Patent Application: US-37764707-A

Abstract:
briefly , the present invention refers to the transcription factors pax2 and pax8 expressed in solid tumours and haematologic malignancies , and their utility as a target in immunotherapy and molecular therapy . in more detail , the invention refers to a method for identifying an immunogenic t - cell epitope from pax2 and / or pax8 . furthermore , the invention refers to a use of immunogenic t - cell epitopes , e . g . identified by said method , and their use as targets for the recognition by targeting means , e . g . t - cells or antibodies . the invention also refers to peptides representing immunogenic t - cell epitopes and their uses for the preparation of a pharmaceutical composition for immunotherapy of pax2 and / or pax8 expressing malignancies .

Description:
identification of hla - a2 positive pax2 peptides as targets for t - cell recognition in cancer patients peripheral blood mononuclear cells ( pbmc ) from 19 hla - a2 positive colorectal cancer patients and 10 healthy subjects were collected and cryopreserved . the investigation had been approved by the institutional ethics committee and informed consent was obtained from all individuals . prediction of candidate 9 - mer and 10 - mer epitopes according to the hla - a2 motif from pax2 ( swissprot accession no . aac63385 ) was performed using the syfpeithi algorithm , and 11 - to 13 - mer candidate epitopes , 37 epitopes in total ( seq id no . 1 to 37 ) were calculated according to the same prediction model as described ( 16 ). using this approach , 13 peptides ( seq id no . 1 to 6 , 16 , 20 to 22 , 26 , 29 , and 32 ) were selected as candidate t - cell epitopes ( table i , see left column ). peptides were synthesized in an applied biosystems 432a peptide synthesizer following standard protocols . synthesized products were analysed by high - performance liquid chromatography ( varian star ; zinsser analytics , munich , germany ) and maldi - tof mass spectometry ( g2025a ; hewlett - packard , waldbronn , germany ), and purified by preparative hplc to purities & gt ; 95 %. antigen - specific t - cells were detected by a functional assay . intracellular ifnγ accumulation induced by wt1 peptide was assessed by flow cytometry as described previously ( 2 ). screening of t - cell reactivity to predicted epitopes in colorectal cancer patients using functional flow cytometry detecting peptide - induced induction of intracellular ifnγ we analyzed circulating t - cells from 19 hla - a2 positive colorectal cancer patients for recognition of the 13 pax2 candidate epitopes . results of the flow cytometric analysis of t - cell responses to hla - a2 binding candidate epitopes from pax2 in the 19 patients are summarized in table ii . frequencies of specific cd3 + cd8 + t - cells secreting ifnγ in response to a pool of 2 or 3 pax2 peptides as well as to an irrelevant hla - a2 binding peptide from hiv are shown . of 19 hla - a2 positive patients analysed , 5 patients showed a t - cell response against a mixture of the three peptides 3520 ( seq id no . 16 ), 3550 ( seq id . no . 29 ), and 3553 ( seq id no . 32 ) ( table ii , see right column ). two further patients showed a t - cell response against a mixture of two peptides , namely peptides 3496 ( seq id no . 5 ) and 3497 ( seq id no . 6 ), and peptides 3519 ( seq id no . 20 ) and 3518 ( seq id no . 21 ), respectively . no t - cell response against these peptides was observed in 10 healthy hla - a2 positive subjects ( table iii ). these findings suggest that similar to wt1 , pax2 may be recognized in tumour - bearing patients . in 10 healthy donors ( ifn - γ plus t - cells in % of cd3 + cd8 + t - cells ) these data show for the first time that peptides derived from pax2 epitopes , and thus probably also peptides derived from pax8 epitopes , have the potential to induce a t - cell response . thus , immunogenic t - cell epitopes of pax2 , and probably of pax8 , can serve as targets in immunotherapy . in a patient with renal cancer we were able to induce specific t - cells against the peptide 3496 ( seq id no . 5 ), and could detect by cr release assay specific cytolysis of both t2 - cells loaded with 3496 peptide ( 32 % killing at 10 : 1 e : t versus 8 % killing of hiv peptide loaded t2 ) and colon cancer cell line sw480 ( 28 % which could be inhibited by cold target to 10 %). a panel of colorectal cancer cell lines has been characterized expressing both pax2 and pax8 to function as targets for recognition by specific t - cells ( table iv ). these data confirm that peptides derived from pax2 epitopes , and thus probably also peptides derived from pax8 epitopes , are indeed able to raise a t - cell response in a subject . for vaccination with pax2 or pax8 epitopes , the protocol given in ( 1 ) describing the vaccination with wt1 . 126 - 134 in a hla - a2 ( hla - a * 0201 ) positive patient is applied . an hla - a2 positive patient diagnosed with pax2 positive cancer receives eight biweekly vaccinations with the peptide in a dose of 0 . 2 mg admixed with 1 mg keyhole limpet hemocyanin ( klh , immucothel , biosyn , germany ) as adjuvant i . d . and s . c . on day 0 . gm - csf ( leukomax , essex pharma , germany ) in a dose of 75 μg per day is injected s . c . at the same site ( proximal thigh ) as the wt peptide on days − 2 to + 1 . the combination of both ajuvants is chosen due to the immunological efficacy in melanoma peptide vaccination ( 23 ). vaccination cycles can be repeated if necessary . during the course of therapy , the patient is monitored with respect to common diagnostic parameters such as blood counts and clinical biochemistry . t - cell response to pax2 peptides is performed in a peripheral blood using tetramer and intracellular ifnγ staining by flow cytometry ( 2 ). using the databases for epitope prediction syfpeithi ( www . syfpeithi . de ) and bimas ( http :// bimas . cit . nih . gov / molbio / hla_bind /) and the database paproc for prediction of proteasomal cleavage sites ( www . paproc . de ), 6 potential t - cell epitopes from pax8 for hla - a2 binding were predicted ( table v ). to identify t - cell epitopes , they were screened for recognition by t - cells from patients with renal cell and ovarian cancer which are pax8 positive tumours secreting cytokine ifnγ or tnfα in response to pax 8 or control hiv peptide . by using 4 pools of three peptides each , every peptide was present in two pools . two epitopes , 277 - 285 and 323 - 333 , were recognized in both pools , thus verifying them as epitopes ( table vi ). carcinoma patients ( ifnγ / tnfα plus t - cells in % of cd3 + cd8 + lymphocytes ) 1 . keilholz u , menssen h d , gaiger a et al . wilms &# 39 ; 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