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IMPORTANCE: The anti–PD-1 therapeutic antibody, nivolumab, has demonstrated clinical activity in patients with advanced melanoma. The activity of nivolumab in subgroups of patients with tumors which have wild-type BRAF kinase vs patients with tumors having mutant BRAF has not systematically been explored in a large dataset. OBJECTIVE: To evaluate the efficacy and safety of nivolumab in patients with wild-type BRAF and mutant BRAF metastatic melanoma. DESIGN, SETTING, AND PARTICIPANTS: This was a retrospective analysis of data pooled from 4 clinical trials of nivolumab in 440 adult patients with unresectable stage III or stage IV melanoma, who had been tested for BRAF mutational status while participating in one of the studies. INTERVENTION: The investigational drug, nivolumab, was administered intravenously to study participants over a 60-minute period, at doses of 0.1, 0.3, 1.0, 3.0, or 10.0 mg/kg every 2 weeks until disease progression, discontinuation owing to adverse events, withdrawal, or end of study. Most patients (83%) received nivolumab at a dosage of 3 mg/kg. MAIN OUTCOME AND MEASURE: Best overall response by modified World Health Organization or Response Evaluation Criteria In Solid Tumors criteria and safety profile. RESULTS: Of a total of 440 patients from 4 nivolumab clinical trials included in the analysis, 334 were BRAF wild-type and 106 were positive for BRAF V600 mutation. With the exception of prior BRAF inhibitor therapy, the demographics were well balanced between the 2 cohorts. In patients evaluable for response, the objective response rates were 34.6% (95% CI, 28.3-41.3) for the 217 patients with wild-type BRAF status and 29.7% (95% CI, 19.7-41.5) for the 74 with mutant BRAF status. The objective response rates did not seem to be affected by prior BRAF inhibitor therapy, prior ipilimumab therapy, or PD-L1 status of the tumor. The median duration of objective response was 14.8 months (95% CI, 11.1-24.0 months) for wild-type BRAF and 11.2 months (95% CI, 7.3-22.9 months) for mutant BRAF. Median time to objective response was 2.2 months in both patient groups. The incidence of treatment-related adverse events of any grade was 68.3% in the wild-type BRAF group and 58.5% in the mutant BRAF group, with grade 3 or 4 adverse events in 11.7% and 2.8% of patients, respectively. Treatment-related AEs of any grade that occurred in at least 5% of patients in either group were fatigue, pruritus, rash, and diarrhea. CONCLUSIONS AND RELEVANCE: The results of this retrospective analysis suggest that nivolumab has similar efficacy and safety outcomes in patients with wild-type or mutant BRAF, regardless of prior BRAF inhibitor or ipilimumab treatment.

Basic Research Review for Clinicians: The cellular and molecular principles that furnish the foundation for adoptive cell therapy of melanoma, and their implications for further clinical research. The cellular and molecular principles that furnish the foundation for ACT of melanoma and their implications for further clinical research are reviewed. The parallel advances in basic immunology, preclinical animal studies, and clinical trials over the last two decades have been integrated successfully with improvements in technology to produce an effective ACT strategy for patients with melanoma. From the initial observation that tumors could be treated effectively by the transfer of immune cells to current strategies using preconditioning with myeloablative therapy before adoptive transfer of native or genetically altered T cells, the role of preclinical animal models is discussed. The importance of the pmel transgenic mouse model in the determination of the mechanisms of lymphodepletion, the ongoing work to identify the optimal T cells for adoptive immunotherapy, and the early impact of the emerging discipline of synthetic biology are highlighted. The clinical consequences of the research described herein are reviewed in the companion manuscript.

The existence of tumor-specific T cells, as well as their ability to be primed in cancer patients, confirms that the immune response can be deployed to combat cancer. However, there are obstacles that must be overcome to convert the ineffective immune response commonly found in the tumor environment to one that leads to sustained destruction of tumor. Members of the tumor necrosis factor (TNF) superfamily direct diverse immune functions. OX40 and its ligand, OX40L, are key TNF members that augment T-cell expansion, cytokine production, and survival. OX40 signaling also controls regulatory T-cell differentiation and suppressive function. Studies over the past decade have demonstrated that OX40 agonists enhance antitumor immunity in preclinical models using immunogenic tumors; however, treatment of poorly immunogenic tumors has been less successful. Combining strategies that prime tumor-specific T cells together with OX40 signaling could generate and maintain a therapeutic antitumor immune response.

Cancer immunotherapy centers on modulating the host's tumor‐directed immune response. One promising approach involves augmentation of cell‐mediated immunity by interrupting T‐cell pathways responsible for immune down‐regulation or tolerance. The discovery of cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) and its role as a key negative regulator for T cells has prompted efforts to target this signaling molecule to improve cancer therapy. Activation, or ‘priming’, of naive T cells in response to tumor‐cell invasion comprises a dual‐signaling mechanism. Signal 1 requires tumor‐associated antigen recognition by the T‐cell receptor, while signal 2 occurs through binding of CD80 or CD86 (B7.1 of 2) on the antigen presenting cell (APC) with CD28 on the T cell. Importantly, there is a final step responsible for naturally occurring immune regulation; this occurs in response to competitive binding of CD80/CD86 on the APC by CTLA‐4 on the T cell. This ‘immune checkpoint’ interrupts signal 2 and inhibits the activated T cell. Targeting CTLA‐4 as an anticancer strategy: Following proof‐of‐concept studies in animals, fully human anti‐CTLA‐4 antibodies were developed and 2 are undergoing clinical evaluation. Ipilimumab and tremelimumab have shown promising antitumor activity, initially in patients with advanced melanoma. Class‐specific immune‐related adverse events (irAEs) were common and mostly transient and/or manageable. These events are thought to be mechanism‐of‐action‐related, indicating immune tolerance is broken; this relation may also explain the association between irAEs and response seen in several trials. Interruption of immune inhibitory pathways via CTLA‐4 blockade appears to be a promising strategy for cancer immunotherapy. Cancer 2007. © 2007 American Cancer Society.

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine embryonic stem cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100, which binds to abundant low-density lipoprotein receptors on the cell surface and is internalized. Here we show that in the majority of patients administered 3 different types of cell-based therapies using cells grown in fetal calf serum-containing media, an antibody response to bovine apolipoprotein B-100 develops after the second infusion and is the dominant specificity. The known and potential clinical effects of such antibodies are discussed.

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine embryonic stem cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100, which binds to abundant low-density lipoprotein receptors on the cell surface and is internalized. Here we show that in the majority of patients administered 3 different types of cell-based therapies using cells grown in fetal calf serum-containing media, an antibody response to bovine apolipoprotein B-100 develops after the second infusion and is the dominant specificity. The known and potential clinical effects of such antibodies are discussed.

Recent advances in flow cytometry have resulted in the development of reliable techniques for performing polychromatic (5–17 color) flow cytometry analysis. However, the data reduction and analysis involved in the resolution of hundreds of possible cellular subphenotypes identified, using a single polychromatic flow cytometry staining panel, presents a major obstacle to the successful application of this technology.To generate two distinct collections of T cell populations with differentially expressed surface markers, cryopreserved lymph node cells from 5 melanoma patients vaccinated with the modified gp100209‐2M melanoma peptide were stimulated with cognate peptide and cultured in either IL‐21 + low‐dose IL‐2 or IL‐15 + low‐dose IL‐2. In vitro stimulated (IVS) cells were interrogated using 8‐color flow cytometry. Data were analyzed using Winlist Hyperlog™ and FCOM™ software, and 32 T cell subsets were resolved for each culture condition. Hierarchical clustering analysis was applied to the relative percentages of each subphenotype for both IVS conditions to determine if unique cell surface marker expression signatures were produced for each IVS culture.Sequential data analysis using Hyperlog™ and FCOM™ demonstrated that lymphocytes cultured in IL‐21 + IL‐2 had a distinctively different set of subphenotype signatures compared to cells grown in IL‐15 + IL‐2 for all 5 patients. Importantly, subsequent cluster analysis of all 32 subphenotype frequencies in each IVS test condition for all 5 patients reproducibly demonstrated that cellular subphenotypes produced after IL‐21 + IL‐2 IVS partitioned separately from subphenotypes produced by IL‐15 + IL‐2 IVS.The integrated sequential use of Hyperlog™ and FCOM™ software with cluster analysis algorithms for the reduction and analysis of polychromatic flow cytometry data produces an effective, rapid technique for the assessment of complex patterns of subphenotype expression between and within multiple test samples. This approach to data analysis may enhance the use of polychromatic flow cytometry for both research and clinical applications. © 2006 International Society for Analytical Cytology

MDA-MB-231, an HLA-A2+, HER2/neu+ allogeneic breast cancer cell line genetically modified to express the costimulatory molecule CD80 (B7-1), was used to vaccinate 30 women with previously treated stage IV breast cancer. Expression of CD80 conferred the ability to deliver a costimulatory signal and thereby improved the antigen presentation capability of the tumor cells to patient T cells in vitro. Patients were vaccinated with 107 or 108 irradiated gene-modified tumor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) or BCG, three times at 2-week intervals and then monthly until progressive disease developed. GM-CSF-related flulike symptoms and minor injection site reactions were observed frequently. Prolonged disease stabilization was observed in four patients but no objective tumor regressions were seen. Immune responses were measured in matched peripheral blood samples collected before and after treatment from 9 of 15 patients treated at the 108 tumor cell dose. Four patients exhibited MHC class I-restricted cytokine production in response to the parental breast cancer cell line. One patient maintained an increased number of circulating tumor-specific, interferon γ-secreting CD8+ T cells for 24 months after the last vaccination. One patient exhibited a tumor-specific interleukin 5 response to an autologous tumor cell line. This immunization strategy proved to be safe and feasible, and induced tumor-specific immune responses in a minority of patients; however, no objective tumor regressions were observed.

We report here that breast cancer cells from spontaneous tumors that arise in rat neutransgenic mice produce several chemokines capable of acting upon cells of the immune system. Moreover, mice bearing these spontaneous tumors possess splenic T cells as well as CD11c+, CD11b+and CD19+cells with an altered sensitivity to recombinant chemokines compared to naïve mice. A comparison between T-cell migration and the level of chemokines produced by the tumor cells revealed that the altered chemotactic activity was not a direct consequence of tumor-derived chemokines. These data suggest that a growing tumor may indirectly alter leukocyte chemotactic activity.

Cancer vaccines are currently a major focus of immunotherapy research. The combination of specific targeting and low levels of toxicity makes vaccination an attractive approach. There are a variety of immunogens that can be employed to vaccinate patients in order to induce or enhance an antitumor response. The observation that most T-cell priming occurs via presentation of tumor antigens from tumor cells engulfed by host antigen-presenting cells, rather than by direct presentation by vaccine tumor cells themselves, provides the immunological rationale for an allogeneic tumor cell vaccine approach. Furthermore, there are practical advantages over an autologous tumor cell vaccine approach. We summarize herein the limited experience using allogeneic whole cell vaccines in patients with breast cancer. We also describe in vitro immunological results using peripheral blood mononuclear cells from women with stage IV breast cancer who were enrolled in a phase I trial employing a human leukocyte antigen-A2—matched, CD80-modified, allogeneic, whole cell vaccine. Clinical trials employing allogeneic tumor cell vaccines have achieved encouraging immunological and clinical effects in stage IV patients. Allogeneic tumor cell vaccines are safe, feasible, and associated with low toxicity, and the early clinical results suggest that they are worthy of further study.

In this study we investigated whether T cells expressing high or low levels of CD62L were differentially susceptible to the T cell chemokine lymphotactin. We found that lymphotactin induced preferential migration of antigen-specific (CD62Llo) T cells over the nonspecific (CD62Lhi) T cells in vitro and in vivo. The differing migratory abilities correlated with higher levels of mRNA encoding the lymphotactin receptor (XCR1) on the CD62Llo cells compared to the CD62Lhi cells. Thus, we have identified a coupling mechanism between the activation of T cells and acquisition of new homing properties, in this case conferred by XCR1 expression. These data confirm that at least one function of lymphotactin includes mediating the recruitment of recently activated antigen-specific T cells.

We conducted a phase II randomized trial of recombinant granculocyte-macrophage colony-stimulating factor (GM-CSF) administered before topotecan chemotherapy to determine whether it could prevent myelosuppression and to determine the antitumor activity of this topoisomerase I inhibitor in 53 patients with metastatic malignant melanoma and renal cell cancer. All patients received GM-CSF after topotecan at a dose of 250 μg/m2daily for at least 8 days. Patients randomly assigned to receive GM-CSF priming were treated with GM-CSF at 250 μg/m2twice daily for 5 days before treatment. Twenty-five patients were randomly assigned to receive GM-CSF priming and 28 to receive topotecan without priming. The primary analysis was restricted to the protective effects seen during the first cycle of therapy. Grade 4 neutropenia occurred in 8 of 23 patients (35%) and grade 3 neutropenia in 5 of 23 patients (22%) randomized to GM-CSF priming, whereas 18 of 26 (69%) and 5 of 26 (19%) patients experienced grade 4 or 3 neutropenia, respectively, without GM-CSF priming (P= .0074). The mean duration of neutropenia was reduced by GM-CSF priming: grade 3 neutropenia from 5.2 ± 0.7 to 2.8 ± 0.7 days (P= .0232) and grade 4 neutropenia from 2.7 ± 0.6 to 1.1 ± 0.4 days (P= 0.0332). The protective effects of GM-CSF extended to the second cycle of treatment. The incidence of febrile neutropenia was also reduced. Chemotherapy-induced anemia and thrombocytopenia were similar in both groups. One partial response was seen in a patient with melanoma, and one patient with renal cell cancer had complete regression of pulmonary metastases and was rendered disease-free by nephrectomy.

We conducted a phase II randomized trial of recombinant granculocyte-macrophage colony-stimulating factor (GM-CSF) administered before topotecan chemotherapy to determine whether it could prevent myelosuppression and to determine the antitumor activity of this topoisomerase I inhibitor in 53 patients with metastatic malignant melanoma and renal cell cancer. All patients received GM-CSF after topotecan at a dose of 250 μg/m2 daily for at least 8 days. Patients randomly assigned to receive GM-CSF priming were treated with GM-CSF at 250 μg/m2 twice daily for 5 days before treatment. Twenty-five patients were randomly assigned to receive GM-CSF priming and 28 to receive topotecan without priming. The primary analysis was restricted to the protective effects seen during the first cycle of therapy. Grade 4 neutropenia occurred in 8 of 23 patients (35%) and grade 3 neutropenia in 5 of 23 patients (22%) randomized to GM-CSF priming, whereas 18 of 26 (69%) and 5 of 26 (19%) patients experienced grade 4 or 3 neutropenia, respectively, without GM-CSF priming (P = .0074). The mean duration of neutropenia was reduced by GM-CSF priming: grade 3 neutropenia from 5.2 ± 0.7 to 2.8 ± 0.7 days (P = .0232) and grade 4 neutropenia from 2.7 ± 0.6 to 1.1 ± 0.4 days (P = 0.0332). The protective effects of GM-CSF extended to the second cycle of treatment. The incidence of febrile neutropenia was also reduced. Chemotherapy-induced anemia and thrombocytopenia were similar in both groups. One partial response was seen in a patient with melanoma, and one patient with renal cell cancer had complete regression of pulmonary metastases and was rendered disease-free by nephrectomy.

We found that the murine breast cancer cell line 4T1 constitutively produced several chemokines capable of recruiting T cells. Additionally, supernatants from the tumor cell line mediated chemotaxis of T cells in a pertussis toxin-sensitive manner, indicating that these chemokines were functional. However, we also found an impaired chemotactic ability of splenic T cells in mice bearing these same tumors. The receptors for RANTES, MCP-1, and SLC were desensitized. Thus, the impaired chemotactic ability of T cells in tumor-bearing mice may explain why tumors that secrete chemokines grow progressively in a host.

FVBN202 mice, which are transgenic for the rat neu gene, spontaneously develop mammary carcinomas between 6 and 7 months of age. We investigated whether these spontaneous tumors (spontaneous breast carcinoma cells, SBCC) could elicit an immune response in naive 6- to 8-week-old FVBN202 transgenic and FVBN nontransgenic mice. After s.c. injection of SBCC, the recently activated T cells, which were identified by their reduced expression of CD62L (L-selectin), were isolated from the draining lymph nodes, expanded with anti-CD3 and IL-2, and their cytokine response to tumor cells in vitro was analyzed. Tumor-vaccine draining lymph node lymphocytes (TVDLN) from transgenic mice failed to make IFN-γ in response to the tumor cells. However, TVDLN from the nontransgenic mice exhibited a tumor-specific IFN-γ response against the SBCC. This indicates that the SBCC are immunogenic. The lack of response in transgenic mice could not be attributed to cytokine immune deviation or T-cell signaling defects. Although transgenic mice were tolerant to their own tumors, their immune competence was established by their ability to respond in an allogeneic mixed lymphocyte reaction, to reject an allogeneic breast carcinoma cell line, and to produce a tumor-specific IFN-γ response against a syngeneic cancer cell line. This transgenic mouse model provides the opportunity to investigate the immune response against a primary tumor cell culture rather than cell lines or clones and should prove useful for developing immunotherapies that overcome tolerance to self-tumor antigens. Int. J. Cancer 87:688–694, 2000. © 2000 Wiley-Liss, Inc.

We generated a monoclonal antibody (MAb), H8.98, that recognizes an antigen shared by 50% of examined renal carcinoma cell (RCC) lines and is susceptible to lysis by a Vγ3Vδ1⁺ T-cell clone derived from RCC tumor-infiltrating lymphocytes. H8.98 inhibited Vγ3Vδ1⁺ T-cell clone–mediated lysis of RCC lines. It did not stain normal kidney lines, melanomas, fibroblasts, Burkitt's lymphoma or Epstein-Barr virus–transformed B-cell lines but it did stain 2 of 4 tested breast cancer lines. Through screening of a renal carcinoma cDNA library using H8.98, we isolated a cDNA clone which, upon sequencing, was found to be cytochrome b with 2 point mutations. Int. J. Cancer 82:562–568, 1999. © 1999 Wiley-Liss, Inc.

Abstract: The therapeutic benefit of adding interferon α (IFNα) to established single-agent and combination chemotherapy regimens for the treatment of metastatic melanoma has not been proven. We designed the present study to estimate the response rate of IFNα, dacarbazine, cisplatin and tamoxifen in patients who had not been treated with systemic therapy for advanced disease. Using a schedule similar to that which had previously been shown to favor IFNα plus dacarbazine over dacarbazine alone, we treated patients with an “induction” regimen of IFNα, 15 mU m−2 day−1 intravenously 5 days/week for 3 weeks. Following induction, schedules of IFNα, 5 mU m−2 day−1 subcutaneously three times a week, and tamoxifen, 10 mg orally twice a day, were begun. Dacarbazine, 250 mg m−2 day−1 and cisplatin 33 mg m−2 day−1 for 3 consecutive days were repeated every 4 weeks, and subcutaneous IFNα and oral tamoxifen were continued until the discontinuation of chemotherapy. We treated 25 patients (18 men and 7 women, median age 52 years) and observed only 1 objective response (response rate 4%, 95% confidence interval 0.1%–20%). The toxicities of the regimen consisted of moderate myelosuppression and constitutional side-effects. On the basis of the low antitumor activity of this regimen, we do not recommend it for further study or for use as standard therapy of metastatic melanoma.

This dose-escalation study was performed to evaluate the hematologic activity, biological effects, immunogenicity, and toxicity of PIXY321 (an interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein) administered after high-dose carboplatin (CBDCA) treatment. Patients with advanced cancers received CBDCA at 800 mg/m2 intravenously on day 0 of repeated 28-day cycles. In part A of the study, patients were treated with CBDCA alone during cycle 1 and then received PIXY321 on days 1 through 18 of cycle 2 and later cycles. In part B, patients received 18 days of PIXY321 beginning on day 1 of all CBDCA cycles, including cycle 1. PIXY321 was administered subcutaneously in 2 divided doses. Total doses of 135, 250, 500, 750, and 1,000 μg/m2/d were administered to successive cohorts of 3 to 6 patients in part A. In part B, patient groups received PIXY321 doses of 750, 1,000, and 1,250 μg/m2/d. The hematologic effects of PIXY321 were assessed in the first 2 cycles of therapy. Anti-PIXY321 antibody formation was assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. Of the 49 patients enrolled, 31 were fully evaluable for hematologic efficacy. When comparing the first B cycle (cycle B-1; with PIXY321) with the first A cycle (cycle A-1; without PIXY321), the fusion protein had no significant effect on platelet nadirs or duration of platelets less than 20,000/μL but was able to speed the time of recovery of platelet counts to 100,000/μL (15v 20 days; P = .01). Significant improvements in neutrophil nadir and duration of ANC less than 500 were observed in cycles A-2 and B-1 (with PIXY321) as compared with cycle A-1 (without PIXY321). Initial PIXY321 prophylaxis (cycle A-2 and cycle B-1), enhanced the recovery of ANC to greater than 1,500/μL by an average of at least 8 days as compared with cycle A-1 (without PIXY321;P ≤ .004). However, positive PIXY321 hematologic effects were lost in the second course of PIXY321 among patients treated in part B. ELISA analysis showed that 92% of patients had developed neutralizing anti-PIXY321 antibodies by the completion of 2 PIXY321-containing cycles. The incidental action of PIXY321 to depress serum cholesterol levels was also abrogated during cycle B-2. We conclude that PIXY321 was active in speeding hematologic recovery but that neutralizing anti-PIXY321 antibody formation suppressed the hematologic and biochemical effects by the second cycle of PIXY321 administration. The immunogenicity of this fusion protein provides a cautionary warning that clinical development of bioengineered human molecules requires thorough testing for immune neutralization.

This dose-escalation study was performed to evaluate the hematologic activity, biological effects, immunogenicity, and toxicity of PIXY321 (an interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein) administered after high-dose carboplatin (CBDCA) treatment. Patients with advanced cancers received CBDCA at 800 mg/m2intravenously on day 0 of repeated 28-day cycles. In part A of the study, patients were treated with CBDCA alone during cycle 1 and then received PIXY321 on days 1 through 18 of cycle 2 and later cycles. In part B, patients received 18 days of PIXY321 beginning on day 1 of all CBDCA cycles, including cycle 1. PIXY321 was administered subcutaneously in 2 divided doses. Total doses of 135, 250, 500, 750, and 1,000 μg/m2/d were administered to successive cohorts of 3 to 6 patients in part A. In part B, patient groups received PIXY321 doses of 750, 1,000, and 1,250 μg/m2/d. The hematologic effects of PIXY321 were assessed in the first 2 cycles of therapy. Anti-PIXY321 antibody formation was assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. Of the 49 patients enrolled, 31 were fully evaluable for hematologic efficacy. When comparing the first B cycle (cycle B-1; with PIXY321) with the first A cycle (cycle A-1; without PIXY321), the fusion protein had no significant effect on platelet nadirs or duration of platelets less than 20,000/μL but was able to speed the time of recovery of platelet counts to 100,000/μL (15v20 days; P= .01). Significant improvements in neutrophil nadir and duration of ANC less than 500 were observed in cycles A-2 and B-1 (with PIXY321) as compared with cycle A-1 (without PIXY321). Initial PIXY321 prophylaxis (cycle A-2 and cycle B-1), enhanced the recovery of ANC to greater than 1,500/μL by an average of at least 8 days as compared with cycle A-1 (without PIXY321;P≤ .004). However, positive PIXY321 hematologic effects were lost in the second course of PIXY321 among patients treated in part B. ELISA analysis showed that 92% of patients had developed neutralizing anti-PIXY321 antibodies by the completion of 2 PIXY321-containing cycles. The incidental action of PIXY321 to depress serum cholesterol levels was also abrogated during cycle B-2. We conclude that PIXY321 was active in speeding hematologic recovery but that neutralizing anti-PIXY321 antibody formation suppressed the hematologic and biochemical effects by the second cycle of PIXY321 administration. The immunogenicity of this fusion protein provides a cautionary warning that clinical development of bioengineered human molecules requires thorough testing for immune neutralization.

We describe two patients with a new clinical pathologic syndrome of diffuse osteosclerosis in association with hairy cell leukemia. In both patients bone marrow biopsies could not be obtained due to extremely hard bones and inability to insert the biopsy needle; neither patient had a history of bony pain or fracture. The osteosclerotic process in one patient stabilized after successful treatment of her hairy cell leukemia with interferon alpha and deoxycoformycin suggesting that the osteosclerosis observed was related to the underlying malignant disease. Possible étiologie mechanisms are discussed.

Immune function in patients with hairy cell leukemia (HCL) was examined serially during treatment with alternating monthly cycles of recombinant interferon α-2a and 2'-deoxycoformycin (dCF). At Presentation, most patients had normal numbers of T lymphocytes and their cells had normal proliferative responses to mitogens [Phytohemag-glutinin (PHA) and concanavalin A (Con A)] and alloantigens. Patients had severe monocytopenia, decreased delayed-type hypersensitivity (DTH) reactions, and decreased peripheral blood natural killer (NK) activity. Treatment caused a profound decrease in all lymphocyte subpopulations. T cells were more affected than B cells or NK cells. Numbers of CD4+and CD8+lymphocytes decreased to levels <200 cells/µL in all patients during treatment. This decrease in T cell number was associated with a marked decrease in proliferative responsiveness to PHA, Con A, and alloantigens. These abnormalities persisted throughout the 14 months of treatment and have continued for up to 6 months beyond discontinuation of treatment. NK cell activity increased during treatment, but cycled depending on the phase of treatment; highest activities were observed after interferon (IFN)-αand lower levels of activity were observed after dCF. DTH responses generally did not improve during therapy. Levels of IgM, IgG, IgA, and IgD did not change during treatment, but IgE levels rose in most patients. All immunosuppressive effects were attributable to dCF since patients receiving IFN-α2a alone did not exhibit these same immunosuppressive effects, and patients receiving dCF alone after IFN failure exhibited similar abnormalities. Despite this severe immunosuppression from dCF, life-threatening opportunistic infections have not been observed in our patient population. Six patients developed localized Herpes zosterinfection among 21 patients who had received dCF. Pending the results of long-term follow-up, we recommend that dCF be reserved for patients who have failed splenectomy and IFN therapy.

We describe a patient with the so-called “prolymphocytic variant” form of hairy cell leukemia (HCL) resistant to treatment with interferon-α(IFN-α). Analysis of immunoglobulin (Ig) and T-cell receptor-β(TCRβ) gene rearrangements from serial peripheral blood mononuclear cell specimens (MNCs) confirmed not only the B-cell nature of the disease, but also the subsequent emergence of a morphologically indistinguishable population of cells with a clonal TCRi rearrangement in addition to the original Ig gene rearrangement. With the exception of a transient increase in peripheral blood T cells during treatment with deoxycoformycin (DCF), the MNCs remained essentially constant throughout therapy with no evidence of a co-existing T-cell clone to account for the TCRβrearrangement. Although NCs from this patient bound significantly less IFN-αthan did MNCs from other HCL patients, the binding was of high affinity with a kd similar to that of control cells. The number of IFN-γreceptors on our patient’s MNCs was four times higher than the number of IFN-αreceptors and was similar to the number of IFN-αreceptors on MNCs from HCL patients responsive to IFN-α. While various treatments including IFN-α, DCF, chlorambucil, splenectomy, leukopheresis, and IFN-γwere not able to change the clinical progression of the disease, they may have provided an opportunity for the divergent TCRβrearranged clone to expand and displace the initially dominant clone.

Production of indirect TNP-specific plaque-forming cells (PFC) in response to immunization with 2, 4, 6-trinitrophenyl (TNP) conjugated to autogenous mouse serum albumin (MSA) in complete Freund's adjuvant (CFA) is underH-2 control. On the C57BL/10 (B10) background,H-2b andH-2d strains of mice are high responders, whereasH-2a,H-2k orH-2y2 strains yield low levels of indirect TNP-specific PFC. An unusual pattern of inheritance has been revealed in B10 congenic mice: high responsiveness controlled byH-2b is inherited recessively, while high responsiveness controlled byH-2d is inherited dominantly. On the C3H and A strain backgrounds, high responsiveness controlled byH-2b is partially recessive;H-2b/H-2a F1 mice respond with 20%-40% of the high responderH-2b response. Yet, high responsiveness controlled by theH-2d haplotype remains dominant on the C3H background. A hierarchy of haplotypes in order of decreasing immune responsiveness to TNP-MSA is evident as follows:H-2d>H-2b>H-2k,H-2a orH-2y2. The unusual patterns of inheritance in the TNP-MSA system reveal graded regulation of responsiveness attributable to bothH-2 and non-H-2 genes.

The antibody response to the hapten 2,4,6-trinitrophenyl (TNP) conjugated to autogenous mouse serum albumin (MSA) is regulated by anIr gene(s) located within the major histocompatibility complex (MHC). Both the qualitative and quantitative ability of congenic strains to produce TNP-specific antibodies are functions of theH-2 haplotype. Thus, mouse strains may be classified as high (H-2d), intermediate (H-2b,H-2s), and low responders (H-2a,H-2k,H-2n,H-2p,H-2q). Antibody responses, as measured by antigen-binding capacities in modified Farr assays, were compared among strains carrying recombinantH-2 haplotypes and their hybrid progenies. Distinct high- and low-responder phenotypes were evident throughout the time course of both primary and secondary antibody responses. The gene locus controlling specific responsiveness to TNP-MSA, now designatedIr-6, was mapped within theI-B subregion of theH-2 complex. Recessive inheritance of high responsiveness was confirmed in hybrid progenies of three different low × high-responder crosses.

We examined signaling molecules of peripheral blood T lymphocytes obtained from women with breast cancer. In 6 of 14 patients, T lymphocytes displayed an impaired ability to translocate NFêB p65 (Rel-A) following activation by anti-CD3 and IL-2. This observation was made despite normal cytoplasmic levels of the Rel-A protein. We also detected abnormally low levels of the signaling molecules T-cell receptor (TCR)-ζ, ZAP-70 and p56^lck in 4 of 14 breast cancer patients, i.e., defects in T-cell signaling molecules. T lymphocytes from 6 of the 14 patients also exhibited an increased expression of the dual specificity phosphatase, map kinase phosphatase-1 (MKP-1). MKP-1 inactivates MAP kinase and therefore may interfere with the activation of c-jun and c-fos. Abnormalities of 1 or more signaling molecules were found in 9 of 14 patients; however, only 3 patients had T cells that exhibited all 5 defects. Our data have implications for the detection of potentially dysfunctional T cells in patients with cancer. For example, the analysis of only 1 signaling molecule may allow patients with significant defects in T-cell signaling to go unnoticed. Finally, despite impaired Rel-A translocation, T cells were capable of transcribing IL-2. Impairments in the translocation of Rel-B and c-Rel further suggest that the NFκB family members Rel-A, Rel-B and c-Rel are not required for the transcription of IL-2 in the peripheral T lymphocytes of patients with breast cancer. Int. J. Cancer 78:16–20, 1998.© 1998 Wiley-Liss, Inc.

Interferons are not curative in hairy cell leukemia (HCL), and retreatment is necessary in most patients whose therapy is stopped. In an attempt to maintain or improve responses, we administered recombinant interferon-α2a (rlFN-α2a) continuously to patients with HCL who initially responded to this therapy. Of 53 evaluable patients enrolled in this study, 32 have received rlFN-α2a continuously for a median of 5 years. Patients received 3 million units of rlFN-α2a subcutaneously (SC) daily for 6 months, followed, in responding patients, by the same dose three times weekly. Twenty-one patients (40%) discontinued IFN after a median of 29 months, seven of whom developed resistant disease in association with anti-IFN antibodies. Treatment produced high response rates: complete response plus partial response (CR + PR) = 40 of 53 (76%), CR + PR + minor response (MR) = 43 of 53 (82%), with no differences in response rates between patients with and without splenectomy. Sixteen patients who had MR at 18 months had PR with prolonged treatment, nine of whom had a significant further reduction in the hairy cell infiltrate in the bone marrow (BM). The median granulocyte and platelet counts have continued to increase and the median serum soluble interleukin-2 receptor (slL-2R) level has continued to decrease with prolonged treatment. Two patients developed erythrocytosis that may be treatment related, but no other new toxicities were noted with prolonged treatment. We conclude that prolonged, continuous rlFN-α2a treatment has acceptable toxicity, is not associated with late development of IFN resistance, and results in continued hematologic improvement with time on treatment. This is a US government work. There are no restrictions on its use.

Administration of intravenous interleukin-2 (IL-2), followed by intraperitoneal IL-2 and autologous lymphokine-activated killer (LAK) cells to six patients with colonic, ovarian, or endometrial carcinoma restricted to peritoneal spread increased significantly the ascitic fluid concentrations of the neuropeptides substance P (SP) and calcitonin-gene related peptide (CGRP). After intravenous IL-2 alone, the level of SP rose 10- to 140-fold, without a change in that of CGRP. Intraperitoneal IL-2 and LAK cells led to elevations in the concentrations of SP and CGRP to respective maximal means of 319 and 175 pM after 8 hr, which were maintained for 24–48 hr without alterations in the levels of vasoactive intestinal peptide or somatostatin. SP and CGRP from peritoneal fluid were chromatographically indistinguishable from synthetic neuropeptides. The increases in concentrations of SP and CGRP after IL-2 and LAK-cell therapy are the first demonstration of a neural response to a human cellular immunological reaction. The time course and magnitude of the neuropeptide response suggest a role in the vascular side effects of this form of treatment.

Inbred mice injected intravenously with 5 × 106cells of Cryptococcus neoformansshowed two patterns of survival: sensitive (A/WySn, A.BY, A/J, DBA/2J, NZB/B1NJ, and SWR/J) and resistant [C57BL/10Sn, B10.A, B10.A (2R), B10.S (7R),C57BR/cdJ, C58/J, C3H/HeJ, BALB/c, DBA/1J, and SJL/J]. Relative susceptibility based on survival time was shown to correspond to differences obtained for 50% lethal dose values. Either decreasing the dose of organisms or changing to the intraperitoneal route of inoculation resulted in prolonged survival times, but neither change affected the observed patterns of survival. F1 hybrids between different sensitive strains were also sensitive, whereas F1 hybrids between sensitive and resistant strains were resistant, indicating a dominant mode of inheritance. Sensitivity and resistance were shown to be under single gene control by segregation analysis in F2 progeny produced by inbreeding (B10.A × A/WySn)F1 hybrids and in (F1 × A/WySn) backcross progeny. Blood obtained from parental strains, F1, F2, and backcross hybrids was tested for the presence or absence of hemolytic complement. Mice lacking hemolytic complement activity in their sera are homozygous for the Hc0allele at the Hclocus on chromosome 2 and are deficient in the complement component C5. A 1:1 correspondence was found between C5 deficiency and sensitivity to C. neoformans.Resistance was shown to cosegregate with the presence of hemolytic complement in the F2 and the backcross progenies.

•Manual cold-cap training may improve efficacy and subjective patient experience•Recurrent subjective effects of manual cold capping were identified across patients•These subjective effects provided insight on determining clinical benefit from harm•The proposed clinical instrument promotes informed decision-making

It is generally believed that most tumor antigens are passively released from either health or dying tumor cells as intact soluble antigens, peptide fragments complexed with heat shock proteins (HSPs), or packaged in secretary vesicles in the form of microparticles or exosomes. The passive release of tumor antigens is generally non-inflammatory and non-immunogenic; however, results from others and our laboratories suggest that autophagy is critically involved in immunogenic cell death.

ABSTRACTThe immune system plays an essential role in eradicating cancer in concert with various treatment modalities. In the absence of autologous tumor material, no standardized method exists to assess T cell responses against the many antigens that may serve as cancer rejection antigens. Thus, development of methods to screen for therapy-induced anti-tumor responses is a high priority that could help tailor therapy. Here we tested whether a tumor-derived antigen source called DRibbles®, which contain a pool of defective ribosomal products (DRiPs), long-lived and short-lived proteins (SLiPs) and danger-associated molecular patterns (DAMPs), can be used to identify tumor-associated antigen (TAA)-specific responses in patients before or after immunotherapy treatment. Protein content, gene expression and non-synonymous – single nucleotide variants (ns-SNVs) present in UbiLT3 DRibbles were compared with prostate adenocarcinomas and the prostate GVAX vaccine cell lines (PC3/LNCaP). UbiLT3 DRibbles were found to share proteins, as well as match tumor sequences for ns-SNVs with prostate adenocarcinomas and with the cell lines PC3 and LNCaP. UbiLT3 DRibbles were used to monitor anti-tumor responses in patients vaccinated with allogeneic prostate GVAX. UbiLT3-DRibble-reactive CD8+T-cell responses were detected in post-vaccine PBMC of 6/12 patients (range 0.85–22% of CD8+cells) after 1 week in vitro stimulation (p = 0.007 vs. pre-vaccine). In conclusion, a cancer-derived autophagosome-enriched preparation, packaging over 100 proteins over-expressed in prostate cancer into microvesicles containing DAMPs, could be used to identify CD8+T cells in peripheral blood from patients after prostate GVAX vaccination and may represent a general method to monitor anti-cancer T cell responses following immunotherapy.

Cross-presentation of exogenous antigens by host professional antigen-presenting cells (APCs) plays a pivotal role in the initiation and development of T-cell immune responses to tumor-associated antigens, including self or mutated self-antigens derived from tumor cells, and foreign antigens derived from infectious agents. Cross-presentation requires multiple steps that involve the antigens’ synthesis and compartmentalization in donor cells, packaging and delivery, and processing and presentation by MHC class I molecules on professional APCs. The intricate pathways that lead to protein degradation and the formation of MHC I-peptide complexes inside the APC are well documented for both soluble and particulate antigens.  However, much less is known about how cross-presentation is regulated by the protein degradation pathways in antigen-donor cells (ADCs), including autophagy-mediated lysosomal proteolysis and proteasomal degradation. The exact nature or form of the antigens derived from donor cells at the time of delivery to the APC for cross-presentation is very controversial.

Stereotactic body radiation therapy enhances tumor response rate to high-dose interleukin-2 in a phase 1 study.

Natural killer cell activity and in-terferon (IFN) production were measured in 6 patients receiving flavone acetic acid for treatment of cancer. Natural killer cell activity was significantly increased in 3 of 6 patients receiving 6.4 g of flavone acetic acid/m2 by 3-hour iv infusion. Analysis of cell surface markers failed to reveal significant changes in any cell population. There was no evidence of induction of IFN-gamma, but 3 of 4 patients tested had evidence of induction of type I IFN, as measured in a virus neutralization assay.

The investigational drug flavone acetic acid (FAA) systemically augments natural killer (NK) cell activity in normal and tumor-bearing mice and in human cancer patients. The results from the present investigation demonstrate that in vivo administration of FAA induces in a dose-dependent manner high levels of serum interferon (IFN) within 4 hours in BALB/c, C57BL/6, and BALB/c nude mice. Antibody neutralization studies indicated that FAA induced IFN of the α/β type, while molecular hybridization studies demonstrated that FAA rapidly stimulated the production of IFN a mRNA in splenic leukocytes. In vivo administration of anti-IFN α/β antibodies to FAA-treated mice inhibited the FAA-induced augmentation of splenic NK cell activity at 4 hours. These results suggest that FAA mediates its anti-tumor effects indirectly by immunomodulation as well as directly by antiproliferative or cytotoxic activity. [J Natl Cancer Inst 1988;80:1226–1231]

Lymphokine-activated killer (LAK) cells and interleukln-2 (IL-2) were administered by the ip route to patients with intra-abdominal malignancies. Pharmacokinetic studies of IL-2 revealed 10-to 100-fold higher concentrations of IL-2 in peritoneal fluid versus serum. Ip levels of IL-2 were maintained well above those required to generate and maintain LAK cells in vitro. LAK cell activity was detectable in the peritoneal fluid for the duration of each treatment cycle and did not disappear until IL-2 was discontinued. Detection of interferon-gamma (IFN-γ) in the peritoneal fluid of all patients was consistent with production in situ by activated lymphocytes. In some patients, low but detectable levels of IFN-γ were also found in the serum. In vivo activation of monocytes in the peritoneal fluid as measured by in vitro production of hydrogen peroxide was documented in the majority of patients. Neither interleukin-1 nor tumor necrosis factor-alpha was detected in the peritoneal fluid. We found no correlation between the presence or levels of IL-2, IFN-γ, or LAK cell lytic activity in peritoneal fluid or serum and response or nonresponse to therapy. [J Natl Cancer Inst 81:602–611, 1989]

Background Experiments in animal tumor models suggest that the antitumor effects of interleukin-2 (IL-2) or IL-2 in combination with lymphokine-activated killer (LAK) cells can be enhanced by chemotherapy agents such as cyclo-phosphamide or doxorubicin or by the biologic agent interferon α. Purpose We determined the toxicity and clinical response rate of an IL-2-LAK cell regimen modified by the addition of moderate, immunomodulatory doses of chemotherapy and sequenced with interferon alfa-2a (IFN α-2a) in patients with metastatic melanoma and renal cell carcinoma. Methods IL-2(3–6 million units/m2 per day) was administered by continuous infusion on days 0–5 and days 11–16. LAK cells were infused on days 11 and 12 or on days 11, 12, and 14. Low doses of cyclophosphamide (300 mg/ m2) and doxorubicin (25 mg/m2) were given on day 9 before the LAK cell infusions. Following the IL-2-LAK cell infusion, IFN α-2a (12 million units/m2) was administered for a total of nine doses to complete a cycle of treatment. A total of 89 patients were enrolled in the study. Results For each histology, there were eight partial responses in 40 assessable patients, for an overall response rate of 20% (90% confidence interval = 10%–33%). The median response duration was 5 months, although two patients with renal cell carcinoma and one patient with metastatic melanoma had almost complete disappearance of tumor and are still responding after 26+, 22+, and 26+ months, respectively. Toxic effects were severe in patients receiving the highest dose of IL-2 administered in this study and similar to those reported with other high-dose IL-2-LAK cell regimens. Although toxic effects were completely reversible in most patients, there were four treatment-related deaths. Conclusions This regimen is active in patients with metastatic melanoma and renal cell carcinoma and produces meaningful responses in a small percentage of these patients; however, it is not clear whether cyclophosphamide, doxorubicin, and IFN a-2α as used in this protocol appreciably augmented the antitumor activity of the IL-2-LAK cell regimen.

Background: Toxicity to interleukin-2 (IL-2) tumor immunotherapy is manifested principally by the vascular leak syndrome, hypotension, and a hyper-dynamic response with low systemic vascular resistance. Nitric oxide(·N = O), a recently discovered biological mediator of vascular smooth muscle relaxation, is produced in increased amounts by numerous cell types exposed to a number of inflammatory cytokines. Purpose: Our purpose was to determine if there is an increased production of ·N = O in patients receiving IL-2 tumor immunotherapy, and, if so, whether increases in ·N = O production correlate with hemodynamic instability. Methods: Twelve patients undergoing immunotherapy trials with IL-2 and anti-CD3 monoclonal antibody-activated lymphocytes (T-AK cells) were studied. Plasma levels of nitrate (NO3−), the stable end metabolic product of ·N = O synthesis, were measured before and at the end of IL-2 treatment cycles. Results: We observed a ninefold increase in plasma levels of NO3− in patients after 7 days of treatment (P<.0001). A significant decrease in both systolic and diastolic blood pressures was observed in all patients (P<.001). Conclusions: We propose that mediated induction of ·N = O synthase enzyme leads to progressive increases in ·N = O production which, in turn, produces clinically significant hypotension. Implications: Since ·N = O synthesis can be competitively inhibited by L-arginine analogues, a possible pharmacologic modulation of N = O production could potentially contribute to better management of toxic side effects seen in IL-2 cancer therapies. [J Natl Cancer Inst 84: 864–867, 1992]

Background: Dolastatins 10 and 15 are small peptides isolated from the marine sea hare Dolabella auricularia. In vitro studies of these peptides have demonstrated antimitotic and antiproliferative activity and growth inhibition in hematopoietic progenitor cells. Purpose: The purpose of our in vitro study was to determine the biological effects of these marine peptides on growth of human lymphoma cell lines and to investigate mechanisms by which the dolastatins may act. Methods: Cell lines DB, HT, RL, and SR were grown from the ascites or pleural effusion of four patients with lymphoma. The DB, HT, and RL cell lines are of B-cell origin, and the SR cell line appears to be a less differentiated lymphoid cell type. Cells from these lines were cultured in the presence of vincristine or dolastatin 10 or 15. [3H]Thymidine-uptake assays were used to measure effects on DNA synthesis. Cell cycle analysis using propidium iodide was performed to measure drug-induced cell-cycle arrest. DNA fragmentation was used as an assay for drug-induced apoptosis and was measured by agarose gel electrophoresis. Results: In the three B cell lines, dolastatin 10 was more effective than dolastatin 15. Values for concentrations required for inhibition of proliferation by 50% (IC50) were.00013-.0013 nM for dolastatin 10 in each cell line; values for dolastatin 15 were approximately.13 nM in DB and HT cells and.0013-.013 nM in RL cells. SR cells were more sensitive to dolastatin 15 than to dolastatin 10 (IC50 = .00013-.0013 nM versus.0013-.013 nM). Both dolastatins arrested more than 70% of cells in mitosis in all cell lines. This effect was reversed if the drug was removed by 4 hours, but by 8 hours of exposure, reversal was not possible. Both dolastatins 10 and 15 produced apoptosis in DB and HT cells but not in the other two cell lines. Conclusions: We have demonstrated that dolastatins 10 and 15 have a profound antiproliferative effect on four different human lymphoma cell lines and that the dolastatins are approximately 3–4 logarithms more effective as anti-proliferative compounds, on a molar basis, than vincristine—a clinically useful, antiproliferative agent. These data support the hypothesis that apoptosis, as measured by DNA fragmentation, appears to be a cell-specific response and may not be directly related to the antimitotic effect of the dolostatins. Implications: Our results suggest that these compounds may be good candidates for development as antineoplastic agents. [J Natl Cancer Inst 85: 483–488, 1993]