DOSE DETERMINATION FOR IMMUNOTHERAPEUTIC AGENTS
20200191798 · 2020-06-18
Inventors
- Stefan Josef Christof Friedrich Strobl (Mainz, DE)
- Roman Peter Rösemann (Mainz, DE)
- Ugur Sahin (Mainz, DE)
- Veronika JAHNDEL (Mainz, DE)
- Doreen Schwarck-Kokarakis (Mainz, DE)
- Yves Hüsemann (Mainz, DE)
- Kathrin Dorer (Mainz, DE)
- Robert Jabulowsky (Mainz, DE)
Cpc classification
A61K31/7105
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
G01N2800/52
PHYSICS
International classification
A61K31/7105
HUMAN NECESSITIES
Abstract
The present invention relates to methods for determining suitable doses for administration of immunotherapeutic compounds, whose effectiveness and toxicity can vary at the same dose between individuals due to natural variations within individual subjects, such as variations in the reaction of the immune system in response to administration of such immunotherapeutic compounds.
Claims
1. A method for determining a suitable dose of an immunotherapeutic agent for administration to an individual, comprising: (a) separately contacting multiple different doses of the immunotherapeutic agent with immune-reactive material of the individual, and (b) measuring at least one immunological reaction caused by the multiple different doses of the immunotherapeutic agent.
2. The method according to claim 1, wherein step (b) is characterized by qualitatively and/or quantitatively measuring at least one immunological reaction, preferably quantitatively measuring at least one immunological reaction.
3-5. (canceled)
6. The method according to claim 1, wherein the immunotherapeutic agent comprises at least one immunoreactive peptide or protein, or a nucleic acid encoding at least one immunoreactive peptide or protein.
7. The method according to claim 6, wherein the nucleic acid comprises RNA.
8-9. (canceled)
10. The method according to claim 1, wherein the multiple different doses comprise at least one dose that is below the standard dose range for the immunotherapeutic agent, or wherein the multiple different doses comprise at least one dose that lies within the standard dose range for the immunotherapeutic agent.
11. (canceled)
12. The method according to claim 1, wherein steps (a) and (b) are preformed sequentially.
13. (canceled)
14. The method according to claim 1, wherein the at least one immunological reaction comprises the production of at least one cytokine.
15. The method according to claim 14, wherein the at least one cytokine is selected from the group consisting of interleukin 6 (IL-6), tumor necrosis factor- (TNF-), interferon- (IFN-), interferon- (IFN-), interferon gamma-induced protein 10 (IP-10), interleukin-1 (IL-1), interleukin-2 (IL-2) and interleukin-12p70 (IL-12p70).
16. (canceled)
17. The method according to claim 1, which is an in vitro method.
18-21. (canceled)
22. The method according to claim 17, wherein a dose where the at least one immunological reaction indicates an acceptable therapeutic effect for the immunotherapeutic agent, reflects a suitable dose for administration of the immunotherapeutic agent to the individual.
23. The method according to claim 1, wherein step (a) is carried out in vivo and is characterized by separately contacting multiple different doses of the immunotherapeutic agent with immune-reactive material of the individual in separate administration steps, each characterized by administration of one dose of the immunotherapeutic agent to the individual.
24. The method according to claim 23, wherein the separate administration steps are carried out subsequently and are separated from each other by time intervals of between 2 and 30 days, between 7 and 28 days, 7 days, 14 days, 21 days or 28 days.
25. The method according to claim 23, wherein measuring at least one immunological reaction is separately carried out following each separate administration step.
26. The method according to claim 23, wherein the first of the separate administration steps is characterized by administration of a dose of the immunotherapeutic agent that is below the standard dose range for the immunotherapeutic agent, and wherein the dose administered in the subsequent of the separate administration steps is optionally higher than the dose administered in the first of the separate administration steps.
27. The method according to claim 23, further comprising (c) detecting the presence or absence of at least one side effect.
28-29. (canceled)
30. The method according to claim 27, wherein where at least one side effect is detected following administration of one of the multiple different doses, all subsequent doses are administered with at least one antitoxic agent.
31. The method according to claim 30, wherein where at least one side effect is detected following administration of one of the multiple different doses that is not administered with at least one antitoxic agent, the next dose of the immunotherapeutic agent to be subsequently administered is identical to or less than the dose administered in the previous administration step.
32. The method according to claim 31, wherein the subsequent administration step that directly follows the previous administration step is further followed by one or more further administration steps that optionally represent a dose escalation scheme from step to step.
33. The method according to claim 30, wherein where at least one side effect is detected following administration of one of the multiple different doses that is administered with at least one antitoxic agent, the next dose of the immunotherapeutic agent to be subsequently administered is less than the dose administered in the previous administration step.
34-35. (canceled)
36. The method according to claim 23, wherein where no side effect is detected following administration of any of the multiple different doses, a dose where the at least one immunological reaction indicates an acceptable therapeutic effect for the immunotherapeutic agent reflects a suitable dose for administration of the immunotherapeutic agent to the individual.
37. The method according to claim 23, wherein where at least one side effect is detected following administration of any of the multiple different doses, a dose in a subsequent administration step that is administered with at least one antitoxic agent and where the at least one immunological reaction indicates an acceptable therapeutic effect for the immunotherapeutic agent reflects a suitable dose for administration of the immunotherapeutic agent to the individual.
38. The method according to claim 37, wherein where no side effect is detected following administration of any of the multiple different doses administered with at least one antitoxic agent, a dose where the at least one immunological reaction provides the strongest indication of an acceptable therapeutic effect is a suitable dose for administration of the immunotherapeutic agent to the individual.
39. The method according to claim 37, wherein where at least one side effect is detected following administration of any of the multiple different doses administered with at least one antitoxic agent, the highest dose where the side effect is not detected or is least severe or is otherwise deemed acceptable in light of the severity of the disease is a suitable dose for administration of the immunotherapeutic agent to the individual.
40-41. (canceled)
42. A method of treating an individual with a suitable dose of an immunotherapeutic agent, comprising: a. separately contacting multiple different doses of the immunotherapeutic agent with immune-reactive material of the individual, b. measuring at least one immunological reaction caused by the multiple different doses of the immunotherapeutic agent, wherein a dose where the at least one immunological reaction indicates an acceptable therapeutic effect for the immunotherapeutic agent reflects a suitable dose for administration to the individual, and c. administering the immunotherapeutic agent to the individual at the suitable dose.
43. (canceled)
44. The method according to claim 42, wherein the suitable dose of the immunotherapeutic agent is administered with at least one antitoxic agent.
45. The method according to claim 42, wherein the immunotherapeutic agent is a nucleic acid encoding one or more neoepitopes.
46. (canceled)
Description
FIGURES
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EXAMPLE 1
[0237] Within an approved interventional Phase I clinical trial (NCT02410733) fifteen human patients with malignant melanoma were treated on days 1 (V2), 8 (V3), 15 (V4), 22 (V5), 29 (V6), 36 (V7), 50 (V8) and 64 (V9) (patients 1, 2, and 3 only on days 1, 8, 15, 22, 29, 43) with increasing amounts of a nanoparticulate liposome formulated RNA-based immunotherapeutic by intravenous administration. The immunotherapeutic comprised four individual RNA-lipoplex (RNA.sub.(LIP)) products, each encoding for one melanoma-associated antigen, which after intravenous administration results in efficient TLR7-triggered type-I-interferon-driven immune activation and T-cell stimulation. Patients were treated with increasing dose levels, starting with 7.2 g total RNA for the first vaccination cycle, 14.4 g total RNA for the second vaccination cycle, and up to 29, 50, 75 or 100 g total RNA, respectively, for the remaining vaccination cycles.
[0238] Vital signs and adverse events/serious adverse events (side effects) were assessed and reported prior to and after each vaccination cycle. Blood samples were obtained for hematological analyses and systemic cytokine measurements at individual vaccination cycles (at 0 (pre-vaccination), 2, 6, 24, and 48 hours (h) after RNA.sub.(LIP) administration). Lymphocyte counts (
[0239] The first patient (female, born 1982) experienced symptoms typically associated with immune system activation such as headache, fatigue, shivering, and fever, within hours after administration of the immunotherapeutic. These symptoms were dose-dependent and were observed at a dose of 14.4 g (2.sup.nd vaccination cycle). After treatment with 29 g RNA (3.sup.rd vaccination cycle), moderate fever-associated tachycardia and hypotension were additionally observed. The observed symptoms were readily manageable by administration of paracetamol but nevertheless led to a dose reduction to 14.4 g total RNA for the remaining vaccination cycles for this patient. Hematological changes observed included a reversible dose-dependent reduction of systemic lymphocytes and thrombocytes, as well as a minor transient increase of systemic IFN-, IL-6, IFN- and strong secretion of IP-10. These observations are in line with the believed mode of action for RNA.sub.(LIP) immunotherapy, and confirm results observed from extensive preclinical studies.
[0240] The second patient (female, born 1947) tolerated administration of the immunotherapeutic (vaccination) at all three dose levels very well with no observed adverse events related to the immunotherapeutic. Moreover, only minor hematological changes were detected in addition to slight transient increases of IFN- and IL-6 as well as substantial IP-10 secretion in a dose-dependent manner. However, total secreted cytokine amounts were significantly less as compared to the first patient, as depicted in
[0241] The third patient (male, born 1950) tolerated administration of the immunotherapeutic at all three dose levels very well with co-administration of paracetamol given prior and post administration at the discretion of the investigator. For this patient, mild fever after the 3.sup.rd vaccination cycle (29 g) that was resolved within 24 h was the only clinical symptom observed. As with the first patient, a dose-dependent transient decrease of systemic lymphocytes, although to a lesser extent, and a dose-dependent transient increase of IFN-, IFN-, and IP-10 were observed, whereas amounts of systemic IL-6 were significantly higher than in the first and second patients but fully reversed within 24 h as well.
[0242] The fourth patient (female, born 1971) experienced symptoms typically associated with immune system activation such as headache, fatigue, and chills within hours after administration with either 7.2 g, or 14.4 g total RNA, respectively. At both dosages, a slight transient decrease of circulating lymphocytes was detected and moderate transient dose-dependent cytokine induction of IFN-, IP-10, and IFN- comparable to the first patient was observed, whereas IL-6 increase after administration with 14.4 g was slightly higher and rather comparable to cytokine levels for the third patient.
[0243] The fifth patient (male, born 1980) tolerated administration of the immunotherapeutic very well with slightly increased body temperature after the 2.sup.nd vaccination cycle (14.4 g) and mild joint pain after the 3 vaccination cycle (14.4 g). Whereas platelet count was not significantly affected, a moderate but fully transient lymphocytopenia was observed after the 2.sup.nd vaccination cycle with 14.4 g. Almost no increase of systemic cytokines was observed except for a marginal and fully reversible increase of IP-10 secretion after the 2.sup.nd vaccination cycle that was the lowest in comparison to the other five patients.
[0244] The sixth patient (female, born 1974) tolerated administration of the immunotherapeutic very well. With this patient, chills, headache, and pain in the limbs (all mild) were the only clinical observed adverse events reported upon administration of 14.4 g (2.sup.nd vaccination cycle). In addition, fully reversible slight dose-dependent decreases of platelets and lymphocytes were observed. Further, minor dose-dependent increases of systemic IFN- and IFN- after the 2.sup.nd vaccination cycle (14.4 g) were detected, the latter comparable to the first and fourth patients in intensity.
[0245] Likewise, inter-individual sensitivities towards RNA.sub.(LIP) treatment was observed for patients 7, 8, 9, 10, 11, 12, 14, 15, and 16 (age range 27-75 years). This is reflected by the different intensities of hematological changes and the varying transient induction of systemic cytokine levels especially at doses 29 g total RNA and the diverging adverse event profiles related to repetitive RNA.sub.(LIP) dosing. Whereas the majority of patients tolerated repetitive RNA.sub.(LIP) very well also at doses up to 75 and 100 g total RNA, selected patients experienced severe fever after treatment with 100 g RNA.sub.(LIP) (patient 16) or worsening of hypertension after treatment with 7.2 g (patient 11), 14.4 g (patient 10), and 75 or 100 g total RNA.sub.(LIP) (patient 16).
[0246] Based not only on the differences between the observed adverse events experienced at each dose among the fifteen patients, but also on the inter-individual differences in hematological changes and serum cytokine expression levels at various time points after administration of the immunotherapeutic, the data clearly indicate that different individuals vary in their intensity of induced immunological reactions upon the administration of the same immunotherapeutic agent at the same dose.
EXAMPLE 2
[0247] The objective of the following study was to obtain information about the in vitro activation of isolated peripheral blood mononuclear cells (PBMCs) or cells in whole blood by measuring the expression of certain cytokines after the cells have been contacted with RNA molecules complexed with liposomes, which RNA molecules encode certain tumor antigens (RNA.sub.LIP). The encoded antigens were NY-ESO1, a cancer antigen expressed in a wide variety of tumors (RBL001.1), tyrosinase (RBL002.2), MAGE-A3, a melanoma associated antigen (RBL003.1) and TPTE, a tyrosine-protein phosphatase (RBL004.1).
[0248] In the first part of this study, samples of human heparinized whole blood and PBMCs (isolated from heparinized whole blood) obtained from healthy donors were contacted with a mixture of equal portions of liposomally formulated RBL001.1, RBL002.2, RBL003.1 and RBL004.1. To obtain information about the role of dendritic cells (DCs) in relation to cytokine secretion in whole blood, in the second part of the study, heparinized whole blood was enriched with plasmacytoid DCs (pDCs) or monocyte-derived immature DCs (iDCs) and subsequently incubated with the liposomally formulated RNA molecules (RNA-LIP).
[0249] As a primary endpoint in both parts of this study, the activation of these cells was determined 6 h and 24 h after contacting by analysis of cytokine expression in culture supernatant. For first part of the study, the following cytokines have been analyzed: IP-10, IFN-, TNF-, IL-1, IL-2, IL-6, IL-12 and IFN-2. In the second part of the study, only IP-10, IL-6 and IFN-2 were analyzed.
[0250] In cultured PBMCs contacted with the four different RNA-LIP compositions, a dose-dependent induction of all detectable cytokines was observed (
[0251] In the study with dendritic cell-enriched whole blood, a dose-dependent induction of the IP-10, IFN-2, and IL-6 was also observed.
[0252] The results observed from isolated PBMCs showed some differences compared to whole blood suggesting a higher sensitivity of the test system with isolated PBMCs. With isolated PBMCs, increased cytokine levels were observed for cytokines tested, whereas with PBMCs in whole blood, cytokine detection was restricted to IFN-2, IP-10, and IL-6. In the second part of the study, cytokine secretion in whole blood was increased by enrichment with different types of DCs, suggesting that DCs are a major cell type for uptake of the RNA-liposome complexes. Based on this data it could be assumed that pDCs are the major cell type for liposomally formulated RNA uptake in whole blood since enrichment with fresh pDCs lead to increased IFN-2 secretion.
[0253] Moreover, these results clearly show significant individual variations in various immunological reactions in response to contacting an immunotherapeutic agent (RNA-LIP) with immune-reactive material of an individual (PBMCs, whole blood, and whole blood enriched with DCs) at the same dose.
[0254] Materials:
[0255] The materials used in the studies and their individual sources are as follows: Customer 7-plex (Cat.: L5002JFHHC), IFN-2 single Plex (Cat.: 171-B6010M), IP-10 single Plex (Cat.: 171-B5020M), IL-6 single Plex (Cat.: 171-B5006M), Cytokine Standards Group II (Cat.: 171-D60001), Cytokine Standards Group I (Cat.: 171-D50001), Bio-Plex Pro Reagent Kit (Cat.: 171-304070M), all obtained from Bio-Rad Laboratories GmbH; Sodium Pyruvate (Cat.: 11360-039), Non-Essential Amino Acids (Cat.: 11140-035), Penicillin-Streptomycin (Cat.: 15140-122), HEPES (Cat.: 15360-056), RPMI-1640+ Glutamax (Cat.: 61870-010), all obtained from Invitrogen, GIBCO; Human Serum Type AB obtained from LONZA; IL-4 (Cat. No.: 130-093-924), CD14-Beads (Cat.: 130-050-201), CD304-Beads (Cat.: 130-090-532), all obtained from Miltenyi Biotech GmbH; Leucomax, Molgramostim (rHuman GM-CSF) obtained from Novartis; and Ficoll-Paque PLUS (Cat.: 17-1440-03) obtained from GE Healthcare.
[0256] Methods:
[0257] Whole blood was collected from healthy volunteers in sterile syringes. Heparin was used as anticoagulant. Heparinized whole blood was used to generate PBMCs by density centrifugation on Ficoll-Paque. iDCs were isolated by using freshly prepared PBMCs isolated from whole blood and the isolation of CD14+ monocytes was by magnetic bead based separation. pDCs were isolated by using freshly prepared PBMCs isolated from whole blood and the isolation of pDCs was by magnetic bead based separation.
[0258] For the first part of this study, heparinized whole blood was collected from healthy donors (n=4). One part of whole blood was used to generate PBMCs. Subsequently, PBMCs were resuspended in medium and seeded in a 96-well-plate. In detail, per dose group 510.sup.5 PBMCs were seeded in 180 L per well. Then 20 L of the solutions containing the liposome-complexed RNA (RNA-liposome mixture) was added to reach a final volume of 200 L (1:10 dilution of each solution) and a final cell density of 2.510.sup.6 PBMCs/mL. For all doses tested, data was generated from triplicates. The remaining whole blood obtained from the same donors was pipetted directly into the wells of a 96-well-plate. In detail, 1804 whole blood was seeded in triplicates for all dose groups and 20 L of the solutions containing the liposome-complexed RNA was added to reach a final volume of 200 L and a 1:10 dilution of spike solutions. Tables 1 and 2 below summarize the individual test samples:
TABLE-US-00001 TABLE 1 Dose groups for the first part of the study (isolated PBMCs) Final Dose Dose Group Test System Test Item/IVT RNA (g RNA/ml) #1 PBMCs RNA-liposome mixture 3.33 #2 PBMCs RNA-liposome mixture 1.111 #3 PBMCs RNA-liposome mixture 0.370 #4 PBMCs RNA-liposome mixture 0.123 #5 PBMCs RNA-liposome mixture 0.041 #6 PBMCs RNA-liposome mixture 0.014 ctrl PBMCs Diluent/NaCl (0.9%) 0
TABLE-US-00002 TABLE 2 Dose groups for the first part of the study (whole blood) Final Dose Dose Group Test System Test Item/IVT RNA (g RNA/ml) #1 Whole blood RNA-liposome mixture 3.33 #2 Whole blood RNA-liposome mixture 1.111 #3 Whole blood RNA-liposome mixture 0.370 #4 Whole blood RNA-liposome mixture 0.123 #5 Whole blood RNA-liposome mixture 0.041 #6 Whole blood RNA-liposome mixture 0.014 ctrl Whole blood Diluent/NaCl (0.9%) 0
[0259] For the second part of the study, heparinized whole blood was collected twice from the same donors (n=2). The first time, heparinized whole blood was used to generate PBMCs and subsequently to isolate CD14+ monocytes. The isolated monocytes were cultivated for five days to generate iDCs. Cytokines IL-4 and GM-CSF (1000 U/ml of each) were added to the culture medium, to stimulate the generation of iDCs. After three days, the cells were fed with fresh medium including cytokines. Then, heparinized whole blood from the same donors was collected for a second time. One part of this blood was used to generate PBMCs and subsequently to isolate pDCs. Afterwards, the rest of whole blood was seeded into wells as described above: 180 L per well in a 96-well-plate. For the highest dose group, 100 L whole blood was pipetted. Adding autologous pDCs or iDCs was as follows: 10,000 DCs were added to whole blood samples as indicated in Table 3. After addition of DCs, 20 L of solutions containing the liposomally formulated RNAs were added to reach a final volume of 200 L and a 1:10 dilution of the solutions.
TABLE-US-00003 TABLE 3 Dose groups for the second part of the study Spiking with 10,000 Final Dose Dose Group Test System DCs (type) Test Item/IVT RNA (g RNA/ml) #1 Whole blood RNA-liposome mixture 50 #2 Whole blood RNA-liposome mixture 10 #3 Whole blood RNA-liposome mixture 2 #4 Whole blood RNA-liposome mixture 0.4 #5 Whole blood RNA-liposome mixture 0.08 #6 Whole blood RNA-liposome mixture 0.016 ctrl Whole blood Diluent/NaCl (0.9%) 0 #1 Whole blood iDCs RNA-liposome mixture 50 #2 Whole blood iDCs RNA-liposome mixture 10 #3 Whole blood iDCs RNA-liposome mixture 2 #4 Whole blood iDCs RNA-liposome mixture 0.4 #5 Whole blood iDCs RNA-liposome mixture 0.08 #6 Whole blood iDCs RNA-liposome mixture 0.016 ctrl Whole blood iDCs Diluent/NaCl (0.9%) 0 #1 Whole blood pDCs RNA-liposome mixture 50 #2 Whole blood pDCs RNA-liposome mixture 10 #3 Whole blood pDCs RNA-liposome mixture 2 #4 Whole blood pDCs RNA-liposome mixture 0.4 #5 Whole blood pDCs RNA-liposome mixture 0.08 #6 Whole blood pDCs RNA-liposome mixture 0.016 ctrl Whole blood pDCs Diluent/NaCl (0.9%) 0
[0260] Experimental Timeline
[0261] First part of study:
[0262] Day 1: Collection of heparinized whole blood (n=4)
[0263] Preparation of PBMCs of each donor
[0264] Seed PBMCs and whole blood
[0265] Addition of solutions of RNA-LIP and incubate
[0266] Harvest supernatants/plasma at the 6 h time point and freeze at 65 to 85 C.
[0267] Day 2: Harvest supernatants/plasma at the 24 h time point and freeze at 65 to 85 C.
[0268] Perform analysis of the frozen supernatant on any following day
[0269] Second part of study:
[0270] Day 1: Collection of whole blood (n=2)
[0271] Preparation of PBMCs from each donor
[0272] Isolation of CD14+ monocytes from PBMCs
[0273] Cultivation of isolated CD14+ monocytes to generate iDCs
[0274] Day 4: Feeding iDCs
[0275] Day 6: Harvest iDCs
[0276] Collection of whole blood (n=2; same donors)
[0277] Preparation of PBMCs of each donor
[0278] Isolation of pDCs from PBMCs
[0279] Seed whole blood
[0280] Adding iDCs or pDCs, respectively
[0281] Addition of solutions containing RNA-LIP
[0282] Harvest supernatants/plasma at the 6 h time point and freeze at 65 to 85 C.
[0283] Day 7: Harvest supernatants/plasma at the 24 h time point and freeze at 65 to 85 C.
[0284] Perform analysis of the supernatants/plasma on any following day
[0285] Results:
[0286] After incubation of PBMCs with the RNA-LIP mixture for 24 h, a dose-dependent secretion of all cytokines was detected. However, high variations in concentration levels of the cytokines (20-60,000 pg/ml) were observed. The cytokine response was dominated by five out of the eight selected cytokines, namely IP-10, IFN-, TNF-, IL-1, and IL-6 (see Table 4). Additionally, differences in the time point of secretion were also observed: IL-1, IL-6 and TNF- already were detected after 6 h of incubation (at high RNA concentrations) and the levels were not increased remarkably after 24 h, indicating variation in the ability of cells from the different donors to respond to the addition of the RNA-LIP composition.
TABLE-US-00004 TABLE 4 Summary of Results from Isolated PBMCs Cytokine Result IFN-2 Elevated secretion detectable after 24 h in samples obtained from all donors Dose-dependent induction Values on a low level and not elevated remarkably in doses 0.014-0.37 g/mL IP-10 Elevated secretion detectable after 24 h in samples obtained from all donors Dose-dependent induction In 4/4 donors elevated levels detectable at 0.37 g/mL In 3/4 donors elevated levels detectable at 0.12 g/mL IL-6 Elevated secretion detectable after 6 h and 24 h in samples obtained from all donors Dose-dependent induction In 4/4 donors elevated levels detectable at 0.37 g/mL In 3/4 donors elevated levels detectable at 0.12 g/mL after 24 h IFN- Elevated secretion detectable after 24 h in samples obtained from all donors Dose-dependent induction In 4/4 donors elevated levels detectable at 0.37 g/mL TNF- Elevated secretion detectable after 6 h and after 24 h in samples obtained from all donors Dose-dependent induction In 4/4 donors elevated levels detectable at 0.37 g/mL only after 6 h IL-1 Elevated secretion detectable after 6 h and 24 h in all donors Dose-dependent induction In 4/4 donors elevated levels detectable at 0.37 g/mL In 2/4 donors elevated levels detectable at 0.12 g/mL after 24 h IL-2 Elevated secretion detectable after 6 h and after 24 h in samples obtained from all donors Dose-dependent induction Values on a low level and not elevated in doses 0.014-0.37 g/mL IL-12 Elevated secretion detectable after 24 h in samples obtained from all donors Dose-dependent induction In 2/4 donors elevated levels detectable at 0.37 g/mL
[0287] In addition to studies with isolated PBMCs, analogous experiments in whole blood were performed. In these studies, no elevated cytokine secretion to a remarkable concentration was detectable after incubation with the RNA-LIP composition for the following cytokines: IFN-, TNF-, IL-1, IL-2, and IL-12 (see Table 5 and
TABLE-US-00005 TABLE 5 Summary of Results for Whole Blood Cytokine Result IFN-2 Secretion detectable on a very low level in some samples obtained from three of four donors No dose-dependency and no distinct elevation detectable compared to control in any dilution IP-10 Elevated secretion detectable after 24 h in samples obtained from all donors Dose-dependent induction In samples obtained from two out of the four donors, elevated levels were detectable at 0.37 g/mL IL-6 Elevated secretion detectable at a very low level and only at highest dose in samples obtained from two out of the four donors IFN- No elevated secretion detectable TNF- No elevated secretion detectable IL-1 No elevated secretion detectable IL-2 No elevated secretion detectable IL-12 No elevated secretion detectable
[0288] To test the role of DCs for activation and cytokine secretion in whole blood, the second part of the study was performed. Here, heparinized whole blood, enriched with autologous iDCs or pDCs, was incubated with an RNA-LIP composition in a dose range of 0.016 to 50 g/mL. The results presented in
[0289] Discussion and Conclusion
[0290] Regarding the first part of the study, in both test systems, isolated human PBMCs and whole blood, several cytokines were secreted after incubation with the RNA-LIP composition. However, differences between the test systems suggest that PBMCs have a higher sensitivity as test system. On the one hand, increased cytokine levels in isolated PBMCs for all eight tested cytokines could be detected. Using whole blood as a test system, cytokine detection was restricted to IFN-2, IP-10, and IL-6. The differing results could be caused by different cultivation conditions since isolated PBMCs were cultivated with culture medium supplemented with serum (FCS) and antibiotics whereas cultivation in whole blood means that PBMCs were cultured with human plasma and red blood cells.
[0291] In the second part of the study it was shown that IFN-2 secretion in whole blood could be increased with addition of pDCs. It is known that pDCs secrete IFN- upon TLR-7 activation (Kwissa et al., 2012, Distinct TLR adjuvants differentially stimulate systemic and local innate immune responses in nonhuman primates, Blood 119:2044-2055; Schiller et al., 2006, Immune response modifiersmode of action, Exp. Dermatol. 15:331-341, Hornung et al., 2005, Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7, Nat. Med. 11(3):263-270). Since TLR-7 is activated by RNA within endosomes, this indicates that pDCs are a major cell type for uptake of RNA.
[0292] The results from these experiments also show that the same immune-reactive material isolated from different individuals contacted with the same immunotherapeutic agent at the same concentration (same dose) results in a significantly different immunological reaction, thus further supporting the conclusion that the immunotherapeutic agents at the same dose have different effects in different individuals such and that there is no single dose that is therapeutically effective and/or tolerated in all individuals.
EXAMPLE 3
[0293] The objective of the following study was to obtain information about the activation in vitro of isolated peripheral blood mononuclear cells (PBMCs) or cells in fresh whole blood by measuring the secretion of certain cytokines and the induction of a general activation marker (CD69) after the cells were incubated with various concentrations of small TLR-7 agonist compounds, namely N-(4-(4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl)butyl)-N-(tetrahydro-2H-pyran-4-yl)acetamide (designated herein as SM1) and N-{4-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]butyl}-N-(1,1-dioxothietan-3-yl)acetamide (designated herein as SM2). As discussed in more detail below, human heparinized whole blood and PBMCs (isolated from buffy coats) obtained from healthy donors were incubated for 24 hours with equimolar amounts of agonist compounds SM1 or SM2.
[0294] PBMCs used for the experiment were isolated from buffy coats by density centrifugation on Ficoll-Paque. Subsequently, PBMCs were resuspended in cell culture medium and seeded in a 96-well-plate. In detail, per dose group 510.sup.5 PBMCs were seeded in 1900 per well. Then 10 L of agonists at specified concentrations was added to reach a final volume of 200 L (1:20-dilution of each solution) and a final cell density of 2.510.sup.6 PBMCs/mL. Plates were incubated at 37 C. and 5% CO.sub.2 for 24 hours. Subsequently, supernatants were harvested and immediately analyzed or frozen and kept at 80 C. until analysis. For all doses of the agonist tested, data was generated from 10 individuals (for both study parts), each in biological duplicates.
[0295] Whole blood was collected from healthy volunteers in sterile syringes. Heparin was used as anticoagulant. The fresh heparinized whole blood was pipetted directly into a 96-well-plate. In detail, 190 L whole blood from different individuals (n=10 donors for CBA; n=8 for flow cytometry) was seeded in duplicates for all dose groups and 10 L of test-item-solutions was added to reach a final volume of 200 L and a 1:20 dilution of spike solutions. Plates were incubated at 37 C. and 5% CO.sub.2 for 24 hours. Subsequently, supernatants were harvested and immediately analyzed or frozen and kept at 80 C. until analysis.
[0296] For measuring cellular activation by CD69 expression, cell pellets were harvested and flow cytometry staining and measurements were performed immediately.
[0297] Each agonist was prepared in a serial dilution with the diluent DMSO (dimethyl sulfoxide): 5-fold in 8 steps. Agonist compound concentrations incubated with the PBMCs or whole blood were 10 M, 2 M, 0.4 M, 0.08 M, 0.016 M, 0.0032 M, 0.0006 M, and 0.0001 M.
[0298] As primary endpoint of the first part of the study, activation of the cells was determined by analysis of induction of cytokine secretion in cell culture supernatant (PBMCs) and plasma (whole blood) via cytometric bead assay (CBA). The following cytokines/chemokines were analyzed (IFN-, IP-10, IFN-, TNF-, IL-6, IL-8, IL-10, IL-12p70 and IL-2). For the second part of the study, cellular activation was determined by analysis of the expression of the general activation marker CD69 in several types of immune cells via flow cytometry. The following types of immune cells were investigated: plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), monocytes, B cells and NK cells.
[0299] In particular, for determination of cytokine concentrations a cytometric bead assay (Multiplex-Kit, (ProcartaPlex; eBioscience), was used, which included all ten cytokines/chemokines (IFN-, IP-10, IFN-, TNF-, IL-6, IL-8, IL-10, IL-12p70 and IL-2). Analysis was performed with a Luminex system.
[0300] For flow cytometry cell pellets were stained with an antibody mixture, combining the surface markers, CD3, CD16, CD19, CD14, BDCA2 and BDCA3, and the activation marker CD69. With this flow panel it was possible to analyze the activation of B cells, NK cells, monocytes, plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs). Measurements were performed with the BD FACSCanto II.
[0301] In cultured PBMCs incubated with the TLR7-agonists, a consistent and dose-dependent induction of 8 out of 10 measured cytokines was observed (
[0302] After incubation of whole blood with each agonist compound (
[0303] Cellular activation after incubation of PBMCs and whole blood with TLR7-agonists was also analyzed via flow cytometry (expression of CD69). Comparably to the observations made with cytokine secretion, there is a consistent dose-dependent activation of all analyzed immune cell populations (pDCs, mDCs, monocytes, B-cells and NK cells) for both agonist compounds, for PBMCs (
[0304] The above-described results clearly show that different individuals vary significantly in their intensity of induced cellular immunological reactions in response to an immunotherapeutic agent, in the present case a TLR7-agonist.
EXAMPLE 4
[0305] The objective of the following study was to observe the effect of the in vivo administration of different amounts of small molecule agonists of TLR-7, N-(4-(4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl)butyl)-N-(tetrahydro-2H-pyran-4-yl)acetamide (SM1) and N-(4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl)-N-(1,1-dioxidothietan-3-yl)acetamide (SM3), on expression of various cytokines in the blood in a cynomolgus monkey model. The various cytokines whose expression was determined include interferon alpha, interleukin 1 receptor antagonist, interleukins 4, 6, 8, 10, 12, 15, 18, monocyte chemoattractant protein 1, granulocyte-colony stimulating factor, macrophage inflammatory protein 1 beta, tumor necrosis factor alpha, and vascular endothelial growth factor.
[0306] A defined single dose of one of the agonist compounds was administered intravenously to cynomolgus monkeys in a thirty minute infusion. At several time points after initiation of the administration, blood samples (0.5 mL for approximately 0.25 mL plasma) were collected from the monkeys via the vena cephalica antebrachii or vena saphena blood vessel into K3EDTA tubes. The blood samples were stored on crushed ice prior to centrifugation. Plasma was obtained by centrifugation at 4 C. and approximately 1800 g for 10 minutes and was aliquoted into labeled micro tubes and stored frozen at 70 C. or below. Prior to cytokine determination, the frozen plasma samples were thawed, diluted.
[0307] Cytokines levels were determined using an interferon alpha Elisa kit (e.g., VeriKine Cynomolgus/Rhesus IFN ELISA Kit) and a Luminex non-human primate cytokine/chemokine kit (e.g., Milliplex Non-Human Primate Cytokine/Chemokine Magnetic Premixed 23 Plex Panel), in accordance to the manufacturers' instructions. First, a low dose of the agonist compound (30 [only animals receiving SM1], 100 [only animals receiving SM1] or 300 g/kg) was administered to the monkeys. Later, at 14 days, a second, higher dose of the same agonist compound (1, 3 or 10 mg/kg) was administered to the same monkeys. Results are depicted in
[0308]
[0309] Agonist compound SM1 was administered by an intravenous infusion to cynomolgus monkeys denoted as individuals P0101 (male), P0102 (male), P0501 (female), P0502 (female), P0201 (male), P0202 (male), P0601 (female), P0602 (female), P0301 (male), P0302 (female), P0701 (female), P0702 (female) at doses of 30 g/kg, 100 mg/kg, 300 g/kg and 1 mg/kg, 3 mg/kg, and 10 mg/kg, as explained below. Cytokine secretion into blood was measured at various time points until 168 hours after administration. The plasma concentration for various cytokines is shown in the figures at up to 12 or 24 hours after starting the infusion.
[0310] Each monkey was given the same agonist twice. The first administration was at one of the low doses, 30 g/kg, 100 g/kg or 300 pig/kg and the second administration was at one of the higher doses, 1 mg/kg, 3 mg/kg or 10 mg/kg. Monkeys receiving 30 g/kg as the first dose were given a second dose of 1 mg/kg. Monkeys receiving 100 g/kg as the first dose were given a second dose of 3 mg/kg. Monkeys receiving 300 g/kg as the first dose were given a second dose of 10 mg/kg. [0311]
[0328]
[0342]
[0348]
[0349] Agonist compound SM3 was administered by an intravenous infusion to male cynomolgus monkeys denoted as individuals 16962, 17477, 17479, 17607, 16988, 30018, 16669, 17613, 14030, 16216 in doses of 300 g/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg. Cytokine secretion into blood was measured at various time points until 168 hours after administration. The plasma concentration for various cytokines is shown in the figures at up to 12 or 24 hours after starting the infusion. [0350]
EXAMPLE 5
[0363] The objective of the following study was to observe the effect of two small molecule agonists of TLR8, 2-ethyl-1-(4-((2-methyltetrahydrofuran-3-yl)amino)butyl)-1H-imidazo[4,5-c]quinolin-4-amine (SM4) and 1-(4-(cyclohexylamino)butyl)-2-ethyl-1H-imidazo[4,5-c]quinolin-4-amine (SM5) on cytokine secretion by human PBMCs in vitro. The various cytokines whose expression was determined include tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, 8, 10 and 12p70, interferon gamma, interleukin 10, and interferon gamma inducible protein 10.
[0364] PBMCs were isolated from fresh blood samples withdrawn from four human voluntary blood donors. The PBMCs were isolated according to standard protocols, resuspended in cell culture medium containing 10% fetal calf serum at a cell count of 210.sup.6/mL, seeded into 96-well plates at 100 l per well and subsequently incubated for 6 hours at 37 C. Appropriate stock solutions of each agonist compound were produced by dissolving the agonist compound in DMSO and subsequently diluting in one or several steps with DMSO to a concentration of 1000-fold of the final test concentration. Appropriate pre-dilutions of the agonist were prepared with medium; in a first step the agonist was diluted 1:100, and in a second step 25 L of pre-dilution and 125 L of medium were added to 1004, of cells in the wells. The cells were incubated for 24 hours at 37 C. and supernatants were then harvested and analyzed with a Luminex bead assay or an ELISA assay specific for the particular human cytokines according to the manufacturers' instructions.
[0365] Results, which are depicted in
[0366]
[0367] Agonist compound SM4 was added in an in vitro assay to freshly prepared human PBMCs from four blood donors denoted as individuals 130325, 100621, 110126, 110125 at various concentrations, i.e., 0.1 M, 0.3 M, 1 M, 3 M, 10 M, and 30 M. 24 hours after addition of the agonist compound, cytokine secretion into the supernatant was measured as described above. The concentrations of the various cytokines in the supernatant after 24 hour incubation with the different amounts of the agonist are shown. [0368]
[0374]
[0375] Agonist compound SM5 was added in an in vitro assay to freshly prepared human PBMCs from four blood donors denoted as individuals 131105, 130618, 130325, 131120 at various concentrations of 0.1 M, 0.3 M, 1 M, 3 M, 10 M, and 30 M. 24 hours after addition of the agonist compound, cytokine secretion into the supernatant was measured as described above. The concentrations of the various cytokines in the supernatant after 24 hour incubation with the different amounts of the agonist are shown. [0376]