philadelphia positive acute lymphoblastic leukemia ( ph+ all ) is a high - risk , aggressive form of acute leukemia , affecting primarily adults and the elderly .
the hallmark of this disease is the presence in all leukemia cells of a reciprocal translocation termed t(9 ; 22)(q34 ; q11 ) resulting in a chimeric bcr - abl ( e1a2 breakpoint ) fusion gene that encodes a 190  kd protein ( p190 ) with constitutively active tyrosine kinase activity that can alter multiple signaling pathways , contributing to tumor growth and proliferation . before the advent of tyrosine kinase inhibitors ( tkis ) , the outcome of ph+ all patients not eligible for allogeneic stem cell transplant ( allo - sct ) was characterized by an extremely poor prognosis , a weak response to most chemotherapy combinations , short remission durations , and poor survival rates .
the introduction of imatinib , a selective inhibitor of the abl tyrosine kinase , has revolutionized the treatment and the outcome of this subset of patients . however , a substantial proportion of imatinib - treated ph+ all patients develop resistance to imatinib .
second - generation tkis have demonstrated promising efficacy in the treatment of imatinib - resistant ph+ all patients , but despite these results , the relapse rate of ph+ all patients remains very high with an overall survival still unsatisfactory .
the persistence of a measurable residual disease at molecular level appears to be the key issue for treatment failure [ 35 ] .
the development of alternative strategies that could selectively target ph+ all cells and synergistically work in combination with tki may have a crucial impact on disease control and ultimately patients ' survival . on this matter , a p190-specific active immune approach like a vaccine could meet these requirements . due to bcr - abl fusion
, the corresponding p190 joint region contains an amino acid sequence unique to the oncoprotein in addition to a novel amino acid , not belonging to either bcr or abl sequences , created at the exact fusion point .
thus , from an immunologic point of view , peptides derived from p190-breakpoint area are leukemia - specific antigens that may be employed as therapeutic vaccine with the purpose to induce a t cell response toward p190 + leukemia cells .
recently  natural  bcr - abl breakpoint - specific cytotoxic t lymphocytes ( ctls ) were found in the bone marrow of ph+ all patients treated with imatinib correlating with a better response to this tki .
these findings suggest a potential activity of the immune system against this lethal disease and the crucial role of p190 itself as target . in the present work we searched for p190-derived breakpoint peptides
previously , we have developed a p210-breakpoint derived penta - peptide vaccine for controlling minimal residual disease in chronic myeloid leukemia ( cml ) patients treated with imatinib . in this setting , we found that the best antileukemia immune response was mediated by cd4 + t cells specific for an hla class ii 
p210-breakpoint peptide - specific cd4 + t cells isolated from vaccinated patients were found to be either perforin+ or cd25+/foxp3 + : in both cases they exerted direct cytotoxic activity against a cml cell line . based on these premises , in our vaccine strategy for ph+ all , we focused our efforts in the search for p190 breakpoint peptides as strong inducers of a peptide - specific cd4 + t cell response .
our results show a promising p190-derived breakpoint peptide suitable for a peptide vaccine therapeutic approach in these patients .
to pursue our vaccine strategy for ph+ all we investigated the fusion region of p190 in search of novel 25-mer p190 breakpoint peptides with strong hla class ii binding prediction and thus potentially able to induce a strong cd4 + t cell stimulation .
the length of 25 amino acids has been chosen as maximum length that should contain all possible hla class ii molecules binding epitopes , usually from 13 to 23 amino acids long , always including the breakpoint and the new amino acid produced at the fusion point .
we analysed all 25 possible 25-mer long peptides that include the fusion point ( table 1 ) .
we employed syfpeithi database for mch ligands and peptides motifs and bimas database which allow to estimate the ligation strength to a defined hla type for a sequence of amino acids .
the level of binding was compared with our  binding prediction mean database  for hla class ii bcr - abl - derived peptides already employed in cml patients .
promising peptides will be synthesized on f - moc solid phase synthesis and purified by hplc for in vitro use . in order to evaluate the capability of p190-derived peptides to induce a peptide - specific cd4 + t cell in vitro
cd4 + t cells freshly isolated from pbmc were cultured for 21 days in 5% ab human serum media while undergoing to 3 rounds of stimulation with autologous cd14 + cells previously isolated and frozen .
cd4 + t cells were seeded in 24-well plates at 1  10  cell / ml together with cd14 + cells at 0.25  10  cell / ml ( 4  :  1 ratio ) and maintained in a humidified incubator with 5% co2 and 95% air at 37c for 7 days .
each test peptide was added at 20  g / ml and il-15 was added at final concentration of 10  ng / ml . after this time cd4 + t cells underwent the second round of stimulation :
the cells were collected , counted , and replaced in the same conditions with fresh cd14 + cells , peptides , and il-15 for other 7 days .
the peptide - specific cd4 + t cell proliferation was measured by standard thymidine incorporation assay and expressed by a stimulation index ( si = cpm cd4 + t cells plus test peptides / cd4 + t cell alone or cd4 + t cells plus control peptides ) .
briefly , cd4 + t cells were collected and incubated at 2  10 cells per well for 96 hours and maintained for 4 days in a humidified incubator with 5% co2 and 95% air at 37c with 20  g / ml peptide under the following experimental conditions : ( 1 ) no peptide , ( 2 ) each test peptide alone , and ( 3 ) wt1-derived 25 amino acid peptide alone as negative control .
the level of p190-peptide specific t cell proliferation was compared with our  stimulation index mean database  for hla class ii bcr - abl - derived peptides already employed in cml patients .
the latter allowed us to identify peptides with good probability to be sufficiently immunogenic and suitable for vaccination studies in vivo .
among all 25 possible peptide in the fusion region , we identified three promising 25-mer long p190 fusion peptides : pdngegafhgdaealqrpvasdfep ( p190 - 13 ) , dgegafhgdaealqrpvasdfepqg ( p190 - 15 ) , and egafhgdaealqrpvasdfepqgls ( p190 - 17 ) with strong hla binding properties for hla - drb1 * 0101 , hla - drb1 * 0401 , hla - drb1 * 1101 , and hla - drb1 * 0301 ( dr17 ) . subsequently p190 - 13 , p190 - 15 , and p190 - 17 have then been synthesized and purified for in vitro t cell stimulation testing . in vitro p190-derived peptide cd4
+ t cell stimulation was first performed in 5 normal subjects . in each healthy donor p190 - 13
, p190 - 15 , and p190 - 17 peptides were tested in separated experiments .
only p190 - 13 peptide was able to induce in all 5 subjects , regardless of their hla - dr phenotype , a peptide - specific cd4 + t cell proliferation as measured by standard thymidine assay , with an si ranging from 2.0 to 2.7 .
all patients had previously received a standard induction treatment followed by tkis therapy and were at least in hematologic remission at the time of in vitro cd4 + t cell experiment .
three of six were receiving dasatinib at 100  mg / day and 3/6 were receiving imatinib at 400  mg / day ( see table 2 ) .
again p190 - 13 peptide induced peptide - specific cd4 + t cell proliferation in 4/6 ph+ all patients with an si value of 2.2 , 2.0 , 2.2 , and 2.1 , respectively ( table 2 ) .
the ph+ all is a high - risk acute leukemia with poor prognosis , in which the specific t(9 ; 22)(q34 ; q11 ) translocation results in a chimeric bcr - abl ( e1a2 breakpoint ) and in a 190  kd protein ( p190 ) with constitutive tyrosine kinase activity . despite the promising results of tkis , the relapse rate of ph+ all patients remains very high with an overall survival still unsatisfactory . in ph+ all , leukemic cells could be targeted also by means of an active specific immune response raised against bcr - abl - derived p190 fusion protein that is indeed a leukemia - specific intracellular antigen . thus vaccinations with p190-derived peptides could induce in ph+ all patients peptide - specific t cell response that could control or even eradicate minimal residual disease thus reducing the probability of relapse in these patients . in an antitumor vaccine strategy
the choice of the appropriate peptide is crucial and we decided to focus our search on longer , hla class ii binder p190-breakpoint peptides , with the intent mainly to prompt a peptide - specific and possibly leukemia - specific cd4 + t cell response .
the present study identified p190 - 13 , a novel p190-derived 25-mer peptide that met these requirements .
in fact p190 - 13 was able to easily induce , in vitro , a peptide - specific cd4 + t cell response in all healthy donors and in 4/6 ph+ all patients tested .
of note , this peptide includes in its sequence all the epitopes for which p190-specific ctls were naturally found in all patients   and thus p190 - 13 could as well mediate a peptide - specific ctls response in this setting of patients . regarding the choice of a breakpoint peptide for developing a peptide vaccine strategy in ph+ all we are aware that all has a much more instability than cml and thus p190 has more probability to undergo mutations . even if the latter is true , it has to be underlined that p190 mutations do not usually appear in the breakpoint region which sequence is very stable through all the phases of the disease .
the little number of patients studied and the variability of age , chemotherapy induction treatment , and type of tki do not allow us to speculate further about the quality and intensity of the in vitro peptide - specific immune response observed .
however , it is encouraging that 4/6 ph+ all patients were able to mount an in vitro cd4 + t cell response against p190 - 13 while being treated with imatinib but also with second generation tkis , dasatinib .
the magnitude of p190-derived peptide - specific t cell response , as measured by the stimulation index , was the same in patients and healthy donors and was in the range of what we previously found in cml patients , confirming that all patients treated with tkis maintain a functional and reactive immune system even against a poorly immunogenic self oncoprotein like p190 .
for all these reasons our peptide could be a good candidate for developing an immune target vaccine strategy possibly synergizing with tkis for remission maintenance and reducing the probability of relapse in ph+ all patients .
these results support the rationale to offer to ph+ all elderly patients or adult patients not eligible for intensive chemotherapy and allo - sct , a line treatment without chemotherapy , by using a tki as debulking induction treatment and subsequently by adding a potentially synergic leukemia - specific immune approach such as a p190-derived peptide vaccine .
in conclusion novel identified p190 - 13 25-mer peptide is able to induce in vitro a peptide - specific cd4 + t cell response in ph+ all patients .
thus there appears a good candidate for developing an immune target vaccine strategy possibly synergizing with tkis for remission maintenance . a p190 - 13 peptide vaccine trial in ph+ all patients in complete hematologic remission during tkis is currently in planning .
 ph+ acute lymphoblastic leukemia ( ph+ all ) is a high - risk acute leukemia with poor prognosis , in which the specific t(9;22)(q34;q11 ) translocation results in a chimeric bcr - abl ( e1a2 breakpoint ) and in a 190  kd protein ( p190 ) with constitutive tyrosine kinase activity . 
 the advent of first- and second - generation tyrosine kinase inhibitors ( tkis ) improved the short - term outcome of ph+ all patients not eligible for allo - sct ; yet disease recurrence is almost inevitable . 
 peptides derived from p190-breakpoint area are leukemia - specific antigens that may mediate an antitumor response toward p190 + leukemia cells . 
 we identified one peptide named p190 - 13 able to induce in vitro peptide - specific cd4 + t cell proliferation in ph+ all patients in complete remission during tkis . 
 thus this peptide appears a good candidate for developing an immune target vaccine strategy possibly synergizing with tkis for remission maintenance . 