Treatment of Refractory Hodgkin's Disease With an Anti-CD16/CD30 Bispecific Antibody

Fifteen patients with refractory Hodgkin's disease were treated in a phase I/II trial with the natural killer (NK)-cell–activating bispecific monoclonal antibody HRS-3/A9, which is directed against the Fcγ-receptor III (CD16 antigen) and the Hodgkin's-associated CD30 antigen, respectively. Median counts of NK cells and of all lymphocyte subsets were considerably decreased in the patients before therapy. HRS-3/A9 was administered 4 times every 3 to 4 days, starting with 1 mg/m2. The treatment was well tolerated, and the maximum tolerated dose was not reached at 64 mg/m2, the highest dose administered because of the limited amounts of HRS-3/A9 available. Side effects were rare and consisted of fever, pain in involved lymph nodes, and a maculopapulous rash. A total of 9 patients developed human antimouse Ig antibodies, and 4 patients developed an allergic reaction after attempted retreatment. A total of 1 complete and 1 partial remission (lasting 16 and 3 months, respectively), 3 minor responses (1 to 11+ months), and 1 mixed response were achieved. There was no clear-cut dose-side effect or dose-response correlation. Our results encourage further clinical trials with this novel immunotherapeutic approach and emphasize the necessity to reduce the immunogenicity of the murine bispecific antibodies.

CD16+ monocytes in patients with cancer: spontaneous elevation and pharmacologic induction by recombinant human macrophage colony- stimulating factor

The small subset of circulating monocytes that express the maturation-associated CD16 antigen has recently been reported to be elevated in patients with bacterial sepsis. We now show that this novel CD16+ monocyte population is also spontaneously expanded in patients with cancer. We studied 14 patients with metastatic gastrointestinal carcinoma enrolled ina clinical trial of recombinant human macrophage colony-stimulating factor (rhMCSF) plus monoclonal antibody D612. We found that before any cytokine treatment, 12 of 14 patients constitutively displayed significant elevations in both the percentage and the absolute number of CD16+ monocytes, as compared with both normal subjects and ill patients with elevated monocyte counts but without malignancy. CD16+ monocytes accounted for 46% +/- 22% of total monocytes in the patients with cancer versus 5% +/- 3% for controls (P < .01). The increase was not attributable to infection or intercurrent illness and appeared to be associated with the underlying malignancy itself. A similar spontaneous elevation of CD16+ monocytes was observed in 35 of 44 additional patients diagnosed with a variety of other solid tumors. When patients with gastrointestinal carcinoma were treated with rhMCSF, there was a marked further increase in the percentage of CD16+ monocytes (to 83% +/- 11%), as well as in the absolute number of CD16+ cells and the level of CD16 antigen expression. In every case, the patients with cancer showed a greater CD16+ monocyte response than the maximal response obtained in normal volunteer subjects treated witha similar regimen of rhMCSF (n = 5, P < .001), suggesting that the presence of malignancy primed patients for enhanced responsiveness to rhMCSF. We hypothesize that spontaneous expansion of the CD16+ monocyte population may represent a novel biologic marker for a widespread and previously unsuspected host immune response to malignancy.

The novel subset of CD14+/CD16+ blood monocytes is expanded in sepsis patients

Staining with CD14 and CD16 monoclonal antibodies will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the CD14 antigen (CD14++), and a minor population with weak expression of CD14 and expression of the CD16 antigen (CD14+/CD16+ cells). As shown herein, the latter cells account for 45 +/- 22 cells/microL and 9% +/- 5% of the monocytes in healthy control donors (n = 35). In septicemia patients, the CD14+/CD16+ cells can become a major population, with more than 50% of all monocytes in 3 of 18 patients and with more than 500 cells in 4 of 18 cases. There was no correlation of CD14+/CD16+ cells to any clinical parameter except for CD14+/CD16+ percentage and body temperature (P = .013). The CD14++ regular monocytes showed a substantial decrease in CD14 antigen density in 9 of 11 patients. Three-color immunofluorescence shows that the CD14+/CD16+ monocytes in septicemia patients when compared with the CD14++ monocytes exhibit a higher level of class II antigen and a lower level of CD11b and CD33 antigens, consistent with a more mature nature of the CD14+/CD16+ cells. Levels of interleukin-6 (IL-6) were increased in septicemia patients; 3 of 5 patients with high numbers of CD14+/CD16+ cells (> 200/microL) had high levels of IL-6 (> 250/U/mL). These data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as IL-6.

The novel subset of CD14+/CD16+ blood monocytes is expanded in sepsis patients

Staining with CD14 and CD16 monoclonal antibodies will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the CD14 antigen (CD14++), and a minor population with weak expression of CD14 and expression of the CD16 antigen (CD14+/CD16+ cells). As shown herein, the latter cells account for 45 +/- 22 cells/microL and 9% +/- 5% of the monocytes in healthy control donors (n = 35). In septicemia patients, the CD14+/CD16+ cells can become a major population, with more than 50% of all monocytes in 3 of 18 patients and with more than 500 cells in 4 of 18 cases. There was no correlation of CD14+/CD16+ cells to any clinical parameter except for CD14+/CD16+ percentage and body temperature (P = .013). The CD14++ regular monocytes showed a substantial decrease in CD14 antigen density in 9 of 11 patients. Three-color immunofluorescence shows that the CD14+/CD16+ monocytes in septicemia patients when compared with the CD14++ monocytes exhibit a higher level of class II antigen and a lower level of CD11b and CD33 antigens, consistent with a more mature nature of the CD14+/CD16+ cells. Levels of interleukin-6 (IL-6) were increased in septicemia patients; 3 of 5 patients with high numbers of CD14+/CD16+ cells (> 200/microL) had high levels of IL-6 (> 250/U/mL). These data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as IL-6.

Alloimmune neonatal neutropenia due to an antibody to the neutrophil Fc- gamma receptor III with maternal deficiency of CD16 antigen

Antibodies to the neutrophil-specific antigens NA1 and NA2 are associated with alloimmune neonatal neutropenia (ANN), autoimmune neutropenia of childhood, and acute pulmonary transfusion reactions. These antigens have been found to be located on the neutrophil Fc-gamma receptor III (FcRIII). The mother of a child with ANN was found to lack both NA antigens and to produce an antibody that reacted with all normal neutrophils tested. We used maternal antibody and a CD16 monoclonal antibody (MoAb) that has specificity for FcRIII to immunoblot and immunoprecipitate neutrophil membranes of various NA phenotypes. Both antibodies immunoblotted an approximately 40- to 70-Kd glycoprotein (GP) on NA1, NA2-positive membrane, an approximately 40- to 55-Kd GP on NA1-homozygous membranes, and an approximately 55- to 70- Kd GP on NA2-homozygous membranes. Both antibodies also immunoprecipitated a 50- to 80-Kd GP from NA1, NA2-positive cells, a 50- to 60-Kd GP from NA1-homozygous cells, and a 55- to 80-Kd GP from NA2- homozygous cells. To further examine the specificity of the maternal antibody, sequential immunoprecipitation studies were performed using maternal antisera and a CD16 MoAb. After extracts of 125I surface- labeled neutrophils were precleared with maternal serum, CD16 MoAbs no longer immunoprecipitated any GP. Neither the CD16 MoAb nor a rabbit polyclonal antibody specific for FcRIII detected any GP in maternal neutrophil membranes by immunoblotting. Neutrophil FcRIII is a glycosyl- phosphatidylinositol anchored membrane GP as is decay accelerating factor and both are absent from neutrophils of patients with paroxysmal nocturnal hemoglobinuria (PNH). Maternal neutrophil membranes were probed with antibody specific for DAF and an 80-Kd GP was detected. This woman also has had no clinical evidence of PNH. These studies provide further evidence that the NA1 and NA2 antigens are on FcRIII and identify a healthy person whose neutrophils lack not only the neutrophil specific antigens NA1 and NA2 but multiple other epitopes of FcRIII and, therefore, likely lack FcRIII entirely.

Alloimmune neonatal neutropenia due to an antibody to the neutrophil Fc- gamma receptor III with maternal deficiency of CD16 antigen

Antibodies to the neutrophil-specific antigens NA1 and NA2 are associated with alloimmune neonatal neutropenia (ANN), autoimmune neutropenia of childhood, and acute pulmonary transfusion reactions. These antigens have been found to be located on the neutrophil Fc-gamma receptor III (FcRIII). The mother of a child with ANN was found to lack both NA antigens and to produce an antibody that reacted with all normal neutrophils tested. We used maternal antibody and a CD16 monoclonal antibody (MoAb) that has specificity for FcRIII to immunoblot and immunoprecipitate neutrophil membranes of various NA phenotypes. Both antibodies immunoblotted an approximately 40- to 70-Kd glycoprotein (GP) on NA1, NA2-positive membrane, an approximately 40- to 55-Kd GP on NA1-homozygous membranes, and an approximately 55- to 70- Kd GP on NA2-homozygous membranes. Both antibodies also immunoprecipitated a 50- to 80-Kd GP from NA1, NA2-positive cells, a 50- to 60-Kd GP from NA1-homozygous cells, and a 55- to 80-Kd GP from NA2- homozygous cells. To further examine the specificity of the maternal antibody, sequential immunoprecipitation studies were performed using maternal antisera and a CD16 MoAb. After extracts of 125I surface- labeled neutrophils were precleared with maternal serum, CD16 MoAbs no longer immunoprecipitated any GP. Neither the CD16 MoAb nor a rabbit polyclonal antibody specific for FcRIII detected any GP in maternal neutrophil membranes by immunoblotting. Neutrophil FcRIII is a glycosyl- phosphatidylinositol anchored membrane GP as is decay accelerating factor and both are absent from neutrophils of patients with paroxysmal nocturnal hemoglobinuria (PNH). Maternal neutrophil membranes were probed with antibody specific for DAF and an 80-Kd GP was detected. This woman also has had no clinical evidence of PNH. These studies provide further evidence that the NA1 and NA2 antigens are on FcRIII and identify a healthy person whose neutrophils lack not only the neutrophil specific antigens NA1 and NA2 but multiple other epitopes of FcRIII and, therefore, likely lack FcRIII entirely.