A Phase 1/2 Trial of Rituximab, Imprime PGG, and Alemtuzumab in the Early Treatment of Patients with High Risk Chronic Lymphocytic Leukemia (CLL)

Nandita Bose,1 Clive Zent,2 George J. Weiner,3 Betsy LaPlant,4 Nadine Ottoson,1 Steven M. Leonardo,1 Adria Jonas,1 Richard Dale Huhn,1 Ada H. Braun,1 Stephen Maxted Ansell4

1Biothera, Inc., Eagan, MN; 2University of Rochester, Rochester, NY; 3University of Iowa, Iowa City, IA; 4Mayo Clinic, Rochester, MN

Background

Objectives

Results

Hypothesis

Methods

References

Early treatment of high-risk chronic lymphocytic leukemia. Certain molecular features of CLL cells predict poor response to therapy and an aggressive clinical course (rapid progression of disease) and define patient groups with poor prognosis irrespective of whether they meet clinical criteria for treatment, as described in criteria of the NCI-IWCLL (see Methods below).1 Previous clinical trials have shown that early treatment of high-risk patients with rituximab and alemtuzumab can:

• increase the rate of complete response (CR) compared with either rituximab or alemtuzumab alone, and• delay the need for treatment with cytotoxic chemotherapy.2

Achievement of complete response, especially in the absence of residual disease (as measured by immunohistochemistry) was specifically associated with prolongation of time to need for treatment.2,3 Treatment of high-risk CLL with rituximab and alemtuzumab induced a 37% complete response rate and a median time to need for retreatment of 4.4 yr.2 Rituximab and alemtuzumab are both known to activate complement upon opsonization of target cells.4,5

Imprime PGG. Imprime PGG is a novel innate immune cell modulator consisting of soluble 1,3/1,6 beta-glucan derived from a proprietary strain of Saccharomyces cerevisiae. Imprime PGG is a pathogen-associated molecular pattern (PAMP) that binds complement receptor 3 (CR3) on innate immune cells (macrophages, monocytes and neutrophils), priming them to exert anti-tumor activity against complement (iC3b)-opsonized tumor cells.6-9 Naturally occurring anti-beta glucan antibodies (ABA) are required for binding of Imprime PGG to innate immune cells and ABA concentrations are correlated with cell binding and activation.7,10

Primary objective: to assess the safety and efficacy of the combination of Imprime PGG 4 mg/kg in combination with rituximab and alemtu-zumab for early treatment of patients with high-risk CLL

• Adverse event (AE) profile• Overall response rate (ORR)• Complete (CR) and partial (PR) rates

Exploratory objectives:• To assess whether there is a relationship between endogenous anti-beta-glucan antibody levels and Imprime PGG binding to neutro-

phils and monocytes in CLL patients• To assess relationships between endogenous anti-beta-glucan antibody levels and clinical responses to treatment with rituximab,

alemtuzumab and Imprime in CLL patients

Patient characteristics. Fourteen patients were enrolled at the Imprime PGG dose level of 4 mg/kg. Clinical characteristics of the subjects are shown in Table 1.

ABA levels and clinical outcomes. Using a cutoff value of 235 RAU/mL for “positive” ABA level, the relative rate of CR was compared between positive and negative subjects. As shown in Figure 2, there was no apparent difference.

Macrophage antibody-dependent cellular phagocytosis. Phagocytosis of rituximab-treated CD20+ tumor cell lines by macrophages in vitro was enhanced by Imprime PGG, as shown in Figure 3.

Table 1: Figure 1:

• Imprime PGG in combination with rituximab and alemtuzumab will be safe and well tolerated • The addition of Imprime PGG to early treatment with rituximab and alemtuzumab will increase the CR rate in patients with high-risk CLL• Increase in CR rate in patients with high-risk CLL will extend the time to need for subsequent treatment

Trial design. The clinical investigation described herein represents the results of early treatment of high-risk CLL patients with Imprime PGG obtained from a Phase 1/2 study. Results of Phase 1, which evaluated escalating Imprime PGG doses of 1, 2 and 4 mg/kg have been previ-ously reported.11 The 4 mg/kg Imprime PGG dose was chosen for further evaluation in Phase 2, and subjects treated at the Phase 2 dose of 4 mg/kg combined with rituximab and alemtuzumab are reported here. Results reflect those available as of the date of data cutoff on Sep-tember 23, 2014.Subjects. Previously untreated patients with Rai stage 0-2 high-risk CLL (as diagnosed by standard immunophenotypic criteria) and none of the standard NCI-IWCLL criteria for requiring treatment1 were eligible for inclusion in this trial conducted at the Mayo Clinic, Rochester, MN. All subjects gave informed consent. High risk status was defined as one or more of the following poor prognostic factors:

• chromosome 17p13 deletion [del(17p)]• chromosome 11q22.3 deletion [del(11q)]• unmutated IgHV (<2%) (u-IgHV) or VH3-21 gene segment usage (irrespective of IgHV mutation status) together with either CD38 expres-

sion (≥30%) (CD38+) and/or ZAP70 expression (≥20%) (ZAP70+) Adequate renal and hepatic function and ECOG performance status 0-2 were required.

Exclusion criteria included New York Heart Association Class III or IV heart disease, recent (<1mo.) myocardial infarction, pregnancy, un-controlled infection, HIV/AIDS virus infection, serological evidence of active hepatitis B or C infection, active autoimmune complications, or other active primary malignancy requiring treatment or limiting life expectancy to <2 years.Therapy. Imprime PGG was administered intravenously (IV) at 4 mg/kg on days 1, 5, 10, 17, 24 and 31. The first dose was accompanied by pre-medication with acetaminophen, diphenhydramine and hydrocortisone. Alemtuzumab was administered subcutaneously beginning with test doses of 3-10-30 mg/day on days 3-5 as tolerated with premedication with acetaminophen and diphenhydramine, then at 30 mg on day 8 and thereafter 3x/week. Rituximab was given at 375 mg/m2/week IV for four doses beginning on day 10 with standard premedi-cation, antiviral and anti-pneumocystis prophylaxis according to the package insert. The total period of treatment was 5 weeks which was followed by observation visits at 3, 6, 9 and 12 months and every 6 months thereafter for up to an additional 4 years. Response evaluation. Clinical responses were evaluated according to the NCI-IWCLL criteria of 2008 without imaging.1

• Complete response (CR): (1) resolution of palpable lymphadenopathy and hepatosplenomegaly on physical examination; (2) no consti-tutional symptoms; (3) CBC: neutrophils >1.5x109/L, platelets >100x109/L, hemoglobin >11.0g/dL, absolute lymphocyte count <4x109/L; (4) normocellular 3-month post-treatment bone marrow exam with <30% lymphocytes and no CLL nodules

• Complete response with incomplete marrow recovery (CRi): all of the above criteria for CR but with persistent cytopenia not related to CLL1

• Stringent CR (sCR): all of the above criteria for CR with no detectable minimal residual disease on histochemical examination of marrow • Nodular PR (nPR): all of the above criteria for CR but with nodules of CLL cells in the marrow• Partial response (PR): (1) ≥50% decrease in absolute lymphocyte count, and (2) ≥50% decrease in sum of the products of perpendicular di-

ameters of enlarged lymph nodes and size of liver and/or spleen by physical exam, and (3) ≥50% improvement in ANC, platelet count and hemoglobin above baseline, or ANC >1.5x109/L, platelet count >100x109/L and hemoglobin >11 g/dL

• Progressive disease (PD): (1) ≥50% increase in sum of products of at least 2 palpable lymph nodes on 2 consecutive determinations 2 weeks apart, or (2) the appearance of new palpable lymph nodes >1.5 cm, or (3) ≥50% increase in the size of the liver and/or spleen as determined by measurement below the respective costal margin on 2 consecutive determinations 2 weeks apart, or appearance of hepatomegaly or splenomegaly which was not previously present at baseline, or (4) a ≥50% increase in the ALC above the lowest ALC recorded since the start of treatment and with an ALC of at least 5x109/L. For patients who achieved CR or nodular PR, progression was defined as recurrence of a circulating leukemia cell close with ALC >5x109/L or recurrence of adenopathy >1.5 cm not due to tumor flare.

• Stable disease (SD): criteria not met for response or PD.

Clinical statistical methods. On the basis of the results of a previous study of the same antibody regimen in a comparable population in which the observed CR rate was 37%, it was proposed that a CR rate of 30% with the addition of Imprime PGG to rituximab and alemtuzum-ab would not be of interest while a CR rate of ≥50% would be of interest. A minimum of 17 and maximum of 39 subjects were needed to test the null hypothesis that the CR rate was ≤30% using a one-stage three-outcome design with an interim analysis.12 Enrollment into the phase 2 portion was terminated at 14 subjects because of diminishing accrual related to competing clinical trials for this patient population.

Time-to-event endpoints were estimated using the Kaplan-Meier method:• Time to disease progression: time from registration to the earliest date of documented PD• Time to subsequent treatment: time from registration to the date of initiation of subsequent treatment

Evaluation of patient ABA levels and possible relationship to clinical response. Baseline sera collected from patients treated with Imprime PGG, rituximab and alemtuzumab were analyzed for ABA levels by a qualified enzyme-linked immunosorbant assay.10 Titers were converted mathematically to “relative antibody units” (RAU) by comparison to standard dilutions. Based previous evaluations of binding of Imprime PGG to neutrophils of healthy volunteers detected by flow cytometry and correlation with ABA levels, an ABA level of ≥ 235 RAU/mL was considered predictive of binding, i.e., “positive”. Patients were categorized as CR (sCR, CR or CRi) or PR (nPR, PR) plus SD and ABA levels were compared between the two groups.

Evaluation of phagocytosis of CD20+ cells by M2c macrophages derived from Imprime-treated monocytes. M2c were prepared by CD14 cell selection from whole blood of healthy volunteers and cultured in the presence of M-CSF and IL-10. Antibody-dependent cell-mediat-ed phagocytosis (ADCP) by M2c was evaluated with violet-labeled CD20+ tumor cell lines Raji and Z138 in the presence of anti-CD20 mAb rituximab with or without Imprime PGG. After mixing with macrophages and incubation at 37°C for 4 hours, treated cells were immunos-tained for CD11b, fixed and analyzed by flow cytometry with counting beads. ADCP was interpreted as cells positive for both CD11b and violet (double positive), divided by the total tumor cells.

1. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diag-nosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leu-kemia updating the National Cancer Institute-Working Group 1996 Guidelines. Blood 111(12):5446-56, 2008

2. Zent CS, Call TG, Shanafelt TD, et al. Early treatment of high-risk chronic lymphocytic leukemia with alemtuzumab and rituximab. Cancer 113(8):2110-2118, 2008

3. Strati P, Keating MJ, O’Brien SM, et al. Outcomes of first-line treat-ment for chronic lymphocytic leukemia with 17p deletion. Hae-matologica 99(8):1350-5, 2014

4. Kennedy AD, Beum PV, Solga MD, et al. Rituximab infusion pro-motes rapid complement depletion and acute CD20 loss in chron-ic lymphocytic leukemia. J Immunol. 172:3280-3288, 2004

5. Baig NA, Taylor RP, Lindorfer MA, et al. Complement dependent cytotoxicity in chronic lymphocytic leukemia: ofatumumab en-hances alemtuzumab complement dependent cytotoxicity and reveals cells resistant to activated complement. Leuk Lymphoma 53(11):2218-27, 2012

6. Bose N, Chan AS, Guerrero F, et al. Binding of soluble yeast β–glucan to human neutrophils and monocytes is complement-de-pendent. Front Immunol 4:230, 2013

7. Antonysamy M, Bose N, Chan A, et al. Endogenous anti β-glucan antibodies, a potential predictive biomarker for the efficacy of

soluble yeast β-1,3/1,6 glucan (Imprime PGG) immunotherapy in cancer patients. J Immunol 192:73.9 (abst), 2014

8. Li B, Allendorf DJ, Hansen R et al. Yeast beta-glucan amplifies phagocyte killing of iC3b-opsonized tumor cells via complement receptor 3-Syk-phosphatidylinositol 3-kinase pathway. J Immunol 177(3):1661-9, 2006

9. Qi C, Cai Y, Gunn L, et al. Differential pathways regulating innate and adaptive antitumor immune responses by particulate and soluble yeast-derived β-glucans Blood 117(25):6825-36, 2011

10. McMurray D, Harrison B, Ertelt K, et al. Distribution, cutoff, and functional significance of a potential biomarker for Imprime PGG, an experimental cancer immunotherapeutic, in a healthy subject population. In: Procedings of AACR meeting on Tumor Immunolo-gy and Immunotherapy: A New Chapter, Dec 1-4, 2014, Orlando FL, Abst. A08

11. Zent CD, LaPlant BE, Call TG, et al. Early treatment of high-risk chronic lymphocytic leukemia with alemtuzumab, rituximab and PGG beta glucan: A phase 1 clinical trial. Blood (ASH Annual Meeting Abstracts) 120:1792, 2012

12. Sargent DJ, Chan V, Goldberg RM. A three-outcome design for phase II clinical trials. Control Clin Trials 22(2):117-25, 2001

Abstract 7078

Adverse events. All 14 subjects had adverse events. Table 2 shows the number of subjects with identified adverse events considered by in-vestigators to be possibly, probably or definitely related to study treatment by treatment period vs. post-treatment observation period. AEs considered by investigators to be unlikely or unrelated to study are not shown.

Clinical treatment response. Clinical response to treatment, duration of response (DOR), the status of response as of the date of data cutoff (Status), the time to need for retreatment (TTR) and the retreatment status are shown in Table 3. Of the 14 subjects, 13 responded to treatment resulting in an ORR of 93% (CR=7, CRi=2, nPR=1, PR=3). With a median follow-up time of 18.0 mo. (range 9.5 – 28.0), the median DOR and the median TTR have not been reached. Kaplan-Meier analysis of TTR is shown graphically in Figure 1. All subjects were alive as of the date of data cutoff.

Table 2:

Table 3:

* Subjects who had not progressed or who were not retreated were censored as of the last date known to be event-free. § PST = pentostatin; CTX = cyclophosphamide

Comments• The combination of Imprime PGG 4 mg/kg with rituximab and alemtuzumab in treatment of high-risk CLL was safe and well-tolerated.• The rate of complete response (sCR+CR+CRi) to treatment with Imprime PGG, rituximab and alemtuzumab was 64% which compares fa-

vorably to the previously observed CR rate of 37% with rituximab and alemtuzumab in a group of patients with similar risk characteristics.2

• Responses of high-risk CLL to the regimen of Imprime PGG, rituximab and alemtuzumab were durable. Seven of 9 patients who obtained complete responses were progression-free as of the date of data cutoff. Follow-up time is yet insufficient to compare TTR to prior studies.

• The combination of Imprime PGG with rituximab and alemtuzumab may be a useful approach to extending TTR in high-risk patients who would be unlikely to tolerate chemotherapy, e.g., due to age or co-morbidities.

• While ABA levels correlated with binding of Imprime PGG to neutrophils and monocytes in CLL patients (data not shown), correlation with clinical response was not seen in this study. It is not yet understood whether healthy volunteer ABA levels for Imprime PGG binding to neu-trophils are relevant for cancer patients, in general, or CLL patients, in particular.

• Antibody-dependent cellular phagocytosis of rituximab-treated CD20+ cells by M2c macrophages in vitro was increased by Imprime PGG. This may contribute to anti-tumor mechanisms in B cell malignancies.

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