Patent Application
20050222060
The present invention relates generally to motifs that are useful in inducing an immune response. Specifically, the present application is directed to non-coding RNA motifs that are used in conjunction with an antigen or without an antigen to induce, enhance or modulate an immune response that comprises a B cell (antibody) and optionally a T cell component.
A significant number of viral infections are associated with, or result in production of, RNA species that are not normally encountered in normal states. Such RNAs are either genomic fragments (in case of viruses containing double-stranded RNAs), replicative intermediates or stem-and-loop structures that are recognized by innate immune receptors such as Toll-like receptor 3 (TLR3) and trigger production of IFN-I and other soluble mediators. In addition, certain dsRNA motifs such as polyl:polyC (pI:pC or pI:C) have been shown to activate immature dendritic cells to a stage where they act as professional APC. Despite the fact that polyI:polyC and IFN-I have been shown to influence the antibody response to a protein antigen, most of the information obtained relative to dsRNA-immune modulatory motifs resulted from models of innate immunity, limited to natural killer cells, macrophages and other cell subsets devoid of specific antigen-receptors. Thus, it has not been demonstrated whether motifs associated with double-stranded or other RNA species have only a limited effect on the adaptive immune response, or act as potent danger signals that prevent immune tolerance and direct the differentiation of specific T cells. In addition, the critical question as to whether there is a multiplicity of RNA-associated danger motifs with potential differential impacts on immune response has not been addressed. Further, it has not been defined whether non-coding RNA motifs can facilitate the induction of class I-restricted immune responses during viral infections, thought until recently to occur, in the most part, subsequent to abortive or productive infection of antigen presenting cells (APC).
During viral infection, specific T lymphocytes are exposed to foreign epitopes displayed by MHC molecules and the B lymphocytes recognize antigens in soluble form. The proliferation and differentiation of lymphocytes define the adaptive immune response consisting of specific effector cells and memory cells. During the initial phase of the immune response, the innate immunity recognizes microbial associated motifs as well as lesion-triggered endogenous danger signals that direct the subsequent differentiation of specific lymphocytes and the overall profile of immune response. In the absence of danger signals, the T and B cell responses are reduced in magnitude and immune tolerance results, particularly at moderate to high doses of antigen. It has been proposed that this is a critical mechanism in discriminating between innocuous and ‘dangerous’ antigens associated with infection. This mechanism also sheds a different light on the strategy of immune system to discriminate between self and non-self, previously thought to be determined exclusively at the level of antigen-receptor repertoire.
The adaptive immune response is triggered by recognition of T and B cell epitopes and shaped by “danger” signals that act via innate immune receptors. In the present application it is shown that motifs associated with non-coding double stranded or single stranded RNA provide essential features to the immune response, reminiscent of viral infection, such as, rapid induction of pro-inflammatory chemokine expression, recruitment and activation of antigen presenting cells (APC), modulation of regulatory cytokines, differentiation of Th1 cells, isotype switching and stimulation of cross-priming, consisting in induction of MHC class I-restricted immune responses. The present application demonstrates the heterogeneity of RNA-associated motifs resulting in the differential impact on the profile of the immune response. Based on the ability of specific RNA-motifs to block tolerance induction and to effectively organize the immune defense during viral infection, this demonstrates that such RNA species are potent “danger signals.” The present application teaches using selected RNA motifs as adjuvants for optimizing the immune response to subunit vaccines. In conclusion, RNA-associated motifs that are produced during viral infection not only have a short-term impact on innate immunity, but also bridge the early response with the late adaptive phase comprising activation and differentiation of antigen specific B and T cells.
In the present application, it is demonstrated that in addition to the single versus double stranded nature of RNA, the oligonucleotide composition is a critical determinant for recognition of non-coding RNA motifs by innate immune receptors. In addition, heterogenous RNA motifs have potent and differential impact on the adaptive immunity, mediating most of the features of the immune response during viral infection. Finally, it is also shown that the described RNA-motifs effectively turn on defense mechanisms with prophylactic or therapeutic use in infectious diseases or cancers.
The present application claims priority to and incorporates by reference PCT US03/07995 filed Mar. 14, 2003, U.S. Ser. No. 60/364,490 filed Mar. 15, 2002 and U.S. Ser. No. 60/412,219 filed Sep. 20, 2002.
The mechanisms of immune response during microbial infection became a major area of investigation subsequent to advances in understanding the potential role of innate immunity. It quickly became evident that the innate immune cells are endowed with multiple categories of receptors that discriminate between various microbial-associated motifs or “exogenous” danger signals. Communication between innate and adaptive immunity subsequent to such pattern recognition events decisively influences the magnitude and profile of T and B cell responses. The innate immunity is rapid though less discriminative, but instructs the adaptive immunity that evolves slower and is composed of more potent effectors, with a vast repertoire acquired by somatic mutation. This multi-patterned recognition strategy that employs jointly, the innate and adaptive immunity, shifted the immune discrimination paradigm from self/non-self to dangerous/non-dangerous cognition. The poor immunogenicity of purified proteins, induction of immune mediators by microbial motifs and characterization of the activity of such mediators (cytokines, chemokines and co-stimulatory molecules) on adaptive immunity, all support this concept.
As described in the present application, a rational approach in delineating the role of non-coding RNA motifs as danger signals was taken, with direct implications for understanding their role in controlling the adaptive immunity during viral infections. In addition, their use as adjuvants in conjunction with vaccination is explored.
A library of synthetic RNAs and a two-tier strategy was employed, using as read-out, the effect on the adaptive, rather than innate, immunity. By this method, it was surprisingly found that the oligonucleotide composition in addition to the double stranded nature of RNA plays a role in this concern. It is shown that A:U-based motifs have the ability to turn on the Th1 immunity, isotype switching to IgG2a (
Based on the data, it is concluded that the newly characterized pA:pU-associated motifs can induce a large number of features of the adaptive immune response, that are usually noted only subsequent to viral infection. Induction of T1 responses (both Th1 and Tc1) was documented with protein antigens (OVA and gp140) and inactivated influenza virus (
Since potent danger motifs should influence the outcome in terms of immune responsiveness versus tolerance, it was studied whether dsRNAs prevent high-zone tolerance to human IgG, a well-characterized model of immune unresponsiveness. It is shown that both pA:pU and pI:pC are potent inhibitors of immune tolerance (
The potency of pA:pU as a danger motif is illustrated by its ability to control primary infection from the influenza virus (
Based on the results stated herein, dsRNA motifs are logical candidates for adjuvants in conjunction with subunit, recombinant or inactivated vaccines. In particular, pA:pU appears likely to provide some of the beneficial features of live vaccines in the absence of vector replication. The present application describes immunological complexes for mucosal and systemic vaccination that allowed co-formulation of antigen and dsRNA. As shown in the
In conclusion, by using a rational approach of selecting RNA motifs that impact the adaptive immune responses, an unexpected heterogeneity and novel RNA-associated danger motifs were defined. A systematic study of the adaptive immune response demonstrated that selected RNA motifs orchestrate a broad range of features that are reminiscent of natural infection. Finally, the present application defines novel formulations containing such RNA motifs that are of potential use in mucosal or systemic vaccination, as well as immunotherapy, by eliminating immune unresponsiveness or immune tolerance.
A) Materials and Methods
1) Antigens and Immunomodulator
A panel of 18 single-stranded and double stranded synthetic RNAs (see Table 1) was purchased from Sigma and dissolved in sterile PBS. The RNAs were used as pools or individually. Ovalbumin (OVA, low endotoxin) was purchased from Sigma (A7641). Cholera toxin subunit B (CTB) from Calbiochem (catalog # 227039), Complete Freund's adjuvant (CFA) from DIFCO (catalog #263810) and human IgG (hIgG) from Sigma (catalog # 14506). The recombinant gp140 HIV antigen that retains conformational epitopes and has the ability to trimerize, was derived from gp160 envelope protein of the strain IIIB by introducing a stop mutation. The antigen was expressed by a vaccinia virus vector generously provided by Dr. Bernard Moss (N.I.H.), in BS-C-1 (ATCC) cells and purified by lentil lectin sepharose chromatography (Pharmacia, Piscataway, N.J.). The identity of gp140 antigen was confirmed by Western blot analysis using HIV envelope-specific antibodies purchased from Fitzgerald (Catalog # 20-HG81). Influenza virus (strain A/WSN/32 H1N1) was grown on MDBK cells and purified from supernatants by sucrose-gradient centrifugation. For virus-inactivation, the virions were exposed to short-wave UV light for 15 minutes under stirring. The inactivation was confirmed by virus titration on permissive MDCK cells. Recombinant mouse IgG2b bearing the I-Ed-restricted hemagglutinin-derived peptide SFERFEIFPKE (IgHA) [Seq. I.D. No. 1] within the variable region was obtained and purified as characterized previously.
*p = “poly”
2) Animals
C57BL/6, BALB/c and TLR4−/− C3H/HeJ female mice, between 6-8 weeks of age, were purchased from the Jackson Laboratories (Bar Harbor, Mass.) and housed under specific pathogen conditions at Alliance Pharmaceutical Corp. Key observations in C57BL/6 and BALB/c mice were reproduced in C3H/HeJ mice that have deficient responsiveness to endotoxin. Female Sprague. Dawley rats (250-330 grams) were purchased from Taconic farms and housed in similar conditions.
3) Immunization, Challenge and Measurement of Virus Titers
Mice and rats were primed by intratracheal instillation or aerosolization respectively, as described before and in the case of mice, boosted twice intranasally, at 2 weeks intervals. For induction of high-zone tolerance, the mice were primed by intravenous injection. Finally, for the induction of strong immune responses, mice were immunized subcutaneously with antigen emulsified in CFA. The amounts of antigens used for priming, boosts or induction of tolerance were: OVA-100 μg; HIV gp140-10 μg; hIgG-200 μg; and sucrose-purified UV-WSN-20 μg. The amount of synthetic RNA used was 40-50 μg/dose, with or without antigen, incorporated or not in short chain lipid (SCL) complexes. The amount of CTB/dose was of 10 μg. The antigens were either delivered in saline or when formulated, in perfluorocarbon (perflubron [neat perfluorooctyl bromide], Liquivent®, Alliance Pharmaceutical Corp.) which is an inert vehicle that is compatible with the SCL matrix (total volume of instillation or aerosolization, of 40-45 μl).
For virus challenge, C57BL/6 and TLR4−/− C3H/HeJ mice under Metofane anesthesia were infected with sublethal doses (104 tissue culture infective doses 50%-TCID50) of live WSN virus, via the nasal route. On day 5 after infection, 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). The endpoint titers were estimated in triplicate measurements by interpolation and expressed as TCID50/organ.
4) Immunological Complexes
The technological process of obtaining short chain lipid (SCL) complexes (or “immunological complexes”) with phospholipid as major excipient was spray drying. A more simple version of this process was used herein. In brief, the phospholipid was homogenized in water (to form liposomes or micelles) and mixed with the excipients and the actives, followed by spray drying, as described in detail: an aqueous preparation was prepared by mixing two preparations, A and B, immediately prior to spray-drying. Preparation A was comprised of a micellar preparation in which 0.14 g of dioctanoylphosphatidylcholine (Avanti Polar Lipids) by dissolving the phospholipid in 23 mL of hot DI water. 0.0357 g of CaCl2.2H2O and 0.714 g of lactose was dissolved in the phospholipid micellar preparation. Preparation B was comprised of 20 mg of Ovalbumin (Sigma) and 4 mg of pA:pU (endotoxin free) which was dissolved in 5 mL of PBS. The combined feed preparation (2 mL of preparation A with preparation B) was spray dried with a standard B-191 Mini spray drier under the following conditions: inlet temperature=70° C., outlet temperature=43° C., aspirator=90%, pump=2.2 mL/min, nitrogen flow=2400 L/h. The resulting complex had a PL:OVA:pApU:CaCl2.2H2O:Lactose weight ratio of 12:20:4::3:61.
5) Measurement of Antibody and T Cell Response
The antibody response was measured by ELISA. In brief, wells were coated with antigen (2 μg/ml of gp140, 8 μg/ml of sucrose purified virus, 10 μg/ml of hIgG or OVA, respectively) and blocked with SeaBlock (Pierce, Rockford, Ill., catalog # 37527). Serial dilutions of serum and bronchoalveolar lavage fluid were incubated for at least 2 hours at room temperature. After washing, the assay was developed with anti-mouse IgG antibody coupled with alkaline phosphatase (Sigma, cat# A7434) followed by addition of substrate (PNPP, Sigma, cat# N2765) and measurement by using an automatic ELISA reader (Molecular Devices, ThermoMax) equipped with SoftMax software.
For the measurement of cellular response, 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: 96-well 45 micron mixed cellulose ester plates (Millipore, cat#MAHA S4510) were coated with 4 μg/ml of rat anti-mouse anti-IFNγ, 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 5×105 cells/well together or without antigens or peptides. In the case of pulmonary lymphocytes, effector cells were 1:1 mixed with mitomycin-treated, splenic stimulator cells before stimulation. For stimulation, graded amounts of antigen were used (OVA, gp140, hIgG or sucrose-purified WSN virus) or peptides: class II-restricted HA SFERFEIFPKE [Seq. I.D. No. 1]; or class I-restricted SIINFEKL [Seq. I.D. No. 2] and HIV V3-derived R10K peptide described previously. At 72 hours after stimulation, 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).
6) Measurement of Chemokine Gene-Expression
The level of chemokine expression in the lungs of mice treated 1 day previously with synthetic RNA or controls was measured by DNA array technology as follows: 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 further 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.).
7) Flow Cytometry
Cell suspensions from lungs of mice, treated one day previously with synthetic RNA or saline, were prepared by collagenase digestion and Ficoll gradient centrifugation, as above. The cells were resuspended in phosphate buffered saline containing 1% (v:v) mouse serum (Sigma. cat# M5905) and 1% (w:v) bovine albumin, fraction V (Sigma, cat# A3059) and stained with PE-labeled rat anti-mouse CD11b (PharMingen, cat# 01715A), PE-labeled hamster anti-mouse CD11c (PharMingen, cat# 09705A) or the appropriate PE-labeled isotype control (PharMingen, cat# 11125A or 11185A) at a concentration of 1 μg antibody/106 cells, for 40 minutes on ice. The analysis was carried out on a Becton Dickinson FacsCalibur instrument. The non-viable cells were gated out using propidium iodide.
8) Magnetic Separation and Adoptive Transfer
Professional APC such as CD11c+ dendritic cells were separated from spleens of BALB/c mice by using magnetic beads coupled to rat anti-mouse anti-CD11c antibodies (Miltenyi Biotech). In brief, single cells suspensions were resuspended in MACS buffer (BSA and EDTA) at 107 cells/ml, incubated for 15′ on ice with magnetic beads, washed and passed through magnetic columns. The columns were washed three times before elution, followed by two consecutive washings and in vitro overnight pulsing with 100 μg/ml of IgHA together or without 50 μg/ml RNA motifs, or 5 ng/ml rIL-12 (Biosource Int., Camarillo, Calif.). Alternatively, the cells were incubated overnight with IgHA on wells previously coated with rat anti-mouse CD40 monoclonal antibody (BD-PharMingen). The cells were washed, resuspended in balanced sterile saline and adoptively transferred by subcutaneous injection into naïve BALB/c mice (2.5×105 APC/mouse). The T cell response was measured at 14 days by IL-2 ELISPOT analysis subsequent to stimulation with HA class II-restricted peptide, as described above.
9) Statistical Analysis
Magnitudes of immune responses were compared by using the t test, assuming a normal distribution of the values and equal variances.
B. Results
1) Systematic Definition of RNA Motifs that Modulate Immune Responses
During viral infection, transient, unusual RNA species are created and act as “danger” signals. It was therefore postulated that multiple, various RNA motifs are being recognized by the innate immune cells and profoundly regulate the adaptive immune response. To address this hypothesis on a systematic basis, a library of synthetic, single-stranded and double-stranded RNA motifs was screened for the ability to modulate the specific IgG response to a protein antigen (OVA) administered via the respiratory tract. To simplify the process, the screening was organized into two rounds: (1) round one involving pools of RNA species (Table 1); and, (2) the second round dissecting components within pools endowed with maximal impact on the immune response.
The double stranded RNA (dsRNA) or single stranded RNA (ssRNA) of the present invention is made by Sigma according to the following method:
The various 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.
Impact of various pools of RNA motifs on antibody response to a standard antigen (OVA).
The impact of various RNA pools (Table 1) on the adaptive immunity was measured in C57B1/6 mice co-immunized with OVA via the respiratory tract. As described in the “Materials and Methods” section, the antibody response was expressed as mean±SEM of IgG endpoint titers (n=4/group). As controls, OVA in sterile PBS, OVA with cholera toxin subunit B (CTB) and PBS alone, respectively. As shown in the
This shows that both the nature of residues and secondary structure of RNA determine their ability to act as “danger” motifs, with effect on the specific B cell response.
Effect of various individual dsRNA motifs on the induction of antibody response to OVA.
Experiments were carried out as in the previous example and as described in the “Materials and Methods” section, but instead of pools of RNAs, we used individual dsRNAs were used. The results are expressed in
Thus, individual RNA motifs display different ability to impact the antibody response in terms of magnitude and profile.
The magnitude and profile of T cell response induced by OVA together with various, individual dsRNA motifs.
The results obtained by ELISPOT analysis are expressed as mean±SEM of the number of IFN-γ and IL-4 spot-forming-colonies (SFC) per spleen (n=4/group). As is shown in
In conclusion, different dsRNA motifs have different impact on the magnitude and profile of T helper response.
The effect of a defined dsRNA motif on the antibody response to a viral antigen, the HIV recombinant gp140 protein.
As shown in
Thus, antibody responses to a viral antigen of potential practical use are enhanced by use of a novel dsRNA motif.
The effect of a defined dsRNA motif on the antibody response to whole UV-inactivated influenza virus, strain A/WSN/32 H1N1 (UV-WSN).
As in Example 4, the influenza virus-specific IgG antibodies were measured after mucosal immunization with UV-inactivated WSN virus (UV-WSN) (20 μg, see “Materials and Methods”) alone or together with dsRNA motifs (50 μg). As a control, the antibody response subsequent to infection with the same strain of influenza virus (n=4/group) was used. The results are expressed as mean±SEM of IgG endpoint titers in
Thus, antibody responses to a viral antigen in context of whole inactivated microbe, are enhanced by use of a novel dsRNA motif.
The enhancement of T cell response to whole-inactivated influenza virus by co-administration of a defined dsRNA motif.
The T cell response to whole influenza virus was studied by ELISPOT analysis of splenocytes and expressed as IFN-γ, IL-4 and IL-2 SFC (mean±SEM, n=4/group), after the subtraction of background. The T cell response to antigen together with pA:pU was compared to that subsequent to immunization with antigen alone or influenza virus infection (see
Thus as demonstrated in Examples 4-6, the impact of dsRNA on antibody response was independently confirmed with foreign antigens, namely HIV envelope protein (recombinant gp140) and whole-inactivated influenza virus (FIGS. 2A,B). In fact, pA:pU, rather than pI:pC, restored the titer of specific antibodies to the influenza virus, to a level similar to that triggered by infection (
2) Motifs Associated with dsRNA Regulate the Recruitment and Activation of Professional Antigen Presenting Cells
It was hypothesized that dsRNA-associated danger motifs such as pA:pU and pI:pC influence the T cell response indirectly, via components of innate immunity. To test this hypothesis, the expression of chemokine genes was defined in the pulmonary lymphoid tissue, subsequent to administration of RNAs.
Local Uu-regulation of chemokine gene-expression by dsRNA motifs.
Local up-regulation of chemokine gene-expression by dsRNA motifs was measured by DNA array technique (see Materials and Methods “Measurement of Chemokine Gene Expression”) one day subsequent to treatment via the respiratory tract. The results are expressed as fold-increase over expression levels measured in the pulmonary tissue of non-treated mice. The pattern of chemokine expression triggered by dsRNA was contrasted with that induced by LPS. The chemokines that selectively bind to receptors on Th1 and Th2 cells respectively, were indicated with continuous and interrupted contours (
The DNA array technique showed that IP-10, MIG, MIP-1α, MIP-1β and MCP-1 were strongly induced by both pA:pU and pI:pC (see
Recruitment of professional APC in the lungs of mice treated with dsRNA.
Recruitment of professional APC in the lungs of mice treated with dsRNA motifs was assessed by FACS analysis one day after the treatment. The results are expressed in
Activation of professional APC (dendritic cells) by the dsRNA motifs.
Activation of professional APC by the dsRNA motifs was ascertained by ex vivo pulsing of CD11c+ cells with antigen together with dsRNA, followed by an adoptive transfer experiment into naïve BALB/c mice and measurement of T cell response (
Thus, dsRNA motifs activate APC in addition to the recruiting effect.
Cross-priming of MHC class I-restricted T cells against a viral antigen, stimulated by dsRNA motifs in BALB/c mice.
Cross-priming (referring to special circumstances when APC acquire the ability to prime class I restricted T cells without infection) stimulated by dsRNA motifs was studied in BALB/c mice treated with recombinant-engineered HIV gp140 antigen (10 μg) together with pA:pU, by ELISPOT analysis, using in vitro stimulation with MHC class I-restricted cognate peptide (see “Materials and Methods”). As a control, dose-matched gp140 antigen was used. The results are expressed in
In conclusion, dsRNA motifs facilitate the induction of MHC class I-restricted to non-infectious antigens of potential practical use.
Cross-priming of MHC class I-restricted T cells against OVA, stimulated by dsRNA motifs in C57BL/6 mice.
Cross-priming stimulated by dsRNA motifs was studied in C57BL/6 mice treated with whole OVA together with pA:pU by ELISPOT analysis, using in vitro stimulation with MHC class I-restricted peptide (see “Materials and Methods”). As a control, dose-matched OVA antigen or sterile PBS was used. The results are expressed in
In conclusion, dsRNA motifs facilitate the induction of MHC class I-restricted to non-infectious antigens of potential practical use.
FACS analysis of pulmonary interstitial cells subsequent to mucosal administration of pA:pU and pI:pC showed prompt recruitment of CD11b+ monocytes and in the second case, of CD11c+ dendritic cells (
3) dsRNA Motifs Block the Induction of High-Zone Tolerance and Trigger Protective Anti-Viral Immunity
Danger molecules participate to discriminate between innocuous antigens and antigens associated with infectious processes. In high doses, non-infectious purified protein antigens induce unresponsiveness or immunological tolerance. Central ways of achieving tolerance to self or innocuous antigens is “immunological ignorance” and “immunological tolerance”. In the first scenario, antigens are not accessible to APC due to spatial segregation. In the second scenario, the antigens are accessible to APC, are internalized, processed and the resulting epitopes are presented in context of poor co-stimulation. The net outcome can be the induction of immune unresponsiveness or tolerance at the level of T cells. In the case of infection or immunological challenge, there are mechanisms that prevent “immunological ignorance” and “tolerance”. Such mechanisms occur via inducing novel migration patterns for APC together with activation of expression of co-stimulatory molecules and proinflammatory chemokines and cytokines. The outcome will be strong immune response rather than ignorance or tolerance to any antigens to which the immune system is exposed in such circumstances defined by the presence of “danger-molecules.” As an example, tumor-associated antigens are often ignored by immune effectors or presented in a toleragenic context. Means to restore the immune competence against such antigens have direct practical implications in anti-cancer therapy. In order to test the danger-signal competence of pA:pU and pI:pC motifs, the model of tolerance achieved by intravenous inoculation of hIgG was used.
dsRNA motifs prevent high-zone tolerance in mice injected with human IgG.
The mice were initially injected intravenously with a standard toleragenic dose (200 μg) of hIgG alone (closed symbols) or together with pI:pC or pA:pU (40-50 μg) (open symbols; see
The results demonstrate that co-inoculation of either pA:pU or pI:pC together with hIgG in saline prevented the induction of B cell unresponsiveness, as shown by antibody titers subsequent to boost with hIgG in CFA (
dsRNA motifs display differential ability to mobilize immune defense against influenza virus infection.
It was postulated that danger motifs have the capability of rapidly mobilizing protective arms of immune defense. Thus, it was tested whether pA:pU and pI:pC have any impact on the evolution of primary infection with influenza virus.
Mice were treated via the respiratory route with either pI:pC, pA:pU or saline one day before and after pulmonary infection with a sublethal dose of influenza virus. On day 5, the virus titer in the lung tissue was estimated and represented as total infectious units/organ (means±SEM; n=6/group; results representative of two independent studies in C3H/HeJ TLR-4−/− and competent mice).
As shown in
4) Enhancement of Immunity to Protein Antigens by Co-Encapsulation with RNA Danger Motifs
Since the immune response to non-formulated subunit vaccines and in general, to purified protein antigens is minimal, it was tested whether in vivo co-exposure of APC to antigen and dsRNA danger motifs within the same microstructure results in a more favorable outcome. To this aim, a prototype antigen (OVA) was coformulated with pA:pU or pI:pC in short chain lipid complexes (“SCL”)(see “Materials and Methods”) with biocompatible and immunologically inert phospholipids (e.g., dioctanoylphosphatidylcholine) and lactose, as excipients. Upon delivery to the respiratory tract of C57BL/6 mice with either OVA+pA:pU or pI:pC formulated in short-chain lipid complexes, antibody responses were measured that were considerably higher than those of mice immunized with non-formulated antigen in saline, or antigen formulated in SCL complexes devoid of dsRNA motifs (
Short chain lipid complexes loaded with model antigen (OVA) alone or together with dsRNA motifs.
Short chain lipid complexes composed of short-chain phospholipids and loaded with model antigen (OVA) alone or together with dsRNA motifs have been generated and tested in C57BL/6 mice as shown in
Local (lung) and systemic (splenic) T cell response in C57BL/6 mice to whole OVA antigen or class I-restricted dominant OVA peptide, subsequent to immunization with OVA co-formulated with dsRNA motifs.
Interestingly, CTB had only a limited adjuvant effect in context of short chain lipid complex co-formulation. Consistent with previous results, induction of T1 immunity was measured only with pA:pU particles as shown in
Systemic and local antibody response of Sprague-Dawley rats to mucosal vaccination with SC-lipid complexes loaded with model antigen (OVA) co-formulated with dsRNA motifs.
Rats were immunized with lipid-complexes co-formulated with OVA and dsRNA. As controls, SCL complexes devoid of antigen, SCL complexes loaded with OVA but devoid of dsRNA motifs and dose-matched amounts of OVA in saline were used, respectively. The results are expressed as endpoint titers (means±SEM; n=4/group) of OVA-specific antibodies measured by ELISA in serum (
A similar enhancement of the antibody response has been measured in case of Sprague-Dawley rats aerosolized with SCL complexes loaded with OVA together with pA:pU or pI:pC (
Thus, co-formulation of RNA-associated danger motifs and protein antigen by using a novel spray-drying technology preserves the immunomodulatory properties of RNA motifs and results in substantial increase of the specific immune response, both locally and systemically.
Non-replicating dsRNA motifs act as master switch for the adaptive (B and T cell) immune response.
Antigens devoid of danger motifs such as dsRNA are poorly immunogenic or if provided in large doses may induce immunological tolerance. However, dsRNA motifs modify the way the immune system perceives the antigen: instead of poor responsiveness or tolerance, such motifs instruct the adaptive (T and B cell) immunity to mount strong responses to co-existing antigens, as well as prevent or block the immunological tolerance. Thus, innate immunity by virtue of recognition of dsRNA motifs operates as master switch for adaptive B and T cell immunity (
Naturally occurring dsRNA bridges the innate with adaptive immune response. Example 18 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
Permissive MDCK cells were infected with WSN influenza virus (108 TCID50/1×109 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 5μ of S1 nuclease (Ambion, Inc., Austin, Tex.)/μg 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. As a control, material purified and treated similarly, from 109 non-infected MDCK cells, was used. The concentration of nucleic acid was measured by spectrophotometry (A260nm) 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 μg of RNA and 2 μg of antigen in 25 ml of sterile PBS).
After demonstrating lack of infectivity, 40 μg of dsRNA or control RNA were admixed with 40 μg of recombinant truncated antigen (gp140 of HIV envelope) and were administered to BALB/c mice by intranasal instillation (n=3/group). Additional controls were animals immunized with 40 μg of gp140 protein in saline (n=3/group). The mice were boosted once, at 2 weeks after priming. Blood was harvested 2 weeks after the boost, sera prepared and the antibody response against gp140 measured by ELISA. In brief, wells were coated with antigen (2 μg/ml of gp140) and blocked with SeaBlock (Pierce, Rockford, Ill., catalog # 37527). Serial dilutions of serum and bronchoalveolar lavage fluid were incubated for at least 2 hours at room temperature. After washing, the assay was developed with anti-mouse IgG antibody coupled with alkaline phosphatase (Sigma, cat# A7434) followed by addition of substrate (PNPP, Sigma, cat# N2765) and measurement by using an automatic microtiter plate reader (Molecular Devices, ThermoMax) equipped with SoftMax software.
In
Overall, the data in
Effects of selected RNA motifs on the innate immune response: heterogeneous motifs. This Example Shows, Unexpectedly, that Different Synthetic RNA Motifs Have a Distinct Effect on the Adaptive Specific Immune Response to a Protein Antigen
The results in FIGS. 8B-D show that different synthetic RNAs have an enhancing effect on the B and T cell response to a prototype protein antigen. In addition, different motifs, comprising specific nucleotide combinations, have specific effects in terms of T1 versus T2 induction and subsequently, immunoglobulin isotype switching.
Use of selected synthetic RNA motifs facilitates the induction of MHC class I-restricted Tc1 cells, producing IFN-γ.
The results in FIGS. 9A-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-γ-producing MHC class I-restricted T cells.
Example 21 shows that unexpectedly, different synthetic RNA motifs bind to different cellular receptors; in other words, there are multiple receptors that discriminate among RNA Motifs.
In vitro binding of CD11b+ APC, by fluorescently-tagged pA:pU, was measured by FACS analysis. The MACS-separated APC were incubated at 4° C. for 30 minutes with 10 μg/ml of tagged pA:pU ([pA:pU]-F), washed and analyzed. Alternatively, APC were preincubated for 10 minutes with 20 or 100 μg/ml of non-tagged pA:pU, pA or pI:pC respectively, before staining with tagged pA:pU and FACS analysis. The profiles of stained (open area), non-stained (filled area) cells and the percentage of highly stained APC were represented in each panel, with logarithmic x axis. The data are representative of two independent measurements with 10,000 events acquired for each sample.
Materials:
The results in
Example 22 shows that selected synthetic RNA motifs trigger in vivo expression of chemokine genes, of importance for immunological activity.
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 further 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 (
The results in
Example 23 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 μg of pI:pC, pA:pU or 50 μl of saline one day before and after pulmonary infection with a sublethal dose of influenza virus. For virus challenge, C57BL/6 and TLR4−/− C3H/HeJ mice under Metofane anesthesia were infected with sublethal doses (104 tissue culture infective doses 50%-TCID50) of live WSN (A/WSN/H1n1) virus, via the nasal route. On day 5 after infection, 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). The endpoint titers were estimated in triplicate measurements by interpolation and expressed as TCID50/organ (means±SEM; n=6/group; results are representative of two independent studies in C3H/HeJ TLR-4−/− and competent mice). Similar results were obtained in TLR4 competent, C57BL/6 mice.
Thus, the results depicted in
Example 24 shows that co-administration of selected synthetic RNA motifs breaks tolerance to high dose standard antigen
dsRNA motifs prevent high-zone tolerance in mice injected with human IgG. The mice (C57BL/6) were initially injected i.v. with a toleragenic dose of 200 μg of hIgG alone (closed symbols) or together with 100 μg of pI:pC or pA:pU (open symbols) and subsequently boosted subcutaneously with an immunogenic dose of 100 μg of hIgG emulsified in CFA (Complete Freund's Adjuvant). The titer of antibodies against hIgG was measured by ELISA (as detailed in the Example 19), with the difference consisting in use of 10 μg/ml of hIgG for coating) at various intervals after the first injection. As a control, mice immunized with 100 μg of hIgG emulsified in CFA were included and represented the maximal titer on the graph (interrupted line).
The results are represented in
Example 25 shows that selected RNA motifs induce differential cytokine production by human APC.
Human THP-1 monocytic cells, following differentiation, were incubated with different concentrations of synthetic RNA (pA:pU, pI:pC or pA) for 24 hours, and the cell supernatants collected. The concentration of IL-12 and TNF-α were measured by ELISA. The results are expressed in
The results in
Materials:
THP-1 cells were incubated 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
The results in
Materials:
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 0.005EU/μg. 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
The useful fraction comprises synthetic RNA of less than 20 to maximum 100 bp size. After purification, 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 25).
Example 28 shows that unexpectedly, different fractions of a selected synthetic RNA compound are endowed with different biological activity, based on size.
Differentiated human THP-1 monocytic cells were incubated with different concentrations of synthetic RNA (pA:pU, fractionated as described in the Example 27) for 24 hours, and the supernatants collected. The concentration of TNF-α was measured by ELISA using BioSource International kits (Camarillo, Calif.). The results are expressed in
The results depicted in
Selected synthetic RNA motifs have, unexpectedly, a different immune profile in regard to generation of anti-RNA antibodies.
BALB/c mice were immunized intraperitoneally and subcutaneoulsy [i.p.+s.c.] with 50%μg+50 μg of hIgG and synthetic RNA (pI:pC or pA:pU) and serum samples were prepared 1 week later. As a control, 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. In brief, wells were coated with antigen (10 μg/ml of hIgG or synthetic RNAs) and blocked with SeaBlock (Pierce, Rockford, Ill., catalog # 37527). Serial dilutions of serum and bronchoalveolar lavage fluid were incubated for at least 2 hours at room temperature. After washing, the assay was developed with anti-mouse IgG antibody coupled with alkaline phosphatase (Sigma, cat# A7434) followed by addition of substrate (pNPP, Sigma, cat# N2765) and measurement by using an automatic microtiter plate reader (Molecular Devices, ThermoMax) equipped with SoftMax software.
The results are expressed in
In vivo loading of APC by recombinant IgG results in generation of Tc1 type of MHC class I responses only when additional conditions are satisfied.
BALB/c mice were immunized with 50 ug of recIgG-NP(Kd) (see
The next day 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. Louis, Mo.) and stopped by washing the plate twice with tap water. Plates 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.). The frequency of cytokine producing T cells reacting to NP peptide (NP peptide is a protective and conserved epitope of Type A influenza virus) was measured and expressed against the amount of peptide used for stimulation. The results are expressed in
The administration of recombinant IgG bearing the NP MHC class I-restricted epitope resulted in generation of Tc2 immunity but not Tc1 response, implying in vivo formation of class I-peptide complexes with a specific co-stimulation profile. The results in
Effective formation of MHC class I-peptides and instruction of the resulting T cell response by simultaneous manipulation of APC loading via Fcgamma R and activation via RNA receptors.
Splenic APC were isolated from naive BALBc mice and pulsed ex vivo overnight with 1 ug NP peptide, or 50 μg recIgG-NP (Kd) with or without 50 μg/ml selected synthetic dsRNA (pA:pU). The cells were washed and 5×106 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 μg/ml for anti-IL4, and 8 μg/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 μl/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 μl/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 5×105/well together with 30 μg/ml, 10 μg/ml, or 3 μg/ml NP peptide or just with media, to assess the background. Plates were incubated 72 hours at 37° C., 5% CO2. After 3 days, plates were washed 5 times with PBS-tween20 0.05% (washing buffer), and incubated with 100 μl/well of biotinylated anti-cytokine Abs, 2 μg/ml in PBS-tween20 0.05%-FBS 0.1% (ELISPOT buffer) overnight at 4° C. The next day 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. Plates 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.). The results are expressed in
The results in
Example 32 shows that co-priming with IgG-peptide together with a selected co-stimulatory motif resulted in more effective secondary expansion of MHC class I-restricted T cells subsequent of virus infection.
BALB/c mice were injected with recIgG-NP(Kd), pA:pU separately, or in combination (50 ug/injection). As a control, naive mice were used. Three weeks after treatment, the mice were infected with 104 TCID50 of A/WSN/32 H1N1 influenza virus, via the respiratory tract. Four days after infection, the T cell profile in 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 μg/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 μl/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 μl/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 5×105/well together with 20 μg/ml NP peptide or just with media, to assess the background. Plates were incubated 72 hours at 37° C., 5% CO2. After 3 days, plates were washed 5 times with PBS-tween20 0.05% (washing buffer), and incubated with 100 μl/well of biotinylated anti-cytokine Abs, 2 μg/ml in PBS-tween20 0.05%-FBS 0.1% (ELISPOT buffer) overnight at 4° C. The next day 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. Plates 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.). The results are expressed in
The results in
Example 33 shows that the most effective priming of cytotoxic lymphocytes recognizing an MHC class I-restricted epitope occurs by co-administration of selected RNA motif together with peptide epitope inserted within the IgG backbone.
BALBc 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 μg/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.
After expansion, 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 μg/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).
The results in
Example 34 shows that vaccination with an IgG bearing a viral MHC class I-restricted epitope, together with selected synthetic RNA motif, provided protection against infectious challenge with a prototype virus.
BALB/c 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 104 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 2×104/well/200 ul and incubated for 24 hours at 37° C., 5% CO2. The next day, 25 μl of the 10 fold dilutions in DMEM media of the lung homogenates were incubates in briefly tripsinized MDCK plates (1 minute) in triplicates and incubated at 37° C. After one hour 175 ul of the DMEM complete media was added and plates were incubated for 48 hours at 37° C., 5% CO2. After two days the hemagglutination-inhibition was done with chicken red blood cells incubated with the cell culture supernatants from the MDCK plate for 30 minutes at room temperature and the results were expressed as means±SEM of total pulmonary virus (n=4 mice/group). As a control, non-immunized mice were used.
The results in
Balb-c mice (Kd restricted) have been used to establish a tumor model. Tumor cells (1 to 15 million in 100 μL) were typically injected in the flank (see arrow in upper photo). Primary tumors (i.e. those at the sight of injection) were first detected by palpating the area and then quantitated by measuring the tumor size with a caliper. In one series of experiments 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), was used to induce tumors in the mice. Expression of heterologous proteins in the SP2/0 cells provided specific tumor associated antigens (TAA) for testing various anti-tumor strategies in the immunocompetent mice. Typically, 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
This tumor model was extended to include an adenocarcinoma cell line (4T1, ATCC CRL-2539, Kd restricted) previously shown to induce metastatic tumors in Balb-c mice. The 4T-1 cell line was similarly 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.
Example 36 demonstrates successful control and treatment of a tumor after clinical diagnosis, by using a tumor associate T cell epitope within a recombinant IgG, together with a selected co-stimulatory RNA motif.
Balb/c mice were injected with SP2/0 cells (15 million in 100 μL) stably expressing recombinant IgG carrying the MHC I (Kd) NP epitope peptide in the CDR3 region of the heavy chain (IgNP). At day 7 post injection all mice had palpable tumors and the mice were randomized into 3 groups: co-stimulatory motif (i.e. dsRNA comprised of polymeric pApU) alone; purified IgTAA protein (IgNP); both. The time of treatment is indicated by the arrows, and each injection contained 50 μg of the indicated compound. The mice that developed metastatic disease and died are represented with a “D” in the figure.
The data show that the combination of dsRNA (co-stimulatory motif) and IgTAA (IgNP) produced a dramatic protective response in mice that all had primary tumors at the start of therapy. While all mice treated with either compound alone succumbed to disease, 100% of the mice treated with both were still alive 3 weeks after initiation of treatment and they were in good clinical condition at the time of sacrifice for measurement of T cell response. These data show that 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 pA:pU dsRNA highlights the important role co-stimulation plays in treatment of tumors with tumor-associated antigens.
In conclusion, the results in
This example, 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 IgG backbone, together with co-stimulatory motif.
Balb/c mice were injected with SP2/0 cells stably expressing recombinant IgG (IgNP) that contains the MHC I (Kd) epitope (amino acids 147-155) of WSN virus nucleoprotein in the CDR3 of the heavy chain. The cell inoculum was 1 million cells (in 100 μL) 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 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.
Panel A of
The results in
Example 38 shows that therapy of tumor-bearing mice with a tumor epitope within IgG backbone together with co-stimulatory synthetic RNA results in the restoration of the activatory status of tumor infiltrating lymphocytes.
Two BALB/c mice were injected with 10 million sp20 transfectoma expressing the NP-Kd epitope. After tumors developed, one mouse was injected intratumorally with 50 ◯g of selected dsRNA motif (pApU) plus 50◯g of “IgNP”-recIgG-NP(Kd) 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.
Materials:
The results in
Example 39 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 rejected successfully the tumor following treatment with recombinant Ig carrying a tumor associated epitope together with selected synthetic dsRNA motif as explained in Example 37, 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 μg/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 μl/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 μl/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 5×105/well together with various concentrations of NP peptide. Plates were incubated 72 hours at 37° C., 5% CO2. After 3 days, plates were washed 5 times with PBS-tween20 0.05% (washing buffer), and incubated with 100 μl/well of biotinylated anti-cytokine Abs, 2 μg/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. Plates 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.). The results were expressed as number (mean±SEM) of spot forming colonies corresponding to IL-4, IL-2 and IFN-γ. As a control, non-treated mice were used, which failed to reject tumor (n=4/group).
The results in
Example 40 shows induction of effective memory response subsequent to specific treatment of tumor bearing mice with a T cell epitope within IgG backbone, together with a selected co-stimulatory motif.
Mice bearing sp2/0 tumors expressing the NP-Kd TAA were treated as described in the Example 37, 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 naïve 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.
The results in
Example 41 shows that surprisingly, the induction of tumor rejection by an IgG bearing a TAA together with co-stimulator, results in cross-protection against a range of tumor cell variants lacking the TAA or displaying variants of TAA.
The mice protected against homologous challenge as described in Example 40, 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-Kd epitope. In addition, mice were challenged with a different type of tumor cell line (4T-1 adenocarcinoma) as a control, displayed in the table attached to
The status of 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. The ELISPOT plates (Millipore, Molsheim, France) were incubated with purified anti-cytokine Abs (4 ug/ml for anti-IL2 and anti-IL4, and 8 μg/ml for anti-IFN gamma, from BD Pharmingen) in sterile PBS (50 μl/well) at 4° C. overnight. The next day, the plates were washed 2 times with DMEM media and blocked with 200 μl/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 5×105/well together with various concentrations of antigen. Plates were incubated 72 hours at 37° C., 5% CO2. After 3 days, plates were washed 5 times with PBS-tween20 0.05% (washing buffer), and incubated with 100 μl/well of biotinylated anti-cytokine Abs, 2 μg/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. Plates 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.). The results were expressed as number (mean±SEM) of spot forming colonies corresponding to IL-4, IL-2 and IFN-γ. As a control, non-treated mice that failed to reject tumor (n=4/group) were used. As a control, naïve mice were included. The data are expressed as number (mean±SEM) of cytokine producing cells/organ (n=3/group).
The results in
The present application claims priority to PCT US03/07995, filed Mar. 14, 2003 which claims priority to U.S. Patent Application Serial No. 60,412,219, filed Sep. 20, 2002 and U.S. Patent Application Ser. No. 60/364,490, filed Mar. 15, 2002.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US03/07995 | 3/14/2003 | WO | 6/2/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60364490 | Mar 2002 | US | |
| 60412219 | Sep 2002 | US |
