Br. J. Cancer (1987), 56, 722-726
`Br. J. Cancer (1987), 56, 722-726
`
`k(--" The Macmillan Press Ltd., 1987
`
`© The Macmillan Press Ltd., 1987
`
`Sensitivity of newly established colorectal cell lines to cytotoxic drugs
`and monoclonal antibody drug conjugates
`
`L.G. Durrant', M.C. Garnett', J. Gallego3, N.C. Armitage2, K.C. Ballantyne2,
`R.A. Marksman', J.D. Hardcastle2 & R.W. Baldwin'
`
`'Cancer Research Campaign Laboratories, University of Nottingham, University Park, Nottingham NG7 2RD; 2Department of
`Surgery, University Hospital, Queens Medical Centre, Nottingham NG7 2UH, UK; and 3J. Gallego, Simo i Pera 1, 3e, la,
`Badelona, Barcelona, Spain.
`
`Summary A major problem in the chemotherapy of colorectal cancers is their resistance to most cytotoxic
`drugs which may be due to insufficient cellular transport. Drugs conjugated to monoclonal antibodies
`recognising tumour antigens may overcome these difficulties by providing access of active agents to the
`tumour cells. The anti-tumour monoclonal antibody shown to localise in patients with colorectal cancer,
`791T/36, has been investigated as a potential targeting antibody.
`Eight cell lines were established from surgically resected material and were shown to bind 791T/36
`antibody. They were screened for their sensitivity to methotrexate, 5-fluorouracil and daunomycin. Although
`5-fluorouracil is the drug of choice for chemotherapy of colorectal cancer it was the most cytotoxic drug in
`only 2 of the 8 cell lines. Only the 4 cell lines which were resistant to methotrexate showed less cytotoxicity
`with methotrexate than 5-fluorouracil. The cell lines which were resistant to methotrexate were more sensitive
`to 791T/36-methotrexate conjugates. Daunomycin was the most cytotoxic drug in 4 of the 8 cell lines.
`However, a similar cytotoxicity was observed for free drug and 791T/36 daunomycin in the two lines tested.
`Selective monoclonal antibody drug conjugates may offer a solution to treatment of tumours which are
`resistant to classical chemotherapeutic agents. This is the first report to show that newly established cell lines
`that are resistant to classical chemotherapeutic agents are rendered sensitive when the drug enters the cell as a
`drug monoclonal antibody carrier.
`
`Colorectal carcinoma is one of the most common solid
`tumours in humans and is relatively resistant to all forms of
`currently
`chemotherapy.
`available
`Several
`have
`groups
`shown that antitumour antibodies can localise in colorectal
`cancer (Mach et al., 1980; Chatal et al., 1982) and it has
`been proposed that they may be used to direct therapeutic
`drugs to tumour cells. Monoclonal antibody, 791T/36, raised
`al., 1981)
`against osteogenic sarcoma cells (Embleton et
`binds to colorectal cancer cell (Durrant et al., 1986a) and
`has been shown to localise in colorectal cancer (Farrands et
`al.,
`1982; Armitage et al.,
`1984). Conjugates synthesized
`between this antibody and methotrexate or daunomycin have
`been shown to be selectively cytotoxic against an osteogenic
`sarcoma cell line (Garnett et al., 1983; Gallego et al., 1984).
`Dividing cells have been isolated from primary colorectal
`tumours (Durrant et al., 1986b) and their response to free
`drug or 791T/36-drug conjugates studied to assess their
`potential therapeutic usefulness in this common cancer.
`
`Materials and methods
`
`Clinical specimens
`Collection of surgically resected specimens, disaggregation
`vitro isolation of dividing tumour cell
`lines has
`and in
`previously been described (Durrant et al., 1986b).
`
`Cell culture
`The basal medium consisted of Dulbecco's minimal essential
`medium (DMEM) supplemented with insulin (Sigma, Poole,
`Dorset, UK) and pyruvate (Flow Labs.,
`Irving,
`Fife).
`DMEM was enriched with 10% heat inactivated foetal calf
`serum and designated IOFDMEM. Newly established cell
`lines, C146, C168, C170, 223, 224 and 225 are routinely
`passaged
`weekly
`by
`0.025%
`twice
`detachment
`with
`trypsin/EDTA and reseeding in 25cm3 or 75cm3 flasks at
`
`Correspondence: L.G. Durrant.
`Received 30 March 1987; and in revised form, 2 July 1987.
`
`- 106 cells. 277 and 280 are detached by vigorous pipetting
`but are seeded at similar densities to the trypsinised lines.
`
`Indirect immunofluorescence
`Cells were stained by indirect immunofluorescence as previ-
`ously described (Durrant et al., 1984) and analysed on a
`FACS IV (Becton
`Dickinson,
`Sunnyvale,
`Ca., USA).
`Fluorescein fluorescence was excited at 488 nm and collected
`via a 1O nm band with band pass filter centred at 515 nm
`after adjustment for standard conditions using fluorochrome-
`labelled latex beads. Fluorescence intensity, expressed as
`mean linear fluorescence (MLF), was calculated by multi-
`plying the contents of each channel by its channel number
`and dividing by the total number of cells in the distribution
`(Roe et al., 1985). Each cell line was also stained using
`normal mouse Ig, and the MLF in this control was sub-
`tracted from the values obtained with monoclonal antibody.
`The percentage of cells staining was also calculated.
`791T/36 monoclonal antibody recognises a glycoprotein of
`mol. wt. 72,000 which is found in osteogenic sarcomas, colon
`carcinomas and prostate carcinoma (Embleton et al., 1981;
`Price et al., 1983).
`
`Cytotoxicity assays
`
`[75Se]-selenomethionine incorporation assay
`Five thousand
`of IOFDMEM in sterilin M29
`cells were plated in 100y
`ART flat bottomed sterile tissue culture microtitre plates and
`incubated at 37°C for 2-4h to allow cells to become
`adherent. The drug or conjugate at various dilutions was
`added in 1OO,I1 of growth medium. Cells were incubated for
`64 h at 37°C prior to adding 0.1 pl of [75Se]-selenomethionine
`in 50pl of growth medium per well. Cells were incubated for
`a further 8-16 h at 37°C prior to washing gently 3 times with
`PBS,
`drying
`plates,
`spraying
`plastic
`the
`with
`film
`(Nobecutane) separating the wells with a band saw and
`counting the separate wells
`in a gamma counter. The
`surviving fraction was defined as the number of cpm in wells
`containing drug divided by cpm in wells without drug.
`
`IMMUNOGEN 2295, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`SENSITIVITY OF COLORECTAL CELLS TO ANTIBODY DRUG CONJUGATES
`
`723
`
`Clonogenic assay One hundred cells were plated in soft agar
`(Durrant et al., 1986b) in the presence of various dilutions of
`drug or drug antibody conjugates and incubated for 14 days
`prior to counting colonies containing 50 or more cells. The
`surviving fraction was defined by the number of colonies in
`wells containing drugs divided by the number of colonies in
`untreated control wells.
`
`Drug-antibody conjugates
`Methotrexate was obtained from Lederle
`Laboratories,
`Cyanamid, Gosport, Hants. Conjugates were prepared by an
`activated ester method as described previously (Embleton &
`Garnett,
`1985).
`Briefly
`N-hydroxysuccinamide
`of
`ester
`methotrexate was prepared by the method of Kulkarni et al.
`(1981) and reacted with 791T/36 antibody for 1 h. Unreacted
`ester and unwanted small molecular weight products were
`removed by gel filtration on Sephadex G-25 column. A
`conjugate, designated MDC31, had one molecule of 791T/36
`antibody substituted with an average of 2.7 molecules of
`methotrexate and retained 75% of its binding activity
`(Robins et al., 1986). This conjugate was used with cell lines
`C146, C168, C170. A similar conjugate, MDC34, containing
`molecules
`of methotrexate
`antibody
`molecule
`1.9
`per
`retained 84% of its binding activity. This conjugate was used
`with cell lines 223, 224, 225, 277 and 280. 14-bromodauno-
`mycin was kindly provided by Farmitalia Carlo Erba, Italy.
`Conjugates were prepared as described earlier by Gallego et
`al. (1984). Briefly, 791T/36 antibody was mixed with a 25
`molar excess of 14-bromo-daunomycin dissolved in methanol
`for 4h. Following desalting on Sephadex G-25 columns the
`excluded fraction containing conjugate was concentrated by
`dialysis against Aquacide and centrifuged prior to use. Each
`molecule of 791T/36 antibody was substituted with 2-3
`molecules of daunomycin and retained 98% of its original
`binding activity.
`
`Results
`
`Expression of 791Tp72 antigen on newly established
`colorectal cell lines
`Eight primary colorectal tumours have successfully been
`adapted to in vitro culture. Three of these (C146, C168 and
`C170) have previously been characterised in detail (Durrant
`et al., 1986b). Table I shows the clinicopathology of these
`primary tumours and compares the binding of 791T/36
`monoclonal antibody in primary tumours and their in vitro
`culture derived cell lines. All the cell lines expressed 791T-
`p72 antigen. Furthermore 100% of cells growing in early in
`
`vitro culture (passage 5) express the antigen even when the
`primary tumours from which they were isolated had only a
`small weakly positive
`population.
`791T/36 monoclonal
`antibody bound to the cells with varying intensities ranging
`from MLFs 138-454. The level of antigen expression was
`relatively stable in the majority of cell lines (Table II).
`However, expression in line C146 varied from MLF127-
`MLF430 with no obvious correlation with passage number
`or growth characteristics.
`
`Sensitivity of colorectal cell lines to cytotoxic drugs
`All the cell lines were screened by [75Se]-selenomethione
`assays for their
`sensitivity to three cytotoxic drugs,
`5-
`fluorouracil,
`methotrexate and daunomycin (Table
`III).
`
`Table II
`
`Stability of expression of 791T-p72 antigen on colorectal
`cell lines on prolonged in vitro culture
`
`Binding of 791T/36 monoclonal antibody as assessed by indirect
`immunofluorescence (MLF):
`
`Colorectal cell lines
`
`In vitro
`passage no.
`
`5
`20
`50
`100
`150
`
`CJ46 C168 C170
`
`223
`
`224
`
`225
`
`277
`
`280
`
`377
`127
`165
`333
`430
`
`184
`130
`232
`225
`200
`
`454
`300
`278
`153
`138
`443
`NDa ND ND 220
`480
`131
`NAb 398
`185
`356
`460
`180
`396
`180
`164 NA
`187
`178
`200 NA NA NA NA NA
`
`'ND = not done; bNA= not applicable as recently derived cell lines
`have not been in continuous culture for sufficient time.
`
`Table Ill
`
`Cytotoxicity of 5-fluorouracil, methotrexate and dauno-
`mycin to freshly derived colorectal cell lines
`
`Cytotoxicity of drugs as measured by [75Se]-selenomethionine
`incorporation:
`
`IC50 (nM)
`
`Cell lines
`
`5-fluorouracil
`
`Methotrexate
`
`Daunomycin
`
`C146
`C168
`C170
`223
`224
`225
`277
`280
`
`3,800
`3,800
`4,600
`3,800
`2,300
`3,100
`230
`3,700
`
`2,200,000
`2,200,000
`2,200,000
`3,300
`44
`18
`15
`29
`
`5,500
`5,500
`550
`185
`640
`6
`370
`9
`
`Table I
`
`Characteristics of the newly established colorectal cell lines and the tumours
`from which they were derived
`
`Clinicopathology
`
`Patient
`no.
`
`Differ-
`entiationa
`
`Tumour
`gradeb
`
`Binding of 791T/36 as assessed
`by indirect immunofluorescence:
`
`I' tumour
`
`cell line
`
`Site
`
`MLFC % + ve MLF % + ve
`
`C146
`C168
`C170
`223
`224
`225
`277
`280
`
`Adenoma
`M
`M
`M
`M
`W
`M
`P
`
`D
`C
`B
`B
`A
`A
`C
`
`Rectosigmoid
`Sigmoid
`Ascending
`Sigmoid
`Sigmoid
`Rectum
`Hepatic flexure
`
`NEe
`72
`40
`266
`ND
`179
`21
`ND
`
`ND
`ND
`ND
`47
`ND
`51
`3
`ND
`
`377
`184
`153
`443
`454
`138
`278
`300
`
`97
`96
`73
`97
`82
`89
`95
`92
`
`'Tumours were assessed by standard criteria as well (W), moderate (M) or poorly
`(P) differentiated; bTumours were graded by Dukes' classfication (Dukes, 1932)
`metastases; CMLF= Mean linear
`with
`stage D describing
`distant
`fluorescence;
`d% + ve = The percentage
`791T/36;
`of cells
`staining with monoclonal antibody
`eND= Not done.
`
`IMMUNOGEN 2295, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`724
`
`L.G. DURRANT et al.
`
`Seven of the 8 lines had similar sensitivities to 5-fluorouracil
`whereas one was ten times more sensitive (Table III). The
`cell lines varied in their sensitivities to daunomycin, 225 and
`280 were very sensitive whereas C146 and C168 were
`resistant to the cytotoxic effects of daunomycin.
`There was an enormous difference in sensitivity to metho-
`trexate. Three of the lines grew efficiently in 1 mg ml -
`of
`methotrexate whereas other lines were killed by doses as
`small as 7ngml-'. This resistance was also observed when
`C170, C146 and C168 cell lines were assayed by a 14 day
`clonogenic assay (data not shown).
`Resistance to methotrexate was gradually and irreversibly
`lost during continuous culture (Figure 1). Between passages
`0-60 all cell lines were 103 times less sensitive to metho-
`trexate but by passage 200 C146, C168 and C170 cells were
`104-105 times more sensitive to the cytotoxic effects of
`methotrexate. However resistance to daunomycin and 5-
`fluorouracil remained stable (data not shown).
`
`Sensitivity of colorectal cells to 791T/36-drug conjugates
`The instability of the cell lines as regards methotrexate
`sensitivity was not anticipated but as soon as it was observed
`(passage 50) the cell lines were screened for the sensitivity to
`791T/36 conjugated to methotrexate. The C170, C146, C168
`103 fold more reistant than
`cells at this passage were still
`103 fold more resistant than they
`the other cell lines and
`eventually became on further in vitro culturing. Furthermore
`as growing cells
`the loss of resistance was irreversible,
`following exposure to mutagens, in methotrexate failed to
`reverse the trend (unpublished data). C146, C168, C170 cell
`lines were more sensitive to the conjugated methotrexate
`than to the free drug at passage 50-53 (Figure 2). However
`as they continued to lose their resistance to free drug their
`sensitivity to conjugate remained unaltered (Table IV). Cell
`lines which were very sensitive to free drug at early passages
`were less sensitive to 791T/36-methotrexate (Table IV). C170
`cells were injected into nude mice at in vitro passage number
`40 (Durrant et al., 1986b). Following 10 in vivo transplan-
`culture and
`cells were reintroduced to
`tations
`vitro
`in
`immediately assayed for methotrexate and 791T/36 metho-
`trexate sensitivity. These cells had identical sensitivities to
`cells maintained in vitro culture for 50 passages and were
`
`Table IV Sensitivity of a series of colorectal cell lines to 791T/36
`drug conjugates and free drug
`
`Cytotoxicity of drug or conjugates as measured by
`[[75Se]-selenomethionine incorporation:
`
`IC50 ±s.e. (nM)a
`
`Cell lines
`
`Methotrexate
`
`791 T/36-
`Methotrexate
`
`in vitro passage 5-8
`225
`277
`280
`in vitro passage 50-53
`C146
`C168
`C170
`223
`224
`in vitro passage 200-203
`C168
`C170
`
`20+7
`15+2
`26+2
`
`2,140+145
`11,407+2,570
`3,940+370
`46+4
`46+4
`
`18+4
`8+2
`
`432+85b
`447+84
`702+126b
`
`112+24b
`786 +93C
`357+57b
`363+62C
`436+88C
`
`619+90b
`888+ 112b
`
`Cell lines
`
`Daunomycin
`
`791 T/36-Daunomycin
`
`5,500
`550
`
`in vitro passage 50-
`5 Wod
`C168
`500d
`C170
`aIC50 + s.e.
`experiments performed
`are from 3
`at
`separate
`consecutive passages; bp<0.001 when comparing cytotoxicity of free
`drug to conjugated drug; CP<0.01 when comparing cytotoxicity of
`free drug to conjugated drug; dNot significant when comparing
`cytotoxicity of free drug to conjugated drug.
`
`more sensitive to conjugate than free methotrexate (Figure
`3). Only two lines (C168 and C170) were screened with the
`daunomycin-791T/36 conjugate as it was in limited supply.
`Both lines had similar sensitivities to free or conjugated
`daunomycin although one line, C168 was 10 times more
`resistant than C170 (Figure 4).
`
`C146
`
`Cl 68
`
`C170
`
`100l
`
`80 -
`
`60-
`
`40 -
`
`20 -
`
`100
`
`80-
`
`60-
`
`40
`
`20-
`
`E u
`
`S
`+1
`
`16
`
`0
`
`io-8
`
`10-6
`
`io-4
`
`Methotrexate (g ml- ')
`
`0o
`
`I
`
`lo-8
`
`10-616-4
`
`10-8
`
`10-6
`
`10 -4
`
`Methotrexate (g ml-')
`
`Methotrexate (g ml-1)
`
`Loss of resistance of colorectal cell lines C146, C168 and C170 to methotrexate on prolonged in vitro culture. (0)
`Figure 1
`passages 30-40. (A) passages 46-50. (M) passages 60-64. If error bars are not shown it is because they are encompassed by the
`symbol.
`
`IMMUNOGEN 2295, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`SEN'SITIVITY OF COLORECTAL CELLS TO ANTIBODY DRUG CONJUGATES
`
`725
`
`&
`A Methotrexate
`*......4 791T/36 - Methotrexate
`
`C146
`
`C168
`
`C170
`
`80
`
`60
`
`l--
`
`2
`
`1.
`
`I
`
`a
`
`0-
`
`-9
`
`I
`
`I
`
`.,-
`
`7
`
`I
`I
`10-5
`
`I
`io-3
`
`80
`
`20
`
`0
`
`I
`I
`
`Io -9
`
`I
`a
`i o - 7
`
`I
`I
`1o-5
`
`I
`10-3
`
`I
`-9
`
`I
`
`I
`
`I
`
`I
`
`3
`
`I c
`
`7
`
`80
`
`-
`
`60
`
`-
`
`_
`
`2-
`
`E U
`
`,
`
`cu
`CD
`
`+l.'
`
`Methotrexate (g ml-')
`Methotrexate (g ml-')
`Methotrexate (g ml- ')
`In vitro cytotoxicity of free methotrexate (A) and 791T 36 methotrexate conjugate (0) to colorectal tumour cell lines
`Figure 2
`C146. C168 and C170 at passages 50-53.
`
`-c--HC
`
`3c.
`
`E U
`
`,
`
`Cu
`
`en
`
`2
`
`i
`
`9
`
`v
`
`-,
`
`7
`
`---I
`
`5
`
`(I!
`
`I
`
`I
`
`Dau-nom9c 7ml' 5
`Daunomycin (gml-')
`Daunomycin (g ml-')
`In vitro cytotoxicity of free daunomycin (0) and
`Figure 4
`791T 36 daunomycin (0) to colorectal tumour cell lines. C170
`and C168.
`
`100.
`
`80-
`
`60-
`
`2C
`
`E
`
`u
`
`C-,
`
`-W
`
`t ^-9
`
`I-7
`I r)-5
`Methotrexate (g ml- ')
`In vitro cytotoxicity of free methotrexate (A) and
`Figure 3
`791T 36 methotrexate (0) to colorectal tumour cell line C170 at
`passage 50. In vitro cytotoxicity of free methotrexate (A) and
`791T 36 methotrexate (0) to C170 cells injected into nude miice
`at passage 40 and grown as xenografts for 10 passages pnror to
`reintroduction into culture.
`
`I1-3
`
`Discussion
`
`Two major problems are associated with chemotherapy of
`human tumours. vi-., drug resistance and the toxic side
`effects of drugs on normal cells. Previous studies have shown
`that 791T,36 monoclonal antibody localises in the tumours
`of patients with colorectal cancer (Farrands et al., 1983;
`Armitage et al., 1984). It can therefore act as a potential
`
`carrier of drugs to tumour cells and reduce normal drug
`toxicity. This monoclonal antibody also binds to the cell
`surface of colorectal tumour cells (Durrant et al., 1986) and
`may therefore act as a transplasma membrane carrier of
`cytotoxic drugs (Garnett & Baldwin, 1986; Shen et al., 1986).
`This hypothesis has been tested by screening 791T/36 drug
`conjugates on eight new colorectal cell lines with varying
`sensitivities to cytotoxic drugs.
`Daunomycin was the most cytotoxic drug in cell lines
`C170, 223, 225 and 280, methotrexate was the most effective
`drug in cell lines 224 and 277 whereas 5-fluorouracil was the
`most cytotoxic drug in only 2 cell lines, C168 and C146.
`Both of these cell lines were resistant to methotrexate and
`daunomycin. Although 5-fluorouracil is at present the drug
`of choice for chemotherapy of colorectal cancer, the overall
`clinical response rate to this drug is less than 25% and its
`use has not significantly improved the survival of patients
`with large bowel cancer (Davis, 1982; Gilbert, 1982). Only
`25% of the cell lines used in this study were more sensitive
`to 5-fluorouracil than methotrexate or daunomycin. They
`as other
`5-fluorouracil
`sensitivities
`also had similar
`to
`
`IMMUNOGEN 2295, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`726
`
`L.G. DURRANT et al.
`
`colorectal lines developed in other laboratories (Dexter et al.,
`1981; Kimball & Brittain, 1980). These results suggest that
`daunomycin may be
`drug
`better
`choice
`of
`for
`a
`chemotherapy of colorectal cancer.
`Half of the cell lines were resistant to the cytotoxic effect
`of methotrexate. However, these lines were rendered sensitive
`to this drug when it was attached to 791T/36. In fact, all of
`the cell lines had a similar sensitivity to 791T/36-metho-
`trexate despite their varying sensitivities to free drug. This
`supports the concept that monoclonal antibody methotrexate
`conjugates enter the cell by a separate mechanism to the
`normal active transport system for methotrexate uptake
`(Shen et al., 1986). As one of the major causes of metho-
`trexate resistance is defective drug transport (Curt et al.,
`1984), monoclonal antibody methotrexate conjugates should
`be much more effective in therapy of either naturally or
`acquired drug resistant tumours.
`Two of the cell lines which were resistant to daunomycin
`were treated with a 791T/36-daunomycin conjugate. How-
`ever, unlike their response to 791T/36-methotrexate, they
`were as resistant to 791T/36-daunomycin as to free drug.
`Increased active transport of daunomycin out of cells has
`resulted in resistance to this drug (Dano, 1973). Whether free
`drug was taken up by diffusion, or conjugated drug internal-
`
`ised as a monoclonal antibody conjugate, enhanced extra-
`cellular transport would result in similar resistance to free
`drug or monoclonal antibody drug conjugate.
`The methotrexate resistant cell lines became increasingly
`sensitive to this drug in in vitro culture. However, expression
`of 791T p72 antigen,
`sensitivities
`to
`5-fluorouracil and
`daunomycin, and their ability to grow as xenografts in nude
`mice remained unaltered.
`Similarly if the cell
`lines were
`passaged as xenotransplants in nude mice they still lost their
`methotrexate resistant at a similar rate to cells in in vitro
`culture. It must therefore be assumed that the environment
`of the human colon favours the growth of methotrexate
`resistant tumours, whereas in vitro culture or xenotransplan-
`tation in the flanks of nude mice favours the growth of
`methotrexate sensitive tumour cells.
`Monoclonal antibody drug conjugates may not only
`reduce toxic side effects of drugs on normal cells but offer an
`alternative intracellular transport mechanism for cytotoxic
`drugs.
`
`These studies were supported by the Cancer Research Campaign,
`UK. The skilful technical assistance of Mr. O. Roberts is gratefully
`acknowledged.
`
`References
`
`ARMITAGE, N.C., PERKINS, A.C. PIMM, M.V., FARRANDS, P.A.,
`BALDWIN, R.W. & HARDCASTLE, J.D. (1984). The localisation of
`an antitumour monoclonal antibody (791T/36) in gastrointestinal
`tumours. Br. J. Cancer, 71, 407.
`CHATAL, J.F., SACCAVINI, J.C., FUMOLEAU, P. & 4 others (1982).
`Photoscanning localisation of human tumours using radioiodin-
`ated monoclonal antibodies to colorectal carcinoma. J. Nucl.
`Med., 23, 8.
`CURT, G.A., CHENENINN, N.J. & CHABNER, B.A. (1984). Drug
`resistance in cancer. Cancer Treatment Reports, 68, 87.
`DANO, K. (1973). Active outward transport of daunomycin in
`resistant Ehrlich ascites tumor cells. Biochim, Biophys. Acta., 323,
`1466.
`DAVIES, H.L. (1982). Chemotherapy of large bowel cancer. Cancer
`Treat. Rev., 9, 195.
`DEXTER, D.L., SPREMULLI, F.N., FLIGIEL, Z. & 4 others (1981). The
`of cancer cells from a single human colon
`heterogeneity
`carcinoma. Am. J. Med., 71, 949.
`DUKES, C.E. (1932). The classification of cancer of the rectum. J.
`Path. Bact., 35, 323.
`DURRANT, L.G., PARKER, M., KENWORTHY, N. & TAYLOR, G.M.
`(1984). Characterisation of a human x mouse T-cell hybridoma
`and identification of a clone secreting and binding interleukin-2.
`Immunology, 52, 117.
`DURRANT, L.G., ROBINS, R.A., ARMITAGE, N.C., BROWN, A.,
`BALDWIN, R.W. & HARDCASTLE, J.D. (1986a). Association of
`and DNA-ploidy in human colorectal
`expression
`antigen
`tumours. Cancer Res., 46, 3543.
`DURRANT, L.G., ROBINS, R.A., PIMM, M.V. & 4 others (1986b).
`Antigenicity of newly established colorectal carcinoma cell lines.
`Br. J. Cancer, 53, 37.
`EMBLETON, M.J., GUNN, B., BYERS, C.S. & BALDWIN, R.W. (1981).
`Antitumour reactions of monoclonal antibody against a human
`osteogenic sarcoma cell line. Br. J. Cancer, 43, 582.
`EMBLETON, M.J. & GARNETT, M.C. (1985). Antibody targeting of
`anti
`Monoclonal Antibodies for
`Cancer
`agents.
`In
`cancer
`Detection and Therapy, Baldwin, R.W. & Byers, V.S. (eds) p.
`159. Academic Press: London.
`FARRANDS, P.A., PERKINS, A.C., PIMM, M.V., HARDY, J.G.,
`BALDWIN, R.W. & HARDCASTLE, J.D. (1982). Radioimmuno-
`detection of human colorectal cancers using an anti-tumour
`monoclonal antibody. Lancet, ii, 397.
`
`GALLEGO, J. & PRICE, M.R. (1984). Preparation of four daunomycin
`monoclonal antibody
`conjugates
`with
`anti-tumour
`791T/36
`activity. Int. J. Cancer, 33, 737.
`GARNETT, M.C. & BALDWIN, R.W. (1986). Endocytosis of a mono-
`clonal antibody recognising a cell surface glycoprotein antigen
`visualised using fluorescent conjugates. Eur. J. Cell Biol., 41, 214.
`GARNETT, M.C., EMBLETON, M.J., JACOBS, E. & BALDWIN, R.W.
`(1983). Preparation and properties of a drug-carrier-antibody
`conjugate showing selective antibody-directed cytotoxicity
`in
`vitro. Int. J. Cancer, 31, 661.
`(1982). Adjuvant chemotherapy of large bowel
`GILBERT, J.M.
`cancer. Cancer Treat. Rev., 9, 195.
`KIMBALL, P.M. & BRITTAIN, M.G. (1980). Isolation of a cellular
`sub-population from a human colonic carcinoma cell line. Cancer
`Res., 40, 1574.
`(1981). Covalent
`KULKARNI, P.N., BLAIR, A.H. & GHOSE, T.I.
`binding of methotrexate to immunoglobulins and the effect of
`antibody-linked drug on tumour growth in vivo. Cancer Res., 41,
`2700.
`MACH, J.-P., CARREL, S., FORNI, M., KITSCHARD, J., DONATH, A.
`& ALBERTO, P. (1980). Tumour localisation of radiolabelled
`antibodies against carcinoembryonic antigen in patients with
`carcinoma. N. Engl. J. Med., 303, 5.
`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.
`ROBINS, R.A., LAXTON, R.R., GARNETT, M.C., PRICE, M.R. &
`of tumour reactive
`(1986).
`BALDWIN, R.W.
`Measurement
`antibody and antibody conjugate by competition, quantitated by
`flow cytofluorimetry. J. Immunol. Methods, 90, 165.
`ROE, R., ROBINS, R.A., LAXTON, R.R. & BALDWIN, R.W. (1985).
`Kinetics of divalent monoclonal antibody binding to tumour cell
`antigens
`using flow cytometry:
`surface
`Standardization and
`mathematical analysis. Molecular Immunol., 22, 11.
`SHEN, W.-C., BALLOU, B., RYSER, H.J.-P. & HAKALA, T.R. (1986).
`Targeting,
`internalization and cytotoxicity
`of methotrexate-
`stage-specific embryonic antigen-1
`monoclonal anti
`antibody
`conjugates in cultured F-9 terato carcinoma cells. Cancer Res.,
`46, 3912.
`
`IMMUNOGEN 2295, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676