GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving β cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC).

Engineered red blood cells as an off-the-shelf allogeneic anti-tumor therapeutic.

Checkpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12.

Artificial Anti-Tumor Opsonizing Proteins with Fibronectin Scaffolds Engineered for Specificity to Each of the Murine FcγR Types.

We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins.

Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs.

The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset.

Protein Engineering and Selection Using Yeast Surface Display.

Yeast surface display is a powerful technology for engineering a broad range of protein scaffolds. This protocol describes the process for de novo isolation of protein binders from large combinatorial libraries displayed on yeast by using magnetic bead separation followed by flow cytometry-based selection. The biophysical properties of isolated single clones are subsequently characterized, and desired properties are further enhanced through successive rounds of mutagenesis and flow cytometry selections, resulting in protein binders with increased stability, affinity, and specificity for target proteins of interest.

Engineering fibronectin-based binding proteins by yeast surface display.

Yeast surface display (YSD) presents proteins on the surface of yeast through interaction of the agglutinin subunits Aga1p and Aga2p. The human 10th type III fibronectin (Fn3) is a small, 10-kDa protein domain that maintains its native fold without disulfide bonds. A YSD library of Fn3s has been engineered with a loop amino acid composition similar to that of human antibody complementarity-determining region heavy chain loop 3 (CDR-H3) and varying loop lengths, which has been shown to improve binding ability.

Engineering new protein-protein interactions on the β-propeller fold by yeast cell surface display.

REINVENTING THE WHEEL: The β-propeller domain folds like a wheel to provide key protein-protein interactions in the cell. Here we used high-throughput yeast sorting to "invent" β-propellers of new binding specificities with cellular targets.

Triepitopic antibody fusions inhibit cetuximab-resistant BRAF and KRAS mutant tumors via EGFR signal repression.

Dysregulation of epidermal growth factor receptor (EGFR) is a hallmark of many epithelial cancers, rendering this receptor an attractive target for cancer therapy. Much effort has been focused on the development of EGFR-directed antibody-based therapeutics, culminating in the clinical approval of the drugs cetuximab and panitumumab. Unfortunately, the clinical efficacy of these drugs has been disappointingly low, and a particular challenge to targeting EGFR with antibody therapeutics has been resistance, resulting from mutations in the downstream raf and ras effector proteins.

Role of dimerization in the catalytic properties of the Escherichia coli disulfide isomerase DsbC.

The bacterial protein-disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two alpha-helical linkers, and two opposing thioredoxin fold catalytic domains. The functional significance of the two catalytic domains of DsbC is not well understood yet. We have engineered heterodimer-like DsbC derivatives covalently linked via (Gly(3)-Ser) flexible linkers.