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8-K - 8-K - Enumeral Biomedical Holdings, Inc.s102453_8k.htm
EX-99.2 - EXHIBIT 99.2 - Enumeral Biomedical Holdings, Inc.s102453_ex99-2.htm

Exhibit 99.1

B Discovery and Functional Characterization of Novel Anti-PD-1 Antibodies Using Ex VivoCell-Based Assays, Single-Cell Immunoprofiling, and In Vivo Studies in Humanized Mice Abstract Given recent approvals of anti-PD-1 inhibitors Keytruda (pembrolizumab)andOpdivo(nivolumab)fortreatmentofmetastatic melanomaandnon-smallcelllungcancerNSCLC,weaskedwhether additionalanti-PD-1inhibitorswithdifferentepitopesorpotentially differentiated mechanisms of action can provide clinical benefit beyondthetwomarketedtherapies.UsingEnumeral’sproprietary single cell technology, we generated more than 300 anti-PD-1 monoclonalantibodiesfromprimaryBcells.Bioinformaticsanalysisof sequences show these antibodies comprise 26 distinct clades, or families, that bind to PD-1. Results from binding studies further indicatewehavediscoveredafamilyofnovelantibodiesthatdonot appeartocompetewithcurrentlymarketedantibodiesforbindingto PD-1, nor do they appear to compete with PD-L1, suggesting a differentiatedmechanismofaction.Wehavehumanizedtwolead antibodiesforpreclinicaltestingandinpreparationforclinicalstudies. Here we describe functional characterization of the two lead antibodies using cell-based ex vivo assays, patient-derived tumor profilingassaysusingoursingle-cellplatform,andinvivostudiesin humanized NSG mice. Results show that the two lead anti-PD-1 antibodies exhibit a higher level of T cell activation in mixed lymphocytereaction(MLR)assaysusingprimaryhumanimmunecells. Second, these antibodies also demonstrated dose-dependent increasesinTcellCD25expression.Next,totesttheeffectofthetwo leadanti-PD-1antibodiesontumorgrowth,weconductedastudy usingapatient-derivedtumor(PDX)modelonhumanizedNSGmice. NSG mice reconstitute a full human immune systemincluding a functionalT,Bcellrepertoire,enablingthedirect,invivoevaluationof ourhumananti-PD-1antibodies. Figure 1. (A) Enumeral TargetDiscovery and (B) AntibodyDiscoveryPlatformsOverview (A)Analysisoftumorinfiltratedlymphocytes(TILs)ICexpression atthesinglecellfromhumantumorbiopsyusingEnumeral’s proprietarytechnology.Eachsinglecellisprofiledforsurface markerexpression,cytokinesecretion,andsubmittedforNGS. (B)Aftertargetidentification,immunizedanimalsarescreened using Enumeralmicro-engravingtechnology. Afterretrievalof antibody-secretingcells,heavyandlightchainsarerecoveredby single cell RT-PCR. Then, antibodies are cloned, tested for binding,andcharacterizedbiochemicallyandfunctionally. Conclusion Enumeral developed novel, fully humanized PD-1 blocking monoclonal antibodies, 388D4 and 244C8. 244C8 family of antibodies appears to have a novel mechanism of action, as they neither compete with currently marketed PD-1 antibodies for binding to PD-1 nor with PD-L1 binding, but induce expression of the high affinity IL-2 receptor alpha chain CD25. Both 388D4 and 244C8 can reverse T cell suppression in ex vivo TIL experiments in a subset of patient samples at least as potently as nivolimumab. Importantly, both 388D4 and 244C8 exhibited significant anti-tumor efficacy when tested in a NSCLC lung PDX model in vivo. These results demonstrate both ex vivo and in vivo function by our novelanti-PD-1 antibodies. B Figure 2. In Vitro Blockade with Enumeral’s anti-PD-1 AntibodiesCanRestoreTcellImmunity Enumeralanti-PD-1leadcandidatesweretestedfordifferentialTcell activationinresponsetoPD-1blockade invitro.Top2leadswere humanizedandtestedinamixedlymphocytereactionafter6daysof co-incubation of HLA-mismatched T cells and monocyte derived dendritic cells. T cell activation was assessed as inferred by secretion of effector cytokines such as interferon gamma (IFN?)(A)andupregulationofthehighaffinityIL-2receptorCD25 (B). Figure4.StudyoutlineforNSGPDXstudy 75humanized-NSGmicewereimplantedwithalungtumorPDX derived from a patient with metastatic non