THE LANCET
`
`No. 8275
`
`LONDON SATURDAY 3 APRIL 1982
`
`VOL. I FOR 1982
`
`ORIGINAL ARTICLES
`Autoimmunity to Anterior Pituitary Cells and the Pathogenesis of
`Insulin-dependent Diabetes Mellitus
`Rita Mi rakian, M.D., A. G. Cudwort h, F.R.C. I'., G. F. Bottazzo, M.R.C. I'ATH .,
`Anne Richardson, B.sc .. Prof. Deborah Doniach, F.R.C. I'.
`Colonic Absorption ·of Secondary Bile-acids in Patients with Adenomatous
`Polyps and in Matched Controls
`S. D. J. va n der We rf, M D , F. M . Nage ngast, M. D , G. P. va n Berge Henegouwe n, M.D.,
`A. \XI. M. Huij brcgts, !' II. D., ] . H. M . van Tongercn, M.D.
`Phase-I Clinical Trial of Monoclonal Antibody in Treatment of
`Gastrointestinal Tumours
`H . F. Scars, M.D .. Barbara Atkinson, M.D., Jeffrey Mattis, M.D,
`Carolyn Ernst, MD ., Dorothec Hcrlyn, D.V.M., Zenon Stcplewski, M.D.,
`Pekka 1-Hiyry, M.D., Prof. Hilary Koprowski, M.D.
`Morphological Identification of the Agent of Korean Haemorrhagic Fever
`(Hantaan Virus) as a Member of the Bunyaviridae
`] . B. McCormick, M.D., D . R. Sasso, B.s., E. L. Palme r, 1'1-l .D., M . P . Kiley, I'H .D.
`tlantaa n Virus, Aetiological Agent of Korean Haemorrhagic Fever, Has
`Bunyaviridae-like Mo~phology
`] . D . Whit e, I' H.D., Frances Shirey, B.A., G. R. French, l' h.D. ]. W . Huggins, PH .D ,
`0. M . Brand, M.S, Ho Wa ng Lee, M.D.
`
`755
`
`759
`
`762
`
`765
`
`768
`
`PRELIMINARY COMMUNICATION
`Serotonin Immunoreactivity in
`Carcinoid Tumours Demonstrated
`by a Monoclonal Antibody
`A. C. Cuel lo, M D.,
`C. Wells, M .B.,
`A. ] . Chaplin, F I.M .L.S.,
`C. Milstein, !' II .D., F.R.s
`METHODS AND DEVICES
`Porous Containers for Small
`Biopsy Specimens
`S. ] . Colley, M R c P •
`T. Gkdhill, F R C.S .•
`R. Bone, I' .I.M .L.s .•
`] . Burston, F.R.C.I'AT H.,
`D. G. Colin-Jones, F R.C. I' .
`
`REVIEWS
`Noticcsof'B ooks
`
`OCCASIONAL SURVEY
`Patterns of Hepatocyte Injury in
`Man
`Prof. Sheila Sherlock, DB . E., F.R.C. I' .
`
`HOSPITAL PRACTICE
`Five-year Study ofCimetidine or
`Surgery for Severe Duodenal Ulcer
`Dyspepsia
`G . R. Gray, I'.R.C.S,
`D. McWhinnic, I' R.c.s .•
`I. S. Smith, I· R.c.s.,
`G. Gilles pie, I' .R. c.s.
`
`77 1
`
`773
`
`774
`
`782
`
`787
`
`DISABILITIES AND HOW TO
`LIVE WITH THEM
`22 Years a Tetraplegic
`R. G. Coc
`
`ROUND THE WORLD
`Unit edStates
`
`789
`
`790
`
`MEDICINE AND THE LAW
`Extent of Su rgeon's Duty to Explain
`Particular Risks Inherent in Ope rat ton 808
`
`COMMENTARY FROM
`WESTMINSTER
`M r C larke on the Effects of Cuts
`Standards of Performance from Region
`to Region
`
`809
`
`809
`
`Traveller's Diarrhoea
`T Lymphocytes
`Cryoa na1gcsia
`Clonogenic Assays for the
`Chemotherapeutic Sensitivity
`ofHu ma n T umours
`a 2 Adrenergic Receptors in
`Depression
`
`777
`778
`779
`
`780
`
`78 1
`
`79 1
`
`79 1
`
`792
`
`792
`
`793
`
`793
`
`794
`
`LETTERS TO THE EDITOR
`Passive Smoking
`M r 1'. N. Lee, M.A.
`Cotinine in Amniotic Fluids from
`Pass ive Srnokcrs
`Dr B. D. And rese n and others
`Cholecystokinin to Treat Paralytic Ileus
`D r I ngc r Ma gnusson, D r Thomas lhrc
`Experien ce with a Practical Food
`Pattern to Reduce Heart Disease
`Dr Hele n Brow n, Dr I. 1-1 . !'age
`Caffeine for Allergic Rhinitis
`Mr Phi li p Shapiro
`Diagnostic Contribution of
`Echoca rdiography
`Dr M G . St J. Sutton
`Effect of S teroids on Placental Transfer
`ofl>latelet Autoantibodies
`Dr A. H. Wa ters,
`M iss R. M . Mi nchinton , B.Appl. Sc.
`Taxonomic Features of Cau sative Agent
`of Hacmorrhagic Fever with Renal
`Syndrome
`Dr M. A. Donets and ot hers
`Ockclbo Disea se: Epidemic Arthritis(cid:173)
`cxanthcrna Syndrome in Sweden
`C aused by Sindbis-virus-like Agent
`Dr Ma rcus Skogh, Prof. Akc Espmark
`Disposition of Morphine in
`Cerebrospinal Fluid after Epidural
`1\.drninistration
`Dr I .. L. Gu stafsson :tnd ot hers
`Storing Oral Rehydration Solution
`Dr M . Sa nt osham and others
`J ~gi o nnairc s ' Disease Related to Gastric
`La vage with Tap Water
`Ur E. Dourn on and 01hcrs
`hnpo rt a n cc of P a th ological E xamination
`of Products of Conception which arc
`Delivered after Prenatal Screening
`Pro cedures
`l' rol Hope l' unnett, Prof. D. S. Hun·
`Bee Stin gs
`Dr R. Urbanek and others
`
`799
`
`799
`
`800
`
`800
`
`80 1
`
`80 1
`
`80 1
`
`802
`
`802
`
`802
`
`802
`
`803
`
`803
`
`803
`
`804
`
`lmmunoregulation in Patients with
`Rheumatoid Arthritis
`ll> 1'. M . Lydyard and ot hers
`Alcohol and Breast C ancer
`Dt T im Bye rs, Dr Donna Funch
`Rena l Disturbance in Cystic Fibrosis
`D1 Birgitta St ra nd vik
`Clost ridium difficile, Enterocolitis, and
`Hirschsprung's Disease
`Dr M S. Coopcrstock
`Mental Health Legislation
`M r Larry Gostin, LI.B .,
`Mrs Gwynneth Hemmings, B.Sc.
`Ramptonlnquiry
`M r Peter Thompson
`Pharmacological Defences against
`Shoplifting
`D r B. C. Dean, M r C. L. Bywater
`How LongWilliBein, Doctor?
`M r A. D. B. Chant, F. R.C.S.
`How Long Has He Got, Doctor?
`M r C.- B. Wa lle mark, M.S.
`U.S. Gun Laws
`D r JefT Murray
`Career Prospects in Medical
`Gastroenterology
`Dr Dav id Pyke
`Endotoxin Reference Standard
`Dr A. S. Out schoorn
`Standardisation of hCG Immunoassays
`and Pregnancy Kits
`Prof. S. L.. J eHi:oate
`Need for Antibiotics in Children with
`Asthma
`Dr Vanessa G raha m and others
`Bacterial Meningitis after Influenza
`Dr J. V. S. !'ether;
`Prof. M iklos Degrc
`Cakitonin Secretion and
`Postmenopausal Osteoporosis
`Dr J. C. Stevenson, Dr M . I. Whit ehead
`Primary Prevention and Low
`Birthweight
`D r T'imothy Johnstone;
`Dr Fiona Stanley, D r Sue At ki nson
`Necrotising Otitis Externa and Diabetic
`Control
`M r A. Resou ly, F.R.C.S. , and others
`Age-specific Immunisation Schedules
`Prof. G. T . Stewart;
`Dr R. M . Ande rson, Prof. R. M . May
`Intrauterine Devices for Diabetics
`M . Lawless, Prof. M. P. Vessey
`Kctoconazole and the Actinomycetales
`D r M . V. Marti n
`Thyrotoxicosis and Asthma
`Dr D. A. 1-lofl'man, Dr W. M . M cConahcy
`Missing Meals in Pregnancy
`Dr C. Lowy
`
`804
`
`805
`
`805
`
`806
`
`807
`
`807
`
`808
`
`B08
`
`8 10
`8 10
`
`8 10
`
`8 11
`
`8 11
`
`81 1
`8 11
`
`8 12
`8 12
`8 12
`
`8 12
`
`8 12
`
`OBITUARY
`Ge rtrude Dearnley
`Peter Audae r Overend Wilson
`
`INTERNATIONALDIARY
`
`NOTES AND NEWS
`Controlled Words from Mount Sinai
`Public Opinion and Severe Neonatal
`Handicap
`Bcnoxaprofcn Photosensitivity, a T est
`for Prescri ption Monitoring
`Smoke rs and Smoking
`Prescribed Diseases to Include
`Occupational Asthma
`Arth ritis Fou ndationAwards
`T obacco Adve rtising
`Disastrous Consequences of Neglected
`Cyanosis During Anaesthesia
`CORRECTION
`Adult T -cell Lymphoma-leukaemia in
`Blacks from the West Indies
`
`794
`
`795
`
`796
`
`797
`
`797
`
`798
`
`798
`
`Offices: 7 Adam Street, London WC2N 6AD T elephone: 01 -836 7228 Telegrams: Lancet, London WC2 . ISSN 0140-67 36. Founded
`1823. Published weekly. Registered as a N ewspaper at the Post Office. Annual Subscription £34.00 (Overseas £50). The whole of the
`Price £1.50
`literary matter in The Lancet is copyright. © The Lancet Ltd, 1982.
`
`BIOEPIS EX. 1028
`Page 1
`
`

`

`762
`
`Tl i H .i\NCl"' ', i\l'RII . 3, 198 2
`
`PHASE-I CLINICAL TRIAL OF
`MONOCLONAL ANTIBODY IN TREATMENT
`OF GASTROINTESTINAL TUMOURS
`
`H ENRY F. SEARS
`J EFFR EY MATTIS
`D OROT'HEE H ER LY N
`PE KKA H AYRY
`
`B ARBARA AT KINSON
`CARO LYN ERNST
`ZE NON STEP! .EWSKI
`H!Li\ RY K OPROWSKI
`
`The Fox Chase Cancer Cem er, Philadelphia, Penmy lvania, U. S. A .;
`Departmem of Pathology, Hospital of the University of Pennsy lvania,
`Philadelphia; Centocor Inc., M alvern, Pennsy lva nia; and The
`Wistar lnstiwte of A110tomy and Biology, Philadelphia
`
`Summary
`
`trial of a murine
`A phase-I clinical
`monoclonal
`antibody
`that
`specifically
`suppresses growth of human gastrointestinal tumours in
`athymic mice was conducted in four patients, who were given
`15-200 mg purified antibody. The monoclonal antibody
`persisted in the circulation for more than a w eek when more
`than 15 mg was given. Antibodies against mouse
`immunoglobulin developed in three of the four patients . In
`one patient who received autologous mononuclear cells that
`had been mixed with monoclonal antibody by way of a
`hepatic-artery catheter, h epatic metastas es became smaller
`and their echogenic characteristics changed, and there was
`heavier monocyte infiltration in the histological appearanc~
`of a resected metastasis .
`
`Introduction
`
`WE have developed a series of monoclonal antibodies that
`bind selectively to malignant cells of human gastrointestinal
`tract tumours. 1
`2 One of these antibodies, secreted by
`•
`hybridoma 1083-17-1 A (antibody 17-lA), mediates lysis of
`colorectal carcinoma cells by human or mouse effector cells 3
`and specifically
`inhibits the growth of human colon
`· carcinomas xenografted in athymic (nu/nu) mice.4 The
`antigen detected by antibody 17-1A is not sh ed during culture
`by tumour cells . 5
`Monoclonal antibody 17-lA perfused through fr eshly
`resected human colons containing adenocarcinomas binds
`selectively to cells of some ofthese tumours.6 W e have u sed
`this purified antibody in a phase-I clinical trial to assess its
`persistence in the systemic circulation, binding to tumour
`tissue, toxicity, and immunogenicity.
`
`Patients and Methods
`
`Patients
`Four patients with metastatic gastrointestinal cancer, scheduled
`for palliative surgery at Fox Chase Cancer Center, gave informed
`consent to take part . Two patients had one ureter obstructed by
`tumour, three had hepatic metastases, and one had only local pelvic
`
`DR VAN DERWERF AN D OTHERS: REFERE NCES-continued
`
`28. Linos DA, Beard CM, O' Fallon WM, Brockc rt y MB, Bean RW, Kur land LT.
`Cho lecystectomy and carcin oma oft he colon. Lancet 198 I; ii: 379 - 8 1.
`29. Vernick LJ, Kullcr LH . Cholecystectomy and riglu sidcd colon ca n c ~.: r : an
`epidemiological study. Loucel 198 1; ii: 38 1- 83.
`30 . Liu K, Stamler J, Moss D, Garside D, 1\:rskc:y V, Solt ero I. Dietary cholr.:stcrol, fat,
`fibr e and colon cam:cr mortality . An analysis of imcrnational data. Lancet 1979; ii:
`782-85 .
`31. Cruse P, Lewin M, Clark CG. Dietary cholesterol is co·car<.:inogenic for human colon
`cancer. Lancet 1979; i: 752 - 55.
`)2. Hcpn<:r GW. EfTcct of decreased !;Jllbladder stimula tion on ent crohcpatic cycling and
`kinetics of bile ad ds. Gasrrol'lltcrolngy 197 5; 68: 1571l-8 1.
`33 . Northfield TC, H ofm;~nn AI:. Bili<try lipid secretion in gallstone patient s. Lancer 1973;
`i: 747- 48.
`
`recurrence . One patient, who died 2 months after surgery, had liver
`metastasis, obstructed ureter and colon, and an entcrovaginal fistula
`after rad iation therapy. Patient 4, a 54-year-old man, underwe nt
`subtotal gastrectomy fo r a poorly differentiated ade nocarcinoma of
`the stomach 6 weeks befo re this trial. Seri al computerised
`tomography (CAT) scans showed enlarging li ve r metastases .
`
`Prepara rion of M onoclonal A ntibody
`M ur ine monoclonal antibody against human colorectal
`carcinoma (anti body 17- IA) of y2a isorype has been descri bed
`previously.1•2 Ascitic flu id was collected aseptical ly, was allowed to
`clot at 37°C, and was then centrifuged and filtered under sterile
`conditions through 0 · 22 1-1m 'Millex' fi lters (M illipore, Bedford,
`Massac husetts). The filtrate was dil uted with an equa l volume of
`sterile 0 · I mol/1 " tris" -bufTcr, pl-1 8 · 0, and applied to a sterile
`'protein-A-Sepharose' (Pharmacia, Piscataway, New Jersey) column
`(10 ml) for isolation of the IgG2, immunoglobulin . The colu mn was
`then washed thoroughly with 0 · 1 mol/1 " tris" buffe r, pH 8 · 0; the
`adsorbed IgG2, was eluted with 0 ·I mol /I citrate, pH 4 · 5. The pH
`of the eluate was ad justed to neut ral, and the eluate was di alysed
`against sali ne. The immu noglobulin was judged to be 95% pure in
`sodium-dodecyl-sulphate/polyacrylarni cle-ge l electrophoresis and
`gave negative results in the L imulus amoebocyte lysate assay (M.A .
`Bioprod ucts, Bethesda, Ma ryland) at a concentration of 500 1-1g/ml.
`The immunoglobulin was quantified by absorba nce at 280 nm.
`
`Trea tmem wi1h Monoclonal A l1l ibody
`to mouse
`Patients were
`tested
`for
`hypersensitivity
`immunoglobulin; patient 1 received 1 5 mg purified, pyrogen-free
`monoclonal antibody 17- IA in trave nously, and patients 2 and 3
`received 180 mg and 150 mg, respectively. Patient 4 was given a first
`injection of' 200 mg ant ibody int rave nous]~ on day 0. On day I
`mononuclear cells (app roximutcly 7 · 5 x 10 ), separated from one
`unit of his blood by gradi ent centrifugation, were incu bated with 67
`mg antibody 17- l A for 30 min at room temperatu re and return ed to
`patient 4 by way of a hepatic-artery catheter. He was given a further
`38 mg antibody 17- IA on day 3 and another 30 mg on day 7; the final
`inj ection was attempted on day I 0, but the patient received onl y half
`the 30 mg dose.
`Detection of Immunoglobulins
`Radioimmunoassay was car ried out as descri bed bcfore. 1
`2
`5 To
`•
`•
`detect mouse immunoglobulin, rabbit anti-mouse-IG antibody
`was exposed to patients' serum or urine samples, and the binding
`125!-labelled
`rabbit anti-mouse F(ab'h
`was determined by
`immunoglobulin. Circulating specific anti-colorectal-carcinoma
`ac tivity of mouse immunoglobulin in patients' serum was detected
`with live SW 111 6 colon carcinoma cells as the target cells. 7 To
`detect human antibodies aga inst mouse
`immunoglobu lin
`in
`patients' serum, mouse monoclonal anti-colon-carcinoma antibody
`was allowed to react with patients' serum samples, and 125!-labc lled
`rabbit antibodies against human F(ab'h immunoglobulin we re used
`to detect the binding.
`
`lmmunoperox idase Assay with Monoclonal A mibodies
`The immunoperoxidasc assay was carried out by the method of
`Kolcher et al. 8 Fixed, deparaffinised tissue sam ples were assayed for
`binding with monoclonal antibodies (see table): 17-IA, 19-9
`
`BI N DI NG O F MONOCLONA l. ANTIBODIES AS DET ECTED BY
`IMMUNO PEROX IDASE ASSAY ON TISSUE SPEC IM ENS FROM
`PATI EN T 4
`
`Primary
`stom"ch
`lesion
`
`Live r
`mc:t::lstasis
`after in fusion
`
`13onc
`metastasis
`after infusion
`
`±
`±
`++
`++
`++
`++
`++
`++
`- - - - - ------
`
`±
`
`+ +'
`+ +
`
`Antibody
`
`17- IA
`!9-9
`l0-17
`29- l
`!'3 x 63Ag8
`
`•Focal.
`
`BIOEPIS EX. 1028
`Page 2
`
`

`

`Tlll ' I.,\ NC ET,,\l'Rll.3, 1982
`
`763
`
`...:.. ,.. '" ,_;a:: j>,
`2
`4
`6
`
`1--\\r'-...... '+-- -+---'
`,....,.-· --.,
`I
`8
`14
`20 50
`11 0
`10
`12
`Days after antibody injection
`
`12 000
`
`10 00 0
`
`a ooo
`
`~ 6000
`
`4 000
`
`2 000
`
`:\
`
`·---
`
`\,
`---~·- .-'
`'•;_ _
`·-----\<\
`·~ :/ \,/
`
`. /""
`~-·, ·""· :\ ,/ \,~
`. .
`.
`-~\ .. _,/ __ :,..: ·f==-.-\0.··,,,'',,,
`
`AOHINiflHA TION
`or 17- IA
`Fig. 1-J>rcscncc of mouse immunoglobulin (--.-) and of
`antibody against mouse irnmunoglobulin (- -
`- -) ut serum of
`patients 1 ( • ), 2 (.&)and 3 ( • )-
`- -- -- -- -
`(di rected against a monosialoga nglioside present in serum of
`pat ient s with gastroint~stinal tumours), 9
`10 ant ibody I 0- 17 with Leb
`•
`sp~cifi ci ty, 11 and 29- 1 (ra ised against fres hly tsolated gastnc
`ca rcinoma cells and directed against la-1,31 fucosyl-p-globostde
`prese nt
`in gastrointestinal tumour cells). Immunoglobulin of
`P3 x 63Ag8 mouse myeloma was used as a control.
`
`Results
`Mouse immunoglobulin was found in the circulation of
`patient I for only 48 h after he received 15 mg monoclonal
`antibody (fig. 1 ). Mouse immunoglobulin was detectable for
`considerably longer in the blood of pauents 2_ and 3, who
`received 180 mg and !50 mg antibody, respecu vely (fig. I).
`immunoglobulin were first
`Antibodies against mouse
`detected 6 to 8 days after treatment in patients I and 3 and
`reached peak levels 11 - 14 days after treatment; they dropp<:d
`to zero 110 and 50 days after treatment, respecuvely, m
`patients 1 and 3 (fig.
`I). Antibodies against mouse
`immunoglobu li n did not develop in patient 2 dunng the 40
`days after treatment . Patient 4, who recetved repeated
`injections of antibody 17-1 A (fig. 2), had the htghest seru m
`levels of mouse immunoglobulin 24 h after admmtstratwn of
`monoclonal antibody . The injections 3 and 10 days after
`treatment were followed by rises in circulating mouse
`immunoglobulin : the levels then fell in a linear fashion from
`the 1 1 th until the 14th day after treatment.. Anubodtes agamst
`mouse immunoglobu lin were first detected 9 days after
`treatment and increased steadily throughout 21 days after
`treatment (fig . 2).
`
`/ \ ./'-o-o
`0
`
`""o ?\
`--0
`~
`
`12 000
`
`8000
`
`4000
`
`]
`
`~ 2800 ' /
`
`2000
`
`6 1 8
`.)() rng
`
`12
`
`14
`
`16
`
`18
`
`20
`
`o I 2
`I 4
`l s 7mg 38 mg
`15mg
`200 mg
`' - - - - -v - - - - - - '
`AOHINI5TRA TION Or 17- IA
`Ooys after start of treatment
`F ig. 2-Presence of mouse immunoglobulin (O---O) and of
`a ntibody against mouse immunoglobulin (0 -- --0 ) and binding
`activity to SW 1116 target cells (e- - -e) in serum of patient 4.
`
`The serum of patient 4 also showed a strong binding
`capacity of circulating mouse monoclonal antibody 1 7-1A to
`cultured colon carcinoma SW 1116 cells (fig. 2). The binding
`increased greatly immediately after each administration of
`the monoclonal antibody (except on day 7 when no blood
`sample could be obtained). The highest values for binding of
`circulating monoclonal antibody to cultured SW 1 1 16 cells
`by sera of patients 2 and 3 were observed 1-3 days after
`administration of monoclonal antibodies.
`In patients 1, 2, and 3 there were no immediate or delayed
`side-effects after administration of murine monoclonal
`antibody 17-IA. It was not possible to measure the effect of a
`single injection of monoclonal antibodies on the tumour
`because of the need for surgical intervention. Since we were
`seeking data to indicate the lack of adverse effects of murine
`immunoglobulins, we will not give a detailed description. In
`patient 4, however, the study was extended to include an
`evaluation of
`the
`therapeutic effect after
`repeated
`administration of monoclonal antibody, given either alone or
`together with the patient's peripheral-blood mononuclear
`cells.
`Levels of carcinoembryonic antigen were normal, and
`levels of a circulating tumour antigen detected with antibody
`19-9 against monosialoganglioside 9
`10 were high and
`•
`remained high during the course of immunotherapy in
`patient 4.
`Patient 4 was given a first injection of 200 mg purified
`antibody 17-I A intravenously over 30 min . The next day (day
`I) the mixture of mononuclear cells and antibody was infused
`through a hepatic-artery catheter over 15 min . Small
`aggregates in the preparation were noted towards the end of
`the injection . The flow in the hepatic artery, which was
`sluggish at the start of the infusion, temporarily stopped at
`the end of the infusion. The next day (day 2) the patient's
`temperature was 38 · 5°C and he complained of right
`epigastric discomfo rt and hiccups. Abdominal examination
`was unremarkable; however, the patient's serum aspartate
`aminotransferase (AAT) level, which had been 86 IU on
`admission, rose to 259 IU then fe ll rapidly to 195 IU by that
`to decrease
`throughout
`the
`afternoon and continued
`remainder of the treatment . Lactic dehydrogenase levels also
`increased (to 429 IU) on day 2 but were almost normal by day
`7.
`
`On day 3, 38 mg 17-1A antibody was given intravenously.
`At laparotomy on day 4 three hepatic metastases with
`surrounding normal heparic parenchyma were resected, and
`the nodal metastases in the retrocaval area were biopsied. On
`day 7 another 30 mg antibody was given . On day 10 the
`patient received less than half of the 30 mg dose, since he
`became flushed and complained of mild bronchospasm.
`Symptoms were relieved when administration of antibody
`was discontinued, and 0 -3 ml adrenaline (1 :10 000) was
`given intravenously. 4 days later the clavicular metastasis was
`biopsied. The patient showed no signs of serum sickness
`when examined for the nexr 2 weeks as an outpatient. He had
`no proteinuria, and renal function was normal. Liver
`ultrasound examination 3 weeks after administration of
`monoclonal antibodies showed that the metastases were
`much smaller, and their echogenic characteristics had
`changed. No change was noted in the bone metastasis during
`rhe same period.
`The material fro m the original gastric resection of patient 4
`showed a poorly differentiated adenocarcinoma that widely
`infiltrated the mucosa, muscularis, and serosa. The linitis
`plastica invasion of the tumour extensively involved nerves .
`
`BIOEPIS EX. 1028
`Page 3
`
`

`

`764
`
`TIIEJ.,\ NC ET,AI'RII.3, 1982
`
`and vascular spaces . The liver metastasis resected 4 days after
`the first infusion of antibody was a well-defin ed nodule with
`total necrosis in the centre. At the periphery of the nodu le
`there was a rim of viable tumour cells interspersed among an
`inflammatory infiltrate composed mainly of mononuclear
`cells. Sections from the bone metastasis resected 14 days after
`the first infusion of antibody showed bone marrow with
`of metastatic
`poorly
`differentiated
`several
`foci
`adenocarcinoma similar to the primary tumour but without
`tumour necrosis and only focal mononuclear inflammatory
`infiltrate.
`Sections from the stomach tumour and liver and bone
`metastases were studied by immunoperoxidase assay for
`binding of four monoclonal antibodies with a variety of
`specificities. Antigen detected by monoclonal antibody
`17-I A was present on the tumour cells of the primary gastric
`carcinoma and in the liver and bone (clavicle) metastases (sec
`table); the staining of the specimens, however, was very weak.
`Antigens detected by antibody I 0- 17 (which defines an Leb
`specificity 11
`) and antibody 29-1 (against [c:r-1 ,3 ] fucosyl-p(cid:173)
`globoside) were present in all three specimens, and the
`strong. The
`immunoperoxidase
`reaction was
`very
`monosialoganglioside antigen detected by monoclonal
`10 was expressed by the tumour cells of the
`antibody 19-99
`•
`primary stomach lesion (fig. 3) and its liver metastasis (fig. 4),
`but not by tumour cells of the bone metastasis (see table).
`
`-......
`
`Fig. 3-0riginal biopsy of gastric adenocarcinoma.
`
`Immunopcroxidasc countastaincd wit h hacmmoxylin only. Poorly
`difTercnt iatcd tumour nests infiltrati ng the gastric muscu laris demonstrate
`strong straining
`(arrow) wit h 19-9 monoclonal amibody. Origin al
`magnification x 640, reduced by one third.
`
`Fig. 4-Livcr metastasis.
`
`Immunopcroxidase coum crs1aincd wi th hacmatoxylin only. Cytoplasmic
`loca lisat ion of monosialogan gli osidc dc~ cctcd by .19-9 monoclonal antibody in
`malignant cells. Original magnification x 640, reduced by one third.
`
`Discussion
`Our aim was to identify potential hazards of further
`immunotherapy or immunodiagnostic efforts by means of a
`that specificall;-' destroys human
`monoclonal antibody
`gastrointestinal tumours implanted in animals. 1 We were
`particularly concerned with binding of the antibody to
`tumour and to normal tissues, sensitisation of the host to
`mouse immunoglobulin, and potential antigenic modulation
`secondary to exposure to ant ibody. Though the patients
`showed no evidence of serum sickness, the data suggest that
`whole mouse immunoglobulins will induce an anti-mouse(cid:173)
`immunoglobulin response.
`
`In other attempts at immunotherapy against human
`tumours 12- 1'1 antibody against a normal lymphocyte ant igen
`was used in smaller amounts; it may therefore have been
`bound rapidly by antigen on circulating cells. 13 By contrast,
`antibody 17 -I A does not react with antigens shed by the
`tumour cells. 5 This may explain why functiona l antibody
`could be detected
`for
`a
`considerable
`time
`after
`administration . Administration of 15 mg antibody 17-IA
`results in the transient appearance of mouse immunoglobulin
`in the patient's circulation immediately after injection. When
`larger amounts (!50 mg) of antibody 17-1 A were injected, the
`intact mouse immunoglobulin was present in the circulation
`for longer periods of time and was also found transiently in
`the urine of one patient.
`
`The fraction of the circulating mouse immunoglobul in that
`binds in vitro to colorectal carcinoma target cells and
`represents the active 17-IA antibody persisted in the serum of
`patients 2 and 3 for as long as the mouse immunoglobulin did .
`In patient 4 the specific binding decayed by day 10 after
`treatment, whereas mouse immunoglobulin persisted for a
`longer time . As with mouse immunoglobulin, binding
`activity to colorectal carcinoma cells was highest 2 to 4 days
`after treatment started.
`
`In three of our four patients an antibody response to the
`mouse immunoglobulin developed within 6 to 1 0 days. The
`lack of antibody response in patient 2 might be attributed to
`her debilitated condition and to radiation and chemotherapy
`before the antibody therapy. Development of antibody
`against mouse immunoglobulin in patient 4 led to a large fall
`immunoglobulin;
`this change
`circulating mouse
`in
`accompanied the patient's adverse clinical reaction to the last
`injection ofantibody 17-1A.
`
`Miller eta!. 12
`• 11 have described a T-cellleukaemic patient
`in whom antibody against mouse immunoglobulin was
`detected
`transiently 5 days after administration of
`monoclonal antibody, but who showed no clinical signs after
`a second dose of antibody 7 days after the first. The lack of
`clinical signs may be due to the smaller dosage of monoclonal
`antibody used ( 1-5 mg) or to the reduced ability of a patient
`with advanced leukaemia to mount an adequate immune
`response.
`
`Purified peripheral-blood mononuclear cells exposed to
`17- 1 A monoclonal antibody effectively destroy colorectal
`carcinoma cells 4 Destruction ofcolorectal carcinoma cells in
`a thymic nude mice inj ected with antibody 17-1A is attributed
`to effector cells exposed to circulating antibody. 15 As an
`ad junct to our immunotherapeutic trial we therefore isolated
`peripheral-blood lymphocyte's of patient 4, exposed them to
`antibody 17-1 A, and returned them to the patient. Liver
`metastases of patient 4 were affected by the treatment, as
`
`BIOEPIS EX. 1028
`Page 4
`
`

`

`T JII : JN,iCET ,i\ I' R II. 3, 1982
`
`765
`
`shown by histology of the resected metastases and by
`u ltrasound scanning of the two metastases remaining in situ.
`Dur ing the admimstration of mononuclear cells and antibody
`17- 1 A, small aggregates fo rmed which interfered with arterial
`b lood su pply to liver; lysis of tumour cells may therefore have
`been due to ischaemia. T he transient elevation of AAT levels
`immediately after infusion of the mixture of peripheral-blood
`mononuclear cells and antibody may also have indicated
`hepatic dysfunction resulting from ischaemia. However,
`hepatic-artery flow observed 2 days later at surgery appeared
`normal, and the liver tissue appeared to be well vascularised.
`Furthermore, histology showed heavy infiltration of the
`necrotic area by monon uclear cells, implying that the
`peripheral-blood mononuclear cells played an active part in
`the destruction of tumour metastases .
`Although the bone metastasis apparently became smaller
`during the treatment, there was no histological evidence of
`tumour destruct ion . The lack of evidence of tumour
`destruction might be attributed to antigenic modulation of
`the metastatic cells, as indicated by the absence of 19-9
`antigen in this lesion .
`We have demonstrated that a mouse monoclonal antibody
`against a human tumour antigen can be safely administered
`directly to the affected organ and that the antibody persists in
`the circulation for long periods of time . An anti-mouse(cid:173)
`immunoglobu lin response develops; this may limit repeated
`adm inistration
`of whole molecules
`of mouse
`immunoglobu lins . The efficacy of this immunotherapeutic
`approach may be enhanced by exposing the patient's own
`effector cells to monoclonal antibody and administering these
`cells directly into the metastatic site.
`
`We thank ] . Smith and K. O'Nei ll for technical assistance. The s!lldy was
`; upport ed by grant s Ci\· 108 1'5 and C/\·2 11 24 from the Nat ional Cancer
`Institute and gram RR·055-10 fro m the Division of Resea rch Resources.
`
`Correspondence should be addressed to H . K ., The Wistar Institute, 36th
`St reet at Spruce, Phil adelphia, 1'/\ 19 104, U.S./\.
`
`RE FERE NC ES
`
`J. lfcrlyn M, Stq >lcwski z, I krl yn D, Koprowski l-1. Color ec t :~ l carcinoma-specific
`nntigc n: detection by rncons of monoclon:~l ru11ibodics. Proc Nat! Acad Sci US/I
`19"19; 76: 1438-·12.
`2. Koprowski II , Stcplcwski Z, Mi tchel l K, lkrlyn M, 1-Jcrlyn D, Fuhrer P. Colorcctal
`carcinoma .mtigcns dctcCh:d by hybridom:. ant ibodies. Soma t Cell Gc:m:r 1979; 5:
`957-72.
`J. lf ..:rlyn D, Herlyn M, Steplcwski Z, Koprowski II. Monoclonal ant ibodies in cell·
`mcdi tued cytowx icit y :~ga inst humun melanoma and colorcctal carcinomn . l:.'ur J
`lmmunol 1979; 9: 657-59.
`4. ll erlyn D, Steplcw5ki Z, ll erlyn M, Kop rowski H . Inhibition of growth ofcolorcctal
`carcinoma in nude mice by monodon:li antibody. Cancer ReJ 1980; 40: 719-2 1.
`5. Stcplcwski Z, Ch:~ng T lf , ll crlyn M, Koprowski H. Rdcuse of monoclonal antibody
`defin ed antigens by human coloreculi carcinoma and mclonoma ct:lls. Ca ncer Ru
`198 1; 4 1: 2723-27.
`6. Sca rs HI:, ll crl yn D, I Jerlyn M, ct ol.l:.'.t·vivo perfusion oftumor- comainingcolon with
`monoclonal ant ibody. J Surg ReJ 198 1; 31: I ~ 5-50.
`7. Koprowsk i H, Stcplcwski Z, 1-l crlyn D, Hcrlyn M. Study of antibodies against human
`melanoma produced by somatic cel l hybrids. Proc Na t/ Acad Sci USA 1978; 75:
`3405-09.
`8. Kolchtr D, 1-Iova nhand P, Tcramoto YA, Wu nderlich D, Sclliom j . UsC of monoclonal
`antibodies to define a diversit yofmammory tumor vira l gent produ ~.:ts in virions and
`mammary tumors of the genus MUS. Ca ncer ReJ 198 1; 414: 1451-59.
`9. Koprowski J-1, Hcrlyn M , Stcplcwski Z, Sears HF. Specific antigen in serum of pat ients
`wit h colon ca rcinoma. Sde"u 1981; 212: 53-55 .
`10. Magnani jJ., Brockhaus M, Smith DF, et al. A monosia logangliosidc is a monoclonal
`antibody defin ed antigen of colon carcinoma . Sciena 198 1; 212: 55-56.
`II . Brockhaus M, Magnani ]I ., Blaszczyk M, ct al. Monoclonal antibodies directed against
`the human J.eO blood group omigcn. J Bioi Ch.:m 198 1; 256: 13223-25.
`12. Miller RA, Levy R. Response of cutaneous T cell lymphoma to therapy with
`hybridoma monoclonal antibody. Lmtcer 198 1; ii : 226- JO.
`J J. Ritl. J, Pcsnndo J M, Sn llan SE, et nl. Serotherapy or acute lymphoblastic leukemia with
`monoclonal antibody. Blood 198 1; 58: 14 1-52 .
`14. Miller RA , Maloney DG, McKillop Y, Levy R. /n vivo cflCcts of murine hybridoma
`monoclonal ant ibody in 3 patient with 'l '.cclllcukcmia. Blood 198 1; 58: 78-86.
`15. Hcrlyn D, Koprowski H. Monoclonal antibodies against solid human tumors inhibit
`tumor growth in nude mice. 1-Jy bridoma 1982; 1: 206.
`
`MORPHOLOGICAL IDENTIFICATION OF THE
`AGENT OF KOREAN HAEMORRHAGIC FEVER
`·(HANTAAN VIRUS) AS A MEMBER OF THE
`BUNYA VIRIDAE
`
`J. B. M CCORM IC K
`E . L. Pi\LJ\1\ER
`
`D . R. SASSO
`M. P. KI L EY
`
`Vira l Disease Di-vision, Cenrer for l nfccr iom Diseases,
`Cencersfor Disease Conrrol, ! lrlanra, Georgia, U.S.A.
`
`Summary
`
`(KHF)
`feve r
`haemorrhagic
`Korean
`(Hantaan virus), a rodent-borne viral ill ness,
`is an important cause of human disease throughout much of
`Asia and Eastern Europe. The agent responsible for KHF has
`not yet been conclusivel y identified . Plaque-purified KHF
`virus was concentrated and then banded in a potassiu m
`tartrate gradient. Material from the I· 17-1 · 19 g/ml band
`was examined by electron microscopy a nd particles with a
`morphology ident ical to that· of the fam ily Bunyaviridae were
`found. The panicles were agg regated by KHF serum but not
`by saline solution or non- immune serum. Identification of
`KHF virus as a member of the family Bunyaviridae
`implies a potential for spread by arthropod vectors.
`
`Introduction
`
`KOREAN haemorrhagic fever (KHF) was described several
`decades ago and is known by many names throughout Asia
`and Europe. 1 It is a severe, not uncommon disease, found in a
`geographic area from Japan to Europe w hich is occupied by
`about one-half of the wo rld's population . The agent was
`isolated in 1978 by Lee eta!. 2 and was grown in tissue culture
`by French in 198J. l The virus has not, however, been
`satisfactorily purified for morphological identification . We
`describe the purification and morphological characteristics of
`H antaan virus.
`The strain of Hantaan virus used for this study, designated
`76- 11 8, has beeri registered in the Working C atalogue of
`Arthropod-Borne Viruses.·1 Since it is a direct descendent of
`the 76- 11 8 isolate described by Lee et al. in the origina l
`isolation of the virus, 2 and by F rench for growth in A-549
`cells, 3 it has a well-defin ed pedigree . It is also I