~
Cancer Imrnunol Immunother (1990) 32: 2 9 - 3 7
anceF
mmunology mmunotherapy
© Springer-Verlag 1990
Humoral and eellular responses of coloreetal eaneer patients treated with monoclonal antibodies and interferon y* Herve M. BlottierO, Jean-Yves Douillard2, Hilary Koprowski3, and Zenon Steplewski3 i INSERM U211 Faculte de Medecine, 44 034 Nantes, France 2 Center Rene Gauducheau, 44 034 Nantes, France 3 The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19 104, USA Received 2 January 1990/Accepted 17 April 1990
Summary. Fifteen patients with metastatic gastrointestinal adenocarcinomas were treated with low doses of recombinant human interferon y (rh-IFNy) and a mixture of monoclonal antibodies (mAb) that bind to tumor cells. All antibodies were of the IgG2a isotype and interact with human effector cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Natural killer lysis against K562 cells by peripheral blood mononuclear cells purified from patients' blood was enhanced in all patients at day 3 during IFNy treatment. Monocytes from two patients had increased ADCC levels. Increase in the percentage of monocytes able to bind mouse IgG2a was detected by Fc receptor flow cytometry analysis 24 h after the first IFNy infusion. However, 3 days later, the percentage of fluorescent cells had fallen below baseline levels. The analysis of patients' sera showed that at day 2 after mAb infusion, only 50% of the circulating mouse IgG was immunoreactive, and after 1 week, only traces of immunoreactive mouse IgG were detected. All patients developed a human anti-(mouse Ig) response of IgG, IgM and IgA isotypes, although only low levels of anti-idiotypic antibodies were detected at the time of testing (up to 9 weeks) after mAb infusion. No difference in the IgG subclasses of anti(mouse Ig) antibody was observed between patients treated with mAb and IFNy and patients treated with mAb alone.
Introduction The expression by tumor cells of mAb-defined tumor-associated antigens has been utilized for diagnostic purposes [6, 25] and as a target for immunotherapy [8, 30]. Several mAb have been developed against tumor-associated an-
tigens expressed by colorectal carcinomas [21]. One mAb in particular (CO17-1A) has been used extensively for therapy in patients with gastrointestinal cancer [8, 9, 13, 23, 30, 31, 35, 39]. mAb are known to participate in antibody-dependent cell-mediated cytotoxicity (ADCC) of targer cells depending on their isotype. This process involves the binding of the mouse mAb Fc portion to Fc receptor on effector cells [14, 24]. Human monocytes/macrophages have been shown to express Fc receptors [24] that bind with high-affinity routine IgG2a and to be responsible for tumor cell destruction in ADCC [16, 34]. In addition to its antiviral and anti-proliferative activities [28, 37], IFNyhas been shown to enhance the tumoricidal properties of and Fc receptor expression by human monocytes [1]. A drastic increase in the expression of Fc receptor by effector cells was associated with enhanced ADCC activity [1]. Recent advances in molecular biology and recombinant DNA technology have made recombinant human IFN (rh-IFNy) available in large quantities for clinical trials. Cancer patients have been treated with rh-IFNy in an attempt to generate anti-tumor activity via effector cell activation with [401 and without mAb [11, 38]. In our previous pilot trials of mAb therapy [8, 30], the patients were in advanced stages of disease and offen immunodeficient. In one such triat, rh-IFNy was used to boost the patient's immune system and to enhance tumor cell destruction with mAb. Fifteen patients were treated with a mixture of mAh; the mixture was determined according to the expression of tumor-associated antigen in biopsies, as detected in immunoperoxidase staining. We describe hefe the modifications in the cellular and humoral immune response in these patients during and after the mAb and rh-IFNy treatment course.
Materials and methods * This work was supported by a fellowship from the Minister of Foreign Affairs, France, and National Institutes of Health Grants CA10815, CA25874 and CA21124. Offprint requests to: Z. Steplewski
Patiets. Fifteen patients with metastatic spread of gastrointestinal adenocarcinoma were treated with rh-IFNy (Roussel-UCLAF, France) and mAb (Table 1). At days 1, 2, 3, 4, 7 and 9, 1 x 106 units IFN/m 2 were given i.v. At day 5, 5 x 109- 1 x 1010 peripheral blood mononuclear
30 Table 1. Clinical characteristics of patients a Patient
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sex
F M M F M F M F F F M M F M F
Site primary lesion Peritoneal Ca Rectum Colon Colon Colon Colon Colon Colon Colon Rectum Colon Pancreas Biliary tract Colon Colon
Site metastasis Peritoneal Liver Liver Liver Liver Liver Liver Liver Lung Liver Liver Liver Liver Liver Lung
Number of mAb 3 2 3 2 2 2 3 2 2 3 3 1 3 3 3
Total dose of mAb (mg) 600 400 600 400 400 400 600 400 500 600 550 500 600 500 600
Amount (mg) of 17-1A
CA19-9
BR55-2
GA73-3
200 200 200 200 200 200 150 500 200 200
-
200 200 200 200 200 200 200 200 250 200 200 200 250 200
200 200 200 200 200
200 -
250 200 200 200 250 200
3-month response
Survival (months)
pb P P P P S P P P S S P S P P
9 12 18 5 9 17 7 5 >28 >29 >28 2 28 9 6
a Patients received 1 x 106 U/m 2 recombinant human interferon 7 (rh-IFN7) at days 1, 2, 3, 5 and 6. At day 4, autologous peripheral blood mononuclear cells (PBMC) were incubated with 5 x 106 U IFN, then with a mixture of mAbs and reinfused into the patients b p~ disease progression; S, stable disease
cells (PBMC) were collected by leukapheresis, incubated with 1 x 106 units rh-INF? for 1 h at room temperature and then with a cocktail of mAb for 1 h and the mixture was reinfused into the patient. Six patients ( A - F ) with metastatic spread of gastrointestinal adenocarcinoma, treated with a mixture of mAb but not with rh-IFN 7, served as controls. The two groups of patients were matched for age, immunological status and the amount and type of antibody given.
Cells and sera. Blood samples (50 tal) were collected at days 0, 1, 2, 3, 4, 6, and 8 always preceding each IFN infusion. PBMC from each patient and control healthy donors were isolated on Ficoll/Itypaque gradients [5], frozen and stored in liquid nitrogen. An additional 10 ml patients' blood was collected on the same days and then weekly until the 9th week. Sera were separated flora cell components and kept frozen a t - 3 0 ° C. Monoclonal antibodies. Four mAb were tested for binding in an immunoperoxidase staining assay (Vectastain ABC Kit, Vector Laboratories, Burlingame, Calif) on paraffin-embedded biopsies. CO17-1A [21], CA19-9 [21], BR55-2 [2] and GA73-3 [ 17] were generated at The Wistar Institute, Philadelphia, Pa. All were of the IgG2a isotype, m a h H24B5 (anti-influenza) of the IgG2a isotype was supplied by Dr. Walter Gerhard (The Wistar Institute).
Cytotoxicity assays. RPMI-1640 medium supplemented with 10% fetal bovine serum was used throughout the assays. Spontaneous cytotoxicity was assessed in an 18-h min-release assay using K562 human myeloid leukemia cells [20] as a target for PBMC obtained from patients before and during treatment and separated on Ficoll/Hypaque gradients [5]. Target cells (2 x 106) were centrifuged and 20 gCi 11qn oxine (Amersham, Ill) were added to the pellet. After a 10-min incubation, cells were washed three times and resuspended at 5 x 104 cell/ml. An aliquot (100 gl) was placed in each well of U-bottomed 96-well plates, to which 100 gl PBMC suspension (2.5 × 106 cells/ml) was added. The effectorto-target ratio was 50: 1. Plates were then incubated for 18 h at 37°C in a humidified atmosphere of 5% CO2 and the supematant was collected using a superuatant collection system (Skatron, Lier, N o r w a y ) f o r determination of radioactivity. The percentage of U q n release was calculated as: E-S M-X
x 100
in which E is the radioactivity (cpm) in experimental wells, S is the spontaneous radioactivity release in wells containing target cells only, and M is the maximum radioactivity reiease in wells in which target cells were incubated with Triton X-100. Results are expressed as means of triplicate wells. ADCC was assessed using SW948 (22) or LS180 colon carcinoma cells [29] as targets, and PBMC or monocytes, separated by adherence to gelatin/plasma-coated plastic flanks [ 12], as effectors. In all experiments, 1 x 105 effector cells/wel] were incubated with 10 gg/well mAb CO17l A or BR55-2. MAb H24B5 [anti-(influenza virus)] was used as a control. After an 18-h incubation, the supernatant was collected and radioactivity was counted as described above. The percentage lysis was calculated by subtracting the percentage cytotoxicity obtained with H24B5 from percentage cytotoxicity with CO17-1A or BR55-2 monoclonal antibodies.
Immunofluorescence analysis. To detect the level of Fc receptor expression on the cell sufface, 2.5 x 10 ~ cells were incubated with mAb CO17lA, BR55-2 or H24B5 for 30 min at 0 ° C in phosphate-buffered saline (PBS) containing 0.1% gelatin. Cells were then washed three times and 50~1 fluorescein-isothiocyanate (FITC)-labeled goat anti:[mouse F(ab')2] was added. After 30 min at 4 ° C, cells were washed again three times and resuspended in 0.3 ml medium. Samples were analyzed with a Cytofluorograph 50H (connected to a 2150 data handling system, Ortho Diagnostic, Westwood, Mass). Results are expressed as the percentage of fluorescent cells.
Detection ofcirculating mouse IgG. Circulating mouse IgG was detected in enzyme-linked immunosorbent assay (ELISA) using slight modifications of techniques previously described [4]. Briefly, 100 ~tl goat anti(mouse IgG) (Zymed, Sah Francisco, Calif) diluted 1:1000 in carbonate/bicarbonate buffer pH 9.6 was adsorbed to duplicate wells of microtiter plates (Immulon 2, Dynatech Laboratories, Chantilly, Va). Non specific binding sites were blocked with 2% bovine serum albumin in PBS. Patients' sera diluted 1 : 10 and 1 : 100 were added (50 ~1) to each well and incubated for 1 h at room temperature. Plates were washed, incubated with 50 ~1 alkaline-phosphatase-conjugated goat anti-(mouse IgG) (Zymed) for 1 h, washed again, and 100 ~1 substrate (Sigma, St. Louis, Mo) was added. Absorbance was read at 405 nm in a Titertek Multiscan ELISA reader (Flow Labs, McLean, Va).
31 Table 2. Cytotoxicity of PBMC from normal donors and patients treated with rh-IFNy and mAb
Action
NK lysis of K562 Normal donors Patients
ADCC of LS180 b with mAb CO17-1A Normal donors Patients
ADCC yLS180 with mAb BR55-2 Normal donors Patients
Day
n
Cytotoxicity(%)a
Statistical analysis
Mean _+ SD
Range
8 10 10 10 10 10
27.4a + 10.1 5.7 _+ 6.8 8.0 _+ 6.6 18.6 + 10.5 12.8 _-!- 6.7 13.4 +11.3
11.1-42.0 0.0-21.0 1.3-23.8 2.0-36.8 0.0-20.1 0.0-30.5
P
Cancer Imrnunol Immunother (1990) 32: 2 9 - 3 7
anceF
mmunology mmunotherapy
© Springer-Verlag 1990
Humoral and eellular responses of coloreetal eaneer patients treated with monoclonal antibodies and interferon y* Herve M. BlottierO, Jean-Yves Douillard2, Hilary Koprowski3, and Zenon Steplewski3 i INSERM U211 Faculte de Medecine, 44 034 Nantes, France 2 Center Rene Gauducheau, 44 034 Nantes, France 3 The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19 104, USA Received 2 January 1990/Accepted 17 April 1990
Summary. Fifteen patients with metastatic gastrointestinal adenocarcinomas were treated with low doses of recombinant human interferon y (rh-IFNy) and a mixture of monoclonal antibodies (mAb) that bind to tumor cells. All antibodies were of the IgG2a isotype and interact with human effector cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Natural killer lysis against K562 cells by peripheral blood mononuclear cells purified from patients' blood was enhanced in all patients at day 3 during IFNy treatment. Monocytes from two patients had increased ADCC levels. Increase in the percentage of monocytes able to bind mouse IgG2a was detected by Fc receptor flow cytometry analysis 24 h after the first IFNy infusion. However, 3 days later, the percentage of fluorescent cells had fallen below baseline levels. The analysis of patients' sera showed that at day 2 after mAb infusion, only 50% of the circulating mouse IgG was immunoreactive, and after 1 week, only traces of immunoreactive mouse IgG were detected. All patients developed a human anti-(mouse Ig) response of IgG, IgM and IgA isotypes, although only low levels of anti-idiotypic antibodies were detected at the time of testing (up to 9 weeks) after mAb infusion. No difference in the IgG subclasses of anti(mouse Ig) antibody was observed between patients treated with mAb and IFNy and patients treated with mAb alone.
Introduction The expression by tumor cells of mAb-defined tumor-associated antigens has been utilized for diagnostic purposes [6, 25] and as a target for immunotherapy [8, 30]. Several mAb have been developed against tumor-associated an-
tigens expressed by colorectal carcinomas [21]. One mAb in particular (CO17-1A) has been used extensively for therapy in patients with gastrointestinal cancer [8, 9, 13, 23, 30, 31, 35, 39]. mAb are known to participate in antibody-dependent cell-mediated cytotoxicity (ADCC) of targer cells depending on their isotype. This process involves the binding of the mouse mAb Fc portion to Fc receptor on effector cells [14, 24]. Human monocytes/macrophages have been shown to express Fc receptors [24] that bind with high-affinity routine IgG2a and to be responsible for tumor cell destruction in ADCC [16, 34]. In addition to its antiviral and anti-proliferative activities [28, 37], IFNyhas been shown to enhance the tumoricidal properties of and Fc receptor expression by human monocytes [1]. A drastic increase in the expression of Fc receptor by effector cells was associated with enhanced ADCC activity [1]. Recent advances in molecular biology and recombinant DNA technology have made recombinant human IFN (rh-IFNy) available in large quantities for clinical trials. Cancer patients have been treated with rh-IFNy in an attempt to generate anti-tumor activity via effector cell activation with [401 and without mAb [11, 38]. In our previous pilot trials of mAb therapy [8, 30], the patients were in advanced stages of disease and offen immunodeficient. In one such triat, rh-IFNy was used to boost the patient's immune system and to enhance tumor cell destruction with mAb. Fifteen patients were treated with a mixture of mAh; the mixture was determined according to the expression of tumor-associated antigen in biopsies, as detected in immunoperoxidase staining. We describe hefe the modifications in the cellular and humoral immune response in these patients during and after the mAb and rh-IFNy treatment course.
Materials and methods * This work was supported by a fellowship from the Minister of Foreign Affairs, France, and National Institutes of Health Grants CA10815, CA25874 and CA21124. Offprint requests to: Z. Steplewski
Patiets. Fifteen patients with metastatic spread of gastrointestinal adenocarcinoma were treated with rh-IFNy (Roussel-UCLAF, France) and mAb (Table 1). At days 1, 2, 3, 4, 7 and 9, 1 x 106 units IFN/m 2 were given i.v. At day 5, 5 x 109- 1 x 1010 peripheral blood mononuclear
30 Table 1. Clinical characteristics of patients a Patient
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sex
F M M F M F M F F F M M F M F
Site primary lesion Peritoneal Ca Rectum Colon Colon Colon Colon Colon Colon Colon Rectum Colon Pancreas Biliary tract Colon Colon
Site metastasis Peritoneal Liver Liver Liver Liver Liver Liver Liver Lung Liver Liver Liver Liver Liver Lung
Number of mAb 3 2 3 2 2 2 3 2 2 3 3 1 3 3 3
Total dose of mAb (mg) 600 400 600 400 400 400 600 400 500 600 550 500 600 500 600
Amount (mg) of 17-1A
CA19-9
BR55-2
GA73-3
200 200 200 200 200 200 150 500 200 200
-
200 200 200 200 200 200 200 200 250 200 200 200 250 200
200 200 200 200 200
200 -
250 200 200 200 250 200
3-month response
Survival (months)
pb P P P P S P P P S S P S P P
9 12 18 5 9 17 7 5 >28 >29 >28 2 28 9 6
a Patients received 1 x 106 U/m 2 recombinant human interferon 7 (rh-IFN7) at days 1, 2, 3, 5 and 6. At day 4, autologous peripheral blood mononuclear cells (PBMC) were incubated with 5 x 106 U IFN, then with a mixture of mAbs and reinfused into the patients b p~ disease progression; S, stable disease
cells (PBMC) were collected by leukapheresis, incubated with 1 x 106 units rh-INF? for 1 h at room temperature and then with a cocktail of mAb for 1 h and the mixture was reinfused into the patient. Six patients ( A - F ) with metastatic spread of gastrointestinal adenocarcinoma, treated with a mixture of mAb but not with rh-IFN 7, served as controls. The two groups of patients were matched for age, immunological status and the amount and type of antibody given.
Cells and sera. Blood samples (50 tal) were collected at days 0, 1, 2, 3, 4, 6, and 8 always preceding each IFN infusion. PBMC from each patient and control healthy donors were isolated on Ficoll/Itypaque gradients [5], frozen and stored in liquid nitrogen. An additional 10 ml patients' blood was collected on the same days and then weekly until the 9th week. Sera were separated flora cell components and kept frozen a t - 3 0 ° C. Monoclonal antibodies. Four mAb were tested for binding in an immunoperoxidase staining assay (Vectastain ABC Kit, Vector Laboratories, Burlingame, Calif) on paraffin-embedded biopsies. CO17-1A [21], CA19-9 [21], BR55-2 [2] and GA73-3 [ 17] were generated at The Wistar Institute, Philadelphia, Pa. All were of the IgG2a isotype, m a h H24B5 (anti-influenza) of the IgG2a isotype was supplied by Dr. Walter Gerhard (The Wistar Institute).
Cytotoxicity assays. RPMI-1640 medium supplemented with 10% fetal bovine serum was used throughout the assays. Spontaneous cytotoxicity was assessed in an 18-h min-release assay using K562 human myeloid leukemia cells [20] as a target for PBMC obtained from patients before and during treatment and separated on Ficoll/Hypaque gradients [5]. Target cells (2 x 106) were centrifuged and 20 gCi 11qn oxine (Amersham, Ill) were added to the pellet. After a 10-min incubation, cells were washed three times and resuspended at 5 x 104 cell/ml. An aliquot (100 gl) was placed in each well of U-bottomed 96-well plates, to which 100 gl PBMC suspension (2.5 × 106 cells/ml) was added. The effectorto-target ratio was 50: 1. Plates were then incubated for 18 h at 37°C in a humidified atmosphere of 5% CO2 and the supematant was collected using a superuatant collection system (Skatron, Lier, N o r w a y ) f o r determination of radioactivity. The percentage of U q n release was calculated as: E-S M-X
x 100
in which E is the radioactivity (cpm) in experimental wells, S is the spontaneous radioactivity release in wells containing target cells only, and M is the maximum radioactivity reiease in wells in which target cells were incubated with Triton X-100. Results are expressed as means of triplicate wells. ADCC was assessed using SW948 (22) or LS180 colon carcinoma cells [29] as targets, and PBMC or monocytes, separated by adherence to gelatin/plasma-coated plastic flanks [ 12], as effectors. In all experiments, 1 x 105 effector cells/wel] were incubated with 10 gg/well mAb CO17l A or BR55-2. MAb H24B5 [anti-(influenza virus)] was used as a control. After an 18-h incubation, the supernatant was collected and radioactivity was counted as described above. The percentage lysis was calculated by subtracting the percentage cytotoxicity obtained with H24B5 from percentage cytotoxicity with CO17-1A or BR55-2 monoclonal antibodies.
Immunofluorescence analysis. To detect the level of Fc receptor expression on the cell sufface, 2.5 x 10 ~ cells were incubated with mAb CO17lA, BR55-2 or H24B5 for 30 min at 0 ° C in phosphate-buffered saline (PBS) containing 0.1% gelatin. Cells were then washed three times and 50~1 fluorescein-isothiocyanate (FITC)-labeled goat anti:[mouse F(ab')2] was added. After 30 min at 4 ° C, cells were washed again three times and resuspended in 0.3 ml medium. Samples were analyzed with a Cytofluorograph 50H (connected to a 2150 data handling system, Ortho Diagnostic, Westwood, Mass). Results are expressed as the percentage of fluorescent cells.
Detection ofcirculating mouse IgG. Circulating mouse IgG was detected in enzyme-linked immunosorbent assay (ELISA) using slight modifications of techniques previously described [4]. Briefly, 100 ~tl goat anti(mouse IgG) (Zymed, Sah Francisco, Calif) diluted 1:1000 in carbonate/bicarbonate buffer pH 9.6 was adsorbed to duplicate wells of microtiter plates (Immulon 2, Dynatech Laboratories, Chantilly, Va). Non specific binding sites were blocked with 2% bovine serum albumin in PBS. Patients' sera diluted 1 : 10 and 1 : 100 were added (50 ~1) to each well and incubated for 1 h at room temperature. Plates were washed, incubated with 50 ~1 alkaline-phosphatase-conjugated goat anti-(mouse IgG) (Zymed) for 1 h, washed again, and 100 ~1 substrate (Sigma, St. Louis, Mo) was added. Absorbance was read at 405 nm in a Titertek Multiscan ELISA reader (Flow Labs, McLean, Va).
31 Table 2. Cytotoxicity of PBMC from normal donors and patients treated with rh-IFNy and mAb
Action
NK lysis of K562 Normal donors Patients
ADCC of LS180 b with mAb CO17-1A Normal donors Patients
ADCC yLS180 with mAb BR55-2 Normal donors Patients
Day
n
Cytotoxicity(%)a
Statistical analysis
Mean _+ SD
Range
8 10 10 10 10 10
27.4a + 10.1 5.7 _+ 6.8 8.0 _+ 6.6 18.6 + 10.5 12.8 _-!- 6.7 13.4 +11.3
11.1-42.0 0.0-21.0 1.3-23.8 2.0-36.8 0.0-20.1 0.0-30.5
P
