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Patent US8084592 - Multivalent entrain-and-amplify immunotherapeutics for carcinoma - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe present invention provides a method of treating a cell proliferative disease such as cancer by providing to a subject in need thereof an immunogenic composition comprising plasmid and peptide(s) or analogues thereof. In embodiments of the present invention there is provided methods and compositions...http://www.google.com/patents/US8084592?utm_source=gb-gplus-sharePatent US8084592 - Multivalent entrain-and-amplify immunotherapeutics for carcinomaAdvanced Patent SearchPublication numberUS8084592 B2Publication typeGrantApplication numberUS 11/455,279Publication dateDec 27, 2011Filing dateJun 16, 2006Priority dateJun 17, 2005Also published asCA2612518A1, CN101687020A, EP1901774A2, EP1901774B1, EP2465530A1, US20070003563, US20120148613, WO2006138568A2, WO2006138568A3Publication number11455279, 455279, US 8084592 B2, US 8084592B2, US-B2-8084592, US8084592 B2, US8084592B2InventorsAdrian Ion Bot, Chih-Sheng Chiang, David C. Diamond, Jian Gong, Kent Andrew Smith, Liping Liu, Xiping Liu, Zhiyong QiuOriginal AssigneeMannkind CorporationExport CitationBiBTeX, EndNote, RefManPatent Citations (63), Non-Patent Citations (23), Classifications (21), Legal Events (2) External Links: USPTO, USPTO Assignment, EspacenetMultivalent entrain-and-amplify immunotherapeutics for carcinoma
US 8084592 B2Abstract
Embodiments of the disclosed invention are directed to the use of combinations of tumor-associated antigens (TuAAs) for the immunotherapy of patients with various types of cancer. In preferred embodiments, the TuAAs are antigens expressed by the cancer cell itself. Examples of such TuAAs are Melan-A, tyrosinase, SSX-2, NY-ESO-1, and PRAME. In alternate embodiments, the TuAAs are antigens associated with non-cancerous components of the tumor, such as tumor-associated neovasculature or other stroma. An example of such an antigen is PSMA�though, in prostate cancer PSMA is expressed by cancerous cells. In particularly preferred embodiments both types of antigen are targeted. Different aspects of the invention include the immunogenic compositions, their collection into defined products, and methods for their use.
Still other embodiments can include alternate epitopes (such as those described in the U.S. patent application Ser. No. 10/117,937, entitled �Epitope Sequences,� filed on Apr. 4, 2002 (Publication No. 20030220239 A1), which is hereby expressly incorporated by reference) substituted in similar combination as the epitopes expressed in the pSEM (SEQ ID NO. 19), pBPL (SEQ ID NO. 20), and pRP12 (SEQ ID NO. 21) plasmids and corresponding peptide immunogens administered as the amplification portion of the immunization strategy.
FIG. 1. Tetramer analysis of pSEM/pBPL, primed animals prior to peptide boost. Group 1, 2, and 3 animals (n=60) were primed with four injections of the pSEM/pBPL plasmid mixture on days 1, 4, 15, and 18 (100 μg/day) in bilateral inguinal lymph nodes. Tetramer analysis was performed on day 25, 10 days following the final plasmid injection and compared to untreated na�ve littermate controls (n=5). Tetramer values (Melan A, Tyrosinase, SSX-2, NY-ESO-1) represent the average+/−SEM.
FIG. 2. Melan-A/Tyrosinase, SSX-2/NY-ESO-1 tetramer analysis was performed on day 39 demonstrating a tetravalent immune response in individual animals. Animals were primed with a plasmid mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 μg/day) in bilateral inguinal lymph nodes followed by a peptide boost consisting of SSX241-49 A42V (SEQ ID. NO. 5) in the left lymph node and Tyrosinase369-377 V377Nva (SEQ ID. NO. 12) on in the right lymph node on days 28 and 32 (25 μg/day). Representative animals (n=3) from Group 2 are shown and compared to tetramer values for an untreated na�ve littermate control.
FIG. 3. Tetramer analysis of pSEM/pBPL primed, SSX-2/Tyrosinase boosted animals. Melan-A/Tyrosinase, SSX-2/NY-ESO-1 tetramer analysis was performed on day 39, 7 days following the last peptide injection. Group 1 animals (n=10) were primed with a plasmid mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 μg/day) followed by a boost with a plasmid mixture of pBPL+pSEM on days 28 and 32 (100 μg/day). Group 2 and 3 animals (n=50) were primed with a plasmid mixture of pBPL+pSEM similar to Group 1 followed by a peptide boost consisting of SSX-241-49 A42V (SEQ ID. NO. 5) in the left lymph node and Tyrosinase369-377 V377Nva (SEQ ID. NO. 12) in the right lymph node on days 28 and 32 (25 μg/day). Average tetramer values (Melan A, Tyrosinase, SSX-2, and NY-ESO-1) were compared to untreated na�ve littermate controls (n=5) and represent the average+/−SEM.
FIGS. 4A-4B. IFN-γ ELISpot analysis following a first peptide boost (FIG. 4A). ELISPOT analysis was performed on day 41. Group 1 animals (n=3 sacrificed) were primed with a plasmid mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 mg/day) followed by a boost with a plasmid mixture of pBPL+pSEM on days 28 and 32 (100 μg/day). Group 2 and 3 animals (n=6 sacrificed) were primed with a plasmid mixture of pBPL+pSEM similar to Group 1 followed by a peptide boost consisting of SSX-241-49 A42V (SEQ ID. NO. 5) in the left lymph node and Tyrosinase369-377 V377Nva (SEQ ID. NO. 12) in the right lymph node on days 28 and 32 (25 μg/day). Antigen specific (Melan A, Tyrosinase, SSX-2, and NY-ESO-1) interferon-γ spot forming cells per spleen were compared to untreated na�ve litternate controls (n=3); FIG. 4A. IFN-γ ELISpot analysis was performed in triplicate, values represent average+/−Stdev. Peptide stimulating concentration was at 10 μg/ml and incubated for 42 hrs. FIG. 4B.�IFN-γ ELISpot analysis following the second peptide boost. ELISPOT analysis was performed by sacrificing representative animals on day 63. Group 1 animals (n=3 sacrificed) received injections of a mixture of pBPL+pSEM on Days 1, 4, 15, 18, 28, 32, 49, and 53 (100 μg/day). Group 2 animals (n=4 sacrificed) received injections of a mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 μg/day) followed by a peptide boost consisting of SSX-241-49 A42V (SEQ ID. NO. 5) in the left lymph node and Tyrosinase369-377 V377Nva (SEQ ID. NO. 12) in the right lymph node on days 28, 32, 49, and 53 (25 μg/day). Group 3 animals (n=7 sacrificed) received injections of a mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 μg/day) in bilateral inguinal lymph nodes followed by a peptide boost consisting of SSX-241-49 A42V (SEQ ID. NO. 5) in the left lymph node and Tyrosinase369-377 V377Nva (SEQ ID. NO. 12) in the right lymph node on days 28 and 32 (25 μg/day) and a second peptide boost consisting of NY-ESO-1157-165 L158Nva (SEQ ID. NO. 6), C165V (12.5 μg on days 49 and 53) in the left lymph node and Melan A76-35 A27Nva (SEQ ID. NO. 11) (25 μg on days 49 and 53) in the right lymph node. Antigen specific (Melan A, Tyrosinase, SSX-2, and NY-ESO-1) interferon-γ spot forming cells per spleen were compared to a untreated na�ve littermate control (FIG. 4B). IFN-γ ELISpot analysis was performed in triplicate, values represent average+/−SEM. Peptide stimulating concentration was at 10 μg/ml and incubated for 42 hrs.
FIG. 5. Depicts tetramer levels, IFN-γ ELISPOT and carboxy-fluorescein diacetate, succinimidyl ester (CFSE) histograms from in vivo studies where animals were challenged with human melanoma tumor cells expressing all four tumor associated antigens. Na�ve control (top left panel); two animals with tetravalent immunity (top right panel and lower left panel); and an animal with a monovalent response to Melan A (lower right panel).
FIG. 6. Tetramer analysis of the �original� versus the �expanded� protocol. Animals were injected based on a �original protocol� (Groups 1-3) or an �expanded protocol� (Groups 4-6) with 4 injections of D1 (pRP12) plasmid (4 mg/ml) in the right inguinal lymph node and 4 injections of D2 (pBPL) plasmid (4 mg/ml) in left inguinal lymph node. Animals were subsequently boosted with PSMA, SSX-2, PRAME and NY-ESO-1 peptides. Animals were primed with D1 (pRP12) plasmid and D2 (pBPL) plasmid (4 mg/ml) on days 1, 4, 15, and 18, followed by boosting with PSMA788-297 (I297V) peptide (RLN) (SEQ ID. NO. 8) and SSX-241-49 (A42V) peptide (LLN) (SEQ ID. NO. 5) on days 29 and 31 for the original protocol; and boosting with PRAME425-433 (L426Nva, L433Nle)) peptide (RLN) (SEQ ID. NO. 7) and NY-ESO-1157-165 (L158Nva, C165V) peptide (LLN) (SEQ ID. NO. 6) on days 42, 45 for the expanded protocol (Groups 4-6). Values represent average+/−SEM from individual animals after peptide boost and are compared to untreated na�ve littermate controls (n=5).
FIG. 8. IFN-γ ELISPOT analysis of the �original� versus the �expanded� protocol. Total antigen specific (SSX-2, NY-ESO-1, PRAME, and PSMA) interferon-γ spot forming cells per spleen are shown comparing the �original� and �expanded� protocols comprised of low, medium and high peptide boosts. IFN-γ ELISpot analysis was performed in triplicate, values represent average+/−SEM after peptide boost. Splenocytes (3�105 cells per well) were stimulated, ex vivo in 96 well ELISpot plates, with peptide (SSX-2, NY-ESO-1, PRAME, and PSMA) at a concentration of 10 μg/ml for 72 hrs. Values are extrapolated from total nucleated splenocytes and normalized per spleen from each animal.
In some embodiments all or a subset of the compositions of the product are packaged together in a kit. In some instances, the inducing and amplifying compositions targeting a single epitope, or set of epitopes, can be packaged together. In other instances, multiple inducing compositions can be assembled in one kit and the corresponding amplifying compositions assembled in another kit. Alternatively, compositions may be packaged and sold individually along with instructions, in printed form or on machine-readable media, describing how they can be used in conjunction with each other to achieve the beneficial results of the indicated immunization protocol. Further variations will be apparent to one of skill in the art. The use of various packaging schemes comprising less than all of the compositions that might be employed in a particular protocol or regimen facilitates the personalization of the treatment, for example, based on tumor antigen expression, or observed response to the immunotherapeutic or its various components, as described in U.S. Provisional Application Ser. No. 60/580,969, filed on Jun. 17, 2004; U.S. patent application Ser. No. 11/155,288 (Publication No 20060008468) filed Jun. 17, 2005, and U.S. patent application Ser. No. 11/323,964 filed Dec. 29, 2005, all entitled �COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN DIAGNOSTICS FOR VARIOUS TYPES OF CANCERS�; and U.S. Provisional Patent Application Ser. No. 60/580,964, and U.S. patent application Ser. No. 11/155,928 (Publication No. 20050287068), both entitled �IMPROVED EFFICACY OF ACTIVE IMMUNOTHERAPY BY INTEGRATING DIAGNOSTIC WITH THERAPEUTIC METHODS�, each of which is hereby incorporated by reference in its entirety.
SSX-241-49 KASEKIFYV
NY-ESO-1157-165 SLLMWITQC
PRAME425-433 SLLQHLIGL
PSMA288-297 GLPSIPVHPI
SSX-2 Analogue
KVSEKIFYV
NY-ESO-1 Analogue
SNvaLMWITQV
PRAME Analogue
SNvaLQHLIGNle
PSMA Analogue
Melan-A26-35 ELAGIGILTV
Tyrosinase369-377 YMDGTMSQV
Melan-A Analogue
ENvaAGIGILTV
Tyrosinase Analogue
YMDGTMSQNva
pBPL plasmid
IKASEKIFYV SLLM liberation sequence
WITQC
KASEKIFY
pRP12 plasmid
KR-SLLQHLIGL-
liberation sequence
GDAAY-
pSEM plasmid
MLLAVLYCL-
ELAGIGILTV-
pBPL encoded immuno-
MSLLMWITQCKA
genic polypeptide
SEKIFYVGLPSIPV
pRP12 encoded immuno-
MNLLHETDSAVA
TARRPRWLCAGA
DAAYSLLQHLIGL ISPEKEEQYIASLL QHLIGLKRPSIKR
pSEM encoded immuno-
MLLAVLYCLELA
GIGILTVYMDGT
Melan-A26-35
As discussed above, the present invention provides immunogenic compositions for the treatment of cancer comprising plasmid(s) used in combination with synthetic peptide(s). Such an immunogenic protocol elicits a strong cell-mediated immune response to target a particular cancer thereby eliminating, eradicating or ameliorating the cancer in a subject. Preferred plasmids employed in the present invention are the pRP12 plasmid (SEQ ID NO. 21) (U.S. Provisional Patent Application No. 60/691,579 and the corresponding U.S. patent application Ser. No. 11/454,616 (Publication No. 20070004662), filed on the same date as the present application) both entitled �METHODS AND COMPOSITIONS TO ELICIT MULTIVALENT IMMUNE RESPONSES AGAINST DOMINANT AND SUBDOMINANT EPITOPES EXPRESSED ON CANCER CELLS AND TUMOR STROMA�), the pBPL plasmid (SEQ ID NO. 20), and the pSEM plasmid (SEQ ID NO. 19) disclosed in U.S. Provisional Patent Application No. 60/691,579 and U.S. patent application Ser. No. 10/292,413 (Publication No. 20030228634) respectively; each of which is incorporated herein by reference in its entirety (Note that in those documents pSEM is referred to as pMA2M). Additional plasmids that can be used are disclosed in these references and in U.S. patent application Ser. No. 10/225,568 (Publication No. 20030138808).
Therefore, in one particular embodiment of the present invention there is provided an assemblage comprising the pBPL plasmid (described in detail in U.S. application Ser. No. 10/292,413 (Publication No. 20030228634), entitled �EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN,� which is hereby expressly incorporated by reference in its entirety) expressing the NY-ESO-1157-165 (SEQ ID NO. 2) and SSX-241-49 (SEQ ID NO. 1) epitopes and the pRP12 plasmid (described in U.S. Provisional Application No. 60/691,579 U.S. patent application Ser. No. 11/454,616 (Publication No. 20070004662), filed on the same date as the present application, both entitled �METHODS AND COMPOSITIONS TO ELICIT MULTIVALENT IMMUNE RESPONSES AGAINST DOMINANT AND SUBDOMINANT EPITOPES EXPRESSED ON CANCER CELLS AND TUMOR STROMA,� which are hereby expressly incorporated by reference in their entirety) expressing the PRAME425-433 (SEQ ID NO. 3) and PSMA288-297 (SEQ ID NO. 4) epitopes. The liberation sequence for the pBPL and pRP12 plasmids are represented herein as SEQ ID NO. 13 and 14 respectively, and are also disclosed in U.S. patent application Ser. No. 10/212,413 (Publication No. 20030228634), incorporated herein by reference. The plasmids encode the epitopes in such a manner that they can be expressed and presented by pAPC.
In another particular embodiment of the present invention there is provided an assemblage comprising the pSEM plasmid, (described in detail and referred to as pMA2M in U.S. patent application Ser. No. 10/292,413 (Publication No. 20030228634) incorporated herein by reference) expressing the A27L analogue of Melan-A26-35 epitope (SEQ ID NO. 9) and the native tyrosinase369-377 (SEQ ID NO. 10) epitope. The peptide analogues Melan-A26-35 A27Nva (SEQ ID NO. 11) and tyrosinase369-377 V377Nva (SEQ ID NO. 12) are disclosed in U.S. patent application Ser. No. 11/156,369, and U.S. Provisional Patent Application Ser. No. 60/691,889, both entitled �EPITOPE ANALOGS�, each of which is hereby incorporated by reference in its entirety. The liberation sequence of this plasmid is represented herein as SEQ ID NO. 15 and is also disclosed in U.S. Provisional Patent Application No. 60/691,579, filed on Jun. 17, 2005; and U.S. patent application Ser. No. 11/454,616 (Publication No. 20070004662), filed on the same date as the present application, both entitled �METHODS AND COMPOSITIONS TO ELICIT MULTIVALENT IMMUNE RESPONSES AGAINST DOMINANT AND SUBDOMINANT EPITOPES, EXPRESSED ON CANCER CELLS AND TUMOR STROMA.� The pSEM plasmid encodes the Melan-A and tyrosinase epitopes in a manner that allows for their expression and presentation by pAPCs.
Exemplary methodology for obtaining a profile of antigen expression of a particular tumor that can be used to determine which antigen or combination of antigens are useful in treating a particular cancer can be is found in U.S. Provisional Application Ser. No. 60/580,969, filed Jun. 17, 2004; U.S. patent application Ser. No. 11/155,288 (Publication No. 20060008468), filed Jun. 17, 2005; and U.S. patent application Ser. No. 11/323,964, also filed on Jun. 17, 2005, all entitled �COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN DIAGNOSTICS FOR VARIOUS TYPES OF CANCERS�; each incorporated herein by reference in its entirety. Specific antigenic combinations of particular benefit in directing an immune response against particular cancers are disclosed in U.S. Provisional Application Ser. No. 60/479,554, filed on Jun. 17, 2003, U.S. patent application Ser. No. 10/871,708 (Publication No. 20050118186), filed on Jun. 17, 2004, (both entitled �COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN COMPOSITIONS FOR VARIOUS TYPES OF CANCERS�), and PCT Patent Application Publication No. WO 2004/112825, filed Jun. 17, 2004; each of which is incorporated herein by reference in its entirety.
One embodiment of the current invention relates to a tetravalent entrain-and-amplify therapeutic for carcinoma. Therefore, in one particular embodiment of the present invention there is provided an assemblage comprising the pBPL plasmid expressing the NY-ESO-1157-165 (SEQ ID NO. 2) and SSX-241-49 (SEQ ID NO. 1) epitopes and the pRP12 plasmid expressing the PRAME425-433 (SEQ ID NO. 3) and PSMA288-297 (SEQ ID NO. 4) epitopes (referred to herein as the PP regimen), each administered as the entraining immunogens of an immunization strategy. An �entraining� immunogen as contemplated in the present invention includes in many embodiments an induction that confers particular stability on the immune profile of the induced lineage of T cells.
Additionally, four peptide compositions corresponding to the NY-ESO-1, SSX-2, PRAME and PSMA epitopes are administered as the amplification portion of the same immunization strategy as that of the entraining immuogen. In a preferred embodiment, the peptide analogues NY-ESO-1157-165 L158Nva, C165V (SEQ ID NO. 6); SSX-241-49 A42V (SEQ ID NO. 5); PSMA288-297 I297V (SEQ ID NO. 8); and/or PRAME425-433 L426Nva, L433Nle (SEQ ID NO. 7) are utilized in the amplification step. As contemplated in the present invention, the term �amplifying or amplification�, as of a T cell response, includes in many embodiments a process for increasing the number of cells, the number of activated cells, the level of activity, rate of proliferation, or similar parameter of T cells involved in a specific response.
The entrain-and-amplify protocol employed in the present invention is described in greater detail in U.S. Provisional Application No. 60/640,402, U.S. patent application Ser. No. 10/871,707 (Publication No. 20050079152), and U.S. patent application Ser. No. 11/323,572, each entitled �METHODS TO ELICIT, ENHANCE AND SUSTAIN IMMUNE RESPONSES AGAINST MHC CLASS I-RESTRICTED EPITOPES, FOR PROPHYLACTIC OR THERAPEUTIC PURPOSES� each of which is incorporated herein by reference in their entirety.
To introduce the immunogenic composition into the lymphatic system of the patient the composition is preferably directed to a lymph vessel, lymph node, the spleen, or other appropriate portion of the lymphatic system. In some embodiments each component is administered as a bolus. In other embodiments one or more components are delivered by infusion, generally over several hours to several days. Preferably, the composition is directed to a lymph node such as an inguinal or axillary node by inserting a catheter or needle to the node and maintaining the catheter or needle throughout the delivery. Suitable needles or catheters are available made of metal or plastic (e.g., polyurethane, polyvinyl chloride (PVC), TEFLON, polyethylene, and the like). In inserting the catheter or needle into the inguinal node for example, the inguinal node is punctured under ultrasonographic control using a Vialon� Insyte W� cannula and catheter of 24G3/4 (Becton Dickinson, USA) which is fixed using Tegaderm� transparent dressing (Tegaderm�, St. Paul, Minn., USA). This procedure is generally done by an experienced radiologist. The location of the catheter tip inside the inguinal lymph node is confirmed by injection of a minimal volume of saline, which immediately and visibly increases the size of the lymph node. The latter procedure allows confirmation that the tip is inside the node. This procedure can be performed to ensure that the tip does not slip out of the lymph node and can be repeated on various days after implantation of the catheter. In the event that the tip does slip out of location inside the lymph node, a new catheter can be implanted.
The therapeutic composition(s) of the present invention may be administered to a patient in a manner consistent with standard vaccine delivery protocols that are well known to one of ordinary skill in the art. Methods of administering immunogenic compositions of the present invention comprising plasmids and peptides or peptide analogues of TuAAs include, without limitation, transdermal, intranodal, perinodal, oral, intravenous, intradermal, intramuscular, intraperitoneal, and mucosal administration, delivery by injection or instillation or inhalation. A particularly useful method of vaccine delivery to elicit a CTL response is disclosed in Australian Patent No. 739189; U.S. Pat. Nos. 6,994,851 and 6,977,074 both entitled �A METHOD OF INDUCING A CTL RESPONSE�.
TABLE 2 Immunization Schedule Peptide (boost) Peptide and Plasmids (prime) Lymph Node Group N* Plasmids Days Each Dose (R/L) Days Each Dose 1 10 pSEM + pBPL 1, 4, 15, 18, 100 μg � � � 28, 32, 49, 53 2 25 pSEM + pBPL 1, 4, 15, 18, 100 μg Tyrosinase (R) 28, 32, 49, 53 25 μg SSX-2 (L) 28, 32, 49, 53 25 μg 3 25 pSEM + pBPL 1, 4, 15, 18 100 μg Tyrosinase (R) 28, 32 25 μg SSX-2 (L) 28, 32 25 μg NY-ESO-1 (R) 49, 53 12.5 μg Melan A (L) 49, 53 25 μg Tetramer Analysis
Spleens were isolated on days 27 and 62 from euthanized animals, subjected to the plasmid/peptide immunization schedule as described above. The mononuclear cells, after density centrifugation (Lympholyte Mammal, Cedarlane Labs, Burlington, N.C.), were resuspended in HL-1 medium. Splenocytes (5�105, or 3�105 cells per well) were incubated with 10 μg of Melan-A26-35 (A27L) (SEQ ID NO. 9), Tyrosinase369-377 (SEQ ID NO. 10), SSX-241-49 (SEQ ID NO. 1), or NY-ESO-1157-165 (SEQ ID NO. 2) natural peptide in triplicate wells of a 96 well filter membrane plates (Multiscreen IP membrane 96-well plate, Millipore. Boston, Mass.). Samples were incubated for 42 hours at 37� C. with 5% CO2 and 100% humidity prior to development. Mouse IFN-γ coating antibody (IFN-γ antibody pair, U-CyTech Biosciences, The Netherlands) was used as a coating reagent prior to incubation with splenocytes, followed by the accompanied biotinylated detection antibody. GABA conjugate and proprietary substrates from U-CyTech were used for IFN-γ spot development. The CTL response in immunized animals was measured 24 hours after development on the AID International plate reader using ELISpot Reader software version 3.2.3 calibrated for IFN-γ spot analysis.
Immunization with Plasmids pSEM and pBPL Prior to Peptide Boost
Three groups of female HHD animals (H-2 class I-negative (knockout) HLA-A2.1-transgenic HHD mice, 8-12 weeks of age) were immunized with a mixture of pSEM/pBPL (100 μg/day) to the bilateral inguinal lymph nodes. Group 1 (n=10 mice) received plasmid injections on Days 1, 4, 15, 18, 28, 32, 49, and 53; Group 2 and Group 3 (n=25 mice per group) received plasmid injections on Days 1, 4, 15, and 18 respectively (Table 2; above). On day 25, blood was collected from the immunized animals, and CD8+ T cell analysis was performed using a tetramer assay as discussed elsewhere herein. Responses were compared to na�ve littermate control mice (n=5).
Individual Immunization with Plasmid Primed SSX-2/Tyrosinase
It was assessed whether boosting with the subdominant epitope peptides alone following plasmid priming was sufficient to achieve a tetravalent immune response. Therefore, animals from Group 2 above, were boosted with the sub-dominant epitopes, tyrosinase V377Nva (SEQ ID NO. 12) and SSX-2 A42V (SEQ ID NO. 5) peptide analogues and immune responses were compared to a na�ve control.
FIG. 2 shows the tetravalent responses from peripheral blood on day 39 following the Tyrosinase and SSX-2 peptide boost, generated in three representative immunized animals as compared to a selected na�ve control animal using a tetramer flow cytometry assay. For example, animal 2 demonstrated tetramer responses specific to SSX-2 (5.8%), NY-ESO-1 (4.1%), tyrosinase (8.7%) and Melan A (10.8%). These data taken together, represent specific CTL responses comprised of 29.4% of the total CD8+ T cell repertoire. Furthermore, the results show that boosting with the subdominant epitope peptides alone, following plasmid priming, was sufficient to achieve a tetravalent immune response.
Immunization with Plasmid Primed SSX-2/Tyrosinase
In order to generate a more balanced tetravalent immune response, animals were boosted with the sub-dominant peptide epitopes Tyrosinase369-377 (V377Nva) (SEQ ID NO. 12) and SSX-241-49 (A42V) (SEQ ID NO. 5) (Groups 2 and 3, n=50) and immune responses were compared to animals boosted with a mixture of pSEM/pBPL plasmid (Group 1, n=10) or na�ve controls (n=10).
Average tetramer values for Melan A, Tyrosinase, SSX2, and NY-ESO-1 were compared to untreated na�ve littermate controls (n=5) and represent the average+/−SEM. FIG. 3 shows the immune responses prior to and following the tyrosinase and SSX-2 boost for Groups 2 and 3 (n=50) compared to Group 1 (n=10; plasmid alone).
IFN-γ ELISPOT Analysis of First and Second Peptide Boost
ELISPOT analysis, as described elsewhere herein, was performed by sacrificing representative animals on day 41, nine days following the last peptide boost. Group 1 animals (n=3 sacrificed) were primed with a plasmid mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 μg/day) followed by a boost with a plasmid mixture of pBPL+pSEM on days 28 and 32 (100 μg/day) in bilateral inguinal lymph nodes. Group 2 animals (n=6 sacrificed) were primed with a plasmid mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 μg/day) in bilateral inguinal lymph nodes followed by a peptide boost consisting of SSX241-49 A42V (SEQ ID NO. 5) in the left lymph node and Tyrosinase369-377 V377Nva (SEQ ID NO. 12) in the right lymph node on days 28 and 32 (25 μg/day). Antigen specific (Melan A, Tyrosinase, SSX2, and NY-ESO-1) interferon-γ spot forming cells per spleen were compared to untreated na�ve littermate controls (n=3), FIG. 4A.
Following the second peptide boost, ELISPOT analysis was performed by sacrificing representative animals on day 63, ten days following the second peptide boost. Group 1 animals (n=3 sacrificed) received injections of a mixture of pBPL+pSEM on days 1, 4, 15, 18, 28, 32, 49, and 53 (100 μg/day) in bilateral inguinal lymph nodes. Group 2 animals (n=4 sacrificed) received injections of a mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 μg/day) in bilateral inguinal lymph nodes followed by a peptide boost consisting of SSX241-49 A42V in the left lymph node and Tyrosinase369-377 V377Nva in the right lymph node on days 28, 32, 49, and 53 (25 μg/day). Group 3 animals (n=4 sacrificed) received injections of a mixture of pBPL+pSEM on days 1, 4, 15, and 18 (100 μg/day) in bilateral inguinal lymph nodes followed by a peptide boost consisting of SSX241-49 A42V (SEQ ID NO. 5) in the left lymph node and Tyrosinase369-377 V377Nva (SEQ ID NO. 12) in the right lymph node on days 28 and 32 (25 μg/day) and a second peptide boost consisting of NY-ESO-1157-165 L158Nva, C165V (SEQ ID NO. 6) (12.5 μg on Days 49 and 53) in the left lymph node and Melan A26-35 A27Nva (SEQ ID NO. 11) (25 μg on Days 49 and 53) in the right lymph node. Antigen specific (Melan A, Tyrosinase, SSX2, and NY-ESO-1) interferon-γ spot forming cells per spleen were compared to a untreated na�ve littermate control FIG. 4B.
Overall, the data obtained from the above Examples (2-5), depict the successful generation of a tetravalent immune response in animals immunized with the NS and/or MT regimens of the present invention. A comparison of the immune responses (tetramer and IFN-γ ELISPOT analysis) in na�ve animals or animals boosted with a mixture of pSEM/pBPL plasmid alone (Group 1) to animals boosted with the sub-dominant peptide epitopes tyrosinase and SSX-2 (Groups 2 and 3) on days 28 and 32 confirmed the successful generation of a tetravalent immune response in animals immunized with this regimen. Similar results were obtained following the second peptide boost on days 49 and 53 in where Group 3 (n=25) was boosted with the dominant epitope peptides, Melan A26-35 (A27Nva) (SEQ ID NO. 11) and NY-ESO-1157-165 (L158Nva, C165V) (SEQ ID NO. 6) and Group 2 (n=25) was boosted again with the sub-dominant epitope peptides Tyrosinase369-377 (V377Nva) (SEQ ID NO. 12) and SSX-241-49 (A42V) (SEQ ID NO. 5).
Generation of an Immune Response to Human Melanoma
On day 61, two animals from each group (Group 1, 2, and 3) were selected based on high tetramer levels, and injected intravenously with CFSE labeled tumor cells. More precisely, human 624.38 cultured melanoma tumor cells (10�106), expressing all four tumor associated antigens for SSX-2, NY-ESO-1, Tyrosinase, and Melan A, were stained with CFSEhi (Vybrant CFDA SE cell tracer kit, Molecular Probes) fluorescence (1.0 μM for 15 minutes) and co-injected intravenously into Group 1, 2, or 3 immunized mice (N=2/group) or into na�ve HHD mice (N=2) with an equal ratio of 624.28 HLA-A2 negative control cells stained with CFSElo fluorescence (0.1 μM). Animals received a second injection of target cells two hours later.
[(1−% CFSEhi/% CFSElo) in immunized−(1−% CFSEhi/% CFSElo) in na�ve]�100
FIG. 5 shows tetramer levels, IFNγ ELISPOT results, and two peak CFSE histograms from a na�ve control (top left panel), two animals with tetravalent immunity (top right and lower left panel), and an animal with a monovalent response to Melan A (lower right panel). As expected, the na�ve control animal was unable to clear the target cells as demonstrated by the maintenance of an equal ratio of both histogram peaks as was the case in the animal demonstrating the monovalent immune response. On the other hand, animals displaying an immune response to all four antigens were much more capable of clearing the human melanoma tumor target cells with 71% and 95% specific lysis.
Generation of an Immune Response by a Original Vs. Expanded Protocol
Two different boosting strategies were tested with regard to their ability to enhance the desired immune responses. The first approach (the �original� protocol) utilized a single injection of each peptide during the boosting procedure. The second approach (the �expanded� protocol) tested two injections of each peptide. Three dosage levels of each peptide (low, mid, and high) were tested in an effort to determine a dose-response relationship and to help define the optimum peptide concentration.
Six groups of 10 female HHD-1 animals/group were immunized with plasmids D1 and D2 injected directly into the bilateral inguinal lymph nodes. Animals from Groups 1-3 were boosted using the �original� protocol, and Groups 4-6 animals were boosted using the �expanded� protocol.
Animals on the �original protocol� (Groups 1-3, n=10 per group) received 4 injections of D1 (pRP12 (SEQ ID NO. 21)) plasmid (100 μg per dose) in the right inguinal lymph node and 4 injections of D2 (pBPL (SEQ ID NO. 20)) plasmid (100 μg/dose) in left inguinal lymph node on days 1, 4, 15 and 18. This was followed by a boost with PSMA288-297 (I297V) (SEQ ID NO. 8) in the right lymph node and SSX-241-49 (A42V) (SEQ ID NO. 5) in the left lymph node on day 29, and with PRAME425-433 (L426Nva, L433Nle) (SEQ ID NO. 7) in the right lymph node and NY-ESO-1157-165 (L158Nva, C165V) (SEQ ID NO. 6) in the left lymph node on day 32.
Animals on the �expanded protocol� (Groups 4-6, n=10 per group) received 4 injections of D1 (pRP12 (SEQ ID NO. 21)) plasmid (100 μg/dose) in right inguinal lymph node and D2 (pBPL (SEQ ID NO. 20)) plasmid (100 μg/dose) in left inguinal lymph node on days 1, 4, 15, and 18. The animals were subsequently boosted with PSMA288-297 (I297V) (SEQ ID NO. 8) in the right lymph node and SSX-241-49 (A42V) (SEQ ID NO. 5) in the left lymph node on days 29 and 32 and with PRAME425-433 (L426Nva, L433Nle) (SEQ ID NO. 7) in the right lymph node and NY-ESO-1157-165 (L158Nva, C165V) (SEQ ID NO. 6) in the left lymph node on days 43 and 46.
Blood was collected from each group in both protocols, 7 days following the last peptide boost, and CD8+ T cell analysis was performed using a tetramer assay (FIG. 6). Responses were compared to na�ve littermate control mice (n=5). SSX-2, NY-ESO-1, FRAME, and PSMA tetramer values are shown comparing the original and expanded protocols comprised of low, medium and high peptide boosts in FIG. 6.
IFN-Γ ELISPOT of an Immune Response by a Original Vs. Expanded Protocol
Spleens were isolated on day 68 from euthanized animals and the mononuclear cells, after density centrifugation (Lympholyte Mammal, Cedarlane Labs, Burlington, N.C.), were resuspended in HL-1 medium. Splenocytes (3�105 or 1.5�105 cells per well) were incubated with 10 μg of PSMA288-297 (SEQ ID NO. 4), PRAME425-433 (SEQ ID NO. 3), SSX-241-49 (SEQ ID NO. 1), or NY-ESO-1157-165 (SEQ ID NO. 2), natural peptide in triplicate wells of a 96 well filter membrane plates (Multi-screen IP membrane 96-well plate, Millipore, Mass.). Samples were incubated for 72 hours at 37� C. with 5% CO2 and 100% humidity prior to development. Mouse IFN-γ coating antibody (IFN-γ antibody pair, U-CyTech Biosciences, The Netherlands) was used as coating reagent prior to incubation with splenocytes, followed by the accompanied biotinylated detection antibody. GABA conjugate and proprietary substrates from U-CyTech Biosciences were used for IFN-γ spot development. The CTL response in immunized animals was measured 24 hours after development on the AID International plate reader using ELISpot Reader software version 3.2.3 calibrated for IFN-γ spot analysis.
The IFNγ ELISPOT results shown in FIG. 7 correlate well with the tetramer data (FIG. 6) and confirm a robust immune response to PRAME475-433 (SEQ ID NO. 3), PSMA288-433 (SEQ ID NO. 4), SSX-241-49 (SEQ ID NO. 1), and NY-ESO-1157-165 (SEQ ID NO. 2) elicited by the �original� therapeutic protocol. The �expanded� protocol did not appear to offer any apparent advantage over the �original� protocol as measured by IFN-γ ELISPOT analysis.
Tetravalent Immune Response Generated by the PP/NS Therapeutic Regimen
It was assessed whether a tetravalent immune response can be elicited by first boosting with the subdominant epitopes PSMA and SSX-2 followed by boosting with the dominant epitopes PRAME and NY-ESO-1. A representative animal from Group 1 (�original protocol�; high dose) received 4 injections of D1 (pRP12 (SEQ ID NO. 21)) plasmid (100 μg/dose) in the right inguinal lymph node and 4 injections of D2 (pBPL (SEQ ID NO. 20)) plasmid (100 mg/dose) in left inguinal lymph node on days 1, 4, 15 and 18. This was followed by a boost with peptides, PSMA288-297 (I297V) (SEQ ID NO. 8) in the right lymph node (25 μg) and SSX-241-49 (A42V) (SEQ ID NO. 5) in the left lymph node (25 μg) on day 29 and with PRAME425-433 (L426Nva, L433Nle) (SEQ ID NO. 7) in the right lymph node (20 μg) and NY-ESO-1157-165 (L158Nva, C165V) (SEQ ID NO. 6) in the left lymph node (25 μg) on day 32. The data (FIG. 8) shows a tetravalent immune response as measured by two separate assays, tetramer and ELISpot analyses.
51Chromium-Release Assay Measuring CTL Activity to PRAME, PSMA, NY-ESO and SSX-2
Briefly, mice were sacrificed and the spleens were removed. The spleens were homogenized and the cell suspension was strained to yield a single-cell suspension. Quantities of 5�106 cells/well were plated in 24 well tissue culture plates and 1.5�106 peptide-pulsed, γ-irradiated and LPS (lipopolysaccharide) blasted B cells were added to each well. Mouse recombinant IL-2 was also added at a concentration of 1 ng/ml. The cells were incubated for 4 days for the PRAME group and 6 days for each of the PSMA, SSX-2 and NY-ESO-1 groups.
After the ex vivo stimulation, CTLs were collected from the plates, washed, and plated into 96 well U-bottom micro-titer assay plates at concentrations of 106, 3.3�105, and 1.1�105 cells/well in a total of 100 μL per well. To assess peptide specific lysis, T2 cells were labeled with 51Cr and pulsed with 20 μg/mL of each peptide (SSX-2, NY-ESO-1, PSMA, or PRAME) at 37� C. for 1.5 hours. After the incubation, the cells were washed and resuspended. Ten thousand 51Cr-labeled and peptide-pulsed T2 cells were added to each well. The cells were then incubated at 37� C. for 4 hours.
After incubation, supernatants were harvested and the cytolytic activity was measured in triplicate samples using a gamma counter. The corrected percent lysis was calculated for each concentration of effector cells, using the mean cpm for each replicate of wells (FIG. 8). Percent specific lysis was calculated using the following formula: Percent release=100�(Experimental release−spontaneous release)/(Maximum release−spontaneous release). Data are presented as follows: the x-axis shows the effector to target ratio; the γ-axis shows the corresponding percentage specific lysis.
The results (FIG. 8) show 51Chromium release assay (CRA) data for CTL from each group against T2 cells pulsed with PRAME415-433 (SEQ ID NO. 3) (panel 1), PSMA288-297 (SEQ ID NO. 4) (panel 2), NY-ESO-1157-165 (SEQ ID NO. 2) (panel 3), or SSX-241-49 (SEQ ID NO. 1) (panel 4) peptides as targets. Specific lysis values were compared to un-pulsed T2 control cells. Given that the ELISA analysis (data not shown) indicated that immunogenicity of the PRAME group is very strong and to avoid antigen-induced cell deaths, the CRA for the PRAME group was pursued following a 4-day IVS protocol. The CRA was done following 6 days IVS for the other peptide groups. It was found that after in vitro re-stimulation, T cells isolated from all immunized groups specifically killed T2 cells pulsed with peptide in contrast with those from na�ve animals. CTL responses to PRAME425-433 (SEQ ID NO. 3), PSMA288-297 (SEQ ID NO. 4), SSX-241-49 (SEQ ID NO. 1) and NY-ESO-1157-165 (SEQ ID NO. 2) were induced in all groups, as assessed by 51Cr cytotoxicity assays. These CTLs had no effect on T2 control cells without peptide. The results demonstrated that T2 target cell lysis by the CTLs isolated from immunized mice is peptide specific. Compared to the �original� protocol, the �expanded� protocol offered no significant enhancement of the lysis percentage, further suggesting that the �original� protocol is sufficient for eliciting a substantial immune response against multiple antigens. Furthermore, due to the increased sensitivity of the CRA assay, the specific NY-ESO-1 responses from each group were more prevalent as compared to the tetramer and ELISPOT assays.
Employing Multiple Therapeutic Cycles
Animals were injected with plasmid vehicle (N=16 animals/group); peptide vehicle (N=16 animals/group); plasmid (400 μg total dose) at high dose (N=16 animals/group); peptide (25 μg total dose) at high dose (N=16 animals/group); plasmid (400 μg total dose) at high dose+peptide (5 μg total dose) at low dose (N=16 animals/group); plasmid at low dose (100 μg total dose)+peptide (25 μg total dose) at high dose (N=14 animals/group); or plasmid (400 μg total dose) at high dose+peptide (25 μg total dose) at high dose (N=16 animals/group) and compared to the na�ve control group N=7 animals/group.
The rationale for various substitutions has been set forth above. The particular substitutions investigated for the PRAME425-433 epitope follow the same logic and are disclosed in the Examples 17-20 and FIGS. 16-18. Substitutions were made at the primary anchor positions P2 and PO(P9), the secondary anchor positions P1 and PO-1 (P8). Substitutions were also made in the TCR interacting positions (in addition to secondary anchor positions) P3 and P6. Selected substitutions have impact on binding and/or stability of MHC class I�peptide complexes: a key feature in determining the immunological properties of peptides. In addition, due to T cell repertoire considerations and to circumvent mechanisms responsible for the limited immunity to native epitopes, substitutions that retain the capability of analogs to interact with T cell receptors recognizing native peptides can be of practical value.
Evaluation of Immunologic Properties of Analogs: Cross-Reactivity and Functional Avidity
1. Minimal required concentration of peptide analog to trigger effects indicative of T cell activation (e.g. cytokine production); 2. Maximal (peak value) effect (e.g. cytokine production) at any analog concentration; 3. Analog concentration at peak value of activating effect (e.g., cytokine concentration) For example, analogs that result in reduced values associated with parameters #1 and 3 but increased #2, can be useful. Use of natural epitope and unrelated non-cross reactive peptides as references is valuable in identifying classes of analogs of potential value. Analogs that display properties quantitatively comparable to or even modestly attenuated from those of natural epitopes are still deemed useful in light of the fact that while they retain cross-reactivity, they may display immunologic properties that are distinct from those of the natural peptide�for example, lower propensity to induce AICD or ability to break tolerance or restore responsiveness in vivo.
The method used for the generation of T cell lines was the following: HHD transgenic mice carrying an A2 human allele (Pascolo et al., J. Exp Med. 185(12):2043-51, 1997, which is hereby incorporated herein by reference in its entirety) were immunized with 50 μg of SSX-2 natural epitope (41-49) admixed with 25 μg of plpC at day 0, 4, 14 and 18 by bilateral administration into the inguinal lymph nodes. At 7 days after the last boost, the mice were sacrificed and a suspension of splenocytes prepared at 5�106 million cells/ml in complete HL-1 medium. Cells were incubated with different concentrations of peptide for 48 hours in flat-bottomed 96-well plates (200 μg/well) and for an additional 24 hours with rIL-2 at 10 U/ml added to the wells. The supernatant was harvested and the concentration of IFN-gamma assessed by standard methods such as ELISA.
Testing of PSMA288-297 Analogs
Cross-Reactivity and Functional Avidity of Analogs Substituted at Single Position
Cross-Reactivity and Functional Avidity of Analogs Substituted at Two Positions
Cross-Reactivity and Functional Avidity of Analogs Substituted at Three Positions
Cross-Reactive Immunogenicity of Various Analogs
Testing of PRAME425-433 Analogs
The analogs listed in FIGS. 16-18 were tested for various properties such as improved affinity and stability of binding, cross-reactivity with the native epitope, and immunogenicity as follows in Examples 17-20. Using the procedures described in application Ser. Nos. 11/455,278 and 11/454,633, each of which is hereby incorporated by reference in its entirety, the HLA-A*0201 binding characteristics of PRAME425-433 and 69 analogs were assessed in comparison to each other. The positive control for binding was melan-A26-35 A27L. The observed affinities of the analogs are resorted as % binding (compared to the positive control) and ED50, and stability of binding as half time of dissociation. Cross reactivity with the native epitope was assessed by using the analog peptides to stimulate IFN-gamma secretion from a T cell line specific for the native epitope, essentially as described in Example 12. The data shown in FIGS. 16-18 were generated by stimulating with analog peptide at approximately 0.3 μM. The results were collected from three separate experiments and were normalized to the amount of IFN-γ elicited by the native peptide in each. In some cases, the reported values are the average of two determinations. An asterisk �*� indicates that IFN-γ production was not distinguishable from background.
Cross-Reactivity and Functional Avidity of Analogs Substituted at a Single Position (FIG. 16)
Cross-Reactive Immunogenicity of the L426Nva L433Nle Analog
Mice were sacrificed at 10 days after the last boost, and splenocytes prepared and assessed for IFN-γ production after in vitro stimulation at 0.5�106 cells/well, with 10 μg/ml of native peptide. At 48 hours after incubation, the supernatant was harvested and the concentration of IFN-γ produced in response to the PRAME425-433 peptide was measured by ELISA. The data are presented in FIG. 19 and show a significant enhancement of IFN-γ production in mice boosted with the PRAME425-433 L426Nva L433Nle analog.
In addition to those already disclosed in this application, the following applications are hereby expressly incorporated by reference in their entireties. Useful methods for using the disclosed analogs in inducing, entraining, maintaining, modulating and amplifying class I MHC-restricted T cell responses, and particularly effector and memory CTL responses to antigen, are described in U.S. Pat. Nos. 6,994,851 (Feb. 7, 2006) and 6,977,074 (Dec. 20, 2005) both entitled �A Method of Inducing a CTL Response�; U.S. Provisional Application No. 60/479,393, filed on Jun. 17, 2003, entitled �METHODS TO CONTROL MHC CLASS I-RESTRICTED IMMUNE RESPONSE�; and U.S. patent application Ser. No. 10/871,707 (Pub. No. 2005 0079152) and Provisional U.S. Patent Application No. 60/640,402 filed on Dec. 29, 2004, both entitled �Methods to elicit, enhance and sustain immune responses against MHC class I-restricted epitopes, for prophylactic or therapeutic purpose�. The analogs can also be used in research to obtain further optimized analogs. Numerous housekeeping epitopes are provided in U.S. application Ser. Nos. 10/117,937, filed on Apr. 4, 2002 (Pub. No. 20030220239 A 1), and 10/657,022 (20040180354), and in PCT Application No. PCT/US2003/027706 (Pub. No. WO04022709A2), filed on Sep. 5, 2003; and U.S. Provisional Application Nos. 60/282,211, filed on Apr. 6, 2001; 60/337,017, filed on Nov. 7, 2001; 60/363,210 filed on Mar. 7, 2002; and 60/409,123, filed on Sep. 5, 2002; each of which applications is entitled �Epitope Sequences�. The analogs can further be used in any of the various modes described in those applications. Epitope clusters, which may comprise or include the instant analogs, are disclosed and more fully defined in U.S. patent application Ser. No. 09/561,571, filed on Apr. 28, 2000, entitled EPITOPE CLUSTERS. Methodology for using and delivering the instant analogs is described in U.S. patent application Ser. No. 09/380,534 and U.S. Pat. No. 6,977,074 (Issued Dec. 20, 2005) and in PCT Application No. PCTUS98/14289 (Pub. No. WO9902183A2), each entitled A �METHOD OF INDUCING A CTL RESPONSE�. Beneficial epitope selection principles for such immunotherapeutics are disclosed in U.S. patent application Ser. Nos. 09/560,465, filed on Apr. 28, 2000, 10/026,066 (Pub. No. 20030215425 A1), filed on Dec. 7, 2001, and 10/005,905 filed on Nov. 7, 2001, all entitled �Epitope Synchronization in Antigen Presenting Cells�; 6, 861, 234 (issued 1 Mar. 2005; application Ser. No. 09/561,074), entitled �Method of Epitope Discovery�; Ser. No. 09/561,571, filed Apr. 28, 2000, entitled EPITOPE CLUSTERS; Ser. No. 10/094,699 (Pub. No. 20030046714 A1), filed Mar. 7, 2002, entitled �Anti-Neovasculature Preparations for Cancer�; application Ser. No. 10/117,937 (Pub. No. 20030220239 A1) and PCTUS02/11101 (Pub. No. WO02081646A2), both filed on Apr. 4, 2002, and both entitled �EPITOPE SEQUENCES�; and application Ser. No. 10/657,022 and PCT Application No. PCT/US2003/027706 (Pub. No. WO04022709A2), both filed on Sep. 5, 2003, and both entitled �EPITOPE SEQUENCES�. Aspects of the overall design of vaccine plasmids are disclosed in U.S. patent application Ser. Nos. 09/561,572, filed on Apr. 28, 2000, entitled �Expression Vectors Encoding Epitopes of Target-Associated Antigens� and 10/292,413 (Pub. No. 20030228634 A1), filed on Nov. 7, 2002, entitled �Expression Vectors Encoding Epitopes of Target-Associated Antigens and Methods for their Design�; 10/225,568 (Pub No. 2003-0138808), filed on Aug. 20, 2002, PCT Application No. PCT/US2003/026231 (Pub. No. WO 2004/018666), filed on Aug. 19, 2003, both entitled �EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS�; and U.S. Pat. No. 6,709,844, entitled �AVOIDANCE OF UNDESIRABLE REPLICATION INTERMEDIATES IN PLASMID PROPAGATION�. Specific antigenic combinations of particular benefit in directing an immune response against particular cancers are disclosed in Provisional U.S. patent Application No. 60/479,554, filed on Jun. 17, 2003 and U.S. patent application Ser. No. 10/871,708, filed on Jun. 17, 2004 and PCT Patent Application No. PCT/US2004/019571 (Pub. No. WO 2004/112825), all entitled �Combinations of tumor-associated antigens in vaccines for various types of cancers�. Antigens associated with tumor neovasculature (e.g., PSMA, VEGFR2, Tie-2) are also useful in connection with cancerous diseases, as is disclosed in U.S. patent application Ser. No. 10/094,699 (Pub. No. 20030046714 A1), filed Mar. 7, 2002, entitled �Anti-Neovasculature Preparations for Cancer�. Methods to trigger, maintain, and manipulate immune responses by targeted administration of biological response modifiers are disclosed U.S. Provisional Application No. 60/640,727, filed on Dec. 29, 2004. Methods to bypass CD4+ cells in the induction of an immune response are disclosed in U.S. Provisional Application No. 60/640,821, filed on Dec. 29, 2004. Exemplary diseases, organisms and antigens and epitopes associated with target organisms, cells and diseases are described in U.S. Pat. No. 6,977,074 (issued Dec. 20, 2005) filed Feb. 2, 2001 and entitled �METHOD OF INDUCING A CTL RESPONSE�. Exemplary methodology is found in U.S. Provisional Application No. 60/580,969, filed on Jun. 17, 2004, and U.S. Patent Application No. 2006-0008468-A1, published on Jan. 12, 2006, both entitled �COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN DIAGNOTISTICS FOR VARIOUS TYPES OF CANCERS�. Methodology and compositions are also disclosed in U.S. Provisional Application No. 60/640,598, filed on Dec. 29, 2004, entitled �COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN COMPOSITIONS FOR VARIOUS TYPES OF CANCER�. The integration of diagnostic techniques to assess and monitor immune responsiveness with methods of immunization including utilizing the instant analogs is discussed more fully in Provisional U.S. Patent Application No. 60/580,964 filed on Jun. 17, 2004 and U.S. Patent Application No. US-2005-0287068-A1, published on Dec. 29, 2005) both entitled �Improved efficacy of active immunotherapy by integrating diagnostic with therapeutic methods�. The immunogenic polypeptide encoding vectors are disclosed in U.S. patent application Ser. No. 10/292,413 (Pub. No. 20030228634 A 1), filed on Nov. 7, 2002, entitled Expression Vectors Encoding Epitopes of Target-Associated Antigens and Methods for their Design, and in U.S. Provisional Application No. 60/691,579, filed on Jun. 17, 2005, entitled �Methods and compositions to elicit multivalent immune responses against dominant and subdominant epitopes, expressed on cancer cells and tumor stroma�. Additional useful disclosure, including methods and compositions of matter, is found in U.S. Provisional Application No. 60/691,581, filed on Jun. 17, 2005, entitled �Multivalent Entrain-and-Amplify Immunotherapeutics for Carcinoma�. Further methodology, compositions, peptides, and peptide analogs are disclosed in U.S. Provisional Application Nos. 60/581,001 and 60/580,962, both filed on Jun. 17, 2004, and respectively entitled �SSX-2 PEPTIDE ANALOGS� and �NY-ESO PEPTIDE ANALOGS.� Each of the applications and patents mentioned in the above paragraphs is hereby incorporated by reference in its entirety for all that it teaches. Additional analogs, peptides and methods are disclosed in U.S. Patent Application Publication No 20060063913, entitled �SSX-2 PEPTIDE ANALOGS�; and U.S. Patent Publication No. 2006-0057673 A1, published on Mar. 16, 2006, entitled �EPITOPE ANALOGS�; and PCT Application Publication No. WO/2006/009920, entitled �EPITOPE ANALOGS�; all filed on Jun. 17, 2005. Further methodology and compositions are disclosed in U.S. Provisional Application No. 60/581,001, filed on Jun. 17, 2004, entitled �SSX-2 PEPTIDE ANALOGS�, and to U.S. Provisional Application No. 60/580,962, filed on Jun. 17, 2004, entitled �NY-ESO PEPTIDE ANALOGS�; each of which is incorporated herein by reference in its entirety. As an example, without being limited thereto each reference is incorporated by reference for what it teaches about class 1 MHC-restricted epitopes, analogs, the design of analogs, uses of epitopes and analogs, methods of using and making epitopes, and the design and use of nucleic acid vectors for their expression. Other applications that are expressly incorporated herein by reference are: U.S. patent application Ser. No. 11/156,253 (Publication No. 20060063913), filed on Jun. 17, 2005, entitled �SSX-2 PEPTIDE ANALOGS�; U.S. patent application Ser. No. 11/155,929, filed on Jun. 17, 2005 entitled �NY-ESO-1 PEPTIDE ANALOGS� (Publication No. 20060094661); U.S. patent application Ser. No. 11/321,967, filed on Dec. 29, 2005, entitled �METHODS TO TRIGGER, MAINTAIN AND MANIPULATE IMMUNE RESPONSES BY TARGETED ADMINISTRATION OF BIOLOGICAL RESPONSE MODIFIERS INTO LYMPHOID ORGANS�; U.S. patent application Ser. No. 11/323,572, filed on Dec. 29, 2005 entitled �METHODS TO ELICIT ENHANCE AND SUSTAIN IMMUNE REPONSES AGAINST MCH CLASS 1 RESTRICTED EPITOPES, FOR PROPHYLACTIC OR THERAPEUTIC PURPOSES�; U.S. patent application Ser. No. 11/323,520, filed Dec. 29, 2005, entitled �METHODS TO BYPASS CD4+ CELLS IN THE INDUCTION OF AN IMMUNE RESPONSE�; U.S. patent application Ser. No. 11/323,049, filed Dec. 29, 2005, entitled �COMBINATION OF TUMOR-ASSOCIATED ANTIGENS IN COMPOSITIONS FOR VARIOUS TYPES OF CANCERS�; U.S. patent application Ser. No. 11/323,964, filed Dec. 29, 2005, entitled �COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN DIAGNOSTICS FOR VARIOUS TYPES OF CANCERS�; U.S. Provisional Application Ser. No. 60/691,889, filed on Jun. 17, 2005 entitled �EPITOPE ANALOGS.�
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Title: Multivalent Entrain- and-Amplify Immunotherapeutics for Carcinoma.23Zhong L, et al., "Recombinant adenovirus is an efficient and non-perturbing genetic vector for human dendritic cells," Eur J Immunol. 29(3):964-972, 1999.* Cited by examinerClassifications U.S. Classification536/23.1, 530/328International ClassificationC07H21/02Cooperative ClassificationA61K2039/545, C07K14/4748, A61K39/00, A01K2217/05, A01K67/0275, A61K2039/53, A01K2207/15, A01K2227/105, A61K39/0011, C07K14/70539, A01K2217/00, C12N15/8509, A01K2267/03European ClassificationC07K14/705B28, A61K39/00D6, C07K14/47A34, A01K67/027M, C12N15/85ALegal EventsDateCodeEventDescriptionFeb 20, 2007ASAssignmentOwner name: MANNKIND CORPORATION, CALIFORNIAFree format text: CORRECTED COVER SHEET TO CORRECT NOTICE OF RECORDATION, PREVIOUSLY RECORDED AT REEL/FRAME 018760/0044 (ASSIGNMENT OF ASSIGNOR S INTEREST);ASSIGNORS:BOT, ADRIAN ION;CHIANG, CHIH-SHENG;DIAMOND, DAVID C.;AND OTHERS;REEL/FRAME:018919/0175;SIGNING DATES FROM 20060802 TO 20060821Free format text: CORRECTED COVER SHEET TO CORRECT NOTICE OF RECORDATION, PREVIOUSLY RECORDED AT REEL/FRAME 018760/0044 (ASSIGNMENT OF ASSIGNOR S INTEREST);ASSIGNORS:BOT, ADRIAN ION;CHIANG, CHIH-SHENG;DIAMOND, DAVID C.;AND OTHERS;SIGNING DATES FROM 20060802 TO 20060821;REEL/FRAME:018919/0175Jan 10, 2007ASAssignmentOwner name: MANNKIND CORPORATION, CALIFORNIAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BOT, ADRION ION;CHIANG, CHIH-SHENG;DIAMOND, DAVID C.;ANDOTHERS;REEL/FRAME:018760/0044;SIGNING DATES FROM 20060802 TO 20060821Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BOT, ADRION ION;CHIANG, CHIH-SHENG;DIAMOND, DAVID C.;ANDOTHERS;SIGNING DATES FROM 20060802 TO 20060821;REEL/FRAME:018760/0044RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services