I
`
`G 3
`
`A MONTHLY JOURNAL DEVOTED TO THE ART AND SCIENCE OF SURGERY
`
`
`1989
`
`SEPTEMBER
`Volume 706 Number 3
`
`EDITORS IN CHIEF
`
`WALTER F. BALLINGER, ST. LOUIS
`
`GEORGE D. ZUIDEMA, ANN ARBOR
`
`CHARLES M. BALCH, HOUSTON
`F. WILLIAM BLAISDELL, SACRAMENTO
`MURRAY BRENNAN, NEW YORK
`GREGORY B. BULKLEY, BALTIMORE
`JOHN L. CAMERON, BALTIMORE
`LARRY C. CAREY, COLUMBUS
`D. C. CARTER, GLASGOW
`ORLO H. CLARK, SAN FRANCISCO
`JOHN A. COLLINS, STANFORD
`ROBERT E. CONDON, MILWAUKEE
`JEROME J. DeCOSSE, NEW YORK
`TOM R. DBMEESTER, OMAHA
`CALVIN B. ERNST, DETROIT
`RONALD M. FERGUSON, COLUMBUS
`
`EDITORIAL BOARD
`
`ERIC W. FONKALSRUD, LOS ANGELES
`LAZAR J. GREENFIELD, ANN ARBOR
`ALDEN H. HARKEN, DENVER
`ROBERT E. HERMANN, CLEVELAND
`GORDON L. HYDE, LEXINGTON
`BERNARD M. JAFFE, BROOKLYN
`CRAWFORD W. JAMIESON, LONDON
`GORDON L. KAUFFMAN, JR., HERSHEY
`KEITH A. KELLY, ROCHESTER
`JOHN A. MANNICK, BOSTON
`FRANK G. MOODY, HOUSTON
`DAVID L. NAHRWOLD, CHICAGO
`JOHN S. NAJARIAN, MINNEAPOLIS
`GEORGE L. NARDI, BOSTON
`RICHARD A. PRINZ, MAYWOOD
`
`JUDSON G. RANDOLPH, WASHINGTON
`KEITH REEMTSMA, NEW YORK
`ROBERT S. RHODES, JACKSON
`NORMAN M. RICH, BETHESDA
`WALLACE P. RITCHIE, JR., PHILADELPHIA
`WILLIAM SILEN, BOSTON
`THOMAS E. STARZL, PITTSBURGH
`GLENN D. STEELE, JR., BOSTON
`RONALD J. STONEY, SAN FRANCISCO
`NORMAN W. THOMPSON, ANN ARBOR
`DONALD D. TRUNKEY, PORTLAND
`JEREMIAH G. TURCOTTE, ANN ARBOR
`SAMUEL A. WELLS, JR, ST. LOUIS
`ROBERT ZEPPA, MIAMI
`
`
`
`
`
`~>”_Jrx;UsLusffiqnvU'"V6’F$'U’ofWvsconsin
`
`OFFICIAL PUBLICATION
`
`SOCIETY OF UNIVERSITY SURGEONS
`NEXT MEETING FEBRUARY 8-10, 1990
`LOS ANGELES, CALIFORNIA
`
`JTRAL SURGICAL ASSOCIATION
`NEXT MEETING MARCH 1.3, 1990
`CHICAGO, ILLINOIS
`
`AMERICAN ASSOCIATION OF
`ENDOCRINE SURGEONS
`NEXT MEETING APRIL 22-24, 1990
`CLEVELAND, OHIO
`
`PUBLISHED BY
`
`»
`
`5-,
`S?
`V"""
`
`.
`{EL
`
`U?)
`
`_ §
`.
`65
`,.
`5‘, vi
`
`I
`__,
`*0 5
`
`" ru
`2
`
`THE,C.V. MOSBY COMPANY, ST. LOUIS, MO. 63146, U.S.A.
`ISSN 0039-6060
`IMMUNOGEN 2315, pg.1
`Phigenix v. Immunogen
`|PR2014-00676
`
`
`
`IMMUNOGEN 2315, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`C ONTENTS continued
`
`517
`
`525
`
`533
`
`Microfluorometric measurements of cytoplasmic calcium in chief
`and oxyphil parathyroid cells of adenomatous and hyperplastic
`glands and of normal-sized glands associated with adenomas
`H. johansson, MD, PhD, J. Rastad, MD, PhD, G. Bjerneroth, MD,
`E. Gylfe, PhD, and G. Akerstriim, MD, Uppsala, Sweden
`
`The influence of the surgical wound on local tumor recurrence
`D. G. Baker, PhD, T. M. Masterson, MD, R. Pace, BSC, W. C.
`Constable, MD, and H. Wanebo, MD, C/iarlottesoille, Va.
`
`Phase I clinical trial of drug-monoclonal antibody conjugates in
`patients with advanced colorectal carcinoma: A preliminary
`report
`J.
`Tjandra, FRCS, G. A. Pietersz, PhD, J. G. Teh, BSc(Hon),
`A. M. Cuthbertson, FRACS,
`R. Sullivan, FRACP, C. Penfold,
`FRACS, and I. F. C. McKenzie, PhD, FRACP, FRCPA, Par/wille
`and East Z\/Ielbourne, Victoria, Australia
`
`546
`
`Esophageal blood flow in the rabbit: Response to calcium chan-
`nel blockers
`
`Gloria Duda, MD, Jo Ellen Huesken, MS, Barbara L. Bass, MD,
`and john W. Harmon, MD, Washington, D.C.
`
`555
`
`Proteases and protease inhibitor balance in peritonitis with dif-
`ferent causes
`
`Magnus Delshammar, MD, Ake Lasson, MD, and Kjell Ohlsson,
`MD, Malmii, Sweden
`
`continued on page 6A
`
`
`
`
`
`
`
`
`
`""7ijtja:l..‘1§§°-fig};-.1!‘LE!1*...1.¢.‘-i¥-an‘:1new4-.x;~2;.
`
`Vol. 106, No. 3, September 1989; SURGERY (ISSN 0()3‘)—6()6()) is published monthly (six issues per volume, two volumes per year) by The CV.
`Mosby Company, '1 1830 \/Vestline Industrial Drive, St. Louis, MO 63146-3318. Second class postage paid at St. Louis, M0,, and additional mailing
`otfices. POSTMASTER: Send address changes to 11830 VVcstline Industrial Drive, St. Louis, MO 63146-3318, (314) 872-8370, ext. 351.
`Internaiional
`International (az'rmoz'[)*
`{surface mail)
`I
`All regions
`R3310” 7
`$130.75
`$155.50
`iii 73.75
`$ 98.50
`$ 51.25
`3% 76.00
`
`Canada
`$148.50
`3 91.50
`YB 69.00
`
`_
`‘MMZEO
`$150.50
`35 93.50
`11% 71.00
`
`_
`Rggwn 2
`$177.50
`$120.50
`55 98.00
`
`V
`Region 3
`$200.00
`$143.00
`$120.50
`
`1939 Annual subscription rates
`lnstitutionalf
`Individual:
`Student, residenti
`
`U.S.A.
`$111.50
`54.50
`1% 32.00
`
`Subscription rates include supplements. Single copies are $6.50. Remittances should be made by check, draft, post office or express money order,
`in US. funds drawn through a U.S. bank, payable to this JOURNAL. Claims for missing issues will be serviced onlyiwithin 6 months of cover date.
`Single copy prices will be charged on missing issue claims older than 6 months from cover date. Back issues generally are available for the previous
`5 years. Contact the publisher to confirm availability of specific issues.
`*Az'r-nzatl /man/ao’own—I)omestu:: First class and Priority rates for the U.S. and possessions are available upon request. Canada: All provinces of
`Canada. Jtlsxicros All localities within Mexico. Region 7: Colombia, Venezuela, Central America, Caribbean Islands, Bahamas, Bermuda, and St.
`Pierre & Miquelon (also, from American Samoa to Western Samoa, and from Guam to the Philippines). Region 2: South America (except
`Colombia & Venezuela), Europe (except Estonia, Latvia, Lithuania & USSR), and North Africa (Morocco, Algeria, Tunisia, Libya, and Egypt).
`Region 3: Estonia, Latvia, Lithuania, USSR, Asia, Australia, New Zealand, Pacific Ocean Islands, Africa (other than North Africa), Indian Ocean
`Islands, and the Middle East.
`ylnstitutional (multiple-reader) subscriptions are available to public and private libraries, schools, hospitals, and clinics; city, county, state,
`provincial, and national government bureaus and departments; and all commercial and private institutions and organizations.
`ztlndividual subscriptions and all student-rate subscriptions must be in the names of, billed to, and paid by individuals. All student-rate requests
`must indicate training status and name of institution.
`Subscriptions may begin at any time.
`Printed in the USA. Copyright © 1989 by The CV. Mosby Company.
`
`5A
`
`IMMUNOGEN 2315, pg. 2
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2315, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Phase I clinical trial of drug—monoclonal
`antibody conjugates in patients with advanced
`colorectal carcinoma: A preliminary report
`
`G. Teh, BSc(Hon),
`Tjandra, FRCS, G. A. Pietersz, PhD,
`J.
`A. M. Cuthbertson, FRACS,
`R. Sullivan, FRACP, C. Penfold, FRACS, and
`I. F. C. McKenzie, PhD, FRACP, FRCPA, Parkville and East hfelbourne, Victoria, Australia
`
`Melphalan (JWEL), an alkylating agent, has been modified to a derivative,
`N-acetylrnelphalan (N-ACMEL), which can be conjugated to anticolon cancer
`monoclonal antibodies (ll/Io/lbs 30.6, I- 7, and ]GT) and usedfor
`immunochemotherapy. The final immunoconjugates possess potent cytotoxicity and
`specificity in preclinical studies. In a phase I clinical study, N-ACMEL-MoAb
`conjugates were administered via the hepatic artery to 70 patients, nine of whom had
`disseminated colorectal cancer (including the liver) and one of whom had Dukes’ C
`colon cancer that had been resected. The selection of .MoAb was based on the
`immunoperoxidase staining of the primary colon cancer tissue. Thus far doses of 7000
`mg/m2 MoAb conjugated to 20 mg/m2 of N-Acl\4EL have been administered with no
`significant side efiects, whereas MEL unconjugated to monoclonal antibodies would
`have caused myelosuppression in a proportion ofpatients at the same dosage. Serum
`antimouse antibody responses were noted in all of the patients; febrile reactions were
`noted with higher doses but were easily controlled with antipyretics, antihistamines
`and,
`necessary, steroids. Serum sickness developed in one patient who was given a
`second course of treatment in the presence of human antimouse antibody, but the
`episode was self-limiting. Eight of the 70 patients had evaluable disease. Subjective
`improvement was noted in almost all of the patients examined, and 33%, or 3 of 9, of
`the treatments (nine courses of treatment in eight patients with evaluable disease; one
`of the patients had two courses of treatment) led to antitumor responses (minor
`response) by objective assessment with computed tomography of the liver. It is
`important to note that treatment with N-AcMEL-l\/IoAb conjugates was safe at a dose
`of 20 mg/m2 of N-AcMEL, whereas the eflicacy of such aform of treatment remains to
`be determined.
`(SURGERY 7989;706:533-45.)
`
`From the Research Centre for Cancer and Transplantation, Department of Pathology, The
`University of Alelbourne, and the Colorectal Unit, Royal .Melbourne Hospital, Par/tville, and
`the Department of lwedical Oncology, St. Andrew’s Hospital, Clarendon Place, East
`Ale/bourne, Victoria, Australia
`
`CANCER or THE COLON and rectum is one of the most
`
`common forms of malignancy in Western countries,
`with approximately 120,000 new cases reported annu-
`ally in the United States.‘ Hepatic metastases are
`present on initial diagnosis of colorectal cancer in 25%
`to 30% of patients? After curative resection of colorec-
`
`Accepted for publication Nov. 22, 1988.
`Reprint
`requests:
`Ian F. C. McKenzie, MD, PhD,
`Research Centre for Cancer and Transplantation, Depart-
`ment of Pathology, The University of Melbourne, Park-
`ville, Victoria 3052, Australia.
`
`tal primary tumors, the liver is again the most frequent
`site of relapse in 40% to 50%.“ Once hepatic metasta-
`ses have developed,
`the prognosis is poor, with an
`expected median survival of 6 to 9 months} 5 the extent
`of the tumor being the most
`important prognostic
`factor.“ Many different forms of treatment, including
`systemic chemotherapy, have been used for colorectal
`hepatic metastases, without much success.“ The only
`patients who may achieve 5-year survival are the
`highly select group suitable for surgical resection—
`usually those patients with less than four hepatic
`metastatic lesions.” It should be recognized, however,
`
`SURGERY 533
`
`IMMUNOGEN 2315, pg. 3
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2315, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`534 Tjandra et al.
`
`Surgery
`September 7989
`
`Table I. Characteristics and clinical features of patients treated with N-ACMEL-MoAb conjugates
`D0Se*(Mg/W12) of
`Performance status
`Age
`Previous therapy
`N~AciM EL:.WOAb
`(ECOG)
`Sex
`(yr)
`Patient
` ___
`HA1 of cis-platinum
`5mg/m2:120mg/mg
`1
`59
`M
`2
`10mg/m2:160mg/ml
`10mg/mZ:98Omg/m2
`
`Partial hepatectomy
`Adjuvant chemotherapy
`
`61
`
`M
`
`57
`58
`62
`57
`46
`62
`38
`64
`
`9
`10
`
`HA1 of cis-platinum
`
`L>3>—‘bJ’vJl\)UJ[\JlQ
`
`10mg/m2:250mg/m2
`15mg/rn2:340mg/m2
`15mg/m2:38Omg/m2
`15mg/m2:50Omg/m2
`20mg/m2:440mg/mg
`20mg/m2:1O00mg/m2
`20mg/m2:820mg/Inz
`20mg/m2:10()Omg/mg
`
`Legezzd: ECOG, Eastern Cooperative Oncology Group; liAl, hepatic artery infusion.
`The amount (mg) of N-ACIVIEL conjugated to l\/1oAb and administered was expressed as mg/m2 of body surface area of the patient.
`
`that patients suitable for resection make up a very
`small percentage of all patients with colorectal hepatic
`metastases. More recently there have been encouraging
`reports of response to regional perfusion with chemo-
`therapy, especially 5-fiuoro-2-deoxyuridine (FUDR);
`however, this is still limited by complications related to
`chemotherapy.‘ '“
`It is with this background of unsuccessful therapeu-
`tic maneuvers that alternative therapeutic avenues with
`monoclonal antibodies
`(MoAbs) are explored. By
`means of the hybridoma technique,“ murine mono-
`clonal antibodies have been produced against almost all
`of the major types of human cancer.” However, no
`truly tumor-specific MoAb has been derived thus far,
`but in most cases the antigens recognized are present on
`tumors in greater concentrations than on normal tis-
`sues.” There are several reports of clinical response to
`antitumor monoclonal antibodies used alone, mostly in
`malignant melanoma, neuroblastoma,
`leukemia, and
`lymphoma.”"" However, the therapeutic effects are not
`dramatic, presumably because murine antibodies do
`not adequately incite appropriate host effector mecha-
`nisms to destroy tumors. It is therefore believed that the
`greatest therapeutic potential for MoAbs lies in the
`targeting of
`anticancer
`agents
`(chemotherapeutic
`drugs,
`toxins, or
`radioactive substances)
`to tumors
`rather than their use in unmodified form. By using a
`“prodrug” approach, a potent immunoconjugate was
`produced by covalently conjugating an inactive N-
`acetyl derivative of melphalan (N-ACMEL)
`(“pro-
`drug”) to murine MoAbs.” The procedure removed
`the ability of the melphalan to enter cells by its usual
`active transport via the amino acid transport systems;
`however, the MoAb provided the alternative route of
`cell entry via endocytosis, and such N-ACMEL-MoAb
`
`conjugates, on binding to tumor antigen on the tumor
`cell surface, exert their effects after internalization and
`lysosomal degradation within the target tumor cell to
`release melphalan.” The immunoconjugates have dis-
`played in vitro and in vivo specificity and cytotoxicity
`and specifically inhibit
`the growth of human colon
`carcinomas xenografted in athymic mice when injected
`intravenously.”
`We have described a murine MoAb 30.6 that reacted
`with > 90% of colon cancer tissue“ and could preferen-
`tially localize human colorectal
`tumor xenograft
`in
`nude mice“ and in primary and secondary colon
`carcinoma in patients?” Two additional anticarci-
`nogenic embryonic antigen MoAbs (I-1 and JGT) had
`been developed, and they reacted strongly with 80% of
`colon carcinoma on immunoperoxidase
`staining.“
`Immunoconjugates of N-ACMEL to these MoAbs
`(30.6, I-1, JGT) have been developed by means of the
`same principles.” We now report a phase I clinical
`study with N-ACMEL-MoAb conjugates administered
`via hepatic artery infusion in 10 patients: nine with
`disseminated (including liver) colorectal cancer and one
`with resection of Dukes’ C colon carcinoma.
`
`MATERIAL AND METHODS
`
`Patients. Ten patients with advanced colorectal
`carcinoma were included in this study. They were
`estimated to have at least a 3-month expected survival,
`a performance status (Eastern Cooperative Oncology
`Group) less than or equal
`to 3, and had no other
`cytotoxic therapy for at least 1 month before adminis-
`tration of immunoconjugate and during the 3-month
`evaluation phase of the study. Table I summarizes the
`characteristics and clinical features of the patients.
`Ages ranged from 38 years to 64 years. Nine of 10
`
`IMMUNOGEN 2315, pg. 4
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2315, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Volume 706
`Number 3
`
`Drug-A/I0Ab conjugates and coloreclal carcinoma
`
`535
`
`patients had extensive hepatic metastases from colorec-
`tal carcinoma, and the remaining patient had a locally
`advanced colon cancer
`(Dukes’ C)
`that had been
`resected and did not have demonstrable hepatic metas-
`tases by laparotomy or computed tomography (CT).
`Two of nine patients with hepatic metastases also had
`pulmonary metastases, and one of nine patients had a
`primary colon carcinoma that had not been resected
`because of the poor general medical condition of the
`patient. Two of the nine patients previously had failed
`intensive chemotherapy (hepatic artery infusion of
`cis-platinum), and one of these two patients had two
`courses of immunoconjugates separated by a 2-month
`interval. One of the nine patients had recurrent hepatic
`metastases after a previous partial hepatectomy and
`adjuvant 5-fiuorouracil. All patients (except patient 9)
`had progressive metastatic disease at
`the time they
`entered the study, and the hepatic metastases were too
`extensive for hepatic resection. All patients were fol-
`lowed for at
`least 3 months after therapy (except
`patient 7 who died 4 weeks after therapy with a
`generalized debility and patient 10—see below); they
`were evaluated at weekly intervals for 6 weeks, then
`monthly. This phase I study was approved by the
`Medical Research Board of the Royal Melbourne
`Hospital, and written informed consent was obtained
`from all patients.
`Monoclonal antibodies. l\/Iurine MoAbs used were
`
`an anti-
`IgG2b antibody 30.6, directed against
`gen present on human colon secretory epithelium but
`also reactive against a number of colon carcinoma cell
`lines,” and IgG1 antibodies
`I-1
`and JGT (both
`anticarcinoembryonic antigen), which were produced
`by means of a novel
`immunization technique with
`whole serum of patients with advanced colorectal
`cancer;
`they reacted with human colon carcinoma,
`malignant tumors of noncolonic origin (breast,
`thy-
`roid), and a number of colon carcinoma cell lines but
`not with normal tissue or benign lesions (24, unpub-
`lished observations). The antibodies were purified on
`protein A-superose (Pharmacia,
`Inc., Piscataway,
`After elution, l\/IoAbs were concentrated by 45%
`ammonium sulfate precipitation, dialyzed against
`phosphate-buffered saline (PBS), aliquoted, and stored
`at —70° C. The concentration of IgG was estimated by
`absorbance at 280 nm. The prepared antibodies were
`retested for activity after all procedures (see below),
`filtered through a 0.22 pm Millex-GV filter (Milli-
`pore, Befored, Ann Arbor, Mich.), and batch tested for
`purity by sodium dodecyl sulphate polyacrylamide gel
`electrophoresis (SDS—PAGE).
`Preparation of N-ACMEL-IgG conjugates. The
`MoAbs used included 30.6 (IgG2b), I-1, and JGT
`
`(lgG1). The N-acetyl derivative of melphalan was
`prepared and conjugated to whole IgG.” Briefly, MEL
`was acetylated with acetic anhydride and an active
`ester of this N-ACMEL derivative was then coupled to
`the amino groups of the MoAb. Any precipitated
`protein was removed by centrifugation, and free N-
`ACMEL was removed by gel filtration chromatography
`with a Sephadex G-25 column (PD-10; Pharmacia).
`N-ACMEL incorporated in the drug-MoAb conjugates
`was determined by absorbance spectrophotometry at
`258 nm (E253 = 1 X 10‘M‘1cm"‘) after subtracting the
`protein contribution following its estimation by the
`Bradford dye-binding assay.” The alkylating activity
`of the conjugate was determined by a modification of
`the method of Epstein et al.26 The final preparation
`after drug conjugation was batch tested for pyrogens
`and sterility (Department of Pharmacology, University
`of Melbourne, and Sigma Pharmaceuticals, Clayton,
`Victoria, Australia).
`The antibody activity of N-ACMEL-IgG conjugates
`was demonstrated in a rosetting assay” and in immu-
`noperoxidase
`staining with
`formalin-fixed
`(N-
`ACMEL-I-1, N-ACMEL-JGT) and snap-frozen (N-
`ACMEL-30.6) sections of human colon cancer tissue
`(data not shown).
`Administration of drug-MoAb conjugates. By
`means of the Seldinger technique,
`the catheter was
`introduced percutaneously into the left axillary or high
`brachial artery. The catheter was placed in the com-
`mon hepatic artery, and when multiple hepatic arteries
`were found supplying the liver, the catheter was placed
`in the largest vessel. The immunoconjugate was admin-
`istered via hepatic artery infusion with an oxymetric
`pump in 100 ml of normal saline solution for 2 hours
`per day for 2 days. All patients had three doses of the
`immunoconjugates (t = 0, t = 24 hours, t = 48 hours).
`Between infusions of the immunoconjugates, the paten-
`cy of the catheter was accomplished with heparinized
`saline solution (5000 IU aqueous heparin in 1 L
`normal saline solution at the rate of 50 ml/hr) with the
`oxymetric pump. At
`the end of the 2-day infusion
`period, the indwelling catheter was removed. Patients
`were given dexamethasone, 8 mg intravenously, just
`before each infusion of immunoconjugates and oral
`prednisolone, 10 mg daily for 7 days after completion
`of infusion as prophylaxis for allergic reactions. The
`dose escalation protocols used (Table I) were as
`follows: one patient received 5 mg/m3 and 2 months
`later, 10 mg/m2; two received 10 mg/m3; three received
`15 mg/ml; and four received 20 mg/m2 N-ACMEL
`conjugated to MoAbs. The study was closed at the 20
`mg/m2 dose of N-AcMEL conjugates because of the
`cost incurred in producing such a large quantity of
`
`IMMUNOGEN 2315, pg. 5
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2315, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`536
`
`7:jandm et al.
`
`Su1"ge7‘y
`Septem ber 7989
`
`Table II. Binding of MoAb (30.6, 1-1, JGT) as detected by immunoperoxidase staining on primary
`colon cancer
`
`Smmmg gmde*
`ll/IoAb.s‘ used’ in
`
`Patient
`Colon cancer li.s'.vue
`30.67‘
`I-7
`_/GT
`imrnunoconjugates
`
`1::
`
`2
`3
`
`4
`5
`
`6
`7
`
`Fixed
`
`Fixed
`Fixed
`
`Fresh/fixed
`Fixed
`
`Fixed
`Fixed
`
`3
`
`4
`
`4
`4
`
`4
`3
`
`3
`3
`
`3
`
`4
`2
`
`4
`3
`
`4
`3
`
`1-1
`1-1, _]GT
`30.6, I-1, JGT
`I-1
`
`30.6, l-1, JCT
`I-1
`
`1-1, JGT
`30.6
`
`4
`Fresh/fixed
`8
`4
`Fresh/fixed
`9
`3
`Fixed
`10
`*Staining score was graded based on the proportion of carcinoma cells stained: O = no staining;
`100%.
`’r3().6 lVloAh tested on fresh colon Cancer tissue only.
`:tPatient '1 had two courses of treatment.
`
`3
`2
`
`30.6, l-l, JCT
`4
`I-1, ‘]GT
`4
`1-1, JGT
`4
`1 = up to 23%; 2 = 26% to 50%; 3 = 51% to 75%; 4 = 76% to
`
`antibodies and the concern that the maximum tolerated
`
`dose of such a form of treatment may not be practicably
`achieved (see below).
`
`Patients were monitored clinically for changes in
`temperature, pulse, blood pressure, and respiratory
`function during and after the infusion. Blood studies
`were also done before, during, and weekly for 6 weeks
`after the therapy to assess potential hematologic (full
`blood examination), renal (urea and electrolytes), or
`hepatic toxicity (liver
`function test) and to detect
`human immune responses
`stimulated by murine
`immunoglobulin (human antimouse antibody).
`Human antimouse antibody response. Human
`antibodies against the murine MoAbs were measured
`by an enzyme-linked immunosorbent assay (ELISA)
`modified from that previously described.” Ninety-six
`well flexible polyvinyl chloride plates (Costar, Cam-
`bridge, Mass.) were coated with 50 pl/well of admin-
`istered MoAb (5 /.Lg/ml of purified 30.6, 1-1, or JGT
`MoAbs) in a 0.1M carbonate buffer, pH 9.6, and
`nonspecific binding blocked with 1% bovine serum
`albumin/PBS, pH 7.6. Serial dilutions of patient sera
`and pooled normal human serum (50 /.Ll/well)
`in
`PBS/0.05% Tween 20 to a final dilution of 1/256 were
`performed and added to the coated wells (50 pl /well).
`Plates were then washed with PBS/0.05% Tween 20
`and then reacted with 50 pl/well of phosphatase
`labeled aflinity purified goat antihuman IgM and IgG
`(Kirkegaard and Parry, Gaithersburg, Md.). The color
`reaction was developed with alkaline phosphatase
`substrate and read with an ELISA plate reader (Ti-
`tretek, Multiscan, MC) at a wavelength of 405 nm.
`
`Results were expressed as the absorbance value of
`patient serum compared with pooled normal human
`serum, and a positive test result was considered to be a
`value at least twice the control.
`
`Immunoperoxidase
`Immunoperoxidase staining.
`staining was performed” on 6 am tissue sections of
`colon cancer tissue from all patients with 1-1 and JGT
`MoAbs; if possible staining was also performed with
`30.6 MoAb. The 30.6 MoAb only reacts with snap-
`frozen but not
`formalin-fixed colon cancer
`tissue,
`whereas I-1 and JGT MoAbs react with both snap-
`frozen and formalin-fixed sections. A nonreactive con-
`
`trol antibody was used in all cases. The sections were
`then assessed by light microscopy to estimate the
`percentage of colon carcinoma cells stained with each of
`the antibodies; results were expressed on a scale of 0 to
`4 according to whether nil (0), up to 25% (1), 26% to
`50% (2), 51% to 75% (3), or >75% (4) of carcinoma
`cells stained. This is a semiquantitative assay and is
`highly reproducible.” The intensity of stain, the distri-
`bution of stain in the cancer cells, and the staining of
`extracellular material were not taken into account. The
`
`MoAbs selected for use in drug conjugation for an
`individual patient had to have a staining score of 3
`or 4.
`
`Evaluation of tumor responses. Patients were
`evaluated clinically and biochemically (liver function
`test, carcinoembryonic antigen [CEAl level), and the
`measurable lesions were measured at 1 and 2 months
`
`after therapy by CT scans of the abdomen performed
`with the same technique by the same radiographers
`and radiologists as that used for the pretherapy evalu-
`
`IMMUNOGEN 2315, pg. 6
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2315, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Volume 706
`Number 3
`
`Drug-MoAb conjugates and colorectal carcinoma
`
`537
`
`ation (performed within 2 weeks before therapy).
`Complete response is defined as the disappearance of
`all evidence of tumor. Partial response is defined as a
`reduction of at least 50% in the sum of the products of
`the two greatest diameters of measured lesions. Minor
`response is a reduction of more than 25% but less than
`50% in the size of measurable tumors in the absence of
`
`progression or occurrence of new lesions elsewhere.
`Stable disease is an objective regression of measurable
`lesions less than that required to meet the criteria for
`minor or partial response or an increase of less than
`25% in the size of one or more measurable lesions for at
`
`least 4 weeks. Progressive disease is the appearance of
`new lesions or increase in size of one or more measur-
`
`able lesions by at least 25%.
`
`RESULTS
`
`Immunohistochemical testing on primary colon
`cancer. A selection of MoAbs for conjugation with
`N-ACMEL was made for each patient, based on the
`binding of the particular MoAb (30.6, I-1, JGT) to
`sections of the primary colon cancer by immunoperox-
`idase staining (Table II).
`In general, MoAb was
`selected only if it had a staining score of 3 or 4. When
`multiple antibodies were used for drug conjugation, the
`final preparation of the immunoconjugates had equal
`proportions of the MoAbs. A combination of at least
`two MoAb conjugates (N-ACMEL-30.6, N-AcMEL-
`I-1, N-ACMEL-JGT) was used in 7 of 11 treatments
`(Table II).
`It was considered that
`the use of a
`combination of MoAb conjugates would ensure
`maximal immunoreactivity and help to overcome the
`potential problem of tumor heterogeneity within and
`between tumor masses. We were unable to obtain
`tissue from the liver metastases itself for immunohisto-
`
`chemical testing before treatment.
`Toxicity. Tables III and IV summarize the effects
`of hepatic artery infusion of N-ACMEL-MoAb conju-
`gates. In general, the therapy was well tolerated with
`no disturbance in gastrointestinal, renal, or cardiac
`parameters, and there was no evidence of myelosup-
`pression. There was neutrophilia during the time of
`treatment, which restabilized after completion of treat-
`ment. The external arterial catheter was well tolerated
`
`with no complications, and all patients maintained
`good mobility for the duration of therapy. Patient 1 had
`two courses of therapy separated by a 2-month interval,
`despite the presence of a high titer of human antimouse
`antibody (see below). During the second course of
`therapy he had pain in the lower back, fever (39° C),
`urticaria, and bronchospasm. These reactions occurred
`about
`1 hour after immunoconjugate infusion was
`
`Table III. Toxicities
`
` Parameters N0. of patient:
`
`Pain
`1
`Febrile >38° C
`5
`
`Allergic phenomena
`(urticaria, bronchospasm)
`Hematologic
`V/hite cell count
`
`<4OO0/rnm3
`Platelets <100,000/mm‘
`Gastrointestinal
`
`Nausea/vomiting/
`dyspepsia
`Diarrhea
`Bilirubin elevation >50%
`
`AST/ALT elevation
`>507}
`
`Alkaline phosphatase
`elevation >25%
`Renal
`Urea elevation >25%
`Creatinine elevation
`>25%
`
`Proteinuria/hematuria
`Cardiac
`
`Rhythm changes
`Rate <60 or >110/min
`Diastolic blood pressure
`elevation >30%
`
`Catheter-related,
`(thromboembolism,
`hemorrhage,
`displacement, intimal
`tears)
`
`1
`
`0
`
`0
`
`0
`
`1
`0
`
`2
`
`0
`
`0
`0
`
`0
`
`0
`0
`0
`
`0
`
`legend." AST, Aspartatc transaminase; ALT, alanine transaminase.
`
`begun on the second day of the second course of
`therapy, despite prior administration of dexametha-
`sone; treatment was required with antihistamine and
`an additional dose of dexamethasone. The broncho-
`spasm, urticaria, and pain rapidly resolved with such
`additional measures, but the fever persisted for another
`4 hours after the completion of infusion of immunocon-
`jugates. There was, however, no reaction when further
`infusion of
`immunoconjugates was given.
`In four
`patients (patients 7, 8, 9, and 10) a temperature of 38
`to 38.5° C was noted during the second and third day
`of antibody infusion, starting about 1 hour after the
`infusion was begun and continuing for 1 hour after the
`infusion had ended. It therefore appears that febrile
`reactions were more common in patients receiving
`higher doses of N-ACMEL-MoAb conjugates.
`
`IMMUNOGEN 2315, pg. 7
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2315, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`538 Tjandra et at.
`
`Surgery
`September 7989
`
`Table IV. Results of hepatic artery infusion of N-ACMEL conjugates
`Serum CEA* level
`(ng/mt relative to
`Res (mse
`if bypCT
`After
`Alteration
`scan of
`(4 wk)
`liver
`
`Re-
`xponxe
`duration
`(mo)
`
`Status
`(time from
`treatment)
`
`SW01-ml fmm
`dia notis
`oféiiz/er
`metastases
`(mo)
`
`HA3/IA7‘
`
`Patient
`
`Known disease
`site:
`
`Before
`
`1
`
`2
`
`3
`
`4
`
`5
`
`Hepatic
`metastases
`Hepatic
`metastases
`Hepatic and
`pulmonary
`metastases
`
`Hepatic and
`pulmonary
`metastases
`
`Hepatic
`metastases
`
`3920
`1,160
`200
`
`26
`
`270
`
`920
`1,050
`221
`
`15
`
`50
`
`5,425
`
`4,300
`
`77
`9
`10
`
`42
`
`81
`
`21
`
`5.0:1.0
`6.2:1.0
`321.0
`
`MR
`SD
`SD
`
`3.0210
`
`MR
`
`2.4:1.0
`
`SD
`
`3
`2
`3
`
`12
`
`6
`
`Deceased
`(12 mo)
`Alive with disease
`(9 mo)
`Alive with disease
`(12 mo)
`
`Alive with disease
`(10 mo)
`
`5.0:1.0
`
`PD
`
`—
`
`Deceased (6 mo)
`
`24
`
`17
`
`9
`
`22
`
`12
`
`8
`
`6
`
`7
`
`8
`
`Hepatic
`metastases
`Hepatic
`metastases
`
`Hepatic
`metastases
`and
`unresected
`
`32
`
`62
`
`325
`
`12
`
`144
`
`42
`
`38
`
`132
`
`87
`
`4.6:1.0
`
`l\'IR
`
`4.1:1.0
`
`PD
`
`4.6:1.0
`
`—
`
`6
`
`—
`
`—
`
`Alive with disease
`(6 mo)
`Deceased (5 mo)
`
`Deceased (1 mo)
`
`6
`
`3
`
`9
`
`10
`
`<1
`
`<1
`
`1,850
`
`2,000
`
`0
`
`8
`
`2.0:1.0
`
`—
`
`4.0:1.0
`
`SD
`
`primary
`colon
`carcinoma
`Resected
`Dukes’ C
`Hepatic
`metastases
`Legend: l\4R, l\/Iinor response; SD, stable disease; PD, progressive disease.
`*Carcinoembryonic antigen (normal <5 ng/ml).
`-H-luman antimouse antibody (IgG) response at 4 weeks after treatment expressed as the ratio of absorbance value of patient scrum compared with pooled normal human
`serum; the higher the ratio, the higher the HAMA response. A positive test result was considered to be a value 2201.0.
`
`—
`
`1
`
`Alive with disease
`(10 mo)
`Alive with disease
`(6 wk)
`
`11
`
`4
`
`Patient 1 also had mild diffuse arthralgia and fever
`that developed 11 days after completion of the second
`course of therapy; both slowly resolved over 2 months.
`Patient
`8, who had unresected primary colon
`carcinoma and multiple hepatic metastases, died 4
`weeks after receiving 20 mg/m2 N-ACMEL conjugated
`to 1000 mg/m2-MoAb of general debilitating disease.
`There was worsening of diarrhea that started 2 weeks
`after the treatment was given; however, the patient had
`unresected advanced colon cancer causing subacute
`bowel obstruction, which could have accounted for the
`
`diarrhea. Patients 7 and 10 had transient increases in
`the aspartate transaminase level by 70% (from 71
`IU/L to 120 IU/L) and 100% (40 IU/L to 81 IU/L),
`respectively, during treatment, which rapidly returned
`
`to pretreatment levels within 3 days of completion of
`treatment.
`All patients had human antimouse antibody with
`raised level (absorbance value twice normal) as from
`the twelfth day after the first antibody exposure and
`was of IgM as well as
`IgG response. The peat:
`response usually occurred within 30 days after therapy.
`The geometric means of the human antimouse anti»
`body titer were not higher in patients receiving higher
`doses of MoAbs.
`Antitumor effects. Table IV summarizes the tumor
`
`responses evaluated by CT scan in patients treated
`with N-ACMEL-MoAb conjugates. Minor antitumor
`responses were seen in three patients (patients 1, 3, and
`6).
`
`IMMUNOGEN 2315, pg. 8
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2315, pg. 8
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Volume 706
`Number 3
`
`Drug-MOA/3 conjugates and colorectal carcinoma
`
`539
`
`
`
`Fig. 1. CT scans of patient 1 with multiple hepatic metastases before treatment (A) and 1 month after treatment
`(B)-
`
`Patient 1 had subtotal colectomy 24 months previ-
`ously for synchronous carcinoma of the transverse and
`sigmoid colon. At laparotomy multiple large metasta-
`ses in the liver were noted. The hepatic metastases
`were too extensive for surgical
`resection and were
`treated with two courses of hepatic artery infusion of
`czis-platinum with no response. The clinical condition
`continued to deteriorate over the following 10 months,
`with anorexia, nausea,
`lethargy, and severe hepatic
`pain. Results of
`liver
`function tests were grossly
`deranged with elevated transaminase (AST = 136 IU/
`L) and CEA (3920 ng/ml) levels. The abdominal CT
`scan showed multiple large metastases in both lobes
`occupying about 60% of the liver. Within 3 weeks after
`treatment with N-ACMEL-MoAB conjugates,
`there
`was a dramatic improvement
`in his constitution; he
`regained his appetite and had good relief of the hepatic
`pain; he felt so well that he returned to work as a
`headmas