Maintenance therapy with ex vivo expanded lymphokine-activated killer cells and rituximab in patients with follicular lymphoma is safe and may delay disease progression.

Anti-cluster of differentiation 20 (CD20) monoclonal antibodies (mAbs) have shown promise in follicular lymphoma (FL) as post-induction therapy, by enhancing antibody-dependent cellular cytotoxicity (ADCC). However, cytotoxic cells are reduced after this treatment. We hypothesised that ex vivo expanded lymphokine-activated killer (LAK) cells administered to FL-remission patients are safe and improve anti-CD20 efficacy. This open, prospective, phase II, single-arm study assessed safety and efficacy of ex vivo expanded LAK cells in 20 FL-remission patients following rituximab maintenance. Mononuclear cells were obtained in odd rituximab cycles and stimulated with interleukin 2 (IL-2) for 8 weeks, after which >5 × 108 LAK cells were injected. Patients were followed-up for 5 years. At the end of maintenance, peripheral blood cells phenotype had not changed markedly. Natural killer, LAK and ADCC activities of mononuclear cells increased significantly after recombinant human IL-2 (rhIL-2) stimulation in all cycles. Rituximab significantly enhanced cytotoxic activity. No patients discontinued treatment. There were no treatment-related serious adverse events. Three patients had progressed by the end of follow-up. After a median (interquartile range) follow-up of 59.4 (43.8-70.9) months, 85% of patients remained progression free. No deaths occurred. Quality-of-life improved throughout the study. Post-induction LAK cells with rituximab seem safe in the long term. Larger studies are warranted to confirm efficacy.

Functional specific-T-cell expansion after first cytomegalovirus reactivation predicts viremia control in allogeneic hematopoietic stem cell transplant recipients.

The use of preemptive antiviral therapy to prevent cytomegalovirus (CMV) disease in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients might result in over-treatment, inducing drug-related toxicity and viral resistance. A search for predictive markers is needed to determine requirement for antiviral therapy. Clinical follow-up, in combination with the use of streptamers (STs) and cytokine-intracellular staining, could help to identify patients at high risk for CMV reactivations. To study the immune response and reactivation control by CMV-specific CD8+ T-cell (CMV-CTL) populations, we monitored 25 patients who have undergone allo-HSCT by using ST multimer and intracellular cytokine staining. Our study has revealed that the presence of functional CMV-specific T cells, determined by early interferon γ production or by significant T-cell expansion after first CMV reactivation, correlated with short CMV viremia duration and low number of CMV reactivations. By contrast, the absence of functional CMV-CTLs does correlate with CMV recurrence. These results support that behavior of CMV-specific subpopulations after reactivation influences reactivations and can guide preemptive therapy.

Phase II trial of ofatumumab plus ESHAP (O-ESHAP) as salvage treatment for patients with relapsed or refractory classical Hodgkin lymphoma after first-line chemotherapy.

The management of recurrent/refractory (R/R) Hodgkin lymphoma (HL) remains challenging. Previously published data have shown some efficacy of rituximab in this setting. The purpose of this phase II trial was to investigate the activity of ofatumumab in combination with etoposide, steroids, cytarabine and cisplatin (O-ESHAP) in 62 patients with R/R classical HL. Treatment consisted of ESHAP plus ofatumumab 1000 mg on days 1 and 8 of the first cycle and day 1 of the second and third cycles. O-ESHAP was well tolerated with only 3% of patients requiring treatment discontinuation because of adverse events. Overall response rate was 73% (44% complete metabolic response). In multivariate analysis, early relapse (P < 0·001), bulky disease (P < 0·001) and B symptoms (P < 0·001) were the most important prognostic factors for response. No failures of stem cell mobilization were observed. The high response rate, particularly the complete metabolic response rate, the low toxicity profile, and the high mobilizing potential of the O-ESHAP regimen suggest that patients with R/R HL may benefit from this salvage regimen. However, with the encouraging results observed with other new therapeutic agents in HL, the O-ESHAP regimen could be restricted to patients failing these agents or to those with R/R nodular lymphocyte-predominant HL.

Follicular lymphoma: in vitro effects of combining lymphokine-activated killer (LAK) cell-induced cytotoxicity and rituximab- and obinutuzumab-dependent cellular cytotoxicity (ADCC) activity.

Follicular lymphoma (FL) is a disease of paradoxes-incurable but with a long natural history. We hypothesized that a combination of lymphokine-activated killer (LAK) cells and monoclonal antibodies might provide a robust synergistic treatment and tested this hypothesis in a phase II clinical trial (NCT01329354). In this trial, in addition to R-CHOP, we alternated the administration of only rituximab with rituximab and autologous LAK cells that were expanded ex vivo. Our objective was to determine the in vitro capability of LAK cells generated from FL patients to produce cytotoxicity against tumor cell lines and to determine rituximab- and obinutuzumab-induced cytotoxicity via antibody-dependent cellular cytotoxicity (ADCC) activity. We analyzed the LAK cell-induced cytotoxicity and rituximab (R)- and obinutuzumab (GA101)-induced ADCC activity. We show that LAK cells generated from FL patients induce cytotoxicity against tumor cell lines. R and GA101 enhance cytolysis through ADCC activity of LAK cells. Impaired LAK cell cytotoxicity and ADCC activity were detected in 50 % of patients. Percentage of NK cells in LAK infusions were correlated with the R- and GA101-induced ADCC. Our results indicate that the combination of R or GA101 and LAK cells should be an option as frontline maintenance therapy in patients with FL.

Impact of T cell selection methods in the success of clinical adoptive immunotherapy.

Chemotherapy and/or radiotherapy regular regimens used for conditioning of recipients of hematopoietic stem cell transplantation (SCT) induce a period of transient profound immunosuppression. The onset of a competent immunological response, such as the appearance of viral-specific T cells, is associated with a lower incidence of viral infections after haematopoietic transplantation. The rapid development of immunodominant peptide virus screening together with advances in the design of genetic and non-genetic viral- and tumoural-specific cellular selection strategies have opened new strategies for cellular immunotherapy in oncologic recipients who are highly sensitive to viral infections. However, the rapid development of cellular immunotherapy in SCT has disclosed the role of the T cell selection method in the modulation of functional cell activity and of in vivo secondary effects triggered following immunotherapy.

The abrogation of TCR-independent interactions with human serum ensures a selective capture of therapeutic virus-specific CD8+ T-cells by multimer technology in Adoptive Immunotherapy.

Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8(+) T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes. Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8(+) T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy.

Prolonged idiotypic vaccination against follicular lymphoma.

During the last 2 decades, idiotypic vaccination has provided proof of principle of biological efficacy, clinical efficacy and clinical benefit in small follicular lymphoma trials. However, with the exception of anecdotal reports, most patients have received no more than 10 doses of their customised idiotype (Id) vaccine. Therefore, it is not known whether prolonged usage of idiotypic vaccination is safe. Since 2002, 18 previously treated patients with follicular lymphoma have received extended idiotypic vaccination at our institution outside clinical trials. Vaccination was provided as a compassionate alternative to no further treatment, and was meant to be stopped only upon complete consumption of the available patient- and tumor-specific vaccine [Id-keyhole limpet hemocyanin + granulocyte-macrophage colony-stimulating factor (Id-KLH + GM-CSF)], or in case of disease relapse or any serious non-local toxicity. So far, 18 patients have received an average of 18 doses of Id vaccine (median: 17; mean: 18; range: 10-31). Eleven patients are still actively receiving idiotypic vaccination: some of them are now over more than 6 years. Toxicity has been systematically negligible and mostly local. No patient has abandoned the vaccination program because of toxicity. Prolonged idiotypic vaccination with the soluble protein Id-KLH + GM-CSF formulation is safe and well tolerated.

Human follicular lymphoma cells contain oligomannose glycans in the antigen-binding site of the B-cell receptor.

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

Clinical benefit associated with idiotypic vaccination in patients with follicular lymphoma.

BACKGROUND: Follicular lymphoma is considered incurable, although cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy can induce sequential remissions. A patient's second complete response is typically shorter than that patient's first complete response. Idiotype vaccines can elicit specific immune responses and molecular remissions in patients with follicular lymphoma. However, a clinical benefit has never been formally proven. METHODS: Thirty-three consecutive follicular lymphoma patients in first relapse received six monthly cycles of CHOP-like chemotherapy. Patients who achieved a second complete response were vaccinated periodically for more than 2 years with autologous lymphoma-derived idiotype protein vaccine. Specific humoral and cellular responses were assessed, and patients were followed for disease recurrence. Statistical tests were two-sided. RESULTS: Idiotype vaccine could be produced for 25 patients who had a second complete response. In 20 patients (80%), a humoral (13/20) and/or a cellular (18/20) idiotype-specific response was detected. The median duration of the second complete response has not been reached, but it exceeds 33 months (range = 20+ to 51+ months). None of the 20 responders relapsed while undergoing active vaccination. All responders with enough follow-up for the comparison to be made experienced a second complete response that was statistically significantly (P<.0001) longer than both their first complete response (18 of 18 patients) and than the median duration of a CHOP-induced second complete response, i.e., 13 months (20 of 20 patients). The five nonresponders all had a second complete response that was shorter (median = 10 months; range = 8-13 months) than their first complete response (median = 17 months; range = 10-39 months). CONCLUSIONS: Idiotypic vaccination induced a specific immune response in the majority of patients with follicular lymphoma. Specific immune response was associated with a dramatic and highly statistically significant increase in disease-free survival. This is the first formal demonstration of clinical benefit associated with the use of a human cancer vaccine.

Combined vaccination with idiotype-pulsed allogeneic dendritic cells and soluble protein idiotype for multiple myeloma patients relapsing after reduced-intensity conditioning allogeneic stem cell transplantation.

BACKGROUND AND OBJECTIVE: To combine the use of idiotype-pulsed allogeneic dendritic cells (alloDC) and soluble protein Id conjugated with KLH (Id-KLH) in a vaccine strategy for multiple myeloma (MM). DESIGN AND METHODS: Four MM patients received the combined vaccine after having experienced disease relapse/progression following reduced intensity conditioning (RIC) allogeneic stem cell transplantation (alloSCT) and failure to rescue therapy with donor lymphocyte infusion or chemotherapy (CHT). RESULTS: Vaccination was well tolerated and induced an anti-KLH antibody response in all 4 patients as well as substantial cell proliferation. In contrast, no case showed similar effects against either tumor-specific Id or irrelevant isotype control immunoglobulins (Ig). In turn, vaccination was associated with modulation of biological responses linked to both inflammatory and T-cell activation, with secretion of effector Th1 cytokines. In particular, an important increase in the spontaneous ex vivo secretion of TNFalpha, IL-6 and IFNgamma as well as IL-2 and IL-10 was frequently observed prior to the fourth vaccination. Moreover, in vitro stimulation with Id-KLH and Id-KLH plus alloDC, but not with alloDC alone was associated with an enhanced number of TNF-alpha+ T-cells and an increased secretion of IFNgamma and IL-2 before the third and fourth vaccination. From a clinical standpoint, 2 patients had a transient response and 1 has stable disease after stopping vaccination, while 3 of them ultimately progressed. INTERPRETATION AND CONCLUSIONS: The results show for the first time that the use of Id-pulsed alloDC following RIC alloSCT is safe and feasible. However, crucial strategy improvements are warranted to possibly achieve clinical benefit.

Variations in "rescuability" of immunoglobulin molecules from different forms of human lymphoma: implications for anti-idiotype vaccine development.

Idiotypic (Id) vaccination has shown promising results in patients with follicular lymphoma (FL). However, it still remains unclear whether the same approach might be suitable for the treatment of other B-cell malignancies. For this reason, we recently performed an interim analysis of patients proposed to receive this treatment at our center. The feasibility of employing idiotype vaccines was evaluated for five different B-cell malignancies in their first relapse, both in terms of induction and fusion, as well as overall treatment. Our data suggest that, unlike follicular lymphoma (87%), this approach is not feasible to treat other B-cell malignancies (0-20%) such as mantle cell, small lymphocytic, diffuse large cell and Burkitt's lymphoma (P < 0.01). The main difficulties encountered were technical problems related to the survival of idiotype-producing hybridomas (83%) and the early loss of idiotype production by growing hybridomas (17%). However, it remains possible that an idiotype vaccine might still be produced through molecular means for most, if not all cases of relapsing B-cell malignancies.

Positron emission tomography using 18F-fluorodeoxyglucose for the evaluation of residual Hodgkin's disease mediastinal masses.

Given its obvious prognostic implications, the correct interpretation of the significance of any residual mediastinal mass following Hodgkin's disease (HD) treatment keeps maintaining its paramount importance. In this respect, 18F-fluorodeoxyglucose positron emission tomography (PET) is proving very effective for both active disease detection and relapse prediction. Twenty-nine consecutive HD patients, in whom computed tomography (CT) scan performed after therapy completion had documented a residual mediatinal mass of at least 2 cm, prospectively entered the study and underwent PET within 1 week from CT scan. With a median follow-up of 28 months from PET execution, no relapse was recorded among the 17 patients presenting with a negative PET. On the contrary, 9 of the 12 patients presenting with a positive PET relapsed/progressed within one year from PET execution. PET's negative and positive predictive values at 1 year were 100% and 75%, respectively. A negative PET seems to possibly exclude relapse in HD patient with a residual mediastinal mass. On the contrary, a positive PET result indicates a significantly higher risk of relapse. However, due to possible false positive results, a closer follow-up for all and a pathologic study in few selected patients is warranted.

Acquired potential N-glycosylation sites within the tumor-specific immunoglobulin heavy chains of B-cell malignancies.

BACKGROUND AND OBJECTIVES: Among B-cell malignancies, follicular lymphomas (FL) more frequently show acquired, potential N-glycosylation sites (AGS) within tumor-specific immunoglobulin. The aim of this study was to extend this observation and to evaluate the pattern of presentation of AGS within five different forms of B-cell lymphoma. DESIGN AND METHODS: We sequenced the tumor-specific immunoglobulin heavy chain variable region fragment, including complementarity-determining regions 2 and 3, of forty-seven consecutive patients with a B-cell malignancy enrolled in idiotype vaccine clinical trials. This sequencing approach is known to allow the identification of most AGS. We then statistically analyzed differences in presentation pattern, in terms of tumor histology, immunoglobulin isotype, AGS location and amino acid composition. RESULTS: All twenty-four FL cases presented with at least one AGS, whereas the vast majority of four B-cell lymphoma types other than FL did not. The non- FL group of tumors included four cases of Burkitt's lymphoma, six of diffuse large cell lymphoma, seven mantle cell lymphomas and six small lymphocytic lymphomas. Most IgM-bearing follicular lymphoma cases featured their AGS within complementarity-determining region 2, as opposed to those bearing an IgG, which mostly displayed the AGS within complementarity-determining region 3. The vast majority of AGS located within either complementarity-determining region ended with a serine residue, whereas those located within framework regions mostly featured threonine as the last amino acid residue. INTERPRETATION AND CONCLUSIONS: In our series, all cases of FL had AGS within their tumor-specific immunoglobulin heavy chain variable regions. In contrast, most B-cell malignancies other than FL did not. Further studies are warranted in order to establish the possible meaning of these findings in terms of disease pathogenesis, their diagnostic value in doubtful cases and their potential implications for immunotherapy.

Clinical implications of antigen transfer mechanisms from malignant to dendritic cells. exploiting cross-priming.

Expansion and activation of cytolytic T lymphocytes bearing high-affinity T-cell receptors specific for tumor antigens is a major goal of active cancer immunotherapy. Physiologically, T cells receive promitotic and activating signals from endogenous professional antigen-presenting cells (APC) rather than directly from malignant cells. This phenomenon fits with the broader concept of cross-presentation that earlier was demonstrated for minor histocompatibility and viral antigens. Many mechanisms have been found to be capable of transferring antigenic material from malignant cells to APC so that it can be processed and subsequently presented by MHC class I molecules expressed on APC. Dendritic cells (DC) are believed to be the most relevant APC mediating cross-presentation because they can take up antigens from apoptotic, necrotic, and even intact tumor cells. There exist specific molecular mechanisms that ensure this transfer of antigenic material: 1) opsonization of apoptotic bodies; 2) receptors for released heat shock proteins carrying peptides processed intracellularly; 3) Fc receptors that uptake immunocomplexes and immunoglobulins; and 4) pinocytosis. DC have the peculiar capability of reentering the exogenously captured material into the MHC class I pathway. Exploitation of these pieces of knowledge is achieved by providing DC with complex mixtures of tumor antigens ex vivo and by agents and procedures that promote infiltration of malignant tissue by DC. The final outcome of DC cross-presentation could be T-cell activation (cross-priming) but also, and importantly, T-cell tolerance contingent upon the activation/maturation status of DC. Artificial enhancement of tumor antigen cross-presentation and control of the immune-promoting status of the antigen-presenting DC will have important therapeutic implications in the near future.

An anti-ICAM-2 (CD102) monoclonal antibody induces immune-mediated regressions of transplanted ICAM-2-negative colon carcinomas.

Monoclonal antibodies (mAbs) can mediate antitumor effects by indirect mechanisms involving antiangiogenesis and up-regulation of the cellular immune response rather than by direct tumor cell destruction. From mAbs raised by immunization of rats with transformed murine endothelial cells, a mAb (EOL4G8) was selected for its ability to eradicate a fraction of established colon carcinomas that did not express the EOL4G8-recognized antigen. The antigen was found to be ICAM-2 (CD102). Antitumor effects of EOL4G8, which required a functional T-cell compartment, were abrogated by depletion of CD8(+) cells and correlated with antitumor CTL activity, whereas only a mild inhibition of angiogenesis was observed. Interestingly, we found that EOL4G8 acting on endothelial ICAM-2 markedly enhances leukotactic factor activity-1-independent adhesion of immature dendritic cells to endothelium-an effect that is at least in part mediated by DC-SIGN (CD209).

Upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12.

OBJECTIVE: Injection of dendritic cells (DC) engineered with recombinant adenoviral vectors to produce interleukin-12 (IL-12) inside experimental murine tumors frequently achieves complete regressions. In such a system the function of CD8(+) T cells has been shown to be an absolute requirement, in contrast to observations made upon depletion of CD4(+) T cells, which minimally affected the outcome. The aim of this work was to study the possible involvement of natural killer (NK) cells in this setting. MATERIALS, METHODS, AND RESULTS: Depletions with anti-AsialoGM1 antiserum showed only a small decrease in the proportion of complete regressions obtained that correlated with induction of NK activities in lymphatic tissues into which DC migrate, whereas combined depletions of CD4(+) and NK cells completely eliminated the antitumor effects. Likewise in vivo neutralization of interferon-gamma (IFN-gamma) also eliminated those therapeutic effects. Trying to define the cellular role played by NK cells in vivo, it was observed that injection of cultured DC inside the spleen of T- and B-cell-deficient (Rag1(-/-)) mice induced upregulation of NK activity only if DC had been adenovirally engineered to produce IL-12. In addition, identically transfected fibroblasts also activated NK cells, indicating that IL-12 transfection was the unique requirement. Equivalent human DC only activated in vitro the cytolytic and cytokine-secreting functions of autologous NK cells if transfected to express human IL-12. CONCLUSIONS: Overall, these results point out an important role played by NK cell activation in the potent immunotherapeutic effects elicited by intratumoral injection of IL-12--secreting DC and that NK activation under these conditions is mainly, if not only, dependent on IL-12.

Effective tumor immunotherapy: start the engine, release the brakes, step on the gas pedal,...and get ready to face autoimmunity.

Cellular immune responses can destroy cancer cells, achieving the cure of experimental malignancies. An expanding wealth of knowledge on the molecular basis of how to prime and amplify a T cell response has fueled a number of strategies successful at treating established tumors (rather than merely preventing tumor grafting). The most efficacious approaches operate at different stages, including: 1) priming the immune response using tumor antigen-expressing dendritic cells or tumor cells transfected with genes that render them immunogenic, 2) sustaining and amplifying immunity using agonistic monoclonal antibodies against costimulatory molecules or immune-potentiating cytokines, and 3) eliminating mechanisms that self-regulate the strength of the immune response, such as inhibitory receptors or regulatory T cells. A rational combination of such approaches holds great hope for cumulative and synergistic effects, but there is also evidence that they can open the flood-gates for unwanted inflammatory reactions. The next decade can be envisioned as the time when the first reproducibly efficacious combination regimes for cancer immunotherapy will become available and widely used in the clinic, as clinicians learn the best strategies and try to harness their potentially damaging effects.