Phase I Study of rhIL7 in Humans

Based on the large body of preclinical evidence pointing to its multiple immune-enhancing properties, investigations of the possible roles of rhIL-7 in clinical immunotherapy were initiated through several phase I trials in varied subject populations as part of a long-standing collaboration for the clinical development of rhIL-7 between Cytheris, Inc. (Rockville, MD), and the National Cancer Institute (NIH,

HHS, Bethesda, MD). Three phase I clinical trials were initiated at the National Cancer Institute. The experience from a trial in adults with incurable malignancy is discussed below.

2.1. Subject Population and Study Design

Adults diagnosed with incurable nonhematologic malignancy (except primary carcinoma of the lung) were included and standard phase I eligibility criteria such as life expectancy greater than 3 months, adequate Karnofsky performance status, heart, lung, liver, kidney, and marrow functions were required. In addition, a mean value of 4 peripheral CD3+ cell count determinations over a 2-week period was required to be above 300/mm3, with a coefficient of variation of less than 20% attesting to the stability of the peripheral lymphocyte count before therapy.

The study followed a classic phase I dose escalation design with four successive cohorts of three subjects to be expanded to six in the event of one occurrence of predefined dose-limiting toxicity (DLT). If two or more patients experienced DLT, the maximum tolerated dose (MTD) was deemed exceeded. The occurrence of neutralizing IL-7 antibodies in more than one subject would have been deemed unacceptable toxicity and prompted termination of the trial. Based on preclinical animal data, the four tested doses of "CYT 99 007" (rhIL-7; manufactured and provided by Cytheris, Inc., Rockville, MD) were 3, 10, 30, and 60 ^g/kg of body weight per dose. It was given subcutaneously, every other day for 2 weeks (eight injections).

The primary study objective was the determination of safety and DLT. Secondary objectives aimed at the characterization of biological activity in humans and determination of a range of biologically active doses, based on known IL-7 properties from preclinical studies such as enlargement of lymphoid organs, increase in number of various peripheral blood lymphocyte subsets, and evidence of T cell effects by flow cytometry: (1) increase in T cell cycling (Ki67 expression), (2) down-regulation of the IL-7R (CD127 expression), and (3) Bcl-2 upregulation. Possible antitumor effects and lymphoid organ enlargement were evaluated by CT scan at baseline, at the end of therapy (day 14), and at day 28, at a minimum. Subjects with stable disease at day 28 had the possibility of longer follow-up until disease progression. rhIL-7 effects on various bone marrow mononuclear subsets were studied by H&E and immunostaining on biopsy and flow cytometry on aspirate at baseline and at the end of treatment. Those with significant changes noted at day 14 underwent a follow-up biopsy between 4 and 8 weeks.

2.2. Results

Twelve men and 4 women, ages ranging from 20 to 71 years (median 49), were treated. "CYT 99 007" was overall well tolerated clinically and MTD was not reached. Serum from all subjects was tested for the presence of anti-IL-7 antibodies on day 28. Three subjects developed non-neutralizing antibodies, but none developed detectable anti-IL-7-neutralizing antibodies. One subject showed a possible antitumor effect, albeit minimal and questionable due to the somewhat unpredictable natural history of the rare disease. This subject with resected primary CNS hemangiopericytoma and recurrent abdominal metastatic disease had a substantial relief of his abdominal pain and 20% shrinkage of the mass was noted 3 months after treatment. The abdominal disease remained stably decreased until and beyond CNS recurrence, 9 months after rhIL-7. All other subjects showed disease progression at day 28 or at the 6-week follow-up.

Biological activity, defined per protocol as 50% increase over baseline of peripheral blood CD3+ T cells, was seen with a clear dose-response effect starting with 10 ^g/kg/dose, with already a trend suggesting activity at the 3^g/kg dose (Figure 1). A 50-80% transient drop of total circulating lymphocytes following the first injection, likely representing early trafficking changes and tissue redistribution as observed in most preclinical models, was observed in all individuals at all four dose levels. Other IL-7 clinical and biological ffects expected from murine and nonhuman primate preclinical models were seen in all subjects receiving 10 ^g/kg/dose or more: spleen and lymph node enlargement (on CT scan), increase in circulating CD3+, CD4+, CD8+ populations, IL-7R a-chain (CD127) downregulation, Bcl-2 up-regulation, and marked increase in lymphocyte proliferation and activation markers (Ki67, CD71) by flow cytometry. Consistent with animal data, the IL-7Ra down-regulation was observed after 1 week of treatment, not only by decreased CD127 expression on flow cytometry but also by a greater than 50% decline of IL-7Ra mRNA copy number in CD4+ and CD8+ cell-sorted populations.

In addition, more detailed evaluation of naive, memory, and effector subsets in the CD4+ and CD8+ cell-sorted populations of T cells as well as of mature and transitional B cells was performed by multicolor flow cytometry at baseline, week 1, 2 and 3.

Figure 1. Kinetics of circulating lymphocytes during and after IL-7 treatment. X axis: days from start of treatment (treatment: days 0-14); Y axis: mean cohort value of absolute counts per mm3 for top (total lymphocytes) and middle panels, left (CD4+) and right (CD8+). Lower panels: percentage of circulating cells in cycle (Ki67+); CD4+ (left) and CD8+ (right) Cohort 1: 3 ^g/kg • ; cohort 2: 10 ^g/kg ♦---; cohort 3: 30 ^g/kg ▲-; cohort 4: 60 ^g/kg

Figure 1. Kinetics of circulating lymphocytes during and after IL-7 treatment. X axis: days from start of treatment (treatment: days 0-14); Y axis: mean cohort value of absolute counts per mm3 for top (total lymphocytes) and middle panels, left (CD4+) and right (CD8+). Lower panels: percentage of circulating cells in cycle (Ki67+); CD4+ (left) and CD8+ (right) Cohort 1: 3 ^g/kg • ; cohort 2: 10 ^g/kg ♦---; cohort 3: 30 ^g/kg ▲-; cohort 4: 60 ^g/kg

Cell numbers, cycling (Ki67+), and Bcl-2 expression were evaluated within each subset. RTEs, the most naïve circulating CD4+ cells, were defined as CD45RA+/CD31+. Among CD4+ and CD8+ cells, the main naïve, memory, and effector populations were defined respectively as CD45RA+/CD27+, CD45RA-/CD27+, and CD45RA /CD27-. rhIL-7 induced marked proliferation in all T cell subsets, but, although significant proliferation was induced in effector and memory CD4+ and CD8+ subsets, it resulted in a smaller net population expansion than for the CD4+ RTE and other naïve subsets (Figure 2). The magnitude of the resulting biological effects was variable, but the kinetics were similar in all T cell subsets and at all effective doses. After 1 week of therapy, up to 70% of circulating CD4+ RTE and naïve CD4+ and CD8+ T cells were in cycle and expressed elevated levels of Bcl-2. The combination of high proliferation rates, for the first week, and Bcl-2 up-regulation, sustained throughout the 2 weeks of treatment, resulted in net T cell expansion (up to 20-fold in some individuals), persisting one to several weeks after treatment (Figure 1). Interestingly, in spite of continuing exposure to the pharma-cologic doses of rhIL-7 during the second week of treatment and in the absence of IL-7 antibodies, the proliferation rates were halved by the end of treatment in all subsets, coinciding with IL-7Ra downregulation, then returned to baseline by week 3 (Figure 1), coinciding with the recovery of IL-7Ra surface expression and normalization of Bcl-2 expression after the cessation of treatment.

The T cell increase was primarily due to IL-7-induced peripheral expansion. There was no dilution effect of TREC numbers per 105 CD4+ sorted cells, which, consistent with preclinical data, is more suggestive of cell recruitment or expansion but does not exclude a thymic contribution. Most importantly, this large expansion of naïve cells resulted in a broadening of T cell repertoire diversity demonstrated on spectratyping analysis of the TCR VP genes and, unlike what is observed with IL-2

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Figure 2. Expansion of CD4+ and CD8+ T cell subsets in cohorts 3 and 4 (30 and 60^g/kg). Y axis: mean cohort values of percentage increase over the baseline absolute number/mm3; X

axis: Days from start of treatment. CD4+ subsets (left): RTE *______; Main naïve o-;

Memory x----- ; Effectors □ ; CD8+ subsets (right): Naïve o- ; Memory x----- ;

Figure 2. Expansion of CD4+ and CD8+ T cell subsets in cohorts 3 and 4 (30 and 60^g/kg). Y axis: mean cohort values of percentage increase over the baseline absolute number/mm3; X

axis: Days from start of treatment. CD4+ subsets (left): RTE *______; Main naïve o-;

Memory x----- ; Effectors □ ; CD8+ subsets (right): Naïve o- ; Memory x----- ;

Figure 3. Kinetics of CD20+ B cell. X axis: Days from start of treatment; Y axis: mean cohort values of absolute number/mm3 (left panel) and percentage increase over baseline absolute number/mm3 (right panel); cohort 1: 3 ^g/kg o; cohort 2: 10 ^g/kg cohort 3: 30 ^g/kg A; cohort 4: 60 ^g/kg □.

therapy, this IL-7-induced expansion resulted in a relative decrease in the proportion of CD4+/CD25Hi/FoxP3+ T-regs.

Mature CD20+ B cells showed a 40-70% decrease in absolute number of circulating cells during therapy, probably the result of trafficking alterations, with a gradual return to baseline numbers over the 2 weeks following treatment (Figure 3).

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