Leukemia Research 26 (2002) 721–724
`
`Pilot study of recombinant human soluble tumor necrosis
`factor receptor (TNFR:Fc) in patients with low risk
`myelodysplastic syndrome
`Craig S. Rosenfeld a,∗
`, Cindi Bedell b
`a Suite D-220, 7777 Forest Lane, Dallas, TX 239, USA
`b MSN, OCN, US Oncology, Dallas, TX, USA
`
`Received 22 October 2001; accepted 29 November 2001
`
`Abstract
`
`In low risk myelodysplastic syndrome (MDS), increased apoptosis of marrow cells is a reproducible finding. Cytokines may drive this
`apoptosis. Several studies have demonstrated elevated levels of tumor necrosis factor-alpha (TNF-␣) in MDS. Soluble tumor necrosis
`factor receptor (TNFR:Fc) can eliminate biologically active TNF in vivo. This data provided the rationale for a clinical trial of TNFR:Fc
`in low risk MDS. Eligibility was limited to cytopenic MDS patients with < 10% marrow blasts. Secondary MDS was an exclusion. The
`study design was to administer 25 mg TNFR:Fc twice a week for 10 weeks. Toxicity did not exceed grade 1. No responses were observed
`in the 10 treated patients and one had disease progression. At this dosing schedule, TNFR:Fc is unlikely to ameliorate cytopenias in low
`risk MDS. © 2002 Elsevier Science Ltd. All rights reserved.
`
`Keywords: Myelodysplastic syndrome; Tumor necrosis factor; Cytogenetics; Acute non-lymphocytic leukemia
`
`1. Introduction
`
`Despite more that two decades of myelodysplastic syn-
`drome (MDS) clinical trials, there is no FDA approved
`therapy. Recent advances in the understanding of the patho-
`physiology of MDS may provide a platform for rational
`drug development [1]. One important concept is that MDS
`can be divided into low (<10% marrow blasts) and high risk
`disease (>10% blasts) [2]. Some characteristics of low risk
`disease include lack of DNA hypermethylation, increased
`apoptosis and worse prognosis compared to patients with
`> 10% marrow blasts [2–4]. Clinical trials may be more
`productive if focused on these risk groups compared to
`French–American–British (FAB) cytologic groups. Other
`prognostic factors, such as cytogenetics, are also important.
`Elevated serum levels of tumor necrosis factor receptor
`(TNF-␣) in MDS have been reported by several investigators
`[5–8]. Increased TNF-␣ production by blood mononuclear
`cells was observed in several patients with refractory anemia
`(RA) and refractory anemia with ringed sideroblasts (RARS)
`but not RAEB or RAEB-t [9]. Furthermore, overexpression
`of TNF-␣ mRNA from marrow was detected in most cases
`
`∗
`
`Corresponding author. Tel.: +1-972-566-7790; fax: +1-972-566-3034.
`E-mail address: craig.rosenfeld@usoncology.com (C.S. Rosenfeld).
`
`of MDS but not in normal controls or AML patients [10].
`One probable source of TNF-␣ is marrow macrophages
`which are increased in MDS patients [11]. The physiological
`significance of TNF-␣ in MDS may be appreciated by (1)
`enhanced formation of CFU-GM in vitro when anti-TNF-␣
`was added to the MDS in vitro cultures (2) inverse correla-
`tion between serum TNF-␣ concentration and hemoglobin
`(3) inverse correlation between clinical response to ery-
`thropoietin and TNF-␣ levels and (4) positive correlation
`between TNF-␣ positive cells in the marrow (by immunohis-
`tochemistry) and apoptosis (by in situ end labeling of frag-
`mented DNA) [6–8,12]. The therapeutic implication is that
`inhibition of TNF-␣ activity should improve blood counts.
`Pentoxifylline can inhibit TNF-␣ mRNA transcription.
`Combination therapy with pentoxifylline + ciprofloxacin
`was not effective in one study but a triple drug regimen of
`pentoxifylline + ciprofloxacin + dexamethasone produced
`hemopoietic responses in 35% (18/51) and 28% (5/18) of
`responders demonstrated a cytogenetic response [13,14].
`These observations indicate that TNF-␣ may have an
`important role in the cytopenias of low risk MDS. The
`implication is that therapies which ameliorate the action of
`TNF may be effective in low risk MDS. As noted earlier,
`transcriptional inhibition of TNF-␣ secretion was effective
`in one study [14]. Another approach may be to directly
`
`0145-2126/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
`PII: S 0 1 4 5 - 2 1 2 6 ( 0 1 ) 0 0 2 0 1 - 6
`
`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
`Exhibit 2025, Page 1
`
`

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`722
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`C.S. Rosenfeld, C. Bedell / Leukemia Research 26 (2002) 721–724
`
`remove TNF-␣ by administration of soluble TNFR:Fc. In
`vitro, incubation of MDS marrow with TNFR:Fc enhanced
`CFU-GM formation [8]. These two observations (TNF-␣
`may be related to cytopenias and enhancement of CFU-GM
`by TNFR:Fc) formed the preclinical basis for this clinical
`trial of TNFR:Fc in low risk MDS.
`
`a sterile lyophilized powder containing 25 mg TNFR:Fc;
`40 mg mannitol, USP; 10 mg sucrose, NF; and 1.2 mg
`tromethamine (TRIS), USP per vial. TNFR:Fc was recon-
`stituted with 1.0 ml bacteriostatic water for injection, USP
`(0.9% benzyl alcohol).
`
`2. Patients, materials and methods
`
`2.1. Patients
`
`Eligibility was limited to MDS patients with < 10%
`marrow blasts. In addition, patients were required to be red
`cell transfusion dependent as defined by a requirement for
`at least two red cell transfusions in the month prior to study
`initiation, absolute neutrophil count (ANC) < 1000/␮l, or a
`platelet count < 50,000/␮l. Exclusions included ECOG per-
`formance status > 2, myelosclerosis, therapy related MDS,
`any prior transplant, or administration of MDS disease mod-
`ifying therapy in the 4 weeks prior to starting TNFR:Fc.
`All marrows were reviewed independently by a consulting
`pathologist and the principal investigator. Patients in this
`study provided written informed consent that had been app-
`roved by the Institutional Review Board of PRN Research,
`Inc.
`
`2.2. Study drug
`
`two
`Recombinant human TNFR:Fc is a dimer of
`molecules of the extracellular portion of the p75 TNF
`receptor each consisting of 235 amino acids. The two
`receptors are fused to the Fc portion of human IgG1 con-
`sisting of 232 amino acids. The gene fragments encoding
`the truncated TNFR and the Fc portion of human IgG1 are
`fused using an nucleotide expressed in a Chinese hamster
`ovary (CHO) expression vector. TNFR:Fc was supplied as
`
`Table 1
`Response criteria
`
`2.3. Treatment plan
`
`The dose of TNFR:Fc was fixed at 25 mg twice weekly.
`The justification for this dose was that there was little expe-
`rience with higher doses at the time of study initiation and
`25 mg twice weekly was known to have superior biological
`effectiveness in blocking TNF activity compared to lower
`doses. In the absence of safety data at higher doses, 25 mg
`twice weekly was chosen for this study. TNFR:Fc was ad-
`ministered subcutaneously. The planned treatment duration
`of 10 weeks was based on the delay to detect a response
`with antithymocyte globulin was 65 days (median) and 3–9
`months for cyclosporine [15,16]. Since, TNFR:Fc therapy
`could be considered a more specific type of immunosup-
`pression compared to ATG or cyclosporine, it seemed rea-
`sonable that the duration of TNFR:Fc should be similar to
`the time required for a response with ATG or cyclosporine.
`TNFR:Fc would be discontinued for grade 3 or 4 toxicity or
`disease progression.
`
`2.4. Definitions
`
`2.4.1. For response
`Disease progression was defined as the appearance ≥ 5%
`blasts on at least two occasions or a marrow with > 10%
`blasts. Toxicity was evaluated by NCI common toxicity crite-
`ria. IPSS scores were calculated retrospectively [2] (Table 1).
`
`2.4.2. Study design
`This was a phase II study that was limited to 10 evaluable
`patients.
`
`Response category
`
`Patient category
`
`Major response
`
`Minor response
`
`Red cell
`
`Red cell transfusion dependent
`
`Non red cell transfusion dependent
`
`Platelet
`
`Platelet transfusion dependent
`
`Non platelet transfusion dependent
`
`ANC
`
`Transfusion-independent
`throughout
`the study period or ≥ 2.0 g/dl rise in
`hemoglobin without transfusion
`
`> 2.0 g/dl elevation in hemoglobin
`sustained throughout the study period
`
`A sustained platelet count at or above the
`baseline value with a decrease in platelet
`transfusion requirements of at least 50%
`≥ 50% increase in platelet count and
`incremental net increase ≥ 20000/␮l
`ANC < 1500/␮l at screening: ANC
`increase exceeding 2X baseline, and
`absolute increase ≥ 500/␮l
`
`< 2.0 g/dl incremental rise in hemoglobin
`with a decrease in transfusion requirements
`of at least 50% compared to the mean
`5-week pre-study period
`≥ 1.0 g/dl elevation in hemoglobin
`sustained throughout the study period
`
`≥ 50% increase in platelet count
`with net increase < 20000/␮l
`
`ANC increase > 2X baseline, but
`absolute increase < 500/␮l
`
`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
`Exhibit 2025, Page 2
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`

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`C.S. Rosenfeld, C. Bedell / Leukemia Research 26 (2002) 721–724
`
`723
`
`Table 2
`Characteristics of the patients under studya
`FABb
`
`Age
`
`Sex
`
`Karyotype
`
`Patient
`number
`
`IPSSc
`
`Blood and marrow counts
`
`Transfusion
`dependence
`
`RBC
`
`Platelet
`
`Marrow
`blasts (%)
`
`Hgb
`(g/dl)
`
`7.3
`
`ANC
`(×103/␮l)
`3354
`
`Platelets
`(×106/␮l)
`45
`
`Int-2
`
`Int-1
`
`11.1
`
`Int-1
`Int-1
`
`Low
`Int-1
`
`Int-1
`
`6.4
`8.9
`
`9.2
`10.1
`
`10.6
`
`4535
`
`1116
`836
`
`1798
`936
`
`286
`
`335
`
`290
`64
`
`289
`31
`
`258
`
`0
`
`6.0
`
`0
`4.0
`
`2.0
`0.5
`
`2.0
`
`(cid:1)
`
`(cid:1)
`
`(cid:1)
`(cid:1)
`
`(cid:1)
`
`(cid:1)
`
`(cid:1)
`(cid:1)
`
`79
`
`76
`
`36
`76
`
`82
`73
`
`62
`
`M
`
`F
`
`F
`M
`
`F
`M
`
`F
`
`RA
`
`RARS
`
`RA
`RA
`
`RARS
`RA
`
`RA
`
`1
`
`2
`
`3
`4
`
`5
`6
`
`7
`
`8
`9
`10
`
`46, XY, del(5) (q1?5q3?3),
`r(7) (p?22q?27) [19]/47, XY,
`idem, +8[2]
`46, XX, t(2;3) (p23;q21)
`[25/25]
`46, XX
`46, XY, del(20) (q11.2q13.3)
`[4]/46, XY [3]
`46, XX
`46, XY, del(20) (q11.2q13.3)
`[5]/46, XY [2]
`46, XX, add(17) (q25)
`[15]/46, XX, del(12)
`(p12)[2]/46, XX [3]
`61
`M
`RAEB
`46, XY
`Int-1
`13.9
`1836
`21
`8.5
`58
`M
`RA
`46, XY
`Low
`8.4
`2144
`173
`2.5
`84
`M
`RA
`46,XY
`Int-1
`11.0
`1058
`38
`1.0
`a Hgb: hemoglobin; Retic: reticulocyte count; ANC: absolute neutrophil count; RBC: red blood cells; M: male; F: female. Low: low risk; Int: intermediate.
`b FAB: French–American–British classification [17]; RA: refractory anemia; RARS: refractory anemia with ringed sideroblasts.
`c Classified according to international prognostic score as described by Greenberg et al. [2].
`
`3. Results
`
`3.1. Patient characteristics
`
`The median age of the 10 patients was 74.5 years (range
`36–84) (Table 2). There were six males and four females.
`According to the IPSS, two patients were classified as
`low risk, six intermediate-1, and two intermediate-2. Five
`patients had cytogenetic abnormalities.
`
`3.2. Compliance
`
`Nine patients received all planned 20 doses of TNFR:Fc.
`TNFR:Fc was discontinued after nine doses in patient 8 due
`to leukemic progression.
`
`3.3. Toxicity
`
`There were no injection site reactions. Three patients had
`grade 1 fatigue and two of these also had grade 1 arthra-
`gia/myalgia. There were no grade 2–4 toxicities.
`
`3.4. Response
`
`There were no red cell, platelet or ANC responses. Patient
`8 progressed to AML as noted by 23% blood blasts.
`
`4. Discussion
`
`The lack of hemopoietic response to TNFR:Fc suggests
`that TNF alone is not responsible for cytopenias in MDS.
`
`including Fas ligand, IL-1␤,
`Other apoptotic cytokines,
`interferon-␥ and TGF-␤, are increased in MDS and may
`contribute to persistent cytopenias during TNFR:Fc therapy.
`Our clinical results are consistent with in vitro studies which
`demonstrated that
`inhibition of the increased caspase-3
`activity in low risk MDS did not increase colony formation.
`A potential consideration for treatment failure is that
`the dose of TNFR:Fc in this study did not eliminate TNF
`bioactivity. TNF bioactivity was not assayed in this trial.
`However, at TNFR:Fc doses approximately half of the
`dose administered in this protocol, TNF bioactivity was
`eliminated in humans given OKT3 or endotoxin [18,19].
`It is also possible that the small sample size of this study
`precluded detection of a low response rate. Responses to
`TNFR:Fc have been reported in two other MDS pilot trials.
`Partial responses in hemoglobin, platelet counts or ANC
`were observed in 10/18 evaluable patients [20]. Responses
`correlated with increased marrow cellularity and normal
`cytogenetics. In another trial, 6/14 MDS patients had
`“moderate” improvements in hemoglobin, platelet counts,
`or neutrophil counts [21]. The heterogeneity of MDS pa-
`tients may explain the responses observed in these other
`series. For example, the median age of patients in these two
`other series was about a decade younger than in this trial.
`There was a low incidence of adverse events. The most
`common toxicities were grade 1 arthralgia and myalgia.
`Interestingly,
`rheumatoid arthritis patients treated with
`TNFR:Fc often have resolution of arthalgia [22]. The fre-
`quency of disease progression (1/10) is comparable to other
`MDS trials. Thus, TNFR:Fc does not appear to accelerate
`MDS progression.
`
`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
`Exhibit 2025, Page 3
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`

`

`724
`
`C.S. Rosenfeld, C. Bedell / Leukemia Research 26 (2002) 721–724
`
`The lack of response to TNFR:Fc provides insights to the
`activity of effective agents for MDS. For example, transfu-
`sion dependence is eliminated in some MDS patients treated
`with thalidomide [23]. One of the proposed therapeutic
`activities of thalidomide is TNF-␣ inhibition. Absence of
`response to TNFR:Fc in MDS suggests that thalidomide
`functions through non TNF-␣ mechanisms.
`In summary, TNFR:Fc was well tolerated in MDS pa-
`tients. Single agent TNFR:Fc has a low likelihood of
`reversing cytopenias in low risk MDS patients.
`
`Acknowledgements
`
`The author wish to thank Jeremy Day for assistance with
`data collection.
`
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`Dr. Reddy’s Laboratories, Inc. v. Celgene Corp.
`IPR2018-01504
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