Patent Application: US-201314417752-A

Abstract:
peptides and vaccine compositions comprising peptides based upon egfrviii and lacking a glycine at the splice junction are disclosed . the vaccines can induce immune responses against egfrviii . methods of inhibiting formation or growth of egfviii tumors , methods of inducing regression of egfviii tumors , methods of immunizing against egfviii tumors and methods of treating a subjects who have egfviii tumors are disclosed .

Description:
vaccines which comprise a peptide sequence similar to the fusion junction of egfrviii including a substitution of the glycine residue formed at the splice junction of the egfrviii receptor are provided . the peptides used in the vaccine are sufficiently similar to egfrviii such that an immune response generated against the peptides cross - reacts to egfrviii expressed on cancer cells . generally , the peptides contain portions similar to each of the sequences from the two formerly distant portions of the normal egf receptor . it is preferred that the vaccine comprises a peptide conjugated to a hapten / carrier such as keyhole limpet hemocyanin ( klh ), bovine serum albumin ( bsa ) or human serum albumin ( has ). in some embodiments , the vaccines comprise a peptide selected from the group consisting of leekkanyvvtdh ( seq id no : 3 ), leekkvnyvvtdh ( seq id no : 4 ), and leekkpnyvvtdh ( seq id no : 5 ). in some embodiments , peptides comprise a c - terminal cysteine . in some embodiments , the vaccines comprise a peptide selected from the group consisting of leekkanyvvtdhc ( seq id no : 6 ), leekkvnyvvtdhc ( seq id no : 7 ), and leekkpnyvvtdhc ( seq id no : 8 ). in some embodiments , the vaccines comprise a peptide linked to klh and are selected from the group consisting of : leekkanyvvtdhc : klh ( seq id no : 6 conjugated to keyhole limpet hemocyanin ), leekkvnyvvtdhc : klh ( seq id no : 7 conjugated to keyhole limpet hemocyanin ), and leekkpnyvvtdhc : klh ( seq id no : 8 conjugated to keyhole limpet hemocyanin ) other peptides based upon the egfrviii sequence with substitutions of the splice junction glycine may also be used in the vaccines and methods . in some embodiments , peptides , which are optionally linked to a carrier such as for example klh , bsa or has , have the formula : α is absent , acyl , leu - glu ; glu ; or leu ; xaa is ala , arg , asn , asp , cys , gln , glu , his , ile , leu , lys , met , phe , pro , ser , thr , trp , tyr or val ; and β is absent , asn , asn - tyr , asn - tyr - val , asn - tyr - val - val , asn - tyr - val - val - thr ( seq id no : 9 ), asn - tyr - val - val - thr - asp ( seq id no : 10 ), asn - tyr - val - val - thr - asp - his ( seq id no : 11 ), asn - tyr - val - val - thr - asp - his - cys ( seq id no : 12 ); tyr , tyr - val , tyr - val - val , tyr - val - val - thr ( seq id no : 13 ), tyr - val - val - thr - asp ( seq id no : 14 ), tyr - val - val - thr - asp - his ( seq id no : 15 ), tyr - val - val - thr - asp - his - cys ( seq id no : 16 ), val , val - val , val - val - thr , val - val - thr - asp ( seq id no : 17 ), val - val - thr - asp - his ( seq id no : 18 ), val - val - thr - asp - his - cys ( seq id no : 19 ), val - thr , val - thr - asp , val - thr - asp - his ( seq id no : 20 ), val - thr - asp - his - cys ( seq id no : 21 ), thr , thr - asp , thr - asp - his , or thr - asp - his - cys ( seq id no : 22 ), asp , asp - his , asp - his - cys , his , his - cys , or cys . in some embodiments a carrier is optionally linked to a terminal residue such as cys and if present the carrier is preferably a hapten . in some embodiments , the carrier is klh , bsa or hsa . in some embodiments , the vaccines comprise compounds which are optionally linked to a carrier such as for example klh , bsa or has and have the formula : l1 is absent or leu ; e2 is absent or glu ; xaa6 is ala , arg , asn , asp , cys , gln , glu , his , ile , leu , lys , met , phe , pro , ser , thr , trp , tyr or val ; n7 is absent or asn ; y8 is absent or tyr ; v9 is absent or val ; v10 is absent or val ; t11 is absent or thr ; d12 is absent or asp ; h13 is absent or his ; c14 is cys or a linking moiety that can link the peptide to carrier ; and carrier is optional and if present is preferably a hapten . in some embodiments , the carrier is klh , bsa or hsa . in some embodiments the peptide of the vaccine has formula ii wherein l1 - e2 - glu - lys - lys - xaa6 - n7 - y8 - v9 - v10 - t11 - d12 - h13 - c14 and the vaccine comprises at least 4 amino acids , at least 5 amino acids , at least 6 amino acids , at least 7 amino acids , at least 8 amino acids , at least 9 amino acids , at least 10 amino acids , at least 11 amino acids , at least 12 amino acids , at least 13 amino acids or at least 14 amino acids . in some embodiments , two or more of l1 , e2 , n7 , y8 , v9 , v10 , t11 , d12 , h13 and c14 are absent . in some embodiments , l1 and e2 are absent . in some embodiments , l1 and n7 are absent . in some embodiments , l1 and y8 are absent . in some embodiments , l1 and v9 are absent . in some embodiments , l1 and v10 are absent . in some embodiments , l1 and t11 are absent . in some embodiments , l1 and d12 are absent . in some embodiments , l1 and h13 are absent . in some embodiments , l1 and c14 are absent . in some embodiments , e2 and n7 are absent . in some embodiments , e2 and y8 are absent . in some embodiments , e2 and v9 are absent . in some embodiments , e2 and v10 are absent . in some embodiments , e2 and t11 are absent . in some embodiments , e2 and d12 are absent . in some embodiments , e2 and h13 are absent . in some embodiments , e2 and c14 are absent . in some embodiments , n7 and y8 are absent . in some embodiments , n7 and v9 are absent . in some embodiments , n7 and v10 are absent . in some embodiments , n7 and t11 are absent . in some embodiments , n7 and d12 are absent . in some embodiments , n7 and h13 are absent . in some embodiments , n7 and c14 are absent . in some embodiments , y8 and v9 are absent . in some embodiments , y8 and v10 are absent . in some embodiments , y8 and t11 are absent . in some embodiments , y8 and d12 are absent . in some embodiments , y8 and h13 are absent . in some embodiments , y8 and c14 are absent . in some embodiments , v9 and v10 are absent . in some embodiments , v9 and t11 are absent . in some embodiments , v9 and d12 are absent . in some embodiments , v9 and h13 are absent . in some embodiments , v9 and c14 are absent . in some embodiments , v10 and t11 are absent . in some embodiments , v10 and d12 are absent . in some embodiments , v10 and h13 are absent . in some embodiments , v10 and c14 are absent . in some embodiments , t11 and d12 are absent . in some embodiments , t11 and h13 are absent . in some embodiments , t11 and c14 are absent . in some embodiments , d12 and h13 are absent . in some embodiments , d12 and c14 are absent . in some embodiments , h13 and c14 are absent . in some embodiments , three or more of l1 , e2 , n7 , y8 , v9 , v10 , t11 , d12 , h13 and c14 are absent . in some embodiments , four or more of l1 , e2 , n7 , y8 , v9 , v10 , t11 , d12 , h13 and c14 are absent . in some embodiments , five or more of l1 , e2 , n7 , y8 , v9 , v10 , t11 , d12 , h13 and c14 are absent . in some embodiments , six or more of l1 , e2 , n7 , y8 , v9 , v10 , t11 , d12 , h13 and c14 are absent . in some embodiments , seven or more of l1 , e2 , n7 , y8 , v9 , v10 , t11 , d12 , h13 and c14 are absent . in some embodiments , eight or more of l1 , e2 , n7 , y8 , v9 , v10 , t11 , d12 , h13 and c14 are absent . in some embodiments , nine or more of l1 , e2 , n7 , y8 , v9 , v10 , t11 , d12 , h13 and c14 are absent . in some embodiments , each of l1 , e2 , n7 , y8 , v9 , v10 , t11 , d12 , h13 and c14 are absent . xaa may be ala , arg , asn , asp , cys , gln , glu , his , ile , leu , lys , met , phe , pro , ser , thr , trp , tyr or val . the manufacture of peptides is well known . automated peptide synthesizers may be employed to produce the peptides using techniques that are well known to those having ordinary skill in the art . one having ordinary skill in the art can generate a nucleic acid molecule that encodes a peptide or a protein comprising a peptide and insert it into an expression vector using standard techniques and readily available starting materials . the cloning and expression of proteins is well known as is their purification using for example immunoaffinity , charge or size exclusion . in some embodiments , the peptide may be linked to a carrier or haptenized to increase immunogenicity . in some cases , the haptenization is the conjugation of a larger molecular structure to the peptide . haptenization is well known and can be readily performed . haptenization methods which may be adapted to be used to prepare haptenized peptides include those described in the following u . s . patents which are each incorporated herein by reference : u . s . pat . no . 5 , 037 , 645 issued aug . 6 , 1991 to strahilevitz ; u . s . pat . no . 5 , 112 , 606 issued may 12 , 1992 to shiosaka et al . ; u . s . pat . no . 4 , 526 , 716 issued jul . 2 , 1985 to stevens ; u . s . pat . no . 4 , 329 , 281 issued may 11 , 1982 to christenson et al . ; and u . s . pat . no . 4 , 022 , 878 issued may 10 , 1977 to gross . peptide vaccines and methods of enhancing immunogenicity of peptides which may be adapted to modify peptides are also described in francis et al . 1989 methods of enzymol . 178 : 659 - 676 , which is incorporated herein by reference . sad et al . 1992 immunolology 76 : 599 - 603 , which is incorporated herein by reference , teaches methods of making immunotherapeutic vaccines by conjugating gonadotropin releasing hormone to diphtheria toxoid . peptides may be similarly conjugated to produce an immunotherapeutic vaccine of the present invention . maclean et al . 1993 cancer immunol . immunother . 36 : 215 - 22 . 2 , which is incorporated herein by reference , describes conjugation methodologies for producing immunotherapeutic vaccines which may be adaptable to produce an immunotherapeutic vaccine of the present invention . the hapten is keyhole limpet hemocyanin which may be conjugated to a peptide . pharmaceutical formulations comprising peptides and conjugated peptides may be routinely formulated by one having ordinary skill in the art . suitable pharmaceutical formulations and components are described in remington &# 39 ; s pharmaceutical sciences , a . osol , a standard reference text in this field , which is incorporated herein by reference . in some embodiments , for example , the vaccine can be formulated as a solution , suspension , emulsion or lyophilized powder in association with a pharmaceutically acceptable vehicle . examples of such vehicles are water , saline , ringer &# 39 ; s solution , dextrose solution , and 5 % human serum albumin . liposomes and nonaqueous vehicles such as fixed oils may also be used . the vehicle or lyophilized powder may contain additives that maintain isotonicity ( e . g ., sodium chloride , mannitol ) and chemical stability ( e . g ., buffers and preservatives ). an injectable composition may comprise the peptide or conjugated peptide in a diluting agent such as , for example , sterile water , electrolytes / dextrose , fatty oils of vegetable origin , fatty esters , or polyols , such as propylene glycol and polyethylene glycol . the vaccines may also comprise an adjuvant . adjuvants useful in vaccine are well known to those of skill in the art , thus , selection of an appropriate adjuvant can be performed routinely by one of skill in the art upon this disclosure . examples of useful adjuvant include , but are not limited to , complete and incomplete freund &# 39 ; s , mineral gels such as aluminum hydroxide , surface active substances such as lysolecithin , pluronic polyols , polyanions , peptides and oil emulsions . in some embodiments , the vaccine is an injectable composition that is sterile , pyrogen free , formulated to be isotonic and free of particulates . the standards of purity required for injectable compositions are well known as are the production and purification methods used to prepare injectable compositions . the vaccines may be administered by any means that enables the immunogenic agent to be presented to the body &# 39 ; s immune system for recognition and induction of an immunogenic response . pharmaceutical injectable compositions may be administered parenterally , i . e ., intravenous , subcutaneous , intramuscular . in some embodiments , pharmaceutical vaccine compositions may be administered intranasally or to tissue in the oral cavity such as by administration sublingually or to buccal tissue . dosage varies depending upon known factors such as the pharmacodynamic characteristics of the particular agent , and its mode and route of administration ; age , health , and weight of the recipient ; nature and extent of symptoms , kind of concurrent treatment , frequency of treatment , and the effect desired . an amount of immunogen is delivered to induce a protective or therapeutically effective immune response . those having ordinary skill in the art can readily determine the range and optimal dosage by routine methods . the vaccines can be used to treat or prevent tumors that express egfrviii . examples of tumor types that are known to express egfrviii include but are not limited to glioblastoma , pediatric brain tumors , non - small cell carcinoma of the lung , ovarian tumors , prostate tumors , head and neck cancers , and breast tumors among several others . in some embodiments , egfrviii expression may be confirmed prior to treatment by in vitro detection of egfrviii expression in patient samples , in vitro evaluation of patient samples including tumor sample or other samples containing tumor cells , in vivo imaging or detection of egfrviii expression or other means to indicate that the patient &# 39 ; s cancer expresses egfrviii . in some embodiments , treatment with vaccines is part of a comprehensive treatment protocol which includes surgical resection and / or radiation therapy and / or chemotherapy with anti - cancer compounds , antibodies and the like . in some embodiments , vaccines are administered following resection . in some embodiments , vaccines are administered following radiation therapy . in some embodiments , vaccines are administered together with chemotherapy . in some embodiments , vaccines are administered together with chemotherapy using temozolomide . in some embodiments , vaccines are administered together with chemotherapy using anti - egfr antibodies . in some embodiments , vaccines are delivered ex vivo to cells which are then administered to the individual . in some embodiment , the vaccines are delivered as part of an autologous cell therapy protocol whereby cells removed from an individual as treated ex vivo with vaccine and reintroduced in the individual . dendritic cells and other immune cells may be treated ex vivo and used in cell therapy / vaccine protocols . the vaccines can be used as antigen targets for producing antibodies including monoclonal antibodies , using any technique which provides for the production of antibodies by continuous cell line in culture . such techniques are well known to those of skill in the art and include , but are not limited to , the hybridoma technology originally described by kohler and milstein , nature 1975 , 256 , 495 - 497 , the human b - cell hybridoma technique described by kosbor et al ., immunology today 1983 , 4 , 72 and the ebv - hybridoma technique described by cole et al ., monoclonal antibodies and cancer therapy , alan r . liss , inc ., pp 77 - 96 . antibodies , including monoclonal antibodies , humanized antibodies , and human antibodies can be prepared and used as therapeutics . the following nonlimiting examples are provided to further illustrate the invention . studies were undertaken to improve the anti - tumor efficacy of the egfrviii vaccine . vaccines comprising egfrviii peptide variations produced and tested show greatly increased tumor regression following vaccination . these egfrviii peptide variations included substitutions in the splice junction glycine . a structural study of the egfrviii peptide bound to a single chain recombinant antibody revealed that the novel glycine makes no contacts with the antibody ( landry et al j mol biol . 2001 ; 308 ( 5 ): 883 - 93 ). thus , this glycine may not be essential for immune recognition . however , the glycine might be important for the flexibility of the peptide as the structure of this peptide makes a turn at this amino acid . since the glycine was not essential for immune recognition but might contribute to structure , peptides vaccines were prepared with amino acid substitutions for the glycine to determine if such modification enhance the anti - tumor effects of the peptide vaccine . the peptides tested were : the peptides were conjugated to klh to produce the following conjugated peptide vaccines . the conjugated peptide vaccines were tested in tumor regression experiments and survival results were compared among animals treating with one of the conjugated peptide vaccines or klh only . the data is shown in table 1 . peptide a : klh , peptide v : klh , and peptide p : klh each showed better survival rates than those observed with the original conjugated peptide vaccine peptide g : klh . peptide no g : klh was less effective than peptide g : klh which was only slightly more effective than klh only . these vaccines appear to be faster acting , i . e ., induce tumor regression in a shorter period of time . they also appear to be more effective , i . e ., more animals showed regression using vaccines having g substitutions than with peptide g : klh . thus the data indicate that the central glycine , thought to be essential for activity , can be modified to give superior vaccines . additional experiments were performed repeating the experiments described above . following completion of additional experiments the data was compiled and set forth in table 2 , which shows the overall survival data and total number of animals . survival over time is shown in the figure . in the overall survival data , use of each of peptide a : klh , peptide v : klh and peptide p : klh resulted in a higher percent survival compared to the use of peptide g : klh and peptide g : klh was moderately more effective than klh only . for all experiments , the peptides were synthesized with the sequence as indicated with the cysteine at the carboxy terminus added for the purposes of conjugation . peptides were then conjugated at a 1 : 1 w / w ratio to maleimide activated keyhole limpet hemocyanin ( klh ) for 24 hours . following conjugation , the peptide : klh conjugate was dialyzed against pbs to remove unconjugated peptide nih swiss mice were inoculated subcutaneously in the right hand flank with 2 × 10 6 hc2 20d2 / c cells , an nih - 3t3 cell line engineered to overexpress egfrviii . this cell line has been previously used to study anti - tumor responses to seq id no : 2 conjugated to klh ( moscatello et al ., cancer res . 57 : 1419 ). on the 7 th day following inoculation , mice were immunized with 100 μg of conjugated peptide in 100 μl of pbs emulsified with 100 μl of freund &# 39 ; s complete adjuvant . on the 14 th day , mice were immunized with 100 μg of peptide in 100 μl of pbs emulsified with 100 μl of freund &# 39 ; s incomplete adjuvant . newly diagnosed gbm preferably undergo at least a 95 % resection of the t1 - gadolinium enhancing component of the tumor . prior to vaccination all patients preferably receive at least standard of care external beam radiation . vaccine administered directly to patients by treating autologous dendritic cells ( dcs ) ex vivo with vaccine and then reintroducing the vaccinated dcs into the patient . in some embodiments , vaccines is peptide a : klh , peptide v : klh , or peptide p : klh . vaccine may be loaded onto autologous dcs , which are matured and used for immunization . patients undergo leukapheresis to obtain peripheral blood mononuclear cells in preparation for dc generation . dcs are pulsed for two hours with 500 μg of vaccine . patients receive vaccination using autologous vaccinated dcs administered intradermally for examples into the upper thigh , 10 cm below the inguinal ligament , every 2 weeks beginning 2 weeks following completion of radiation therapy . in some embodiments , patients may receive about 3 × 10 7 dcs per injection . vaccine may be administered directly to patients . in some embodiments , vaccines is peptide a : klh , peptide v : klh , or peptide p : klh . newly diagnosed egfrviii - positive gbm patients may be treated with vaccine administered given intradermally in gm - csf without accompanying dcs . in some embodiments , two weeks after completing standard external beam radiation therapy , patients receive 3 vaccinations at 2 week intervals of 500 μg of vaccine in 0 . 8 ml of saline with gm - csf . subsequent vaccines may be continued monthly . vaccine may be administered directly to patients in combination with chemotherapy . in some embodiments , the vaccine is peptide a : klh , peptide v : klh , or peptide p : klh . the vaccine may be given in coordination with concurrent daily temozolomide ( tmz ) in monthly cycles after completion of radiation . prior to receiving the vaccine , patients undergo & gt ; 95 % volumetric tumor resection , along with standard of care radiation therapy with concurrent tmz . newly diagnosed egfrviii - positive gbm patients may be treated with vaccine given intradermally in gm - csf . vaccine may be administered in a 500 μg dose with gm - csf near the inguinal region in the upper thigh , on alternating sides . patients receive tmz at a dose of 200 mg / m 2 for 5 days of a 28 day cycle or at a dose of 100 mg / m 2 for 21 days of a 28 day cycle . in some embodiments , patients are vaccinated on day 21 of each cycle until progression . first three vaccines may be given biweekly , followed by monthly injections . the specification includes recitation of the sequence listing found in ascii text file name seqlistingtxt . txt , created and submitted to the united states patent and trademark office on jan . 27 , 2015 ( 4 kilobytes ), which is incorporated by reference in its entirety 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