Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

Gene editing can be used to overcome allo-recognition, which otherwise limits allogeneic T cell therapies. Initial proof-of-concept applications have included generation of such "universal" T cells expressing chimeric antigen receptors (CARs) against CD19 target antigens combined with transient expression of DNA-targeting nucleases to disrupt the T cell receptor alpha constant chain (TRAC). Although relatively efficient, transgene expression and editing effects were unlinked, yields variable, and resulting T cell populations heterogeneous, complicating dosing strategies. We describe a self-inactivating lentiviral "terminal" vector platform coupling CAR expression with CRISPR/Cas9 effects through incorporation of an sgRNA element into the ΔU3 3' long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid ΔU3-sgRNA, delivery of Cas9 mRNA resulted in targeted TRAC locus cleavage and allowed the enrichment of highly homogeneous (>96%) CAR (>99%) TCR populations by automated magnetic separation. Molecular analyses, including NGS, WGS, and Digenome-seq, verified on-target specificity with no evidence of predicted off-target events. Robust anti-leukemic effects were demonstrated in humanized immunodeficient mice and were sustained longer than by conventional CARTCR T cells. Terminal-TRAC (TT) CAR T cells offer the possibility of a pre-manufactured, non-HLA-matched CAR cell therapy and will be evaluated in phase 1 trials against B cell malignancies shortly.

! 1! Generation) of) human) islet0specific) regulatory) T) cells) by) TCR) gene) transfer.) )Caroline)M.)Hull 1,5) *,)Lauren)E.)Nickolay 1,5 ,)Megan)Estorninho 1,5 ,)Max)W.) Richardson 2 ,)James)L.)Riley 2 ,)Mark)Peakman 1,3,4,5 ,)John)Maher 5,6,7 ,)Timothy) I.M.)Tree 1,5) *) ) 1 Department)of)Immunobiology,)Faculty)of)Life)Sciences)&)Medicine,)King's) College)London,)London)SE1)9RT,)UK) ) 2 Department)of)Microbiology,)University)of)Pennsylvania)School)of)Medicine,) Philadelphia,)Pennsylvania,)USA) ) 3 Peak)Therapeutics,)London)SE24)9LG,)UK ) 4 Division)of)Diabetes)and)Nutrition,)King's)College)London)SE1)1UL,)UK) ) 5 NIHR)Biomedical)Research)Centre,)Guy's)and)St)Thomas')NHS)Foundation) Trust)and)King's)College)London,)London)SE1)9RT,)UK) ) 6) CAR)Mechanics)Group,)Division)of)Cancer)Studies,)Guy's)Hospital)Campus,) King's)College)London)School)of)Medicine,)London)SE1)1UL,)UK) ) 7 Department)of)Allergy)and)Immunology,)King's)College)Hospital)National) Health)Service)Foundation)Trust,)London)SE5)9RS,)UK) ) ) Short)Running)Title:)Generation)of)human)islet[specific)Tregs.) ) ) ) *Corresponding)authors) ) caroline.hull@kcl.ac.uk) timothy.tree@kcl.ac.uk) ) ! 2! Abstract) )Based)on)the)success)in)animal)models)of)type)1)diabetes)(T1D),)clinical)trials) of) adoptive) regulatory) T) cell) (Treg)) therapy) are) underway) using) ex# vivo) expanded)polyclonal)Tregs.)However,)pre[clinical)data)also)demonstrate)that)islet[specific) Tregs) are) more) potent) than) polyclonal) Tregs) at) reversing) T1D.)Translation) of) this) approach) into) man) will) require) methods) to) generate) large) populations) of) islet[specific) Tregs) which,) to) date,) has) proved) to) be) a) major)hurdle.)Here)we)demonstrate)the)feasibility)of)lentiviral[mediated)T)cell)receptor) (TCR))gene)transfer)to)confer)antigen)specificity)on)polyclonal)human)Tregs.)Targeting) has) been) achieved) using) TCRs) isolated) from) human) islet [specific) and) viral[specific) CD4+) T) cell) clones.) Engineered) T) cells) demonstrated) expression)of)ectopically[delivered)TCRs,)resulting)in)endowment)of)cognate)antigen[specific) responses.) This) enabled) antigen[specific) suppression) at) increased)potency)compared)to)polyclonal)Tregs.)However,)cells)transduced) with) islet[specific) TCRs) were) less) responsive) to) cognate) antigen) than) viral[ specific) TCRs,) and) in) some) cases,) required) additional) methods) to) isolate) functional)antigen[specific)Tregs.)This)study)demonstrates)the)potential)of)TCR) gene)transfer)to)develop)islet[specific)Treg)therapies)for)effective)treatment)of) T1D,)but)also)highlights)that)additional)optimisation)may)be)required)to)achieve)Populations) of) Tregs) found) in) the) periphery,) including) those) expressing) the)with)T1D.)This)abnormality)is)evident)before)clinical)diagnosis,)at)the)time)of) diagnosis) and) many) years) following) onset) of) T1D [4[7].) Since) defective) Treg) function) appears) to) be) central) to) the) pathogenesis) of) T1D,) it) is) logical) to) hypothesise) that) correction) of) this) imbalance) may...

In the Guillain-Barré syndrome subform acute motor axonal neuropathy (AMAN), Campylobacter jejuni enteritis triggers the production of anti-ganglioside Abs (AGAbs), leading to immune-mediated injury of distal motor nerves. An important question has been whether injury to the presynaptic neuron at the neuromuscular junction is a major factor in AMAN. Although disease modeling in mice exposed to AGAbs indicates that complement-mediated necrosis occurs extensively in the presynaptic axons, evidence in humans is more limited, in comparison to the extensive injury seen at nodes of Ranvier. We considered that rapid AGAb uptake at the motor nerve terminal membrane might attenuate complement-mediated injury. We found that PC12 rat neuronal cells rapidly internalized AGAb, which were trafficked to recycling endosomes and lysosomes. Consequently, complement-mediated cytotoxicity was attenuated. Importantly, we observed the same AGAb endocytosis and protection from cytotoxicity in live mouse nerve terminals. AGAb uptake was attenuated following membrane cholesterol depletion in vitro and ex vivo, indicating that this process may be dependent upon cholesterol-enriched microdomains. In contrast, we observed minimal AGAb uptake at nodes of Ranvier, and this structure thus remained vulnerable to complement-mediated injury. These results indicate that differential endocytic processing of AGAbs by different neuronal and glial membranes might be an important modulator of site-specific injury in acute AGAb-mediated Guillain-Barré syndrome subforms and their chronic counterparts.