High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics

Abstract

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 transduction 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 platform could revolutionize next-generation cancer immunotherapy drug development and advance medical research.

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.

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.

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-γ (F) and IL-2 (G) in the culture supernatant were measured by ELISA.

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.

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-κB-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.

Fig. 6. Characterization of the identified CD40 agonist antibody.

(A) The FcγRIIB dependency of antibody C04. Jurkat/NF-κB-GFP-hCD40 reporter cells were stimulated with C04 antibody or anti-HEL antibody in coculture with FcγRIIB-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 FcγR/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. FcγR/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.