Clin. exp. Immunol. (1989) 75, 258-264
`
`Humoral immune responses to XMMCO-791-RTA immunotoxin in
`colorectal cancer patients
`
`L. G. DURRANT, V. S. BYERSt P. J. SCANNONt R. RODVIEN,4 K. GRANT,t R. A ROBINS,
`R. A. MARKSMAN & R. W. BALDWIN Cancer Research Campaign Laboratories, University of Nottingham,
`Nottingham, UK, tXOMA Corporation, 2910-Seventh Street, Berkeley and JPacific Medical Center, San Francisco, CA.
`
`(Acceptedfor publication 14 October 1988)
`
`SUMMARY
`Monoclonal antibody 791 (XMMCO-791) recognizes a colorectal tumour-associated antigen.
`Antibody 791-ricin A chain immunotoxin (XMMCO-791-RTA) inhibits growth of human tumour
`xenografts and it is therefore being evaluated for the treatment of colorectal cancer. One of the
`problems with therapy with mouse monoclonal antibodies is they stimulate humoral responses in
`patients. However antigens linked to ricin are cytotoxic for B cells and therefore XMMCO-791-RTA
`may not be immunogenic. The humoral antibody response to murine monoclonal antibody
`XMMCO-791 (IgG2b) conjugated to the plant toxin, ricin A chain (RTA), was measured in
`colorectal cancer patients in a phase I clinical trial. All patients produced strong responses to the
`XMMCO-791 immunoglobulin and to RTA. The predominant response to the antibody was against
`the idiotypic determinant although anti-subclass and anti-mouse antibodies were also detected. A
`component of the anti-idiotypic immunoglobulin response in the colorectal cancer patients was
`directed against the combining site of XMMCO-791. These antibodies inhibited in-vitro binding of
`XMMCO-791 to target 791 cells and so may be inhibitors of repeated immunotoxin therapy.
`Immunotoxins do not abrogate the immune response to mouse immunoglobulin in vivo but instead
`are highly immunogenic.
`
`Keywords human anti-mouse antibodies (HAMA) immunotoxin
`
`INTRODUCTION
`
`Monoclonal antibody XMMCO-791 recognizes a 72 kD glyco-
`protein on tumour cells (Price et al., 1983; Campbell, Price &
`Baldwin, 1984) and this antibody, labelled with 131-iodine or
`I 11-indium, has been used extensively to gamma camera image
`primary and metastatic colorectal cancers (Farrands et al., 1982;
`Armitage et al., 1984; Ballantyne et al., 1986). Flow cytometry
`analysis of tumour cells derived by collagenase disaggregation
`of surgically resected colorectal carcinomas also showed that
`XMMCO-791 antibody reacted with two-thirds of tumours
`(Durrant et al., 1986). Based upon these studies, XMMCO-791
`antibody has been used to construct an immunotoxin by
`conjugation to ricin A chain (RTA) (Embleton et al., 1986).
`Immunotoxin XMMCO-791-RTA is specifically cytotoxic in
`vitro for tumour cells expressing the gp72 antigen recognized by
`the antibody moiety (Embleton et al., 1986), and it specifically
`and effectively inhibits growth of human tumour xenografts
`(Byers et al., 1987b). Based upon these studies the immunotoxin
`is being evaluated for the treatment of colorectal cancer.
`
`Correspondence: L. G. Durrant, Cancer Research Campaign
`Laboratories, University of Nottingham, Nottingham NC7 2RD, UK.
`
`Murine monoclonal antibodies are known to stimulate a
`human humoral antibody response. Anti-murine antibodies
`have been detected in patients treated with radiolabelled
`monoclonal antibody (1 to 5 mg) for tumour imaging (Pimm et
`al., 1985; Rowe, Pimm & Baldwin, 1985) as well as in patients
`treated with larger doses (up to 1-2 g) for tumour therapy
`(Meeker et al., 1985; Schroffet al., 1985; Courtenay-Luck et al.,
`1986). Furthermore, responses in vivo to murine monoclonal
`antibody OKT3 have been extensively documented in renal
`allograft patients (Chatenoud, 1986; Chatenoud et al., 1986;
`Jaffers et al., 1986). This study was therefore designed to analyse
`the spectrum of antibody responses to murine immunoglobulin
`in colorectal cancer patients treated with ricin A chain immuno-
`toxin. In addition patients were monitored for antibody re-
`sponses to the ricin A chain polypeptide component.
`The objective of this study was to define the temporal
`changes in the anti-murine immunoglobulin responses, particu-
`larly the anti-idiotypic responses which have been reported to
`have the most significant influence on biological activity in renal
`allograft patients (Chatenoud et al., 1986). The generation of
`anti-RTA responses may also be important in terms of antibody
`targeting of the immunotoxin. Finally the influence of the
`cytotoxic moiety in terms of cytotoxicity for antibody-produc-
`ing B cells is important.
`
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`MATERIALS AND METHODS
`
`Patients
`The colorectal cancer patients in this study were entered into a
`phase I clinical trial of XMMCO-791-RTA immunotoxin.
`Patients' ages ranged from 30-70 years and all had colorectal
`cancer metastases either in liver, lung or both organs. Routine
`clinical tests indicated that patients had good function in all
`organs at the time of entry into the study.
`
`Immunotoxin Treatment
`Immunotoxin XMMCO-791-RTA was prepared by conjugat-
`ing highly purified ricin A chain to the murine monoclonal
`antibody XMMCO-791 (IgG2b) via a disulphide linkage
`(Embleton et al., 1986). After purification it was produced for
`clinical use in a standard form at a concentration of I mg
`protein/ml in phosphate-buffered saline (PBS), pH 7 5.
`XMMCO-791-RTA immunotoxin treatment was given as
`five daily intravenous infusions diluted in approximately 100 ml
`normal saline at doses ranging from 0-05 to 0-2 mg/kg/day (total
`doses 6 8-52 8 mg). Patients were tested for presensitization to
`mouse immunoglobulin prior to treatment by intradermal skin
`testing with 200 ug of native XMMCO-791 immunoglobulin. In
`one patient (EW) treatment was stopped after the first dose.
`
`Detection ofhuman immunoglobulins recognizing XMMCO-791-
`RTA
`The presence of anti XMMCO-791-RTA antibodies was
`screened in parallel by ELISA assays. ELISA microplates were
`incubated for 18 h at 4°C with purified XMMCO-791 (5 pg/ml,
`250 ng/well in PBS), or RTA (100 yig/ml, 5 dug/well in PBS) or
`purified myeloma IgG2a (5 pg/ml, 250 ng/well in PBS, Sigma,
`Poole, UK) prior to washing in PBS-Tween (0-01 M phosphate,
`0 005% Tween 20, Sigma, Poole, UK).
`The plates were incubated for I h at room temperature with
`serial dilutions (10-1-104) of patient's serum diluted in 50 mM
`sodium citrate buffer, pH 4 5, containing 5% BSA. Following
`extensive washing the plates were incubated for I h at RT with a
`I in 1000 dilution of alkaline-phosphatase-conjugated goat
`anti-human IgG (anti-Fcy) or anti-human IgM (anti-Fcp)
`antiserum (Sigma, Poole, UK). After washing the assay was
`developed with p-nitrophenolphosphate (Sigma, Poole, UK) as
`the alkaline phosphatase substrate (I mg/ml diluted in 01l M
`glycine buffer, pH 10 4, containing 0 001 M MgCl2 and 0-001 M
`ZnCl2). The optical densities of each well were read (Multiscan,
`Titertek, Flow Labs, Irving, UK) at 405 nM.
`All sera were assayed on the same day and a maximum
`optical density was noted for each ELISA. The serum titration
`producing 50% of this maximum value was calculated since it
`was the most sensitive area of the assay where very small
`increases in antibody concentrations produced large changes in
`optical density. The 50% serum titre value allows comparison of
`results between the different ELISA assays. Sera were only
`considered positive if serial titration produced a significant
`decrease in optical density.
`
`Detection of anti-combining site antibodies
`Serial dilutions (undiluted to 10-2) of serum in PBS containing
`1% BSA were incubated for 1 h at RT with 0 1 jIg of fluorescein
`isothiocyanate (FITC)-labelled XMMCO-791 prior to incuba-
`tion with 2 x 105 791T cells. Under these conditions of tumour
`
`antigen excess, XMMCO-791 FITC was particularly sensitive
`to inhibition of binding to its target cell by human anti-
`combining site antibodies. Similar dilutions of serum were also
`incubated for I h at room temperature with 0-1 HIg of
`biotinylated SRL-3 (Serotech, Oxford, UK) prior to incubation
`with avidin FITC for I h at 4°C and then added to 791T cells
`(2 x 105). SRL-3 is an IgG2b monoclonal antibody which
`recognizes B2 microglobulin expressed by 79 IT cells. As it has a
`different recombining site to XMMCO-791, human anti-com-
`bining site antibodies in patients' serum should not interfere
`with binding of SRL-3 to 791T cells. However, if anti-subclass
`or anti-mouse antibodies can prevent antigen-antibody binding
`both XMMCO-79 1 and SRL-3 should be equally inhibited. The
`tests were assayed by flow cytometry (Robins et al., 1986).
`Fluorescence was excited at 488 nm and collected via a 10 nm
`band with band pass filter centred at 515 nm after adjustment for
`standard conditions using fluorochrome-labelled latex beads.
`Fluorescence intensity expressed as a mean linear fluorescence
`(MLF) was calculated by multiplying the contents of each
`channel by its channel number and dividing by the total number
`of cells in the distribution (Roe et al., 1985).
`Anti-combining site antibodies in individual patients at
`different times were compared by calculating the serum titre
`which produced a 50% inhibition of XMMCO-791 FITC
`binding to target cells.
`
`Detection of anti- (anti-combining site) antibodies
`791T cells were incubated with patients' sera (50 ul, 10-0
`dilution) for I h at 4°C. The cells were washed in PBS and
`incubated with FITC-labelled goat anti-human Ig antisera for
`1 h at 4°C. Cells were analysed by a FACS IV as described
`above.
`
`RESULTS
`
`Detection of anti-XMMCO-791-RTA immunotoxin antibodies
`All patients produced both IgM and IgG antibodies recognizing
`XMMCO-791 immunoglobulin (Fig. I a,b). The IgM response
`was first detected between 7-22 days after initiation of immu-
`notoxin therapy, with peak responses in individual patients
`occurring between 7-32 days. In four patients where sera were
`analysed up to 40-60 days after treatment, the IgM antibody
`levels progressively diminished. The IgG antibody response to
`XMMCO-791 immunoglobulin was initially detected in all but
`one patient 5-18 days after initiation of therapy. In one patient
`(RA) IgG antibody did not become detectable until day 28 and
`coincided with the peak IgM response.
`The IgM and IgG antibody responses to ricin A chain are
`shown in Fig. 2. All but one of the patients produced an IgM
`antibody response (Fig. 2b); the IgG antibody response was
`more pronounced will all patients responding (Fig. 2a). The
`most marked responses were observed in three patients (AF,
`YBP and 1K).
`The relative response of patients to XMMCO-791 immuno-
`globulin and ricin A chain was determined by titrating serial
`dilutions of serum against both components of the immuno-
`toxin and comparing the dilution producing 50% of the
`maximum response (Table 1). In patient AF both the IgG and
`IgM responses to RTA were more pronounced than those
`elicited by XMMCO-791 immunoglobulin. In patient RA the
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`2-0
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`1-8
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`1-6
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`1-4
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`E 1-2
`c
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`1-0
`so
`004
`0 H
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`04
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`0-2
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`10
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`20
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`30
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`4a
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`10
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`20
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`30
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`40
`
`50
`
`6
`
`70
`
`0
`
`s0
`
`60
`
`70
`
`so
`
`Time (days)
`Fig. 1. Anti-murine XMMCO-791 IgG (a) and IgM (b) responses in colorectal cancer patients treated with XMMCO-791-RTA
`immunotoxin. Sera at a dilution of 10- 1 in pH 4 5 buffer were screened by ELISA. Patient LF (0); EW (0); AF (-); RA (0); FC (A),
`YBP (A); IK (v). The shaded area denotes background optical intensity minus two standard deviations.
`
`1-6
`
`1-4
`
`1-2
`
`3 1-0
`8
`
`0 0
`
`w6
`
`04
`
`0-2
`
`1-6
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`1-4
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`1-2
`
`E 1
`
`0 08
`WI
`0
`
`0-4
`
`0-2
`
`10
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`20
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`30
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`40
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`50
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`60
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`0
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`80
`
`10
`
`20
`
`30
`
`40
`
`SO
`
`40
`
`10
`
`w
`
`Time (days)
`Fig. 2. Anti-ricin A chain antibody responses in colorectal cancer patients. (a) IgG, (b) IgM. Sera were screened at a dilution of 10 'in
`pH 4 5 buffer. Patient LF (0); EW (0); AF (U); RA (0); FC (A); YBP (A); 1K (v). The shaded area denotes background optical density
`minus two deviations.
`
`IgG response to XMMCO-791T was much greater than that to
`RTA, whereas the IgM response was greater to RTA.
`Comparing responses in seven patients (Table 1) few
`produced higher IgM titres to RTA compared to XMMCO-79 1;
`two of these patients produced greater IgG responses to RTA.
`Only one patient (LF) produced greater IgG and IgM responses
`to XMMCO-791 when compared to the response to RTA.
`Three patients (AF, LF, YP) had pretreatment IgM anti-
`bodies which recognized RTA. However these patients did not
`produce a particularly strong IgM response to RTA following
`treatment and only one patient produced a strong IgG response.
`One patient had pretreatment IgG antibodies recognizing RTA
`and responded strongly following XMMCO-791-RTA treat-
`ment.
`
`Five patients (LF, FC, IK, EW, RA) had pretreatment IgG
`antibodies which recognized XMMCO-79 1; one (LF) also had a
`similar IgM response. These patients did not respond, either at
`an earlier time or with a response following administration of
`XMMCO-791 stronger than the patients without pretreatment
`antibodies.
`Anti-idiotypic antibodies could not be detected by binding
`to F(ab)2 or Fab fragments of 791T/36, as this antibody is an
`IgG2b subclass and fragments produced from this mouse
`subclass are unstable. Comparative binding assays and inhibi-
`tion assays were performed therefore.
`
`Anti-mouse, anti-isotype and anti-idiotype responses
`Sera were screened for antibodies recognizing XMMCO-791
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`Anti-immunotoxin antibody in colorectal cancer patients
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`261
`
`Table 1. Anti-idiotypic, anti-isotypic and anti-mouse common determinant antibody responses in colorectal
`cancer patients treated with immunotoxin XMMCO-791-RTA
`
`Dose of
`Serum XMMCO-791
`immunotoxin sample
`(days)t
`(mg)*
`
`IgM
`
`IgG
`
`Patient
`
`LF
`
`0 05/13 0
`
`YBP
`
`0 1/25 0
`
`FC
`
`IK
`
`AF
`
`EW
`
`RA
`
`0 1/37 5
`
`0 1/38 0
`
`0 1/42 5
`
`0-1/0-8
`
`0 2/52-8
`
`0
`5
`28
`0
`13
`18
`32
`60
`76
`0
`26
`41
`0
`14
`0
`18
`32
`60
`0
`7
`14
`0
`22
`29
`72
`
`10
`10
`20
`0
`0
`100
`45
`20
`20
`0
`140
`70
`0
`100
`0
`30
`10
`10
`0
`5
`10
`0
`0
`50
`80
`
`50
`50
`500
`0
`10
`2500
`4000
`900
`750
`10
`2500
`7000
`10
`200
`0
`20
`1000
`400
`5
`790
`1000
`10
`20
`670
`7000
`
`Antibody titrest against
`
`Murine IgG2b
`
`Murine IgG2a
`
`RTA
`
`IgM
`
`10
`10
`10
`0
`0
`100
`45
`20
`15
`0
`110
`55
`0
`100
`0
`25
`10
`5
`0
`5
`10
`0
`0
`15
`15
`
`IgG
`
`20
`10
`70
`0
`10
`1 780
`1 500
`450
`250
`4
`1000
`1000
`0
`40
`0
`0
`210
`100
`0
`180
`250
`5
`10
`280
`1600
`
`IgM
`
`IgG
`
`IgM
`
`IgG
`
`10
`10
`10
`0
`0
`100
`45
`17
`15
`0
`30
`30
`0
`40
`0
`15
`5
`5
`0
`5
`5
`0
`0
`5
`5
`
`20
`10
`65
`0
`10
`1700
`1400
`420
`250
`3
`35
`280
`0
`0
`0
`0
`15
`15
`0
`70
`50
`0
`0
`15
`1400
`
`3
`2
`0
`140
`0
`90
`100
`8
`6
`0
`30
`250
`0
`80
`16
`350
`200
`80
`0
`0
`1 780
`0
`100
`180
`5000
`
`0
`0
`20
`0
`25
`31 600
`316000
`3 160
`1250
`0
`6310
`10000
`45
`158 500
`0
`1 600
`2 500
`630
`0
`10
`20
`0
`130
`200
`630
`
`* Daily dose/total dose.
`t Day 0 sample taken immediately prior to immunotoxin treatment (day 0 to 5).
`Serum samples evaluated by ELISA for IgG and IgM antibodies binding to XMMCO-791 (IgG2b),
`murine myeloma IgG2b, murine myeloma IgG2a and RTA. Serum titre expressed as dilution giving 50% of
`maximum response.
`
`immunoglobulin (BALB/c, IgG2b), BALB/c myeloma IgG2b,
`to detect anti-isotype antibody, and BALB/c myeloma IgG2a to
`detect antibodies recognizing mouse immunoglobulin common
`determinants. The relative response to these three immunoglo-
`bulins was compared by titrating serial dilutions of serum and
`determining the dilution producing 50% of the maximum
`response.
`The responses of all seven patients to XMMCO-791,
`myeloma IgG2b and IgG2a are summarized in Table 1. The
`most marked response in all patients was the production of IgG
`antibodies to XMMCO-791, with peak titres ranging from
`1/200 to 1/7000. In all patients the anti-XMMCO-791 response
`was several-fold higher than the response to myeloma IgG2b.
`For example, with patient FC the maximum titres to XMMCO-
`791 and normal IgG2b were 1/2 500 and 1/1 000, respectively.
`Antibody reacting with myeloma IgG2a, recognizing mouse
`common determinants, was detected in all patients. However
`this response was only substantial in one patient (RA) who
`produced a titre greater than 1/1000.
`
`Detection ofanti-combining site antibodies
`Human antibodies recognizing the combining site of XMMCO-
`791 were detected by their ability to prevent binding of
`fluoresceinated XMMCO-791 to 791T cells. Inhibition of
`binding of XMMCO-791-FITC to its target cell, 791T, by serial
`dilutions of sera from patient AF is shown in Table 2.
`Pretreatment and day 8 sera, undiluted, only caused weak
`inhibition of binding when pre-incubated with XMMC-791.
`However, by day 18 significant inhibition was produced by
`undiluted and 1/10 dilution of serum and by day 32 even serum
`diluted 1/100 produced marked inhibition of binding of
`XMMCO-791-FITC to 791T cells. The response began to fall by
`day 60.
`Serum samples were titrated and the dilution which pro-
`duced 50% inhibition of XMMCO-791-FITC binding to 791T
`cells calculated (Fig. 3). All but one patient (1K) produced anti-
`combining site antibodies. The patient who failed to respond
`was only followed for 14 days post therapy. None of the
`patients' sera significantly inhibited binding of the anti-fl2
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`350r
`
`20)
`250 _/
`
`c
`
`0 200
`
`0
`
`150
`
`2 100
`
`50
`
`1
`
`Table 2. Inhibition of binding of XMMCO-791
`antibody or SRL-3 antibody to target cells by
`serial dilutions of serum from patient AF
`
`Serum Sample
`(day)*
`
`Mean linear fluorescence
`
`Dilution XMMCO-791 SRL-3
`
`0
`
`18
`
`32
`
`60
`
`10-0
`10-
`10-2
`10-0
`10-l
`10-2
`10-0
`10'-
`10-2
`10-0
`10-'
`10-2
`
`380
`420
`430
`10
`170
`400
`20
`20
`220
`10
`30
`390
`
`181
`283
`269
`161
`284
`261
`124
`159
`271
`134
`261
`232
`
`* Day 0 sample taken immediately prior to
`immunotoxin treatment.
`
`a)
`(n
`
`0
`
`10
`
`20
`
`50
`40
`30
`Time (days)
`Fig. 3. Serum titre at different times after immunotoxin therapy
`producing 50% inhibition of binding of XMMCO-791 antibody to its
`target cell. Patient LF (0); EW (-); AF (U); RA (0); FC (A); YBP (0).
`
`60
`
`70
`
`80
`
`0
`
`o
`
`I0
`
`20
`
`50
`
`60
`
`70
`
`80
`
`30
`40
`Time (days)
`Fig. 4. Flow cytometry analysis of immunofluorescence binding of
`antibodies from patients serum. Patient LF (0); EW (A); AF (U);
`RA (0); FC (A); YBP (0). Serum samples taken during and following
`treatment with immunotoxin XMMCO-791-RTA.
`
`microglobulin monoclonal antibody SRL-3 to 791T cells. The
`data for patient AF is shown in Table 2. Furthermore, serum
`from a patient injected with radiolabelled antibodies for tumour
`imaging produced human antibodies which reacted equally with
`XMMCO-791 and myeloma IgG2b in the ELISA assays, but
`which failed to inhibit binding of XMMCO-791-FITC to 791T
`cells. Fusion of lymphocytes from this patient with a mouse
`myeloma resulted in the production of a human anti-IgG2b
`specific monoclonal antibody. This antibody also failed to
`inhibit binding ofXMMCO-791T-FITC to target cells (Durrant
`to be published).
`Of the four patients who were followed for more than
`30 days after therapy, two continued to secrete quantities of
`anti-combining site antibodies which increased with time post-
`therapy. The two other patients produced strong responses
`which peaked at days 13 and 32.
`The drop in anti-combining site antibodies could have been
`due to the formation of anti-(anti-combining site) antibodies.
`Anti-(anti-combining site) antibodies could also inhibit binding
`of XMMCO-791 FITC to its target cells and could therefore
`give a misleading impression of anti-combining site antibodies.
`However anti-(anti-combining site) antibodies will bind directly
`to target cells and were detected by their ability to react with
`791T cells. Figure 4 shows the presence of human antibodies
`recognizing 791T cells in the serum from patient YBP and AF.
`These antibodies could be detected at day 18 and slowly
`decreased to negligible levels at day 60. None of the sera from
`other patients bound to 791T cells (Fig. 4).
`The peak inhibition of binding of XMMCO-791-FITC to
`791T cells was at day 32 in sera from patient AF and at day 13 in
`sera from patient YBP. However, peak anti-(anti-combining
`site) antibodies could be detected at day 18 for both patients. If
`anti-(anti-combining site) antibodies alone were responsible for
`inhibition of binding of XMMCO-791-FITC to 791T cells the
`peak responses should have coincided. Furthermore, anti-
`combining site antibodies fail to bind non-specifically to 791T
`cells since the majority of patients producing anti-combining
`site antibodies failed to bind to 791T cells.
`
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`

`Anti-immunotoxin antibody in colorectal cancer patients
`
`263
`
`The response to the idiotypic determinant of XMMCO-791
`(response to XMMCO-791 less the response to myeloma
`IgG2b) was several-fold higher than the response to the anti-
`combining site. However, similar patterns of either increasing
`titre with time, or peaking at days 13-72, were observed for both
`types of antibodies.
`
`DISCUSSION
`The development of human antibodies recognizing mouse
`immunoglobulin represents an obstacle to effective monoclonal
`antibody therapy. This response could result in immune com-
`plex formation, possibly inducing serum sickness or renal
`toxicity, or interfere with the efficacy of treatment, either by
`inhibiting binding of the administered antibody to antigen, or
`by increasing the removal of antibody by the reticuloendothelial
`system. This study demonstrates that colorectal cancer patients
`treated with XMMCO-791 monoclonal antibody conjugated to
`the plant toxin, ricin A chain, not only produce a significant
`response to the mouse immunoglobulin but also generate an
`antibody response to ricin A chain. The production ofanti-RTA
`antibody in melanoma patients treated with an immunotoxin
`has also been observed (Spitler et al., 1987).
`All patients produced strong responses to the idiotypic
`determinant of antibody XMMCO-791T, whereas only one
`patient produced a high response to mouse common deter-
`minants. The anti-idiotypic IgG response was several-fold
`higher than the IgM response and was extremely rapid in onset.
`The IgG antibodies could be detected by day 7 even in one
`patient who received a single dose (6-8 mg) of immunotoxin.
`Renal allograft patients treated with murine monoclonal anti-
`body OKT3 also produced a predominant anti-idiotypic res-
`ponse (Chatenoud et al., 1986). Anti-idiotypic antibodies were
`also detected in 43% of cancer patients treated with a single dose
`(15-849 mg) of murine antibody 17-1A, the frequency rising to
`78% following multiple injections (Koprowski et al, 1984).
`There are multiple immunogenic idiotypes on an immuno-
`globulin variable region (Brown & Sealy, 1986). These idiotypes
`may be at the antibody-antigen combining site and antibodies to
`these sites will interfere with antigen recognition. Renal allo-
`graft patients treated with monoclonal antibody OKT3 pro-
`duced antibodies to the combining site of the monoclonal
`antibody and this response correlated with graft failure (Chate-
`noud et al., 1986). A component of the anti-idiotypic immuno-
`globulin response in the colorectal cancer patients was directed
`against the combining site of antibody XMMCO-791. These
`antibodies inhibited in-vitro binding of XMMCO-791 to target
`791T cells and so must be viewed as potential inhibitors of
`immunotoxin cytotoxicity.
`This is further indicated by previous studies where multiple
`injections of low doses (1-5 mg) of I3'I-labelled XMMCO-791
`antibody for gamma camera imaging of colorectal patients
`(Farrands et al., 1982; Armitage et al., 1984; Ballantyne et al.,
`1986) led to the generation of anti-idiotypic antibody, so
`rendering patient imaging ineffective (Pimm et al., 1985). An
`alternative explanation could be that circulating 791T p72
`antigen inhibits binding of XMMCO-791-FITC to 791T cells.
`However, in the ELISA screens patients' antibodies bound to
`XMMCO-791 at a higher titre than they bound to myeloma
`IgG2b. This suggests that if circulating antigen is present anti-
`idiotypic antibodies are produced in excess.
`
`In two patients (YP and AF) anti-combining site antibodies
`stimulated an anti-(anti-combining site antibody) response. As
`anti-combining site antibodies are human antibodies with an
`idiotype similar to the administered mouse XMMCO-791
`antibody they may elicit help for host cellular immune responses
`against tumour. This has been reported to be one of the
`mechanisms involved in anti-tumour responses generated
`against colorectal cancer in patients treated with monoclonal
`antibody 17-lA (Koprowski et al., 1984).
`A stronger IgG anti-mouse immunoglobulin response to
`monoclonal antibody in presensitized patients when compared
`to non-sensitized patients has been reported (Courtenay-Luck
`et al., 1986). Although several of our patients were presensitized
`to mouse Ig common determinants, and to RTA, they showed
`no significant elevation in antibody responses when compared
`non-sensitized individuals following administration of
`to
`XMMCO-791-RTA. The anti-murine antibodies detected prior
`to therapy may have been rheumatoid factors binding via Fc-Fc
`Furthermore, the predominant response to
`interactions.
`XMMCO-791 in presensitized patients was anti-idiotypic.
`The generation of the antibody response to mouse immuno-
`globulin and the equally strong response to ricin A chain in
`patients treated with the immunotoxin are significant in view of
`the proposal that immunotoxins may be used to abrogate
`immune responses to the moiety conjugated to RTA. This has
`been clearly demonstrated in experiments showing that RTA
`conjugated to the acetylcholine receptor (ACh.R) selectivity
`inhibited in vitro antibody responses by rat lymph node cells
`against purified ACh.R (Killen & Lindstrom, 1984). Compara-
`bly, in vitro antibody responses generated against tetanus toxoid
`(Volkman et al., 1982), thyroglobulin (Rennie et al., 1983) and
`nucleosides (Morimoto et al., 1983) by ricin A chain conjugates
`has been reported. The most likely explanation for the failure of
`immunotoxins to have in vivo immunosuppressive effects is that
`they are not able to target effectively to B cell populations
`involved in the generation of immune responses. This may be
`related to the rapid liver uptake of RTA-containing immuno-
`toxins through Kupffer cell recognition of oligosaccharide
`structure of RTA (Bourrie et al., 1986; Thorpe et al., 1985; Byers
`et al., 1987a).
`This rapid uptake of immunotoxin by Kupffer cells may also
`be involved in the rapid stimulation of antibody responses to
`immunotoxin components. Several procedures have been deve-
`loped to limit hepatic uptake of immunotoxins, prolonging
`blood survival and so enhancing target tissue localization. These
`include the use of deglycosylated RTA for immunotoxin
`synthesis and the combined administration of an agent such as
`mannosyl-lysine which is a mannose blocking agent preferen-
`tially taken up into hepatic Kupffer cells (Byers et al., 1987a).
`Although ostensibly designed to effect increased tumour locali-
`zation of immunotoxins, these procedures produce a very
`significant increase in blood survival and so should improve
`targeting of immunotoxins to antibody-producing B cells.
`Control of humoral immune responses in patients to the
`murine immunoglobulin component ofimmunotoxins is necess-
`ary. The predominant response was anti-idiotypic and so the
`generation of anti-idiotypic antibodies to human monoclonal
`antibodies or to constructs with mouse antibody variable region
`on human immunoglobulin must be considered. In this respect
`we have demonstrated the production of anti-idiotypic anti-
`bodies in normal BALB/c mice immunized with the murine
`
`IMMUNOGEN 2328, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`264
`
`L. G. Durrant et al.
`
`immunotoxin XMMCO-791-FITC (unpublished findings).
`From these considerations the design of procedures for abro-
`gating antibody responses in patients to immunoconjugates is
`viewed as the most appropriate pathway so permitting multiple
`treatments of patients to be carried out.
`
`ACKNOWLEDGMENTS
`This research was supported by the Cancer Research Campaign, UK,
`and XOMA Corporation.
`
`REFERENCES
`ARMITAGE, N.C., PERKINS, A.C., PiMM, M.V., FARRANDS, P.A., BALD-
`WIN, R.W. & HARDCASTLE, J.D. (1984) The localization of an anti-
`tumour monoclonal antibody (791T/36) in gastrointestinal tumours.
`Br. J. Surg. 71, 407.
`BALLANTYNE, K.C., DURRANT, L.G., ARMITAGE, N.C., ROBINS, R.A.,
`BALDWIN, R.W. & HARDCASTLE, J.D. (1986) Binding of a panel of
`monoclonal antibodies to primary and metastatic colorectal cancer.
`Br. J. Cancer 54, 191.
`BOURRIE, B.J.P., CASELLAS, P., BLYTHMAN, H.E., JANSEN, F.K. (1986)
`Study on the plasma clearance of antibody-ricin A chain immuno-
`toxins. Evidence for specific recognition sites on the A chain that
`mediate rapid clearance of the immunotoxin. Eur. J. Biochem. 155, 1.
`BROWN, A.R. & SEALY, R.W. (1986) Regulation of the Balb/c anti-p-
`Azophenylarsonate antibody response by monoclonal anti-idiotype.
`J. Immunol. 137, 603.
`BYERS, V.S., PIMM, MV., PAWLUCZYK, I., LEE, H.M., SCANNON, P.J. &
`BALDWIN, R.W. (1987a) Biodistribution of ricin toxin A-chain
`monoclonal antibody 791T/36 immunotoxins and the influence of
`hepatic blocking agents. Cancer Res. 47, 5277.
`BYERS, V.S., PIMM, M.V., SCANNON, P.J., PAWLUCZYK, I.Z.A. &
`BALDWIN, R.W. (1987b) Inhibition of growth of human tumour
`xenografts in athymic mice treated with ricin toxin A chain-
`monoclonal antibody 791T/36 conjugates. Cancer Res. 47, 5042.
`CAMPBELL, D.G., PRICE, M.R. & BALDWIN, R.W. (1984) Analysis of a
`human osteogenic sarcoma antigen and its expression on various
`human tumour cell lines. Int. J. Cancer 34, 31.
`CHATENOUD, L. (1986) The immune response against therapeutic
`monoclonal antibodies. Immunology Today 7, 367.
`CHATENOUD, L., BAUDRIHAYE, M.F., CHKOFF, N., KREIS, H., GOLD-
`STEIN, G. & BACH, J.-F. (1986) Restriction of the human in vivo
`immune response against the mouse monoclonal antibody OKT3. J.
`Immunol. 137, 830.
`COURTENAY-LUCK, N.S., EPENETOS, A.A., MOORE, R., LARCHE, M.,
`PECTASIDES, D., DHOKIA, B. & RITTER, R.A. (1986) Development of
`primary and secondary immune responses to mouse monoclonal
`antibodies used in the diagnosis and therapy ofmalignant neoplasms.
`Cancer Res. 46, 6489.
`DURRANT, L.G., ROBINS, R.A., ARMITAGE, N.C., BROWN, A., BALDWIN,
`R.W. & HARDCASTLE, J.D. (1986) Association of antigen expression
`and DNA ploidy in human colorectal tumors. Cancer Res. 46, 3543.
`EMBLETON, M.J., BYERS, V.S., LEE, H.M., SCANNON, P., BLACKHALL,
`N.W. & BALDWIN, R.W. (1986) Sensitivity and selectivity of ricin
`toxin A chain-monoclonal antibody 791T/36 conjugates against
`human tumor cell lines. Cancer Res. 46, 5524.
`FARRANDS, P.A., PERKINS, A.C., PIMM, M.V., HARDY, J.G., BALDWIN,
`R.W. & HARDCASTLE, J.D. (1982) Radioimmunodetection of human
`
`colorectal cancers using an anti-tumour monoclonal antibody; Lancet
`ii, 397.
`JAFFERS, G.J., FULLER, T.C., CoSIMI, M.D., RUSSELL, P.S., WINN, H.J. &
`COLVIN, R.B. (1986) Monoclonal antibody therapy: anti-idiotypic
`and non-anti-idiotypic antibodies to OKT3 arise despite intense
`immunosuppression. Transplantation 41, 572.
`KILLEN, J.A. & LINDSTROM, J.M. (1984) Specific killing of lymphocytes
`that cause experimental auto-immune myasthenia gravis by ricin
`toxin-acetylcholine receptor conjugates. J. Immunol. 133, 2549.
`KoPROWSKI, H., HERLYN, D., LUBECK, M., DEFREITAS, E. & SEARS,
`H.F. (1984) Human anti-idiotype antibodies in cancer patients: Is the
`modulation of the immune response beneficial for the patients. Proc.
`nati. Acad. Sci. USA. 81, 216.
`MEEKER, T.C., LOWDER, J., MALONEY, D.G., MILLER, R.A., THIELE-
`MANS, K., WARNKE, R. & LEVY, R. (1985) A clinical trial of anti-
`idiotype therapy for B cell malignancy. Blood 65, 1349.
`MORIMOTO, C., MASUKO, Y., BOIEL, Y., STEINBERG, A.D. & SCHLOSS-
`MAN, S.F. (1983) Selective inhibition of anti-nucleoside specific
`antibody production by nucleoside-ricin A conjugate. J. Immunol.
`131, 1762.
`PIMM, M.V., PERKINS, A.C., ARMITAGE, N.C. & BALDWIN, R.W. (1985)
`Implications for immunoscintigraphy of the characteristics of blood-
`borne radiolabel and production of anti-mouse IgG antibodies in
`patients receiving 131I or Illn-labeled monoclonal antibody (791T/
`36). J. Nucl. Med. 26, 1011.
`PRICE, M.R., CAMPBELL, D.G., ROBINS, R.A. & BALDWIN, R.W. (1983)
`Characteristics of a cell surface antigen defined by an anti-human
`osteogenic sarcoma monoclonal antibody. Eur. J. Cancer Clin. Oncol.
`19, 81.
`RENNIE, D.P., MCGREGOR, A.M., WRIGHT, J., WEETMAN, A.P., HALL,
`R. & THORPE, P. (1983) An immunotoxin of ricin A chain conjugated
`to thyroglobulin selectively suppresses the anti-thyroglobuin auto-
`antibody response. Lancet i, 1338.
`ROBINS, R.A., LAXTON, R.R., GARNETT, M., PRICE, M.R. & BALDWIN,
`R.W. (1986) Measurement of tumour reactive antibody and antibody
`conjugate by competition, quantitated by flow cytofluorimetry. J.
`Immunol. Meth. 90, 165.
`ROE, R., ROBINS,