US20060193855A1 - Methods and compositions to generate and control the effector profile of t cells by simultaneous loading and activation of selected subsets of antigen presenting cells - Google Patents

Methods and compositions to generate and control the effector profile of t cells by simultaneous loading and activation of selected subsets of antigen presenting cells Download PDF

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US20060193855A1
US20060193855A1 US10/527,931 US52793105A US2006193855A1 US 20060193855 A1 US20060193855 A1 US 20060193855A1 US 52793105 A US52793105 A US 52793105A US 2006193855 A1 US2006193855 A1 US 2006193855A1
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cells
peptide
antigen
epitope
cell
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Adrian Bot
Lilin Wang
Dan Smith
Bill Phillips
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Priority claimed from PCT/US2003/007995 external-priority patent/WO2003078595A2/en
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Definitions

  • the present invention is generally directed to methods and compositions to generate an immune response. More specifically, the present invention is directed to methods and compositions of loading an antigen presenting cell to display a delivered epitope on a MHC class I molecule in a context appropriate for the generation of desired T cell responses.
  • Fc ⁇ R Fc gamma receptors
  • the present invention demonstrates, contrary to expectations, that in vivo and ex vivo loading of APC via monovalent engagement of Fc ⁇ R, using peptide epitopes covalently attached to the IgG backbone without modification of the Fc portion, results in access of the epitope to the MHC I processing and presentation pathway, with effective loading of MHC class I molecules. Unexpectedly, this results in generation of robust Tc2 responses characterized by IL-4, but not IL-2 or IFN- ⁇ -producing, MHC class I restricted T cells that recognize the epitope within IgG backbone.
  • compositions that result in effective redirection of class I-immunity to Tc1 effectors that take advantage of the unexpected loading of MHC I by peptide within IgG backbone.
  • Such compositions are able to transform seemingly ineffective MHC class II and class I-restricted peptides into highly effective ones.
  • Fc ⁇ R-mediated loading of APC associated with stimulation of APC by novel synthetic polynucleotides result in generation of class I-restricted cytolytic cells and IFN- ⁇ , IL-2 producing T cells, further associated with protection against a highly virulent microbe or recovery from malignant tumoral process.
  • RNA is dsRNA strand and is pA:pU.
  • the peptide epitope is selected from the group consisting of: influenza virus M1 or M2; hepatitis C virus NS3; hepatitis B virus core antigen; human papilloma virus HPV 18-E7, HPV 16-E7, HPV 18 E6, HPV 16 E6; melanoma-gp100; MART-1; TRP-2; carcinoembryonic antigen precursor; Her-2; tetanus toxin universal T helper epitope; HIV-1: reverse transcriptase; HIV1: gag; insulin precursor-human; human Gad 65; prostate tumor antigens; mucin 1; herpes simplex antigens; and, respiratory syncytial virus antigens.
  • the antigen presenting cell is selected from the group consisting of dendritic cells, monocytes, macrophages and B cells.
  • T cell response is enhanced by co-stimulation with one of the following selected from the group consisting of anti-CD40mAb, recombinant IL-12 or synthetic dsRNA.
  • RNA motifs are ssRNA selected from the group consisting of p(A), p(C), p(G), p(I) and p(U).
  • a method of immunization of a patient comprised of loading an antigen presenting cell by use of at least one peptide epitope of an antigen attached to an Ig backbone or portion thereof thereby forming an Ig-peptide molecule and administering to the patient in vivo the Ig-peptide molecule in conjunction with a dsRNA motif wherein the epitope is effectively processed and presented by the MHC I pathway resulting in effective loading of MHC class I molecules and thereby resulting in an effective secondary expansion of MHC class I-restricted T cells subsequent to in vivo exposure to the antigen.
  • T cells are cytotoxic T lymphocytes.
  • a method of controlling and treatment of a tumor after clinical diagnosis by loading an antigen presenting cell by use of at least one tumor associated T cell epitope attached to an IgG backbone or portion thereof thereby forming an IgG-peptide molecule and administering the Ig-peptide molecule in vivo in conjunction with dsRNA.
  • tumor associated T cell epitope is selected from the group consisting of melanoma-gp100, MART-1, TRP-2, carcinoembryonic antigen precursor XP 064845/NCB1, Her-2, prostate tumor antigens, and MUC 1.
  • compositions for enhancing an immune response to an antigen wherein the composition is a polynucleotide wherein the polynucleotide is made up of compounds selected from the group consisting of adenine, uracil, guanine, cytosine and inosine.
  • composition of paragraph 67 wherein the polynucleotide is dsRNA.
  • composition of paragraph 68 wherein the dsRNA is selected from the group consisting of pA:pU and pI:pC.
  • composition of paragraph 69 wherein the antigen is a virus.
  • composition of paragraph 69 wherein the antigen is attached to an immunoglobulin or portion thereof and administered in vivo.
  • composition of paragraph 72 wherein the antigen is protein or a peptide.
  • composition of paragraph 74 wherein the antigen is a T cell epitope.
  • composition of paragraph 67 wherein the polynucleotide is dsRNA and is coadministererd with the antigen.
  • composition of paragraph 67 wherein the antigen is administered in a pharmaceutically acceptable carrier.
  • dsRNA in the manufacture of a medicament for enhancing an immune response to an antigen in a patient, comprising administering said dsRNA to a patient in conjunction with said antigen.
  • dsRNA consists of bases selected from the group consisting of adenine, cytosine, uracil, guanine and inosine.
  • composition and antigens are administered by one of the following selected from the group consisting of mucosal administration, respiratory administration, intravenous administration, subcutaneous administration, and intramuscular administration.
  • a method of preventing high zone tolerance in a patient to an antigen comprising administering said antigen together with a dsRNA composition wherein the dsRNA composition comprises at least one compound selected from the group consisting of poly-adenine, poly-uracil, poly-guanine, poly-cytosine, poly-inosine.
  • a method of enhancing the immune system in a patient exposed to a pathogen comprising the administration of dsRNA to the patient.
  • dsRNA is selected from the group consisting of pA:pU and pI:pC.
  • a method of enhancing an immune response in a patient in need thereof comprising loading an antigen presenting cell by use of at least one peptide epitope of an antigen attached to an Ig backbone thereby forming an Ig-peptide complex or molecule and administering the Ig-peptide complex or molecule in vivo in conjunction with a dsRNA motif wherein the epitope is effectively processed and presented by the MHC pathway of the antigen presenting cell resulting in effective loading of MHC molecules and thereby resulting in an effective secondary expansion of MHC molecules subsequent to in vivo exposure to the antigen.
  • a method of generating an immune response to an antigen in a patient comprising:
  • T cells are cytotoxic T lymphocytes.
  • dsRNA segment is selected from the group consisting of pA:pU and pI:pC.
  • the peptide epitope is selected from the group consisting of influenza virus M1 or M2; hepatitis C virus NS3; hepatitis B virus core antigen; human papilloma virus HPV 18-E7, HPV 16-E7, HPV 18 E6, HPV 16 E6; melanoma-gp100; MART-1; TRP-2; carcinoembryonic antigen precursor; Her-2; tetanus toxin universal T helper epitope; HIV-1: reverse transcriptase; HIV1: gag; insulin precursor-human; human Gad 65; prostate tumor antigens; mucin 1; herpes simplex antigens; and, respiratory syncytial virus antigens.
  • FIG. 1A shows (a) representation of natural IgG (light chain-heavy chain heterodimer); (B) antigen (Ag) derived peptide inserted within CDR (complementarity determining region) 3, 2, 1 or framework region; (C) VH (heavy chain, variable region) segment replaced with an antigen or fragment; (D) VH and CH1 segments replaced with antigen or antigen fragment;
  • FIG. 1B illustrates diagramatically the IgG-peptide and Fc-peptide
  • FIG. 1C shows properties of selected human IgG backbone
  • FIG. 1D shows the sequence of the constant region of the heavy chain as well as schematic depiction of a prospective construct
  • FIGS. 1E-1M show the sequences of various antigens and epitopes discussed in the present application and which can be inserted into an immunoglobulin [sequences can be accessed on the internet at ncbi.nlm.nih.gov (add the proper address prefix: http://www.) by searching the “proteins” section by use of the provided accession number. The content of this database is hereby incorporated by reference in its entirety.];
  • FIGS. 2A-2B show that while the injection of the peptide epitope in saline was not immunogenic, a similar dose of peptide used for ex vivo loading of APC effectively triggered a substantial immune response upon adoptive transfer;
  • FIG. 3 shows that delivery of epitope within Ig backbone considerably favored its stability in the systemic circulation
  • FIGS. 4A-4B show that pre-incubation of peptide with serum resulted in decreased TcH activation
  • FIGS. 5A-5B show that the relative efficiency of MHC-peptide complex formation greatly varied depending on the nature of antigen and APC;
  • FIGS. 6A-6B show that the peptide epitope within IgG backbone was more effective on a molar basis (1 order of magnitude) than the peptide alone in inducing TcH activation when handled by blood-derived APC;
  • FIGS. 7A-7B show that the use of oil-in-water adjuvant (incomplete Freund's adjuvant, IFA) only modestly enhanced the in vivo formation of MHC-peptide complexes on APC of lymph nodes but not the spleen or thymus;
  • IFA incomplete Freund's adjuvant
  • FIGS. 8A-8D show that use of Fc ⁇ R mediated delivery of peptides results in preferential formation of immunogenic MHC II-peptide complexes on CD11c+ and CD11b+ APC;
  • FIGS. 9A-9C show long lasting expression of peptide onto endogenous MHC II, on both DC (dendritic cells) and monocytes;
  • FIG. 10 shows that formation of MHC II-peptide complexes on dendritic cells and monocytes, subsequent to IgG mediated delivery of peptide epitope, is critically dependent on ITAM+ Fc ⁇ R that encompass the gamma chain;
  • FIG. 11 shows that results show that the expression of the gamma chain of ITAM+ Fc ⁇ R isoforms is necessary for the induction of T cell response to APC loaded with peptide within the IgG backbone;
  • FIGS. 12A-12D show that unexpectedly and in contrast with the potency/cell basis (Example 8), at the organism level, the CD11b+ monocytes have the highest impact on the immune response to a peptide epitope delivered within the IgG backbone;
  • FIGS. 13A-13B shows that Fc ⁇ R-mediated delivery of a T cell epitope within the recombinant Ig backbone results in Th2 rather than Th1 response;
  • FIG. 14 shows that Fc ⁇ R-mediated delivery of T cell epitope within recombinant Ig backbone results in Th2 rather than Th1 response;
  • FIG. 15 shows that a peptide epitope within the IgG backbone triggers a cellular response of Th2 profile that is enhanced but not switched by a conventional adjuvant (CFA);
  • CFA conventional adjuvant
  • FIG. 16 shows that peptide presentation by APC, subsequent to loading with antigen by using recombinant IgG as delivery platform, occurs in context of limited co-stimulation
  • FIGS. 17A-17B show that the activity of HA (110-120 hemagglutinin peptide) specific IL-4 producing T cells triggered by administration of recHA(I-Ed)-IgG is dependent on CD4 rather than CD8;
  • FIG. 18 shows that the IgG mediated delivery of T cell epitope has a profound and differential effect on the expansion and cytokine production by activated T cells: IL-2, IFN- ⁇ and surprisingly IL-4, were down-regulated in a dose-related manner;
  • FIGS. 19A-19B show that in contrast to viral immunization with an influenza virus strain bearing the cognate peptide, Ig-mediated peptide delivery was ineffective in triggering cytotoxic response;
  • FIGS. 20A-20D show that co-administration of MBP and PLP epitopes by using recombinant IgG curbed the chronic progression of disease
  • FIG. 21 summarizes the impact of IgG/Fc ⁇ R-mediated delivery of epitopes on the T cell response, based on data provided in Examples 2-20;
  • FIG. 22 shows that shows that natural, non-infectious double stranded RNA produced during infection with influenza virus, has substantial effects on the specific immune response to a protein antigen
  • FIG. 23A shows an extensive library of synthetic RNA motifs
  • FIGS. 23B-23D show that different synthetic RNAs have an enhancing effect on the B and T cell response to a prototype protein antigen
  • FIGS. 24A-24B show effects of selected RNA motifs on the innate immune response
  • FIG. 25 shows that distinct RNA motifs bind to different receptors on antigen presenting cells
  • FIG. 26 shows that distinct RNA motifs induce differential upregulation of chemokines
  • FIG. 27 shows that the control of replication of influenza virus can be achieved by using selected synthetic RNA motifs
  • FIG. 28 shows that selected synthetic RNA motifs pI:pC and pA:pU largely prevent high zone tolerance that is usually associated with administration of large amounts of purified protein;
  • FIG. 29 shows that selected synthetic RNA motifs effect on human monocytic cells
  • FIGS. 30A-30B show that non-tagged pA:pU, but not non-tagged pI:pC, was able to compete out the binding of tagged pA:pU to human THP-1 monocytic cells;
  • FIG. 31 shows the purification and fractionation steps of dsRNA
  • FIG. 32 shows that lower molecular weight fractions of a selected synthetic RNA compounds are endowed with different biological activity
  • FIG. 33 shows that pI:pC but not pA:pU induced antibody response against itself, with a cross-reactive component against another RNA motif;
  • FIGS. 34A-34B show that co-use of selected synthetic RNAs promote effective induction of IL-2 and IFN-gamma subsequent to IgG mediated delivery of an MHC class I-restricted epitope;
  • FIG. 35 shows that ex vivo APC loading by recombinant IgG is more effective in formation of MHC class I-peptide complexes and generation of Tc response, compared to use of free peptide itself;
  • FIG. 36 show that IgG mediated delivery of a class I restricted epitope is most effective in priming class I restricted Tc1 responses when co-administration of selected synthetic RNA was carried out;
  • FIG. 37 shows that effective priming of anti-viral cytotoxic T cells requires both effective i* vivo loading of APC with class I restricted epitope delivered via IgG, together with appropriate instruction by selected synthetic RNA motif;
  • FIG. 38 shows that immunization with a recombinant IgG bearing a viral class I restricted epitope together with selected synthetic dsRNA, resulted in priming of an immune response capable of limiting the replication of a virus subsequent to infectious challenge;
  • FIG. 39 describes the tumor models used for testing the efficiency of Ig-peptide-based molecules
  • FIG. 40 shows that both effective in vivo loading of APC with tumor associated antigen, together with simultaneous activation by selected synthetic RNA motifs, are necessary and sufficient for effective control of tumor growth and induction of tumor rejection;
  • FIG. 41 shows that both effective in vivo loading of APC with tumor associated antigen, together with simultaneous activation by selected synthetic RNA, can trigger an effective immune response to tumor-associated antigens;
  • FIG. 42 shows that tumor infiltrating lymphocytes displaying the T cell receptor marker TCR ⁇ acquired expression of the activation marker CD25 upon treatment with recombinant immunoglobulin bearing tumor associated epitope, together with selected synthetic dsRNA motif;
  • FIG. 43 shows that the treated mice that successfully rejected the tumor developed Tc1 responses against the tumor-associated epitope on the therapeutic Ig, along with Tc2 immunity;
  • FIG. 44 shows that successful rejection of tumor induced by indicated treatment is followed by effective protection against subsequent challenge with the same tumor, indicating development of effective immune memory
  • FIGS. 45A-45B show that the emerging immunity, subsequent to the indicated treatment that results in tumor rejection, protects against challenge with loss of antigen variants and is associated with overall expansion of cytokine producing cells.
  • adjuvant a substance that enhances the adaptive arm of the immune response to an antigen
  • adoptive transfer transfer of a cell population from one animal to another of the same haplotype
  • antigen a molecule that can be specifically recognized by the adaptive elements of the immune system (B cells, T cells or both);
  • antigen presenting cell heterogeneous population of leukocytes with very efficient immunostimulatory capacity
  • B cell a type of lymphocyte developed in the bone marrow. Each B cell encodes a surface receptor specific for a particular antigen. Upon recognition of a specific antigen, B cells multiply and produce large amounts of antibodies which in turn bind to the antigen which activated the B cell;
  • B cell unresponsiveness antigen-specific lack of response by B cell
  • CDR Complementarity Determining Region
  • hypervariable regions in an immunoglobulin which create the antigen binding site. There are three CDR regions: CDR1, CDR2 and CDR3;
  • chemokines a group of at least 25 small cytokines, all of which bind to heparin;
  • cross primed—antigen presenting cells that have acquired antigens from infected tissues and then present them to cognate T cells;
  • Dendritic Cells A subtype of antigen presenting cells (i.e. CD11c+);
  • epitope parts of an antigen which contact the antigen binding site of the antibody or T cell receptor;
  • heterodimer dimeric protein consisting of 2 different protein sequences
  • high zone tolerance a state of unresponsiveness specific to a particular antigen that is induced upon challenge with a high concentration of said antigen
  • IL-2 refers to interleukin-2
  • IL-4 refers to interleukin-4
  • Immunoglobulin a group of glycoproteins present in the serum and tissue fluids of all mammals and are located on the surface of B cells and serve as antibodies free in the blood or lymph. There are five classes of immunoglobulins: IgG (70-75%), IgM (10%), IgA (15-20%), IgD (>1%) and IgE (found on basophils and mast cells in all individuals). IgG has four human subclasses (IgG1, IgG2, IgG3 and IgG4);
  • Immunoglobulin backbone refers to an immunoglobulin molecule or portion thereof wherein at least one CDR region is able to receive an inserted peptide epitope
  • immunoglobulin isotype switching stimulation of B cells to switch production from one immunoglobulin isotype to another;
  • incomplete Freund's adjuvant an oil-in-water emulsion not containing mycobacterial cell wall components
  • the innate immune system provides broad relatively nonspecific host defenses that lack antigenic specificity but have the ability to guide acquired immunity.
  • the cells types involved are dendritic cells and macrophages;
  • isoforms different glycosylation, phosphorylation, deamidation and other posttranslational modifications of proteins
  • ITAM immunodeceptor tyrosine-based activation motifs
  • ITIM immunodeceptor tyrosine-based inhibitory motifs
  • macrophages Any mononuclear, actively phagocytic cell arising from monocytic stem cells in the bone marrow;
  • MHC refers to the Major Histocompatibility Complex
  • modified immune response enhanced or diminished immune response
  • monocytes Mononuclear leukocytes found in lymph nodes, spleen, bone marrow and loose connective tissue
  • peptide a compound consisting of two or more amino acids joined together by a peptide bond
  • polynucleotide a polymer of nucleotides
  • self-antigens antigens that are derived from the host
  • Tc1 immunity Cytotoxic T cell type 1, CD 8+;
  • Th1 cells T helper 1 cells which are involved in cell mediated inflammatory reactions, identified by production of IFN ⁇ , TNF ⁇ and IL-2;
  • Th2 cells T helper 2 cells which encourage production of antibodies and are identified by production of IL-4 and IL-5;
  • Th3 cells T helper regulatory cell, known to produce transforming growth factor (TGF)-beta;
  • TR1 cells T regulatory cell, known to produce interleukin 10;
  • upregulation enhancement of expression or activity of a particular compound or effect
  • FIG. 1A For selective in vivo loading of antigen presenting cell subsets, the use of compounds described schematically in the FIG. 1A are used: (A) representation of natural IgG (light chain-heavy chain heterodimer); (B) antigen (Ag) derived peptide inserted within CDR 3, 2, 1 or framework region; (C) VH segment replaced with an antigen or fragment; and, (D) VH and CH1 segments replaced with antigen or antigen fragment.
  • This type of molecules are engineered using methods known in the art and as stated as follows:
  • PCR Polymerase chain reaction
  • VH and inserted epitopes were confirmed by DNA sequencing.
  • the human IgG backbone was obtained from IgGA1 myeloma cell line by RT-PCR.
  • the recombinant human IgG was cloned by inserting the stated epitopes to replace the CDR2 or CDR3 regions of the human IgG1 backbone. Briefly, T cell epitopes were created by PCR mutagenesis and subcloned into the CDR2/CDR3 region.
  • the recombinant heavy chains were then subcloned into pMG vector (Invivogen, San Diego, Calif.) by BamHI and XbaI sites. The heavy chain expression was controlled by the hCMV promoter.
  • the human kappa light chain was subcloned into the pMG vector by StuI and NheI sites.
  • the expression of the light chain was controlled by an EF-1 alpha and HTLV-1 LTR hybrid promoter.
  • the double expression vector carrying both the recombinant heavy chain and light chain were then transfected into expression cell lines.
  • the Fc-peptides were constructed by cutting off the VH and CH1 fragment and replacing it with stated viral or tumor antigens (8-150 Aas). Briefly, the human IgG1 heavy chain was subcloned into pCDNA3 vector by EcoRI and XhoI sites. Then the stated antigens are inserted between the leader sequence and hinge region of IgG1 by PCR mutagenesis. To increase the flexibility of the fused antigens, an oligo-glycine linker (5 glycines) was added after the antigen. The expression of human IgG recombinant molecules can be performed by using either one of the strategies displayed in FIG. 1B .
  • the human IgG backbone has been selected rationally, based on the ability to bind to Fc ⁇ R, complement and cytokine activation in various states. Properties of selected human IgG backbone are shown in the FIG. 1C and the sequence of the constant region of the heavy chain as well as the schematic depiction of a prospective construct, is shown in FIG. 1D .
  • Epitopes used for model recombinant IgG are shown in FIG. 1E (mouse MHC class II-restricted HA epitope and mouse MHC class I restricted NP epitope).
  • the nomenclature of recombinant constructs is recIgG-epitope (HA or NP)-restriction element (I-Ed or Kd, respectively). In short, they may be referred to as IgHA or IgNP.
  • Model molecules comprising defined mouse self epitopes (MBP or PLP derived) were similarly constructed.
  • the sequence of the variable region of the heavy chain of anti-arsonate antibody used as the backbone has been depicted in FIG. 1E and the technology is well known in the art (Zaghouani et al., Science Jan. 18, 1993;259(5092):224-7) the contents of which is hereby incorporated by reference.
  • FIGS. 1E-1M examples of antigens and epitopes (in bold) are provided that could be inserted (larger parts up to 150 AA spanning one or multiple epitopes) or attached to the backbone. Such constructs comprising the shown antigens/epitopes may be used as drugs against infectious or tumoral diseases.
  • FIG. 1I there is the HLA-A2 anchor motif displayed, that allows the prediction of location of potentially therapeutic cytotoxic epitopes in any protein, facilitating the selection of the antigen fragment to be used in the recombinant immunoglobulin.
  • FIG. 1J examples of “universal” T helper epitopes (Kumar et al. J Immunol Mar. 1, 1992;148(5):1499-505) are provided, both dominant and promiscuous from the point of view of MHC restriction, that could be used for construction of composite molecules for the purpose of inducing or enhancing immunity to MHC class I-restricted epitopes, using compounds such as:
  • FIG. 1K bottom.
  • FIG. 1K examples of human self antigens with epitopes bolded are shown, that could be used to generate recombinant IgG molecules against autoimmune/inflammatory disorders.
  • FIGS. 1L and 1M other antigen sequences that could be used for the construction of above mentioned immunoglobulin constructs are shown.
  • the antigen fragments of interest could be defined by using methods to predict MHC class I epitopes (Lim et al., Mol Immunol. February 1996;33[2]:221-30).
  • the SP2/0 cell line (American Type Culture Collection) is used for the production of all the recombinant IgGs (rIgG) discussed in this patent application.
  • Stable expressing cell lines i.e. transfectomas
  • plasmids encoding the heavy and light chains of an anti-arsenate mouse IgG.
  • Each transfectoma differs only in the sequence of the CDR3 region of the heavy chain.
  • the SP2/0 transfectomas were initially grown in Quantum Yield media (BD Biosciences) supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 0.5 mg/mM gentamicin and 2.5 ⁇ g/mL Fugizone. Cultures were maintained at 37° C. in a humidified CO2 incubator. Efforts were made to adapt each of the cell lines to growth in different commercially available serum-free medias (Lymphocyte Growth Media 2, Clonetics; Cell MAb Growth Media Serum Free, BD Biosciences; Animal Component Free Cell Media, BD Biosciences). Each of the serum-free medias was supplemented with antibiotics as above. Culture media containing secreted IgG was produced from each media noted above. No difference in the IgGs produced in the different medias was observed over the course of this work (molecular weight analysis by SDS PAGE [see below], ELISPOT assays, and immune responses in mice).
  • the amount of secreted rIgG was quantitated using an ELISA: capture antibody was a goat anti-mouse IgG (Sigma) and secondary antibody was an anti-mouse IgG HRP conjugate (Sigma). Purified mouse IgG (Sigma) was used as a standard.
  • CM conditioned media
  • flasks flasks, stirred vessels, packed bed bioreactors (New Brunswick Cellagen), CELLine flasks (BD Biosciences).
  • CM conditioned media
  • the cells were fed and/or harvested twice a week and maintained at least 50% viability, but viability was generally greater than 70%.
  • Collected media was filtered and held at 4 C.
  • Stirred vessels (1 L) were seeded at 10 6 cells per mL in 200 mL starting volume. Media was added weekly to keep the cell number between 10 7 and 10 6 per mL until 800 mL of total volume was reached.
  • each flask was seeded with 10 7 to 10 8 cells in a total volume 40 mL in the cell compartment; 1 L of media was added to the feed compartment; CM was harvested from the cell chamber after 2 to 3 weeks, or when viability of the cells fell below 20%.
  • the rIgGs produced by the above methods were purified by one of two methods.
  • CM that contained FBS
  • an anti-mouse IgG immunoaffinity resin was used for CM that contained FBS.
  • the immunoaffinity resin was synthesized using the following protocol: 10 mL of cyanogen bromide-activated Sepharose 4B (Sigma) was washed with 1 mM HCl as per manufacturer's instructions; 10-20 mg of goat anti-mouse IgG (Sigma) was dissolved in coupling buffer (0.1 M sodium carbonate [pH 8.4]/0.5 M NaCl) at a concentration of 2 mg/mL; the IgG solution was added to the washed resin, and the slurry was mixed end-over-end at room temperature; the extent of coupling was monitored using the Bradford assay to determine the amount of remaining soluble IgG; the coupling was quenched by addition of ethanolamine to a final concentration of 10 mM when the amount of soluble IgG was less
  • the immunoaffinity resin was then washed with the following buffers: PBS, 10 mM glycine (pH 2.4), 20 mM Tris/1 M NaCl (pH 8.0), PBS.
  • the resin was stored at 4° C. in PBS.
  • the protocol for purifying rIgG with this resin was initiated by passing CM through the column at 1 to 2 mL/min.
  • the resin was then washed free of nonbound protein using the following protocol: 100 mL PBS/0.5M NaCl followed by 50 mL 1 mM Tris (pH 8). Fractions were monitored for protein using the Bradford assay. Specifically bound rIgG was eluted with a low pH buffer (5 mM glycine (pH 2.4)/0.5 M NaCl). The eluted protein was collected and held at 4° C. for further processing (see below).
  • the rIgG produced in serum-free culture media was purified using Protein A affinity chromatography. Typically, a 5 mL rProtein A column (HiTrap rProtein A FF from Amersham Pharmacia Biotech) was equilibrated with PBS and the sample was run through the column at 2 mL/min using a FPLC unit (Pharmacia). The resin was washed free of nonspecifically bound protein with PBS, followed by 20 mM Tris (pH 8.0)/1 M NaCl, then water. The specifically bound rIgG was eluted with 1 mM glycine (pH 2.4). The eluted peak was collected and held at 4 C for further processing.
  • the rIgG fractions were pooled and concentrated using Centricon ultrafiltration units (Amicon) to a final concentration of 1 to 4 mg/mL (Bradford assay with IgG as standard). The concentrated fraction was then dialyzed into 1 mM glycine (pH 2.4), the final concentration determined by A 280 using an extinction coefficient of 1.4 for a 1 mg/mL IgG solution, and aliquoted into 100 ⁇ l fractions that were stored in the ⁇ 80° C. freezer. The purified rIgGs were analyzed for structural integrity and purity by SDS gel electrophoresis. The gels were stained with Coomassie blue (Pierce Chemical).
  • the rIgGs used in the reported experiments displayed their expected molecular weight (reduced and nonreduced) as compared to protein standards and control IgG.
  • the purified rIgG was greater than 95% pure as determined by visual inspection of the stained bands relative to the bands of known amounts of control IgG run on the same gel.
  • dsRNA double stranded RNA
  • ssRNA single stranded RNA segments of the present invention
  • ssRNA The polynucleotides
  • polyA polyA
  • polyU polynucleotide-phosphorylase
  • dsRNA Annealing of polyadenylic acid (polyA or pA) with polyuridylic acid (polyU or pU).
  • the dsRNA and ssRNA of the present invention are homopolymers with, in the case of dsRNA, a single base or nucleotide (e.g., adenine) consistently forming one strand with its complement consistently forming the other strand.
  • a single base or nucleotide e.g., adenine
  • the single strand is consistently made of the same nucleotide.
  • dsRNA or ssRNA compositions that are made up of mixed nucleotides (and without or without their complements in the case of dsRNA).
  • a polyA:polyU dsRNA segment with occasional substitution by an a non-complementary nucleotide e.g., guanine, cytosine or inosine.
  • the dsRNA and ssRNA compositions of the present invention are comprised of the bases/nucleotides adenine (A), guanine (G), cytosine (C), uracil (U) and inosine (I) and could also be comprised of a small percentage of the DNA base thymine (T).
  • the RNA compositions in Table I and FIG. 8A is descriptive of various RNA compositions used in the Examples.
  • the RNA compositions of the present invention were prepared and purified according to Example 30.
  • RNA strands used in the present invention are generally between 100-2000 base pairs in length but may be between 1-20, 20-40, 40-60, 60-80, 80-100, 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 800-900, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100-2200, 2300-2400, 2400-2500, 2500-3000, 3000-4000, 4000-5000, 5000-10,000 base pairs and greater than 10,000 base pairs in length and/or mixtures thereof.
  • Antigen presenting cells from 1 na ⁇ ve BALB/c mouse were obtained from splenic tissue. Following washing, three million APC were incubated with 13.5 nM HA 110-120 peptide for 3 hours at 37° C., in 1 ml of HL-1 medium. The cells were washed, divided into three equal inoculi and injected (1 ⁇ 2 subcutaneously+1 ⁇ 2 intraperitoneally) into 3 na ⁇ ve BALB/c mice.
  • mice were sacrificed 2 weeks later and the immune response measured against HA 110-120 peptide, by ELISPOT analysis as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ /well of DMEM complete containing FBS, for an hour at 37° C.
  • Single cell suspension was made from the spleens, red blood cells were lysed, cells washed, counted and incubated at 5 ⁇ 10 5 /well together with 20 ⁇ g/ml HA 110-120 peptide or just with media, to assess the background.
  • mice were then allowed to dry at room temperature for 24 hours.
  • the data were acquired using an automated system (Navitar, Rochester, N.Y.) with ImagePro-Plus) software (Media Cybernetics, Silver Spring, Md.).
  • 3 na ⁇ ve BALB/c mice were each injected with 4.5 nM of HA peptide in sterile PBS, half of it administered subcutaneously and half of it intraperitoneally. The mice were sacrificed 2 weeks later and the T cell response characterized as above, by ELISPOT analysis.
  • FIG. 2 (A) the experimental protocol is described.
  • FIG. 2 (B) the results of the experiment are shown: they were expressed as number of IFN- ⁇ , IL-2 and IL-4 spot forming colonies/spleen, after the subtraction of the background (mean ⁇ SEM).
  • H-APC antigen presenting cells (dendritic cells) loaded ex vivo prior to adoptive transfer.
  • HA corresponds to peptide directly injected into animals.
  • FIGS. 2A-2B show that while the injection of the peptide epitope in saline was not immunogenic, a similar dose of peptide used for ex vivo loading of APC effectively triggered a substantial immune response upon adoptive transfer. This shows that if directly injected, the peptide does not effectively reach APC, a prerequisite for effective induction of an immune response.
  • BALB/c Scid mice (3/group) were injected intravenously with 60 nM of SFERFEIFPKE (“HA”) [Seq. I.D. No. 5] peptide or 2.4 nM of recHA (I-Ed)-IgG (“Ig-HA”) and blood was harvested at various intervals. Serum was immediately separated and promptly frozen at ⁇ 70° C. Later, the serum samples were incubated with 2 ⁇ 10 4 cells/well/50 ⁇ l HA-specific T cell hybridoma (TcH) and 1 ⁇ 10 4 cells/well/50 ⁇ l M12 B cell lymphoma APC, in serum free HL-1 medium at 37° C. and 5% CO 2 for 24 hours.
  • HA SFERFEIFPKE
  • X-gal substrate freshly prepared as follows: 200 ⁇ l of the X-gal stock solution, (40 mg/ml in DMSO) in 10 ml of substrate buffer (5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS).
  • substrate buffer 5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS.
  • the blue activated TcH were scored visually using the microscope.
  • TcH The activation of TcH was represented as function of time post-injection.
  • the epitope could be detected in the blood only in the case of mice injected with recHA(I-Ed)-IgG, for an interval of about one day.
  • the HA peptide injected as is was not detected in the periphery despite being used in large molar excess (25 fold).
  • FIG. 4 (A) shows the detrimental effect of serum on the presentation of a T cell epitope peptide: M12 B cell lymphoma APC were incubated with TcH in the presence of various amounts of SFERFEIFPKE (HA) peptide in serum-free HL-1 medium (“HA+HL-1”) or HL-1 medium supplemented with 20% mouse serum from BALB/c scid mice (“HA+serum”). The number of cells incubated was 2 ⁇ 10 4 M12 and 1 ⁇ 10 4 TcH/100 ⁇ l of HL-1 medium supplemented or not with serum.
  • SFERFEIFPKE HA
  • X-gal substrate freshly prepared as follows: 200 ul of the X-gal stock solution, (40 mg/ml in DMSO) in 10 ml of substrate buffer (5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS).
  • substrate buffer 5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS.
  • the blue activated TcH were scored visually using the microscope.
  • the serum negatively interfered with the formation and/or presentation of immunogenic MHC-peptide complexes.
  • FIG. 4B the serum negatively interfered with the formation and/or presentation of immunogenic MHC-peptide complexes.
  • HA peptide peptide
  • IgHA recHA-IgG
  • X-gal substrate freshly prepared as follows: 200 ⁇ l of the X-gal stock solution, (40 mg/ml in DMSO) in 10 ml of substrate buffer (5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS).
  • substrate buffer 5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS.
  • the blue activated TcH were scored visually using the microscope.
  • the results were represented as percentage of activated T cells (beta-gal + TcH)/well at concentrations of 2 ⁇ g/ml of recHA (I-E d )-IgG (“IgHA”) or 40 ⁇ g/ml of HA peptide (1,000 molar excess relative to the recombinant Ig).
  • results described in the FIG. 4 show that pre-incubation of peptide with serum resulted in decreased TcH activation. Addition of serum after APC pulsing did not have an effect on TcH activation. In contrast, the formation of MHC-peptide complexes was not impaired by serum when the recombinant immunoglobulin carrying the peptide was used instead of the peptide alone.
  • ex vivo formation of MHC-peptide complexes on antigen presenting cells (APCs) from spleen was measured as follows: splenic APC were isolated by magnetic sorting using anti-MHC II antibodies. Separation by using magnetic beads coupled with anti-MHC II was carried out using magnetic cell separators and reagents from Miltenyi Biotec, Germany as follows: spleens were processed to single cell suspension, red blood cells lysed, then cells washed, counted and resuspended in MACS buffer (PBS supplemented with 2 mM EDTA and 0.5% BSA). Magnetically labeled cells were passed through a separation column which is placed in the magnetic field of a MACS separator.
  • MACS buffer PBS supplemented with 2 mM EDTA and 0.5% BSA
  • the magnetically labeled positive fraction is retained in the column while the negative fraction runs through. After removal of the column from the magnetic field, the magnetically retained positive cells are eluted from the column, cells are washed, counted, resuspended in HL1 complete media and they were incubated with specific T cell hybridoma recognizing I-E d +SFERFEIFPKE overnight, in the presence of various amounts of SFERFEIFPKE (“HA”) peptide or recHA(I-Ed)-IgG (“IgHA”). Per well, 2 ⁇ 10 4 APC were incubated with 1 ⁇ 10 4 TcH.
  • HA SFERFEIFPKE
  • IgHA recHA(I-Ed)-IgG
  • the plate was centrifuged for 15 min/4° C./1500 RPM, then the supernatant was flicked, the cells were fixed with cold freshly made fixing solution (2% Formaldehyde, 0.2% Glutaraldehyde in 1 ⁇ PBS) and the plate was again centrifuged for 3 min/4° C./1500 RPM. Fixing solution was flicked off the plate, cells washed once with PBS 200 ⁇ l/well, centrifuging the plate for 3 min/4° C./1500 RPM. PBS was flicked off the plate and cells were incubated overnight at 37° C.
  • fixing solution 2% Formaldehyde, 0.2% Glutaraldehyde in 1 ⁇ PBS
  • the X-gal substrate freshly prepared as follows: 200 ⁇ l of the X-gal stock solution, (40 mg/ml in DMSO) in 10 ml of substrate buffer (5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS).
  • substrate buffer 5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS.
  • the blue activated TcH were scored visually using the microscope. The number of activated TcH was quantified and the results expressed as activation versus molar amount of epitope.
  • the results described in the FIG. 5B show that the relative efficiency of MHC-peptide complex formation greatly varied depending on the nature of antigen and APC.
  • the peptide epitope within the IgG backbone was 10 times more effectively handled by MHC II+ APC from lymphoid organs and 1000 times more effectively handled by transformed B cell lymphoma cells, as compared to the free peptide itself.
  • the cellular handling of the epitope and formation of MHC-peptide complexes subsequent to delivery within IgG greatly varies with the nature of APC.
  • PBMC peripheral blood mononuclear cells
  • FACS analysis for expression of CD11c, CD11b and B220 was carried out.
  • FIG. 6A The results are represented in FIG. 6A as percentage of APC and T cells in blood versus a prototype secondary lymphoid organ (spleen).
  • the number of professional APC such as CD11c+ cells is tremendously (2 logs) decreased in blood as compared to spleen.
  • B220+ and CD11b+ cells were decreased as well (1 order of magnitude). The following materials and methods were used.
  • Ficoll Ficoll-hypaque (1.077, Amersham, cat# 17-1440-02)
  • Antibodies CD11b cat#01715A, CD11c cat# 557401, B220 cat#01125A, all PE conjugated (BD PharMingen)
  • FACS Buffer PBS, 1% FCS, 0.1% sodium azide.
  • PBMC peripheral blood mononuclear cells
  • SFERFEIFPKE HA-specific TcH
  • I-Ed recHA
  • IgG cognate peptide or recHA-IgG
  • the cells were co-incubated for 24 hours (2 ⁇ 10 4 APC+1 ⁇ 10 4 TcH). The next day the plate was centrifuged for 15 min/4 C/1500 RPM, then the supernatant was flicked, the cells were fixed with cold freshly made fixing solution (2% Formaldehyde, 0.2% Glutaraldehyde in 1 ⁇ PBS) and the plate was again centrifuged for 3 min/4° C./1500 PM.
  • FIGS. 6A-6B show that the peptide epitope within IgG backbone was more effective on a molar basis (1 order of magnitude) than the peptide alone in inducing TcH activation when handled by blood-derived APC, suggesting that in suboptimal conditions associated with limiting numbers of professional APC, the Ig backbone greatly facilitates the creation of MHC-peptide complexes.
  • the local (mesenteric) lymphoid nodes (LN), spleen and thymus were harvested, single cell suspensions were made, red blood cells lysed from the spleens, LN and thymus were collagenase digested. All cells were washed, counted and incubated with TcH recognizing I-Ed+SFERFEIFPKE (MHC class II-HA) complexes. The number of TcH was 1 ⁇ 10 4 /well. The formation of such MHC-peptide complexes was evaluated by titrating the number of APC with constant number of TcH and measuring TcH activation after overnight incubation.
  • X-gal substrate freshly prepared as follows: 200 ⁇ l of the X-gal stock solution, (40 mg/ml in DMSO) in 10 ml of substrate buffer (5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS).
  • substrate buffer 5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS.
  • the blue activated TcH were scored visually using the microscope.
  • the data are expressed as TcH activation versus APC number ( FIG. 7A ) and as estimated percentage of APC expressing MHC-peptide complexes ( FIG. 7B ), based on in vitro standard curve obtained as depicted in the previous Examples, 5 and 6.
  • FIGS. 7A-7B show that the use of oil-in-water adjuvant (IFA) modestly enhanced the in vivo formation of MHC-peptide complexes on APC of lymph nodes but not spleen or thymus.
  • IFA oil-in-water adjuvant
  • Substantial dose escalation of peptide in saline or in emulsion is not paralleled by proportional enhancement in the generation of loaded APC and/or MHC-peptide complexes on APC in vivo.
  • use of peptide within Ig backbone enhances the formation of MHC peptide complexes considerably, on APC from secondary lymphoid organs such as lymph nodes and spleen.
  • MHC II-peptide complexes on APC from thymus remained limited, similar to that conferred by peptide alone.
  • the enhancement factor conferred by incorporation of peptide within the IgG was unexpectedly high (approximately 2-3 orders of magnitude), indicating that other factors, in addition to cellular handling (e.g. the above described pharmacokinetics and protective effects), were involved. Even 100 fold dose escalation of peptide alone, in saline or IFA, could not restore the in vivo loading of APC noted with peptide within IgG backbone.
  • EXAMPLE 7 SHOWS THAT AMONG THE THREE MAJOR APC SUBSETS (DC, MONOCYTES/MACROPHAGES AND B CELLS) RATHER THAN B CELLS ARE THE MOST POTENT ON A PER CELL BASIS IN PRESENTING THE PEPTIDE EPITOPE SUBSEQUENT TO IN VIVO DELIVERY VIA IgG BACKBONE. THE EFFICIENCY OF APC LOADING AND RESULTING PRESENTATION IS SUBSTANTIALLY HIGHER THAN THAT RESULTING FROM DELIVERY OF FREE PEPTIDE
  • X-gal substrate freshly prepared as follows: 200 ⁇ l of the X-gal stock solution, (40 mg/ml in DMSO) in 10 ml of substrate buffer (5 nM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS).
  • substrate buffer 5 nM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS.
  • the blue activated TcH were scored visually using the microscope.
  • results are expressed in FIG. 8B as number of activated TcH/well.
  • MHC II+ APC from naive BALB/c mice were incubated in vitro, overnight, with an optimal concentration of HA peptide (50 ug/ml), extensively washed and incubated in different numbers with TcH as above.
  • the results show that the formation of MHC II-peptide complexes on splenic APC is at least 2 orders of magnitude more effective when the epitope is delivered within IgG backbone.
  • FIGS. 8A-8D show that use of FcgR mediated delivery of peptides results in preferential formation of immunogenic MHC II-peptide complexes on CD11c+ and CD11b+ APC.
  • EXAMPLE 8 SHOWS A PROLONGED PERSISTENCE IN VIVO OF MHC-PEPTIDE COMPLEXES ON APC (DC AND MONOCYTES) FOLLOWING ADMINISTRATION VIA A IgG BACKBONE
  • MHC II-peptide complexes were measured by magnetic separation of CD11c+ DC and CD11b+ monocytes at various intervals subsequent to intravenous injection of 2 uM of recHA (I-Ed)-IgG.
  • magnetic separation was carried out using magnetic cell separators and reagents from Miltenyi Biotec, Germany as follows: spleens were processed to single cell suspension, red blood cells lysed, then cells washed, counted and resuspended in MACS buffer (PBS supplemented with 2 mM EDTA and 0.5% BSA). Magnetically labeled cells were passed through a separation column which is placed in the magnetic field of a MACS separator.
  • the magnetically labeled positive fraction is retained in the column while the negative fraction runs through. After removal of the column from the magnetic field, the magnetically retained positive cells are eluted from the column, cells are washed, counted, resuspended in HL1 complete media and incubated. Different numbers of separated APC (A—CD11b+ monocytes, B—CD11c+ dendritic cells, C—whole splenocyte population) were incubated overnight with 1 ⁇ 104 TcH specific for the HA peptide.
  • APC from naive mice were used that were in vitro loaded with optimal amounts of HA peptide (50 ⁇ g/ml), overnight and washed prior to incubation (“ctrl”). The next day the plate was centrifuged for 15 min/4° C./1500 RPM, then the supernatant was flicked, the cells were fixed with cold freshly made fixing solution (2% Formaldehyde, 0.2% Glutaraldehyde in 1 ⁇ PBS) and the plate was again centrifuged for 3 min/4° C./1500 RPM. Fixing solution was flicked off the plate, cells washed once with PBS 200 ⁇ l/well, centrifuging the plate for 3 min/4° C./1500 RPM.
  • fixing solution 2% Formaldehyde, 0.2% Glutaraldehyde in 1 ⁇ PBS
  • PBS was flicked off the plate and cells were incubated overnight at 37° C. with 200 ⁇ l/well of the X-gal substrate freshly prepared as follows: 200 ⁇ l of the X-gal stock solution, (40 mg/ml in DMSO) in 10 ml of substrate buffer (5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS).
  • substrate buffer 5 mM Potassium Ferrocyanide, 5 mM Potassium Ferricyanide, 2 mM MgCl 2 in 1 ⁇ PBS.
  • the blue activated TcH were scored visually using the microscope and the number of activated TcH/well was plotted against the number of APC harvested at various intervals after treatment.
  • FIGS. 9A-9C show that the MHC-peptide complexes on selected APC formed subsequent to in vivo delivery of epitope via Ig are long-lived.
  • Separation by using magnetic beads coupled with anti-CD11c and anti-CD11b antibodies was carried out using magnetic cell separators and reagents from Miltenyi Biotec, Germany as follows: spleens were processed to single cell suspension, red blood cells lysed, then cells washed, counted and resuspended in MACS buffer (PBS supplemented with 2 mM EDTA and 0.5% BSA). Magnetically labeled cells were passed through a separation column which is placed in the magnetic field of a MACS separator. The magnetically labeled positive fraction is retained in the column while the negative fraction tuns through.
  • the magnetically retained positive cells are eluted from the column, cells are washed, counted, resuspended in HL1 complete media and they were incubated in different numbers with 1 ⁇ 10 4 TcH specific for the HA peptide, overnight.
  • APC from FcR gamma competent BALB/c mice were used. The next day the plate was centrifuged for 15 min/4° C./1500 RPM, then the supernatant was flicked, the cells were fixed with cold freshly made fixing solution (2% Formaldehyde, 0.2% Glutaraldehyde in 1 ⁇ PBS) and the plate was again centrifuged for 3 min/4° C./1500 RPM.
  • EXAMPLE 10 SHOWS THAT THE EFFICIENCY OF T CELL ACTIVATION BY A PEPTIDE DELIVERED WITHIN THE IgG BACKBONE IS DEPENDENT ON THE EXPRESSION OF ⁇ CHAIN+ Fc ⁇ R (THAT PROMOTE ACTIVITY) AND Fc ⁇ RIIB (THAT LIMIT THE ACTIVITY) ON APC. IN ADDITION, THIS EXPERIMENT SHOWS THAT ITIM-BEARING Fc ⁇ RIIB KEEPS IN CHECK THAT IMMUNE RESPONSE TO A PEPTIDE DELIVERED WITHIN IgG BACKBONE
  • FcR gamma+ versus gamma ⁇ isoforms The differential role of FcR gamma+ versus gamma ⁇ isoforms to the immune response triggered by peptide epitope within IgG backbone, was studied by ex vivo loading of APC followed by adoptive transfer.
  • Splenocytes from wild type, FcR gamma ⁇ or FcRIIB-BALB/c mice were incubated for 3 hours at 370° C. as follows: 10 million cells/1 ml of serum free HL-1 medium were admixed with 50 ug/ml of HA 110-120 peptide or 10 ug/ml of recHA(I-Ed)-IgG.
  • mice (1 million cells suspended in 200 ul serum free HL-1 and divided into 2 equal inoculi administered subcutaneously and intraperitoneally).
  • the recipient mice were sacrificed, spleens harvested and the T cell response to the HA 110-120 peptide measured by ELISPOT analysis as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C.
  • FIG. 11 show that the expression of the gamma chain of ITAM+ FcgR isoforms is necessary for the induction of T cell response to APC loaded with peptide within IgG backbone. This was not necessary for the immunogenic effect of APC pulsed with peptide. Conversely, absence of ITIM+ FcgRII results in profound increase of the T cell response to APC pulsed with recombinant IgG but not HA peptide. Together, these data show that the T cell response to recombinant IgG bearing a peptide epitope is determined by a complex interplay between ITAM+ and ITIM+ Fcgamma receptors on APC.
  • mice Four BALB/c mice were injected intravenously with 2 ⁇ M of recHA (I-Ed)-IgG. One day later, the spleens were harvested and APC were isolated by MACS using anti-CD11c, anti-CD11b or anti-CD19 monoclonal antibodies coupled with magnetic beads. Separation by using magnetic beads coupled with anti-CD11b, anti-CD11c and anti-CD19 mAb is carried out using magnetic cell separators and reagents from Miltenyi Biotec, Germany as follows: spleens were processed to single cell suspension, red blood cells lysed, then cells washed, counted and resuspended in MACS buffer (PBS supplemented with 2 mM EDTA and 0.5% BSA).
  • MACS buffer PBS supplemented with 2 mM EDTA and 0.5% BSA.
  • Magnetically labeled cells were passed through a separation column which is placed in the magnetic field of a MACS separator.
  • the magnetically labeled positive fraction is retained in the column while the negative fraction runs through.
  • the magnetically retained positive cells are eluted from the column, cells are washed, counted, resuspended in serum free HL-1 medium as follows: 3 ⁇ 10 6 /ml CD11c + DC, 28 ⁇ 10 6 /ml CD11b + or 84 ⁇ 10 6 /ml of CD19 + B cells. This numerical distribution respects the proportion of the APC subsets isolated from the splenic tissue.
  • ELISPOT IL-4 and IFN- ⁇
  • IL-10 kit Biosource international, cat#KMC0104
  • results are expressed in FIG. 12 as number of spot forming colonies/spleen (average of duplicates; panels A, B) or amount of cytokine measured in supernatants (pg/ml, average of duplicates; panels C, D) at various concentrations of HA peptide used for restimulation.
  • FIG. 12 panels A-D
  • the results clearly show that unexpectedly, and in contrast with the potency/cell basis (Example 8), at the organism level, the CD11b + monocytes have the highest impact on the immune response to a peptide epitope delivered within the IgG backbone.
  • the CD11b + APC subset induced both Th2, Tr1 and Th3 cells.
  • the CD11c + DC induced Th3 cells and more reduced Th2 response.
  • the CD19 + B cells were poor inducers of T cell immunity to the peptide epitope within the IgG backbone. No significant Th1 responses were induced by either of the APC subsets tested.
  • EXAMPLE 12 SHOWS THAT THE LOADING OF APC IN VIVO WITH A PEPTIDE DELIVERED WITHIN IgG BACKBONE RESULTS IN INDUCTION OF Th2 BUT NOT Th1 IMMUNITY
  • mice were immunized with 100 ⁇ g of recHA (I-Ed)-IgG (“IgHA”), or a molar equivalent amount of HA peptide epitope (2 ⁇ g), by subcutaneous injection and sacrificed 2 weeks later.
  • IgHA recHA-Ed-IgG
  • the immune response was measured by ELISPOT analysis using splenocytes from treated mice as responders, and mitomycin-treated splenocytes; from na ⁇ ve mice as stimulators, as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • Single cell suspension was made from the spleens, red blood cells were lysed, cells washed, counted and incubated at 5 ⁇ 10 5 /well together with 20 ⁇ g/ml HA 110-120 peptide or just with media, to assess the background.
  • Stimulator cells were prepared from na ⁇ ve mice as follows: single cell suspension was prepared from spleens, red blood cells were lysed, cells were washed, resuspended in HL1 complete and mitomycin treated for 30 minutes. Afterwards, cells were washed 3 times, counted and resuspended in serum free HL1 media. The plates were incubated 72 hours at 37° C., 5% CO2.
  • EXAMPLE 13 SHOWS THAT THE REPEATED LOADING OF APC IN VIVO WITH A PEPTIDE DELIVERED WITHIN IgG BACKBONE RESULTS IN INDUCTION OF Th1 AND Tr1 IMMUNITY
  • mice were immunized with 40 ug of heat aggregated (15 mins at 63° C.) of recHA (I-Ed)-IgG (“IgHA”) administered by intranasal instillation boosted 2 weeks later by subcutaneous injection with 100 ug of recombinant immunoglobulin in saline.
  • IgHA recHA
  • mice primed with heat aggregated IgG2b isotype control were used.
  • mice were sacrificed and T cell response assessed by in vitro restimulation of splenocytes with HA peptide by ELISPOT analysis as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • the data were acquired using an automated system (Navitar, Rochester, N.Y.) with ImagePro-Plus) software (Media Cybernetics, Silver Spring, Md.).
  • the TGF-beta and IL-10 production were measured by ELISA TGF- ⁇ 1 kit (R&D Systems, cat # DY240) and IL-10 kit (Biosource international, cat#KMC0104). The results are expressed as cytokine concentration (average of triplicates) after subtraction of background.
  • the data show that mucosal priming with epitope bearing recombinant immunoglobulin resulted in differentiation of Th3 and Tr1 cells that were expanded subsequently by systemic boosting.
  • mice were immunized intraperitoneally with 100 ug of recHA (I-Ed)-IgG in saline, emulsified in Complete Freund's Adjuvant (“CFA”) or with 105 TCID50 of influenza virus strain WSN, that bears the HA epitope.
  • CFA Complete Freund's Adjuvant
  • Single cell suspension was made from the spleens, red blood cells were lysed, cells washed, counted and incubated at 5 ⁇ 10 5 /well together with 20 ⁇ g/ml HA 110-120 peptide or just with media, to assess the background.
  • the data were acquired using an automated system (Navitar, Rochester, N.Y.) with ImagePro-Plus) software (Media Cybernetics, Silver Spring, Md.). The results are represented as mean ⁇ SEM of frequency of cytokine producing colonies in the spleen.
  • FIG. 15 show that a peptide epitope within the IgG backbone triggers a cellular response of Th2 profile that is enhanced but not switched by a conventional adjuvant (CFA).
  • CFA conventional adjuvant
  • EXAMPLE 15 SHOWS THAT THE PRESENTATION OF PEPTIDE EPITOPE SUBSEQUENT TO IgG MEDIATED DELIVERY RESULTS IN A T CELL RESPONSE THAT COULD BE FURTHER MANIPULATED BY DECREASING CO-STIMULATION WITH ANTI-CD40mAB, RECOMBINANT IL-12 OR SYNTHETIC dsRNA
  • Dendritic cells from naive BALB/c mice were harvested by MACS from splenic cell suspensions as follows: Separation by using magnetic beads coupled with anti-CD11c was carried out using magnetic cell separators and reagents from Miltenyi Biotec, Germany as follows: spleens were processed to single cell suspension, red blood cells lysed, the cells washed, counted and resuspended in MACS buffer (PBS supplemented with 2 mM EDTA and 0.5% BSA). Magnetically labeled,.cells were passed through a separation column which is placed in the magnetic field of a MACS separator. The magnetically labeled positive fraction is retained in the column while the negative fraction runs through.
  • the magnetically retained positive cells are eluted from the column, cells are washed, counted, resuspended in HL1 complete media and were pulsed ex vivo in serum free HL-1 medium for 2 hours, at a concentration of 3 million/ml, with 50 ug/ml of recHA(I-Ed)-IgG alone or supplemented with 5 ng/ml of recIL-12, 50 ug/ml of double stranded RNAs (pA:pU or pI:pC).
  • the cells were incubated with recombinant Ig and wells precoated with 10 ug/ml of anti-CD40 mAb.
  • the cells were harvested, washed and adoptively transferred to naive BALB/c mice (300,000 delivered half subcutaneously and half intraperitoneally) in serum free HL-1 medium.
  • mice were sacrificed and T cell responses measured against HA by ELISPOT analysis as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • FIG. 16 show that peptide presentation by APC, subsequent to loading with antigen by using recombinant IgG as delivery platform, occurs in context of limited co-stimulation.
  • IL-12, anti-CD40 or synthetic dsRNA can all enable APC loaded with antigen via FcgR, to prime IL-2 and enhanced IL-4 producing T cell immunity against the cognate (HA) peptide.
  • EXAMPLE 16 THE ACTIVITY OF THE LONG-LIVED IL-4 PRODUCING Th2 CELLS TRIGGERED BY IN VIVO LOADING OF APC WITH IgG-PEPTIDE IS DEPENDENT ON THE CONTINUOUS INTERACTION WITH ENDOGENOUS APC AND REQUIRES COMPETENT CD4
  • mice were immunized with 100 ug of recHA (I-Ed)-IgG or HA peptide subcutaneously, sacrificed at 2 weeks and the T cell response measured by ELISPOT analysis as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml anti-IL-4, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plate was washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • Single cell suspension was made from the spleens, red blood cells were lysed, cells washed, counted and incubated at 5 ⁇ 10 5 /well together with 20 ⁇ g/ml HA 110-120 peptide or just with media, to assess the background.
  • the plate was incubated 72 hours at 37° C., 5% CO2. After 3 days, the plate was washed 5 times with PBS-tween20 0.05% (washing buffer) and incubated with 100 ⁇ l/well of biotinylated anti-cytokine Abs, 2 ⁇ g/ml in PBS-tween20 0.05%-FBS 0.1%(ELISPOT buffer) overnight at 4° C.
  • Magnetically labeled cells were passed through a separation column which is placed in the magnetic field of a MACS separator.
  • the magnetically labeled positive fraction is retained in the column while the negative fraction runs through.
  • the magnetically retained positive cells are eluted from the column, cells are washed, counted, resuspended in HL1 complete media and were incubated in the ELISPOT assay, protocol to follow.
  • the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • Single cell suspension was made from the spleens, red blood cells were lysed, cells washed, counted and incubated at 5 ⁇ 10 5 /well together with 50 ⁇ g/ml HA 110-120 peptide or just with media, to assess the background.
  • FIGS. 17A-17B show that the activity of HA specific IL-4 producing T cells triggered by administration of recHA(I-Ed)-IgG is dependent on CD4 rather CD8.
  • the long lived IL-4 production by primed T cells depends on stable interaction with endogenous APC.
  • Activated SFERFEIFPKE-specific T cells were separated from BALB/c mice immunized 2 weeks previously with 100 ⁇ g peptide in CFA. They were incubated with mitomycin treated splenocytes in the presence of various amounts of recHA(I-Ed)-IgG or corresponding peptide.
  • the expansion and cytokine production (IFN- ⁇ , IL-4, IL-2) was estimated by ELISPOT analysis as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour, at 37° C.
  • Single cell suspension was made from the spleens, red blood cells were lysed, cells washed, counted and incubated at 5 ⁇ 10 5 /well together with 20 ⁇ g /ml HA 110-120 peptide or just with media, to assess the background.
  • the plates were incubated 72 hours at 37° C., 5% CO2. After 3 days, the plates were washed 5 times with PBS-tween20 0.05% (washing buffer) and incubated with 100 ⁇ l/well of biotinylated anti-cytokine Abs, 2 ⁇ g/ml in PBS-tween20 0.05%-FBS 0.1%(ELISPOT buffer) overnight at 4° C. The next day, the plates were washed five times with washing buffer, and incubated for an hour with 1:1000 Streptavidin-HRP diluted in ELISPOT buffer. The reaction was developed with 3-amino-9-ethylcarbazole substrate (Sigma, St. Luis, Mo.) and stopped by washing the plate twice with tap water. The plates were then allowed to dry at room temperature for 24 hours.
  • TGF- ⁇ and IL-10 production were measured by ELISA at 48 hours after incubation using TGF- ⁇ 1 kit (R&D Systems, cat # DY240) and IL-10 kit (Biosource international, cat#KMC0104). The results are expressed as frequency of spot forming cells (SFC) or concentration of cytokine versus amount of antigen added in vitro.
  • SFC spot forming cells
  • the results in FIG. 18 show that the IgG mediated delivery of a T cell epitope has a profound and differential effect on the expansion and cytokine production by activated T cells: IL-2, IFN- ⁇ and surprisingly IL-4, were down-regulated in a dose-related manner.
  • the Ig-peptide was substantially more effective in modulating the cytokine production, as compared to the peptide itself.
  • only the Ig-peptide turned on effectively the production of IL-10 and TGF-beta in a dose-dependent manner.
  • the T cell epitope in context of Ig backbone, but not separately, differentially modulated the function of activated cells.
  • EXAMPLE 18 SHOWS THAT SURPRISINGLY, A PEPTIDE DELIVERED WITHIN THE IgG BACKBONE, THAT IS NOT AN IMMUNE COMPLEX NOR IS A RECEPTOR CROSS-LINKING ANTIBODY, RESULTS IN INDUCTION OF A CLASS I RESTRICTED IMMUNE RESPONSE.
  • THIS RESPONSE HAD A DIFFERENT PROFILE FROM THAT TRIGGERED BY LIVE VIRUS (Tc2 TYPE CONSISTING IN IL-4 BUT NOT IFN- ⁇ PRODUCTION
  • mice were injected with 50 ⁇ g of recNP(Kd)-IgG encompassing the MHC class I-restricted peptide TYTQTRALV (Seq. I.D. No. 6) by subcutaneous injection.
  • the mice were sacrificed 2 weeks later and peptide-specific cytokine production was measured by ELISPOT analysis as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • FIG. 19A-19B show that in contrast to viral immunization with an influenza virus strain bearing the cognate peptide, Ig-mediated peptide delivery was ineffective in triggering IFN- ⁇ producing Tc1 cells.
  • Ig-peptide administration still resulted in formation of MHC class I-peptide complexes and induced significant NP-specific MHC class I-restricted T cell immunity consisting in IL-4 producing Tc2 cells.
  • mice were injected subcutaneously with 200 ⁇ l of rat brain homogenate emulsified in Complete Freund's Adjuvant and boosted with 50 ng of pertussis toxin at 6 hours and 2 days.
  • the mice developed an aggravated, progressive form of paralytic disease.
  • Half of the mice received via subcutaneous injection a combination of recombinant immunoglobulins bearing the MBP and the PLP epitopes (recMBP(I-As)-IgG; recPLP(I-As)-IgG), respectively (150 ⁇ g/molecule, on day 8, 12, 18 after induction of disease).
  • recMBP(I-As)-IgG recombinant immunoglobulins bearing the MBP and the PLP epitopes
  • recPLP(I-As)-IgG recPLP(I-As)-IgG
  • mice After a period of observation of 70 days, the mice were sacrificed, spleens harvested and elispot analysis carried out as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ /well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • FIG. 20B The results ( FIG. 20B ) were expressed as frequency of IL-4 producing T cell colonies in the absence of added PLP peptide plotted against the frequency of IFN- ⁇ -producing T cells in condition of peptide stimulation. Mice progressing to full-blown limb paralysis (score equal to or higher than 1.5) were represented with closed symbols. Mice that did not progress to limb paralysis were represented with open symbols.
  • FIG. 20C the total number of IL-4 spot forming colonies/spleen (mean ⁇ SEM) in condition of in vitro stimulation was represented with nil, MBP or PLP peptide.
  • mice treated with IgG2b isotype control An additional control, consisting of splenocytes from mice treated with IgG2b isotype control, has been included.
  • in vitro culture was carried out in the presence of neutralizing anti-IL-4 mAb (40 ⁇ g/ml) and the number of IFN-y ⁇ producing T cells was represented in the panel D.
  • FIGS. 20 A-D show that co-administration of MBP and PLP epitopes by using recombinant IgG significantly curbed the chronic progression of disease.
  • the mice protected from paralysis developed unexpectedly, an enhanced reactivity to self-epitopes MBP and PLP, manifested by increased basal and peptide-stimulated IL-4 or IFN-Y production, respectively.
  • the reactivity of IFN- ⁇ -producing T cells is kept in check by IL-4 suggesting a complex immunomodulatory mechanism triggered by IgG-mediated delivery of epitopes.
  • ITIM and ITAM Fc gamma + )-bearing receptors on APC.
  • ITIM + Fc ⁇ RIIB limits the degree of activation of T cells and gamma + FcRs are required for effective formation of MHC-peptide complexes when epitopes are delivered via the IgG backbone.
  • ITIM + Fc ⁇ RIIB limits the degree of activation of T cells and gamma + FcRs are required for effective formation of MHC-peptide complexes when epitopes are delivered via the IgG backbone.
  • Such in vivo delivery of epitope results in effective formation of MHC-peptide complexes on peripheral CD11c + and CD11b + APC, but not thymic APC.
  • ITAM + Fc ⁇ Rs makes the nature and magnitude of resulting T cell response difficult to predict without experimentation.
  • the data in FIG. 21 show that IgG-delivery of peptide epitope results in exposure of T cells to peptide-loaded APC in context of limited co-stimulation, having a differential effect on na ⁇ ve versus activated T cells: 1) de novo induction of Th2, Tc2, Th3, Tr1 cells and, 2) downregulation of activated Th1, Th2 cells with stimulation of activated Tr1 and Th3 cells.
  • the overall effect is immunomodulatory, rather than pro-inflammatory (associated with Th1 and Tc1 immunity).
  • Permissive MDCK cells were infected with WSN influenza virus (10 8 TCID 50 / 1 ⁇ 10 9 cells) and after 24 hours, the cells were harvested, washed and the total RNA extracted using an RNA separation kit (Qiagen, Valencia, Calif.). The RNA was further purified by treatment with RNAse-free DNAseI (Stratagene, San Diego, Calif.). The single stranded RNA in the samples was then removed by 30 minutes incubation at 37° C. with 5 U of S1 nuclease (Ambion, Inc., Austin, Tex.)/ ⁇ g of RNA. The RNA was analyzed prior to and subsequent to the digestion by gel electrophoresis.
  • the absence of infectious properties of the purified dsRNA was confirmed by standard influenza virus titration.
  • material purified and treated similarly, from 10 9 non-infected MDCK cells was used.
  • the concentration of nucleic acid was measured by spectrophotometry (A 260nm ) and the absence of endotoxin confirmed by Limulus assay.
  • the purified dsRNA and control RNA were used individually, or as a mixture with gp140 recombinant antigen (25 ⁇ g of RNA and 2 ⁇ g of antigen in 25 ml of sterile PBS).
  • gp140 recombinant truncated antigen
  • FIG. 22A the general principle of the experiment is illustrated.
  • FIG. 22B the absorption after assay development is represented, corresponding to various serum dilutions, in case of whole IgG.
  • FIG. 22B the absorption at 1/50 serum dilution, in case of IgG2a and IgG1 antibody isotypes, is represented.
  • FIGS. 22 A-B show that natural, non-infectious dsRNA from influenza virus-infected MDCK cells, has an unexpected enhancing effect on the adaptive response to a prototype antigen. Both IgG1 and IgG2a antibody responses were increased showing that a strong T helper1 and T helper 2 response was induced.
  • FIG. 23A shows an extensive library of synthetic RNA motifs, that was grouped in pools and used for a two-tier screening process as follows:
  • mice were immunized intratracheally with RNA pools, followed by 2 boosts two weeks apart, carried out by intranasal instillation.
  • dose-matched OVA in sterile PBS was used, OVA with cholera toxin subunit B (CTB) and PBS alone, respectively.
  • wells were coated with antigen (10 ⁇ g/ml of OVA) and blocked with SeaBlock (Pierce, Rockford, Ill., catalog # 37527).
  • FIG. 23C The effect of various dsRNA motifs on the induction of antibody response to OVA: the results are expressed as in FIG. 23C .
  • the data are representative for two independent experiments.
  • INSET the ratio between mean IgG2a and IgG1 titers to OVA.
  • biotin-conjugated anti-mouse IgG1 and IgG2a antibodies were used followed by incubation with streptavidin-AKP conjugate.
  • the order from left to right is similar as in the main panel in FIG. 23C : PBS OVA, CTB OVA, pC:pG OVA, pI:pC OVA and pA:pU OVA.
  • C The magnitude and profile of T cell response induced by OVA together with various dsRNA motifs, in female C57BL/6 mice.
  • splenic cell suspensions were obtained by passing the organ through 70 micron nylon Falcon strainers (Becton Dickinson, cat# 352350) followed by lysis of red blood cells with red blood cell lysis buffer (Sigma, cat# R7757).
  • the lymphocytes from the pulmonary associated lymphoid tissue were isolated by collagenase (Sigma, cat# C9891) digestion of lung tissue followed by Ficoll-Paque (Amersham Pharmacia, cat# 17-1440-02) gradient centrifugation.
  • the T cell response was measured by ELISPOT analysis as follows: 96-well 45 micron mixed cellulose ester plates (Millipore, cat#MAHA S4510) were coated with 4 ⁇ g/ml of rat anti-mouse anti-IFN ⁇ , IL-2 or IL-4 monoclonal antibodies (BD-PharMingen, cat#554430, cat#18161D, cat# 554387 respectively). After blocking with 10% FCS in sterile saline for 1 hour at 37° C., spleen cell suspensions were added at 5 ⁇ 10 5 cells/well, with or without antigens/peptides. For stimulation, graded amounts of antigen (OVA) were used.
  • OVA antigen
  • the assay was developed with biotinylated rat anti-mouse cytokine antibodies (BD-PharMingen) followed by streptavidin-HRP (BioSource Int., Camarillo, Calif.) and insoluble AEC substrate.
  • the results were measured using an automatic imaging system (Navitar/Micromate) equipped with multiparametric-analysis software (Image Pro, Media Cybernetics).
  • FIGS. 23 B-D show that different synthetic RNAs have an enhancing effect on the B and T cell response to a prototype protein antigen.
  • different motifs comprising specific nucleotide combinations, have specific effects in terms of T1 versus T2 induction and subsequently, immunoglobulin isotype switching.
  • EXAMPLE 23 USE OF SELECTED SYNTHETIC RNA MOTIFS FACILITATES THE INDUCTION OF MHC CLASS I-RESTRICTED Tc1 CELLS, PRODUCING IFN- ⁇
  • FIGS. 24 A-B show that a selected synthetic RNA motif was able to promote increased T cell immunity to different MHC class I-restricted peptides encompassed within larger antigens (polypeptides).
  • This immune response comprised a Tc1 component, consisting in IFN- ⁇ -producing MHC class I-restricted T cells.
  • FACS Buffer PBS, 1% FCS, 0.1% sodium azide
  • MACs buffer PBS, 2 mM EDTA, 0.5% BSA;
  • Collagenase Buffer 0.225 mg BSA, 0.0062 mg collagenase in 50 ml RPMI; and,
  • each RNA motif was tagged with the ULYSIS Alexa 488 label.
  • FIG. 25 show that pA:pU and pI:pC bind to different cellular receptors. Since pI:pC binds to TLR3, it results that additional receptors distinct from TLR3 are involved in RNA recognition immune function.
  • RNA array technique Local up-regulation of chemokine gene-expression by dsRNA motifs was measured by DNA array technique using RNA from the pulmonary tissue, extracted one day after the administration via the respiratory tract. Total RNA was isolated from lungs using an RNeasy kit (Qiagen, Valencia, Calif.). The RNAs were further purified by treatment with RNase-free DNase I (Stratagene, San Diego, Calif.). DNA array was performed by using the Nonrad-GEArray kit from SuperArray Inc. (Bethesda, Md.). Briefly, cDNA probes were synthesized using MMLV reverse transcriptase with dNTP mix containing biotin-16-dUTP. The GEArray membranes were prehybridized at 68° C. for 1-2 hours.
  • the hybridization was carried out by incubation of the membranes with biotin-labeled cDNA.
  • the hybridized membranes were washed in 2 ⁇ SSC-1% SDS twice and 0.1 ⁇ SSC-0.5% SDS twice.
  • the membranes were fuirther incubated with alkaline phosphatase-conjugated streptavidin (BioSource Int., Camarillo, Calif.) and finally developed with CDP-Star chemiluminescent substrate.
  • the intensity of signal was measured with Image-Pro analysis system equipped with Gel-Pro software (Media Cybernetics, Silver Springs, Md.).
  • the results are expressed as fold-increase of gene expression, over expression levels measured in the pulmonary tissue of non-treated mice.
  • the pattern of chemokine expression triggered by dsRNAs (50 ⁇ g of pA:pU and pI:pC, respectively) was compared to that induced by 1 ⁇ g of LPS.
  • the chemokines that selectively bind to receptors on Th1 and Th2 cells were indicated with continuous and interrupted contours, respectively.
  • FIG. 26 show that pA:pU and pI:pC trigger expression of a wide range of chemokines and that the expression pattern is motif-dependent and different from that elicited by LPS (endotoxin).
  • EXAMPLE 26 SHOWS THAT SELECTED SYNTHETIC RNA MOTIFS MOBILIZE AN IMMUNE DEFENSE THAT IS CAPABLE TO CONTROL INFECTION WITH A PULMONARY VIRUS
  • dsRNA motifs display differential ability to mobilize immune defense against influenza virus infection.
  • C3H/HeJ mice were treated via the respiratory route with 50 ⁇ g of pI:pC, pA:pU or 50 ⁇ l of saline one day before and after pulmonary infection with a sublethal dose of influenza virus.
  • C57BL/6 and TLR4 ⁇ / ⁇ C 3 H/HeJ mice under Metofane anesthesia were infected with sublethal doses (10 4 tissue culture infective doses 50%-TCID 50 ) of live WSN virus, via the nasal route.
  • sublethal doses (10 4 tissue culture infective doses 50%-TCID 50 ) of live WSN virus, via the nasal route.
  • the mice were sacrificed, lungs retrieved, homogenized and stored at ⁇ 70° C.
  • the virus titers were measured by 48-hour incubation of serial dilutions of samples with permissive MDCK cells, followed by standard hemagglutination with chicken red blood cells (From Animal Technologies).
  • results depicted in FIG. 27 show that the control of replication of influenza virus can be achieved by using selected synthetic RNA motifs (dsRNA1 is pA:pU and dsRNA2 is pI:pC).
  • mice immunized with 100 ⁇ g of hIgG emulsified in CFA were included and represented the maximal titer on the graph (interrupted line).
  • results in FIG. 28 show that selected synthetic RNA motifs pI:pC and pA:pU largely prevent high zone tolerance that is usually associated with administration of large amounts of purified protein.
  • THP-1 Human monocytic cell line ATCC, cat # TIB-202;
  • IL-12 Cytokine Human ELISA, IL-12 ultra sensitive (US) cat# KHC0123;
  • TNF alpha Cytokine Human ELISA, TNF alpha cat# KHC3012;
  • THP-1 cells were allowed to differentiate following addition of 10 ng/ml PMA in media containing 10% FCS.
  • RNA motifs and controls were added at concentrations of from 3 to 100 ⁇ g/ml on top of adherent THP-1 cells.
  • FIG. 29 show selected synthetic RNA motifs effect on human monocytic cells; in addition, this effect is heterogeneous, depending on the chemical structure of the motifs (nucleotide composition). Selected but not all synthetic RNA motifs are able to trigger IL-12 production, an important T1 regulatory cytokine, by human monocytic cells.
  • THP-1 cells were incubated at for 15 minutes at room temperature with different amounts of non-labeled synthetic RNA. Subsequently, tagged pA:pU was added for 30 minutes at 4° C., cells washed and the fluorescence quantified by FACS analysis. The results are expressed in FIGS. 30A-30B as histograms corresponding to the large cell subset (A) and total cell population (B). Percentages of stained cells were represented on each Figure.
  • ULYSIS Nucleic acid fluorescent label (Molecular Probes, cat# U-21650).
  • the pA:pU was precipitated using sodium acetate and ethanol at ⁇ 70° C.;
  • the pA:pU was heat denatured and labeled with the Alexa Fluor 488 reagent at 90° C.; and,
  • THP-1 cells were suspended at 2 ⁇ 10 6 cells/ml;
  • Non-tagged pA:pU or pI:pC were added to the THP-1 cells at a concentration of either 20 or 100 ⁇ g/ml and incubated 15 minutes;
  • THP-1 cells were washed once and suspended in FACS buffer followed by flowcytometric analysis to determine relative fluorescent differences between different treatment populations.
  • FIGS. 30A-30B show that non-tagged pA:pU but not non-tagged pI:pC was able to compete out the binding of tagged pA:pU to human THP-1 monocytic cells, both at the level of large cell subset and whole population.
  • the bulk synthetic RNA material is obtained by standard methods of organic synthesis. Afterwards, the material is dissolved in sterile endotoxin-free saline, passed through endotoxin removal columns until the concentration of LPS is below 0.005 EU/ ⁇ g. The measurement of LPS is carried out by standard Limulus assay. Subsequently, the material is fractionated by a series of centrifugation steps through filters of defined porosity (see FIG. 31 ).
  • a useful fraction comprises synthetic RNA of less than 20 to maximum 100 bp size, however, larger RNA fragments may be used.
  • the material is measured and validated on standard assays: spectrophotometry (OD260 nm); gel electrophoresis; endotoxin quantitation by Limulus assay; bioactivity on human THP-1 cells (as in Example 28).
  • Differentiated human THP-1 monocytic cells were incubated with different concentrations of synthetic RNA (pA:pU, fractionated as described in the Example 30) for 24 hours, and the supernatants collected.
  • concentration of TNF- ⁇ was measured by ELISA using BioSource International kits (Camarillo, Calif.). The results are expressed in FIG. 32 as pg/ml (concentration) for each culture condition.
  • results depicted in FIG. 32 show that lower molecular weight fractions of a selected synthetic RNA compound are endowed with higher biological activity, in terms of cytokine production, by human monocytic THP-1 cells.
  • EXAMPLE 32 SELECTED SYNTHETIC RNA MOTIFS, HAVE UNEXPECTEDLY, A DIFFERENT IMMUNE PROFILE IN REGARD TO GENERATION OF ANTI-RNA ANTIBODIES
  • mice were immunized intraperitoneally and subcutaneously with 50 ⁇ g+50 ⁇ g of hIgG and synthetic RNA (pI:pC or pA:pU) and serum samples were prepared 1 week later.
  • mice injected with hIgG in saline were used.
  • the anti-hIgG, and dsRNA IgG antibody titers against pA:pU, pI:pC, pA and hIgG were measured by ELISA.
  • wells were coated with antigen (10 ⁇ g/ml of hIgG or synthetic RNAs) and blocked with SeaBlock (Pierce, Rockford, Ill., catalog # 37527).
  • the results in FIG. 33 show that pI:pC but not pA:pU induced antibody response against itself, with a cross-reactive component against another RNA motif.
  • mice were immunized with 50 ug of recIgG-NP(Kd) subcutaneously, admixed with 50 ug of selected synthetic RNA (pA:pU or pI:pC).
  • pA:pU or pI:pC synthetic RNA
  • the T cell response was measured by ELISPOT analysis as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • the data were acquired using an automated system (Navitar, Rochester, N.Y.) with ImagePro-Plus) software (Media Cybernetics, Silver Spring, Md.).
  • FIGS. 34A and 34B show that co-use of selected synthetic RNAs promoted effective induction of IL-2 and IFN-gamma subsequent to IgG mediated delivery of an MHC class I-restricted epitope (dsRNA1 is pA:pU and dsRNA2 is pI:pC).
  • Splenic APC were isolated from naive BALBc mice and pulsed ex vivo overnight with 1 ug NP peptide, or 50 ⁇ g recIgG-NP (Kd) with or without 50 ⁇ g/ml selected synthetic dsRNA (pA:pU). The cells were washed and 5 ⁇ 10 6 cells were administered by s.c. and i.p. injection equal amount, to naive BALB/c mice.
  • the response was measured 3 weeks later by ELISPOT analysis as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ⁇ g/ml for anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • Single cell suspension was made from the spleens, red blood cells were lysed, cells washed, counted and incubated at 5 ⁇ 10 5 /well together with 30 ⁇ g/ml, 10 ⁇ g/ml, or 3 ⁇ g/ml NP peptide or just with media, to assess the background. Plates were incubated 72 hours at 37° C., 5% CO2. After 3 days the plates were washed 5 times with PBS-tween20 0.05% (washing buffer) and incubated with 100 ⁇ l/well of biotinylated anti-cytokine Abs, 2 ⁇ g/ml in PBS-tween20 0.05%-FBS 0.1%(ELISPOT buffer) overnight at 4° C.
  • the data were acquired using an automated system (Navitar, Rochester, N.Y.) with ImagePro-Plus) software (Media Cybernetics, Silver Spring, Md.).
  • the mean area/colony versus the concentration of peptide used for stimulation is plotted, for both IFN-gamma and IL-4 (arbitrary units).
  • results in FIG. 35 show that ex vivo APC loading by recombinant IgG is significantly more effective in formation of MHC class I-peptide complexes and generation of Tc response, compared to use of peptide itself.
  • the mere formation of MHC class I-peptide complexes subsequent to epitope delivery via IgG/FcgammaR results in differentiation of Tc2 cells producing IL-4 but not IFN-gamma.
  • Simultaneous treatment of APC with selected synthetic RNA results in broadening of the T cell profile, to IFN-gamma producing Tc1 cells.
  • mice were injected with recIgG-NP(Kd), pA:pU separately, or in combination (50 ug/injection).
  • naive mice were used.
  • the mice were infected with 104 TCID50 of A/WSN/32 H1N1 influenza virus, via the respiratory tract.
  • the T cell profile in the spleen was measured by ELISPOT analysis subsequent to ex vivo stimulation with NP peptide as follows: the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • Single cell suspension was made from the spleens, red blood cells were lysed, cells washed, counted and incubated at 5 ⁇ 10 5 /well together with 20 ⁇ g/ml NP peptide or just with media, to assess the background. Plates were incubated 72 hours at 37° C., 5% CO2. After 3 days, the plates were washed 5 times with PBS-tween20 0.05% (washing buffer) and incubated with 100 ⁇ l/well of biotinylated anti-cytokine Abs, 2 ⁇ g/ml in PBS-tween20 0.05%-FBS 0.1%(ELISPOT buffer) overnight at 4° C.
  • FIG. 36 show that IgG mediated delivery of a class I restricted epitope is most effective in priming class I restricted Tc1 responses when co-administration of selected synthetic RNA was carried out. Such primed precursors were rapidly expanded subsequent to infection with influenza virus.
  • mice were immunized and challenged with recIgG-NP (Kd) as in the previous Example and sacrificed 4 days after influenza virus infection.
  • the splenocytes were prepared, suspended in HL-1 medium at 5 million/ml and co-incubated for 5 days with 10 ⁇ g/ml of NP 147-155 peptide and in presence of 5 U/ml of recombinant IL-2. Splenocytes from 4 mice/group were pooled and incubated in flasks.
  • viable cells were recovered by Ficoll gradient centrifugation, washed and incubated for 5 hours in V-bottom plates, in various numbers, with a fixed number of sp20 target cells with or without NP peptide (20 ⁇ g/ml). The supernatants were harvested after plate centrifugation, and the concentration of LDH measured by using a Promega kit (cat # G1780). The results are expressed as percent specific lysis at different E:T ratios (Effector to Target ratio).
  • FIG. 37 show that effective priming of anti-viral cytotoxic T cells requires both effective in vivo loading of APC with class: I restricted epitope delivered via IgG, together with appropriate instruction by selected synthetic RNA motif, namely pA:pU.
  • mice were immunized with 50 ug of recIgG-NP (Kd) together with 50 ug of selected synthetic RNA (pA:pU), by subcutaneous injection. Three weeks after immunization, the mice were challenged with 10 4 TCID 50 of infectious WSN influenza virus and sacrificed 5 days later. The pulmonary virus was titrated in lung homogenates by standard MDCK hemagglutination assay as follows: on day one MDCK cells were plated in 96 well plates at 2 ⁇ 10 4 /well/200 ul and incubated for 24 hours at 37° C., 5% CO 2 .
  • results in FIG. 38 show that immunization with a recombinant IgG bearing a viral class I restricted epitope together with selected synthetic dsRNA (pA:pU) resulted in priming of an immune response capable to limit the replication of a virus subsequent to infectious challenge.
  • FIG. 39 DESCRIBES THE TUMOR MODELS USED FOR TESTING THE EFFICIENCY OF A Ig-PEPTIDE-BASED MOLECULES
  • mice (K d restricted) have been used to establish a tumor model.
  • Tumor cells (1 to 15 million in 100 ⁇ L) were typically injected in the flank to the mouse (see arrow in upper photo in FIG. 39 ).
  • Primary tumors i.e. those at the sight of injection
  • the mouse myeloma cell line SP2/0
  • either untransfected cells or cells stable transfected expressing heterologous protein recombinant IgG expressing different epitope peptides in the CDR3 region of the heavy chain or the complete NP protein
  • heterologous proteins in the SP2/0 cells provided specific tumor associated antigens (TAA) for testing various anti-tumor strategies in the immunocompetent mice.
  • TAA tumor associated antigens
  • untreated mice developed palpable solid primary tumors 1 week post injection that led to morbidity and death over the next 4 weeks.
  • Postmortem examination of the injected mice revealed metastatic lesions (see FIG. 39 ).
  • Sp2/0 cells were cultured from primary tumor tissue as well as spleen taken from tumor-bearing mice (data not shown).
  • SP2/0 cells were stably transfected with a recombinant IgG-expressing plasmids that were all identical except for the specific epitope sequence introduced into the CDR3 region of the heavy chain, for example, the MHC I restricted NP epitope (amino acids 147-155, see FIG. 39 ).
  • SP2/0 cells were also stably transfected with a plasmid containing the coding sequence for the entire NP protein of WSN virus under control of the CMV promoter. All transfected cell lines produced primary tumors over the same frame as wild type SP2/0 cells.
  • This tumor model was extended to include an adenocarcinoma cell line (4T1, ATCC CRL-2539, K d restricted), previously shown to induce metastatic tumors in Balb-c mice.
  • the 4T-1 cell line was similar to that described above for the SP/0 line. Injection of 1 to 15 million 4T-1 cells into the flank of Balb-c mice produced a palpable primary tumor over a time frame similar to injections of SP2/0 cells eventually leading to death. Postmortem collection of tissue from various organs showed that 4T-1 could be recovered from spleen, lungs as well as the primary tumor (not shown). 4T-1 cells were stably transfected with a NP-expressing plasmid described above. As with SP2/0 cells, transfection of the 4T-1 cell did not affect the course of tumor growth and lethality of disease.
  • mice were injected with SP2/0 cells (15 million in 100 ⁇ L) stably expressing recombinant IgG carrying the MHC I (Kd) NP epitope peptide in the CDR3 region of the heavy chain (IgNP).
  • co-stimulatory motif i.e. dsRNA comprised of polymeric pApU
  • IgNP purified IgTAA protein
  • both dsRNA pA:pU and purified IgTAA protein The time of treatment is indicated by the arrows in FIG. 40 , and each injection contained 50 jig of the indicated compound.
  • the mice that developed metastatic disease and died are represented with a “D” in the figure.
  • mice treated with either the dsRNA or IgTAA compound alone succumbed to disease 100% of the mice treated with both were still alive 3 weeks after initiation of treatment and were in good clinical condition at the time of sacrifice for measurement of T cell response.
  • TAA in vivo loading of APC with TAA (accomplished by uptake of IgNP via the Fc receptor of APC) is not sufficient for a potent anti-tumor response.
  • the tumor rejection and survival displayed by mice treated with IgNP in combination with pApU dsRNA highlights the important role co-stimulation plays in treatment of tumors with tumor-associated antigens.
  • results in FIG. 40 show that both effective in vivo loading of APC with tumor associated antigen, together-with simultaneous activation by selected synthetic RNA motifs, are necessary and sufficient for effective control of tumor growth and induction of tumor rejection.
  • EXAMPLE 40 IN CONTEXT OF SUBLETHAL INOCULATION OF TUMOR CELLS, SHOWS THAT THE SUBOPTIMAL RESPONSE TO TUMOR ANTIGENS COULD BE CORRECTED BY THERAPY WITH PEPTIDE EPITOPE WITHIN AN IgG BACKBONE, TOGETHER WITH CO-STIMULATORY MOTIF
  • mice were injected with SP2/0 cells stably expressing recombinant IgG (IgNP) that contains the MHC I (K d )epitope (amino acids 147-155) of WSN virus nucleoprotein in the CDR3 of the heavy chain.
  • IgNP recombinant IgG
  • K d MHC I
  • Epitope amino acids 147-155
  • the cell inoculum was 1 million cells (in 100 ⁇ L) per mouse. The mice were observed until such time as palpable tumors were detected at the site of injection. At this point the tumors were measured and 8 mice were left untreated (control) while 6 were injected intratumorally with purified IgTAA (i.e. purified IgNP, 2 mg/kg) and dsRNA (pApU, 4 mg/kg) weekly. Weekly measurements of the tumors were taken.
  • IgTAA i.e. purified IgNP, 2 mg/kg
  • dsRNA pAp
  • Panel A of FIG. 41 shows that in 6 of 8 of the control mice the induced tumor was progressive and ultimately lethal whereas 2 of the mice completely rejected the tumor spontaneously.
  • Panel B of FIG. 41 shows that the 3 weekly treatments with IgNP/dsRNA (indicated by the arrows) stimulated complete tumor rejection in 4 of the 6 mice and significant remission in another.
  • FIG. 41 shows that both effective-in vivo loading of APC with tumor associated antigen, together with simultaneous activation by selected synthetic RNA, can trigger an effective immune response to tumor-associated antigens.
  • mice Two BALB/c mice were injected with 10 million sp20 transfectoma expressing the NP-K d epitope. After tumors developed, one mouse was injected intratumorally with 50 ⁇ g of selected dsRNA motif (pApU) plus 50 ⁇ g of “IgNP”-recIgG-NP(K d ) in saline. The mice were sacrificed 24 hours later, tumors excised, digested with collagenase, filtered through 70 um filter and viable cells isolated on Ficoll gradient. Cells were stained with mAbs against TCR ⁇ , CD25 or isotype control and assessed by FACS analysis. The results were expressed as histograms, with percentage stained cells indicated.
  • mice (Harland Sprague Dawley);
  • Collagenase buffer 0.225 m BSA+0.00625 gm in 50 ml RPMI;
  • FACS Buffer 1% fetal calf serum+0.1% azide in PBS;
  • results in FIG. 42 show that tumor infiltrating lymphocytes displaying the T cell receptor marker TCR ⁇ acquired expression of the activation marker CD25 upon treatment with recombinant immunoglobulin bearing tumor associated epitope, together with selected synthetic dsRNA motif.
  • EXAMPLE 42 SHOWS THAT SUCCESSFUL THERAPY OF TUMOR BEARING MICE WITH A PEPTIDE EPITOPE WITHIN THE IgG BACKBONE TOGETHER WITH A SELECTED CO-STIMULATORY MOLECULE IS ASSOCIATED WITH A SPECIFIC DIFFERENTIATION PATTERN OF Tc, COMPRISING Tc1 IN ADDITION TO Tc2
  • mice that successfully rejected the tumor following treatment with recombinant Ig carrying a tumor associated epitope together with selected synthetic dsRNA motif as explained in Example 40 were sacrificed and the T cell response against tumor associated epitope measured by ELISPOT analysis.
  • the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • mice that successfully rejected the tumor developed Tc1 responses against the tumor associated epitope on the therapeutic Ig, along with Tc2 immunity.
  • the mice that failed to reject the tumor developed only Tc2 immunity.
  • mice bearing sp2/0 tumors expressing the NP-K d TAA were treated as described in the Example 40, by injection with recombinant Ig bearing TAA together with selected synthetic RNA motifs. After tumor rejection, the mice were challenged by subcutaneous injection administered contralaterally, with 15 million SP2/0 cells expressing NP-Kd epitope. In parallel, 4 control na ⁇ ve mice were similarly injected with a tumorigenic/lethal dose of same type of cells. The development and size of the tumors was monitored and represented as diameter (mm) versus time since challenge.
  • results in FIG. 44 show that successful rejection of the tumor induced by indicated treatment is followed by effective protection against subsequent challenge with the same tumor, indicating development of effective immune memory.
  • EXAMPLE 44 SHOWS THAT SURPRISINGLY, THE INDUCTION OF TUMOR REJECTION BY AN IgG BEARING A TAA TOGETHER WITH A COSTIMULATOR dsRNA MOTIF, RESULTS IN CROSS-PROTECTION AGAINST A RANGE OF TUMOR CELL VARIANTS LACKING THE TAA OR DISPLAYING VARIANTS OF TAA
  • mice protected against homologous challenge as described in Example 43 were subjected to sequential challenge with 15 million tumor cells representing the same tumor cells devoid of TAA (loss of antigen mutants) or bearing variants of TAA lacking the NP-K d epitope.
  • mice were challenged with a different type of tumor cell line (4T-1 adenocarcinoma) as a control, displayed in the table attached to FIG. 45A . In every case, na ⁇ ve controls were included.
  • T cell immunity of mice protected against multiple challenges with tumor variants has been assessed by ELISPOT analysis using splenic cell suspensions stimulated with TAA (NP-Kd peptide), HA (MHC class II-restricted peptide), or protein extracts from cell lysates.
  • TAA NP-Kd peptide
  • HA MHC class II-restricted peptide
  • the ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs ( 4 ug/ml for anti-IL2 and anti-IL4, and 8 ⁇ g/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 ⁇ l/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 ⁇ l/well of DMEM complete containing FBS, for an hour at 37° C.
  • FIG. 45A-45B show that the emerging immunity, subsequent to the indicated treatment that results in tumor rejection, protects against challenge with loss of antigen variants and is associated with overall expansion of cytokine producing cells. This indicates a broadening of the repertoire of anti-tumor lymphocytes, promoted by the proposed regimen, to tumor associated antigens that are not borne by the immunotherapeutic molecule.

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AU2003276932A1 (en) 2004-04-08
EP2258712A2 (en) 2010-12-08
WO2004027049A2 (en) 2004-04-01
ATE460944T1 (de) 2010-04-15
EP1539819B1 (en) 2010-03-17
JP2006514920A (ja) 2006-05-18
EP2258712A3 (en) 2011-05-04
EP1539819A2 (en) 2005-06-15
WO2004027049B1 (en) 2004-07-22
EP1539819A4 (en) 2007-08-08
WO2004027049A3 (en) 2004-05-21
US20110274705A1 (en) 2011-11-10
US8809290B2 (en) 2014-08-19
CN1703427A (zh) 2005-11-30

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