Two randomized, double-blind, placebo-controlled, dose-escalation phase 1 studies evaluating BTH1677, a 1, 3–1,6 beta glucan pathogen associated molecular pattern, in healthy volunteer subjects

Summary Background BTH1677 is a beta glucan pathogen associated molecular pattern (PAMP) currently being investigated as a novel cancer therapy. Here, the initial safety and pharmacokinetic (PK) results of BTH1677 in healthy subjects are reported. Subjects and Methods In the Phase 1a single-dosing study, subjects were randomized (3:1 per cohort) to a single intravenous (iv) infusion of BTH1677 at 0.5, 1, 2, 4, or 6 mg/kg or placebo, respectively. In the Phase 1b multi-dosing study, subjects were randomized (3:1 per cohort) to 7 daily iv infusions of BTH1677 at 1, 2, or 4 mg/kg or placebo, respectively. Safety and PK non-compartmental analyses were performed. Results Thirty-six subjects (N = 24 Phase 1a; N = 12 Phase 1b) were randomized to treatment. No deaths or serious adverse events occurred in either study. Mild or moderate adverse events (AEs) occurred in 67 % of BTH1677-treated subjects in both studies. Treatment-related AEs (occurring in ≥10 % of subjects) included dyspnea, flushing, headache, nausea, paraesthesia, and rash in Phase 1a and conjunctivitis and headache in Phase 1b. BTH1677 serum concentration was linear with dose. Clearance, serum elimination half-life (t1/2) and volume of distribution (Vss) were BTH1677 dose-independent. In Phase 1b, area under the curve, t1/2, and Vss values were larger at steady state on days 6–30 versus day 0. Conclusions BTH1677 was well tolerated after single doses up to 6 mg/kg and after 7 daily doses up to 4 mg/kg.


Introduction
Microbial beta glucans are polymers of glucose extractable from yeasts and fungi [1]. These conserved molecules function as pathogen associated molecular patterns (PAMPs) that are efficiently recognized by the mammalian innate immune system as non-self molecules and assist in elimination of microbial pathogens [2,3]. PAMP recognition drives enhanced killing capacity of innate immune effector cells (e.g., macrophages, monocytes, neutrophils), repolarization of macrophages from an M2 to a more M1 state, and the maturation and activation of dendritic cells-professional antigenpresenting cells that cross talk to cells of the adaptive immune system (i.e., T-cells, B-cells). BTH1677 (Biothera Pharmaceutical Inc., Eagan, MN, USA) is an intravenous (iv) formulation of a yeast-derived, uncharged, water-soluble, 1,3-1,6 beta glucan purified from the cell wall of a proprietary, non-recombinant, strain of Saccharomyces cerevisiae. BTH1677 is a PAMP that can orchestrate a coordinated anticancer immune response, involving both the innate and adaptive immune system, when administered with other anticancer therapies.
When BTH1677 enters the blood, it is bound by endogenous plasma anti-beta glucan antibodies (ABA) resulting in complement activation and opsonization with complement protein iC3b [4]. The BTH1677/ABA/iC3b complex initially binds to innate immune effector cells through complement receptor 3 and Fc gamma receptor IIA (FcγIIA) [4][5][6], activating innate immune cell function and direct killing of antibody-targeted tumor cells [7,8]. BTH1677 also enables re-education of the tumor microenvironment, shifting the normally suppressive M2-state macrophages to a more M1 (tumor attack) state, [9,10] and promotes depletion and/or maturation of myeloid-derived suppressor cells in the tumor microenvironment [11]. BTH1677 treatment additionally activates antigen-presenting cells, driving co-stimulatory marker expression on macrophages and dendritic cells, as well as dendritic cell maturation, CD4 and CD8 T-cell expansion, and production of key anti-tumor cytokines (e.g., interferon gamma) [10,[12][13][14][15]. In murine syngeneic and xenogeneic tumor models, the administration of BTH1677 with various tumor-targeting monoclonal antibodies has resulted in greater suppression of tumor growth and longer survival than with either agent alone [16][17][18]. Similar effects have been observed with BTH1677 combined with anti-angiogenic [11,19,20] and checkpoint inhibitor [13] antibodies.
Here, we report the initial safety and pharmacokinetic (PK) results of dose-escalating single-and multiple-dose Phase 1 studies of BTH1677 in healthy volunteer subjects.

Study objectives
The primary objective of each study was to evaluate the safety profile and tolerability of BTH1677 versus placebo by assessing the frequency, type, and severity of adverse events (AEs), as well as by assessing changes in physical exam findings, vital signs, electrocardiogram (ECG) responses, and clinical laboratory results. The secondary objective of each study was to evaluate the PK profile of BTH1677. The Phase 1b study was initiated following completion of the Phase 1a study.

Subjects
Subjects, 18 to 45 years of age (inclusive), were eligible for study inclusion if they provided written informed consent and had a body weight of 45 to 125 kg (inclusive), and a body mass index ≤30 kg/m 2 . Females could not be pregnant or nursing and, if premenopausal, were required to have a negative urine pregnancy test prior to enrollment and be practicing at least 2 methods of birth control. All subjects were determined to be healthy by the principal investigator on the basis of medical history, physical examination, ECG, and clinical laboratory test results. Subjects enrolled in the study understood the study requirements, agreed to abide by the study restrictions and to return for required assessments, and provided written authorization for use and disclosure of protected health information.
Subjects were excluded from the study if they had either a known hypersensitivity to baker's yeast; a history of tobacco use within 3 months of the screening period; an active yeast infection, a positive hepatitis B, hepatitis C, or human immunodeficiency virus test (conducted during the screening period); or were a known or suspected abuser of alcohol or other drugs/substances of abuse. In addition, subjects were excluded if they had donated or lost more than a unit of blood within 30 days of the last day of the screening period; had any clinical condition that, in the opinion of the principal investigator, warranted exclusion from the study for either a scientific, procedural, or safety perspective; had taken any prescription medication within 14 days (30 days for Phase 1b); had taken any over-the-counter medication, herbal preparation, or vitamin within 7 days (acetaminophen [maximum 3 g/day], female hormone replacement therapy, and oral contraceptives were allowed); or had participated in an investigational drug study within 30 days or 5 half-lives (whichever was longer). Phase 1b also excluded subjects who had ever participated in a study with BTH1677.

Study design
B o t h P h a s e 1 a ( N C T 0 0 5 4 2 2 1 7 ) a n d P h a s e 1 b (NCT00542464) were single-center, randomized, doubleblind, placebo-controlled, dose-escalation studies that evaluated infusion of BTH1677 in healthy volunteer subjects.
The Phase 1a, single-dosing study consisted of a 1-day treatment period and an additional 6-day follow-up period that enrolled 24 subjects in 5 sequential BTH1677 dose cohorts (0.5, 1, 2, 4, and 6 mg/kg). The Phase 1b multiple-dosing study consisted of a 7-day treatment period and an additional 23-day follow-up period and enrolled 12 subjects in 3 sequential dose cohorts (1, 2, and 4 mg/kg). The initial BTH1677 dose chosen for the Phase 1a study was based on safety margins calculated from animal toxicology studies according to regulatory guidelines [21]. For each cohort, in a double-blind manner, 3 subjects were randomized to BTH1677 and 1 subject was randomized to placebo. AEs were assessed as mildawareness of sign, symptom, or event, but easily tolerated; moderate-discomfort enough to cause interference with usual activities and may warrant intervention; severe-incapacitating with inability to do usual activities or significantly affecting clinical status, and warranting intervention; or life threatening-immediate risk of death. Each dose increase was dependent on the approval of an AE taskforce (AETF), based on review of safety data from the previous cohort. The AETF consisted of the principal investigator and 3 members of the Biothera clinical team, including the medical monitor. Dose escalation of study drug was to be discontinued if dose-limiting toxicity (DLT) was observed in the previous cohort. DLT was defined as 2 or more subjects experiencing at least 1 moderate AE judged to be study drug related or 1 or more subjects experiencing at least 1 severe AE or serious AE (SAE) judged to be study drug related.
It was the original intent of the Phase 1a protocol to dose all subjects over a 1-h (hr) period; however, during the study the protocol was amended to revise dosing times resulting in enrollment of an additional 4 subjects to an amended dosing schedule in the 4 mg/kg cohort (see additional information provided in the Results section). Planned dose cohorts and infusion information for the Phase 1a and Phase 1b studies are shown in Table 1. Figure 1 illustrates the design of the Phase 1a and Phase 1b studies.

Pharmacokinetic analysis
In both studies, serum BTH1677 levels were analyzed using an enzyme-linked immunosorbent assay (ELISA) method that was qualified at Biothera Pharmaceutical Inc. (Eagan, MN, USA). The assay was based on galactosylceramide (GalCer; CAS #85305-88-0) capture of BTH1677 and detection with a beta-glucan-specific IgM monoclonal antibody (BFD IV). Test serum samples were incubated in 96-well microtiter plates coated with GalCer to allow for BTH1677 binding to the GalCer coating. The bound BTH1677 was then detected by sequential incubations with unlabeled BFD IV, then biotin-conjugated goat anti-mouse IgM, and finally with streptavidin-conjugated horseradish peroxidase and peroxidase substrate. Color development was quenched by adding the substrate stop solution to each well and absorbance was read at 450 nm. The lower limit of detection for the assay was 1.2 ng/mL and the lower limit of quantitation was 4.7 ng/mL. For PK analysis in the Phase 1a study, blood samples were collected before dosing and at 1, 1.25, 1.5, 2, 3, 4, 8, 12, 24, 48, 72, 96, and 168 h relative to the start of the infusion. For PK analysis in the Phase 1b study, on the first day (day 0) and last day (day 6) of dosing, blood samples were collected before dosing, at the end of infusion, and at 0.25, 0.5, 1, 2, 3, 7, and 12 h relative to the end of infusion. Additional blood samples were collected at 24,48,72,96,120,168,192,216,240,336, and 720 h relative to the start of infusion on day 0. After blood collection, serum was separated, frozen, and analyzed for BTH1677 concentrations. For both studies, PK parameters were calculated using non-compartmental analysis (NCA) methods.

Statistical analysis
Because of the nature of the Phase 1a and Phase 1b studies, statistical analyses were descriptive only. In each study, data for all subjects randomized to placebo were pooled to create the placebo treatment group. Categorical data were summarized in tables listing the frequency and the percentage of subjects in each treatment group. Continuous data were summarized in tables listing the mean, standard deviation, median, minimum, and maximum in each treatment group. All statistical computations were performed using the SAS® system.

Results
Phase 1a study

Subject demographics
Baseline demographics of subjects in the Phase 1a singledosing study are shown on the left-hand side of Table 2.

BTH1677 drug administration
In this study, 24 subjects were randomized and dosed with single doses of placebo (n = 6) or escalating doses of BTH1677 at 0.5, 1, 2, 4, or 6 mg/kg (n = 18). It was originally intended in the protocol to dose all subjects over a 1-h period. However, after initiating dosing of 3 subjects in the 4-mg/kg cohort, 2 subjects reported moderate AEs of flushing, pruritus, tachycardia, dyspnea, and hyperhidrosis within 5 to 10 min of initiation of infusion. At the onset of the AEs, study drug administration was stopped in all 3 subjects (which included 1 subject randomized to placebo who had experienced no AEs) and initiation of dosing in the fourth subject planned for this cohort (a subject randomized to BTH1677) was suspended. The dosed subjects experiencing AEs were administered diphenhydramine (i.e., Benadryl®) intravenously and their AEs resolved within approximately 10 to 15 min after discontinuation of the infusion. At the time study drug administration was discontinued in the 4-mg/kg (1-h infusion) treatment cohort, the subjects exhibiting the abovementioned AEs had received approximately 9 % to 14 % of the total targeted infusion volume. Thus, the total study drug dose administered to these subjects was far less than the total dose safely administered to the 2-mg/kg treatment cohort. Although the 4-mg/kg (1-h infusion) dose was diluted in a 500-mL volume of normal saline versus the 250-mL volume of normal saline used for the previous 2-mg/kg dose, the infusion time was kept constant at 1 h. As a result, the concentration of the study drug infused was similar between the 4-mg/kg (1-h infusion) and 2-mg/kg treatment cohorts (mean concentration of 0.64 mg/mL for the 2-mg/kg treatment cohort compared to 0.73 mg/mL for the 4-mg/kg [1-h infusion] treatment cohort). However, the rate of administration of the 4-mg/kg (1-h infusion) dose was approximately twice that of 2-mg/kg dose. The rate of drug administration for the 4-mg/kg (1-h infusion) cohort varied from 5.7 to 6.5 mg/min, whereas the rate of drug administration for the 2-mg/kg cohort varied from 1.9 to 3.4 mg/min. After review of the clinical and safety data, it was concluded that the AEs observed at the 4-mg/kg (1-h infusion) dose level were most likely not the result of the BTH1677 drug dose, but rather were a result of the rapid infusion rate and were    not considered DLTs. A protocol amendment was submitted and approved to allow enrollment of 4 additional subjects into the 4-mg/kg treatment cohort with a 2-h, rather than 1-h, infusion time to allow an infusion rate similar to that of the previous 2-mg/kg treatment cohort in which BTH1677 had been well tolerated. In support of the conclusion that the observed AEs were the result of infusion rate rather than dose level, there were no treatment-related AEs reported in subjects treated with 4 mg/kg BTH1677 administered over a 2-h infusion. The protocol amendment also modified the infusion time for the 6-mg/kg dose cohort to 3 h, which was also generally well tolerated.

Safety
An overview of the safety summary is shown on the left side of Table 3. Twelve of the 18 BTH1677 subjects (66.7 %) and 3 of 6 placebo subjects (50.0 %) experienced AEs. AEs were assessed as treatment related in 9 of 18 BTH1677 subjects (50.0 %) and in none of the 6 placebo subjects (0.0 %). All AEs were mild or moderate in intensity, with no AEs classified as severe. No SAEs or deaths occurred in the study.
Excluding the 4-mg/kg 1-h infusion group, the mean number of treatment-related AEs per BTH1677 subject in the 0.5-, 1-, 2-and 4-mg/kg groups ranged from 0.0 to 1.0 and increased to 2.7 in the 6-mg/kg group. Four subjects (2 in the 4-mg/kg 1-h infusion group) discontinued the study prematurely because of AEs that were considered treatment related; these AEs are further discussed below. The highest frequency of AEs was reported among subjects receiving 6-mg/kg BTH1677 over a 3-h infusion period ( The treatment-related AEs reported by the 9 BTH1677 subjects are also reported in Table 4. In the 4-mg/kg 1-h infusion group, all AEs but 1 (moderate decreased white blood cell count) were considered treatment related. Across all subjects, treatment-related AEs reported by more than 1 subject included dyspnea (3 events [1 mild and 2 moderate]) and 2 events each of nausea (1 mild and 1 moderate), headache (both mild), paraesthesia (both mild), rash (both moderate), and flushing (both moderate). Treatment-related AEs leading to premature study discontinuation consisted of moderate rash in a 2-mg/kg subject; moderate tachycardia, moderate nausea, moderate dizziness, moderate dyspnea, moderate hyperhidrosis, moderate pruritus and moderate flushing in the two 4-mg/kg 1-h infusion subjects (only dyspnea and flushing were experienced by both subjects); and mild paraesthesia, mild hypoaesthesia, and moderate rash in a 6-mg/kg subject. Overall, moderate treatment-related AEs experienced by subjects other than those undergoing infusion reactions in the 4 mg/kg 1-h infusion group consisted of only 1 subject at 2 mg/kg (rash) and 1 subject at 6 mg/kg (rash), hence, DLT was not observed at any dose tested.
Overall, only minor deviations from the normal range were observed for hematology, chemistry, or urinalysis parameters at various times during the study. These deviations were observed in subjects treated with both placebo and BTH1677, and there were no consistent mean changes from baseline among the treatment groups. As indicated above, only 1 laboratory abnormality was considered clinically significant and reported as an AE. This AE, decreased white blood cell count, occurred in a subject dosed with BTH1677 in the 4-mg/kg 1-h infusion group and had resolved by the end of the study (day 6). Other than physical changes captured as AEs above, all of which had resolved by the end of the study, no additional abnormal physical examination findings were observed after BTH1677 administration nor were changes from baseline noted in vital signs or ECGs.

BTH1677 drug administration
Twelve subjects were randomized and dosed with 7 consecutive daily doses of BTH1677 at 1, 2, or 4 mg/kg. Based on experience from the Phase 1a study, the 1-and 2-mg/kg doses were infused over 1 h and the 4-mg/kg dose was infused over 2 h.

Safety
As shown on the right-hand side of Table 3, 6 of 9 BTH1677 subjects (66.7 %) and 2 of 3 placebo subjects (66.7 %) experienced AEs. AEs were assessed as treatment related in 2 of 9 BTH1677 subjects (22.2 %) and in 1 of 3 placebo subjects (33.3 %). As in the Phase 1a study, all AEs were mild or moderate in intensity. Furthermore, no SAEs or deaths occurred in the study. All AEs designated as treatment related occurred in subjects in the 4-mg/kg BTH1677 group. Two    subjects discontinued the study prematurely because of AEs that were considered treatment related; these AEs are discussed further below. The highest frequency of AEs was reported in the 4-mg/kg group (Table 5). Only 1 AE, headache, was reported by more than 1 subject; this AE was reported by 1 subject in both the BTH1677 1-mg/kg group (mild) and the BTH1677 4-mg/kg group (mild). AEs assessed as treatment-related in the Phase 1b study are also reported in Table 5. Three subjects experienced treatment-related AEs. In separate subjects in the BTH1677 4-mg/kg group, these included conjunctivitis (moderate) and headache (mild) and, in a placebo subject, moderate increase in blood creatinine kinase (CK). The conjunctivitis and increased CK led to premature study discontinuation in the subjects experiencing these events. DLT was not observed at any dose tested.
No clear trends in mean changes from baseline among the treatment groups were noted for hematology, clinical chemistry, or urinalysis parameters. In addition, other than conjunctivitis captured, as an AE above and which had resolved by end of study, no additional abnormal physical examination findings or changes in vital signs from baseline were observed after BTH1677 administration. There were some abnormal ECG results (or normal ECG results with variants) observed after study drug administration compared with baseline; however, these changes were not deemed clinically significant.

Pharmacokinetic analysis
Key PK results from the Phase 1a single-dose study based on NCA methods are shown in Table 6. BTH1677 exposure was generally linear in the range of 0.5 mg/kg to 6.0 mg/kg for   single iv doses based on area under the curve (AUC) values for AUC  , AUC (0-last) , and AUC (0-∞) . The PK parameter for maximum drug concentration (C max ) in this study was generally linear in the dose range of 0.5 mg/kg to 2 mg/kg, in which all subjects were dosed over 1 h, but not in the 4 mg/ kg and 6 mg/kg doses given over longer periods. This was attributed to the different infusion times used among treatments. PK parameters for clearance (CL), serum elimination half-life (t 1/2 ) and volume of distribution (V ss ) were BTH1677 dose independent, but time to maximum concentration (T max ) was dose dependent.  Table 7. BTH1677 concentration was generally linear in the range of 1.0 mg/kg to 4.0 mg/ kg for multiple iv doses on day 0 and at steady state (days 6-30), based on C max , AUC (0-24) , AUC (0-Tau) , AUC (0-last) , and AUC (0-∞) values. PK parameters CL, t 1/2 , V ss , and T max were dose independent for BTH1677 on day 0 and at steady state (days 6-30). Among the different treatment groups, comparisons were evaluated on the PK parameters for day 0 versus days 6-30. True K e on day 0 could not be estimated because of protocol design, thus t 1/2 , V ss , and CL on day 0 were calculated based on the 0-24-h serum concentration versus time profile. AUC, t 1/2 , and V ss values all tended to be larger on days 6-30 versus day 0.

Discussion
BTH1677 is a novel beta glucan PAMP being developed for the treatment of cancer in combination with tumor-targeted antibodies, as well as anti-angiogenic and checkpoint inhibitor antibodies. Here, the results of initial safety and tolerability studies of BTH1677 administered alone in healthy volunteer subjects are reported.
In healthy volunteer subjects, the safety and tolerability of single doses and of 7 consecutive daily doses of BTH1677 were investigated by monitoring AEs as well as changes in clinical laboratory parameters, physical exam findings, vital signs, and ECG responses. After adjusting for infusion rate, single doses of BTH1677 up to 6 mg/kg and repeated doses of BTH1677 up to 4 mg/kg (highest doses tested) demonstrated an acceptable safety profile and were well tolerated based on the aforementioned evaluations. Regarding the infusion reactions, it is difficult to distinguish whether the administration of diphenhydramine or simply cessation of infusion (i.e., fluid  volume distribution) was responsible for resolution. All observed AEs were mild or moderate in intensity and no DLTs were observed. Although the maximum tolerated dose (MTD) was not reached, an increase in the total number of treatment-related AEs between the 4-mg/kg (2-h infusion) dose group and the 6-mg/kg dose group in the Phase 1a study suggested that higher doses may approach a MTD and, hence, the maximum dose chosen for the Phase 1b multi-dose study was 4 mg/kg. The PK evaluations in the single-dose study demonstrated linearity with dose and a t 1/2 of approximately 30 h. The multi-dose PK evaluations demonstrated linearity with dose; however, AUC, t 1/2 and V ss values tended to be larger on days 6-30 versus day 0. In particular, mean t 1/2 values were noticeably different across studies following single-dose administration, with mean values ranging from 23.36 h to 32.75 h when blood samples were collected over 168 h in the Phase 1a study, and from 11.66 h to 13.83 h when blood samples were collected over only 24 h in the Phase 1b study. Additionally, mean t 1/2 values under steady-state conditions (after 7 daily consecutive doses of BTH1677) were noticeably different than those observed under single-dose conditions, with mean t 1/2 values ranging from 80.97 h to 109.51 h when blood samples were collected over 24 days (Phase 1b study). When using NCA methods, if blood is sampled over an insufficient time span, the apparent terminal elimination phase may be representative of a distribution half-life, as opposed to the true elimination half-life. As such, extending the timing of the blood sampling strategy may be expected to result in different assessments of the terminal elimination phase, as was observed at the first dose in the Phase 1b study (t last = 24 h, t 1/2 = 11.66-13.83 h), the only dose of the Phase 1a study (t last = 168 h, t 1/2 = 23.36-32.75 h), and the final dose of the Phase 1b study (t last = 24 days, t 1/2 = 80.97-109.51 h). Overall, the above results suggest that the true elimination phase of BTH1677 was not well characterized using NCA methods and in future studies additional models will be explored.
There were no clear dose correlations of AE occurrence or AE type with PK parameters in the multi-dose study, but in the single-dose study (excluding the 4 mg/kg 1-h infusion group, which prompted infusion rate modification for the 4 mg/kg and 6 mg/kg doses), the greatest number of AEs occurred in the 6-mg/kg group, which also had the greatest exposure to BTH1677.
Based on the favorable safety and tolerability results observed in the above described safety studies in healthy volunteer subjects, Phase 2 investigations dosing BTH1677 in combination with anti-tumor monoclonal antibodies have proceeded in cancer patients [22][23][24][25][26] and will be reported separately.