Despite remarkable contribution of green fluorescent protein and its variants for better understanding of various biological functions, its application for anaerobic microorganisms has been limited because molecular oxygen is essential for chromophore formation. To overcome the limitation, we engineered a plant-derived light, oxygen, or voltage (LOV) domain containing flavin mononucleotide for enhanced spectral properties. The resulting LOV variants exhibited improved fluorescence intensity (20 and 70% higher for SH3 and 70% for BR1, respectively) compared to iLOV, an LOV variant isolated in a previous study, and the quantum yields of the LOV variants (0.40 for SH3 and 0.45 for BR1) were also improved relative to that of iLOV (Q = 0.37). In addition to fluorescence intensity, the identified mutations of SH3 enabled an improved thermostability of the protein. The engineered LOV variants with enhanced spectral properties could provide a valuable tool for fluorescent molecular probes under anaerobic conditions.

Multimer formation is indispensable to the intrinsicbiologicalfunctions of many natural proteins. For example, the human immunoglobulin G (IgG) antibody has two variable regions (heavy chain variable domain [VH] and light chain variable domain [VL]) that must be assembled for specific antigen binding, and homodimerization of the antibody's Fc domain is essential for eliciting therapeutic effector functions. For the more efficient high-throughput directed evolution of multimeric proteins with ease of cultivation and handling, here we report a membrane protein drift and assembly (MPDA) system, in which a multimeric protein is displayed on a bacterial inner membrane by drifting and auto-assembling membrane-anchored subunit polypeptides. This system enabled the auto-assembly of membrane-tethered Fv domains (VH and VL) or the monomeric Fc domain into a functional hetero- or homodimeric protein complex on the bacterial inner membrane. This system could also be used to enrich a desired engineered Fc variant from a mixture containing a million-fold excess of wild-type Fc domain, indicating the applicability of the MPDA system for the high-throughput directed evolution of a variety of multimeric proteins, such as cytokines, enzymes, or structural proteins.

The immunoglobulin G (IgG) molecule has a long circulating serum half‐life (~3 weeks) through pH‐ dependent FcRn binding‐mediated recycling. To hijack the intracellular trafficking and recycling mechanism of IgG as a way to extend serum persistence of non‐antibody therapeutic proteins, we have evolved the ectodomain of a low‐affinity human FcγRIIa for enhanced binding to the lower hinge and upper CH2 region of IgG, which is very far from the FcRn binding site (CH2–CH3 interface). High‐throughput library screening enabled isolation of an FcγRIIa variant (2A45.1) with 32‐fold increased binding affinity to human IgG1 Fc (equilibrium dissociation constant: 9.04 × 10−7 M for wild type FcγRIIa and 2.82 × 10−8 M for 2A45.1) and significantly improved affinity to mouse serum IgG compared to wild type human FcγRIIa. The in vivo pharmacokinetic profile of PD‐L1 fused with engineered FcγRIIa (PD‐L1–2A45.1) was compared with that of PD‐L1 fused with wild type FcγRIIa (PD‐L1–wild type FcγRIIa) and human PD‐L1 in mice. PD‐L1–2A45.1 showed 11.7‐ and 9.7‐fold prolonged circulating half‐life (t1/2) compared to PD‐L1 when administered intravenously and intraperitoneally, respectively. In addition, the AUCinf of PD‐L1–2A45.1 was two‐fold higher compared to that of PD‐L1–wild type FcγRIIa. These results demonstrate that engineered FcγRIIa fusion offers a novel and successful strategy for prolonging serum half‐life of therapeutic proteins.

4-1BB and PD-1 are both T cell costimulatory receptors/immune checkpoint regulators. 4-1BB/4-1BBL provides stimulatory signals while PD-1/PD-L1, suppressive ones to T lymphocytes. Various forms of agonists to 4-1BB or blockers to PD-1 have shown a potent anti-cancer activity by modulating mainly CD8+ T cells. Thus, synergistic or additive anti-cancer effect may be achieved by combining the agonists to 4-1BB and blockers to PD-1. We have tested various combinations of antibodies and proteins with consideration of overall size of the therapeutic candidates. The best anti-cancer activities were obtained by combination of an agonistic anti-4-1BB antibody and soluble PD-1 that binds to PD-L1 with high affinity. To obtain molecularly-evolved PD-1 we first used the 3D complex between PD-1 and PD-L1 to select the major contributing PD-1 amino acids, a library targeting selected amino acids and random mutations were constructed and screened using yeast surface display. The affinity-matured resulting PD-1 is called euPD-1. We finally constructed EU505 that composed of a scFv of anti-4-1BB and euPD-1. Through cell binding and dual antigen assays, we confirmed that EU505 binds both targets independently at the same time. The result of 4-1BB bioassays, a biologically relevant MOA-based assay, showed that potent PD-L1-dependent T cell activation with EU505. In-vitro killing assays showed that EU505 selectively activated T cells which in turn killed high PD-L1-expressing cells, but not low-PD-L1-expressing ones. EU505 demonstrated much stronger tumor-killing effect compared with each component alone when we tested against human PD-L1-expressing tumor cells in a 4-1BB Knock-in mouse model. Furthermore, it was observed that number of effector CD8+ T cells increased in the peripheral blood upon EU505 injection and consequently tumor size was reduced. EU505, a promising anti-cancer drug, appears to enhance CD8+ T cell infiltration and activate T cells in situ at the tumor sites by binding two different targets of 4-1BB and PD-L1 simultaneously. Citation Format: Byoung S. Kwon, Hanna Lee, Jin Sung Park, Yeonji Oh, SeungHee Han, Jin Kyung Choi, Bora Hwang, Sun Woo Im, Seunghyun Lee, HoonSung Jeh. Anti-4-1BB x PD-1, a bispecific anti-cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB176.

Since the Spike protein on the surface of SARS coronavirus 2 (SARS-CoV-2) binds to the ACE2 receptor in human cells, the development of neutralizing proteins or antibodies targeting the receptor binding domain (RBD) of the spike protein is an important strategy for SARS-COV-2 therapy. We chose to develop molecularly-evolved soluble ACE2 protein on three grounds; 1) it can trap and neutralize the SARS-CoV-2 as neutralizing antibodies do, 2) it can supplement angiotensin II-converting enzyme activities that protect lung, heart, and kidneys of severe cases of infections and patients with underlying diseases, and 3) it may trap effectively SARS-CoV-2 mutants even though the mutations compromise the protection by neutralizing antibodies or vaccine. For the enhancement of ACE2 binding affinity to RBD, we used the 3D complex structure between ACE2 and RBD to select the major contributing ACE2 amino acids, a library targeting selected amino acids and random mutations were constructed and screened using yeast surface display. The engineered ACE2, EU129, was fused with the human IgG1 Fc for long half-life and viral clearance. The binding affinity of EU129 to RBD was increased by about 500-folds compared to ACE2 wild-type in SPR analysis, and the neutralizing activity was also increased by about 130-folds compared to ACE2 wild-type in surrogate virus neutralization test (sVNT). In addition, it was confirmed that the enzymatic activity of ACE2, which prevents organ damage due to SARS-CoV-2 infection in the human, is maintained at a level similar to that of ACE2 wild-type. In vitro assays using live SARS-CoV-2 virus and Vero E6 cells, EU129 was shown to be more effective in inhibiting viral infection and amplification than ACE2 wild-type, which was confirmed through protein and RNA level and cell morphology change of the live virus. In vivo stability assays using BALB/c mice, EU129 showed enhanced binding to the RBD and maintained enzymatic activity similar to ACE2 wild-type. We generated EU129 with the improved binding affinity and neutralizing activity through ACE2 receptor engineering. It has angiotensin II-converting enzymatic activity for organ protection, thus EU129 is a better therapeutic candidate for severe cases of SARS-CoV-2 infection and patients with underlying diseases such as cancers. Citation Format: Byoung S. Kwon, Seunghyun Lee, Jin-Kyung Choi, Bora Hwang, Sun-Woo Im, Yun-Sook Lim, Bumseok Kim, Soon B. Hwang, HoonSung Jeh. Development of SARS-CoV-2 neutralizing protein by ACE2 receptor engineering for severe infection and patients with underlying diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB075.