Presentation_1_Reprogramming of IL-12 secretion in the PDCD1 locus improves the anti-tumor activity of NY-ESO-1 TCR-T cells.pptx

IntroductionAlthough the engineering of T cells to co-express immunostimulatory cytokines has been shown to enhance the therapeutic efficacy of adoptive T cell therapy, the uncontrolled systemic release of potent cytokines can lead to severe adverse effects. To address this, we site-specifically inserted the interleukin-12 (IL-12) gene into the PDCD1 locus in T cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing to achieve T-cell activation-dependent expression of IL-12 while ablating the expression of inhibitory PD-1.MethodsNew York esophageal squamous cell carcinoma 1(NY-ESO-1)-specific TCR-T cells was investigated as a model system. We generated ΔPD-1-IL-12 -edited NY-ESO-1 TCR-T cells by sequential lentiviral transduction and CRISPR knock-in into activated human primary T cells.ResultsWe showed that the endogenous PDCD1 regulatory elements can tightly control the secretion of recombinant IL-12 in a target cell-dependent manner, at an expression level that is more moderate than that obtained using a synthetic NFAT-responsive promoter. The inducible expression of IL-12 from the PDCD1 locus was sufficient to enhance the effector function of NY-ESO-1 TCR-T cells, as determined by upregulation of effector molecules, increased cytotoxic activity, and enhanced expansion upon repeated antigen stimulation in vitro. Mouse xenograft studies also revealed that PD-1-edited IL-12-secreting NY-ESO-1 TCR-T cells could eliminate established tumors and showed significantly greater in vivo expansion capacity than control TCR-T cells.DiscussionOur approach may provide a way to safely harness the therapeutic potential of potent immunostimulatory cytokines for the development of effective adoptive T cell therapies against solid tumors.</p

Synthesis of Bispecific Antibodies using Genetically Encoded Unnatural Amino Acids

Bispecific antibodies were constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. A two-step process afforded homogeneous products in excellent yield. Using this approach, we synthesized an anti-HER2/anti-CD3 bispecific antibody, which efficiently cross-linked HER2+ cells and CD3+ cells. <i>In vitro</i> effector-cell mediated cytotoxicity was observed at picomolar concentrations

A Genetically Encoded aza-Michael Acceptor for Covalent Cross-Linking of Protein–Receptor Complexes

Selective covalent bond formation at a protein–protein interface potentially can be achieved by genetically introducing into a protein an appropriately “tuned” electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNA_CUA pairs that genetically encode three aza-Michael acceptor amino acids, <i>N</i>^ε-acryloyl-(<i>S</i>)-lysine (AcrK, <b>1</b>), <i>p</i>-acrylamido-(<i>S</i>)-phenylalanine (AcrF, <b>2</b>), and <i>p</i>-vinylsulfonamido-(<i>S</i>)-phenylalanine (VSF, <b>3</b>), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 <i>in vitro</i> or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein <i>in vitro</i> or in living cells

Self-Assembled Antibody Multimers through Peptide Nucleic Acid Conjugation

With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here, we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody–oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency, and geometry. With this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and <i>in vitro</i> characterization of antibody constructs with unique specificities and molecular architectures

Engineering Bifunctional Antibodies with Constant Region Fusion Architectures

We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies have excellent <i>in vitro</i> activity, physiochemical properties, and stability. Among these, a Her2 × CD3 bispecific antibody (BsAb) was constructed by inserting an anti-Her2 single-chain variable fragment (ScFv) into an anti-CD3 Fab. This bispecific antibody efficiently induces targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations <i>in vitro</i>. Moreover, the Her2 × CD3 BsAb shows potent <i>in vivo</i> antitumor activity in mouse Her2²⁺ and Her2¹⁺ xenograft models. These results demonstrate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multifunctional antibodies for therapeutic applications

Redirection of Genetically Engineered CAR‑T Cells Using Bifunctional Small Molecules

Chimeric antigen receptor (CAR)-engineered T cells (CAR-Ts) provide a potent antitumor response and have become a promising treatment option for cancer. However, despite their efficacy, CAR-T cells are associated with significant safety challenges related to the inability to control their activation and expansion and terminate their response. Herein, we demonstrate that a bifunctional small molecule “switch” consisting of folate conjugated to fluorescein isothiocyanate (folate-FITC) can redirect and regulate FITC-specific CAR-T cell activity toward folate receptor (FR)-overexpressing tumor cells. This system was shown to be highly cytotoxic to FR-positive cells with no activity against FR-negative cells, demonstrating the specificity of redirection by folate-FITC. Anti-FITC-CAR-T cell activation and proliferation was strictly dependent on the presence of both folate-FITC and FR-positive cells and was dose titratable with folate-FITC switch. This novel treatment paradigm may ultimately lead to increased safety for CAR-T cell immunotherapy

An Immunosuppressive Antibody–Drug Conjugate

We have developed a novel antibody–drug conjugate (ADC) that can selectively deliver the Lck inhibitor dasatinib to human T lymphocytes. This ADC is based on a humanized antibody that selectively binds with high affinity to CXCR4, an antigen that is selectively expressed on hematopoietic cells. The resulting dasatinib–antibody conjugate suppresses T-cell-receptor (TCR)-mediated T-cell activation and cytokine expression with low nM EC₅₀ and has minimal effects on cell viability. This ADC may lead to a new class of selective immunosuppressive drugs with improved safety and extend the ADC strategy to the targeted delivery of kinase inhibitors for indications beyond oncology