Spatial Organization of Biological Fuctions | BPS Thematic Meeting

Spatial Organization of Biological Functions Meeting

Poster Abstracts

3-POS Board 3 DESIGN AND CHARACTERIZATION OF DE NOVO MINIPROTEIN BINDERS TARGETING IMMUNE CHECKPOINT PROTEIN PD-L1 Truptimayee Barik 1,2 ; Ashwani Kumar 2 ; Sujay Gaikwad 2 ; Gagan D Gupta 1,2 ; Ravindra D Makde 1,2 ; 1 Homi Bhabha National Institute, Mumbai, India 2 Bhabha Atomic Research Center, BDAS, Mumbai, India Cancer is characterized by abnormal cell division and remains one of the leading causes of mortality worldwide. Current treatment strategies including surgery, chemotherapy, radiation, and immunotherapy aim to control disease progression. Among targeted therapies, monoclonal antibodies (mAbs) have shown significant success. However, they face limitations such as high production cost, post-translational modification challenges, limited tissue penetration, and immunogenicity. Miniprotein scaffolds (4– 12 kDa) offer an attractive alternative to mAbs due to their small size, fewer secondary structural elements, and improved stability and designability. De novo design of such mini-protein binders with high affinity is an emerging strategy for targeted therapy. In this study, we utilized RFdiffusion, a generative AI model, to design de novo mini-protein binders targeting the immune checkpoint protein PD-L1. Hotspot residues on PD L1 were identified based on interactions with a known monoclonal antibody. Binder sequences of 70– 80 amino acid long, predominantly α -helical in structure, were computationally designed through 1000 RFdiffusion simulation runs, using a predicted aligned error (pAE) score threshold of 5 and a predicted local distance difference test (pLDDT) score of 90. From these simulations, 40 candidate binders were generated, and the top 10 designs (based on pAE and pLDDT) were chosen for experimental validation. These binders were gene-synthesized, cloned, expressed, and purified. Circular dichroism (CD) spectroscopy was used to assess secondary structure and thermostability, revealing six binders with high thermostability. Subsequently SPR-based binding assays confirmed that one binder displayed nanomolar affinity toward PD-L1. Future work will focus on improving the affinity of this lead binder using partial diffusion or BindCraft optimization methods and in-vitro cell-based studies.

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