&D Systems), and IL-4 (10 ng/ml;
R&D Systems). Suspended cells were harvested and confirmed to be
DCs by CD11c expression.
B cells were isolated from PBMCs by magnetic selection using
5
CD19 beads (Miltenyi). A total of 1 × 10 DCs or B cells were
incubated with different concentrations of C04 with or without
goat anti-human Fc antibody (SouthernBiotech) for 48 hours. Upregulation of the activation marker CD86 was analyzed by flow
cytometry (BioLegend). Flow cytometry results were analyzed with
FlowJo X software. The MFI of cells was plotted against the antibody concentrations using GraphPad Prism software.
$$
1\times{10}^{5}
$$
## CD40 in vivo experiment
FcgR/CD40-humanized mice have been described previously and were
provided by J. Ravetch (Rockefeller University). Mice were bred
and maintained in a specific pathogen–free environment at the Department of Laboratory of Animal Science, Shanghai Jiao Tong University
School of Medicine (SJTUSM). All animal care and study were performed in compliance with institutional and National Institutes of
Health guidelines and were approved by the SJTUSM Institutional
Animal Care and Use Committee (protocol registry number: A-2015-014).
## + OVA-specific CD8 T cell response model
FcgR/CD40-humanized mice were adoptively transferred with
+ 6
CD45.1 splenic OT-I cells (2 × 10 cells in 200 ml of PBS per mouse)
via tail vein injection 1 day before immunization with 2 mg of CD205
(DEC)-OVA antibody in the presence of the CD40 agonist antibody
or the isotype control by intraperitoneal injection. On day 6, spleen
cells were harvested. After red blood cell lysis, the single-cell suspension was stained with anti-CD4 (clone RM4-5), anti-CD8 (clone
53-6.7), anti-CD45.1 (A20), and anti–TCR-Va2 (B20.1) to quantify
+ +
OVA-specific OT-I CD8 T cells. OT-I CD8 T cells were defined as
+ + +
CD45.1 CD8 TCR-Va2 cells.
$$
\left(2\times{0}^{6}\right.
$$
$$
\mathrm{O T-I\,C D8^{+}\,T}
$$
$$
\mathrm{C D45.1^{4}C D8^{+}T C R.V a2^{+}}
$$
## Syngeneic mouse model
FcgR/CD40-humanized mice were subcutaneously inoculated with
6
2 × 10 MC38 cells. When tumor volumes reached 50 to 100 mm³,
mice were randomly assigned to different groups (*n* = 5). MC38-
bearing FcgR/CD40-humanized mice were intraperitoneally treated
with C04, CP-870,893, or anti-HEL (3 mg/kg, q3d × 2). Tumor
growth was monitored every 3 days by measuring the length (*L*) and
width (*W*) with calipers, and tumor volume was calculated using
the formula (*L* × *W²*)/2.
$$
2\times{10}^{6}
$$
$$
(L\times W^{2})/2
$$
Downloaded from https://www.science.org on February 01, 2023
---
## SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/7/24/eabe3839/DC1
View/request a protocol for this paper from *Bio-protocol*.
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## Acknowledgments
**Funding:** This work was supported by the National Key Research and Development plan of
China (grant number 2017YFA0504801), the National Natural Science Foundation of China
(grant number 81872787), China Postdoctoral Science Foundation (grant number
2020M670625), the Fundamental Research Funds for the Central Universities, Nankai
University (grant numbers ZB19100123 and 63191212), the Natural Science Foundation of
Tianjin (grant number 19JCZDJC32900), the Key Laboratory of Immune Microenvironment and
Disease open funding (grant number 20180102), Shanghai Municipal Science and Technology
Commission (project no. 19431902900), and the National Key Research and Development plan
of China (grant number 2018YFE0200401). F.L. and Y.Z. are also supported by the Innovative
Research Team of High-level Local Universities in Shanghai (SSMU-2DCX20180100) and
Shanghai Science and Technology Commission Projects (grant numbers 18140902600,
19431902900). The work was also sponsored by HiFiBiO Therapeutic funding. **Author**
**contributions:** H.Z. designed all the experiments. Y.W., R.J., B.S., W.W., Y.Z., M.H., P.F., S.W.,
P. Mary, N.L., R.W., P. Ma, and R.L. performed the experiments. H.Zho., Y.W., B.S., H.Zha., Y.T.,
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# SCIENCE ADVANCES
# | RESEARCH ARTICLE
L.S., F.L., and Y.C. analyzed all the data and wrote the manuscript with the input of all authors.
**Competing interests:** Y.W., R.J., H.Z., B.S., M.H., and L.S. are inventors on a pending patent
(Functional Screening Using Droplet-based Microfluidics) related to this work filed by HiFiBiO
(Shanghai) Limited and Nankai University, serial no. PCT/CN2020/110616, filed 21 August
2020. All inventors of the patent are disclosed, and no other authors on this paper are also the
inventors on the patent. The authors declare that they have no other competing interests.
**Data and materials availability:** All data needed to evaluate the conclusions in the paper are
present in the paper and/or the Supplementary Materials. The tabulated underlying data for
indicated figures are included in the attached CSV file. Additional data related to this paper
may be requested from the authors.
Submitted 19 August 2020
Accepted 23 April 2021
Published 11 June 2021
10.1126/sciadv.abe3839
**Citation:** Y. Wang, R. Jin, B. Shen, N. Li, H. Zhou, W. Wang, Y. Zhao, M. Huang, P. Fang, S. Wang,
P. Mary, R. Wang, P. Ma, R. Li, Y. Tian, Y. Cao, F. Li, L. Schweizer, H. Zhang, High-throughput
functional screening for next-generation cancer immunotherapy using droplet-based microfluidics.
*Sci. Adv.***7**, eabe3839 (2021).
Downloaded from https://www.science.org on February 01, 2023
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# High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics
Yuan Wang, Ruina Jin, Bingqing Shen, Na Li, He Zhou, Wei Wang, Yingjie Zhao, Mengshi Huang, Pan Fang, Shanshan
Wang, Pascaline Mary, Ruikun Wang, Peixiang Ma, Ruonan Li, Yujie Tian, Youjia Cao, Fubin Li, Liang Schweizer, and
Hongkai Zhang
Sci. Adv.,7 (24), eabe3839.
DOI: 10.1126/sciadv.abe3839
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