Adverse events were described and coded based on NCI CTCAE v4.03. A dose- limiting toxicity (DLT) was defined as any unexpected adverse event that is possibly, probably or definitely related and: (1) ocular ≥grade 1, (2) non- hematologic/non- metabolic ≥Grade 3, or (3) hematologic/metabolic ≥Grade 3. Toxicities with known or likely autoimmune features and affecting vital organs, including colitis, hepatitis, hypoph-ysitis, uveitis and adrenal insufficiency, were recorded and assessed with special interest. These were considered DLTs if ≥grade 2 and probably or definitely related. Grade 2 autoimmune toxicities involving these organs/tissues were not considered DLTs if only possibly related. Vitiligo was not considered a DLT, but was recorded. Hyper - thyroidism or hypothyroidism was considered a DLT if ≥grade 3. All vaccinated participants were followed for a minimum of 4 weeks for assessment of DLTs. Safety assessment The number of vaccinated participants who experienced DLTs guided decisions about safety. In the initial safety phase for arm A or Arm B, one participant was accrued. If that subject did not experience a DLT within 4 weeks, 2 additional participants were accrued. If 0/3 DLTs were observed, then the initial safety criteria for the arm were satisfied; if ≥2/3 participants experienced a DLT, then accrual to that arm would be halted permanently; other - wise three additional participants would be accrued to the arm to assess safety. Accrual to arm C occurred only after the initial safety criteria for arms A and B were satisfied. A maximum of nine participants were accrued in sequential cohorts of 3. Accrual continued as long as no more than 33% of participants experienced a DLT.ELISpot analysis of T-cell response to phosphopeptides Blood (100–140 mL) was drawn on days indicated in figure 1. PBMCs were isolated from 80 mL (except 120 mL week 0) using Ficoll gradient centrifugation and cryopre-served in 10% dimethyl sulfoxide/90% serum. Thawed PBMCs were incubated 2 hours at 37°C with 40 µg/mL each of pBCAR3 126-134, pIRS21097-1105 and pS33-βcat30-39 (control), washed to remove unbound peptide, and resuspended in medium containing 10 ng/mL recom- binant human IL-7 (Peprotech, Rocky Hill, New Jersey, USA) for 3 days. New medium containing recombinant human IL-7 plus 10 ng/mL IL-15 (Peprotech) was added on days 3, 7 and 10. Cells were assayed on day 14 for IFNγ production by ELISpot assay using methods and criteria described previously. 14 15 Briefly, T2- B7 cells were pulsed with each of the three phosphopeptides for each assay date. Negative controls included no peptide, irrelevant peptide (HIV gag peptide restricted by HLA- A2; Atlantic Peptides, LLC, Lewisburg, Pennsylvania, USA), and posi-tive controls included PMA- ionomycin, PHA. Responses were corrected for the fraction of CD8 T- cells in PBMC as determined by flow cytometry of individual samples at culture initiation. T- cell responses were calculated using the following definitions: Nvax=number T- cells responding to vaccine peptide; Nneg=number T- cells responding to maximum negative control; Rvax=Nvax/Nneg. A patient was considered to have a T- cell response to vaccination (binary yes/no), only if all of the following criteria were met: (1) Nvax exceeded Nneg by at least 100 spots/100 000 CD8 cells, (2) (Nvax – 1 SD) ≥ (Nneg +1 SD) and (3) Rvax after vaccination ≥2 x Rvax prevac- cine. Prevaccine Rvax values less than one (eg, control counts exceed number of responding T- cells) were set equal to one to indicate no response and to prevent overinflating adjusted fold- increases due to prevaccine ratios less than one or division by zero. Interassay coeffi-cients of variation (CVs) were calculated for the response of 2 normal donors to a mixture of viral peptides (CEF peptide pool, Proimmune, Sarasota, Florida, USA): for the high responder, mean number of spots per 1 00 000 cells was 309, and CV was 29%. Statistical assessment of immune response Since this was a first- in- humans clinical trial with phospho- peptide antigens for cancer, a first goal was to assess safety with a small study. The biological primary endpoint was evidence of immunogenicity. The prespecified level of interest was two or more patients with T- cell responses. Beyond that, CI for the observed proportions would determine how much the data supported immunoge-nicity. Assuming that the response to one phosphopep-tide is not affected by the presence of the other for those in arm C, the immunogenicity of each phosphopeptide was based on the total number of participants vaccinated with the specific phosphopeptide, alone or in combina-tion with the other (ie, Arm A+Arm C for pBCAR3 126-134; Arm B+Arm C for pIRS21097-1105). Based on the upper limit of a one- sided 90% CI, if the upper limit of the observed 6 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access bound was >35% to any single phosphopeptide, we would conclude that the phosphopeptides was immunogenic and worthy of further study. To define the CD8 T- cell response rate with higher precision would require a much larger sample size, which was not indicated in this first- in- humans trial.