The Interleukin-2 Receptor: A Target for Monoclonal Antibody Treatment of
`Human T-cell Lymphotrophic Virus I-Induced Adult T - c e l l Leukemia
`
`By Thomas A. Waldmann, 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
`
`Adult T-cell leukemia (ATL) is a malignancy of mature lym-
`phocytes caused by the retrovirus human T-cell lympho-
`trophic virus-I (HTLV-I). It is an aggressive leukemia with
`an overall mortality rate of 50% within 5 months; no con-
`ventional chemotherapy regimen appears successful in in-
`ducing long-term disease-free survival in ATL patients.
`However, ATL cells constitutively express high-affinity in-
`terleukin-2 receptors (IL-2%) identified by the anti-Tac
`monoclonal antibody, whereas normal resting cells do not.
`To exploit this difference in receptor expression, we ad-
`ministered anti-Tac intravenously (IV) to 19 patients with
`ATL. In general the patients did not suffer untoward reac-
`tions, and in 18 of 19 cases did not have a reduction in
`normal formed elementsof the blood. Seven patients devel-
`oped remissions that were mixed (1 patient), partial (4 pa-
`
`T stein’ rekindled interest in the use of antibodies tar-
`
`HE HYBRIDOMA TECHNIQUE of Kohler and Mil-
`
`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.* 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 allograft^.^-^ The IL-
`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 a n t i - T a ~ ~ , ~ ; IL-2RP, a 75-Kd
`and
`the recently discovered IL-2Ry, a 64-Kd protein.” Identifi-
`cation and characterization ofthe 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.s Resting T cells, 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 form of IG2Ra released by the abnormal cells is in-
`creased in patients with these di~0rders.l~ 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.I4 Furthermore, malig-
`nant B cells of virtually all patients with hairy cell leukemia
`and a proportion of patients with large- and mixed-cell dif-
`fuse lymphomas express IL-~RCY.’~ The IL-2Re is also ex-
`pressed on Reed-Sternberg cells of patients with Hodgkin’s
`
`tients), or complete (2 patients), with partial and complete
`remissions lasting from 9 weeks to more than 3 years as
`assessed by routine hematologic tests, immunofluores-
`cence analysis, and molecular genetic analysis of T-cell re-
`ceptor gene rearrangements and of HTLV-I proviral inte-
`gration. Furthermore, remission was associated with a
`return to normal serum calcium levels and an improvement
`of liver function tests. Remission was also associated in
`some cases with an amelioration of the profound immuno-
`deficiency state that characterizes ATL. Thus the use of a
`monoclonal antibody that blocks the interaction of IL-2
`with its receptor expressed on ATL cells provides a rational
`approach for treatment of this aggressive malignancy.
`This is a US government work. There are no restrictions on
`its use.
`
`disease and on malignant cells of patients with true histiocy-
`tic lymphoma.16 Similarly, a proportion of patients with
`cutaneous T-cell 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.17-”
`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-2R expression of a distinct form
`of T-cell leukemia, ATL.” ATL is an aggressive malignancy
`of lymphocytes displaying a multilobulated nucleus with
`dense chromatin and expressing a CD3+, CD4+, CD8-,
`
`CD7-, and CD25’ (IL2Ra, Tac+) p h e n ~ t y p e . ~ ~ , ’ ~ The dis-
`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
`HTLV-I 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 Institute, and the Clinical Pathology Department,
`Warren Grant Magnuson Clinical Center, National Institutes of
`Health, Bethesda, MD.
`Submitted March 17, 1993; accepted May 27, 1993.
`Address reprint requests to Thomas A. Waldmann, MO, Metabo-
`lism Branch, National Cancer Institute, Bldg IO, Rm 4N115, Be-
`thesda, MD 20892.
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be hereby marked
`“advertisement” in accordance with 18 U.S.C. section I734 solely to
`indicate this fact.
`This is a Usgovernment work. There are no restrictions on its use.
`0006-49 71 /93/8206-00~2$0.00/0
`
`Blood, Vol82, No 6 (September 15). 1993: pp 1701-1712
`
`1701
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1036 Page 1 of 13
`
`

`
`1702
`
`WALDMANN ET AL
`
`infection with HTLV-I. Principal clinical features include
`moderate lymphadenopathy, hepatosplenomegaly, and
`skin, central nervous system, and pulmonary involve-
`
`ment2L’22; the occurrence of hypercalcemia is characteristic
`of ATL. Patients with acute ATL manifest a striking degree
`of immunosuppression and develop opportunistic infec-
`tions such as Pneumocystis pneumonia and cryptococcal
`meningitis. The experiences of several clinical oncology
`groups using combination chemotherapy regimens in pa-
`tients with this disease have been disappointing. In most
`chemotherapy series, overall mortality is approximately
`50% within 5 months. No conventional treatment program
`appears successful in inducing long-term disease-free sur-
`vival in ATL patients.
`In our clinical trial, we wished to exploit the observation
`that the normal resting cells of patients with ATL do not
`display IL2Ra, whereas the malignant T cells display
`10,000 to 35,000 IL-2Ra/cell that are identified by the anti-
`Tac monoclonal antib~dy.’’~’~ Thus IL-2R-directed immu-
`notherapy using anti-Tac could theoretically eliminate IL-
`2Ra-expressing
`leukemic cells while
`retaining
`the
`Tac-nonexpressing normal T cells and their precursors that
`express the antigen receptors required for normal T-cell-
`mediated immune responses. In our preliminary studies, we
`observed that anti-Tac induced a remission in some patients
`with ATL without associated toxicity.” In the present
`study, the 19 patients with ATL treated with anti-Tac had
`few untoward reactions related to the immunotherapy and
`in general did not have a reduction in normal formed ele-
`ments of the blood. Seven of I9 treated patients had a tran-
`sient mixed (I), partial (4), or complete (2) remission, with
`partial and complete remissions lasting from 2 to more than
`36 months.
`
`MATERIALS AND METHODS
`Patient population. Nineteen patients with histologically con-
`firmed HTLV-I-associated ATL were studied (Table I). Each of the
`patients manifested the following features: ( 1) 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 peripheral
`blood, lymph node, or dermal T cells; (3) antibodies to HTLV-I
`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; 10 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, 4 1 years); demographic factors in the patient group are
`shown in Table 1. Ten patients were male and nine female, 17 were
`black, one was Hispanic, and one was of Japanese origin, and 10
`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 alz3 and Yamaguchi et
`11 patients with
`ATL were in the acute or crisis stage, four manifested ATL lym-
`phoma, and four had chronic ATL.
`Therapeutic study 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 IV on two occasions during the
`
`first week of therapy, followed by 40 mg on two occasions during
`the second week. For the subsequent 10 patients, to achieve rapid
`saturation of the IL-2R, the basic plan involved IV administration
`of 50 mg anti-Tac on two occasions for each of the first 2 weeks of
`therapy. Dosing schedules were modified slightly in some patients,
`and additional 20- or 50-mg doses were administered during subse-
`quent weeks to maintain the saturation of the IL-2R with the anti-
`Tac monoclonal antibody. Furthermore, in such cases sufficient
`anti-Tac was administered to yield measurable levels of circulating
`antibody in the serum of the patient. As noted above, patients with
`malignant cells in the central nervous system received intrathecal
`methotrexate.
`The criteria for response were as follows: ( I ) complete response,
`disappearance of all measurable and assessable disease lasting more
`than I month; (2) partial response, a 50% reduction of leukemic cell
`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-
`ance of a new lesion for 1 month (3) mixed response, identical to
`partial response with the exception that there is the appearance of a
`new lesion within I month in a tissue other than that involved
`initially; (4) stable disease, less than partial response with no new
`lesions or less than a 25% increase in any measurable lesion; ( 5 )
`progressive disease, at least 25% increase in leukemic cell count or
`an increase of 25% or greater in any measurable lesion. A systems-
`oriented microcomputer-based patient data management system
`was devised and implemented in which historical, clinical, and labo-
`ratory data were stored and manipulated for analysis (R.P.J. and
`T.A. W.).
`Approval was obtained from the institutional review board for
`these studies. Informed consent was provided according to the Dec-
`laration of Helsinki.
`The anti-Tac
`Production of anti-Tac monoclonal antibodies.
`monoclonal antibody was produced as described previo~sly’~ by
`fusion of NS-1 mouse myeloma cells with spleen cells of mice that
`had been immunized with a cell line derived from an ATL patient.
`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 IL-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
`IgG2a 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 IgG2a,
`IgG,, and IgM, 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 preparation was sterilized by passage through
`a 0.22-pm 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 he nonpyro-
`genic and sterile by the Bureau of Biologics.
`Immunofluorescence 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 previo~sly.’~ Two antibodies (anti-Tac and 7G7/B6) that
`are directed toward different epitopes of IL-2Ra were used to define
`the expression of this receptor subunit. Other monoclonal antibod-
`ies used include antibodies reacting with HLA-DR (Ia-I; Ortho,
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1036 Page 2 of 13
`
`

`
`ANTI-TAC THERAPY OF ATL
`
`1703
`
`Abnormal
`Liver
`Function
`Tests
`+
`+
`+
`-
`+
`+
`+
`+
`+
`
`-
`
`-
`
`Table 1. Demographic and Clinical Features of ATL Patients
`
`Patient
`No.
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`
`Type of
`ATL
`
`Acute
`Chronic
`Lymphoma
`Acute
`Acute
`Acute
`Acute
`Lymphoma
`Acute
`Acute
`Chronic
`Chronic
`Lymphoma
`Acute
`Acute
`Lymphoma
`Chronic
`Acute
`Acute
`
`Age/Sex/Race
`
`39/F/B
`28/M/B
`25/M/B
`32/F/B
`62/M/B
`44/M/B
`4 1 /M/B
`57/F/A
`42/M/H
`58/F/B
`40/M/B
`55/M/B
`53/M/B
`24/F/B
`42/F/B
`34/M/B
`4 1 /FIB
`26/F/B
`6O/F/B
`
`slL-2R (U/mL)
`147,130
`2,240
`4,660
`2,200
`230,370
`87,710
`56,420
`920
`60,170
`8,060
`31,700
`2,040
`89,830
`31,580
`48,460
`9,910
`2,210
`158,130
`138,680
`
`WBC/pL
`
`42,600
`3,800
`1,500
`20,700
`41,500
`12,700
`75,400
`5,500
`102,800
`15,400
`2 1,300
`9,100
`7.900
`32,100
`26,500
`7.100
`13,200
`177,000
`21,600
`
`Circulating
`IL-2RfTac-
`Expressing
`LymphocytesfpL
`1 1,200
`2,120
`<loo
`12,930
`24,200
`1,600
`37,720
`230
`50,600
`1.900
`13,500
`3,000
`1,800
`12,700
`8,010
`<loo
`4,710
`152,900
`4,600
`
`Serum
`Antibodies
`Ca
`to HTLV-I
`+
`3.50
`+
`3.75
`+
`2.15
`+
`2.35
`+
`8.10
`+
`4.35
`+
`4.60
`i 2.30
`+
`4.40
`+
`4.10
`+
`2.65
`+
`2.40
`+
`2.20
`+
`3.90
`+
`2.80
`+
`2.45
`+
`2.20
`+
`3.80
`+
`3.60
`
`-
`-
`
`-
`
`+
`+
`+
`+
`
`-
`
`Abbreviations: B, black; H, Hispanic; A, Asian.
`
`Raritan, NJ); human T-cell-associated antigens (CD2, CD3, CD4,
`CD5, and CD8; Ortho and Becton-Dickinson, Mountain View,
`CA); CD7 (3A1; 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 (1) 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-I integration. Analysis for clonal Tcr gene rearrangements
`and for HTLV-I integration were performed as described previ-
`ously.25*26 High-molecular-weight DNA was extracted from frozen
`cell suspensions containing approximately 10’ cells. DNA samples
`were digested with the restriction enzymes BamHI, EcoRI, HindIII,
`or PstI (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 p 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 germline positions of the Tcr genes
`examined. The Tcr p gene probe used was a 700-bp EcoRI fragment
`containing the mouse or human Cp region that recognizes both
`human Cp regions. The HTLV-I probe used was the 9-kb Sac1
`fragment containing the entire viral genome with the exception of
`the long terminal repeats (a gift from Dr Flossie Wong-Staal).
`The sera of ATL patients were
`Assay for antibodies to HTLV-Z.
`analyzed for antibodies to disrupted and inactivated HTLV-I using
`an enzyme-linked immunosorbent assay ([ELISA] Cellular Prod-
`ucts, Buffalo, NY).
`
`Assay for mouse Zg. Murine IgG2a anti-Tac levels in the sera of
`patients were assayed with an ELISA technique using affinity-pun-
`tied goat antimouse immunoglobulin absorbed onto polyvinyl
`plates at 100 ng/well and washed.” 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 antimouse 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 a12’ was used, with
`the exception that anti-Tac was used in lieu of the TlOl 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-I-associated ATL were treated with
`IV administered anti-Tac (Table 1). Four patients had lym-
`phoma-type ATL without circulating malignant cells; the
`peripheral blood WBC count in the remaining 15 patients
`before therapy ranged from 3,800 to 177,0OO/~L (geometric
`mean, 26,000). Patients with ATL had pretherapy serum
`soluble IL-2R (sIL-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).** T-cell leukemic populations were
`confirmed to be monoclonal by molecular genetic analysis
`of the rearrangement of genes encoding the Tcr p chain.
`Southern blot analysis of the Tcr p receptor gene rearrange-
`ment using a radiolabeled probe that hybridizes with the
`constant region of the Tcr p chain showed a band that was
`not present in germline tissues, a hallmark of a clonally
`expanded population of T lymphocytes (Fig 1). Further-
`more, Southern blot analysis of HTLV-I proviral integra-
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1036 Page 3 of 13
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`

`
`1704
`
`WALDMANN ET AL
`
`tion in Psi I and EcoRI digests of DNA obtained from the
`patients defined the clonal integration of HTLV-I 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 I3 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', CD4-/
`CD8-, CD25' and CD3-, CD4+/CD8-, CD25'. Circulating
`mononuclear cells ofthe patients showed intense expression
`of the Tac antigen on a relatively homogeneous cell popula-
`tion manifesting high fluorescence intensity (Fig 2). It a p
`pears that all ofthe 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 pattem 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 CD7 (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 paiienls IO lrealment with ami-Tac
`monoclonal anlihodies. The initial basic protocol for anti-
`Tac therapy involved the administration of 20 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 of 20 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 to 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: ( 1) reaction with FITC antimouse IgG; (2) reaction
`with FITC-labeled 7G7/B6 (an antibody that reacts with
`IL-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
`
`Patient
`23 days post
`treatment
`-
`
`-
`
`8.8 Kb
`7.1 Kb +
`
`+
`
`-
`-U-
`
`Patient
`1099 days post
`treatment
`
`- u -
`
`I
`
`*
`
`
`
`- Y-
`
`Placental
`(germline)
`11 Kb - y
`8.5 Kb
`
`I
`
`4 K b - (1
`
`11 Kb-
`
`4
`BamHl EcoRl
`
`mm+
`
`VPn DP1
`
`+
`
`II I I I1
`
`JP1
`
`EcoR I
`
`+
`
`EcoRl
`
`+
`-4
`EcoRl
`111111
`
`I
`
`Kb-
`EcoRl BamHl
`
`+ +
`
`CP2
`
`CPl
`DP2
`JS2
`w C , I , P R O B E *
`...... .....
`........ ...
`
`H
`1 Kb
`Fig 1. Analysis of Tcr g gene rearrangements to monitor anti-Tac monoclonal antibody treatment in patient no. 5 with ATL using a Tcr 6
`constant region probe (Cg). The Tcr g constant region genes are on four and 1 1 -Kb €col71 fragments in placental (germline) DNA as indicated
`(---). The 1 1 -Kb band contains Cg1 , whereas Cg2 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 Cg2 locus. Digests of patient peripheral blood DNA during an active phase of the disease 23 days after initiation of
`therapy yielded a diminished 1 1 -Kb band as well as two nongermline bands (+ that reflect a monoclonal Tcr g pattem of gene rearrange-
`ment. This pattem indicates that both alleles for Tcr g in the leukemic clone rearranged into Cgl . 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 0 chain gene, we indicate the locations of the BemHl and €coRI
`restriction endonuclease sites as well as the Cg regions recognized by the cDNA probe.
`
`......
`
`PETITIONER'S EXHIBITS
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`Exhibit 1036 Page 4 of 13
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`

`
`ANTI-TAC THERAPY OF ATL
`
`1705
`
`Pretherapy
`
`Control -
`
`I
`
`1 0 2
`10'
`103
`Relative Fluorescence Intensity
`
`1
`
`4
`
`Post Therapy
`
`_I_
`
`Control
`Anti Tac
`
`-
`
`5
`a
`E 3 50-
`- -
`
`I 1 I - i
`
`j
`
`0
`
`I
`
`I a m 1 1 ,
`
`I I
`
`x m ~ n & m 1
`
`I I
`
`t n i i a 1 1
`
`I I i i i i i r
`
`C
`100 I
`
`I lA
`
`Normal Cells
`
`- Control - Anti Tac
`
`0
`1 00
`
`I ,,,,I,-
`
`Relative Fluorescence Intensity
`
`vivo administration of this antibody. In light of these early
`observations, to achieve a rapid saturation of 1G2R, 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 of 23 distinct courses of therapy; there were
`three to 1 1 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, 225). 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 ATL 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 of 39.1 "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/
`pL and in the neutrophil count to 380/pL. 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 ATL before
`therapy; (B) the same patient in remission following completion of
`anti-Tac therapy: and (C) a normal individual. Circulating mononu-
`clear cells of the patient showed intense expression of the Tac anti-
`gen on a relatively homogeneous 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.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1036 Page 5 of 13
`
`

`
`1706
`
`Patient
`No.
`1
`2
`
`3
`4
`5
`6
`7
`8
`9
`10
`
`11
`
`12
`
`13
`14
`15
`
`16
`
`17
`
`18
`19
`
`Previous Therapy
`None
`ProMACEJMOPP
`
`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
`
`Dose of Anti-Tac
`Administered (mg)
`150
`1 st course, 160
`2nd course, 60
`300
`200
`400
`1 80
`147
`250
`120
`100
`
`1 st course, 490
`2nd course, 500
`300
`
`200
`200
`180
`
`1 st course, 200
`2nd course, 200
`
`Bleomycin, cytoxan,
`doxorubicin
`ProMACEJMOPP
`None
`
`1 st 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
`
`Table 2. Effect of Anti-Tac Therapy
`
`WALDMANN ET AL
`
`Clinical Response/
`Duration (d)
`PD
`PR 224
`PR 70
`PR 63
`PD
`CR > 1,098
`PD
`PD
`PD
`PD
`PD
`
`CR 357
`PD
`SD
`
`SD
`PD
`MR 13
`
`PR 252
`PD
`
`Development of
`Anti-Tac
`Antibodies
`(HAMA)
`
`-
`
`-
`
`+
`+
`
`-
`
`~
`
`~
`
`-
`
`~
`
`-
`
`-
`-
`
`-
`
`+
`
`-
`
`~
`
`-
`
`PR 168
`SD
`None
`PD
`None
`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 9,30O/pL to less than
`200/pL (normal individuals have 1500 circulating T cells/
`pL that express Tac weakly). This patient was deemed to
`have a transient mixed, rather than a partial, remission,
`because I- 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 ATL 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 4 1,8OO/pL that included 23,000 circulating
`Tac-expressing malignant T cells/pL 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, t