Leukemiu ond Lymphoma, Vol. 30. pp. 525-517
`Reprints available directly from the publisher
`Photocopying permitted by license only
`
`D 1998 OPA (Overseas Publishers Association) Amsterdam B.V.
`Published in the Netherlands by Hanvood Academic Publishers
`Pnnted in Malaysia
`
`Clinical Trials With an Anti-CD25 Rich A-Chain
`Experimental and Immunotoxin (RFTS-SMPT-dgA)
`in Hodgkin’s Lymphoma
`
`R. SCHNELLa, E. VITETTAb, J. SCHINDLERb, S. BARTH’, U. WJNKLER”, P. BORCHMANN”, M. L. HANSMAN“,
`V. DIEHL”, V. GHETIEb and A. ENGERT”.*
`
`“Klinik I fuer Innere Medizin, Univer.ritaet zu Koeln, 50924 Koeln, Germany; bCancer Immunobiology Center and
`Department of Microbiology, The Univer,si@ of Texas, Southwestern Medical Center, Dallas, Texas 75235, U.S.A. and
`“Senkenbergisches Zentrum der Pathologie, Johann Wolfgang Goethe- Universitaet Frankfurt a.M., 60596 Frankfurt a.M., Germany
`
`(Received 20 September 1997)
`
`Immunotoxins (ITS) consisting of a cell-binding component and a potent toxin were developed
`as a new class of biological anti-tumor agents to improve adjuvant therapy. Hodgkin’s lym-
`phoma (HL) has been demonstrated to be an excellent target for ITS because high concentra-
`tions of lymphocyte activation markers such as CD25 and CD30 are expressed on Hodgkin
`and Reed-Sternberg (H-RS). Several ITS against these antigens have shown potent antitumor
`effects against H-RS cells in vitro and in different HL animal models. On the basis of its supe-
`riority in preclinical models, the anti-CD25 IT RFTS-SMPT-dgA was subsequently evaluated
`in a phase I study in patients with refractory Hodgkin’s lymphoma. The IT was constructed by
`linking the monoclonal antibody (Moab) RFT5 via a sterically hindered disulfide linker
`(SMPT) to deglycosylated ricin A-chain (dgA). All 15 patients enrolled in this trial were heav-
`ily pretreated with a mean of five different prior therapies. The IT was administered intra-
`venously over four hours on days 1-3-5-7 for total doses per cycle of 5, 10,15, or 20 mg/m2.
`Side effects were reversible and related to the vascular leak syndrome (VLS), i.e. decrease in
`serum albumin, edema, weight gain, hypotension, tachycardia, myalgia, and weakness. In all
`three patients receiving 20 mg/m2 NCI toxicity grade 111 was observed. Thus, 15 mg/m2 is the
`maximal tolerated dose (MTD) of RFTS-SMPT-dgA. 50% of the patients developed human
`anti-ricin A-chain antibodies (HARA) and/or human anti-mouse antibodies (HAMA). Clinical
`results included two partial remissions (PR), one minor response (MR), three stable disease
`(SD) and nine progressive disease (PD). In an extension of the phase I trial, five additional
`patients have been treated at the MTD.
`
`Keywords: Immunotoxin, RFTS-SMPT-dgA, CD25, Hodgkin’s lymphoma
`
`*Corresponding author. Fax: +49 (0) 221 / 478-63 83.
`
`525
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`

`526
`
`INTRODUCTION
`
`R. SCHNELL et al.
`
`Polychemotherapy and extended field radiotherapy
`have improved the remission rates of Hodgkin's lym-
`phoma (HL) from less than 5% in 1963 to about 80%
`at present."] However, 30-50% of patients with
`advanced stages die due to relapsed disease.[21 Data
`from other malignancies including colorectal
`myeloid leukemia,[41 or non-Hodgkin's lymphoma
`(NHL)['] indicate that small numbers of residual tumor
`cells remaining after first-line treatment are the source
`of relapses. Thus, the elimination of residual tumor
`cells after first-line treatment might improve the out-
`come in malignant diseases including HL.
`There are several approaches to the elimination of
`residual tumor cells including T-cell stimulation, vac-
`cination or selective destruction using Moabs or
`Moab-based constructs.[&*] Since native Moabs have
`been ineffective against H-RS cells thus far,['] con-
`structs consisting of a specific cell-binding moiety and
`a potent toxin have been developed. These immuno-
`toxins (ITS) are ideally suited to kill residual tumor
`cells in HL for several reasons: 1. H-RS cells express
`large numbers of surface antigens such as CD15,[lo1
`IRac,[''] CD30,[12] CD25,[13,141 CD40,[I5] and CD80
`(B7-
`which are present only on a minority of nor-
`mal human cells. 2. The number of H-RS cells that
`need to be killed is relatively small. 3. Hodgkin's
`tumors are well vascularized, suggesting good access
`of the IT to the target cells. 4. The mechanism of cell
`killing of ITS is completely different from that of con-
`ventional agents. 5. ITS are capable of killing dormant
`non-dividing cells.
`There are two lymphoid activation markers, CD25
`and CD30, which have attracted great interest as tar-
`gets for ITS in HL. Our group has evaluated most
`Moabs available against CD25 and CD30 in terms of
`their ability to form ricin A-chain ITS for possible clin-
`ical use in HL. In this paper, we will focus on ITS
`against CD25.
`CD25 is the a-chain of the IL-2 receptor which is
`composed of three different membrane components
`termed a-, p-, and y-chain."" Combinations of these
`chains result in different forms of the IL-2 receptor
`with distinct binding affinities for IL-2. CD25 is a 55 kd
`
`glycoprotein which is not expressed on resting lympho-
`cytes and stem cells but which is efficiently induced
`upon T-cell activation. CD25 binds IL-2 with low
`affinity without signal transduction activity. IL-2
`receptors have been detected in high copy numbers in
`hematopoietic malignancies and autoimmune disorders
`including HL.'I8.I9]
`
`Preclinical Evaluation of Ricin A-Chain ITS
`Against CD25
`
`Twenty-three different Moabs against CD25 were
`tested in an indirect assay"']
`for their potential use as
`ITS against Hodgkin-derived cell lines such as L428
`and L540.["' The five most potent Moabs were subse-
`quently coupled to deglycosylated ricin A-chain via
`SMPT, and the cytotoxicity of the constructed ITS was
`determined in a standard 3H-leucine uptake assay.[221
`The most potent IT, RFT5-SMPT-dgA, inhibited the
`protein synthesis of L540 cells by 50% at a concentra-
`M, which is identical to that of
`tion (IC50) of 7 x
`native ricin under the same experimental condi-
`
`t i o n ~ . [ ~ ~ ] RIT5 itself showed no major crossreactivity
`with any tissues other than lymphoid, where a few
`large cells in tonsils and lymph nodes were stained
`(Table I) .r231
`The anti-tumor activity was evaluated in triple-
`beige nude mice with subcutaneously growing solid
`Hodgkin's tumors of 60-80 mm3. A single i.v. applica-
`tion of 8 pg (in terms of A-chain) RFT5-SMPT-dgA[231
`induced permanent complete remissions in 78% of the
`animals. In contrast, 100% of the control animals
`showed progressive tumor growth. The tumor size at
`the time of IT application significantly influenced the
`response rates: only 37.5% complete remissions
`occurred in animals with larger tumors (10 mm diame-
`ter), whereas 100% of the mice with smaller tumors
`(3 mm) achieved complete remissions. Treatment of
`disseminated growing Hodgkin's lymphoma was per-
`formed in SCID mice:[241 After administration of 8 pg
`RFT.5-SMPT-dgA (in terms of A-chain) intraperi-
`toneally one day after i.v. inoculation of 1 x lo7
`L54OCy Hodgkin-derived cells, complete remissions
`were observed in 95% (22123). In contrast, 92% (34/37)
`of untreated SCID mice showed signs of progressive
`
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`

`ANTI-CD25 RICIN A IT TRIAL
`
`527
`
`TABLE I Normal tissue staining patterns of CD25 antibodies
`
`Tissue
`
`B-B 10
`
`anti-CD25 antibody
`B-F2
`RFT5y2a
`
`Adrenal
`-
`Brain (cortex)
`-
`-
`Brainstem
`Breast
`-
`Cerebellum
`-
`Cervix
`-
`Colon
`-
`Gall bladder
`-
`Heart
`-
`Ileum
`-
`Kidney
`-
`Liver
`-
`Lung
`-
`Lymph node
`-
`Mucosa (nasal)
`-
`Oesophagus
`-
`-
`Ovary
`Pancreas
`-
`Parathyroid
`-
`Spleen
`-
`Stomach (antrum)
`-
`Stomach (body)
`-
`Testis
`-
`Thyroid
`-
`-
`Thyroid (AI)
`Thyroid (Hashimoto’s)
`-
`-*
`Tonsils
`-
`Uterus
`Vagina
`-
`+++
`Hodgkin’s disease
`* Rare cells within lymphoid tissue stain positively.
`
`-
`-
`-
`-
`-
`++
`-
`-
`-
`++
`+++
`-
`-
`-
`-
`-
`-
`-
`-
`-
`+
`+
`-
`-
`-
`-
`-*
`-
`-
`+++
`
`-
`-
`-
`-
`-
`
`-
`-
`-
`-
`
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-*
`-
`-
`+++
`
`RFT5yI
`-
`-
`-
`-
`-
`
`-
`-
`-
`-
`
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`- *
`-
`-
`+++
`
`tumor growth. The mean survival time (MST) of SCID
`mice treated with RFT5-SMPT-dgA one day after
`tumor challenge was >I80 days as compared to 36 days
`in PBS and 48 days in Moab-treated controls (Fig. 1).
`
`Clinical Phase I Trial with the CD25 Rich
`A-chain IT RFT5-SMPT-dgA
`
`RFT5-SMPT-dgA was the most potent IT against
`human Hodgkin’s lymphoma in vitro and in animal
`models combining strong staining of H-RS cells and
`
`little c r o s ~ r e a c t i v i t y . ~ ~ ’ ~ ~ ~ ~ Thus, RFT5-SMPT-dgA
`was selected for an FDA-approved phase I trial in
`patients with relapsed refractory HL.[251 The patient
`formulation and characteristics of the IT are summa-
`
`rized in Table 11. The study design was in accordance
`with the Declaration of Helsinki. The trial was
`approved by the Ethics Committees of the University
`of Cologne and the University of Texas, Southwestern
`Medical Center, and performed under Food and Drug
`Administration Investigational New Drug Application
`(IND No 4989). Before treatment, all patients gave
`written informed consent. The IT was given intrave-
`neously (i.v.) in 100 ml isotonic saline over 4 hours on
`days 1-3-5-7. Cohorts with a minimum of three
`patients were treated with escalating doses of 5, 10,
`15, and 20 mg/m2. If one patient experienced grade I11
`toxicity three additional patients were enrolled at this
`dose level. If three grade I11 toxicities occurred at one
`dose level, then the previous dose level was regarded
`
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`

`528
`
`R. SCHNELL et al.
`
`0
`
`2 0
`
`6 0
`8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0
`4 0
`Days after tumor challenge
`
`FIGURE 1 Mean survival time of SCID mice bearing disseminated Hodgkin-derived LS40Cy tumors. One day after tumor challenge with
`40pg of the native antibody RFT5 (.-.-) or 8pg (in terms of A-chain)
`1 x 10' LS40Cy cells groups of 10 mice were treated with PBS (-),
`of the IT RFT5-SMPT-dgA (- - -).
`
`as the MTD. If two patients at one dose level experi-
`enced a grade I11 toxicity and one patient at the next
`dose level a grade IV toxicity, then the MTD was the
`dose at which the grade I11 toxicities occurred. If one
`patient experienced a grade I11 toxicity and another
`patient experienced a grade IV toxicity, then the previ-
`ous dose level was defined as the MTD.
`A total of 15 patients with refractory or relapsed (22
`relapses) progressive HL were included in the phase I
`trial. Five additional patients treated at the MTD are
`included in this report.
`
`Patient Characteristics and Tumor Pathology
`
`The demographic data of the 15 patients treated in the
`phase I trial and the additional five patients treated at
`MTD are listed in Table 111. Twelve patients were
`male and eight were female. The median age was 29
`(range 19 to 38). Histopathology at first presentation
`was nodular sclerosis in most cases (14), followed by
`mixed cellularity (4), lymphocyte depletion (l), and
`lymphocyte predominance (1). Of 20 patients, eight
`suffered from primary progressive disease. Most
`
`TABLE 11 Patient formulation and characteristics of RFTS-SMPT-dgA
`
`Sterility
`Endotoxin (Limulus amebocyte lysate assay)
`Sodium dodecyl sulfate-polyacrylamide gel
`electrophoresis (% as M, 180.000 band)
`Binding to thiopropyl-Sepharose 6 B
`(free-sulfhydryl groups)
`A-chain activity relative to native dgA
`Phytohemagglutinin activity for human
`peripheral blood mononuclear cells
`Antibody binding activity relative to native antibody
`Antibody subclass
`Target antigen
`Crossreactivity
`ICso against L540
`LDSO
`
`Sterile
`0.3 Endotoxin unitdm1
`
`70%
`
`none
`100%
`
`2.8 x lo-" M
`100%
`IgG 1
`CD25
`Activated lymphocytes
`7 x lo-'* M
`9 pg/g mouse
`
`Abbreviations: IC50, inhibitory concentration of 50%; LD50, lethal dose of 50%; dgA,
`deglycosylated rich A-chain.
`
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`

`ANTI-CD25 RICIN A IT TRIAL
`
`TABLE 111 Characteristics of patients treated with RFTS-SMFT-dgA
`
`Age
`
`Gender
`
`Histology
`
`23
`27
`34
`33
`31
`28
`32
`19
`34
`31
`20
`33
`28
`29
`33
`37
`19
`38
`23
`36
`
`f
`m
`m
`m
`f
`m
`f
`m
`m
`m
`f
`m
`m
`f
`m
`m
`m
`f
`f
`f
`
`LP
`NS
`NS
`MC
`NS
`NS
`NS
`MC
`NS
`MC
`MC
`NS
`NS
`NS
`NS
`LP
`NS
`NS
`NS
`NS
`
`Primary
`resistant
`N
`N
`N
`N
`N
`Y
`N
`Y
`N
`Y
`N
`N
`Y
`Y
`N
`Y
`Y
`N
`Y
`N
`
`Prior
`therapies
`5
`5
`8
`5
`4
`6
`5
`2
`6
`5
`3
`3
`3
`6
`3
`5
`6
`4
`3
`3
`
`ABMTI
`PSCT
`N
`Y
`Y
`Y
`Y
`N
`Y
`N
`Y
`Y
`N
`Y
`N
`N
`N
`N
`Y
`N
`Y
`N
`
`Karnofsky
`index
`50
`70
`70
`50
`90
`60
`80
`80
`70
`90
`90
`80
`60
`90
`90
`70
`70
`80
`80
`80
`
`529
`
`Stage
`
`1VB
`IVA
`IVB
`IVB
`IVA
`IVB
`IVA
`IVA
`IVA
`IVA
`IIA
`IVB
`IVB
`IVA
`IIA
`IIA
`IVB
`IIIA
`IVA
`IVB
`
`ID
`
`I
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`
`dose
`(mglm2)
`5
`5
`5
`10
`10
`10
`15
`15
`15
`15
`15
`15
`20
`20
`20
`15
`15
`15
`15
`15
`
`Abbreviations: f, female; m, male; NS, nodular sclerosis; MC, mixed cellularity; LP, lymphocyte predominance; LD, lymphocyte depletion;
`N, no; Y, yes; ABMT, autologous bone marrow transplantation; PSCT, peripheral stem cell transplantation; ID, identification number.
`
`patients had been heavily pretreated with an average
`of 4.5 different therapies (range 2-8) including high-
`dose chemotherapy (HDCT) and autologous bone
`marrow transplantation in 50%. All but one patient
`(no. 8) had also received extensive radiotherapy. At
`study entry, the median performance status as mea-
`sured by the Karnofsky index was 70 (range: 50-90).
`Most patients presented with advanced disease (stage
`IV: 16/20) and 8 had B symptoms. Six patients were
`on steroids to control excessive fever or sweating.
`The evaluation of CD25 expression on H-RS cells
`was hindered by the difficulty in obtaining sufficient
`biopsy material (Table IV). A total of nine lymph
`node biopsies were performed of which eight con-
`tained more than 30% of H-RS cells expressing the
`CD25 antigen. In two additional patients, material
`from the lung (no. 9) and liver (no. 12) contained
`>30% CD25+ H-RS cells. CD25 expression was less
`pronounced than CD30 except in patient no. 10, who
`demonstrated positive staining in 40% vs 10%. In
`general, the percentage of CD25-positive small lym-
`phoid cells in the tissues analyzed was less than 1%
`(data not shown).
`
`Toxicity
`
`None of the six patients treated at the first two dose
`levels (5 mg/m2 and 10 mg/m2) experienced toxicity
`higher than grade 11. Since patient no. 7 experienced
`grade I11 toxicity (myalgia, CK,,,
`200 U/ml) at the
`15 mg/m2 dose level, three further patients had to be
`treated at that dosage (total of six). Only one of the
`additional patients experienced grade I11 toxicity (dys-
`pnea) (patient no. 12), thus dose escalation was possi-
`ble. Three patients were treated at 20 mg/m2. Patient
`no. 15 received only three of the four planned IT infu-
`sions due to a grade IV myalgia on day 6 with a CK
`max of 1,500 U/ml. This reaction was rapidly
`reversible after the cessation of IT therapy and treat-
`ment with 24 mg/d dexamethasone for four days. At
`least one grade 111 toxicity was observed in all 3
`patients at this dose level. Two patients experienced
`VLS grade I11 with weight gain > 15 Ib; two patients
`had grade I11 nausealvomiting requiring antiemetics,
`and tachycardia (>140 bpm at rest) occurred in two
`patients, making the administration and beta-blocker
`necessary. Thus, the MTD of RlT5-SMPT-dgA was
`
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`530
`
`R. SCHNELL et al.
`
`TABLE IV Staining of biopsy material of patients treated with RFf5-SMPT-dgA and clinical response
`
`~~
`
`ID
`
`I
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`
`~
`
`dose
`(mg/m2)
`5
`5
`5
`10
`10
`10
`15
`15
`15
`15
`15
`15
`20
`20
`20
`15
`15
`15
`15
`15
`
`__
`
`Biopsy
`
`n.d.
`Lymph node
`Lymph node
`n.d.
`n.d.
`Lymph node
`n.d.
`n.d.
`Lung
`Lymph node
`Lymph node
`Liver
`n.d.
`n.d.
`n.d.
`n.d.
`Lymph node
`Lymph node
`Lymph node
`Lymph node
`
`_ _ _ _ _ _ _ _ _ _ ~ __
`
`% CD25’
`H-RS-cells
`n.d.
`230%
`n.e.
`n.d
`n .d
`230%
`n.d
`n.d
`230%
`240%
`235%
`230%
`n.d
`n.d
`n.d
`n.d.
`250%
`240%
`230%
`230%
`
`Courses
`
`Response
`
`2
`2
`2
`I
`2
`1
`2
`2
`2
`4
`2
`2
`I
`2
`2
`2
`4
`2
`2
`2
`
`PD
`MR
`SD
`PD
`SD
`PD
`SD
`PD
`PD
`PR
`PD
`PR
`PD
`PD
`PD
`PD
`MR
`SD
`SD
`SD
`
`Abbreviations: ID, identification number; H-RS-cells. Hodgkin-ReedBternberg-cells; n.d., not done; n.e., not
`evaluable; PD, progressive disease; MR, minor response; SD: stable disease; PR: partial remission; patients
`treated at MTD are marked with bold numbers.
`
`defined at 15 mg/m’. None of the five additional
`patients treated at MTD had a grade 111 toxicity.
`All the adverse events according to standard NCI
`grading criteria are listed in Table V. The most fre-
`quent side effects (zNCI grade I) were myalgia
`(1 9/20), weight gain (19/20), weaknesdfatigue
`(1 9/20), hypoalbuminemia (17/20), nauseahomiting
`(12/20), dyspnea (10/20), hypotension (9/20), and
`tachycardia (7/20). All patients experienced VLS with
`a tendency towards greater severity at higher doses.
`Hematologic toxicity was observed only in patient
`no. 7 showing thrombocytopenia grade 11. Hemoglobin
`and leukocyte count were unaffected in all patients. A
`variety of different antigens was analyzed in peripheral
`blood mononuclear cells (CD3, CD4, CD8, CD16,
`CD19, CD25, CD45R0, CD45RA, CD45RW, CD56,
`CD7 1). Only the CD25-positive peripheral blood
`mononuclear cells demonstrated significant changes
`during treatment with RFT5-SMPT-dgA. CD25/CD3-
`positive and CD25/CD4-positive PBLs showed a sig-
`nificant decrease ( p < 0.001) of median fluorescence
`intensity (MFI) immediately after the start of IT appli-
`
`cation. This persisted during the period of treatment
`but recovery occurred thereafter. The reduction of
`CD25+ cells probably reflects a destruction of CD25+
`cells by the IT. Another explanation could be a modu-
`lation of CD25, since the Moab used for the FACS
`analysis of CD25 (anti-TAC) is cross-blocked by the
`Moab used for construction of the IT. CD25/CD8-
`positive and CD25/cd 19-positive PBLs were affected
`slightly. This might be due, in part, to the significantly
`lower MFI of these cells as compared to that of the
`CD25/CD3-positive and CD25/CD4-positive cells.
`
`Clinical Response
`
`Tumor evaluations were performed 28 to 35 days after
`the end of completion of treatment to document the
`duration of responses. Overall, there were two PRs,
`two MRs, and six SDs (Table 111). Ten patients showed
`progressive disease. Two patients at the 15 mg/m’ dose
`level achieved PRs lasting 2 and 21 months, respec-
`tively. Patient no. 10 presented with involvement of
`the mediastinal lymph nodes and lung. Four cycles of
`
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`

`ANTI-CD25 RICIN A IT TRIAL
`
`531
`
`TABLE V Maximum grade of toxicity in patients treated with RFT5-SMPT-dgA
`
`ID
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`
`Maximum grade of toxicity (NCI)
`dose
`(mg'm2) Nausea/ Weak- Decrease Weight Hypo-
`Tachy- Dyspnea Myal-
`Vomiting
`ness
`in albumin
`gain
`tension
`cardia
`gia
`0
`I
`1
`2
`0
`0
`1
`0
`1
`2
`0
`1
`1
`1
`0
`1
`0
`2
`0
`1
`1
`1
`1
`1
`0
`0
`2
`1
`0
`2
`2
`2
`1
`1
`1
`0
`I
`0
`1
`1
`1
`1
`2
`3
`1
`3
`2
`2
`2
`1
`0
`1
`1
`1
`1
`1
`2
`2
`2
`2
`1
`1
`1
`I
`1
`0
`0
`1
`0
`0
`1
`2
`2
`2
`2
`2
`1
`2
`1
`2
`2
`2
`1
`1
`2
`0
`1
`3
`1
`2
`3
`3
`3
`2
`2
`1
`3
`2
`2
`2
`3
`3
`2
`2
`4
`0
`2
`2
`2
`2
`2
`1
`I
`2
`0
`0
`2
`1
`1
`0
`2
`2
`2
`2
`2
`1
`0
`2
`2
`0
`0
`2
`2
`0
`2
`0
`2
`2
`2
`1
`
`5
`5
`5
`10
`10
`10
`15
`15
`15
`15
`15
`15
`20
`20
`20
`15
`15
`15
`15
`15
`
`0
`0
`0
`0
`0
`1
`3
`0
`2
`0
`2
`3
`2
`3
`3
`2
`0
`2
`1
`2
`
`Joint
`discomfort
`0
`0
`0
`0
`0
`0
`I
`0
`0
`0
`1
`0
`0
`1
`2
`1
`0
`0
`0
`0
`
`Thrombo- Allergic
`penia
`reaction
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`2
`0
`0
`0
`0
`2
`2
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`1
`0
`0
`
`Toxicity was evaluated in the first cycle. Adverse events were graded according to the National Cancer Institute (NCI) Common Toxicity
`criteria as grade I (asymptomatic, easily tolerated), I1 (mild, tolerable), 111 (moderate, poorly tolerated) or IV (severe, life threatening). VLS
`was specifically graded as described elsewhere.'28' In brief, grade I was defined as minimal ankle pitting edema, grade I1 as ankle pitting
`edema and weight gain <15 lb, grade 111 as peripheral edema and weight gain 15-25 lb or pleural effusion without pulmonary dysfunction,
`grade IV as anasarca, pleural effusion or ascites with respiratory deficit or edema >25 lbs, and grade V as pulmonary failure requiring
`mechanical ventilation assistance. Abbreviations: ID. identification number.
`
`treatment led to substantial reduction of all lymph
`nodes in size and number. The PR was maintained for
`26 months. Patient no. 12 had three histologically
`proven HL lesions in the liver measuring 6 x 3.5 cm2,
`4.5 x 4.5 cm2, and 2 x 3 cm2 in diameter. Ten weeks
`after the first treatment with RFTS-SMPT-dgA, these
`lesions vanished (Fig. 2a, b). An additional parailiacal
`lymph node in this patient was not affected by therapy.
`The remaining 9 patients treated at MTD demon-
`strated one MR, four SD and four PD.
`
`Pharmacokinetics
`
`treated with a total dose of 15 mg/m2. In all patients,
`the C,,, was reached at the end of the IT infusion,
`returning to or close to baseline within 12 to 24 hours.
`The C,,,
`correlated only approximately with the
`administered dose. Possible reasons for the low coeffi-
`cient of correlation are differences in tumor mass,
`variability of CD25 expression on tumor cells and
`peripheral blood cells, levels of soluble CD25 in
`the serum of the patients, and accessibility of the
`tumor. The Cmaxs ranged from 0.2 to 9.7 pg/ml (mean
`5.7 pg/ml). The maximum
`values ranged from
`3.97 to 10.53 hours (mean 6.1 hours).
`
`Pharmacokinetic parameters were evaluated in all 15
`patients of the phase I trial. The values for elimination
`half-life
`area under the curve (AUC), clearance
`(CI), and maximum serum concentration (CmaX) are
`listed in Table VI. Figure 3 shows two typical blood
`clearance curves of the IT in two different patients
`
`Measurement of HAMA and HARA
`
`HAMA and HARA were measured as previously
`described.l6'I Of the 15 evaluated patients, six (40%)
`produced HAMA greater than 1 .O pg/ml after the sec-
`ond to fourth cycle. In addition, seven of 15 patients
`
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`

`532
`
`R. SCHNELL et al.
`
`Hodgkin’s tumors tumor-free when examined 42 days
`after treatment. Thus, an extended phase 1/11 clinical
`trial with RFT5-SMPT-dgA was initiated. The aim of
`this study was the evaluation of the MTD and the
`dose-limiting toxicities (DLT) of the IT in patients with
`refractory HL. The major findings of the trial are: 1 .
`The MTD of RFT5-SMPT-dgA is 15 mg/m2. 2. The
`DLTs are related to VLS including hypoalbuminemia,
`weight gain, dyspnea, myalgia, tachycardia, hypoten-
`sion, weakness, and fatigue. Other side effects were
`nausea and vomiting. 3. Responses in heavily pretreated
`patients with refractory HL (4.6 different chemo-
`therapies; ABMT in 8/15) included two PRs, one MR,
`three SDs, and nine PDs. The additional five patients
`treated at MTD showed one MR, three SDs, and one
`PD. 4. The Cmaxs were roughly dose-dependent varying
`from 0.2 to 9.7 pg/ml (mean 5.7 pg/ml). The TIRs of
`RFT5-SMPT-dgA ranged from 3.97 hours to 10.53
`hours (mean 6 hours). 5 . Six of 15 tested patients
`developed HAMA and seven of 15 patients produced
`HARA > 1 .O pg/ml.
`Response rate, side effects, and pharmacokinetics of
`the IT used in the present trial in patients with Hodgkin’s
`lymphoma are comparable to those observed with simi-
`lar ricin A-chain containing ITS against non-Hodgkin’s
`lymphoma (NHL).‘261 Amlot et al. reported one CR and
`five PRs in 24 evaluable patients with relapsed NHL
`treated with the anti-CD22 IT REB4-SMFT-dgA!271 This
`IT was administered using an identical schedule (4-hour
`bolus infusion every second day over seven days). In a
`subsequent phase I trial, RFl34-SMPT-dgA was given as
`continuous infusion over 192 hours with comparable
`clinical response (4/18 PRs) and toxicity.[281 The MTD
`was 19.2 mg/m2/192h. Stone and colleagues compared
`bolus versus continuous infusion using the anti-CD 19 IT
`HD37-SMPT-dgA in patients with NHL.[291 In this trial,
`there was one persisting CR in 23 evaluable patients in
`the bolus regimen (MTD: 16 mg/m2) compared to one
`PR in nine patients treated on the continuous infusion
`(MTD: 19 mg/m2). Peak serum concentrations of the IT
`at MTD and toxicity profile were very similar, suggest-
`ing no advantage for a continuous infusion protocol of
`IgG-based ricin A-chain ITS.
`A recent analysis of all patients treated in clinical
`phase 1/11 protocols suggests that the continuous infu-
`sion of ricin A-chain ITS is statistically more often
`
`FIGURE 2 Response to RFT5-SMPT-dgA treatment of patient
`No 12. Computed tomography scans of a biopsy proven infiltration
`of the liver by Hodgkin’s disease (a). 10 weeks after start of IT
`application the lesion disappeared (b). (See Color Plate XX at the
`back of this issue.)
`
`(47%) showed a HARA titer t 1 .O pg/ml after the sec-
`ond to fourth cycle, including all patients who pro-
`duced HAMA.
`
`DISCUSSION
`
`The anti-CD25 ricin A-chain IT RFT5-SMPT-dgA
`has demonstrated impressive preclinical efficacy
`against human Hodgkin’s lymphoma in vitro as well
`as in two different mouse model^.^^^,^^] RFT5-SMPT-
`dgA inhibits the protein synthesis of Hodgkin-derived
`M. In vivo, this
`L54OCy cells in vitro at 7 x
`IT induced complete remissions of solid human
`Hodgkin’s tumors in nude mice and rendered more
`than 90% of SCID mice with disseminated L54OCy
`
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`

`ANTI-CD25 RICIN A IT TRIAL
`
`533
`
`TABLE VI Pharmacokinetic parameters of RFT'S-SMPT-dgA
`
`~~
`
`ID
`
`CI (ml/h)
`AUC
`TlihX
`dose
`C,,,
`max.
`(mg x h/L),,,"X
`(h)
`( p g W
`(mg/m2)
`4.54
`0.78
`4,700
`3.16
`5
`1
`5
`410
`19.66
`2
`4.04
`5.49
`5
`9,386
`1.12
`4.05
`0.20
`3
`1.23
`4.72
`4
`5,853
`1.49
`10
`5
`9.73
`7.81
`60
`132.98
`10
`90
`112.93
`6
`6.57
`7 .OO
`10
`7
`302
`44.19
`7.83
`7.94
`15
`8
`452
`33.46
`7.16
`3.65
`15
`15
`9
`39 1
`57.30
`3.98
`7.44
`15
`917
`16.78
`3.97
`2.12
`10
`136
`10.53
`6.27
`15
`89.02
`11
`226
`76.19
`4.9
`6.91
`12
`15
`68 51
`9 70
`5.3
`522
`20
`13
`81
`95.36
`8.41
`9.57
`20
`14
`5.52
`8.80
`20
`235
`80.93
`15
`Maximum pharmacokinetic parameters of the first IT course consisting of 4 infusions over a 4
`hour period every other day.
`Abbreviations: ID, identification number; C,,,, peak serum concentration; TliZmax, maximum
`elimination half-life; AUC,,,
`, maximum area under the concentration versus time curve; CI,,,,
`maximum clearance.
`
`associated with treatment-related severe adverse
`events in patients with prior irradiation (Sausville and
`Vitetta, unpublished). In these trials, DLT was always
`due to VLS consisting of decreased serum albumin,
`weight gain, edema, pulmonary edema, and aphasia.
`These side effects were also observed in the present
`
`trial. However, we detected neither major pulmonary
`edema nor aphasia related to VLS. The major and lim-
`iting side effects were myalgia, dyspnea (without pul-
`monary effusion), tachycardia, weakness, and severe
`fatigue. These differences might, at least in part, be
`explained by the fact that the medium age of our
`
`0
`
`150
`100
`50
`Hours after start of therapy
`FIGURE 3 Pharmacokinetics of RFT5-SMPT-dgA in 2 patients treated with 15mg/mz. Patient No 10 (A) received 4 infusions of 7.1 mg IT
`and patient No 1 I (0) four infusions of 5.8 rng IT. Patient No 10 (A) presented high tumor load with diffuse involvement of lung, liver and
`bone marrow with high levels of sCD25 (6,497 Uiml), whereas patient No I 1 (0) demonstrated minimal tumor mass and significant lower
`sCD25 levels (523 U/ml). Both patients had no significant differences in CD25 expression of peripheral mononuclear blood cells.
`
`200
`
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`

`534
`
`R. SCHNELL et al.
`
`The MTD of 0.2 mgkg daily was determined by hyper-
`patients was lower (29 vs 50-60 years) when com-
`sensitivity-like symptoms and reversible transaminase
`pared with the medium age in the NHL trials.[26,27,283291
`elevations. Foss et al.[371 recently reported the results of
`The development of antibodies against the mouse
`a phase I1 trial including 73 patients with different lym-
`protein (HAMA) and against the toxin moiety of ITS is
`phomas treated with DAB3,,-IL-2. None of the 17
`a problem in most clinical trials.'301 These autoantibod-
`patients with HL responded to the recombinant toxin.
`ies form complexes with the IT which are rapidly
`Fatigue was the dose-limiting toxicity. Other toxicities
`cleared, rendering the IT ineffective.[311 In our study,
`included fever, chills, and nausea. The MTD was 27
`six of 15 analyzed patients produced HAMA and
`pglkglday . The disappointing phase I data with recom-
`human anti-rich antibodies (HARA). One patient
`had HARA greater than 1 .0 pg/ml. In general, the anti-
`binant fusion toxins in HL are contributed to the shorter
`half-life of these toxins as compared to IgG-based ITS
`body response is less pronounced in lymphoma
`like RFT5-SMPT-dgA. Another possible explanation is
`
`patients126"7,28.29J than in patients with solid
`the expression of CD25 on H-RS cells without the other
`The influence of antibodies on the pharmacokinetics
`subunits forming the IL2R against which the IL-2-
`of the IT is illustrated by reduced serum peak concen-
`trations in the presence of significant HAMA or HARA
`based constructs are directed.
`titers (data not shown). Another important factor that
`Another possible candidate for selective immuno-
`has been demonstrated to potentially interfere with the
`therapy of patients with HL, the CD30 antigen, was
`pharmacokinetics of Moabs against CD25 in humans is
`originally identified by the Moab Ki-1 .[421 Anti-
`
`the truncated soluble form (sCD25; sIL~Rcx).[~~] These
`CD30 ITS have been constructed by linking the Moab
`variables differed substantially between individual
`Ber-H2 to Saporin-S6, a single-chain ribosome-
`inactivating protein (type I RIP) extracted from the
`patients in this trial. An example of interindividual vari-
`ability is shown in Figure 3 for two patients (nos. 10 and
`seeds of Saponaria oflicinalis (soapworth). The Ber-
`I l), both receiving the same IT dose (15 mg/m2). The
`HYsaporin IT was claimed to be superior to a BerH2
`ricin A-chain IT when compared under the same
`higher IT concentrations in patient 11 can be explained
`experimental conditions in
`in part by the >lO-times lower sCD25 level, smaller
`However,
`tumor load, variability of CD25 expression on tumor
`unconjugated free Saporin-6 used as a control had a
`cells and peripheral blood cells, and possible different
`high unspecific effect itself at a concentration
`accessibility of the tumor.[301
`exceeding 1 x lo-'" M)431 whereas ricin A-chain was
`The IL-2 receptor as a target for a selective
`M.[231 Anti-tumor activity
`similarly toxic at 1 x
`immunotherapy has attracted the attention of several
`of Ber-H2-Sap6 was also reported in SCID mice with
`investigators. Recombinant diphtheria toxin (DT) ba