PMC

Clinical responders to K562/GVAX vaccination make novel high-titer IgG responses to a broad range of LAAs. (a) Residual CML disease burden as determined by BCR–ABL values as determined by RQ-PCR from patient PBMC mRNA before and after vaccination (vertical dashed line represents primary J0345 vaccination series). (b) Number of LAAs recognized by serum IgG in each patient before (‘pre') and after primary vaccination (time on x axis in days). (c) Quantitative measurement of LAA-specific serum IgG. Maximal ‘titer' of induced responses was observed between day 42 and 84 time points (that is, 6–12 weeks). Normalized intensity is color-coded (lower limit of detection, 0.083 in dark blue, maximal in red) according to the scale in .

Discordant patterns of induced antibody response in identically vaccinated patients. (a) Color code for the heatmap of normalized intensity (∼titer) from dark blue (minimum/threshold of detection) to bright red (maximal, equal or greater to level seen for positive control). Antigens unrecognized at timepoint are coded in gray, missing samples are coded in white. (b) Normal donor reactivity against candidate LAAs. Threshold for detection was set at 85% of normal donor distribution to limit false positive results in patient dataset. As predicted by this, experimentally observed normal donor IgG reactivity to these 14 discovered LAAs was rare, and when responses were present they were low titer. (c) Normalized intensity heatmap of CML patient LAA-specific IgG responses before and after both primary and secondary vaccination. All patients received identical K562/GVAX whole-cell vaccine product at identical time points but exhibited markedly divergent responses.

Relationship between antigen mRNA expression levels in CML and K562 to timing of development of IgG antibody response. Secondary ‘booster' vaccination could elicit IgG LAA responses in patients who did not make a response to that antigen after the initial vaccination series. Figure shows percentage of patients making LAA-specific IgG responses before and after either the first vaccination series (n=19 patients, (a) top panel) or booster vaccination series (n=11 patients, (b) middle panel). Number of normal donors (ND; n=19) making responses to the same antigen is shown for comparison in both panels. Antigens more commonly recognized by patients after a secondary ‘boost' vaccination series (CDC25C, RHOXF2, HBG2 and RHAMM) have higher levels of mRNA expression in K562 (shown as fold expression over the mean expression in 8 normal donor samples) compared with levels in peripheral blood levels from eight ND or eight patients with chronic (CP) or accelerated (AP) phase CML (c) lower panel).

Role of patient HLA type in defining LAA IgG responses to K562/GVAX whole-cell vaccine immunotherapy. Molecular HLA typing was obtained from the Clinical Immunogenetics Laboratory of the Johns Hopkins University School of Medicine. The intra-patient variation in the induced IgG responses following vaccination with an identical K562/GVAX whole-cell vaccine product appears likely due not only to prior antigen experience and immunological memory but also to HLA differences between patients. Three examples are highlighted in the figure above; two clinical responders made responses to three of the same antigens (DNAJA1, RPS23 and RHOX). These patients shared both class I (HLA-A*0301) and class II alleles (DRB*1501) in their HLA haplotype. Additionally, 7 of the 19 patients in this study made a response to RHAMM. All had DRB15 and DQB*0602 alleles in their haplotype; indeed 7 of 8 patients with DRB15 and 7 of 7 patients with DQB*0602 in this study made responses to RHAMM. Finally, only two patients made an initial response to the antigen HN1L. They shared an almost identical HLA haplotype (HLA-A*0101, HLA-C*04KBG, HLA-B*035, HLA-DRB*0301 and HLA-DRQ*0201). No other patient in the study had this haplotype.

Clinical trials schema. We report here correlative immunological investigations of two consecutive immunotherapy pilot studies conducted at Johns Hopkins with patient accrual between 2005 and 2008. Left: the first trial (J0345) enrolled 19 patients with suboptimal clinical response after at least 1 year of imatinib for a study consisting of a 12-week pre-enrollment monitoring period, four vaccinations of irradiated K562/GM-CSF whole-cell vaccine given over a 9-week period followed by extended monitoring. Right: the second trial (J0625) offered a subset of patients with suboptimal clinical responses after the first vaccination series (n=11) a second series of 'booster' vaccinations given in the same manner. The mean time between the primary and booster vaccination series was 111 weeks (range 77–199 weeks). In both trials, vaccine product consisted of 1 × 10⁸ irradiated K562/GM-CSF cells (1.0 × 10⁷ cells per injection in 0.5 cc × 10 intradermal sites on the limbs) known to stably express >1000 ng of GM-CSF/10⁶ cells/24 h. Each injection was followed 3 hours later by the application of an adjuvant (toll-like receptor-7 agonist) 5% imiquimod cream (Aldara, Graceway Pharmaceuticals) on 9 of the 10 vaccination sites. All patients remained on imatinib therapy throughout both studies. The boxed time points were key for response assessment in this study.

Development of a quantitative infrared immunoscreening assay, Quan-SEREX. (a) The presence of antibodies from patient serum samples against the candidate antigens were assessed in patient serum at pre-vaccine and week 12 post-vaccine time point using traditional SEREX technology. The response is scored as being ‘no change' (A, top) if there is no difference between the pre- and post-vaccine samples, ‘increased' (A, middle) if it is present in both pre- and post-vaccine samples, but the signal intensity is increased in the post sample, or ‘induced' (A, bottom) if the signal is only observed in the post-vaccine sample. (b) Quantitative dot blot assay: Each of the 14 antigens as well as controls (encoded in phage clone lysate) were doted in triplicates on a bacterial lawn and incubated with an IPTG soaked nitrocellulose membrane. This membrane is subsequently incubated with patient serum at 1:200. Phage binding to serum IgG was detected with infrared labeled anti-human IgG, and intensity of each spot corresponding to a particular phage clone was determined as described in Materials and Methods. (c) Threshold setting: Density of normalized intensities to candidate antigens among 19 normal donor individuals. The bell shape curve in black solid line presented the empirical density of normalized intensities, and the approximating normal distribution was shown in red dash line. Short vertical strips in the bottom indicate raw data of normalized intensities. Threshold was set as 85th percentile of approximating normal distribution and indicated in blue vertical dash line. (d) Normalized intensity determined by Quan-SEREX is a surrogate for antibody titer: Boxplot of normalized intensities at 1:200 titration by maximum dilution levels. The phage clone lysate were dotted on a set of four different nitrocellulose membranes that were later processed with four different dilutions (1:200, 1:3000, 1:10 000, and 1:30 000) of patient serum. Maximum dilution level was defined as the maximum titration where a phage was considered as recognized. <1:200 box indicated those phages that failed to be recognized in any titrations. Normalized intensity at 1:200 titration was explored with an increasing trend by maximum dilution levels (correlation coefficient=0.86 based on Spearman's approach).