We seek to develop a completely new microfluidics-based method (Droplet Compartmentalized Selection - DCS) to isolate cells that produce antibodies with desired binding characteristics. In DCS, individual cells are encapsulated (together with components to assay binding activity) in pico-liter scale droplets, which can then be screened for binding activity at rates of about 1,000 droplets per second. Individual cells will fill the droplets with antibody to (M concentrations within a few hours. A fluorescent read-out can then be used to identify and collect droplets that contain the desired antibody-producing cells. Crucially, there is no need to immortalize the antibody-producing cells. Instead, the antibody-encoding genes can be isolated from individual selected cells by reverse-transcriptase PCR and then cloned into appropriate expression vectors. DCS has three major advantages over typical methods to isolate monoclonal antibodies. 1) Because the screening can be performed on individual, non-immortalized cells, a great deal of time is saved. There is no need to create hybridomas to immortalize the antibody-producing cells, and the time required to grow and assay individual """"""""clones"""""""" is several hours rather than several weeks. 2) DCS allows access to much greater antibody diversity. By eliminating the highly inefficient process of hybridoma generation (typically only 1/100,000 antibody-producing cells are immortalized), and by greatly speeding the screening process, it is possible to screen tens- or hundreds- of thousands of individual cells for the ability to produce desired antibodies. In contrast, a typical monoclonal antibody screen can analyze at most several hundred unique antibody-producing hybridomas. And 3), DCS will allow screening of cell types which are not amenable to typical hybridoma-based methods. For example, cells that produce antibodies against particular targets could be isolated from blood from human patients. This could pave the way for novel diagnostic tests and would facilitate identification of endogenous human antibodies that might serve as building blocks for therapeutic antibodies.
Therapeutic monoclonal antibodies make up the fastest-growing segment of the prescription pharmaceutical market and are the cornerstone of an emerging class of 'targeted'cancer therapies that are designed to prevent tumor growth by more specific actions than older treatments, via the targeting of molecular drivers of carcinogenesis. Our new microfluidics-based method (Droplet Compartmentalized Selection - DCS) to isolate cells that produce antibodies with desired binding characteristics will revolutionize the production of mAbs. It will enable much higher throughput screening, giving far greater access to immune repertoires, thus allowing the isolation of more and better antibody therapeutics. Moreover, because the antibodies from a single cell can be detected, it becomes possible to directly screen non-immortalized cells instead of hybridomas, further increasing the antibody diversity that can be accessed.
| Mazutis, Linas; Gilbert, John; Ung, W Lloyd et al. (2013) Single-cell analysis and sorting using droplet-based microfluidics. Nat Protoc 8:870-91 |
