COMPOSITIONS AND METHODS FOR TREATMENT OF INFLUENZA A INFECTION
20220306728 · 2022-09-29
Inventors
- Phillip S. PANG (San Francisco, CA, US)
- Lynn E. CONNOLLY (San Francisco, CA, US)
- Erik Mogalian (San Francisco, CA)
Cpc classification
A61K47/22
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
C07K2317/94
CHEMISTRY; METALLURGY
International classification
A61K47/22
HUMAN NECESSITIES
A61K47/26
HUMAN NECESSITIES
Abstract
The present disclosure provides antibodies, antibody compositions, and methods for use in prophylaxis and treatment of influenza A infection. In certain embodiments, a single administration of a presently disclosed antibody or antibody composition is useful to protect against and/or treat an influenza A infection for a full flu season.
Claims
1. A method of treating or preventing an Influenza A infection in a subject, the method comprising administering to the subject a single dose of a pharmaceutical composition comprising an antibody, wherein the antibody comprises a light chain amino acid sequence according to SEQ ID NO:10 and a heavy chain amino acid sequence according to SEQ ID NO:9.
2. The method of claim 1, wherein the pharmaceutical composition comprises the antibody at a concentration in a range from 100 mg/mL to 200 mg/mL, such as 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, or 200 mg/mL, preferably 150 mg/mL.
3. The method of claim 1 or 2, wherein the single dose comprises 3, 4, 5, 6, or 7, preferably 5, mg of the antibody per kg of the subject's body weight.
4. The method of any one of claims 1-3, wherein the single dose comprises up to 60 mg, up to 300 mg, up to 1200 mg, up to 1800 mg, or up to 3000 mg of the antibody.
5. The method of any one of claims 1-4, wherein the single dose comprises up to 60 mg, up to 70 mg, up to 80 mg, up to 90 mg, up to 100 mg, up to 200 mg, up to 300 mg, up to 400 mg, up to 500 mg, up to 600 mg, up to 700 mg, up to 800 mg, up to 900 mg, up to 1000 mg, up to 1100 mg, up to 1200 mg, up to 1300 mg, up to 1400 mg, up to 1500 mg, up to 1600 mg, up to 1700 mg, up to 1800 mg, up to 2,000 mg, up to 2,500 mg, or up to 3000 mg, of the antibody.
6. The method of any one of claims 1-5, wherein the antibody is administered at a dose of 60 mg, 300 mg, 1200 mg, or 1800 mg.
7. The method of any one of claims 1-5, wherein the antibody is administered at a dose of 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1100 mg, or 1200 mg.
8. The method of any one of claims 1-7, wherein: (i) the single dose comprises 300 mg of the antibody, wherein the pharmaceutical composition comprises the antibody at 150 mg/mL, and the dose is administered by a single injection comprising 2 mL of the pharmaceutical composition; (ii) the single dose comprises 1200 mg of the antibody, wherein the pharmaceutical composition comprises the antibody at 150 mg/mL, and the dose is administered by two injections each comprising 4 mL of the pharmaceutical composition; (iii) the single dose comprises 1800 mg of the antibody, wherein the pharmaceutical composition comprises the antibody at 150 mg/mL, and the dose is administered by three injections each comprising 4 mL of the pharmaceutical composition; or (iv) the single dose comprises 60 mg of the antibody, wherein the pharmaceutical composition comprises antibody at 150 mg/mL, and the dose is administered by one injection comprising 0.4 mL of the pharmaceutical composition.
9. The method of any one of claims 1-8, wherein the subject is human.
10. The method of any one of claims 1-9, wherein the method comprises intramuscular (IM) injection.
11. The method of any one of claims 1-10, wherein the pharmaceutical composition further comprises water (e.g., USP water for injection, or US sterile water for injection).
12. The method of any one of claims 1-11, wherein the pharmaceutical composition further comprises histidine, optionally at a concentration in a range from 10 mM to 40 mM, preferably 20 mM, in the pharmaceutical composition.
13. The method of any one of claims 1-12, wherein the pharmaceutical composition further comprises a sugar, such as a disaccharide, such as sucrose, optionally in a range from 3.0% to 9.0% (w/v), preferably in a range from 3.6% to 8.6%, more preferably in a range from 4% to 6%.
14. The method of any one of claims 1-13, wherein the pharmaceutical composition further comprises a surfactant or a triblock copolymer, optionally a polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), optionally in a range from 0.01% to 0.05% (w/v), preferably 0.02% (w/v).
15. The method of any one of claims 1-14, wherein the pharmaceutical composition has a pH in a range from 5.5 to 6.5, or in a range from 5.8 to 6.2, or a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, preferably of 6.0.
16. The method of any one of claims 1-15, wherein the single dose comprises from 0.8 mL to 4 mL per injection.
17. The method of claim 16, wherein the single dose comprises or consists of 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.1 mL, 3.2 mL, 3.3 mL, 3.4 mL, 3.5 mL, 3.6 mL, 3.7 mL, 3.8 mL, 3.9 mL, or 4.0 mL of the composition per injection.
18. The method of any one of claims 1-17, wherein at about 4 weeks, at about 12 weeks, and/or about 20 weeks following administering the pharmaceutical composition to the subject, the subject: (i) has a reduced number and/or severity of a respiratory symptom selected from: cough, sore throat;, rhinorrhea; congestion; or any combination thereof, and/or (ii) has a reduced number and/or severity of a systemic symptom selected from: fever [oral temperature >38° C. (100.4° F.)]; chills; myalgia; headache; malaise; fatigue; or any combination thereof, as compared to a reference subject over a same time period who received a placebo or did not receive a therapy or vaccine for influenza A.
19. The method of any one of claims 1-18, wherein the subject is from 18 years to 65 years of age and has a body mass index in a range from 18 kg/m.sup.2 to 32 kg/m.sup.2 or in a range from 18 kg/m.sup.2 to 35 kg/m.sup.2.
20. The method of any one of claims 1-19, comprising administering the single dose comprising the pharmaceutical composition once to the subject during a six-month period.
21. The method of any one of claims 1-20, comprising administering a single dose comprising the pharmaceutical composition once to the subject during a twelve-month period.
22. The method of any one of claims 1-20, comprising administering a single dose comprising the pharmaceutical composition twice to the subject during a six-month period, such as once every three months.
23. The method of any one of claims 1-22, comprising administering the single dose comprising the pharmaceutical composition within 1-2 months (i.e., within 30 days to within 60 days) prior to the beginning of an influenza season, or within the first 1-2 months of the influenza season.
24. The method of any one of claims 1-23, wherein the antibody, or the pharmaceutical composition comprising the antibody, has an in vitro influenza inhibition of infection IC.sub.90 of about 2.17 μg/mL.
25. The method of any one of claims 1-24, wherein: (i) the administered pharmaceutical composition comprises 60 mg of the antibody, and the antibody is present in serum from the subject at a concentration from about 1 μg/mL to about 7 μg/mL for up to 120 days following administration; (ii) the administered pharmaceutical composition comprises 300 mg of the antibody, and the antibody is present in serum from the subject at a concentration from about 8 μg/mL to about 20 μg/mL for up to 120 days following administration; (iii) the administered pharmaceutical composition comprises 1200 mg of the antibody, and the antibody is present in serum from the subject at a concentration from about 50 to μg/mL to about 100 μg/mL for up to 120 days following administration; (iv) the administered pharmaceutical composition comprises 1800 mg of the antibody, and the antibody is present in serum from the subject at a concentration from about 70 to μg/mL to about 110 μg/mL for up to 120 days following administration; and/or (v) the antibody has an in vivo t.sub.1/2 in the subject of from 49 to 68 days.
26. The method of any one of claims 1-25, wherein the antibody of the pharmaceutical composition has an in vivo t.sub.1/2 in a human subject of from 49 to 68 days, such as 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, or 68 days.
27. The method of any one of claims 1-26, wherein the subject does not experience an adverse event (AE), according to the Common Terminology Criteria for Adverse Events (CTCAE), optionally for up to 140 days after the single dose of the pharmaceutical composition is administered.
28. The method of any one of claims 1-27, wherein the subject does not experience a moderate adverse event (AE), according to the Common Terminology Criteria for Adverse Events (CTCAE), optionally for up to 140 days, after the single dose of the pharmaceutical composition is administered.
29. The method of any one of claims 1-28, wherein the subject does not experience a serious adverse event (AE), according to the Common Terminology Criteria for Adverse Events (CTCAE), optionally for up to 140 days, after the single dose of the pharmaceutical composition is administered.
30. The method of any one of claims 1-29, wherein: (i) the single dose comprises 300 mg of the antibody, wherein the pharmaceutical composition comprises the antibody at 150 mg/mL, and the single dose comprises a single injection comprising 2 mL of the pharmaceutical composition; (ii) the single dose comprises 1200 mg of the antibody, wherein the pharmaceutical composition comprises the antibody at 150 mg/mL, and the single dose comprises two injections each comprising 4 mL of the pharmaceutical composition; (iii) the single dose comprises 1800 mg of the antibody, wherein the pharmaceutical composition comprises the antibody at 150 mg/mL, and the single dose comprises three injections each comprising 4 mL of the pharmaceutical composition; or (iv) the single dose comprises 60 mg of the antibody, wherein the pharmaceutical composition comprises antibody at 150 mg/mL, and the single dose comprises 0.4 mL of the pharmaceutical composition.
31. A pharmaceutical composition comprising an antibody that comprises a light chain amino acid sequence according to SEQ ID NO:10 and a heavy chain amino acid sequence according to SEQ ID NO:9, wherein the antibody is present in the composition at a concentration in a range from 100 mg/mL to 200 mg/mL, such as 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, or 200 mg/mL, preferably 150 mg/mL.
32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition further comprises water (e.g., USP water for injection, or US sterile water for injection).
33. The pharmaceutical composition of claim 31 or 32, further the pharmaceutical composition further comprises histidine, optionally at a concentration in a range from 10 mM to 40 mM, preferably 20 mM, in the composition.
34. The pharmaceutical composition of any one of claims 31-33, the pharmaceutical composition further comprises a sugar, such as a disaccharide, such as sucrose, optionally in a range from 3.0% to 9.0% (w/v), preferably from 3.6% to 8.6%, more preferably in a range from 4% to 6%.
35. The pharmaceutical composition of any one of claims 31-34, the pharmaceutical composition further comprises a surfactant or a triblock copolymer, optionally a polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), optionally in a range from 0.01% to 0.05% (w/v), preferably 0.02%.
36. The pharmaceutical composition of any one of claims 31-35, wherein the composition has a pH in a range from 5.5 to 6.5, or in a range from 5.8 to 6.2, or a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, preferably of 6.0.
37. A, preferably glass, vial comprising the pharmaceutical composition of any one of claims 31-36.
38. A syringe comprising the pharmaceutical composition of any one of claims 31-36.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0261] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present disclosure in more detail. However, they are not intended to limit the subject matter of the disclosure in any way.
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EXAMPLES
[0301] In the following, particular examples illustrating embodiments and aspects of the disclosure are presented. However, the present disclosure shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present disclosure. The present disclosure, however, is not limited in scope by the exemplified embodiments. Indeed, various modifications of the disclosure in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the appended claims.
Example 1
Safety and Tolerability of an Antibody According to the Present Disclosure in Cynomolgus Macaques
[0302] An antibody according to the present disclosure, which comprises (i) the CDR sequences as set forth in SEQ ID NOs 1-6 and (ii) the two mutations M428L and N434S in the heavy chain constant regions, was designed and produced. More specifically, the antibody comprises (i) the heavy chain variable region (VH) sequence as set forth in SEQ ID NO: 7 and the light chain variable region (VL) sequence as set forth in SEQ ID NO: 8; and (ii) the two mutations M428L and N434S in the heavy chain constant regions. Even more specifically, the antibody comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 10. This antibody is referred to herein as “FluAB_MLNS”.
[0303] For comparison, antibody “FluAB_wt” was used, which differs from antibody “FluAB_MLNS” only in that it does not contain the mutations M428L and N434S in the heavy chain constant regions. Accordingly, comparative antibody “FluAB_wt” comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 10.
[0304] A single intravenous infusion of 5 mg/kg of either FluAB_MLNS or FluAB_wt in a 2.5 ml/kg volume was given in a 60-minutes intravenous infusion to three female cynomolgus macaques (Macaca fascicularis) per test group. Blood or urine for clinical chemistry and hematological analyses were collected pre-dose and on days 7 and 21 post-dose.
[0305] Following dosing of either FluAB_MLNS or FluAB_wt at 5 mg/kg in a 60-minutes intravenous infusion, the female cynomolgus macaques were closely monitored for health and weight and regularly sampled for blood and urine. No adverse events — other than bruising 24 h and erythroderma 3 days post-dose at the inoculation site in some of the animals — were observed following intravenous inoculation of the antibodies. All animals were generally healthy, showed normal food consumption, and had overall positive weight gain throughout the study. Clinical chemistry, hematology, and urinalysis parameters were normal at 7- or 21-days post dosing, compared to pre-dosing samples.
[0306] In summary, a single intravenous infusion of either FluAB_MLNS or FluAB_wt into cynomolgus macaques did not induce adverse events and was generally well tolerated.
Example 2
Determination of Plasma Concentration and Pharmacokinetics
[0307] These experiments aimed to determine the concentration, establish half-life, and compare the pharmacokinetics of the antibody according to the present disclosure FluAB_MLNS in comparison to comparative antibody FluAB_wt in the plasma following a single intravenous injection.
[0308] Before dosing, the animals were tested to be negative for influenza-specific antibodies using dot immunobinding assay. Seropositive animals were excluded from the study as pre-existing immunity may interfere with this test. In addition, animals developing anti-drug antibody (ADA) response were excluded.
[0309] A single intravenous infusion of 5 mg/kg of either FluAB_MLNS or FluAB_wt in a 2.5 ml/kg volume was given in a 60-minutes intravenous infusion to three female macaques per test group. Blood was collected in tubes containing K.sub.2EDTA pre-dose and processed to plasma for pharmacokinetic testing after approximately 1, 6, 24, 96, 168, 504, 840, and 1344 hours (h) post-dose.
[0310] Plasma concentration of the antibodies was determined in vitro using an ELISA assay. Briefly, IAV-HA antigen (Influenza A virus H1N1 A/California/07/2009 Hemagglutinin Protein Antigen (with His Tag); Sino Biologicals) was diluted to 2 μg/ml in PBS and 25 μl were added to the wells of a 96-well flat bottom ½-area ELISA plate for coating over night at 4° C. After coating, the plates were washed twice with 0.5× PBS supplemented with 0.05% Tween20 (wash solution) using an automated ELISA washer. Then, plates were blocked with 100 μl/well of PBS supplemented with 1% BSA (blocking solution) for 1 h at room temperature (RT) and then washed twice. Plasma samples were centrifuged at 10,000 g for 10 min at 4° C. and then diluted (1:10 and then 1:30) for a final 1:300 dilution in blocking solution in 96-well cell culture plates.
[0311] The minimum dilution (1:300) of the macaque plasma used for quantification was tested and set to ensure that the matrix effect was negligible. Samples were then diluted 1:2 stepwise in triplicates for a total of 12 dilutions. Standards for each antibody to be tested were prepared similarly via diluting the antibodies 1:300 to 1 μg/ml in a pool of pre-inoculation plasma from all test animals, mimicking the matrix of the test samples. Standards were then diluted 1:3 stepwise in blocking solution in triplicates for a total of 12 dilutions. Twenty-five μl of the prepared samples or standards were added to hemagglutinin (HA)-coated wells and incubated for 1 h at RT. After four washes, 25 μl of goat anti human-IgG HRP conjugate (AffiniPure F(ab′).sub.2 Fragment, Fcγ Fragment-Specific; Jackson ImmunoResearch) diluted in blocking solution 1:5,000 (final concentration 0.16 μg/ml) were added per well for detection and incubated at RT for 1 h. After four washes, plates were developed by adding 40 μl per well of SureBlue TMB Substrate (Bioconcept). After ˜7-20 min incubation at RT, when the color reaction reached a plateau (max OD ˜3.8), 40 μl of 1% HCl were added per well to stop the reaction and absorbance was measured at 450 nm using a spectrophotometer.
[0312] To determine the concentration of the antibodies in cynomolgus plasma, OD values from ELISA data were plotted vs. concentration in the Gen5 software (BioTek). A non-linear curve fit was applied using a variable slope model, four parameters and the equation: Y=(A−D)/(1+(X/C){circumflex over ( )}B)+D). The OD values of the sample dilutions that fell within the predictable assay range of the standard curve—as determined in setup experiment by quality control samples in the upper, medium or lower range of the curve—were interpolated to quantify the samples. Plasma concentration of the antibodies were then determined considering the final dilution of the sample. If more than one value of the sample dilutions fell within the linear range of the standard curve, an average of these values was used. Pharmacokinetics (PK) data were analyzed by using WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (8.1.0.3530 Core Version, Phoenix software, Certara) with the following settings: Model: Plasma Data, Constant Infusion Administration; Number of non-missing observations: 8; Steady state interval Tau: 1.00; Dose time: 0.00; Dose amount: 5.00 mg/kg; Length of Infusion: 0.04 days; Calculation method: Linear Trapezoidal with Linear Interpolation; Weighting for lambda_z calculations: Uniform weighting; Lambda_z method: Find best fit for lambda_z, Log regression. Graphing and statistical analyses (linear regression or outlier analysis) were performed using Prism 7.0 software (GraphPad, La Jolla, Calif., USA). Outlier analysis was performed using the ROUT method (Q=1%), with the potential to find any number of outliers in either direction.
[0313] Results are shown in
[0314] In summary, FluAB_MLNS had an extended in-vivo half-live compared to comparative antibody FluAB_wt at least up to day 56 post-inoculation.
Example 3
Long-Term Stability In Vivo
[0315] To test in-vivo stability and functionality of the antigen binding of FluAB_MLNS over time, the pharmacokinetics measurement (as described in Example 2) of the group receiving FluAB_MLNS was extended to days 86 and 113 post-inoculation. On days 1, 21, 56, 86, 113 post-inoculation, functional FluAB_MLNS was quantified using the hemagglutinin (HA) binding ELISA as described in Example 2.
[0316] Further, total human antibodies in macaque plasma was quantified using a specific anti-CH2 ELISA, using a capture mAb that specifically binds the CH2 region of human but not of monkey Abs. To measure total human IgG and thus quantify total inoculated human antibodies in cynomolgus plasma, an ELISA capturing with mouse anti-CH2 domain-specific to human IgG (clone R10Z8E9; Thermo Scientific) was used. It was verified that this mAb does not cross-react with monkey IgG. For coating of 96-well flat bottom ½-area ELISA plates, mouse anti-human IgG CH2 was added in PBS at 0.5 μg/ml and incubated over night at 4° C. Then, plates were washed and 100 μl/well blocking solution with 5% BSA was added for 1 h at RT. Standards of FluAB_MLNS were prepared via diluting FluAB_MLNS to 1 ng/ml in blocking solution. Standards were then diluted 1:1.5 stepwise in blocking solution in duplicates for a total of 12 dilutions. Cynomolgus plasma samples were centrifuged at10,000 g for 10 min at 4° C. and step-wise diluted to a final 1:1,000, 1:5,000 or 1:15,000 in blocking solution. After washing the plate, 25 μl of samples or standard were added to the ELISA plate and incubated for 1 h at RT. After three washes, 25 μl of goat anti human-IgG HRP (AffiniPure F(ab′).sub.2 Fragment, Fcγ Fragment-Specific; Jackson ImmunoResearch) at 0.04 μg/ml were added in blocking solution with 1% BSA for detection and incubated at RT for 45 min. After three washes, plates were developed by adding 40 μl per well of SureBlue TMB Substrate (Bioconcept). After 20 min incubation at RT, 40 μl of 1% HCl were added to stop the reaction, and absorbance was measured at 450 nm.
[0317] Results are shown in
[0318] In summary, FluAB_MLNS demonstrated functional antigen binding and thus good long-term stability in vivo up to day 113 post-inoculation during study extension.
Example 4
Antibody Concentration in Nasal Swabs and Biodistribution
[0319] To determine biodistribution of FluAB_MLNS and of the comparative antibody FluAB_wt between the nasal mucus relative to plasma, the concentration of the antibody was determined in nasal swabs. To this end, Nasal swabs of the macaques described in Example 2 were collected 24, 504, and 1344 hours after administration of FluAB_MLNS or of the comparative antibody FluAB_wt. Concentrations of antibodies FluAB_MLNS and FluAB_wt in nasal swabs were determined essentially as described in Example 2 for determination in plasma with the following minor adaptations: (a) ELISA plates were blocked 2 h at RT; (b) Nasal swab samples were diluted starting at 1:2 with 1% BSA in PBS and then serially diluted step-wise 1:2 for a total of 8 dilution points; (c) nasal swab medium (RT MINI Viral Transport Medium; Copan) was used as assay matrix control.
[0320] To eliminate differences during the swabbing procedure or in the amount of nasal secretions present in each animal and at different time points (days 1, 21, and 56), results from nasal swabs were normalized to urea content. Urea freely diffuses between blood, being present in similar amounts across these plasma or swab samples (Lim et al., 2017, Antimicrob Agents Chemother 61(8):e00279-17). To this end, Urea Nitrogen (BUN) was measured quantitatively using the “Urea Nitrogen (BUN) Colorimetric Detection Kit” (Invitrogen), following the manufacture's procedure. In brief, samples were diluted 1:3 in PBS and mixed with the kit reagents A and B and incubated at room temperature for 30 minutes. The colored product of the redox reaction was read at 450 nm using a 96-well microplate reader. Quantification was performed via comparing samples to BUN standards, which were provided with the kit and treated equivalently.
[0321] Results are shown in
[0322] In summary, nasal swab samples did not reveal any significant differences in biodistribution between the nasal mucus and plasma amongst the mAb variants.
Example 5
Prophylactic Activity of Antibody FluAB_MLNS in PR8-Infected Tg32 Mice
[0323] Next, the prophylactic activity of FluAB_MLNS compared to antibody FluAB_wt was determined in a H1N1 murine model of lethal influenza A infection.
[0324] To evaluate the prophylactic efficacy, 9- to 14-week-old FcRn−/−hFcRn line 32 Tg mice (C57B6 background) were intravenously (i.v.)-injected (via the tail vein) with 5 ml/kg of a solution containing FluAB_MLNS or the comparator antibody FluAB_wt at doses ranging from 0.3 to 1 mg/kg. Twenty-four hours after the i.v. injection, mice were bled from the tail vein to determine the serum antibody levels before infection. Bleedings were also repeated on day 6 and 13 post infection (p.i.). Both antibody-injected and untreated mice were anesthetized (isoflurane, 4% in O.sub.2, 0.3 L/min) and challenged intranasally (i.n.) by slow instillation in both nostrils of 50 μl (25 μl/each) of PBS containing 5 mouse lethal dose fifty percent (5 MLD.sub.50, equivalent to 1200 TCID.sub.50/mouse) of influenza virus A (H1N1, A/Puerto Rico/8/34, as described in Cottey, R., Rowe, C. A., and Bender, B. S. (2001). Influenza virus. Curr Protoc Immunol Chapter 19, Unit19.11-19.11.32). Each mouse was held upright with its head tilted slightly back for about 1 minute to reduce the likelihood of inoculum dripping from the nares. After the procedure and upon righting reflex occurrence, animals were returned to the cage. The mice were monitored daily for weight loss and disease symptoms until day 14 p.i. and euthanized if they lost more than 20% of their initial body weight (whereby 0% is set on the day of infection) or reached morbidity score of 4. Table 1 details the applied morbidity score:
TABLE-US-00001 TABLE 1 Morbidity Score of PR8-infected mice Morbidity Score Clinical signs 1 Healthy 2 Consistently ruffled fur on the neck 3 Piloerection, possible deeper breathing, less alert 4 Labored breathing, tremors and lethargy 5 Abnormal gait, reduced mobility, emaciation, tail-ears cyanosis 6 Death
[0325] All the animals were eventually sacrificed to collect serum and lungs.
[0326] Serum Preparation:
[0327] Approximately 0.05 ml of blood were collected into gel-containing tubes and let stay for 30 min at RT. Tubes were spun for 5 min at 5500 rpm (3200×g), serum was transferred to new tubes and stored at −20° C. until use.
[0328] Two independent experiments were carried out, according to the following designs:
TABLE-US-00002 TABLE 2 Study Design Experiment 1: Group N of animals IV Treatment mAb Dose 1 4 — — 2 8 FluAB_wt 1 mg/kg 3 4 FluAB_wt 0.3 mg/kg 4 8 FluAB_MLNS 1 mg/kg 5 4 FluAB_MLNS 0.3 mg/kg
TABLE-US-00003 TABLE 3 Study Design Experiment 2: Group N of animals IV Treatment mAb Dose 1 9 — — 2 10 FluAB_wt 0.3 mg/kg 3 6 FluAB_MLNS 0.3 mg/kg
[0329] ELISA Quantification of Circulating mAb:
[0330] Sera were assessed for the levels of circulating antibodies on day 0 and 6. Briefly, half-area ELISA plates were coated over night at 4° C. with recombinant hemagglutinin (HA) from H1N1 strain A/California/07/09 (2 μg/ml, in PBS, 25 μl/well). Following blocking (PBS/1% BSA, 100 μl/well, 1 hr RT) and 2 washes (220 μl/well) with ELISA washing solution (PBST), both dilutions of the sera (initial dilution 1:150 for 1 mg/kg, 1:50 for 0.3 mg/kg) and the antibody standards (FluAB_MLNS and FluAB_wt, 0.1 μg/ml) were added (25 μl/well) in duplicate and serially diluted (1:2 by 10 points for serum dilutions, 1:3 by 8 points for antibody standards). After 1.5 hr RT incubation, plates were washed 4 times with PBST and further incubated 1.5 hr at RT with the HRP-labeled anti-human secondary antibody (0.16 μg/ml, 25 μl/well). After 4 washes with PBST, plates were dispensed with substrate solution (25 μl/well), developed for 14 min and blocked with 1% HCl (v/v, 25 μl/well). Plates were finally read at 450 nm with a spectrophotometer for signal quantification. Concentration values were calculated by using a non-linear regression model (variable slope model, four parameters, GraphPad Prism) of log (agonist) versus response.
[0331] Data Analysis:
[0332] Data were plotted and analyzed using GraphPad Prism software version 8.0 for Macintosh, GraphPad Software, La Jolla Calif. USA, www.graphpad.com. Continuous variables were assessed for statistically significant difference (p<0.05, 95% confidence interval) by using ordinary 2-way ANOVA corrected with Bonferroni multiple comparison test. Survival data were compared by using log-rank analysis with Mantel-Cox method (p<0.05 considered statistically significant). The data from the two independent experiments described above were pooled.
[0333] Results:
[0334] Prophylactic activity was tested upon i.v. administration of FluAB_MLNS and FluAB_MLNS (1 and 0.3 mg/kg) in Tg32 mice one day prior to H1N1 PR8 virus challenge via intranasal infection. Results are shown in
[0335] As depicted in
[0336] The better protective activity of FluAB_MLNS as compared to FluAB_wt was confirmed in the survival analysis shown in
[0337] The differences in the efficacy between FluAB_MLNS and FluAB_wt did not correlate with different levels of circulating antibodies in the serum, as measured 1 and 7 days after i.v. administration of the antibodies (
[0338] In summary, FluAB_MLNS demonstrated, in Tg32 mice, a better protective capacity against H1N1 PR8 intranasal virus challenge over the comparative antibody FluAB_wt. The efficacy was independent of the circulating antibody levels. These data suggest that the enhanced interaction of FluAB_MLNS with hFcRn expressed by Tg32 mice also mediates in vivo effects unrelated to the extended antibody half-life, such as increased efficacy regarding the protective activity.
Example 6
Combination of Antibody FluAB_MLNS with Various Antivirals
[0339] Drug combinations offer the clear opportunity to enhance the potency while reducing the probability to select resistances. Moreover, a putative additive or synergic effect may end up to a dose-sparing approach. Influenza medications currently approved by FDA include the neuraminidase inhibitors oseltamivir and zanamivir as well as the recently approved baloxavir marboxil, which belongs to the endonuclease inhibitors class.
[0340] To evaluate the combined activity of FluAB_MLNS with the antivirals oseltamivir, zanamivir or baloxavir marboxil on both H1N1 and H3N2 representative viral strains, in vitro neutralization was performed to evaluate the resulting inhibitory effect. The analysis of the combined effects was carried out by using the median-effect plot and the calculation of the combination index (CI).
[0341] Briefly, MDCK (Madin-Darby canine kidney) cells were seeded at 30,000 cells/well into 96-well plates (flat bottom, black). Cells were cultured at 37° C. 5% CO.sub.2 overnight. Twenty-four hours later, 4× antibody and antiviral (oseltamivir, zanamivir or baloxavir marboxil) dilutions in 60 μl infection medium (MEM (Sigma Aldrich, cat. n. M0644)+Glutamax (Invitrogen, 41090-028)+1 μg/ml TPCK-treated Trypsin (Worthington Biochemical #LS003750)+10 μg/ml Kanamycin) were prepared by using crisscross 1:2 serial dilutions of FluAB_MLNS (starting from 166.7 nM final, 9 horizontal points) and different antivirals (oseltamivir, zanamivir or baloxavir marboxil), starting from 125 (250 for zanamivir) nM by 7 vertical points), according to the plate scheme shown in
[0342] For each combination, three independent plates were prepared, in order to have triplicates of each drug-drug combination ratio. The single compound titration (namely, FluAB_MLNS, 9 points and each antiviral, 8 points) was included in each plate. Virus solution was prepared at concentrations of 120× the TCID50 in 60 μl, further diluted either 1:1 in MEM or mixed 1:1 with FluAB_MLNS dilutions and incubated 1 h at 33° C. Cells were washed 2 times using 200 μl/well MEM without supplements, followed by the addition of either 100 μl of virus alone or 100 μl of FluAB_MLNS/virus mix (100× TCID50/well) and incubated 4 hours at 33° C. 5% CO2. After the addition of 100 μl/well of infection medium, cells were further incubated for 72 hours at 33° C. 5% CO2. On day 3 after infection, 20 μM MuNANA (4-MUNANA (2_-(4-Methylumbelliferyl)-□-D-N-acetylneuraminic acid sodium salt hydrate (Sigma-Aldrich) #69587) solution was prepared in MuNANA buffer (MES 32.5 mM/CaCl.sub.2 4 mM, pH 6.5) and 50 μl/well was dispensed into black 96-well plates. Fifty μk of either neutralization or virus-alone titration supernatant were transferred to the plates and incubated 60 min at 37° C. The reaction was then stopped with 100 μl/well 0.2 M glycine/50% EtOH, pH 10.7. Fluorescence was quantified at 460 nm with a fluorimeter (Bio-Tek).
[0343] The fraction of virus neutralization was calculated according to the formula:
wherein fx=sample fluorescence signal (cells+virus+FluAB_MLNS+antiviral); fmin=minimal fluorescence signal (cells alone, no virus); fmax=maximal fluorescence signal (cells+virus only).
[0344] The neutralized fraction data were used to compute the quantitative analysis of dose-effect relationships for drug-drug combinations according to the Chou and Talalay method (Chou T C, Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984, 22:27-55). The combination Index, the fraction affected (Fa), and isobolograms were obtained by using the CompuSyn software (ComboSyn Inc., Paramus, N.J., USA) (Chou T-C: Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological Reviews 2006, 58:621-681).
[0345] Results are shown in
[0346] Combination of FluAB_MLNS and Oseltamivir
[0347] The relative efficacy of FluAB_MLNS and oseltamivir to neutralize influenza A viruses was compared in vitro on two viral serotype representatives for both H3N2 and H1N1 strains. As shown in
[0348] To test the effect of a combination of FluAB_MLNS and oseltamivir in neutralizing the infection of MDCK cells with H3 and H1 virus, both compounds were serially diluted at different ratios as described above, and assessed for the enzymatic activity of neuraminidase (NA; as a read out of the viral content in the culture) in the presence of the different drug concentrations and compared to the single drug effects. The neutralization effect measured with FluAB_MLNS is greatly enhanced by the concomitant presence of heteromolar concentrations of the second compound, thus suggesting a synergistic effect rather than an addictive one, both on H3 and H1 virus infection (
[0349] To precisely quantify the putative synergistic effects of the various drug combination ratios, the neutralization data were further transformed according to the median-effect principle and analyzed with the CompuSyn software as described above. The effects of several different FluAB_MLNS-oseltamivir combination constant ratios were plotted in the median-effect plot as shown in
[0350] The CompuSyn software applies the logarithmic transformation of the median-effect equation to the experimental data and calculates both the potency (IC.sub.50) and the so-called combination index (CI) of the various drug combinations. The CI is a Chou-Talalay (median-effect) equation-derived parameter that considers the physico-chemical properties of the mass-action law and results from the sum of the two ratios between the portion of the dose of drug 1 combined with drug 2 to achieve a certain effect divided by dose of the single drug 1 and 2 to obtain the same effect. According to this mathematical algorithm, a CI=1 indicates an addictive effect, CI<1 indicates synergism and CI>1 indicates antagonism.
[0351] As shown in
[0352] The same data can be alternatively described with isobolograms plots, which compare the equipotent concentrations of both the single and combined drugs. As shown in
[0353] Combination of FluAB_MLNS and Zanamivir
[0354] The relative efficacy of FluAB_MLNS and zanamivir to neutralize influenza A viruses was also compared in vitro on two viral serotype representatives for both H3N2 and H1N1 strains. As shown in
[0355] For the combined effect of FluAB_MLNS and zanamivir
[0356] The quantification of the synergic effect was similarly computed with CompuSyn and the median effect principle as described above. The median effect plots for the combined effects of FluAB_MLNS and zanamivir are shown in
[0357] Combination of FluAB_MLNS and Bloxavir Marboxil
[0358] The recently approved endonuclease inhibitor baloxavir marboxil was initially compared with FluAB_MLNS alone on both H1 and H3 strains, similarly as described above for oseltamivir and zanamivir. Results are shown in
[0359] Although Baloxavir has a different mechanism of action in inhibiting viral replication compared to the NA inhibitors, the drug is still able to strongly enhance the inhibitory capacity of FluAB_MLNS, clearly indicating a synergistic effect (
[0360] In summary, neutralization capacity of FluAB_MLNS against both H1 and H3 strains is synergistically enhanced by different antivirals, namely, the NA inhibitors oseltamivir and zanamivir as well as the endonuclease inhibitor baloxavir-marboxil.
Example 7
Clinical Study of FluAB_MLNS
[0361] A phase 1/2, randomized, placebo-controlled study is conducted to evaluate the safety, tolerability, pharmacokinetics, immunogenicity, and efficacy of FluAB_MLNS for the treatment and prevention of Influenza A illness. FluAB_MLNS comprises a light chain amino acid sequence according to SEQ ID NO:10 and a heavy chain amino acid sequence according to SEQ ID NO:9. The study consists of 3 parts:
[0362] Part A: A double-blind study to evaluate the safety, tolerability, pharmacokinetics, and immunogenicity of a single ascending dose of FluAB_MLNS in 100 healthy adult subjects.
[0363] Part B: An open-label study to evaluate the safety, pharmacokinetics, and immunogenicity of FluAB_MLNS following a second dose given approximately 1 year after the initial dose (i.e., in Part A) in approximately 30-80 subjects from Part A.
[0364] Part C: A randomized, double-blinded placebo-controlled study to evaluate the safety, tolerability, efficacy, pharmacokinetics, and immunogenicity of FluAB_MLNS in comparison to placebo for the treatment and prevention of Influenza A illness. The number of subjects is up to 2,760. The study evaluates the effect of FluAB_MLNS compared to placebo on Influenza A-related parameters in subjects with confirmed Influenza-A illness. These parameters include severity of illness, duration of illness, magnitude of viral load in nasopharyngeal secretions at time of presentation with Influenza-A illness. In addition, the study also provides for: [0365] evaluating the effect of FluAB_MLNS on potential biomarkers for host response after Influenza A illness; [0366] monitoring emergence of viral resistance to FluAB_MLNS in subjects presenting with Influenza A illness; [0367] evaluating potential relationships between subject genetic polymorphisms and FluAB_MLNS mechanisms of action and/or pharmacokinetics; [0368] measuring the incidence of healthcare visits due to Influenza A illness; [0369] measuring the impact of FluAB_MLNS on time away from work and productivity due to Influenza A illness.
[0370] FluAB_MLNS is evaluated for the prevention of illness due to influenza A in healthy adults at low risk of developing serious influenza-related complications. FluAB_MLNS has high in vitro potency against a wide variety of seasonal and pandemic strains of influenza A virus and is effective in the prevention of otherwise lethal influenza A infection in animal models. In addition, the epitope recognized by FluAB_MLNS is highly conserved among influenza A viruses and has a high barrier to the development of resistance in vitro.
[0371] This randomized, placebo-controlled, first-in-human, combined Phase 1/2 study is designed to evaluate the safety, tolerability, pharmacokinetics (PK), and efficacy of FluAB_MLNS for the prevention of influenza A illness in healthy adults without risk factors for serious complications from influenza infection. Parts A and B of the study are designed to collect information on the safety and tolerability of FluAB_MLNS as well as relevant data on the PK profile and the generation of anti-drug antibodies (ADAs). Part C of the study is designed to assess the efficacy of FluAB_MLNS in the prevention of influenza A illness and to collect additional safety, tolerability, and PK data. Potential risks are based on common safety risks observed with mAbs: anaphylaxis and other serious allergic reactions, immune complex disease, and injection site-related reactions.
[0372] In the presence of influenza A infection, there is a theoretical risk of antibody-mediated enhancement (ADE) of disease. Subjects are monitored for important potential risks, including assessment of safety in the presence of influenza A infection and routine pharmacovigilance and risk minimization activities is performed.
[0373] This study includes healthy adults aged 18 to 64 years of age, without risk factors for serious complications from influenza infection who have not received the upcoming seasonal influenza vaccination.
[0374] Placebo is the Control for this Study
[0375] To adequately assess the ability of FluAB_MLNS to prevent influenza A illness, study drug administration precedes the influenza season. In this context, data collected during Part A of the study are anticipated to provide sufficient observation of safety and PK to expand into a larger population of healthy adults.
[0376] A single fixed dose-escalation range of 60 to 1800 mg (60, 300, 1200, 1800 mg) of FluAB_MLNS administered IM is selected for the study. The recommended highest starting dose in humans is approximately 5 mg/kg or 300 mg fixed dose.
[0377] Study Design
[0378] This is a randomized, placebo-controlled, Phase 1/2 study of FluAB_MLNS administered intramuscularly (IM) to healthy adult volunteers aged 18 to 64 years of age, who have not received an influenza vaccination for the upcoming season. The study is designed to evaluate the safety, tolerability, PK, immunogenicity, and efficacy of FluAB_MLNS in preventing influenza A illness.
[0379] The study is conducted in 3 parts: [0380] Part A: Phase 1, double-blind, single ascending dose of FluAB_MLNS; [0381] Part B: Phase 1, open label, safety and PK following a second dose of FluAB_MLNS; [0382] Part C: Phase 2, double-blinded study to evaluate the efficacy of FluAB_MLNS in prevention of community-acquired influenza A illness.
[0383] Data collected during Part A of the study provide observation of safety and PK to expand into a larger population of healthy adults to establish proof of concept. The safety management plan, including SRC oversight in Part A and an independent DMC in Part C, is designed to ensure thorough evaluation of safety data and early detection of potential safety signals.
[0384] An interim analysis of safety, efficacy, and futility is conducted when data for approximately half of the first influenza season become available.
[0385] In the event subjects experience severe influenza illness, antiviral therapy is offered if deemed appropriate by the Investigator.
[0386] Duration of Study Participation
[0387] Part A: The estimated total time on study for each subject, inclusive of screening and follow-up, is approximately 13 months.
[0388] Part B: The estimated total time on study for each subject is approximately 6 months for Part B and up to 19 months total including Parts A and B together.
[0389] Part C: The estimated total time on study for each subject, inclusive of screening and follow-up, is approximately 8 months.
[0390] Part A
[0391] 4 cohorts of 25 subjects each received a single dose of FluAB_MLNS placebo consisting of one (or more) intramuscular (IM) injection(s) to evaluate the safety and tolerability of FluAB_MLNS compared to placebo. The dosing regimens for the 4 cohorts are shown in Table A.
TABLE-US-00004 TABLE A Summary of Part A cohorts Subjects FluAB_MLNS Dose or Cohort FLUAB_MLNS Placebo Matched Placebo 1 20 5 60 mg (1 injection × 0.4 mL) 2 20 5 300 mg (1 injection × 2 mL) 3 20 5 Up to 1200 mg (2 injections × 4 mL) 4 20 5 Up to 1800 mg (3 injections × 4 mL)
[0392] Eligible subjects were enrolled into 3 sequential cohorts randomized in a 4:1 ratio to receive either FluAB_MLNS or placebo. The first two subjects in each cohort were randomized 1:1 to receive either FluAB_MLNS or placebo. Following 24 hours of sentinel subject observation, all remaining subjects within each cohort were dosed on the same day and remain at the clinical investigative site for 48 hours post-dose.
[0393] Local tolerability symptoms that were not resolved on Day 3 are followed until resolution or Day 14 (whichever occurs first). Local tolerability parameters include pain/tenderness, swelling, redness, bruising, and pruritus at the injection site.
[0394] Subjects are actively monitored for Influenza-like illness (ILI) throughout the study. Starting on Day 3, subjects complete ILI symptom surveillance questions on an electronic device twice a week. Subjects remain in the study approximately one year to complete assessments for safety, PK, and ADA.
[0395] Results:
[0396] One hundred (100) subjects received a single dose of FluAB_MLNS (N=80) or placebo (N=20). Preliminary blinded safety data for all cohorts and PK data for the 300 and 1200 mg cohorts were collected.
[0397] Serum PK samples were collected at specified visits for 52 weeks. FluAB_MLNS serum concentrations were determined using an electrochemiluminescent method validated on the Meso Scale Discovery (MSD) platform. PK parameters were estimated using standard noncompartmental methods in WinNonlin® 8.2 (Certara L.P., Princeton, N.J.). FluAB_MLNS PK parameters were summarized using descriptive statistics.
[0398] Adverse events monitoring, clinical laboratory examination, physical examination and ECG evaluations were performed throughout the study. Injection site tolerability assessment was performed approximately 30 minutes, 2 hrs, 12 hrs, 24 hrs, 48 hrs, and 1 week post-dose.
[0399] Dosing was well tolerated; 6% ( 6/100) of subjects experienced mild injection site reactions, which generally resolved within 48 hrs. Through 12 weeks post-dosing, the majority (110/112124/126; 98.24%) of adverse events (AEs) were mild to moderate in nature, no serious AEs were reported, and no subjects discontinued due to an AE. Based on available data, exposure (Cmax and AUC) between 300 and 1200 mg of FluAB_MLNS increased in a dose-proportional manner. A PK profile of FluAB_MLNS is shown in
[0400] FluAB_MLNS was well-tolerated following single IM doses of up to 1800 mg in healthy subjects. The preliminary PK profile of FluAB_MLNS enables once per season dosing.
[0401] PK data for all dosing groups through Week 20 is shown in
[0402] Part B
[0403] Subjects who complete Part A are unblinded at the End of Study visit. Subjects who received FluAB_MLNS have opportunity to consent to participate in Part B. Eligible subjects (≥30) receive a second dose (IM) of FluAB_MLNS approximately 12 months after the initial dose to assess the immunogenicity, safety, and PK of FluAB_MLNS following repeat dosing.
[0404] Subjects remain at the clinical investigative site for a minimum of 2 hours post-dose to assess safety and local tolerability of FluAB_MLNS and remain in the study for 24 weeks to complete assessments of safety, PK, and ADA.
[0405] Decisions to suspend dosing or discontinue individual subjects from study drug in Part B are made according to predetermined stopping rules. When a stopping rule is met, dosing is paused. Subjects continue to complete symptom surveillance questions on an electronic device twice a week until the end of study to monitor for influenza-like illness (ILI).
[0406] Part C
[0407] Part C is designed to assess the safety and efficacy of FluAB_MLNS in the prevention of community acquired influenza A illness in healthy adults. Dose selection and enrollment in Part C is initiated after available PK and safety data collected from Part A are reviewed including available safety data through a minimum of 45 days from Cohort 1 and a minimum of 21 days from Cohort 2. Available safety data from Cohorts 3 and 4 are also reviewed.
[0408] The PK data from Part A are reviewed to determine dose selection for Part C. Up to two dose levels are evaluated in Part C to allow for exposure-response analyses and dose selection for further Phase 3 studies. Doses selected for evaluation in Part C consider available PK data from Part A in order to maintain a minimum FluAB_MLNS serum concentration of 8 μg/mL for at least 6 months post-dose. Up to 1380 subjects are randomized and enrolled to receive FluAB_MLNS or placebo.
[0409] One or two dose levels may be evaluated in Part C. If two dose levels are evaluated, eligible subjects are randomized to receive either FluAB_MLNS or placebo in a 2(n=460):2(n=460):1(n=230):1(n=230) ratio on Day 1. If one dose level is evaluated, subjects are randomized in a 1(n=460):1(n=460) ratio. Each dose level has a matched placebo for number of injections and volume. Subjects remain at the clinical investigative site for a minimum of 2 hours post-dose to assess safety and local tolerability of FluAB_MLNS at injection site(s) and complete assessments.
[0410] Subjects are actively monitored for ILI throughout the study. Subjects complete ILI symptom surveillance questions twice a week on an electronic device. Subjects experiencing ILI complete in-clinic evaluations, self-reported influenza symptom severity and WPAI questionnaires, and are followed. Subjects experiencing symptoms consistent with ILI, defined as: [0411] ≥1 respiratory symptom (cough, sore throat, rhinorrhea, congestion) AND [0412] ≥1 systemic symptom (fever [oral temperature >38° C. (100.4° F.)], chills, myalgias, headache, malaise, fatigue)
[0413] For all parts of the study, blood samples are collected to determine presence/absence and titers of anti-drug antibody (ADA. Samples may also be characterized for neutralizing potential of anti-FluAB_MLNS antibody, as appropriate.
[0414] All subjects have a follow-up visit approximately 4 and 12 weeks after receiving study drug and approximately 2 weeks after the end of the influenza season for assessment of safety, PK, and ADA. If a dose level requires a second injection of FluAB_MLNS, the second injection is administered at the Week 12 visit. Subjects remain in clinic for a minimum of 2 hours post-dose to assess safety and local tolerability of FluAB_MLNS at injection site(s).
[0415] An interim analysis is conducted when data for approximately half of the first influenza season become available for Part C and form the basis for a decision to conclude the study at the end of the season or continue enrollment in a second influenza season.
[0416] For Part C, End of Study visit is defined based on the approximate end of flu season in each hemisphere. For participating countries in the Southern Hemisphere, end of flu season is defined as September 30, with End of Study visits completed by approximately mid-October. For participating countries in the Northern Hemisphere, end of flu season is defined as April 30, with End of Study visits completed by approximately mid-May.
[0417] For Part C only, blood sample for Fc Receptors for IgG (FcγR) and IgG1 allele genotyping is collected to evaluate potential relationships with FluAB_MLNS mechanism of action or PK. Subjects are provided with an informed consent specifically for genotype assessment.
[0418] Subject Population
[0419] Subjects are aged 18 to 64 years of age at the time of randomization. Subjects are healthy males and females without acute or chronic medical conditions for Parts A and B. For Part C, subjects must be in good health, determined from a medical history (e.g. any chronic conditions such as hypertension, hyperlipidemia, gastroesophageal reflux disease, anxiety, or depression must be on a stable dose of medication), and no clinically significant finding from physical examination, 12-lead ECG, vital signs, and laboratory values. Body mass index of 18 kg/m.sup.2 to 32 kg/m.sup.2 for Parts A and B and 18 kg/m.sup.2 to 35 kg/m.sup.2 for Part C. Females must have negative pregnancy test or confirmation of post-menopausal status. Male subjects with female partners of child-bearing potential must agree to use of contraception until the last follow-up visit, or vasectomy with documentation of azoospermia. Patients in Parts A and B are non-smokers. Patients in Part A must agree to abstain from alcohol for 72 hours and caffeine for 24 hours prior to study drug administration. Patients in Part B receive FluAB_MLNS approximately 12 months prior and completed Part A.
[0420] Study Procedures
[0421] Part A
[0422] Screening:
[0423] Screening was performed no more than 4 weeks prior to the Day 1 visit and included written informed consent, determination of eligibility, collection of demographics and medical history as well as physical examination (including vitals), laboratory tests, 12-lead electrocardiogram (ECG) and other assessments per the Part A Schedule of Assessments (
[0424] All subjects were admitted to the clinical investigative site on Day −1 and confined for at least 12 hours prior to dosing and for 48 hours following dosing for observation, laboratory evaluation, and PK sampling. Eligible subjects were randomized to receive FluAB_MLNS or placebo on Day 1. Serum and nasopharyngeal PK samples were taken per the Schedule of Pharmacokinetic Timepoints (
[0425] Follow-Up Period:
[0426] Subjects returned to the clinical investigative site for in-person assessments per the Part A Schedule of Assessments (
[0427] Part B
[0428] Screening:
[0429] Provided evaluations of safety, PK and efficacy data from Parts A and C of the study support further product development, subjects who received FluAB_MLNS may be able to progress to Part B. Subject randomization is unblinded at the Part A End of Study visit and subjects who received FluAB_MLNS may consent to be evaluated for eligibility to receive a second dose of FluAB_MLNS approximately 12 months after the initial dose. Screening assessments per the Part B Schedule of Assessments (
[0430] Dosing (Day 1):
[0431] Eligible subjects receive a dose FluAB_MLNS on Day 1. Subjects remain in the clinic for a minimum of 2 hours post-dose to assess safety and local tolerability of FluAB_MLNS and complete assessments per the Part B Schedule of Assessments (
[0432] Follow-Up Period: Subjects return to the clinical investigative site for in-person assessments per the Part B Schedule of Assessments (
[0433] Part C
[0434] Screening:
[0435] Screening is performed no more than 4 weeks prior to the Day 1 visit and includes written informed consent, determination of eligibility, collection of demographics and medical history as well as physical examination (including vitals), laboratory tests, 12-lead electrocardiogram (ECG) and other assessments per the Part C Schedule of Assessments (
[0436] Dosing (Day 1):
[0437] If two dose levels are evaluated, eligible subjects are randomized to receive either FluAB_MLNS or placebo in a 2(n=460):2(n=460):1(n=230):1(n=230) ratio on Day 1. If one dose level is evaluated, subjects are randomized in a 1(n=460):1(n=460) ratio. If the chosen dose level requires a second injection of FluAB_MLNS, the second injection is administered at the 12-week visit. Each dose level will have a matched placebo for number of injections and volume. Subjects remain at the clinical investigative site for a minimum of 2 hours post-dose to assess safety and local tolerability of injection site(s) and complete assessments per the Part C Schedule of Assessments (
[0438] Follow-Up Period:
[0439] Subjects return to the clinical investigative site for in-clinic assessments per the Part C Schedule of Assessments (Appendix 4). Subjects complete ILI symptom surveillance questions on an electronic device twice a week through the End of Study. Subjects experiencing symptoms consistent with ILI, defined as ≥1 respiratory symptom (cough, sore throat, rhinorrhea, congestion) AND ≥1 systemic symptom (fever, chills, myalgias, headache, malaise, fatigue) during follow-up should report symptoms on same day as onset and arrange for an in-clinic evaluation per the Influenza-like Illness Monitoring Schedule (
[0440] In-clinic evaluation includes, but is not limited to, physical examination (including vital signs), laboratory testing (including safety), nasopharyngeal swab for virology, blood samples for PK and ADA, and review of AEs and concomitant medications as appropriate. Subjects also self report influenza symptom severity and complete the WPAI questionnaire per the Influenza-like Illness Monitoring Schedule (
[0441] Influenza Symptom Severity Questionnaire
[0442] Subjects perform a self-assessment of systemic and respiratory symptoms associated with influenza (i.e., cough, sore throat, headache, nasal congestion, feverishness or chills, muscle or joint pain, and fatigue). They score the severity of their symptoms using a 4-point rating scale (0, None; 1, Mild; 2, Moderate; 3, Severe). This information is captured in an electronic device twice daily from ILI-D1 to ILI-D10 (10 days inclusive) during the Influenza-like Illness Monitoring period (
[0443] Anti-influenza A Antibody Titer
[0444] In all parts of the study, blood samples are collected to determine anti-influenza A antibody titers using standard methods according to Part A, B and C Schedule of Assessments (
[0445] Monitoring Schedule in
[0446] Resistance Surveillance
[0447] In all parts of the study, resistance surveillance for potential emergence of resistance to FluAB_MLNS are conducted for all subjects who received study drug and present with laboratory confirmed influenza A virus infection, as per the Influenza-like Illness Monitoring Schedule (
[0448] Exploratory Biomarkers
[0449] For Part C and during ILI Monitoring, samples will be collected per the Schedule of Assessments (
[0450] Genotyping
[0451] Subjects' signed and dated informed consent specifically for genotyping assessments are obtained before conducting any sample collections. For part C only, blood sample for Fc Receptors for IgG (FcγR) and IgG1 allele genotyping is collected per the Schedule of Assessments in
[0452] Product
[0453] FluAB_MLNS is provided as 300 mg lyophilized solid in an air-tight stoppered glass vial. Upon reconstitution to 150 mg/mL with USP water for injection, the drug product, as administered, contains 20 mM Histidine, 5.3% sucrose, and 0.02% PS80 at pH 6. FluAB_MLNS is injected intramuscularly. The unit dose is based on volume (0.8 mL to 4 mL per injection). No special procedures for safe handling of FluAB_MLNS are required. The FluAB_MLNS is stored in a secure, temperature-controlled environment.
[0454] Placebo is a sterile, preservative-free normal saline 0.9% solution for IM injection.
[0455] Statistical Methods
[0456] Part A and Part B
[0457] Statistical analyses are primarily descriptive. All study data is presented in data listings. Summary tables will present results by cohort for each FluAB_MLNS dose and placebo, as appropriate. Descriptive statistics are presented for continuous variables, and frequencies and percentages are presented for categorical and ordinal variables. Percentages are based on the number of non-missing values in a dose group. The impact of ADA on PK, and association with AEs and SAEs may be assessed.
[0458] Part C
[0459] The primary analysis population for efficacy analysis is the Full Analysis Set (FAS), which includes all randomized subjects who receive any amount of study drug. The primary efficacy endpoint is the proportion of subjects with laboratory-confirmed (by RT-PCR) influenza A illness, defined as ≥1 respiratory and ≥1 systemic symptom. The primary analysis consists of superiority tests of FluAB_MLNS compared to placebo based on the reduction of protocol-defined influenza A illness rate. If two dose levels are evaluated, the following hypotheses is tested according to the sequential testing principle at the 2-sided 0.05 level. If a null hypothesis is not rejected, formal sequential testing is stopped, and only nominal significance is reported for the remaining hypotheses: [0460] 1. Superiority of FluAB_MLNS dose level 2 (high) compared to placebo based on the protocol defined influenza A illness rate [0461] 2. Superiority of FluAB_MLNS dose level 1 (low) compared to placebo based on the protocol defined influenza A illness rate
[0462] If two dose levels are evaluated in Part C, a sample size of 460 subjects in each FluAB_MLNS group a 230 subjects in each of the matching placebo group provides approximately 80% power to detect a 70% reduction in the protocol defined illness rate (from 4.5% to 1.35%) between the placebo group and the FluAB_MLNS group respectively, using a 2-sided 0.05-level test. A total of approximately 1380 subjects are enrolled.
[0463] If only one dose level is evaluated, a total of approximately 920 subjects are enrolled. A sample size of 460 subjects in FluAB_MLNS group and 460 subjects in the placebo group provides approximately 80% power to detect a 70% reduction in the protocol defined illness rate (from 4.5% to 1.35%) between the placebo group and the VIR-2482 group respectively, using a 2-sided 0.05-level test.
[0464] If the interim analysis triggers the enrollment of additional subjects, approximately 1380 additional subjects are enrolled, if two doses levels are evaluated. The total study sample size is approximately 2760 subjects. If only one dose level is evaluated, approximately 920 additional subjects are enrolled. The total study sample size will be approximately 1840. A sample size of 920 subjects in FluAB_MLNS group and 920 subjects in the placebo group provide approximately 80% power to detect a 70% reduction in the protocol defined illness rate (from 2.25% to 0.675%) between the placebo group and the FluAB_MLNS group respectively, using a 2-sided 0.05-level test.
[0465] Endpoints
[0466] Part A
[0467] Primary endpoint is: [0468] The safety and tolerability of FluAB_MLNS as measured by the incidence of treatment-emergent adverse events (TEAEs) and clinical assessments
[0469] The secondary endpoints are: [0470] Single-dose FluAB_MLNS serum PK parameters (e.g., C.sub.max, C.sub.last, T.sub.max, T.sub.last, AUC.sub.inf, AUC.sub.last, % AUC.sub.exp, t1/2, λ.sub.z, V.sub.z/F, CL/F) [0471] Incidence and titers (if applicable) of serum ADA to FluAB_MLNS
[0472] The exploratory endpoints may include: [0473] Single-dose FluAB_MLNS nasopharyngeal secretion PK parameters (e.g., C, C.sub.last, T.sub.max, T.sub.last, AUC.sub.inf, AUC.sub.last, % AUC.sub.exp, t.sub.1/2, λ.sub.z, V.sub.z/F, CL/F)
[0474] Part B
[0475] The primary endpoint is: [0476] Incidence and titers (if applicable) of ADA to FluAB_MLNS
[0477] The secondary endpoints are: [0478] The safety and tolerability of FluAB_MLNS as measured by the incidence of treatment-emergent adverse events (TEAEs) and clinical assessments [0479] FluAB_MLNS serum PK parameters (e.g., C.sub.max, Clast, T.sub.max, T.sub.last, AUC.sub.inf, AUClast, % AUC.sub.exp, t.sub.1/2, λ.sub.z, V.sub.z/F, CL/F)
[0480] Part C
[0481] The primary endpoints are: [0482] The safety and tolerability of FluAB_MLNS as measured by the incidence of treatment-emergent adverse events (TEAEs) and clinical assessments. [0483] Efficacy: Proportion of subjects with laboratory-confirmed (by RT-PCR) influenza A illness, defined as ≥1 respiratory and ≥1 systemic symptom
[0484] The secondary endpoints are: [0485] Proportion of subjects with culture-confirmed influenza A illness [0486] Severity and duration of subject-reported signs and symptoms of ILI due to influenza A [0487] Quantification of the viral load present in nasopharyngeal secretions at the time of initial symptomatic presentation by RT-qPCR and viral culture [0488] PK of FluAB_MLNS in serum [0489] Incidence and titers (if applicable) of ADA to FluAB_MLNS
[0490] The exploratory endpoints may include: [0491] Effect of FluAB_MLNS on potential biomarkers for host response after ILI presentation [0492] Emergence of viral resistance to FluAB_MLNS in subjects presenting with influenza A illness [0493] FcR polymorphisms as determined by genotyping and potential relationships with FluAB_MLNS mechanisms of action and/or PK [0494] IgG1 allotypes as determined by genotyping [0495] Rate of medically attended healthcare visits during the ILI monitoring period [0496] Measure of Work Productivity and Activity Impairment (WPAI) due to influenza A illness
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS IN THIS EXAMPLE
[0497] ADA anti-drug antibody
[0498] ADE antibody-dependent enhancement
[0499] AE adverse event
[0500] ALT alanine aminotransferase
[0501] ALP alkaline phosphatase
[0502] AST aspartate aminotransferase
[0503] AUC area under the curve
[0504] BLQ below the limit of quantitation
[0505] BMI body mass index
[0506] BUN blood urea nitrogen
[0507] CLcr creatinine clearance
[0508] CMC Chemistry, Manufacturing, and Controls
[0509] CTCAE Common Terminology Criteria for Adverse Events
[0510] DMC Data Monitoring Committee
[0511] EC Ethics Committee
[0512] ECG electrocardiogram
[0513] eCRF electronic case report form
[0514] EOS End of Study
[0515] ET Early Termination
[0516] FcR Fc receptors for IgG
[0517] FDA Food and Drug Administration
[0518] GCP Good Clinical Practice
[0519] GGT gamma glutamyl transferase
[0520] GLP Good Laboratory Practice
[0521] GMP Good Manufacturing Practices
[0522] HA hemagglutinin
[0523] HED human equivalent dose
[0524] Hgb hemoglobin
[0525] ICF informed consent form
[0526] ICH International Conference on Harmonisation
[0527] IgG immunoglobulin G
[0528] ILI influenza-like illness
[0529] IM intramuscular
[0530] IND Investigational New Drug
[0531] INR International Normalized Ratio
[0532] IP investigational product
[0533] IRB Institutional Review Board
[0534] IV intravenous
[0535] IWRS interactive web response system
[0536] LDH lactate dehydrogenase
[0537] LLN lower limit of normal
[0538] LLOQ lower limit of quantitation
[0539] LLT Lower-Level Term
[0540] mAb monoclonal antibody
[0541] MedDRA Medical Dictionary for Regulatory Activities
[0542] NOAEL no observed adverse effect level
[0543] OTC over-the-counter
[0544] PK pharmacokinetics
[0545] POC Proof of concept
[0546] RBC red blood cell
[0547] SAD single ascending dose
[0548] SAE serious adverse event
[0549] SD standard deviation
[0550] SOC System Organ Class
[0551] SRC Safety Review Committee
[0552] SUSAR suspected unexpected serious adverse reaction
[0553] ULN upper limit of normal
[0554] WBC white blood cell
[0555] WHO World Health Organization
[0556] WOCBP women of child-bearing potential
[0557] WPAI Work Productivity and Activity Impairment
Example 8
Binding to Human FcRn at Different pHs
[0558] FluAB_wt and FluAB_MLNS were compared side by side for their ability to bind to neonatal Fc receptor (FcRn) using biolayer interferometry (BLI). To this end, binding of FluAB_wt and FluAB_MLNS to human FcRn was measured on an Octet RED96 instrument (biolayer interferometry, BLI, ForteBio). Biosensors coated with anti-human Fab-CHI were pre-hydrated in kinetic buffer for 10 min at RT. Then, human mAb (FluAB_wt or FluAB_MLNS) was loaded at 1 μg/ml in kinetics buffer at pH 7.4 for 30 minutes onto the Biosensors. The baseline was measured in kinetics buffer (Sterile filtered 0.01% endotoxin-free bovine serum albumin, 0.002% Tween-20 (Polysorbate 20), 0.005% NaN3 in PBS) at pH=7.4 or pH=6.0 for 4 minutes. Human mAb-loaded sensors were then exposed for 7 minutes to a solution of human FcRn at 1 μg/ml in kinetics buffer at pH=7.4 or pH=6.0 to measure association of FcRn-mAb in different milieus (on rate). Dissociation was then measured in kinetics buffer at the same pH for additional 5 minutes (off rate). All steps were performed while stirring at 1000 rpm at 30° C. Association and dissociation profiles were measured in real time as change in the interference patterns.
[0559] As shown in
Example 9
Characterization of Polymorphisms Identified in the Antibody's Extended Epitope
[0560] Historical polymorphisms in the extended epitope were evaluated for their impact on neutralization activity of FluAB_MLNS using viruses generated by reverse genetics with H1 HA or H3 HA on a A/Puerto Rico/8/34 (PR8) background.
[0561] Single nucleotide polymorphisms were introduced into PR8 H1 HA or A/Aichi/2/68 (Aichi) h-IA pHW2000 plasmids using site-directed mutagenesis. Recombinant influenza A virus were rescued with associated H1 or H3 HA on a PR8 backbone using standard methods (e.g., as described in Erich Hoffmann, Gabriele Neumann, Yoshihiro Kawaoka, Gerd Hobom, Robert G. Webster, 2000, A DNA transfection system for generation of influenza A virus from eight plasmids. Proceedings of the National Academy of Sciences May 2000, 97 (11): 61 08-6113; doi: -10.1073/pnas.t 00-133697).
[0562] Neutralization activity was evaluated in MDCK cells using standard methods. For example, neutralization activity may be evaluated in MDCK cells, e.g. in 96 well plates. To this end, MCDK cells may be seeded at 30,000 cells/well 24 hours prior to infection. Antibody FluAB_MLNS maybe incubated with virus for 1 hour at 37° C. prior to addition to MDCK cells.
[0563] To this end, 1:2.5 9-point serial dilutions of FluAB_MLNS may be created in infection media and each dilution may be tested in triplicate (e.g., 50 μg/mL−0.03 μg/mL final concentration) and may be incubated with 120 TCID.sub.50 of virus for 1 hour at 37° C. MDCK cells may be washed twice with PBS, 100 μl/well of virus:antibody solution may be added, and cells may be incubated for 4 hours at 37° C. After 4 hours, an additional 100 μL/well of infection media may be added to cells. After 72 hours of incubation at 37° C., viral RNA may be extracted and measured by qRT-PCR, e.g. using WHO primers (World Health Organization. CDC protocol of real-time RT-PCR for influenza A H1N1. Apr. 28, 2009). The IC.sub.50 is expressed as the antibody concentration in μg/mL that reduces 50% of virus replication and may be calculated using a non-linear 4-parameter logistic fit curve of data normalized to control wells (no virus and virus alone).
[0564] The neutralization activity of FluAB_MLNS to HI and H3 HA polymorphisms in the extended epitope is shown in Table 4 below.
TABLE-US-00005 TABLE 4 FluAB_MLNS Amino Acid Geomean Fold change Changes Neutralization relative Virus in HA IC.sub.50 (μg/mL) to WT virus PR8: Aichi HA wt wild type 5.6 NA PR8: Aichi HA P11S P11S 9.5 1.7 PR8: Aichi HA D46N D46N 3.3 0.6 PR8: Aichi HA N49T N49T 5.0 0.9 PR8 wt wild type 4.7 NA PR8 HA N146D N146D 5.5 1.2 Aichi = A/Aichi/2/68; Geomean = geometric mean; HA = hemagglutinin; NA = not applicable; PR8 = A/Puerto Rico/8/34 H1N1; wt = wild type
[0565] For viruses encoding H3 HA, FluAB_MLNS neutralized viruses with mutations HA1 P11S, HA2 D46N, or HA2 N49T with IC.sub.50 values similar to wild type virus (<2-fold change in IC.sub.50 relative to wild type virus). For viruses encoding H1 HA, FluAB_MLNS neutralized viruses encoding HA2 N146D with IC.sub.50 values similar to wild type virus (<2-fold change in IC.sub.50 relative to wild type virus). Additionally, the PR8 wild type strain used encoded the HA2 polymorphism L38Q and D46N and was neutralized with an IC.sub.50 value of 4.7 ng/mL by FluAB_MLNS. Overall, all polymorphisms evaluated resulted in IC.sub.50 fold changes of <2 relative to the wild type virus for FluAB_MLNS. In summary, FluAB_MLNS effectively neutralized all evaluated historical polymorphisms in the extended epitope (H3 HA: HA1 P11S, HA2 D46N, or HA2 N49T; H1 HA: N146D).
Example 10
Anti-Drug Antibody Response in Tg32 Mice
[0566] With regard to the M428L/N434S mutation, recently concerns were raised that said mutation increases immunogenicity of antibodies comprising this mutation (Brian C. Mackness, Julie A. Jaworski, Ekaterina Boudanova, Anna Park, Delphine Valente, Christine Mauriac, Olivier Pasquier, Thorsten Schmidt, Mostafa Kabiri, Abdullah Kandira, Katarina Radošević & Huawei Qiu (2019) Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life, mAbs, 11:7, 1276-1288; Maeda A, Iwayanagi Y, Haraya K, et al. Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody. MAbs. 2017; 9(5):844-853).
[0567] To assess immunogenicity, in particular the anti-drug response (anti-drug antibodies; ADA), of antibody FluAB_MLNS in comparison to its parental antibody FluAB_wt, two separate groups (n=5) of TG32 mice (transgenic for the human FcRn) were injected i.v. with 5 mg/kg of either FluAB-MLNS or FluAB_wt monoclonal antibodies. To evaluate the circulating levels of the injected mAbs, blood samples were then obtained at different time points. Samples taken at day 14 and 21 post injection were used to evaluate, by specific ELISA, the anti-drug antibody (ADA) response against the injected human monoclonals.
[0568] Briefly, purified FluAB_wt and FluAB_MLNS monoclonal antibodies were coated on 96-well plates at 2 μg/ml. After blocking, the sera from treated animals obtained 14 and 21 days post injection, diluted 1: 180, were incubated 1.5 h at room temperature (RT). After washings, peroxidase-labeled goat anti-mouse IgG F(ab′)2 fragment (0.16 μg/ml) was added to plates and incubated 1.5 h at RT. ADA IgG (murine antibodies against the injected antibodies FluAB_wt and FluAB_MLNS) were then revealed with the appropriate substrate and read with a spectrophotometer. Data shown are the OD values (450 nm) obtained in each individual serum (n=5/group) collected 14 and 21 days after the antibody i.v. administration. Sera from naïve Tg32 mice (ctrl) were used as negative control.
[0569] Results are shown in
[0570] In summary, these data indicate that surprisingly the anti-drug response (anti-drug antibodies; ADA), and, thus, the immunogenicity of FluAB_MLNS was decreased compared to FluAB_wt.
Example 11
Anti-Drug Antibody Response and Immunogenicity After S.C. Administration
[0571] To further confirm this surprising finding in more immunogenic settings, separate groups of TG32 mice (n=5) were injected with either FluAB-MLNS or FluAB_wt (5 mg/kg) subcutaneously (s.c.), which is generally considered a more immunogenic route of administration. Three weeks after s.c. administration, the levels of anti-drug antibodies were measured in the serum by mouse anti-drug specific ELISA (as described above in Example 10) in the serum of mice injected s.c. with either FluAB_wt or FLuAB_MLNS. As negative control, a pool of 10 sera from naïve, untreated animals was used.
[0572] Results are shown in
[0573] These data surprisingly show that antibody FluAB_MLNS exhibits less immunogenicity as compared to its parental antibody FluAB_wt.
Example 12
In Vitro Neutralizing Activity
[0574] The ability of FluAB_WT to broadly neutralize influenza A viruses was evaluated in vitro in two separate studies using a microneutralization assay. A panel of 52 influenza A isolates collected from 1933-2014 was studied, representing 24 group 1 (subtypes H1, H2, H5, H6, and H9) and 28 group 2 viruses (subtypes H3 and H7). In one study, FluAB_WT neutralized all 37 viruses with a median half-maximal inhibitory concentration IC.sub.50 of 0.78 μg/mL (ranging from 0.12 to 3.07 μg/mL). In the other study, FluAB_WT neutralized 15 additional virus strains isolated from 2010 to 2014 with a median IC.sub.50 of 0.199 μg/mL (ranging from 0.067 to 2.69 μg/mL). The median IC.sub.50 value for all group 1 and group 2 viruses in the two studies, excluding two 7:1 recombinant PR8 viruses, was 0.1 μg/mL for group 1 and 0.80 μg/mL for group 2 viruses (n=24 and n=26, respectively). In total, 17 H1N1 viruses were tested with a median IC.sub.50 of 0.28 μg/mL and a IC.sub.90 of 2.17 μg/mL. Accordingly, FluAB_WT can provide consistent neutralization activity despite naturally occurring antigenic drift. These data also support consistent neutralization activity for FluAB_MLNS.
Table of Sequences and Seq Id Numbers (Sequence Listing)
[0575]
TABLE-US-00006 SEQ ID NO Sequence Remarks FluAB_MLNS SEQ ID NO: 1 SYNAVWN CDRH1 SEQ ID NO: 2 RTYYRSGWYNDYAESVKS CDRH2 SEQ ID NO: 3 SGHITVFGVNVDAFDM CDRH3 SEQ ID NO: 4 RTSQSLSSYTH CDRL1 SEQ ID NO: 5 AASSRGS CDRL2 SEQ ID NO: 6 QQSRT CDRL3 SEQ ID NO: 7 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSY VH NAVWNWIRQSPSRGLEWLGRTYYRSGWYND YAESVKSRITINPDTSKNQFSLQLNSVTPEDTA VYYCARSGHITVFGVNVDAFDMWGQGTMVT VSS SEQ ID NO: 8 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYT VL HWYQQKPGKAPKLLIYAASSRGSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSRTFGQGT KVEIK SEQ ID NO: 9 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSY Heavy chain NAVWNWIRQSPSRGLEWLGRTYYRSGWYND YAESVKSRITINPDTSKNQFSLQLNSVTPEDTA VYYCARSGHITVFGVNVDAFDMWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVLHEALHS HYTQKSLSLSPGK SEQ ID NO: 10 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYT Light chain HWYQQKPGKAPKLLIYAASSRGSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSRTFGQGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC FluAB_wt SEQ ID NO: 11 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSY Heavy chain NAVWNWIRQSPSRGLEWLGRTYYRSGWYND YAESVKSRITINPDTSKNQFSLQLNSVTPEDTA VYYCARSGHITVFGVNVDAFDMWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK
[0576] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification or the attached Application Data Sheet are incorporated herein by reference, in their entirety to the extent not inconsistent with the present description. U.S. Provisional Application 62/893,747, filed Aug. 29, 2019, U.S. Provisional Application 62/993,519, filed Mar. 23, 2020, and U.S. Provisional Application 63/040,966 filed Jun. 18, 2020, are incorporated herein by reference, in their entirety. From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.