Serum elevation of B lymphocyte stimulator does not increase regulatory B cells in glioblastoma patients undergoing immunotherapy.

Regulatory B cells that secrete IL-10 (IL-10(+) Bregs) represent a suppressive subset of the B cell compartment with prominent anti-inflammatory capacity, capable of suppressing cellular and humoral responses to cancer and vaccines. B lymphocyte stimulator (BLyS) is a key regulatory molecule in IL-10(+) Breg biology with tightly controlled serum levels. However, BLyS levels can be drastically altered upon chemotherapeutic intervention. We have previously shown that serum BLyS levels are elevated, and directly associated, with increased antigen-specific antibody titers in patients with glioblastoma (GBM) undergoing lymphodepletive temozolomide chemotherapy and vaccination. In this study, we examined corresponding IL-10(+) Breg responses within this patient population and demonstrate that the IL-10(+) Breg compartment remains constant before and after administration of the vaccine, despite elevated BLyS levels in circulation. IL-10(+) Breg frequencies were not associated with serum BLyS levels, and ex vivo stimulation with a physiologically relevant concentration of BLyS did not increase IL-10(+) Breg frequency. However, BLyS stimulation did increase the frequency of the overall B cell compartment and promoted B cell proliferation upon B cell receptor engagement. Therefore, using BLyS as an adjuvant with therapeutic peptide vaccination could promote humoral immunity with no increase in immunosuppressive IL-10(+) Bregs. These results have implications for modulating humoral responses in human peptide vaccine trials in patients with GBM.

BLyS levels correlate with vaccine-induced antibody titers in patients with glioblastoma lymphodepleted by therapeutic temozolomide.

B lymphocyte stimulator (BLyS) is a cytokine involved in differentiation and survival of follicular B cells along with humoral response potentiation. Lymphopenia is known to precipitate dramatic elevation in serum BLyS; however, the use of this effect to enhance humoral responses following vaccination has not been evaluated. We evaluated BLyS serum levels and antigen-specific antibody titers in 8 patients undergoing therapeutic temozolomide (TMZ)-induced lymphopenia, with concomitant vaccine against a tumor-specific mutation in the epidermal growth factor receptor (EGFRvIII). Our studies demonstrate that TMZ-induced lymphopenia corresponded with spikes in serum BLyS that directly preceded the induction of anti-EGFRvIII antigen-specific antibody titers, in some cases as high as 1:2,000,000. Our data are the first clinical observation of BLyS serum elevation and greatly enhanced humoral immune responses as a consequence of chemotherapy-induced lymphopenia. These observations should be considered for the development of future vaccination strategies in the setting of malignancy.

Intracerebral infusion of an EGFR-targeted toxin in recurrent malignant brain tumors.

The purpose of this study is to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and intracerebral distribution of a recombinant toxin (TP-38) targeting the epidermal growth factor receptor in patients with recurrent malignant brain tumors using the intracerebral infusion technique of convection-enhanced delivery (CED). Twenty patients were enrolled and stratified for dose escalation by the presence of residual tumor from 25 to 100 ng/ml in a 40-ml infusion volume. In the last eight patients, coinfusion of (123)I-albumin was performed to monitor distribution within the brain. The MTD was not reached in this study. Dose escalation was stopped at 100 ng/ml due to inconsistent drug delivery as evidenced by imaging the coinfused (123)I-albumin. Two DLTs were seen, and both were neurologic. Median survival after TP-38 was 28 weeks (95% confidence interval, 26.5-102.8). Of 15 patients treated with residual disease, two (13.3%) demonstrated radiographic responses, including one patient with glioblastoma multiforme who had a nearly complete response and remains alive >260 weeks after therapy. Coinfusion of (123)I-albumin demonstrated that high concentrations of the infusate could be delivered >4 cm from the catheter tip. However, only 3 of 16 (19%) catheters produced intraparenchymal infusate distribution, while the majority leaked infusate into the cerebrospinal fluid spaces. Intracerebral CED of TP-38 was well tolerated and produced some durable radiographic responses at doses

Hyaluronic acid based low viscosity hydrogel as a novel carrier for Convection Enhanced Delivery of CAR T cells.

Convection Enhanced Delivery (CED) infuses therapeutic agents directly into the intracranial area continuously under pressure. The convection improves the distribution of therapeutics such as those aimed at brain tumors. Although CED successfully delivers small therapeutic agents, this technique fails to effectively deliver cells largely due to cell sedimentation during delivery. To overcome this limitation, we have developed a low viscosity hydrogel (LVHydrogel), which is capable of retaining cells in suspension. In this study, we evaluated whether LVHydrogel can effectively act as a carrier for the CED of tumor-specific chimeric antigen receptor (CAR) T cells. CAR T cells were resuspended in saline or LVHydrogel carriers, loaded into syringes, and passed through the CED system for 5 h. CAR T cells submitted to CED were counted and the efficiency of delivery was determined. In addition to delivery, the ability of CAR T cells to migrate and induce cytotoxicity was evaluated. Our studies demonstrate that LVHydrogel is a superior carrier for CED in comparison to saline. The efficiency of cell delivery in saline carrier was only ∼3-5% of the total cells whereas delivery by the LVHydrogel carrier was much higher, reaching ∼45-75%. Migration and Cytotoxicity was similar in both carriers in non-infused samples but we found superior cytotoxicity in LVHydrogel group post-infusion. We demonstrate that LVHydrogel, a biodegradable biomaterial which does not cause acute toxicity on preclinical animal models, prevents cellular sedimentation during CED and presents itself as a superior carrier to the current carrier, saline, for the CED of CAR T cells.

Isolation and generation of human dendritic cells.

Dendritic cells are highly specialized antigen-presenting cells (APC), which may be isolated or generated from human blood mononuclear cells. Although mature blood dendritic cells normally represent ∼0.2% of human blood mononuclear cells, their frequency can be greatly increased using the cell enrichment methods described in this unit. More highly purified dendritic cell preparations can be obtained from these populations by sorting of fluorescence-labeled cells. Alternatively, dendritic cells can be generated from monocytes by culture with the appropriate cytokines, as described here. In addition, a negative selection approach is provided that may be employed to generate cell preparations that have been depleted of dendritic cells to be used for comparison in functional studies.