Pigment Cell Research Suppl. 2: 109-112 (1992)
Potential of Genetically Engineered Monoclonal Antibodies for Cancer Immunotherapy RALPH A. REISFELD* Department of Immunology, Research Institute of Scripps Clinic, 10666 N. Torrey Pines Road, IMM13 La Jolla, CA, 92037 *This work was supported, in part, by a grant from the National Institutes of Health, CA42508; this is the Research Institute of Scripps Clinic Manuscript #6744-IMM.
A considerable amount of evidence indicates that antigens which are preferentially expressed on the surface of tumor cells can serve as targets for cancer immunotherapy (I). The relatively slow progress in the clinical treatment of most solid tumors provided much of the impetus for this approach to tumor therapy. During the last ten years, it was largely the development of monoclonal antibodies (mAb), combined with rapid advances in recombinant DNA methodologies that accelerated and expanded these research efforts. These technological advances also catalyzed the rapid development of molecularly-defined cytokines and growth factors and thereby advanced new immunologic concepts and rational approaches toward cancer immunotherapy. This article summarizes some recent results obtained in our laboratory with recombinant and mutant monoclonal antibodies directed to human tumor-associated antigens that may lead to approaches that are beneficial for the treatment of melanoma and neuroblastoma. MOUSE/HUMAN CHIMERIC ANTIBODIES The development of human/mouse chimeric mAb was stimulated by two major problems which complicate the critical evaluation of the therapeutic benefits of mouse mAb reactive with human tumor-associated antigens. First, such antibodies are recognized as foreign by the human immune system and thus evoke a human antimouse antibody response (HAMA). Second, depending on isotype, the Fc portion of a murine mAb may not be able to activate human complement and human effector cells as efficiently as the human Fc Portion. These limitations, together with data suggesting that complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) correlate with growth suppression of human tumor xenografts in immunodeficient mice led to the development of chimeric antibodies which usually contain the mouse variable (V) regions joined to human constant (C) regions (2,3,4). The initial focus of our efforts was to construct a
chimeric antibody from a mouse mAb 14.18 directed against ganglioside GD2, which is preferentially expressed on human melanoma and neuroblastoma cells. Several findings suggested GD2 to be a good target antigen for chimeric antibody-mediated tumor cell killing. First, this chemically defined cell surface molecule is expressed at high levels on tumors of neuroectodermal origin (I). Second, lymphocytes that infiltrate primary melanoma and their metastases were reported to express gangliosides (5). In this regard, previous studies indicated that anti-GD2 and anti-GD3 mAb potentiate lymphocyte responses to various stimuli (6,7). Consequently. the potentiation of an immune response to tumor cells may be an additional benefit of the therapeutic application of these two antibodies.
We developed a mouse anti-GD2 mAb 14.18 of IgG3 isotype that mediates lysis of cultured human melanoma and neuroblastoma cells and suppresses the growth of these tumors in athymic mice (8). The hybridoma secreting mAb 14.18 was used to construct the chimeric anti-GD2 mAb ch14.18 by combining cDNA sequences encoding the variable portions of murine 14.18 with the constant regions of the human y l H and n L chains. The chimeric IgG construct was expressed at a very high level of 180 mg/liter in the spent culture fluid of a murine non-IgG-producing hybridoma cell line (4). CHARACTERIZATION OF MAB CH14.18 Direct saturation binding studies with M21 human melanoma cells showed essentially identical binding for ch14.18 and for mouse 14.G2a, the latter being an isotype switch variant of mouse mAb 14.18. The average number of binding sites per M21 cell is 1 . 4 ~ 1 0for ~ 14.G2a and 1 . 5 4 ~ 1 0for ~ ch14.18. The disassociation constants (KD) were also to be essentially the same, i.e. 1 I .9 nM for 14.G2a and 11.2 nM for ch14.18, indicating that these two mAbs bind to GD2 on the surface of M21 melanoma cells with equal affinity. Further studies of cell surface binding of ch14.18 and 14.G2a by indirect immunofluorescence on three human
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melanoma cell lines (M21, A375 and LOX) revealed that both antibodies stain essentially all M21 cells. Although the other two cell lines contain smaller numbers of GD2’ cells, their staining patterns were the same with both mAbs. A comparison of the targeting ability of radiolabeled mAb ch14.18 and 14.G2a to M21 melanoma xenografts in athymic mice indicated identical kinetics of blood clearance. Typically, 12% of the injected dose was found 4 h after injection, a value that decreased to 7% 4 days after injection. Quite similar biodistribution of the two iodinated mAbs was found in the melanoma tumors and a variety of other tissues of these athymic mice (9). The ability of ch14.18 and 14.G2a to mediate cytolysis with human complement was essentially the same when used at antibody concentrations ranging from 50 pg to 50 ng/ml on M21 and A375 melanoma cell targets. As little as I pg/ml of both mAb achieved 78% lysis of M21 cells, while the more resistant A375 cells required 50 pg/ml of either mAb for 32% specific lysis. The only clear-cut difference between mAb ch14. I8 and 14.G2a was observed in their respective abilities to mediate the cytotoxicity of human effector cells (PBMC). Thus, specific antibody-dependent lysis did occur in a dose-dependent fashion when these two mAb were tested against M21 and A375 melanoma targets, at effector/target cell (E/T) ratios from 50/1 to 200/1, and at concentrations ranging from 50 to 0.001 pg/ml. For each blood donor tested, the higher E/T ratio resulted in a more efficient lysis, although the specific lysis achieved varied from donor to donor. The difference in lytic capability among blood donors remained constant over time. However, the effector cells of each donor at each E/T ratio tested were considerably more potent in mediating ADCC with ch14.18 than with 14.G2a. Thus, at every given antibody concentration, ch14.18 mediated a higher specific “Cr release than 14.G2a and the amount of chl4.18 required to mediate specific lysis was from 50-100 fold less than that of 14.G2a. Between 25 and 55% specific lysis of M21 cells was achieved with ch14.18 at concentrations ranging between 10 and 0.5 pg/ml, whereas it required from 50 to 1 pg of 14.G2a/ml to obtain a maximum specific lysis of 14 to 33%. The A375 melanoma cells proved far more resistant to lysis than M21 cells, since at least 100 ng ch14.18 was necessary to achieve significant specific lysis; however, even in this case a smaller amount of ch14.18 than of 14.G2a was sufficient to achieve maximum lysis, indicating that ch14.18 is from 50- to 100-fold more effective in mediating ADCC than its mouse counterpart, 14.G2a (9). Results from recent phase I clinical trials support the premise that ch14.18 will not evoke the type of HAMA responses that were observed in all of nine and seven neuroblastoma patients, respectively, that were treated with mouse mAb 14.G2a in two independent phase I clinical trials (R. Handgretinger, A.L. Yu, personal communications). In fact, none of ten neuroblastoma patients treated thus far with ch14.18 in another phase I clinical trial presented with a HAMA response, although all of these patients showed an anti-idiotype response directed against the mouse variable domains of this antibody (A.L. Yu, personal communication). In another phase I clinical trial of melanoma patients, none of six patients treated with
ch14.18 presented with a HAMA response, while only two of these patients showed an anti-idiotype response (A.F. LoBuglio, personal communication). Although the significance and duration of several complete remissions and partial clinical responses and those of the anti-idiotype responses seen in these clinical trials remain to be critically evaluated, the data obtained thus far indicate that ch14.18 has overcome the HAMA responses evoked in patients by its mouse counterpart 14.G2a. ROLE OF DIFFERENT EFFECTOR CELLS AND CYTOKINES IN CH14.18-MEDIATED LYSIS OF HUMAN NEUROBLASTOMA CELLS Similarly, as with human melanoma target cells, mAb ch14.18 was found to bind human neuroblastoma cells (NMB-7) equally well as 14.G2a, as determined by indirect immunofluorescence. Again similarly, as with melanoma cells, ch14.18 was more effective in mediating the lysis of NMB-7 neuroblastoma cells with peripheral blood mononuclear cells (PBMC) from normal donors at an E/T of 50:1, since less ch14.18 was required than 14.G2a to achieve the same lytic effect (10). Proportionately, similar results were obtained at E/T ratios of 1OO:l and 200:l and also with other human neuroblastoma cells, such as LAN-1 and IMR-32. Similar results, as those obtained in ADCC with PBMC from normal donors, were also seen when such effector cells were isolated from the blood of neuroblastoma patients where again ch14.18 was more effective than 14.G2a (10). When different subpopulations of human PBMC were separated based on their ability to adhere to fibronectin coated surfaces, effective lysis of NMB-7 neuroblastoma cells, coated with ch14.18 was only obtained with nonadherent PBMC at an E/T of 501. A similar pattern of results was also observed at an E/T of 1001 and 25:l. The addition of macrophage colony stimulating factor (M-CSF) (1 000 U/ml) to this assay failed to increase the ability of adherent PBMC to lyse NMB-7 neuroblastoma cells. In ADCC experiments with enriched NK cells from healthy donors (>go% CD56+), ch14.18 proved more effective than 14.G2a, especially at lower antibody concentrations of 5-2.5 ng/pl and lower E/T ratios of 2.5-1.25/1 (10). A comparison of the effectiveness of ch14.18 VS. 14.G2a mediated lysis of NMB-7 cells by either granulocytes or PBMC indicated that granulocytes were most effective. However, at relatively high antibody concentrations (>O. 1 pg/ml) granulocytes were capable of mediating NMB-7 lysis equally well with either ch14.18 or 14.G2a. Only at low antibody concentrations (
Potential of Genetically Engineered Monoclonal Antibodies for Cancer Immunotherapy RALPH A. REISFELD* Department of Immunology, Research Institute of Scripps Clinic, 10666 N. Torrey Pines Road, IMM13 La Jolla, CA, 92037 *This work was supported, in part, by a grant from the National Institutes of Health, CA42508; this is the Research Institute of Scripps Clinic Manuscript #6744-IMM.
A considerable amount of evidence indicates that antigens which are preferentially expressed on the surface of tumor cells can serve as targets for cancer immunotherapy (I). The relatively slow progress in the clinical treatment of most solid tumors provided much of the impetus for this approach to tumor therapy. During the last ten years, it was largely the development of monoclonal antibodies (mAb), combined with rapid advances in recombinant DNA methodologies that accelerated and expanded these research efforts. These technological advances also catalyzed the rapid development of molecularly-defined cytokines and growth factors and thereby advanced new immunologic concepts and rational approaches toward cancer immunotherapy. This article summarizes some recent results obtained in our laboratory with recombinant and mutant monoclonal antibodies directed to human tumor-associated antigens that may lead to approaches that are beneficial for the treatment of melanoma and neuroblastoma. MOUSE/HUMAN CHIMERIC ANTIBODIES The development of human/mouse chimeric mAb was stimulated by two major problems which complicate the critical evaluation of the therapeutic benefits of mouse mAb reactive with human tumor-associated antigens. First, such antibodies are recognized as foreign by the human immune system and thus evoke a human antimouse antibody response (HAMA). Second, depending on isotype, the Fc portion of a murine mAb may not be able to activate human complement and human effector cells as efficiently as the human Fc Portion. These limitations, together with data suggesting that complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) correlate with growth suppression of human tumor xenografts in immunodeficient mice led to the development of chimeric antibodies which usually contain the mouse variable (V) regions joined to human constant (C) regions (2,3,4). The initial focus of our efforts was to construct a
chimeric antibody from a mouse mAb 14.18 directed against ganglioside GD2, which is preferentially expressed on human melanoma and neuroblastoma cells. Several findings suggested GD2 to be a good target antigen for chimeric antibody-mediated tumor cell killing. First, this chemically defined cell surface molecule is expressed at high levels on tumors of neuroectodermal origin (I). Second, lymphocytes that infiltrate primary melanoma and their metastases were reported to express gangliosides (5). In this regard, previous studies indicated that anti-GD2 and anti-GD3 mAb potentiate lymphocyte responses to various stimuli (6,7). Consequently. the potentiation of an immune response to tumor cells may be an additional benefit of the therapeutic application of these two antibodies.
We developed a mouse anti-GD2 mAb 14.18 of IgG3 isotype that mediates lysis of cultured human melanoma and neuroblastoma cells and suppresses the growth of these tumors in athymic mice (8). The hybridoma secreting mAb 14.18 was used to construct the chimeric anti-GD2 mAb ch14.18 by combining cDNA sequences encoding the variable portions of murine 14.18 with the constant regions of the human y l H and n L chains. The chimeric IgG construct was expressed at a very high level of 180 mg/liter in the spent culture fluid of a murine non-IgG-producing hybridoma cell line (4). CHARACTERIZATION OF MAB CH14.18 Direct saturation binding studies with M21 human melanoma cells showed essentially identical binding for ch14.18 and for mouse 14.G2a, the latter being an isotype switch variant of mouse mAb 14.18. The average number of binding sites per M21 cell is 1 . 4 ~ 1 0for ~ 14.G2a and 1 . 5 4 ~ 1 0for ~ ch14.18. The disassociation constants (KD) were also to be essentially the same, i.e. 1 I .9 nM for 14.G2a and 11.2 nM for ch14.18, indicating that these two mAbs bind to GD2 on the surface of M21 melanoma cells with equal affinity. Further studies of cell surface binding of ch14.18 and 14.G2a by indirect immunofluorescence on three human
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melanoma cell lines (M21, A375 and LOX) revealed that both antibodies stain essentially all M21 cells. Although the other two cell lines contain smaller numbers of GD2’ cells, their staining patterns were the same with both mAbs. A comparison of the targeting ability of radiolabeled mAb ch14.18 and 14.G2a to M21 melanoma xenografts in athymic mice indicated identical kinetics of blood clearance. Typically, 12% of the injected dose was found 4 h after injection, a value that decreased to 7% 4 days after injection. Quite similar biodistribution of the two iodinated mAbs was found in the melanoma tumors and a variety of other tissues of these athymic mice (9). The ability of ch14.18 and 14.G2a to mediate cytolysis with human complement was essentially the same when used at antibody concentrations ranging from 50 pg to 50 ng/ml on M21 and A375 melanoma cell targets. As little as I pg/ml of both mAb achieved 78% lysis of M21 cells, while the more resistant A375 cells required 50 pg/ml of either mAb for 32% specific lysis. The only clear-cut difference between mAb ch14. I8 and 14.G2a was observed in their respective abilities to mediate the cytotoxicity of human effector cells (PBMC). Thus, specific antibody-dependent lysis did occur in a dose-dependent fashion when these two mAb were tested against M21 and A375 melanoma targets, at effector/target cell (E/T) ratios from 50/1 to 200/1, and at concentrations ranging from 50 to 0.001 pg/ml. For each blood donor tested, the higher E/T ratio resulted in a more efficient lysis, although the specific lysis achieved varied from donor to donor. The difference in lytic capability among blood donors remained constant over time. However, the effector cells of each donor at each E/T ratio tested were considerably more potent in mediating ADCC with ch14.18 than with 14.G2a. Thus, at every given antibody concentration, ch14.18 mediated a higher specific “Cr release than 14.G2a and the amount of chl4.18 required to mediate specific lysis was from 50-100 fold less than that of 14.G2a. Between 25 and 55% specific lysis of M21 cells was achieved with ch14.18 at concentrations ranging between 10 and 0.5 pg/ml, whereas it required from 50 to 1 pg of 14.G2a/ml to obtain a maximum specific lysis of 14 to 33%. The A375 melanoma cells proved far more resistant to lysis than M21 cells, since at least 100 ng ch14.18 was necessary to achieve significant specific lysis; however, even in this case a smaller amount of ch14.18 than of 14.G2a was sufficient to achieve maximum lysis, indicating that ch14.18 is from 50- to 100-fold more effective in mediating ADCC than its mouse counterpart, 14.G2a (9). Results from recent phase I clinical trials support the premise that ch14.18 will not evoke the type of HAMA responses that were observed in all of nine and seven neuroblastoma patients, respectively, that were treated with mouse mAb 14.G2a in two independent phase I clinical trials (R. Handgretinger, A.L. Yu, personal communications). In fact, none of ten neuroblastoma patients treated thus far with ch14.18 in another phase I clinical trial presented with a HAMA response, although all of these patients showed an anti-idiotype response directed against the mouse variable domains of this antibody (A.L. Yu, personal communication). In another phase I clinical trial of melanoma patients, none of six patients treated with
ch14.18 presented with a HAMA response, while only two of these patients showed an anti-idiotype response (A.F. LoBuglio, personal communication). Although the significance and duration of several complete remissions and partial clinical responses and those of the anti-idiotype responses seen in these clinical trials remain to be critically evaluated, the data obtained thus far indicate that ch14.18 has overcome the HAMA responses evoked in patients by its mouse counterpart 14.G2a. ROLE OF DIFFERENT EFFECTOR CELLS AND CYTOKINES IN CH14.18-MEDIATED LYSIS OF HUMAN NEUROBLASTOMA CELLS Similarly, as with human melanoma target cells, mAb ch14.18 was found to bind human neuroblastoma cells (NMB-7) equally well as 14.G2a, as determined by indirect immunofluorescence. Again similarly, as with melanoma cells, ch14.18 was more effective in mediating the lysis of NMB-7 neuroblastoma cells with peripheral blood mononuclear cells (PBMC) from normal donors at an E/T of 50:1, since less ch14.18 was required than 14.G2a to achieve the same lytic effect (10). Proportionately, similar results were obtained at E/T ratios of 1OO:l and 200:l and also with other human neuroblastoma cells, such as LAN-1 and IMR-32. Similar results, as those obtained in ADCC with PBMC from normal donors, were also seen when such effector cells were isolated from the blood of neuroblastoma patients where again ch14.18 was more effective than 14.G2a (10). When different subpopulations of human PBMC were separated based on their ability to adhere to fibronectin coated surfaces, effective lysis of NMB-7 neuroblastoma cells, coated with ch14.18 was only obtained with nonadherent PBMC at an E/T of 501. A similar pattern of results was also observed at an E/T of 1001 and 25:l. The addition of macrophage colony stimulating factor (M-CSF) (1 000 U/ml) to this assay failed to increase the ability of adherent PBMC to lyse NMB-7 neuroblastoma cells. In ADCC experiments with enriched NK cells from healthy donors (>go% CD56+), ch14.18 proved more effective than 14.G2a, especially at lower antibody concentrations of 5-2.5 ng/pl and lower E/T ratios of 2.5-1.25/1 (10). A comparison of the effectiveness of ch14.18 VS. 14.G2a mediated lysis of NMB-7 cells by either granulocytes or PBMC indicated that granulocytes were most effective. However, at relatively high antibody concentrations (>O. 1 pg/ml) granulocytes were capable of mediating NMB-7 lysis equally well with either ch14.18 or 14.G2a. Only at low antibody concentrations (
