Jean E. Surfus

Phase I Clinical Trial of the Immunocytokine EMD 273063 in Melanoma Patients

PURPOSE To evaluate the safety, toxicity, in vivo immunologic activation, and maximum-tolerated dose (MTD) of EMD 273063 (hu14.18-IL-2) in patients with metastatic melanoma. PATIENTS AND METHODS Thirty-three patients were treated with EMD 273063, a humanized anti-GD2 monoclonal antibody (mAb) linked to interleukin-2 (IL-2). EMD 273063 was given as a 4-hour intravenous infusion on days 1, 2, and 3 of week 1. Patients with stabilization or regression of disease could receive a second course of treatment at week 5. Dose levels evaluated were 0.8, 1.6, 3.2, 4.8, 6.0, and 7.5 mg/m2/d. RESULTS Nineteen of 33 patients completed course 1 with stable disease and went on to receive course 2. Eight patients had stable disease on completion of course 2. Grade 3 adverse events included hypophosphatemia (11 patients), hyperglycemia (three patients), hypotension (two patients), thrombocytopenia (one patient), hypoxia (three patients), elevated hepatic transaminases (two patients), and hyperbilirubinemia (one patient). Opioids were required for treatment-associated arthralgias and/or myalgias during 17 of 52 treatment courses. No grade 4 adverse events were observed. Dose-limiting toxicities at the MTD included hypoxia, hypotension, and elevations in AST/ALT. Grade 3 toxicities were anticipated based on prior studies of IL-2 or anti-GD2 mAbs, and all resolved. Immune activation was induced, as measured by lymphocytosis, increased peripheral-blood natural killer activity, and cell numbers, and increased serum levels of the soluble alpha chain of the IL-2 receptor complex. CONCLUSION Treatment with the immunocytokine EMD 273063 induced immune activation and was associated with reversible clinical toxicities at the MTD of 7.5 mg/m2/d in melanoma patients.

Anti-renal-cell carcinoma chimeric antibody G250 facilitates antibody-dependent cellular cytotoxicity with in vitro and in vivo interleukin-2-activated effectors

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy, whereas treatment with biologics has achieved limited success. Although monoclonal antibodies able to recognize human RCC have been identified, most induce little complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC), and thus are of limited potential as therapeutic modalities in their natural conformation. We evaluated a human/ mouse chimeric derivative of the previously described G250 murine monoclonal antibody (mAb), reactive with RCC, to identify a reagent for potential immunotherapy. This chimeric antibody (ch-G250) is composed of the murine variable region from the G250 mAb, which recognizes a tumor-associated antigen expressed on 95% of primary and 86% of metastatic renal cell carcinomas. The constant region of the ch-G250 is comprised of the human IgG1 isotype domains. This chimeric antibody does not bind to normal renal tissue or other normal human tissues, with the exception of gastric mucosal cells and large bile-duct epithelium. Clinical radiolocalization studies have demonstrated the relative tumor-targeting potential of this radiolabeled antibody. This ch-G250 antibody facilitated potent ADCC against several RCC lines when using in vitro and in vivo interleukin-2 (IL-2)-activated peripheral blood mononuclear cells obtained from healthy control donors and patients with cancer, respectively. This lymphocyte-mediated ADCC was specific for RCC cells recognized by the ch-G250 antibody. Using flow cytometry, we found that the level of ADCC was directly related to the degree of binding of ch-G250 to the renal cell target. These in vitro data suggest that this antibody may improve efficacy of IL-2 therapy by targeting cytokine-activated effector cells directly to the tumor and facilitating in vivo ADCC. Clinical studies combining this chimeric antibody with IL-2 treatment will be needed to test the antitumor effects of this ADCC effect in vivo.

Pharmacokinetics and stability of the ch14.18-interleukin-2 fusion protein in mice.

Abstract The fusion protein formed from ch14.18 and interleukin-2 (ch14.18–IL-2), shown to exhibit antitumor efficacy in mouse models, consists of IL-2 genetically linked to each heavy chain of the ch14.18 chimeric anti-GD2 monoclonal antibody. The purpose of this study was to determine the pharmacokinetics of ch14.18–IL-2 in mice and assess its stability in murine serum. Following i.v. injection, the fusion protein was found to have a terminal half-life of 4.1 h. Detection of IL-2 following injection of the ch14.18–IL-2 fusion protein showed a similar half-life, indicating that the fusion protein prolongs the circulatory half-life of IL-2. Detection of human IgG1 following injection of ch14.18–IL-2 showed a terminal half-life of 26.9 h. These data suggested that the native fusion protein is being altered in vivo, resulting in a somewhat rapid loss of detectable IL-2, despite prolonged circulation of its immunoglobulin components. In vitro incubation of the ch14.18–IL-2 fusion protein in pooled mouse serum at 37 °C for 48 h resulted in a loss of its IL-2 component, as detected in enzyme-linked immunosorbent assay systems and in proliferation assays. Polyacrylamide gel electrophoresis and Western blot analysis of the fusion protein incubated in mouse serum at 37 °C indicated that the ch14.18–IL-2 is cleaved, resulting in a loss of the 67-kDa band (representing the IL-2 linked to the IgG1 heavy chain) and the detection of a band of more than 50 kDa, slightly heavier than the IgG1 heavy chain itself. This suggests that the fusion protein is being cleaved in vitro within the IL-2 portion of the molecule. These studies show that (1) ch14.18–IL-2 prolongs the circulatory half-life of IL-2 (compared to that of soluble IL-2) and (2) the in vivo clearance of the fusion protein occurs more rapidly than the clearance of the ch14.18 antibody itself, possibly reflecting in vivo cleavage within the IL-2 portion of the molecule, resulting in loss of IL-2 activity.

Systemic interleukin-2 modulates the anti-idiotypic response to chimeric anti-GD2 antibody in patients with melanoma.

The induction of human antimouse antibodies (HAMA) and human anti-idiotypic (anti-Id) responses in cancer patients receiving therapeutic monoclonal antibody (mAb) may limit the effectiveness of the administered mAb. This report evaluates the influence of systemic interleukin-2 (IL-2) on the anti-Id response to anti-disialoganglioside (anti-GD2) antibody given as treatment for patients with melanoma. Twenty-eight patients with melanoma received combined immunotherapy with anti-GD2 antibody and IL-2 at 1.5 x 10(6) U/m2/day given 4 days/week. The anti-GD2 antibody [murine 14.G2a mAb; dose levels of 2-5 mg/m2/day (4 patients); or human-mouse chimeric 14.18 (ch14.18) antibody; dose levels of 2-10 mg/m2/day (24 patients)] was scheduled to be given for 5 days either before, during, or after initial systemic IL-2 treatment. All four patients who received murine 14.G2a developed HAMA anti-isotype antibodies (660-1,000 ng/ml) as well as measurable anti-Id antibodies. All three patients who received initial treatment with ch14.18 alone developed a strong anti-Id antibody response after IL-2 was started 1 week later. The serum level of anti-Id antibody decreased during subsequent ch14.18 infusions, suggesting that the anti-Id antibody may be binding the administered ch14.18. In contrast, measurable anti-Id antibody was detected in only 3 of 14 patients who received IL-2 before, during, and after initial ch14.18 administration. Two of four patients receiving systemic IL-2 before and during initial ch14.18 infusions, and two of three patients receiving systemic IL-2 concurrent with initial ch14.18 infusions developed anti-Id antibodies. These data suggest that the anti-Id response to chimeric anti-GD2 antibody is influenced by the timing of systemic IL-2 in relation to antibody administration and can be suppressed by systemic treatment with IL-2 given before, during, and after the antibody administration.

Addition of interleukin-2 in vitro augments detection of lymphokine-activated killer activity generated in vivo

SummaryThein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with “secondary-like” kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h51Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.

Antibody-dependent cellular cytotoxicity in asthmatics.

Using a 51Cr-labeled, antibody-coated chicken red blood cell assay system, mononuclear cell and granulocyte preparations from infectious asthmatics, noinfectious asthmatics, and normal controls were tested for antibody-dependent cellular cytotoxicity (ADCC) capacity. The corrected cytotoxic indices of mononuclear cell preparations from infectious asthmatics were reduced (34 +/- 10 SD) as compared to noninfectious asthmatiics (47 +/- 7 SD) or normal controls (47 +/- 6 SD). Granulocyte preparations from infectious asthmatics and normal controls had a severity of the disease or in vivo drug treatment.

Lymphocyte subpopulations in the peripheral blood of patients with farmer's lung.

The numbers of mononuclear cells having receptors for sheep red blood cells (T lymphocytes) or complement (B lymphocytes) in the peripheral blood of farmers lung patients were determined. In patients recovering from a clinical episode of farmers lung or exposed to moldy hay or fodder, both the percentage of T lymphocytes and the T lymphocytes per mm3 were reduced, whereas the number of B lymphocytes remained within normal limits. Farmers lung patients having no exposure to moldy hay or fodder had T and B lymphocyte numbers similar to a normal population.

HLA antigen frequencies in allergic bronchopulmonary aspergillosis

The frequency of HLA antigens in twenty‐two Caucasian patients with allergic broncho‐pulmonary aspergillosis (ABPA) and sixty‐nine unrelated Caucasian controls was determined. The results indicated that there was no increased frequency of a specific HLA antigen in patients with ABPA. Moreover, studies in thirteen families of ABPA patients also demonstrated that, within families, there was no consistent association between a specific haplotype and asthma, allergies or hay fever.

HLA antigen frequencies and natural history in Alternaria-sensitive and perennial nonallergic asthmatics.

The frequency of HLA-A, -B, and -C loci antigens in random populations of Alternaria-sensitive (N = 100) and perennial nonallergic asthmatics (N = 87) were compared with age- (+/- 5 yr) and sex-matched controls from the same geographic region. There was no association between HLA antigens as measured by frequency analyses and Alternaria-sensitive or perennial nonallergic asthma. Moreover there was no association between HLA antigens and the age of onset of asthma, associated allergic disorders, various environmental factors provoking asthma, total serum IgE levels, and Alternaria-specific IgE antibody.

ACTIVATION OF HUMAN EFFECTOR CELLS BY A TUMOR REACTIVE RECOMBINANT ANTI-GANGLIOSIDE GD2 INTERLEUKIN-2 FUSION PROTEIN

Cytotoxic effector cells interact with target cells through various mechanisms. CTLs use the antigen-specific T cell receptor, whereas Fc receptor-positive natural killer cells use this receptor to interact with antibody-coated target cells. We evaluated the tumor-binding and lymphocyte-activating capability of a recombinant fusion protein consisting of a tumor-selective human/mouse chimeric anti-ganglioside GD2 antibody (ch14.18) and recombinant human interleukin-2 (IL2) (ch14.18-IL2). This fusion protein bound specifically to GD2-positive melanoma and neuroblastoma tumor cell lines, and its IL2 component stimulated in vitro proliferation of an IL2-dependent cell line, as well as peripheral blood mononuclear cells, in healthy control individuals and in cancer patients receiving continuous infusion of IL2. The IL2 presented by the fusion protein, when bound to tumor cells, induced proliferation of IL2-responsive cells as well as a comparable amount of soluble IL2 did. This suggests that localization of IL2 at the site of contact between tumor and effector cells is an effective way of presenting this cytokine to IL2-responsive cells. The ch14.18-IL2 fusion protein also mediated antibody-dependent cellular cytotoxicity with Fc receptor-positive effector cells to an extent similar to ch14.18. These results, together with those of previous studies documenting antitumor efficacy against human tumor xenografts in SCID mice and GD2-positive murine tumors in immunocompetent syngeneic mice, suggest that the ch14.18-IL2 fusion protein should be tested in Phase I and II trials in patients with GD2-positive tumors.