The Interleukin-2 Receptor: A Target for Monoclonal Antibody Treatment of
`
`
`Human T-Cell Lymphotrophic Virus I-Induced Adult T-Cell Leukemia
`
`By Thomas A. Wa ldmann, Jeffrey D. White, Carolyn K. Goldman, Lois Top, Angus Grant, Richard Bamford, Eric Roessler,
`
`Ivan D. Horak, Sara Zaknoen, Claude Kasten-Sportes, Richard England, Eva Horak. Bibhuti Mishra. Michael Dipre,
`
`Paula Hale, Thomas A. Fleisher, Richard P. Junghans, Elaine S. Jaffe, and David L. Nelson
`
`
`
`and complete tients), or complete (2 patients). with partial
`Adult T-cell leukemia (ATL) is a malignancy of mature lym­
`lasting from 9 weeks to more than 3 years as
`remissions
`
`phocytes caused by the retrovirus human T-cell lympho­
`
`trophic virus-I (HTLV-1). It is an aggressive leukemia with
`
`assessed by routine hematologic tests, immunofluores­
`rate of 50% within 5 months; no con­
`
`
`
`cence analysis, and molecular genetic analysis of T-cell re­
`an overall mortality
`
`
`ceptor gene rearrangements and of HTLV-1 proviral inte­
`
`
`ventional chemotherapy regimen appears successful in in­
`
`gration. Furthermore. remission was associated with a
`
`ducing long-term disease-free survival in ATL patients.
`return to normal serum calcium levels and an improvement
`
`
`However, ATL cells constitutively express high-affinity in­
`
`of liver function tests. Remission was also associated in
`
`
`ter1eukin-2 receptors (IL-2Rs) identified by the anti-Tac
`
`some cases with an amelioration of the profound immuno­
`
`
`monoclonal antibody, whereas normal resting cells do not.
`
`deficiency state that characterizes ATL. Thus the use of a
`
`To exploit this difference in receptor expression, we ad­
`(IV) to 19 patients with
`
`monoclonal antibody that blocks the interaction of IL-2
`
`ministered anti-Tac intravenously
`with its receptor expressed on ATL cells provides a rational
`ATL. In general the patients did not suffer untoward reac­
`tions. and in 1 8 of 1 9 cases did not have a reduction
`
`approach for treatment of this aggressive malignancy.
`in
`This is a US government work. There are no restrictions on
`normal formed elements of the blood. Seven patients devel­
`
`
`that were mixed (1 patient). (4 pa-
`its use.
`oped remissions
`partial
`THE HYBR100MA TECHNIQUE of Kohler and Mil­
`
`stein' rekindled interest in the use of antibodies tar­
`geted to cell surface antigens to treat cancer patients. Im­
`mune-receptor-directed therapy has been applied clinically
`to an array of human disorders. However, effective therapy
`using unmodified murine monoclonal antibodies has been
`elusive because these antibodies are not cytocidal against
`human cells and in most cases are not directed against a
`vital cell surface structure required for tumor cell prolifera­
`tion and survival.2 We readdressed this issue using the inter­
`
`leukin-2 receptor (IL-2R) as the target for immune inter­
`vention. The scientific basis for this approach using the
`IL-2R as a target for immunotherapy is that resting normal
`cells do not express the high-affinity IL-2R, whereas this
`receptor is expressed by a proportion of the abnormal cells
`in certain forms of lymphoid neoplasia, select autoimmune
`disorders, and in individuals rejecting allografts.>-7 The JL-
`2R consists of at least three IL-2-binding peptide chains, as
`follows: IL-2Ra, a 55-Kd peptide identified by the monoclo­
`nal antibody anti-Tac8•9; IL-2RP,
`a 75-Kd subunit10•11; and
`the recently discovered IL-2R-y, a 64-Kd protein.'2 Identifi­
`
`cation and characterization of the IL-2Ra subunit was facili­
`tated by our development of a monoclonal antibody, anti­
`Tac, that binds to IL-2Ra and prevents the interaction of
`IL-2 with this subunit.8 Resting T ceUs, B cells, and mono­
`cytes in the circulation do not display IL-2Ra. In contrast to
`this lack of IL-2Ra expression in normal resting mononu­
`clear cells, this receptor subunit is expressed by a proportion
`of the abnormal cells in certain forms of neoplasia including
`certain T-cell, B-cell, monocytic, and even granulocytic leu­
`kemias. Furthermore, the serum concentration of the solu­
`ble fonn of lL-2Ra released by the abnormal cells is in­
`creased in patients with these disorders.13 Specifically, a
`proportion of the leukemic cells of patients with chronic or
`acute myelogenous leukemia express IL-2Ra identified by
`the anti-Tac monoclonal antibody.14 Furthermore, malig­
`nant B cells of virtually all patients with hairy celJ leukemia
`and a proportion of patients with large- and mixed-cell dif­
`fuse lymphomas express IL-2Ra. •s The IL-2Ra is also ex­
`pressed on Reed-Sternberg cells of patients with Hodgkin's
`
`disease and on malignant cells of patients with true histiocy­
`tic lymphoma.16 Similarly, a proportion of patients with
`cutaneous T-ceU lymphomas express the Tac peptide on
`their malignant cells. Finally, virtually all patients with hu­
`man T-cell lymphotrophic virus-I (HTLV-I)-associated
`adult T-cell leukemia (ATL) constitutively express very
`large numbers of IL-2Ra.11-19
`These results suggested that anti-IL-2R antibodies might
`be effective agents for the treatment of certain neoplastic
`diseases. We have focused our therapeutic studies of malig­
`nancy on the disordered IL-2 R expression of a distinct form
`of T-cell leukemia, ATL.19 A TL is an aggressive malignancy
`of lymphocytes displaying a multilobulated nucleus with
`dense chromatin and expressing a CD3+, CD4+, co&-,
`Tac+) phenotype.17•18 The dis­
`COr, and CD25+ (lL-2Ra,
`ease exhibits a striking clustering of cases in certain geo­
`graphic regions, notably southwestern Japan, the Caribbean
`basin, northeastern South America, Central America, sub­
`Saharan Africa, and to a lesser extent the southeastern
`United States. The retrovirus HTLV-I is clearly associated
`with this disease and appears to play a major role in its
`pathogenesis.20 Patients with ATL have serum antibodies to
`HTLY-1 and the monoclonal integration of this retrovirus
`in their circulating malignant cells. Frank ATL generally
`has its onset in adulthood, 20 to 30 years following initial
`
`From the Metabolism Branch and Laboratory of Pathology, Na­
`tional Cancer lnstitwe. and the Clinical Pathology Department,
`Warren Grant Magnuson Clinical Center, Naiional Institutes of
`Health, Bethesda, MD.
`Submitted March 17, 1993: accepted May 27, 1993.
`Address reprint requests to Thomas A. Waldmann, MD, Metabo­
`lism Branch, Naiional Cancer Institute, Bldg JO, Rm 4Nl 15, Be­
`thesda, MD 20892.
`The publication costs of this article were defrayed in part by page
`charge payment. This article mus1 therefore be hereby marked
`"advertisement" in accordance with 18 U.S.C. section 1734 solely to
`indicate this fact.
`This is a US government work. There are no restrictions on its use.
`0006-4971/93/8206-0032$0.00/0
`
`Blood. Vol 82, No 6 (September 15), 1993: pp 1701-1712
`
`1701
`
`1 of 13
`
`BI Exhibit 1036
`
`

`

`1702
`
`WALDMANN ET AL
`
`infection with HTL V-1. Principal clinical features include
`
`
`
`
`first week of therapy, followed by 40 mg on two occasions during
`moderate lymphadenopathy, hepatosplenomegaly, and
`the second week. For the subsequent I 0 patients, to achieve rapid
`saturation of the IL-2R, the basic plan involved IV administration
`skin, central nervous system, and pulmonary involve­
`of 50 mg anti-Tac on two occasions for each of the first 2 weeks of
`
`ment21·22; the occurrence of hypercalcemia is characteristic
`with acute ATL manifest a striking degree
`therapy. Dosing schedules were modified slightly in some patients,
`of ATL. Patients
`and additional 20- or 50-mg doses were administered during subse­
`of immunosuppression and develop opportunistic infec­
`quent weeks to maintain the saturation of the IL-2R with the anti­
`tions such as Pneumocystis pneumonia and cryptococcal
`Tac monoclonal antibody. Furthermore, in such cases sufficient
`of several clinical
`meningitis. The experiences
`oncology
`anti-Tac was administered to yield measurable levels of circulating
`groups using combination chemotherapy regimens in pa­
`antibody in the serum of the patient. As noted above, patients with
`
`tients with this disease have been disappointing. In most
`malignant cells in the central nervous system received intrathecal
`is approximately
`
`chemotherapy series, overall mortality
`methotrexate.
`50% within 5 months. No conventional treatment progrdm
`The criteria for response were as follows: ( 1) complete response,
`disappearance of all measurable and assessable disease lasting more
`
`
`appears successful in inducing long-term disease-free sur­
`vival in A TL patients.
`than I month; (2) partial response, a 50% reduction of leukemic cell
`In our clinical trial, we wished to exploit the observation
`count and a 50% reduction in the size of measurable lesions and no
`increase in the size of any measurable or assessable lesion or appear­
`that the normal resting cells of patients with A TL do not
`ance of a new lesion for I month; (3) mixed response, identical to
`display IL-2Ra, whereas the malignant T cells display
`partial response with the exception that there is the appearance of a
`I 0,000 to 35,000 IL-2Ra/cell that are identified by the anti­
`
`Tac monoclonal antibody.17•18 Thus IL-2R-directed
`new lesion within I month in a tissue other than that involved
`immu­
`initially; (4) stable disease, less than partial response with no new
`notherapy using anti-Tac could theoretically eliminate
`IL-
`lesions or less than a 25% increase in any measurable lesion; (5)
`
`2Ra-expressing leukemic cells while retaining the
`progressive disease, at least 25% increase in leukemic cell count or
`Tac-nonexpressing normal T cells and their precursors
`that
`an increase of 25% or greater in any measurable lesion. A systems­
`
`express the antigen receptors required for normal T-cell­
`oriented microcomputer-based patient data management system
`
`mediated immune responses. In our preliminary studies, we
`was devised and implemented in which historical, clinical, and labo­
`
`
`observed that anti-Tac induced a remission in some patients
`ratory data were stored and manipulated for analysis (R.P.J. and
`toxicity.19 In the present
`with ATL without associated
`T.A.W.).
`
`
`had study, the 19 patients with A TL treated with anti-Tac
`Approval was obtained from the institutional review board for
`these studies. Informed consent was provided according to the Dec­
`
`few untoward reactions related to the immunotherapy and
`laration of Helsinki.
`in general d.id not have a reduction in normal formed ele­
`Produc1ion of anti-Tac monoclonal antibodies. The anti-Tac
`ments of the blood. Seven of 19 treated patients had a tran­
`monoclonal antibody was produced as described previously19 by
`sient mixed (I), partial ( 4 ), or complete (2) remission,
`with
`fusion of NS-1 mouse myeloma cells with spleen cells of mice that
`
`partial and complete remissions lasting from 2 to more than
`had been immunized with a cell line derived from an ATL patient.
`36 months.
`The antibody does not function in complement-dependent cytotox­
`icity with human plasma nor does it induce antibody-dependent
`cellular cytotoxicity (ADCC) with human mononuclear cells. How­
`ever, anti-Tac blocks the interaction of lL-2 with the high-affinity
`receptors for this lymphokine. Large quantities of the monoclonal
`antibody were produced by inoculating hybridoma cells into the
`peritoneal cavity of BALB/c mice and then purifying the mouse
`lgG2a anti-Tac from the resulting ascites fluid by diethylamino­
`ethyl cellulose chromatography. The purified antibody was dia­
`lyzed against saline, centrifuged, filtered, precipitated with 20% so­
`dium sulfate, and finally diluted in saline at pH 7.4 to a
`concentration of 2 mg/ml. Each lot of the product was assayed by
`immunoelectrophoresis in agar plates using antisera to lgG2a,
`lgG1, and lgM, as well as polyvalent antibodies to most mouse
`proteins. Lots greater than 98% pure as assessed by these analyses
`and by high-performance liquid chromatography and sodium do­
`decyl sulfate-polyacrylamide gel electrophoresis were used. The
`monoclonal antibody preparntion was sterilized by passage through
`a 0.22-µm membrane filter (Millipore, Marlborough, MA) by the
`Pharmaceutical Development Section of the Clinical Center of the
`National Institutes of Health (NIH) and was shown to be nonpyro­
`genic and sterile by the Bureau of Biologics.
`lmmunojluorescence analysis of cell surface phenotype. The
`phenotype of the leukemic cell population was defined by indirect
`and direct immunofluorescence performed with mouse monoclo­
`nal antibodies using a fluorescence-activated cell-sorter as de­
`scribed previously.19 Two antibodies (anti-Tac and 7G7/B6) that
`are directed toward different epitopes of!L-2Ra were used to define
`the expression of this receptor subunit. Other monoclonal antibod­
`ies used include antibodies reacting with HLA-DR (la-I; Ortho,
`
`Patient population. Nineteen patients with histologically con­
`firmed HTLV-1-associated ATL were studied (Table I). Each of the
`patients manifested the following features: (I) a histologically con­
`firmed diagnosis of leukemia or lymphoma of mature T cells with
`polymorphic indented or lobulated nuclei; (2) intense expression of
`the Tac antigen (IL-2Ra) on at least 10% of the patient's pe.ripheral
`blood, lymph node, or dermal T cells; (3) antibodies to HTL V-1
`demonstrable in the serum; and (4) omission of cytotoxic chemo­
`therapy and radiation therapy for at least 3 weeks before entry into
`the trial. Patients with or without previous chemotherapy were eligi­
`ble for inclusion in this study; I 0 patients had failed to respond to
`prior therapy. Patients with symptomatic central nervous system
`disease were excluded; however, patients with malignant cells de­
`monstrable in the cerebrospinal fluid were included and received
`intrathecal methotrexate. The patients ranged in age from 24 to 62
`years (mean, 41 years); demographic factors in the patient group are
`shown in Table I. Ten patients were male and nine female, 17 were
`black, one was Hispanic, and one was of Japanese origin, and lO
`were from the United States, four were from Jamaica, and one each
`was from Japan, Cuba, Trinidad, Haiti, and Guyana. Using the
`criteria of Kawano et al23 and Yamaguchi et al,24 11 patients with
`A TL were in the acute or crisis stage, four manifested A TL lym­
`phoma, and four had chronic A TL.
`Therapelllic swdy plan. Anti-Tac was administered intrave­
`nously (IV) over a 2-hour period in 100 ml normal saline contain­
`ing 5% albumin. In the basic study plan for the initial nine patients,
`20 mg anti-Tac was administered JV on two occasions during the
`
`MATERIALS AND METHODS
`
`2 of 13
`
`BI Exhibit 1036
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`

`

`ANTl-T AC THERAPY OF A TL
`
`Table 1. Demographic and Clinical Features of A TL Patients
`
`Patient
`No.
`
`Type of
`ATL
`
`Age/Sex/Race
`
`sll-2R (U/ml)
`
`WBC/µl
`
`Circulating
`ll-2R/Tac-
`E>cpressing
`Lymphocytes/µl
`
`Antibodies
`to HTLV�
`
`Serum
`Ca
`
`Acute
`Chronic
`Lymphoma
`Acute
`
`147,130
`2,240
`4,660
`2,200
`230,370
`87.710
`56,420
`920
`60.170
`6.060
`31,700
`2,040
`89,830
`31.580
`48.460
`9.910
`2,210
`158, 130
`138,680
`
`42.600
`3.BOO
`1,500
`20.700
`41,500
`12,700
`75.400
`5.500
`102.800
`15.400
`21,300
`9, 100
`7,900
`32,100
`26.500
`7.100
`13,200
`177,000
`21,600
`
`11.200
`2.120
`<100
`12.930
`24.200
`1,600
`37.720
`230
`50.600
`1,900
`13,500
`3,000
`1,800
`12.700
`8,010
`<100
`4,710
`152,900
`4.600
`
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`
`3.50
`3.75
`2.15
`2.35
`B.10
`4.35
`4.60
`2.30
`4.40
`4.10
`2.65
`2.40
`2.20
`3.90
`2.80
`2.45
`2.20
`3.80
`3.60
`
`1703
`
`Abnormal
`Uver
`Func;tion
`Tests
`
`+
`+
`+
`
`+
`+
`+
`
`+
`
`+
`
`+
`
`+
`
`+
`+
`
`Acute
`
`Acute
`
`Acute
`Lymphoma
`Acute
`Acute
`
`Chronic
`
`Chronic
`Lymphoma
`Acute
`
`39/F/B
`1
`28/M/B
`2
`25/M/B
`3
`4
`32/F/B
`62/M/B
`5
`6
`44/M/B
`7
`41/M/B
`57/F/A
`8
`9
`42/M/H
`10
`58/F/6
`11
`40/M/B
`12
`55/M/B
`53/M/B
`13
`24/F/B
`14
`15
`42/F/6
`Acute
`Lymphoma
`34/M/B
`16
`17
`Chronic
`41/F/6
`Acute
`18
`26/F/6
`19
`60/F/6
`Acute
`Abbreviations: 6, black; H, Hispanic; A. Asian.
`
`Raritan, NJ); human T-cell-associated antigens (CD2, CDJ, CD4,
`CD5, and CDS; Ortho and Becton-Dickinson, Mountain View,
`CA); CD7 (3A I; a gift from Dr Barton Haynes); and CD45 and
`CD29 (Coulter Immunology, Hialeah, FL). The fluorescein isothio­
`cyanate (FITC) antimouse IgG reagent was obtained from Coulter
`Immunology. Histograms for each cell type were integrated to de­
`termine the percentage of mononuclear cells that reacted with indi­
`vidual monoclonal antibodies. The absolute number of cells in the
`circulation per cubic millimeter expressing a particular antigen was
`determined from the product of (I) circulating white blood cell
`(WBC) count per cubic millimeter, (2) the proportion of these cir­
`culating WBCs that were mononuclear cells as determined by rou­
`tine hematologic analysis, and (3) the proportion of these mononu­
`clear cells that expressed the antigen under study as assessed by
`immunocytofluorography.
`Molecular genetic analysis of Tcr gene rearrangement and
`HTLV-1 integration. Analysis for clonal Tcr gene rearrangements
`and for HTLV-1 integration were performed as described previ­
`ously.25·26 High-molecular-weight DNA was extracted from frozen
`cell suspensions containing approximately I08 cells. DNA samples
`were digested with the restriction enzymes BamHI, EcoRI, Hindtll,
`or Pstl (International Biotechnologies, New Haven, CT, and New
`England Biolabs, Beverly, MA) and were size-fractionated on 0.5%
`to 0.9% agarose gels. They were transferred by the Southern blot
`technique to reinforced nitrocellulose paper (Schleicher and Schull,
`Keene, NH). Hybridization to randomly primed 32P-labeled DNA
`probes of the constant regions of the Tcr (3 gene and the HTLV -I
`gene were performed, followed by washing at appropriate strin­
`gency and radioautography. Nonlymphoid control DNA was ana­
`lyzed simultaneously to identify germ line positions of the Tcr genes
`examined. The Tcr f3 gene probe used was a 700-bp Eco RI fragment
`containing the mouse or human C{J region that recognizes both
`human C{J regions. The HTLV-1 probe used was the 9-kb Sacl
`fragment containing the entire viral genome with the exception of
`the long terminal repeats (a gift from Dr Flossie Wong-Staal).
`Assay for antibodies to HTLV-1. The sera of ATL patients were
`analyzed for antibodies to disrupted and inactivated HTL Y-1 using
`an enzyme-linked immunosorbent assay ([ELISA] Cellular Prod­
`ucts, Buffalo, NY).
`
`Assay for mouse lg. Murine IgG2a anti-Tac levels in the sera of
`patients were assayed with an ELISA technique using affinity-puri­
`fied goat antimouse immunoglobulin absorbed onto polyvinyl
`plates at JOO ng/well and washed.i9 Bound murine anti-Tac was
`detected with a goat antimouse immunoglobulin conjugated with
`alkaline phosphatase (Sigma, St Louis, MO) and compared with a
`standard curve of anti-Tac antibody.
`Assay for human anti mouse anti-Tac antibody. The serum used
`in all assays was separated from peripheral blood and stored at
`-20°C until used. For detection of human antimouse antibodies
`(HAMA), the technique described by Schroff et al27 was used, with
`the exception that anti-Tac was used in lieu of the T 101 antibody.
`Serum antiglobulin levels for a given patient were considered mean­
`ingfully increased when the antiglobulin level after therapy was
`greater than twice that in the sample obtained before administra­
`tion of the mouse monoclonal antibody.
`
`RESULTS
`Patient characteristics. Nineteen patients with histologi­
`cally confirmed HTLV-1-associated ATL were treated with
`IV administered anti-Tac (Table I). Four patients had lym­
`phoma-type A TL without circulating malignant cells; the
`peripheral blood WBC count in the remaining 15 patients
`before therapy ranged from 3,800 to 177,000/ 1-'L (geometric
`mean, 26,000). Patients with A TL had pretherapy serum
`soluble IL-2R (s1L-2Ra) levels of 920 to 230,370 U/mL
`(mean, 55,744), whereas the upper limit of the normal is
`502 U /mL (mean, 238). 28 T-Cell leukemic populations were
`confirmed to be monoclonal by molecular genetic analysis
`of the rearrangement of genes encoding the Tcr f3 chain.
`Southern blot analysis of the Tcr f3 receptor gene rearrange­
`ment using a radiolabeled probe that hybridizes with the
`constant region of the Tcr f3 chain showed a band that was
`not present in gennline tissues, a hallmark of a clonally
`expanded population of T lymphocytes (Fig 1). Further­
`more, Southern blot analysis of HTL V-1 proviral in tegra-
`
`3 of 13
`
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`
`

`

`1704
`
`WALDMANN ET AL
`
`lion in Pst I and EcoRI digests of DNA obtained from the
`patients defined the clonal integration of HTL Y-1 provi­
`ruses. Clinically, nine patients manifested involvement of
`the skin. Twelve were hypercalcemic, with a serum calcium
`level in these cases ranging from 2.65 to 8.1 mmol/L (nor­
`mal range. 2.05 to 2.5 mmol/L). Mild to moderate liver
`function abnormalities were demonstrable in 12 of 19 cases.
`Using flow cytometric phenotypic analyses of circulating
`mononuclear cells, we demonstrated that in 13 of 15 cases
`with leukemia. the predominant mononuclear cell popula­
`tion expressed the CD3+, CD4+/CD8-. CD25+ phenotype;
`phenotypes in the two remaining cases were CD3+. co4-;
`co&-, co25+ and cor, co4+;co&-, co25+. Circulating
`mononuclear cells of the patients showed intense expression
`of the Tac antigen on a relatively homogeneous cell popula­
`tion manifesting high fluorescence intensity (Fig 2). It ap­
`pears that all of the circulating malignant cells expressed the
`Tac antigen. Although a small proportion of normal periph­
`eral blood mononuclear cells manifest low-level Tac expres­
`sion, the pattern observed within the leukemic population is
`quite distinct from that observed in normal individuals in
`terms of the homogeneity and intensity of Tac antigen ex-
`
`pression (Fig 2). In the 15 leukemic cases, the abnormal cell
`population did not react with a CD? (3A 1) monoclonal an­
`tibody, which reacts with normal T-cell precursors and with
`at least 70% of normal mature T lymphocytes.
`Response of ATL patients ro treatment with anti-Tac
`monoclonal antibodies. The initial basic protocol for anti­
`Tac therapy involved the administration o f 2 0 mg anti-Tac
`on two occasions during the first week and 40 mg anti-Tac
`on two occasions during the second week of therapy for
`each patient (Table 2). After the second week of therapy,
`additional doses of20 or 50 mg anti-Tac were administered
`to patients who had made an initial clinical response to
`anti-Tac therapy. It was noted that from 40 lo 100 mg anti­
`Tac had to be administered to the patients to saturate the
`IL-2Ra expressed by tumor cells. The IL-2Rs on leukemic
`cells were deemed to be saturated by the infused monoclo­
`nal antibody when the cells manifested the following three
`features: (I) reaction with FJTC anti mouse IgG; (2) reaction
`with FJTC-labeled 7G7 /86 (an antibody that reacts with
`1L-2Ra but does not cross-block with anti-Tac); and (3) no
`binding in direct immunofluorescence analysis with FITC­
`labeled anti-Tac, since the target antigen was blocked by in
`
`Placental
`(germline)
`
`Patient
`23 days post
`treatment
`
`11 Kb -
`
`8.5 Kb•
`
`4 Kb -
`
`8.8 Kb-
`7.1 Kb -
`
`Patient
`1099 days post
`treatment
`
`_...,_
`
`EcoRI
`
`--����-11 Kb�����•
`BamHI EcoRI
`EcoRI
`
`t �
`9J [1J I
`
`V(Jn D(J1
`
`111111
`J(J1
`
`t
`*
`�
`111111 !?di
`C(J1
`flJ-- C(J PROBE -ti!
`
`--4Kb-
`EcoRI
`EcoRI BamHI
`
`t t
`
`1---1
`1 Kb
`
`Fig 1. Analysis of Tcr fJ gene rearrangements to monitor anti-Tac monoclonal antibody treatment in patient no. 5 with ATL using a Tcr fJ
`constant region probe (CP). The Tcr fJ constant region genes are on four and 11-Kb EcoRI fragments in placental (germlinet DNA as indicated
`(---).The 11-Kb band contains C{J1. whereas C{J2 is present in the 4-Kb band. "An artifactual band at 8.5 Kb that is a result of an incomplete
`digestion at a site 5' of the C{J2 locus. Digests of patient peripheral blood DNA during an active phase of the disease 23 days after initiation of
`therapy yielded a diminished 11-Kb band as well as two nongermline bands I+ that reflect a monoclonal Tcr fJ pattern of gene rearrange­
`ment. This pattern indicates that both alleles for Tcr fJ in the leukemic clone rearranged into C{J1. Patient DNA obtained in remission 1.099
`days following initiation of therapy did not express the two nongermline bands. thus confirming elimination of the circulating monoclonal
`population. In the schematic diagram of the germline arrangement of the Tcr fJ chain gene, we indicate the locations of the BamHI and EcoRI
`restriction endonuclease sites as well as the C{J regions recognized by the cDNA probe.
`
`4 of 13
`
`BI Exhibit 1036
`
`

`

`ANTI-TAC THERAPY OF ATL
`
`1705
`
`A
`Pre therapy
`·100.....--------------..
`
`- Control
`-��- Anti Tac
`
`Post Therapy
`
`- Control
`-� .. -- Anti Tac
`
`'
`.
`'
`
`I ,,
`'
`l
`f
`
`� ·�
`'\
`\ \
`102
`101
`103
`Relative Fluorescence Intensity
`
`104
`
`104
`
`..... �
`E :::::> 50
`z
`Q)
`(_)
`
`B
`100
`
`..... Q) .0
`E :J 50
`z
`Q)
`(_)
`
`0
`100
`
`c
`
`� E
`� 50
`� ()
`
`Normal Cells
`
`-Control
`-Anti Tac
`
`101
`Relative Fluorescence Intensity
`
`vivo administration of this antibody. In light of these early
`observations, to achieve a rapid saturation of IL-2R, the
`basic dosing schedule was altered for the final 10 patients in
`the group so that 50 mg anti-Tac per patient was adminis­
`tered on two occasions during the first week of therapy and
`on two occasions during the second week of therapy. Addi­
`tional doses of 50 mg anti-Tac to maintain receptor satura­
`tion were administered to patients who made an initial par­
`tial or complete response to therapy. The 19 patients
`received a total of23 distinct courses of therapy; there were
`three to 11 infusions per treatment course (mean, five).
`Treatment courses ranged from 3 to 57 days (mean, 17),
`with a total dose of antibody per treatment course ranging
`from 60 to 500 mg (mean, 22 5). Maximum levels of mouse
`antibody achieved in the serum 24 hours after the last thera­
`peutic dose of a treatment course ranged from 595 to 12 ,230
`ng/mL.
`Toxicity. The 19 patients officially entered into the clin­
`ical trial did not have any untoward acute reactions. How­
`ever, an untoward event that may represent a reaction was
`observed in one patient with A TL who was treated with
`anti-Tac off-study under a compassionate protocol exemp­
`tion. This patient, who was ill on admission with heart fail­
`ure, hepatosplenomegaly, and multiple effusions, died of
`respiratory distress following the fifth infusion of anti-Tac;
`at autopsy, the cause of death was shown to be massive
`hemorrhagic consolidation of the lungs. One patient devel­
`oped hives after the second of four anti-Tac infusions. Two
`patients developed fever to a maximum of39. l °C following
`anti-Tac administration. Five patients in six treatment
`courses manifested an increase in plasma uric acid levels
`without sequelae; however, the patients were experiencing
`progressive disease during five of these treatment periods. In
`general, patients did not manifest hematologic toxicity af­
`fecting the normal formed elements of the blood including
`platelets or polymorphonuclear leukocytes. In the one ex­
`ception to this generalization, a patient developed a hemo­
`lytic anemia and a reduction in the platelet count to 26,000/
`µ.Land in the neutrophil count to 380/µL. This patient had
`leukemic involvement of the bone marrow; he received sub­
`sequent doses of anti-Tac without any further untoward re­
`action noted.
`Tumor response-clinical response. Twelve of the 19
`patients had either a transient response ( <2 weeks) or no
`response to anti-Tac therapy; two of these patients had
`stable disease, whereas the disease progressed in the remain-
`
`Fig 2. Relative fluorescence intensity of Tac antigen expression
`was defined by indirect immunofluorescence on the peripheral
`blood mononuclear cells from (A} patient no. 5 with A TL before
`therapy; (B) the same patient in remission following completion of
`anti-Tac therapy; and (C) a nonnal individual. Circulating mononu­
`clear cells of the patient showed intense expression of the Tac anti­
`gen on a relatively homogeneoU11 cell population manifesting high
`fluorescence intensity before therapy. Following therapy, a small
`proportion of peripheral blood mononuclear cells of the patient
`manifested low-level Tac expression. This pattern is quite distinct
`from that observed during relapse and is comparable to that noted
`in the normal individual in terms of homogeneity and intensity of
`Tac antigen expression.
`
`5 of 13
`
`BI Exhibit 1036
`
`

`

`1706
`
`Patient
`No.
`
`2
`
`3
`4
`5
`6
`7
`8
`9
`10
`
`11
`
`12
`
`13
`14
`15
`
`16
`
`17
`
`18
`19
`
`Table 2. Effect of Anti-Tac Therapy
`
`WALDMANN ET AL
`
`Previous Therapy
`
`None
`ProMACE/MOPP
`
`None
`CHOP
`None
`DCF
`None
`MACOP·B
`Pulse steroids
`Cytoxan. etoposide,
`doxorubicin, MTX
`None
`
`Vincristine, bleomycin,
`cytoxan, decadron
`None
`None
`ProMACE/MOPP. COP
`+ MOPP
`None
`
`Bleomycin, cytoxan,
`doxorubicin
`ProMACE/MOPP
`None
`
`Dose of Anti·Tac
`Administered {mg)
`
`150
`1st course, 160
`2nd course, 60
`300
`200
`400
`180
`147
`250
`120
`100
`
`1st course, 490
`2nd course, 500
`300
`
`200
`200
`180
`
`1st course. 200
`2nd course. 200
`
`1st course. 300
`2nd course, 200
`220
`220
`
`Toxicity
`
`None
`None
`
`None
`None
`None
`Fever (39. 1 °C)
`Fever (38°C)
`None
`None
`None
`
`None
`
`None
`
`Hives
`None
`None
`
`Transient neutropenia
`Thrombocytopenia,
`hemolytic anemia
`None
`
`None
`None
`
`Oevelopment of
`Anti-Tac
`Antibodies
`(HAMAI
`
`+
`
`+
`
`+
`
`Clinical Response/
`Duration (dJ
`
`PD
`PR 224
`PR 70
`PR 63
`PD
`CR> 1.098
`PD
`PD
`PD
`PD
`PD
`
`CR 357
`PD
`SD
`
`SD
`PD
`MR13
`
`PR 252
`PD
`
`PR 168
`SD
`PD
`PD
`
`Abbreviations: ProMACE, prednisone. methotrexate. doxorubicin, cyclophosphamide. etoposide; MOPP. mechlorethamine, vincristine, procarba­
`zine. prednisone; CHOP. cyclohosphamide, doxorubicin, vincristine. prednisone; DCF. pentostatin; MACOP-B, methotrexate, doxorubicin, cyclophos­
`phamide. vincristine. prednisone. bleomycin; MTX, methotrxate; COP, cyclophosphamide, vincristine, prednisone; CR. complete remission; PR, partial
`response; SD, stable disease; PD. progressive disease; MR, mixed response.
`
`ing 10. The seven remaining patients had a more favorable
`response to therapy. One of these patients manifested a
`transient mixed response characterized by a decrease of the
`circulating Tac-expressing cells from 9,300/µL to less than
`200/µL (normal individuals have �500 circulating T cells/
`µL that express Tac weakly). This patient was deemed to
`have a transient mixed, rather than a partial, remission,
`because 1- to 2-cm enlarged lymph nodes shown on biopsy
`to be effaced by malignant T cells appeared in the neck 13
`days after initiation of anti-Tac therapy. Four additional
`patients, including two with chronic ATL and two with
`lymphoma-type ATL, developed a partial remission; the du­
`ration of these partial remissions ranged from 63 to 252
`days (mean, 177). The three patients of this group that had a
`partial remission lasting from 168 to 252 days were re­
`treated with anti-Tac when their disease relapsed. Two of
`these patients did not respond to retreatment, although
`their leukemic cells continued to express the Tac antigen.
`The third patient had a second partial remission lasting 70
`days following re-treatment, but then relapsed. Two addi­
`tional patients, one with chronic A TL and one with acute
`ATL, developed a complete remission, which lasted for 357
`days in the patient with chronic ATL (Fig 3B). The patient
`with acute ATL is still in a complete remission more than 3
`years following entry into remission (Fig 3A). This patient
`
`initially had hepatosplenomegaly and a peripheral blood
`WBC count of 41,800/ µL that included 23,000 circulating
`Tac-expressing malignant T ce!Js/µL as assessed by immu­
`nofluorescence analysis. The soluble IL-2Ra level was
`230,370 U/mL, the highest level observed in this series of
`ATL patients, and the serum calcium level was 8.10 mmol/
`L before therapy, more than three times the upper limit of
`normal. The patient received 400 mg anti-Tac over a 7-
`week period; by 50 days following initiation of therapy, the
`patient had undergone a complete remission. Abnormal
`cells were no longer demonstrable in the circulation. Fur­
`thermore, the serum calcium level returned to normal (Fig
`4). Before anti-Tac therapy, the patient was anergic; how­
`ever, when reassessed 2 months and again I year postther­
`apy during a complete remission, the patient manifested
`positive skin test responses to recall antigens. The patient
`developed HAMA 4