the A1nerican Society of He1natology
`
`lnsect-St,ing Anaphylaxis and Activation of the Fibrinolytic and Contact Systems
`
`American Society of Hematology
`Thirty-Fifth Annual Meeting
`December 3-7, 1 993
`StLouis, MO
`(see page 1 941 for additional information)
`
`BIOEPIS EX. 1036
`Page 1
`
`

`

`The Interleukin-2 Receptor: A Target for Monoclonal Antibody Treatment of
`Human T -Cell Lymphotrophic Virus 1- lnduced Ad ult T -Cell 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(cid:173)
`phocytes caused by the retrovirus human T-cell lympho(cid:173)
`trophic virus-I (HTLV-1). It is an aggressive leukemia with
`an overall mortality rate of 50% within 5 months; no con(cid:173)
`ventional chemotherapy regimen appears successful in in(cid:173)
`ducing long-term disease-free survival in A TL patients.
`However, ATL cells constitutively ellpress high-affinity in(cid:173)
`t erleukin-2 receptors (IL-2Rs) identified by the anti-Tac
`monoclonal antibody, whereas normal resting cells do not.
`To ellploit this difference in receptor expression, we ad(cid:173)
`ministered anti-Tac intravenously (IV) to 19 patients with
`ATL. In general the patients did not suffer untoward reac(cid:173)
`tions, and in 18 of 19 cases did not have a reduction in
`normal formed elements ofthe blood. Seven patients devel(cid:173)
`oped remissions that were mixed (1 patient), partial (4 pa-
`
`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(cid:173)
`cence analys is, and molecular genetic analysis ofT -cell re(cid:173)
`ceptor gene rearrangements and of HTLV-1 proviral inte(cid:173)
`gration. Furthermore, remission was associated with a
`return to normal serum calcium levels and an improve ment
`of liver function tests. Remission was also associated in
`some cases with an amelioration of the profound immuno(cid:173)
`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.
`
`T HE HYBRlDOMA T ECHNIQUE of Ktihler and Mil(cid:173)
`
`stein' rekindled interest in the use of antibodies tar(cid:173)
`geted to cell surface antigens to treat cancer patients. Im(cid:173)
`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 djrected against a
`vital cell surface structure required for tumor cell prolifera(cid:173)
`tion and survival. 2 We readdressed this issue using the inter(cid:173)
`leukin-2 receptor (IL-2R) as the target for immune inter(cid:173)
`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 oflymphoid neoplasia, select autoimmune
`disorders, and in individuals rejecting allografts.J-7 T he IL-
`2R consists of at least tluee IL-2-binding peptide chains. as
`follows: JL-2Ra, a 55-Kd peptide identified by the monoclo(cid:173)
`9
`11
`nal antibody anti-Tac8
`; IL-2R,8, a 75-Kd subunit 10
`; and
`·
`·
`the recently discovered TL-2R-y, a 64-Kd protein.12 ldentifi(cid:173)
`cation and characterization of the IL-2Ra suburut was facili(cid:173)
`tated by our development of a monoclonal antibody, anti(cid:173)
`Tac, that binds to lL-2Ra and prevents the interaction of
`IL-2 with this subunit.8 Resting T cells, B cells, and mono(cid:173)
`cytes in the circulation do not display I L-2Ra. ln contrast to
`this lack of LL-2Ra expression in normal resting mononu(cid:173)
`clear cells. thls 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(cid:173)
`kemias. Furthermore, the serum concentration of the solu(cid:173)
`ble form of 1L-2Ra released by the abnormal cells is in(cid:173)
`creased in patients with these disorders.13 Specifically, a
`proportion of the leukemic cells of patients with chroruc or
`acute myelogenous leukemia express 1L-2Ra identified by
`the anti-Tac monoclonal antibody. 14 Furthermore, malig(cid:173)
`nant B cells of virtually all patients with hairy cell leukemia
`and a proportion of patients with large- and mixed-celJ dif(cid:173)
`fuse lymphomas express IL-2Ra.15 T he IL-2Ra is also ex(cid:173)
`pressed on Reed-Sternberg cells of patients with Hodgkin's
`
`disease and on malignant cells of patients with true histiocy(cid:173)
`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(cid:173)
`man T-cell lymphotrophic virus-1 (HTLV-1)-associated
`adult T-cell leukemia (ATL) constitutively express very
`19
`large numbers of IL-2Ra. 17
`•
`These results suggested that anti-JL-2R antibodies might
`be effective agents for the treatment of certain neoplastic
`diseases. We have focused our therapeutic studies of malig(cid:173)
`nancy on the disordered 1L-2R 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+, cos-,
`CD7-, and CD25+ (1L-2Ra, Tac+) phenotype. 17
`18 The dis(cid:173)
`•
`ease exhibits a striking clustering of cases in certain geo(cid:173)
`graphic regions, notably southwestern Japan. the Caribbean
`basin, northeastern South America, Central America, sub(cid:173)
`Saharan Africa, and to a lesser extent the southeastern
`United States. The retrovirus HTL V-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-1 and the monoclonal integration ofthis retrovirus
`in their circulating malignant cells. Frank A TL generally
`has its onset in adulthood, 20 to 30 years following initial
`
`From the Metabolism Branch and Laboratory of Pathology, Na(cid:173)
`tional Cancer lnstitwe, and the Clinical Pathology Department,
`Warren Grant Magnuson Clinical Cemer. National lmtitllfes of
`Health. Bethesda. MD.
`Submilled March 17. 1993: accepted May 27. 1993.
`Address reprint requests to Thomas A. Waldmann. MD, Metabo(cid:173)
`lism Branch, National Cancer lnstitwe. Bldg 10, Rm 4Nll5, Be(cid:173)
`thesda. MD 20892.
`The publication costs oft his article were def rayed in part by page
`charge pa_vmem. This article must 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. Vo182, No 6 (September 15), 1993: pp 1701 -1712
`
`1701
`
`BIOEPIS EX. 1036
`Page 2
`
`

`

`1702
`
`WALDMANN ET Al
`
`infection with HTLV-1. Principal clinical features include
`moderate lymphade nopathy. hepatosplenomegaly. and
`skin, central nervous system, and pulmonary involve(cid:173)
`ment21.22; the occurrence of hypercalcemia is characteristic
`of ATL. Patients with acute ATL manifest a striking degree
`of immunosuppression and develop opportunistic infec(cid:173)
`tions such as Pneumocystis pneumonia and cryp tococcal
`meningitis. The experiences of several clinical oncology
`groups using combination chemotherapy regimens in pa(cid:173)
`tients with this disease have been disappointing. In most
`chemotherapy series, overaU mortality is approximately
`50% within 5 months. No conventional treatment program
`appears successful in inducing long-term disease-free sur(cid:173)
`vival in ATL patients.
`In our clinical trial, we wished to exploit the observation
`that the normal resting cells of patients with A T L do not
`display IL-2R a , whereas the malignant T cells display
`I 0,000 to 35,000 IL-2Raj cell that are identified by the anti(cid:173)
`Tac monoclonal antibody. 17
`18 Thus IL-2R-directed immu(cid:173)
`•
`notherapy using anti-Tac could theoretically eliminate IL-
`2Ra -expressing
`leukemic cells while
`reta1010g
`the
`Tac-nonexpressing normal T cells and their precursors that
`express the antigen receptors required for normal T-cell(cid:173)
`mediated immune responses. Jn our preliminary studies, we
`observed that anti-Tac induced a remission in some patients
`with ATL without associated toxicity. 19 In the present
`study, the 19 patients with A TL treated with anti-Tac had
`few untoward reactions related to the immunotherapy and
`in general did not have a reduction in normal formed ele(cid:173)
`ments of the blood. Seven of 19 treated patients had a tran(cid:173)
`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
`
`Patiem population. Nineteen patients with histologically con(cid:173)
`firmed HTL V -!-associated A TL were studied (Table I). Each of the
`patients manifested the following features: (I ) a histologically con(cid:173)
`firmed diagnosis of leukemia or lymphoma of mature T cells with
`polymorphic indented or lobulated nuclei: (2) intense expression of
`the Tac antigen (IL-2Ro') on at least 10% of the patient's peripheral
`blood. lymph node. or dermal T cells; (3) antibodies to HTLV-1
`demonstrable in the serum; and (4) omission of cytotoxic chemo(cid:173)
`therapy and radiation therapy for at least 3 weeks before emry into
`the trial. Patients with or without previous chemotherapy were eligi(cid:173)
`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(cid:173)
`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 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 al23 and Yamaguchi et al,24 II patients with
`A TL were in the acute or crisis stage, four manifested A TL lym(cid:173)
`phoma. and four had chronic A TL.
`Therapell/ic sllld)' plan. Anti-Tac was administered intrave(cid:173)
`nously (IV) over a 2-hour period in 100 mL normal saline contain(cid:173)
`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 I 0 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 su~
`quent weeks to maintain the saturation of the IL-2R with the anti(cid:173)
`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 ofleukernic 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(cid:173)
`ance of a new lesion for I month; (3) mixed response, identical to
`pnrtinl 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. Jess 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 of25% or greater in any measurable lesion. A systems(cid:173)
`oriented microcomputer-based patient data management system
`was devised and implemented in which historical, clinical, and labo(cid:173)
`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 stud.ies. Informed consent was provided according to the Dec(cid:173)
`laration of Helsinki.
`Production Q( ami-Tac monoclonal amibodies. The anti-Tac
`monoclonal antibody was produced as described previously19 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(cid:173)
`icity with human plasma nor does it induce antibody-dependent
`cellular cytotoxicity (A DCC) with human mononuclear cells. How(cid:173)
`ever, anti-Tac blocks the interaction of IL-2 with the high-affinity
`receptors for this Iymphokine. Large quantities of the monoclonal
`antibody were produced by inoculating hybridoma cells into tbe
`peritoneal cavity of BALB/c mice and then purifying the mouse
`JgG2a anti-Tac from the resulting ascites fluid by diethylamino(cid:173)
`ethyl cellulose chromatography. The purified antibody was dia(cid:173)
`lyzed against saline, centrifuged, filtered. precipitated 'vith 20% so(cid:173)
`dium sulfate. and finally diluted in saline at pH 7.4 to a
`concentration of 2 mgfmL. Each lot of the product was assayed by
`immunoelectrophoresis in agar plates using antisera to JgG2a,
`lgG, , and JgM, 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(cid:173)
`decyl sulfate- polyacrylamide gel electrophoresis were used. The
`monoclonal antibody preparation was sterilized by passage through
`a 0.22-~tm membrane filter (Millipore, Marlborough, MA) by the
`Pjlarmaceutical Development Section of the Clinical Center of the
`National Institutes of Health (NIH) and was shown to be non pyro(cid:173)
`genic and sterile by the Bureau of Biologics.
`Immunofluorescence analysis of cell suiface phenotype. The
`phenotype of the leukemic cell population was defined by indirect
`and direct immunofluorescence performed with mouse monoclo(cid:173)
`nal antibodies using a fluorescence-activated cell-sorter as de(cid:173)
`scribed previously. 19 Two antibodies (anti-Tac and 7G7/B6) that
`are directed toward different epitopes of1L-2Ra were used to define
`the expression of this receptor subunit. Other monoclonal antibod(cid:173)
`ies used include antibodies reacting with HLA-DR (la-1: Ortho,
`
`BIOEPIS EX. 1036
`Page 3
`
`

`

`Table 1. Demographic and Clinical Features of ATL Patients
`
`Type of
`ATL
`
`Acute
`Chronic
`Lymphoma
`Acute
`Acute
`Acute
`Acute
`Lymphoma
`Acute
`Acute
`Chronic
`Chronic
`Lymphoma
`Acute
`Acute
`Lymphoma
`Chronic
`Acute
`Acute
`
`1
`2
`3
`4
`6
`6
`7
`8
`9
`10
`11
`12
`13
`14
`16
`16
`17
`18
`19
`
`Age/Sex/Race
`
`sll-2R (U/ml)
`
`39/F/ B
`28/M/ B
`25/M/ B
`32/F/ B
`62/ M/ B
`44/ M/B
`41 / 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
`41 /F/ B
`26/ F/ B
`60/F/ B
`
`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
`
`Abbreviations: B. black; H, Hispanic; A. Asian.
`
`Circulating
`IL·2R{Tac-
`Expressing
`Lymphocytes/"L
`
`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
`
`WBC/"L
`
`42,600
`3.800
`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
`
`Antibodies
`to HTLV-1
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`
`Serum
`Ca
`
`3.50
`3.75
`2.15
`2.35
`8.10
`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
`Function
`Tests
`+
`+
`+
`
`+
`+
`+
`
`+
`
`+
`
`+
`
`+
`
`+
`+
`
`Raritan. NJ): human T -<:ell- associated antigens (CD2. CD3, CD4,
`CDS, and CDS; Ortho and Becton-Dickinson. Mountain View.
`CA); CD7 (3AI ; a gift from Dr Barton Haynes); and CD45 and
`CD29 (Coulter Immunology, Hialeah, FL). The nuorescein isothio(cid:173)
`cyanate (FITC) antimouse lgG reagent was obtained from Coulter
`Immunology. Histograms for each cell type were integrated to de(cid:173)
`termine the percentage of mononuclear cells that reacted with indi(cid:173)
`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(cid:173)
`culating WBCs that were mononuclear cells as determined by rou(cid:173)
`tine hematologic analysis. and (3) the proportion of these mononu(cid:173)
`clear cells that expressed the antigen under study as assessed by
`immunocy10nuorography.
`Molecular genetic anal.vsis of Tcr gene rearmngemenl and
`HTLV-1 integration. Analysis for clonal Tcr gene rearrangements
`and for HTLV -1 integration were performe.d as described previ(cid:173)
`ously.u.>• High-molecular-weight D A was extracted from frozen
`cell suspensions containing approximately I 0 8 cells. DNA samples
`were digested with the restriction enzymes Bam HI. EcoRI. Hind Ill.
`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-Iabeled DNA
`Probes of the constant regions of the Tcr {3 gene and the HTL V-I
`gene were performed. followed by washing at appropriate strin(cid:173)
`gency and radioautography. Nonlymphoid control DNA was ana(cid:173)
`ly-zed simultaneously to identify germ line positions of the Tcr genes
`examined. The Tcr {J gene probe used was a 700-bp EcoRl fragment
`containing the mouse or human C{J region that recognizes both
`human C{J regions. The HTL V-I 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 alllibodies to HTLV-1. The sera of ATL patients were
`analyzed for antibodies to disrupted and inactivated HTL V -1 using
`an enzyme-linked immunosorbent assay ([ELISA] Cellular Prod(cid:173)
`ucts, Buffalo, NY).
`
`Assay for mouse !g. Murine IgG2a anti-Tac levels in the sera of
`patients were assayed with an ELISA technique using affinity-puri(cid:173)
`fied goat antimouse immunoglobulin absorbed onto polyvinyl
`plates at 100 ngjwell and washed.19 Bound murine anti-Tac was
`detected with a goat anti mouse immunoglobulin conjugated with
`alkaline phosphatase (Sigma. St Louis, MO) and compared with a
`standard curve ofanti-Tac antibody.
`Assayforhuman afllimouseall/i-Tacamibody. The serum used
`in all assays was separated from peripheral blood and stored at
`- 20"C until used. For detection of human anti mouse antibodies
`(HAMA), the technique described by Schroffet al 27 was used, with
`the exception that anti-Tac was used in lieu of the TIOI antibody.
`Serum antiglobulin levels for a given patient were considered mean(cid:173)
`ingfully increased when the antiglobulin level after therapy was
`greater than twice that in the sample obtained before administra(cid:173)
`tion of the mouse monoclonal antibody.
`
`RESULTS
`
`Patient characteristics. Nineteen patients with histologi(cid:173)
`cally confirmed HTL V -!-associated A TL were treated with
`IV administered anti-Tac (Table I). Four patients had lym(cid:173)
`phoma-type ATL without circulating malignant cells; the
`peripheral blood W BC coLint in t he 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
`(m ean, 55,744), whereas the upper limit of the normal is
`502 U/ mL (mean, 238). 28 T-Cellleukemic populations were
`confirmed to be monoclona.l by mo.lecular genetic analysis
`of the rearrangement of genes encoding the Tcr fJ chain.
`Southern blot analysis of t he Tcr fJ receptor gene rearrange(cid:173)
`ment using a radiolabeled probe that hybridizes with the
`constant region of the T cr fJ chain showed a band that was
`noi present in germ line tissues. a hallmark of a clonally
`expanded population ofT lymphocytes (Fig I). Further(cid:173)
`more. Southern blot analysis of HTL V-1 proviral integra-
`
`BIOEPIS EX. 1036
`Page 4
`
`

`

`1704
`
`WALDMANN ET Al
`
`tion in Pst I and £coR I digests of DNA obtained from the
`patients defined the clonal integration of HTL V-I provi(cid:173)
`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 S.l mmol/ L (nor(cid:173)
`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(cid:173)
`tion expressed the CD3+, C D4+;cos-, CD25+ phenotype;
`phenotypes in the two remaining cases were CD3+, CD4-;
`c os-. ems+ and CD3-, CD4+ ;co s - , C D2s+. Circulating
`mononuclear cells of the patients showed intense expression
`of the Tac antigen on a relatively homogeneous cell popula(cid:173)
`tion manifesting high fluorescence intensity (Fig 2). It ap(cid:173)
`pears that all of the circulating malignant cel.ls expressed the
`Tac antigen. Although a small proportion of normal periph(cid:173)
`eral blood mononuclear cells manifest low-level Tac expres(cid:173)
`sion, the pattern observed within the leukemic population is
`quite distinct from that observed in normal individuals in
`terms of the homogeneity and intensity ofTac antigen ex-
`
`pression (Fig 2). In the IS leukemic cases, the abnormal cell
`population did not react with a CD7 (3A 1) monoclonal an(cid:173)
`tibody, which reacts with normal T-cell precursors and with
`at least 70% of normal mature T: lymphocytes.
`Response of ATL paLienls to treatment with anti-Tac
`monoclonal amibodies. The initial basic protocol for anti(cid:173)
`Tac therapy involved the administration of20 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(cid:173)
`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(cid:173)
`nal antibody when the cells manifested the following three
`features: ( l ) 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(cid:173)
`labeled anti-Tac, since the target antigen was blocked by in
`
`Placental
`(germline)
`
`Patient
`23 days post
`treatment
`
`Patient
`1099 days post
`treatment
`
`11 Kb -
`8.5 Kb •
`
`4 Kb -
`
`8.8 Kb-
`7.1Kb-> -
`
`~ill!
`V(Jn D(J1
`
`1111 11
`J(J1
`
`EcoRI
`
`-
`4Kb -
`EcoRI
`EcoRI BamHI
`+
`t +
`111 111 ~
`
`0
`C(J1
`~C(JPROBE_... I--;
`1 Kb
`
`Fig 1. Analysis of Tcr fJ gene rearrangements t o monitor anti-Tac monoclonal antibody treatment in patient no. 5 with ATL using a Tcr{J
`constant region probe (C{J). The Tcr {J constant region genes are on four and 11 -Kb EcoRI fragments in placental (germline) 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{J21ocus. 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!+ 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 t herapy did not express the two nongerrnline bands, thus confirming elimination of the circulating monoclonal
`population. In the schematic diagram of the gerrnline arrangement of the Tcr {J chain gene, we indicate the locations of the Bam HI and EcoRI
`restriction endonuclease sites as well as the C{J regions recognized by the eDNA probe.
`
`BIOEPIS EX. 1036
`Page 5
`
`

`

`TAC THERAPY OF ATL
`
`1705
`
`Pre therapy
`100~--~--------------------.
`
`--Control
`AntiTac
`
`50
`
`o~~~mr~~~~~~.-rM~
`101
`102
`103
`104
`10°
`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 I 0 patients in
`the group so that 50 mg anti-Tac per patient was adminis(cid:173)
`tered on two occasions during the first week of therapy and
`on two occasions during the second week of therapy. Addi(cid:173)
`tional doses of 50 mg anti-Tac to maintain receptor satura(cid:173)
`tion were administered to patients who made an initial par(cid:173)
`tial or complete response to therapy. The 19 patients
`received a total of23 distinct courses of therapy; there were
`three to II infusions per treatment course (mean, five).
`T reatment 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 m ouse
`antibody achieved in the serum 24 hours after the last thera(cid:173)
`peutic dose of a treatment course ranged from 595 to 12,230
`ngjmL.
`Toxicity. The 19 patients officially entered into the clin(cid:173)
`ical trial did not have any untoward acute reactions. How(cid:173)
`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(cid:173)
`tion. Thi~ patit:nl, whu wa~ ill un admi~iun with bt:art fail(cid:173)
`ure, hepatosplenomegaly, and multiple effusions, died of
`respiratory distress following the fifth infusion of anti-Tac;
`at a utopsy, the cause of death was shown to be massive
`hemorrhagic consolidation of the lungs. One patient devel(cid:173)
`oped hives after the second of four anti-T ac infusions. Two
`patients developed fever to a maximum of39. 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(cid:173)
`fecting the normal formed elements of the blood including
`platelets or polymorphonuclear leukocytes. In the one ex(cid:173)
`ception to this generalization, a patient developed a hemo(cid:173)
`lytic anemia and a reduction in the platelet count to 26,000/
`JLL and in the neutrophil count to 380/JLL. This patient had
`leukemic involvement of the bone marrow; he received sub(cid:173)
`sequent doses ofanti-Tac without any further untoward re(cid:173)
`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 normal individual. Circulating mononu(cid:173)
`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 pattem 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.
`
`Post Therapy
`
`--Control
`Anti Tac
`
`8
`100
`
`50
`
`i
`\
`\
`
`{~
`) ~
`
`0
`100
`
`102
`103
`101
`Relative Fluorescence Intensity
`
`1 Q4
`
`c
`
`Normal Cells
`
`Relative Fluorescence Intensity
`
`BIOEPIS EX. 1036
`Page 6
`
`

`

`1706
`
`Patient
`No
`
`1
`2
`
`3
`4
`5
`6
`7
`8
`9
`10
`
`11
`
`12
`
`13
`14
`15
`
`16
`
`17
`
`18
`19
`
`Tabl e 2. Effect of Anti-Tac Therapy
`
`W ALOMANN ET AL
`
`Previous Therapy
`
`None
`ProMACE/ MOPP
`
`None
`CHOP
`None
`OCF
`None
`MACOP-8
`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 Antl-T ac
`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" CI
`Fever (38" C)
`None
`None
`None
`
`None
`
`None
`
`Hives
`None
`None
`
`Transient neutropenia
`Thrombocytopenia,
`hemolytic anemia
`None
`
`None
`None
`
`Development of
`Anti-Tac
`Antibotlies
`(HAMAl
`
`+
`
`+
`
`+
`
`Chnical ReSJ>Onsef
`Duratoon (d)
`PO
`PR 224
`PR 70
`PR 63
`PO
`CR > 1,098
`PO
`PO
`PO
`PO
`PO
`
`CR 357
`PO
`so
`
`so
`PO
`MR13
`
`PR 252
`PO
`
`PR 168
`so
`PO
`PO
`
`Abbreviations: ProMACE, prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide; MOPP, mechlorethamine, vincristine, procarba·
`zine, prednisone; CHOP, cyclohosphamide, doxorubicin, vincristine, prednisone; OCF, pentostatin; MACOP-B, methotrexate, doxorubicin, cyclophos(cid:173)
`phamide, vincristine, prednisone, bleomycin; MTX, methotr.ate; COP, cyclophosphamide, vincristine, prednisone; CR, complete remission; PR. partial
`response; SO, stable disease; PO, 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/ILL to less than
`200/ILL (normal individuals have !>:500 circulating T cells/
`ILL 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 A TL. developed a partial remission; the du(cid:173)
`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(cid:173)
`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 bad a second partial remission lasting 70
`days followi ng re-treatment, but then relapsed. Two addi(cid:173)
`tional patients, one with chronic A TL and one with acute
`ATL, developed a complete remission, which lasted for 357
`days in the pati