Phase I Clinical Comparative Study of Monoclonal Antibody
`KS1/4 and KS1/4-Methotrexate Immunconjugate in Patients with
`Non-Small Cell Lung Carcinoma
` 
`Darlene J. Elias, Lynn Hirschowitz, Lawrence E. Kline, et al.
`Cancer Res  
`
`1990;50:4154-4159.
`
`Updated version
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`on May 23, 2014. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org on May 23, 2014. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org
`
`
`
`
`
`IMMUNOGEN 2296, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`[CANCER RESEARCH 50, 4154-4159, July 1, 1990]
`
`Phase I Clinical Comparative Study of Monoclonal Antibody KS1/4 and KS1/4-
`Methotrexate Imniunconjugate in Patients with Non-Small Cell Lung
`Carcinoma1
`
`Darlene J. Elias,2 Lynn Hirschowitz,3 Lawrence E. Kline, Joan F. Kroener, Robert O. Dillman,4 Leslie E. Walker,5
`James A. Robb, and Richard M. Timms
`and Oncology [D. J. E., L. E. K., J. F. K.,
`Department of Molecular and Experimental Medicine, Division of Chest and Critical Care Medicine, Division ofHematology
`R. O. D., R. M. T.], and Department of Immunology [L. H., L. E. W.] and Department of Pathology [J. A. R.¡,Scripps Clinic and Research Foundation, La Jalla,
`California 92037
`
`ABSTRACT
`
`trial was undertaken to evaluate and compare
`A Phase la clinical
`murine monoclonal antibody KS1/4 and KSl/4-methotrexate
`immuno-
`conjugate in patients with Stage IIIB or IV non-small cell carcinoma of
`the lung. Six patients received KS1/4 alone and five patients received
`KSl/4-methotrexate
`conjugate. The maximal
`total dose received per
`patient
`in both groups was 1661 mg. Mild to moderate side effects in
`both groups included fever, chills, anorexia, nausea, vomiting, diarrhea,
`anemia, and brief transaminasemia. One patient who received antibody
`alone had an apparent acute immune complex-mediated reaction. Ten of
`11 patients had a human anti-mouse response. Posttreatment carcinoma
`biopsies revealed binding of monoclonal antibody KS1/4 and deposition
`of C3d and C4c complement
`fragments. Monoclonal antibody binding
`and complement deposition correlated with increasing doses of infused
`antibody. There was one possible clinical response.
`
`INTRODUCTION
`Murine Mo Ab6 KS1/4 is an IgG2a antibody that was pro
`duced to the lung adenocarcinoma
`cell
`line UCLA-P3. This
`MoAb recognizes a M, 40,000 and a M, 42,000 antigen found
`on a variety of neoplastic tissues (1). Immunoperoxidase
`stain
`ing of
`fresh frozen carcinoma
`suggests
`that
`the antigen is
`expressed by most
`if not all adenocarcinomas
`and some squa-
`mous cell carcinomas of the lung. The antigen is expressed on
`a number of normal
`epithelial
`cell
`types, suggesting that
`it
`represents an epithelial cell-derived carcinoma marker
`(2). Re
`cent isolation and characterization
`of a cDNA encoding for the
`antigen have been accomplished
`(3). These data suggest
`that
`the antigen is a single M, 40,000 polypeptide
`that has hetero
`geneity in glycosylation,
`is susceptible to specific proteases, and
`contains a cysteine-rich domain. Sequence analysis of the 5'
`and 3' noncoding regions of the KS1/4 cDNA revealed homol-
`ogies to known protooncogenes
`and inflammatory mediators.
`MoAb KS1/4
`has been conjugated
`to methotrexate
`and
`shown to inhibit
`the growth of adenocarcinoma
`of the lung
`
`in vitro and to effectively suppress the growth
`cells, UCLA-P3,
`human
`lung adenocarcinoma
`xenographs
`in
`of established
`athymic mice (4). These results suggested that
`the antigen that
`is recognized by MoAb KS1/4 may be a target antigen for
`antibody-directed
`therapy of lung carcinomas. A clinical study
`was, therefore, undertaken
`to evaluate the safety and toxicity
`of MoAb alone and MoAb KSl/4-methotrexate
`conjugate ad
`ministered
`to patients with non-small
`cell carcinoma
`of the
`lung.
`
`MATERIALS AND METHODS
`
`Preparation of KS 1/4 and KSl/4-Methotrexate
`
`Immunoconjugate
`
`A total of 12.5 g of unconjugated KS1/4 was prepared by Damon
`Biotech, Inc. (Needham Heights, MA) using an in vitro encapsulation
`technique. KS1/4 was covalently conjugated
`to methotrexate
`(4).
`Briefly, methotrexate (Lederle, Pearl River, NY) at 10 mg/ml
`in normal
`saline, pH 6.8, was mixed with EDAC at a molar ratio of 1:4 (metho-
`trexate:EDAC)
`and allowed to react for 15 min at RT. Monoclonal
`antibody KS1/4 was added and the reaction was continued for 1 h. The
`KSl/4:EDAC:methotrexate
`molar ratios were 1:200:50 in a final re
`action volume of 250 ml/g KS1/4. The conjugate was dialyzed (10 mM
`sodium phosphate-150 mM NaCl) and final purification was achieved
`on a hydroxyapatite column (Bio-Gel) using M APS-100 high perform
`ance liquid chromatography
`(Bio-Rad, Richmond, CA). KSl/4-meth-
`otrexate samples (0.5 g) were applied to the column and washed with
`10 mM phosphate buffer at pH 7.4 to remove free methotrexate. KS1/
`4-methotrexate was eluted with a linear gradient of sodium phosphate
`from 10 mM to 500 mM at a flow rate of 5 ml/min. The immunocon-
`jugate eluted at approximately 300 mM sodium phosphate. Fractions
`containing immunoconjugate were pooled, dialyzed against normal
`saline, and concentrated by filtration through a YM30 Amicon filter.
`As measured spectrophotometrically
`at 410 nm, the conjugation ratio
`was 6 mol of methotrexate/mol
`of antibody (17 mg of methotrexate/g
`of KS1/4). Retention of antigenic specificity of the conjugate was
`confirmed by immunoperoxidase
`staining of a panel of normal and
`tumor tissues. Testing was performed for general safety, sterility, py-
`rogens, viruses, and murine DNA. The stability of the methotrexate
`conjugation over time was assessed by repeated dialysis of the immu
`noconjugate to remove free methotrexate,
`and subsequent spectropho-
`tometric
`loss of absorbance
`at 410 nm was determined. Antigenic
`stability of KS1/4 and KSl/4-methotrexate
`was tested by ELISA or
`the ability to bind to UCLA-P3 cells following storage at -20°C for 6
`months. There was no loss of binding activity at 6 months.
`
`Patients
`
`Received 1/4/90.
`The costs of publication of this article were defrayed in part by the payment
`of page charges. This article must
`therefore be hereby marked advertisement
`in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`' This study was funded in part by NIH General Clinic Research Center Grant
`RR00833 and by National Cancer Institute Grant CA34778. We thank Mr. and
`Mrs. Ed Weerts and the Joan Kroc Foundation
`for their generous
`financial
`support.
`should be addressed, at Scripps Clinic and
`a To whom requests for reprints
`Research Foundation, 10666 North Torrey Pines Road. La Jolla, CA 92037.
`3 Present address: University of Bristol, Bristol Royal Infirmary, Marlborough
`Street, Bristol, United Kingdom.
`4 Present address: Hoag Memorial Hospital Presbyterian, 301 Newport Bou
`levard, Newport Beach, CA 92658-8912.
`'Present
`address: Cytel Corporation,
`Jolla. CA 92037.
`6The abbreviations used are: MoAb, monoclonal antibody; cDNA, comple
`mentary DNA; EDAC,
`l-ethyl-3.3-dimethylaminopropylcarbodiimide
`HC1; RT,
`room temperature; ELISA, enzyme-linked
`immunosorbent
`assay; PBS, phos
`phate-buffered saline: BSA, bovine serum albumin; HRP, horseradish peroxidase;
`DAB, diaminobenzidine.
`
`11099 North Torrey Pines Road, La
`
`Eleven patients with Stage IIIB or IV non-small cell carcinoma of
`the lung who had failed conventional
`therapies were selected to receive
`KS1/4 or immunoconjugate. All patients were males between the ages
`of 41 and 70 years. Eligibility criteria required that each patient's
`carcinoma express KSl/4-reactive
`antigen by immunoperoxidase
`stain
`ing. All patients had measurable or évaluabledisease, a performance
`status of 0-2 on the Eastern Cooperative Oncology Group scale,
`carcinoma accessible for repeat biopsy, no chemotherapy or radiation
`therapy for at least 30 days prior to entry into the trial, and adequately
`4154
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`IMMUNOGEN 2296, pg. 2
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`IPR2014-00676
`
`

`

`MONOCLONAL ANTIBODY AND DRUG CONJUGATE CLINICAL TRIAL
`
`(hemoglobin >10 g/dl, WBC >3000/mm3,
`preserved hematological
`platelet count >100,000/mm3),
`renal
`(creatinine <2.0 mg/dl), and
`hepatic (bilirubin <2.5 mg/dl, serum glutamic-oxaloacetic transaminase
`<70 units, alkaline phosphatase <300 units) parameters.
`Informed
`consent was obtained based on protocols on file with the Human
`Subjects Committee of Scripps Clinic and Research Foundation.
`
`Study Plan
`
`Six patients were treated with KS1/4 alone and five were treated
`with KSl/4-methotrexate
`immunoconjugate.
`Patients received KS1/4
`or immunoconjugate
`in escalating doses of 1, 10, 50, 100, 500, and
`1000 mg via a 4-h i.v. infusion in 100 ml normal saline with 5% human
`serum albumin. The infusions were given biweekly for 3 consecutive
`weeks for a total dose of 1661 mg of protein. The total dose of
`methotrexate
`received with the immunoconjugate
`preparation was 28
`mg. During the course of treatment, biopsies of the tumor were obtained
`24 h after a dose of KS1/4 or immunoconjugate
`and examined for
`evidence of in vivo binding of antibody, unbound KS1/4 antigen sites,
`and deposition of complement. Colonie mucosal biopsies were exam
`ined for the presence of KS1/4 antigen and KS1/4 antibody binding
`after
`the 1000-mg infusion. Serial blood samples were obtained at
`multiple time points during and following treatment
`for determination
`of KS1/4 serum levels and human anti-mouse antibody levels. Evalua
`tion of the status of the carcinoma was based on radiographie evaluation
`and physical examination and was performed at time of entry and at
`completion of the study.
`
`Toxicity Monitoring
`
`A clinical research nurse monitored patients closely for any untoward
`reactions. Vital signs were recorded every 15-30 min during the 4-h
`infusions. Serial complete blood cell counts, electrolytes, and serologi-
`cal tests of renal and hepatic function and urinalyses were obtained
`during the study. Performance status and weight were evaluated daily.
`
`goat anti-
`Sections were covered for l h at RT with HRP-conjugated
`mouse antibody. Bound antibody was visualized with DAB (0.1 mg/
`ml) and 0.03% H2O2 and sections were counterstained with 1% méth
`ylèneblue. Anti-complement
`antibodies were omitted for the negative
`controls.
`
`Serum Levels of KS1/4
`
`Monoclonal antibody KS1/4 serum levels were measured by ELISA.
`Aliquots (10, 25, and 50 /^l) of serum were incubated for l h in 96-well
`plates which had been coated with 5 ^g/well of goat anti-mouse anti
`body. The plates were washed with buffer (0.02 M Tris, pH 8, containing
`0.2% Tween 20) and incubated for 0.5 h with HRP-conjugated
`goat
`anti-mouse antibody. Bound antibody was quantitatively measured by
`reaction with o-phenylenediamine
`and measurement of absorbance at
`490 nm in an ELISA reader (BioTek, Burlington, VT). The concentra
`tion of KS1/4 was determined by comparison with a standard curve.
`
`Human Anti-Mouse Antibodies
`
`Patients' sera were incubated for l h with 1 ml of a 10% suspension
`of KS1/4 conjugated to cyanogen-activated Sepharose 4B (5 mg/ml).
`Following washing to remove unbound protein, bound antibody was
`eluted with 0.1 M acetic acid and quantitated using a protein assay
`(Bio-Rad).
`In addition, each eluate was subjected to sodium dodecyl
`sulfate-polyacrylamide
`gel electrophoresis
`to confirm the presence of
`antibody.
`
`RESULTS
`
`Immunoperoxidase Staining
`
`total administered doses
`Clinical Response. Clinical features,
`of KS1/4 or KSl/4-methotrexate
`immunoconjugate,
`human
`anti-mouse
`response, maximum toxicity and clinical response
`are summarized in Table 1. All patients had Stage IIIB or IV
`non-small
`cell carcinoma
`of the lung and had received and
`failed conventional
`therapies. Each patient received a total dose
`of 1661 mg except
`for patient AS-6 who developed Grade 3
`immuno-
`Detection of KSl/4-reactive Antigen. A two-stage indirect
`and fresh
`peroxidase technique (5) was used on paraffin-embedded
`toxicity attributed
`to a probable acute immune complex-me
`diated reaction. Based on comparison of pre-and posttreatment
`frozen tissue blocks. Briefly, paraffin sections were deparaffinized and
`cryostat sections were air-dried, washed in PBS, and incubated for 15
`radiographie
`evaluations and physical examinations,
`there was
`min at RT in PBS containing
`10% goat serum and 1% BSA. The
`no decrease in measurable or évaluabledisease in either group
`sections were incubated for 1 h at RT with KS1/4 (1:20 dilution),
`of patients. However, one patient, EH-7, may have had a clinical
`washed in PBS, and incubated for l h at RT with HRP conjugated to
`response. This patient had Stage IIIB disease and before entry
`goat anti-mouse antibody (Bio-Rad). Bound antibody was visualized
`into the study had received 6210 cGy to the lung and medias
`with DAB (0.1 mg/ml) and 0.03% H2O2 and sections were counter-
`tinum. Four weeks after completion of the radiation, an endo-
`stained with 1% méthylèneblue. Monoclonal antibody Q128 (provided
`bronchial
`abnormality was visualized at bronchoscopy,
`and
`by Dr. V. Quaranta) which reacts with a monomorphic determinant of
`directed endobronchial
`biopsy showed histológica! evidence of
`the major histocompatibility
`complex class I antigen that
`is found on
`all cells was used as the positive control. Antibody-containing
`super-
`carcinoma. After
`the KSl/4-methotrexate
`treatment,
`the en
`natants of the mouse myeloma cell
`line P3x63.Ag8 or the murine
`dobronchial
`abnormality appeared to be smaller, directed en
`hybridoma 10-2.16, which is the same isotype as KS1/4 but does not
`dobronchial biopsy showed necrosis, and no viable carcinoma
`react with human tissues, were used as negative controls.
`could be identified. This patient
`is alive and free of disease 43
`tech
`Detection of Bound KS1/4 Antibody. Direct immunoperoxidase
`months after immunoconjugate
`treatment.
`nique was used to detect KS1/4 antibody which had bound to tumor in
`Toxicity. Toxicities and side effects are summarized in Table
`vivo. Cryostat sections were air-dried, washed with PBS, and incubated
`2. Some degree of toxicity was seen in five of six patients who
`in PBS (10% goat serum-1% BSA). Sections were covered for l h at
`received KS1/4 alone and in four of five patients who received
`RT with HRP-conjugated goat anti-mouse antibody. Negative controls
`KSl/4-methotrexate
`immunoconjugate.
`Fever and chills were
`were stained with HRP-conjugated
`goat anti-rabbit antibody and posi
`seen in both groups. One of six patients
`in the KS1/4 group
`tive controls with Q128 or W6/32 as described above. Bound antibody
`and four of five patients
`in the KSl/4-methotrexate
`group
`was visualized with DAB (0.1 mg/ml) and 0.03% H2O2 and sections
`were counterstained with 1% méthylèneblue.
`experienced mild to moderate fever and chills. Anorexia, nau
`Complement Deposition at the Carcinoma Site. Murine monoclonal
`sea, vomiting, diarrhea,
`as well as mild anemia (hemoglobin
`anti-human
`complement
`components
`(IgGl/i
`isotype) anti-C3d and
`9.5-10.9),
`and brief increases
`in liver transaminases were re
`anti-C4c (Cytotech, San Diego, CA) were used to label bound comple
`ported in both groups. Allergic reactions
`such as pruritus,
`ment. Air-dried sections were rinsed in PBS and incubated for l h at
`urticaria, and anaphylaxis were not seen. The most significant
`RT in rabbit anti-mouse antibody (Accurate, Westbury, NY) in PBS
`toxicity encountered was a probable acute immune complex-
`(10% goat serum-1% BSA). The rabbit anti-mouse antibody was added
`mediated reaction in one patient who received antibody alone.
`to block the murine MoAb KS1/4 which was already bound to the
`Forty-eight h after this patient had received a total dose of 661
`tissue sections. Sections were washed in PBS and incubated for l h at
`mg of KS1/4,
`in the face of high human anti-mouse levels (1.8
`RT with anti-C3d and anti-C4c,
`in PBS (10% goat serum-1% BSA).
`4155
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`
`
`
`on May 23, 2014. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org
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`IMMUNOGEN 2296, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`MONOCLONAL ANTIBODY AND DRUG CONJUGATE CLINICAL TRIAL
`
`immunoconjugate, human anti-mouse response and maximum toxicity
`or KSI/4-methotrexate
`total doses ofKSl/4
`Table 1 Summary of the clinical features,
`Human anti-mouse antibodies were detected in five of six patients who received KS1/4 alone and in five of five patients who received KSl/4-methotrexate
`immunoconjugate. Some degree of toxicity (0 = none, 1 = minimal, 2 = moderate, 3 = severe, 4 = life threatening) was seen in five of six patients who received KS1/
`4 alone and in four of five patients who received KSI/4-methotrexate
`immunoconjugate.
`
`cell
`MoAb or
`
`
`
`conjugateKS1/4KSl/4-mtxPatiententryno.KW-1PM-2CC3LG-4RO-5AS-6EH-7LK-8BB-9SS-10KP-(yr)6667676759437054506453PrevioustreatmentSurgery,
`typeAdenocarcinomaAdenocarcinomaLarge
`XRT,"ChemoXRTXRTChemo,
`
`
`anti-mouse
`Maximum
`dose
`(mg)1661166116611661166166116611661166116611661Human
`
`antibodiestoxicityI+
`
`cellAdenocarcinomaSquamousAdenocarcinomaLarge
`XRTXRTSurgery,
`
`cellAdenocarcinomaAdenocarcinomaSquamousLarge
`
`ChemoXRTChemoChemoSurgery.
`
`11Histológica!
`cellAge
`°XRT, radiation therapy; KSl/4-mtx, KSI/4-methotrexate;
`
`XRTXRTTotal
`
`Chemo, chemotherapy.
`
`I+
`0+
`1+
`2+
`3+
`
`1+
`2-1-
`1+
`0+
`
`1
`
`Table 2 Summary oftoxicities and side effects
`Fever and chills were seen in both groups. Anorexia, nausea, vomiting, diar
`rhea, mild anemia, and brief increases in liver transaminases were reported in
`both groups. Allergic reactions such as pruritus, urticaria, and anaphylaxis were
`not seen. The most significant
`toxicity encountered was a probable acute immune
`complex-mediated reaction in one patient who received antibody alone.
`KSI/4
`KSI/4-methotrexate
`Total
`(no. of
`(no. of
`(no. of
`
`patients of 1patients of 5)patients of 6)Toxicities1)FeverRigor/chillsAnorexiaNausea/vomitingDiarrheaAnemiaTransaminasemiaUrticariaPruritusDyspneaBronchospasmHypotensionAnaphylaxisSerum
`
`
`
`:
`
`-
`
`
`
`sickness111214300000014413231000000055253740000001
`
`mg/ml), he developed fever to 39°Cfollowed by muscle aches
`and an acute polyarthritis
`and synovitis
`involving the small
`joints of both hands, both elbows, shoulders,
`and knees. No
`infectious etiology was identified,
`erythrocyte
`sedimentation
`rate was 115, creatine phosphokinase was normal, and Raji cell
`assay for circulating
`immune
`complex was elevated at 190
`(reference range <50 ^g aggregated human gamma globulin).
`The patient's
`fever and arthritis
`resolved with discontinuance
`of MoAb infusions and administration
`of aspirin.
`In Vivo Localization of MoAb KS1/4 and Complement Frag
`ments. Pretreatment
`immunoperoxidase
`staining of each pa
`tient's carcinoma demonstrated
`expression of the KSl/4-reac-
`tive antigen. Posttreatment
`carcinoma biopsies obtained within
`24 h after a dose of KS1/4 or immunoconjugate were examined
`for evidence of in vivo binding of antibody, unbound KS1/4
`antigen sites, and deposition of complement. The binding of
`KS1/4 antibody and complement deposition in carcinoma cor
`related with increasing doses of antibody infused. No KS1/4
`antibody could be detected in carcinoma biopsies following the
`biopsies. These
`to that seen in pretreatment
`ing was similar
`1- and 10-mg doses. Binding of KS1/4 to carcinoma as well as
`that
`there was in vivo saturation of antigen sites
`results suggest
`at the higher doses of infused antibody. Additionally,
`there was
`C3d and C4c deposition were demonstrated
`in some patients
`after the 50-mg infusion and in nearly all patients at the 500-
`no loss of expression of the KSl/4-reactive
`antigen and there
`and 1000-mg doses in both groups. There was a correlation
`fore no evidence for antigenic modulation.
`between KS1/4 antibody binding and the deposition of comple
`and immunoperoxi
`Examples of carcinoma histopathology
`dase stains are shown in Figs. 1-3. Nine sigmoidoscopic biop
`ment within the carcinoma. There was no staining of carcinoma
`sies from patients
`treated with KS1/4 alone and immunocon
`with the negative control and no complement deposition in any
`of the pretreatment
`biopsies. All posttreatment
`biopsies were
`jugate were examined within 24 h of completion
`of the 1661
`tested for the KSl/4-reactive
`antigen and the intensity of stain-
`mg total dose. In all of the biopsies,
`the colonie mucosa could
`4156
`
`stain
`carcinoma histopathology and immunoperoxidase
`Fig. 1. Pretreatment
`ing. In a. H & E stain shows poorly differentiated carcinoma
`infiltrating the
`dermis.
`In b. section shows expression of KSl/4-reactive
`antigen by the carci
`noma. Immunostained with MoAb KS1/4. The overlying dermis is not stained.
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`on May 23, 2014. © 1990 American Association for Cancer Research.
`
`IMMUNOGEN 2296, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`MONOCLONAL ANTIBODY AND DRUG CONJUGATE CLINICAL TRIAL
`
`Serum KS1/4 levels did not seem to be altered by high circu
`lating levels of human anti-mouse antibody.
`
`DISCUSSION
`
`The antigen reactive with MoAb KS1/4 is expressed predom
`inantly on epithelial malignancies
`and some normal epithelial
`tissues (1,2). While the function of the antigen is unknown,
`its
`high density on the cell surface and homogeneous
`expression
`on non-small cell lung tumors
`suggested that
`it would be an
`excellent
`target
`for MoAb-drug therapy. We have previously
`shown that KSl/4-methotrexate
`immunoconjugate
`effectively
`inhibits the growth of adenocarcinoma
`of the lung cells, UCLA-
`PS,
`in vitro and suppresses
`the growth of established human
`lung adenocarcinoma
`xenographs
`in athymic mice (4). Re
`cently,
`the sequence of cDNA clones that code for the KS1/4-
`reactive antigen has been determined (3, 6).
`Murine monoclonal
`antibodies CO17-1A and GA733 pro
`duced to a human colorectal adenocarcinoma
`cell
`line and a
`gastric adenocarcinoma
`cell line, respectively, bind to carcino
`mas of the gastrointestinal
`tract and also bind in varying degrees
`to normal epithelial
`tissues. Both CO17-1A and GA733 im-
`munoprecipitate
`a M, 40,000 glycoprotein and the sequence of
`the genomic clone for GA733 has recently been determined (7).
`The predicted sequence for the antigens recognized by KS1/
`4 and GA733 has a greater
`than 50% homology and shows
`
`¿V 2b
`carcinoma biopsies immunostained for MoAb KS 1/4 .
`Fig. 2. Posttreatment
`In a, MoAb KS1/4 is bound to carcinoma.
`Immunostained with goat anti-mouse
`antibody. The overlying dermis is not stained.
`In b, there is no immunostaining
`of carcinoma with negative control goat anti-rabbit antibody.
`
`be immunostained with KS1/4. Bound antibody was detected
`in seven of the nine biopsies (see Fig. 4).
`Pharmacokinetics
`and Human Anti-Mouse Response. To as
`sess the circulating levels of murine MoAb achieved with the
`escalating dose regimen,
`serum levels of MoAb KS1/4 were
`measured at the start of each infusion and at 24, 48, and 72 h
`after completion of each infusion. With each dose escalation,
`postinfusion serum concentrations
`increased at each time point.
`The serum KS1/4 levels between the two groups at each dose
`did not differ significantly. Serum levels increased with each
`escalating dose of antibody at each time point [at the 24-h time
`point,
`fig/ml for KS1/4 alone: 50 mg, 0.58 ±0.32 (SE); 100
`mg, 3.48 ±1.83; 500 mg, 4.60 ±0.83; 1000 mg, 6.82 ±0.93;
`and for KSl/4-methotrexate:
`50 mg, 0.46 ±0.15; 100 mg, 2.68
`±0.51; 500 mg, 4.40 ±1.07; 1000 mg 6 42 ±1.53]. For each
`escalating dose of antibody administered,
`the serum KS1/4
`levels achieved at 24 h declined over the ensuing 48 h (data not
`shown). In all patients there was a low background anti-mouse
`response
`that was present prior
`to initiation
`of treatment.
`Human anti-mouse
`response was found in both groups. A 2-
`fold increase in anti-mouse
`levels was detected within 3 weeks
`of treatment
`in five of six patients who received KS1/4 alone
`and in five of five patients who received KSl/4-methotrexate
`immunoconjugate
`(Table 1). Maximum levels were measured
`in the range of 0.4-2.0 mg/ml. The conjugation therefore did
`not appear to affect the immunogenicity of the KS1/4 antibody.
`
`for complement
`immunostained
`carcinoma biopsies
`Fig. 3. Posttreatment
`deposition.
`In a, anti-C3d antibody shows weak positive immunostaining
`of
`carcinoma. Note that
`the hair follicles are not stained.
`In b, anti-C4c antibody
`shows weak positive immunostaining of carcinoma. Hair follicles are not stained.
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`

`MONOCLONAL ANTIBODY AND DRUG CONJUGATE CLINICAL TRIAL
`
`In
`colonie biopsies.
`Fig. 4. Posttreatment
`a, H & E stain shows normal colonie mucosa.
`In b, there is no immunostaining with negative
`control goat anti-rabbit antibody. In c, KS1/4-
`reactive antigen is expressed by colonie mu
`cosa. Immunostained with MoAb KM 4. In ¡I.
`MoAb KS1/4 bound to colonie mucosa.
`Im
`munostained with goat anti-mouse antibody.
`
`4d
`
`residues. These data
`in 11 of 12 cysteine
`direct alignment
`suggest
`that perhaps a family of closely related tumor-associ
`ated antigens may exist, all sharing structural
`and biochemical
`properties.
`In this report we have shown that KS1/4 and its immuno-
`conjugate KSl/4-methotrexate
`can be given to patients by i.v.
`infusion in cumulative doses of up to 1661 mg. The cumulative
`dose of methotrexate
`given to each patient was 28 mg. While
`it was expected that additional or more severe side effects would
`occur following administration
`of the KSl/4-methotrexate
`con
`jugate when compared to the unconjugated MoAb, we did not
`find this to be the case. Instead the same side effects (fever and
`chills, anorexia, nausea, vomiting, diarrhea, mild anemia, and
`brief increases
`in liver transaminases) were reported in both
`groups. These side effects are similar to those reported in other
`monoclonal antibody clinical trials (8-14) in which unconjugated
`monoclonal
`antibodies were used. Allergic reactions
`such as
`pruritus, urticaria,
`and anaphylaxis were not seen. The most
`significant
`toxicity encountered was a probable acute immune
`complex-mediated
`reaction in one patient who received anti
`body alone. This patient developed fever, acute polyarthritis,
`and circulating immune complexes after a total dose of 661 mg.
`At the time of reaction, human anti-mouse levels were measured
`at 1.8 mg/ml. Two other patients
`in this study had similar or
`higher levels without such systemic reactions. A similar clinical
`reaction characterized
`by fever and arthralgias
`and associated
`with circulating immune complexes has been described after
`administration
`of MoAb 9.2.27 for the treatment of malignant
`melanoma (15).
`in a patient who
`response
`There was one possible clinical
`received KSl/4-methotrexate.
`This patient had Stage IIIB dis
`ease, had received 6210 cGy to the lung and mediastinum and,
`prior
`to entry into the study, a directed biopsy of a visible
`endobronchial mass
`histologically
`documented
`carcinoma.
`After immunoconjugate
`treatment,
`the endobronchial mass ap
`peared smaller, endobronchial
`biopsy showed only necrosis,
`and no histological
`evidence of viable carcinoma
`could be
`identified. This patient
`is alive and free of disease 43 months
`after treatment with KSl/4-methotrexate
`immunoconjugate.
`
`staining provided direct evidence for lo
`Immunoperoxidase
`calization of both the MoAb and the MoAb-drug conjugate as
`well as activated complement
`fragments on the carcinoma cell
`membrane posttreatment. Biopsies taken during and after
`the
`course of immunotherapy
`showed selective localization
`and
`binding of monoclonal
`antibody KS1/4 to the carcinoma
`cell
`surface, confirming
`that antibody injected i.v. reaches carci
`noma in vivo. Additionally, we found a dose-response
`relation
`ship between binding of antibody to carcinoma and the quantity
`of
`i.v. administered
`antibody
`or
`immunoconjugate.
`Similar
`dose-dependent
`localization
`of MoAb to malignant
`cells has
`been described previously for anti-melanoma
`antibodies 9.2.27
`(15) and R24 (13) and anti-T-cell antibodies
`(16, 17).
`Posttreatment
`biopsies demonstrated
`deposition of C3d and
`C4c cleavage fragments on carcinoma
`cells corresponding
`to
`the sites of MoAb KS1/4 or MoAb KSl/4-methotrexate
`bind
`ing. Complement
`components deposit close to the site at which
`they are activated, and in this situation,
`the site of activation
`presumably is the antigen-antibody
`complex at
`the cell mem
`brane. C3d and C4c deposition was found in both patient
`groups. Other MoAb clinical
`trials have described complement
`deposition in posttreatment
`biopsies of the targeted neoplasms.
`C3 deposition has been reported with 17-1A (9) and C3, C5,
`and C9 deposition has been reported with R24 (13).
`Because MoAb KS1/4 cross-reacts with the mucosa of the
`colon, colonie biopsies were taken to determine the presence of
`KS1/4 or its drug conjugate posttreatment. While the antibody
`could be detected in seven of nine colon biopsies from patients
`in both groups,
`it was not possible to correlate the presence of
`KS 1/4 alone or KSl/4-methotrexate
`with the observed gas
`trointestinal
`side effects.
`Higher
`circulating
`serum levels of MoAb KS 1/4 were
`achieved with the administration
`of higher doses of antibody.
`We found no significant change in the serum levels of infused
`MoAb despite increasing levels of human anti-mouse antibodies
`over time. Similar observations have been made by some inves
`tigators (9, 18) but others have found that high circulating levels
`of anti-mouse antibodies were associated with low serum levels
`of circulating MoAb, decreased binding to tumor cells, and less
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`IPR2014-00676
`
`

`

`MONOCLONAL ANTIBODY AND DRUG CONJUGATE CLINICAL TRIAL
`
`depletion of circulating tumor cells (16, 17). The mounting of
`a human anti-mouse
`response may not always be associated
`with toxicity but might be disadvantageous.
`In summary,
`the present findings have defined and compared
`doses and toxicity in both the KS1/4 alone and the KS1/4-
`methotrexate
`patient groups.
`In general,
`the KS1/4 infusions
`were well tolerated. The cross-reactivity of KS1/4 with normal
`epithelial
`tissue needs close monitoring in further human clin
`ical trials. The therapeutic
`efficacy of the immunotherapy
`re
`mains to be assessed. Further
`studies are needed to evaluate the
`effect of the immunotherapy
`on tumor
`response and patient
`survival.
`
`ACKNOWLEDGMENTS
`
`We thank Damon Biotech for the preparation and purification of
`KS1/4, Bio-Rad Laboratories
`for the purification of the MoAb KS1/
`4-methotrexate
`conjugate,
`and Deena Hepburn, Pam Schmidt, and
`Katherine Pekny for their excellent
`technical assistance.
`
`REFERENCES
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