Clinical Application of Monoclonal Antibody-Drug Conjugates
`for lmmunotargeting Chemotherapy of Colorectal Carcinoma
`
`TOSHIO TAKAHASHI, MD,' TOSHIHARU YAMAGUCHI. MD.' KAZUYA KITAMURA. MD.' HlROSHl SUZUYAMA, MD.'
`MITSUYO HONDA, MD,' TAKASHI YOKOTA, MD.' HITOSHI KOTANAGI, MD,t
`MASAHIRO TAKAHASHI, M0.t AND YOSHIYUKI HASHIMOTO, PHDS
`
`Monoclonal antibody-drug conjugates were applied as a clinical trial for patients who, based on the
`experimental study, had colorectal cancer. Monoclonal antibody A7, from a mouse splenocyte immu-
`nized against human colon cancer, was used as a drug carrier for colon cancer. The anti-cancer drugs
`mitomycin C (MMC) and neocarzinostatin (NCS) were bound covalently to A7 to form the conjugates
`A7-MMC and A7-NCS. The in vitro cytotoxic effects of the conjugates on SW1116 cells were stronger
`than those on free MMC or NCS. The conjugate A7-NCS, when administered to nude mice, brought
`about the highest NCS tumor concentration, whereas normal immunoglobulin G (1s)-NCS distributed
`evenly in all tissues. The conjugates showed a strong antitumor effect on colon cancer transplanted into
`nude mice. Forty-one patients with colorectal cancer, including ten patients with postoperative metas-
`tasis, were given A7-NCS. The immunoperoxidase and drug concentration studies of the resected
`specimens showed that NCS was localized specifically in cancer. Patients receiving the conjugate did
`not experience serious adverse effects. Of the eight patients with postoperative liver metastasis, three
`showed evidence of tumor reduction on computed tomography (a) scan and three claimed pain relief.
`The conjugate did not benefit patients with multiple lung metastasis or peritoneal metastasis.
`Cancer 61 :88 1-888, 1 988.
`
`0 therapy is the detrimental effect of an anti-cancer
`
`NE OF THE MAJOR PROBLEMS with cancer chemo-
`
`drug on normal cells. Targeting chemotherapy is one
`way to overcome such adverse effects. We have applied
`clinically targeting chemotherapy using various drug
`carrier systems. 1-3 Of the various drug carriers, the
`monoclonal antibody against cancer cells seems to be
`the most suitable for drug targeting to cancer cells. Thus,
`we have prepared the monoclonal antibody A7,4 which
`is specific highly to colon and rectum carcinoma, and its
`antibody-drug conjugates. This study describes these
`antibody-drug conjugates as drug camer systems and
`their clinical application for colorectal carcinoma pa-
`tients.
`
`From the 'Department of Surgery, Kyoto Prefectural University of
`Medicine, Kyoto, the *Department of Surgery, Akita University,
`Akita, and the $Pharmaceutical Institute. Tohoku University, Sendai,
`Japan.
`Supported by a Grant-in-aid for Cancer Research from the Ministry
`of Health and Welfare of Japan.
`Address for reprints: Toshio Takahashi, MD, Department of Sur-
`gery, Kyoto Prefectural University of Medicine, Kyoto 602, Japan.
`Accepted for publication September 9, 1987.
`
`Materials and Methods
`Monoclonal Antibody
`The monoclonal antibody A7 was produced by a hy-
`bridoma obtained after the fusion of splenocytes from a
`mouse immunized against human colon carcinoma cells
`and murine myeloma P3.X63.Ag8.653 cells as de-
`scribed previ~usly.~ The monoclonal antibody, which
`belonged to the immunoglobulins (Ig) and GI K, was
`purified from ascites fluid in BALB/c mice by chroma-
`tography on Affi-Gel Protein A (Bio-Rad Laboratory,
`Richmond, CA).
`Mitomycin C and Neocarzinostatin Conjugation to the
`Monoclonal Antibody
`A7-mitomycin C: Mitomycin C ([MMC] Kyowa
`Hakko, Tokyo, Japan) was bound covalently to A7
`(IGgl) by cyanogen bromide as Suzuki de~cribed.~
`Briefly, purified A7 (IgG1) was mixed with cyanogen
`bromide for 10 minutes at room temperature. The pH
`of the mixture was maintained at 1 1 by adding 0.5 mol/l
`of sodium hydroxide. The pH was then adjusted to 7.5
`by adding 5% of acetic acid after adding MMC. The
`mixture was stirred for an additional 24 hours under
`
`88 1
`
`IMMUNOGEN 2314, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`882
`
`CANCER March 1 1988
`
`Vol. 6 I
`
`TABLE I . Clinical Cases of A7-Neocarzinostatin Conjugate
`
`No. of
`patients
`
`Administration
`route
`
`Doses [A7 (mg)/NCS (U)]
`
`I5/ I000
`
`30/2000
`
`4514000
`
`90/6000
`
`3
`-
`
`7
`
`18
`
`3
`
`3
`
`1
`
`7
`
`3
`
`1
`* Fourteen patients had intraoperative administration.
`
`nude mice transplanting human colon cancer C-6 that
`was used as the immunogen of A7. The mice were killed
`4 days after intraperitoneal injection of I2'I-A7 or Iz5I-
`normal-IgG. Blood, tumor, and visceral organs were
`counted for radioactivity. Whole body sections of the
`mice were prepared for autoradiograph.
`The NCS concentration in various tissues of the nude
`mice was measured to demonstrate the localization of
`NCS by the passive hemagglutination inhibition (PHAI)
`method.' The nude mice transplanting C-6 were killed
`on days 4 and 8 after intraperitoneal injection of A7-
`NCS (A7: 1 mg; NCS: 150 units), normal IgG-NCS
`(IgG: I mg; NCS: 150 units), or free NCS ( I50 units).
`To investigate antitumor effect, nude mice were in-
`jected with A7 or one of its conjugates or free drugs 7
`days after C-6 transplantation. Tumor volume was eval-
`uated by multiplying one half of the length of the long
`axis by double the length of the short axis after injection
`of the drugs.
`
`Clinical Applications
`
`Forty-one patients with colon and rectum carcinoma,
`including eight patients with postoperative liver metas-
`tasis, one with postoperative lung metastasis, and one
`with postoperative peritoneal metastasis, were given
`A7-NCS. The conjugate was administered intraarter-
`ially to 39 patients, intravenously to one patient with
`lung metastasis, and intraperitoneally to one patient
`with peritoneal metastasis. Of the patients given the
`conjugate intraarterially, 14 were given the conjugate
`intraoperatively from the artery proximal to the tumor.
`and others were given the conjugate by introducing the
`catheter inserted from the femoral artery to the tumor
`artery. The conjugate dose was 15 to 90 mg of antibody
`and 1000 to 6000 units of NCS. Thirty-eight patients
`received the conjugate once and three patients received
`it twice (Table 1).
`All patients were given A7-NCS first. Then, 30 pa-
`tients with primary carcinoma of the colon or rectum
`
`Target lesion
`
`Colon and rectum carcinoma
`(primary)
`Liver metastasis
`(postoperative)
`Lung metastasis
`(postoperative)
`Peritoneal metastasis
`(postoperative)
`
`31
`
`8
`
`1
`
`1
`
`IA"
`
`IA
`
`IV
`IP
`
`A7-NCS: A7-neocarzinostatin: IA: intraarterially; IV: intrave-
`nously; IP: intraperitoneally.
`
`nitrogen at room temperature in the dark. The products
`were fractionated on a Sepharose 6B (Pharmacia, Upp-
`sala, Sweden) column. The extent of substitution in this
`conjugate (A7-MMC) was about 2 mol of drug per mol
`of A7.
`A7-neocarzinostatin: The A7 was conjugated with
`neocarzinostatin ([NCS] Kayaku. Tokyo, Japan) as Fu-
`kuda described.6 The NCS was incubated first with a
`four-fold molar excess of N-succinimidyl 3-(2-
`pyridyldithi0)-propionate (SPDP) in a 0. I mol/l phos-
`phate buffer (pH. 6.5) at 25°C for 30 minutes. The 3-(2-
`pyridyldithi0)-propionated (PDP) NCS was reduced
`with 10 mmol of dithiothreitol (DTT) in a 0.1 mol/l
`acetete buffer (pH, 4.5) at 25°C for 30 minutes. The
`resulting thiol group-introduced NCS(HS-NCS) was
`passed immediately through a Sephadex G-25 (Pharma-
`cia, Uppsala, Sweden) column. The A7 was incubated
`with a ten-fold molar excess of SPDP in a 0.1 mol/l
`phosphate buffer (pH, 6.5) at 25" C for 30 minutes. The
`PDP was purified by gel filtration on a Sephadex G-25
`column. Finally, the PDP was mixed with a six-fold
`molar excess of HS-NCS and allowed to stand at 25°C
`overnight in the dark. Then, the mixture was applied to
`a Sephacryl S-200 (Pharmacia, Uppsala, Sweden) col-
`umn equilibrated with phosphate-buffered saline (PBS)
`(pH, 6.0) and eluted with the same buffer. The peak
`fractions were pooled, concentrated, sterilized, and
`stored at -20" C in the dark. The conjugation ratio was
`2 to 3 mol of NCS per mol of A7.
`I.Jspc.rirn cti t a 1 St iidirs
`In vitro study: The in vitro cytotoxicity of A7-MMC
`and A7-NCS was measured by incubation target cells
`(SW 1 1 16) and in a range of conjugate concentrations in
`microtiter plates at 45 minutes after washing them for
`48 hours and measuring tumor cell survival by trypan
`blue exclusion.
`In vivo study: The localization of A7 was investigated
`using iodine 125 (1251)-radiolabeled A7 or "%radiola-
`beled normal mouse IgG in BALB/c (nu/nu) athymic
`
`IMMUNOGEN 2314, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`No. 5
`
`MONOCLONAL ANTIBODY-DRUG CONJUGATES FOR COLORECTAL CA
`
`- Takahashi et al.
`
`883
`
`\
`\
`
`\, Free NCS
`
`I
`I
`
`L
`
`0.01
`
`I
`0.1
`
`I
`1
`
`I
`10
`
`(U/ml)
`Neocarzinostatin (NCS)
`FK. 2. Cytotoxic effect of A7-NCS on SW 1 1 16 cells. The dose of
`NCS that killed 50% ofSW I 1 16 cells was 0.059 U/ml for A7-NCS and
`0.22 U/ml for free NCS.
`
`compared with 0.57:l for normal IgG. The I2%A7
`levels in other normal organs were comparable with
`those of 1251-normal IgG.
`Whole body sections of the nude mice bearing colon
`cancer C-6 were prepared for autoradiograph 4 days
`after intraperitoneal injection of 12'I-A7. As shown in
`Figure 4, '"LA7 was localized specifically in cancer,
`whereas there was little localization in normal organs.
`Concentration of NCS in the tissues of nude mice:
`NCS was not detected in all tissues at the fourth and
`eighth days after free NCS injection. The A7-NCS gen-
`erated the highest concentration of NCS in the tumor on
`
`2.
`
`1-
`
`0 .-
`c d
`
`0
`0
`0
`B
`7
`i=
`
`9)
`
`FIG. 3. Tissue blood ratio of '"I 4 days after intraperitoneal injection
`of "%A7 or normal lzJI-IgG into nude mice bearing the colon cancer
`I2'1-A7; 0 'z51-normal mouse I&
`(mean ? SE).
`C-6.
`
`I
`
`0.01
`
`1
`I
`0.1
`1
`Mitomycin C (pglrnl)
`FIG 1. Cytotoxic effect of A7-MMC on SW 1 I 16 cells. The dose of
`MMC that killed 50% of the SW1 I16 cells was 0.14 figlml for A7-
`MMC and 10.8 pglrnl for free MMC.
`
`I
`10
`
`underwent surgery for resection of the carcinoma. One
`patient with postoperative liver metastasis underwent
`right hepatectomy of the liver. The remaining ten pa-
`tients with recurrent carcinoma were observed without
`surgery or any other chemotherapy.
`The resected specimens were investigated with immu-
`noperoxidase staining using antibody against NCS for
`evaluation of NCS localization in carcinoma and nor-
`mal tissues. The NCS concentration of the resected spec-
`imens was measured in six colon patients who were
`given A7-NCS (A7: 60 mg; NCS 4,000 units) intraar-
`terially.
`Patients given A7-NCS were followed 5 to 36 months
`after the treatments with serial checks of laboratory data
`and computed tomography (CT) scans.
`
`Results
`
`Experimental Study
`In vitro study: The cytotoxic effect of A7-MMC on
`SW 1 1 16 was much stronger than that of free MMC (Fig.
`1). The dose that killed 50% of SW 11 16 was 0.14 pg/ml
`and 10.8 pg/ml, respectively, for A7-MMC and free
`MMC (ie., A7-MMC showed a 77 times stronger cyto-
`toxic effect than free MMC).
`A7-NCS also was more effective than free NCS (Fig.
`2). The dose that killed 5000 of the target cells was 0.059
`U/ml and 0.22 U/ml, respectively, for A7-NCS and
`free NCS.
`In vivo study
`Monoclonal antibody A7 localization in athymic
`nude mice: There was accumulation of "'LA7
`in colon
`cancer transplanted into nude mice, compared with
`1251-normal mouse IgG and other organs (Fig. 3). The
`tissue-blood ratio of '''I
`in cancer was 2.03:l for A7,
`
`IMMUNOGEN 2314, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`884
`
`CANCER March I 1988
`
`Vol. 61
`
`study of resected specimens showed that NCS was local-
`ized specifically in cancer cells (Fig. 8).
`The NCS concentration in the tumor was higher than
`that of normal mucosa of the colon in all the patients
`given A7-NCS 1 to 72 hours before tumor removal
`(Table 2).
`Of the eight patients with postoperative liver metas-
`tasis who received A7-NCS through the hepatic artery,
`four responded favorably to the conjugate (Table 3).
`Three patients showed evidence of tumor reduction on
`CT scan. Three patients claimed pain relief. Four pa-
`tients died of cancer progression 8 to 25 months after the
`treatment. Three patients are alive for 12 to 26 months
`after treatment with liver metastasis, and two patients
`have had stable liver tumors without regrowth for 12
`and 26 months after treatment, respectively. One pa-
`tient, subjected to right hepatectomy, is alive without
`liver metastasis 10 months after treatment.
`Figure 9A shows the CT scan of a patient with liver
`metastasis 5 years after surgery for colon carcinoma.
`This patient was given A7-NCS (A7: 90 mg; NCS: 6000
`units) once through the hepatic artery. The patient had
`had a temperature of 38.0”C for 3 days after injection.
`The CT scan showed a 67% tumor reduction 3 weeks
`later (Fig. 9B). The patient is alive and in good condition
`26 months after the treatment, without progress of the
`liver metastasis.
`A patient with multiple lung metastasis died of the
`metastasis 1 month after treatment. A patient with peri-
`toneal metastasis died of the metastasis 3 months after
`treatment. Therefore, the conjugate was of no benefit to
`these patients.
`The clinical course of 3 1 patients with primary carci-
`noma of the colon and rectum resected surgically was
`uneventful. The follow-up study is now camed out in
`serial observations.
`
`Colon
`Kidney
`Tumor
`Liver
`Lung
`FIG. 5. Drug concentration (U/g) of NCS in the tissues after injection
`of A7-NCS and normal mouse IgG-NCS. Left on fourth day, right
`on eighth day: 0 left on fourth day, right on eighth day.
`
`FIGS. 4A AND 4B. Autoradiograph of nude mouse bearing C-6 4
`days after intraperitoneal injection of 12’I-A7. (A) Arrows indicate the
`subcutaneously transplanted C-6 in the nude mouse (B) Black grains
`seen at the same site indicate the accumulation of ‘*%A7 at the C-6.
`
`both the fourth and eighth days after injection, whereas
`normal IgG-NCS distributed evenly in the tissues of
`nude mice (Fig. 5).
`Effect of conjugates on tumors transplanted into nude
`mice: Figure 6 shows the tumor volume after intraperi-
`toneal injection of A7 (6mg/kg), A7-MMC (A7: 6mg/
`kg; MMC: 30 pglkg), and free MMC (30 pglkg) twice a
`week with untreated control. The A7 alone had no effect
`on tumor growth. The A7-MMC had the most favor-
`able effect with a T/C (treated/control) of 0.35, com-
`pared with free MMC and free A7 that had a T/C of 0.83
`and 0.8, respectively.
`Figure 7 shows the tumor volume after intravenous
`injection of A7 (3.75 mglkg), A7-NCS (A7: 3.75 mg/kg;
`NCS: 500 units), and NCS (500 U/kg) twice a week with
`untreated control. The A7-NCS exhibited an inhibitory
`effect on tumor growth with a T/C of 0.22, whereas NCS
`alone was toxic but had no effect.
`Clinical application of A7-NCS for patients with
`colon and rectum carcinoma: An immunoperoxidase
`
`IMMUNOGEN 2314, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`No. 5
`
`MONOCLONAL ANTIBODY-DRUG CONJUGATES FOR COLORECTAL CA
`
`- Takahashi et al.
`
`885
`
`T
`
`There were no serious adverse effects on the patients
`who received the conjugate, regardless of the adminis-
`tration route. Of the 41 patients, a fever greater than
`38.0"C was the most common adverse effect and was
`evident in 20 patients. Other side effects included leu-
`kocytosis (leukocyte count of greater than lO,OOO/pl) in
`five patients, slight pain at the portion of tumor region
`and eruption at the site of intracutaneous test injection
`in two patients, and slight hypotension with a systolic
`pressure of 100 in one patient.
`
`Discussion
`
`There have been reports*-'* on the successful applica-
`tion of the murine monoclonal antibody alone in the
`immunotherapy of patients with tumors. However,
`there is little information on the clinical application of
`monoclonal antibody-drug conjugates. Our investiga-
`tions with the monoclonal antibody and its drug conju-
`gates demonstrated that A7, which we prepared for
`human colon carcinoma, can be used effectively to de-
`
`T Ir
`
`2-
`
`1-
`
`I
`I
`
`A
`
`I
`
`i
`
`: 4
`i
`
`4
`i 4
`
`4
`
` 4
`21
`
`4
`
`
`28
`
`Days after Tumor Inoculation
`FIG. 6. Tumor volume after intraperitoneal injection. p A7 (6
`mglkg); $ MMC (30 pg/kg);
`A7-MMC (A7: 6 mg/kg; MMC: 30
`pg/kg); and p saline twice a week (mean -t SE [n = 51).
`
`i.v.
`4
`7
`
`4
`
`+
`
` 4
`14
`
`1
`+
`21
`
`4
`
`
`28
`
`35
`
`Days after Tumor Inoculation
`FIG. 7. Tumor volume after intravenous injection. 6 A7 (3.75
`mg/kg); 5 NCS (500 U/kg);
`A7-NCS (A7; 3.75 mg/kg; N C S 500
`U/kg); and 3 saline twice a week (mean ? SE [n = 51).
`
`liver drugs to human colon and rectum carcinomas in
`both nude mice and patients.
`The A7 was localized specifically to human colon car-
`cinoma transplanted into nude mice. This suggested
`that A7 was a suitable drug camer to colon cancer. In
`our previous study,13-14 A7 alone had no cytotoxic effect
`on S W l l l 6 cells in vitro. Herlyn et al." reported that
`IgG1, IgG2b, IgM, and IgA isotypes were ineffective
`against colorectal carcinomas when used as monoclonal
`antibodies alone, compared with IgG2a and IgG3. Be-
`cause A7 belongs to the IgGl subclass that has no effect
`on cancer, we prepared antibody-drug conjugates for
`targeting chemotherapy of colorectal carcinomas.
`The MMC and NCS were bound covalently to the A 7 .
`For targeting immunochemotherapy, it is essential that
`the conjugate retains the activities of both the antibody
`and the drug. In our previous study: A7-MMC retained
`antibody activity but reduced the antimicrobial activity
`of MMC to only 5% of its spectrophotometrical value.
`This was due presumably to binding with one of the
`three active amino radicals of MMC for can~er'~-'' to
`the Fc portion of the a n t i b ~ d y . ~ Despite such drug activ-
`ity reduction, the cytotoxic effect of A7-MMC on
`
`IMMUNOGEN 2314, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`886
`
`CANCER March I 1988
`
`Vol. 61
`
`FIG. 8. Immunoperoxidase staining (ABC method) of NCS using antibody against NCS in the specimen of colon carcinoma.
`
`SW1116 cells was 77 times greater than that of free
`MMC. The antitumor effect of A7-MMC on trans-
`planted colon carcinoma in nude mice was greater sig-
`nificantly than that of free MMC, but it was not much
`greater than its effect in vitro.
`However, A7-NCS retained the activities of both the
`antibody and the drug completely. The NCS is a pro-
`teinaceous anti-cancer antibiotic with a molecular
`weight of 10,700.18-21
`It consists of a chromophore hav-
`
`TABLE 2 . Concentration of Neocarzinostatin in Resected Specimens
`
`Drug concentration
`
`Patient
`no.
`
`-
`I
`I
`3
`c'
`3
`i
`4
`b
`5
`S
`6
`9
`
`Tumor
`
`30.72
`ZL'OE
`7.68
`89'L
`30.72
`ZL'OE
`3.84
`P8'E
`3.84
`t8'f
`30.72
`i L.OE
`
`Normal
`mucosa
`
`0.00
`000
`I .07
`Z0 1
`1.02.
`ZO' I
`0.96
`96'0
`I .02
`ZO' 1
`3.84
`P8"i
`
`Administration
`time
`
`~
`
`~~
`
`lntraoperative
`2 d preoperative
`3 d preoperative
`2 d preoperative
`3 d preoperative
`In traoperative
`
`ing anti-cancer activity and an apoprotein that makes it
`
` table.'^-^^ J ~ n g ~ ~ reported the method of coupling the
`apoprotein of NCS to the monoclonal antibody after
`dividing it into chromophore and apoprotein, to pre-
`serve the activities of both the drug and the antibody. In
`this study, the A7 was bound to the apoprotein without
`any reduction in drug activity by the SPDP method in
`which NCS was not divided before conjugation. The
`cytotoxic effect of A7-NCS on SW 1 1 16 was about four
`times greater than that of free NCS. The growth of colon
`carcinomas transplanted into nude mice was suppressed
`significantly.
`To ensure that the binding of the conjugate to the
`target cells was specific, the inhibition of the binding of
`A7-NCS by free A7 was evaluated (data not shown).
`The cytotoxicity of A7-NCS on SWl116 was reduced
`by the addition of free A7. This indicates that the cyto-
`toxicity of A7-NCS is related directly to its antibody-
`binding ability.
`To demonstrate the localization of the conjugates in
`tissues, we should have radiolabeled the conjugates.
`
`IMMUNOGEN 2314, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`No. 5
`
`MONOCLONAL ANTIBODY-DRUG CONJUGATES FOR COLORECTAL CA
`
`- Takahashi et a/.
`
`887
`
`TABLE 3. Patients With Postoperative Liver Metastasis Receiving A7-Neocarzinostatin
`
`Patient
`
`1
`2
`
`3
`
`4
`5
`
`6
`7
`
`8
`
`Age
`(Yd
`
`72
`66
`
`71
`
`51
`75
`
`61
`65
`
`28
`
`Sex
`M
`M
`
`M
`
`F
`M
`
`F
`M
`
`F
`
`A7-NCS: A7-nea‘arzinostatin.
`
`A7/NCS
`dose*
`
`30/2,000
`30/2,000
`
`15/1,000 x 2
`
`15/1,000
`90/6,000
`
`30/2,000
`90/6,000
`
`90/6,000
`
`Liver metastask
`changes
`
`Clinical
`features
`
`Pain relief
`
`No change
`Reduced CT level;
`regrowth 10 mo
`later
`58% reduction;
`regrowth 2 mo
`later
`No change
`67% reduction;
`remains stable
`No change
`45% reduction;
`remains stable
`Right
`hepatectorny
`* A7 (mg)/NCS (U).
`
`Pain relief
`
`Pain relief
`
`Side effect
`
`Fever ( 1 d)
`Fever (2 d)
`
`FOIIOW-UP
`
`Dead ( 18 mo)
`Alive (13 mo)
`
`Fever (2 d)
`
`Dead (8 mo)
`
`No
`Hypotension
`fever (3 d)
`No
`Fever ( I d)
`
`No
`
`Dead(l2mo)
`Alive (26 mo)
`
`Dead (25 mo)
`Alive ( I2 mo)
`
`Alive (10 mo)
`
`However, we failed to obtain them without losing their
`activities. Therefore, the NCS concentration in the tis-
`sues was measured to demonstrate the localization of
`A7-NCS compared with normal IgG-NCS. The A7-
`NCS generated the highest concentration of NCS in the
`tumor, whereas normal IgG-NCS distributed evenly in
`the tissues. The result was comparable with the localiza-
`tion of A7 and suggested that A7 delivered NCS to the
`tumor specifically.
`Based on these results, we have applied the antibody-
`drug conjugate to the patients with colon and rectum
`carcinoma. The A7-MMC, which exhibited a strong
`antitumor effect on colon carcinoma, was the first can-
`didate for the treatment of patients. However, it was
`dificult to prepare a large enough dose of MMC for
`clinical use.” Thus, we selected A7-NCS, which has a
`practical drug dose, for clinical use to antibody in the
`conjugation ratio although antitumor effect was weaker
`than that of A7-MMC.
`The 4 1 patients with colorectal carcinoma, including
`ten postoperative patients, were given A7-NCS. Thirty-
`nine patients were given an intraarterial injection that
`seemed to have a less serious adverse effect compared
`with the systemic administration of anti-cancer agents.
`There were no serious side effects in any of the patients
`receiving the conjugate. The most common side effect of
`the conjugate was fever, but it was difficult to differen-
`tiate it from postoperative fever in the patients receiving
`the conjugate intraoperatively.
`To identify the NCS in resected specimens, studies of
`an immunoperoxidase using an antibody against NCS26
`and a NCS concentration were performed. The studies
`showed that NCS was localized specifically in cancer.
`Although the studies do not indicate immediately that
`NCS conjugated to the antibody, such a high concentra-
`tion of NCS may indicate that it is still conjugated to the
`
`antibody because the reduction of half of the free NCS
`injected in the blood vessel occurs in 6 rninute~.~’
`Of the eight patients with postoperative liver metas-
`
`FIGS. 9A AND 9B. (A) ff Scan of the patient with liver metastasis 5
`years after surgery for colon carcinoma. (B) 67% liver metastasis re-
`duction in the area on CT scan 3 weeks after administration of A7-
`NCS (A7: 90 mg; NCS: 6000 U).
`
`IMMUNOGEN 2314, pg. 7
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`

`888
`
`CANCER March 1 1988
`
`Vol. 61
`
`tasis, three showed evidence of tumor reduction on CT
`scan and three claimed pain relief. These effects may be
`ascribed to the accumulation of NCS in metastatic
`cancer cells, because there has been no evidence that free
`NCS can reduce tumor size at a dose as small as 2000 to
`6000 units by any administration route. However. the
`conjugate did not benefit patients with either multiple
`lung metastasis or peritoneal metastasis.
`To evaluate the conjugate for patients with carcinoma
`resected surgically, further studies with large samples
`and randomized control are needed. These studies may
`indicate the possible clinical application of antibody-
`drug conjugates for human cancer to a new approach for
`the targeting of chemotherapy.
`
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`IMMUNOGEN 2314, pg. 8
`Phigenix v. Immunogen
`IPR2014-00676
`
`