Antibody design using LSTM based deep generative model from phage display library for affinity maturation.

Molecular evolution is an important step in the development of therapeutic antibodies. However, the current method of affinity maturation is overly costly and labor-intensive because of the repetitive mutation experiments needed to adequately explore sequence space. Here, we employed a long short term memory network (LSTM)-a widely used deep generative model-based sequence generation and prioritization procedure to efficiently discover antibody sequences with higher affinity. We applied our method to the affinity maturation of antibodies against kynurenine, which is a metabolite related to the niacin synthesis pathway. Kynurenine binding sequences were enriched through phage display panning using a kynurenine-binding oriented human synthetic Fab library. We defined binding antibodies using a sequence repertoire from the NGS data to train the LSTM model. We confirmed that likelihood of generated sequences from a trained LSTM correlated well with binding affinity. The affinity of generated sequences are over 1800-fold higher than that of the parental clone. Moreover, compared to frequency based screening using the same dataset, our machine learning approach generated sequences with greater affinity.

Dissection of Signaling Events Downstream of the c-Mpl Receptor in Murine Hematopoietic Stem Cells Via Motif-Engineered Chimeric Receptors.

Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs.

Quantitative control of intracellular signaling activity through chimeric receptors incorporating multiple identical tyrosine motifs.

Controlling activation levels and durations of native signaling molecules is important for efficiently controlling cellular fates. Previously we developed single-chain Fv (scFv)/cytokine receptor chimeras incorporating tyrosine motifs in the intracellular domain, which artificially control the activation of specific intracellular signaling proteins. In this study, to quantitatively control the activation levels of signaling molecules with an extended dynamic range, we constructed scFv/receptor chimeras incorporating multiple identical motifs at the different positions in the intracellular domain. We used retroviral transduction to express chimeric receptors with multiple STAT3 binding motifs connected with or without flexible linkers in a murine IL-3-dependent pro-B cell line, Ba/F3. Our results showed that the chimeric receptors can control the activation levels of STAT3 depending on ligand concentration and the number of motifs. The existence of linkers between the motifs also affected the signal intensity. Furthermore, the STAT3 activation levels significantly depended on the number of motifs rather than the distance from the JAK-binding region to the tyrosine motif.

Top-down motif engineering of a cytokine receptor for directing ex vivo expansion of hematopoietic stem cells.

The technique to expand hematopoietic stem cells (HSCs) ex vivo is eagerly anticipated to secure an enough amount of HSCs for clinical applications. Previously we developed a scFv-thrombopoietin receptor (c-Mpl) chimera, named S-Mpl, which can transduce a proliferation signal in HSCs in response to a cognate antigen. However, a remaining concern of the S-Mpl chimera may be the magnitude of the cellular expansion level driven by this molecule, which was significantly less than that mediated by endogenous wild-type c-Mpl. In this study, we engineered a tyrosine motif located in the intracellular domain of S-Mpl based on a top-down approach in order to change the signaling properties of the chimera. The truncated mutant (trunc.) and an amino-acid substitution mutant (Q to L) of S-Mpl were constructed to investigate the ability of these mutants to expand HSCs. The result showed that the truncated and Q to L mutants gave higher and considerably lower number of the cells than unmodified S-Mpl, respectively. The proliferation level through the truncated mutant was even higher than that of non-transduced HSCs with the stimulation of a native cytokine, thrombopoietin. Moreover, we analyzed the signaling properties of the S-Mpl mutants in detail using a pro-B cell line Ba/F3. The data indicated that the STAT3 and STAT5 activation levels through the truncated mutant increased, whereas activation of the Q to L mutant was inhibited by a negative regulator of intracellular signaling, SHP-1. This is the first demonstration that a non-natural artificial mutant of a cytokine receptor is effective for ex vivo expansion of hematopoietic cells compared with a native cytokine receptor.

Reconstitution of a cytokine receptor scaffold utilizing multiple different tyrosine motifs.

Cells have intracellular signal transduction systems to control cellular fates. Cytokine receptors initiate the intracellular signaling by recruiting specific signaling molecules to a range of tyrosine-containing motifs. To specifically activate a target signaling molecule, we previously designed single-chain Fv/cytokine receptor chimeras incorporating single tyrosine motifs in the intracellular domain, and each chimeric receptor activated the corresponding signaling molecule by oligomeric antigen stimulation. However, synergistic effects of multiple signaling pathways are indispensable to regulate complex cellular fates. In this study, we extended our approach by incorporating two different motifs in the chimeric receptor, which would result in the activation of multiple signaling molecules. We used retroviral transduction to express chimeric receptors in a murine interleukin-3-dependent pro-B cell line, Ba/F3. Our results indicate that the chimeric receptors incorporating two different motifs can activate both corresponding signaling molecules by the ligand stimulation, and that the signaling intensities are influenced by the distance between two motifs. Moreover, these chimeric receptors transduced downstream signaling, which exerted synergistic effects on cellular proliferation. Our system may be used for efficiently controlling fates of various types of cells, which will be applied to tissue engineering.

Activation of target signal transducers utilizing chimeric receptors with signaling-molecule binding motifs.

Cellular fates such as proliferation, differentiation, and death are controlled by a variety of cytokine receptors, which are crucial in initiating downstream signaling cascades. To initiate signaling, the cytokine receptor cytoplasmic domain recruits specific signaling molecules with a range of tyrosine-containing motifs. Thus, we postulate that it is possible to regulate signal transduction artificially by locating the tyrosine motif of interest into the intracellular domain of specific receptors. Construction of such artificial receptors was based on an anti-fluorescein ScFv/c-Mpl chimera (S-Mpl). We selected several known tyrosine motifs from native cytokine receptors that strongly bind to their target molecule, and located them downstream of the Janus kinase (JAK) binding domain of S-Mpl, which would be necessary for phosphorylation of the receptor. Next, we used retroviral transduction to express chimeric receptors in a murine IL-3-dependent pro-B cell line, Ba/F3, which was stimulated with BSA-fluorescein. The results indicated that each chimeric receptor preferentially activated the corresponding signaling molecule. We also examined whether the position of the tyrosine motif in the receptor could influence the activation levels of the signal transducer, and found that the chimeric receptors could activate the corresponding signaling molecule even when the tyrosine motif was distant from the JAK binding domain.