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Clinical Trial
. 2007 Sep 14:5:43.
doi: 10.1186/1479-5876-5-43.

Adjuvant therapeutic vaccination in patients with non-small cell lung cancer made lymphopenic and reconstituted with autologous PBMC: first clinical experience and evidence of an immune response

Dominik Rüttinger  1 , Natasja K van den Engel, Hauke Winter, Marcus Schlemmer, Heike Pohla, Stefanie Grützner, Beate Wagner, Dolores J Schendel, Bernard A Fox, K-W Jauch, Rudolf A Hatz
Affiliations
Clinical Trial

Adjuvant therapeutic vaccination in patients with non-small cell lung cancer made lymphopenic and reconstituted with autologous PBMC: first clinical experience and evidence of an immune response

Dominik Rüttinger et al. J Transl Med. .

Abstract

Background: Given the considerable toxicity and modest benefit of adjuvant chemotherapy for non-small cell lung cancer (NSCLC), there is clearly a need for new treatment modalities in the adjuvant setting. Active specific immunotherapy may represent such an option. However, clinical responses have been rare so far. Manipulating the host by inducing lymphopenia before vaccination resulted in a magnification of the immune response in the preclinical setting. To evaluate feasibility and safety of an irradiated, autologous tumor cell vaccine given following induction of lymphopenia by chemotherapy and reinfusion of autologous peripheral blood mononuclear cells (PBMC), we are currently conducting a pilot-phase I clinical trial in patients with NSCLC following surgical resection. This paper reports on the first clinical experience and evidence of an immune response in patients suffering from NSCLC.

Methods: NSCLC patients stages I-IIIA are recruited. Vaccines are generated from their resected lung specimens. Patients undergo leukapheresis to harvest their PBMC prior to or following the surgical procedure. Furthermore, patients receive preparative chemotherapy (cyclophosphamide 350 mg/m2 and fludarabine 20 mg/m2 on 3 consecutive days) for induction of lymphopenia followed by reconstitution with their autologous PBMC. Vaccines are administered intradermally on day 1 following reconstitution and every two weeks for a total of up to five vaccinations. Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is given continuously (at a rate of 50 microg/24 h) at the site of vaccination via minipump for six consecutive days after each vaccination.

Results: To date, vaccines were successfully manufactured for 4 of 4 patients. The most common toxicities were local injection-site reactions and mild constitutional symptoms. Immune responses to chemotherapy, reconstitution and vaccination are measured by vaccine site and delayed type hypersensitivity (DTH) skin reactions. One patient developed positive DTH skin tests so far. Immunohistochemical assessment of punch biopsies taken at the local vaccine site reaction revealed a dense lymphocyte infiltrate. Further immunohistochemical differentiation showed that CD1a+ cells had been attracted to the vaccine site as well as predominantly CD4+ lymphocytes. The 3-day combination chemotherapy consisting of cyclophosphamide and fludarabine induced a profound lymphopenia in all patients. Sequential FACS analysis revealed that different T cell subsets (CD4, CD8, CD4CD25) as well as granulocytes, B cells and NK cells were significantly reduced. Here, we report on clinical safety and feasibility of this vaccination approach during lymphoid recovery and demonstrate a patient example.

Conclusion: Thus far, all vaccines were well tolerated. The overall trial design seems safe and feasible. Vaccine site reactions associated with infusion of GM-CSF via mini-pump are consistent with the postulated mechanism of action. More detailed immune-monitoring is required to evaluate a potential systemic immune response. Further studies to exploit homeostasis-driven T cell proliferation for the induction of a specific anti-tumor immune response in this clinical setting are warranted.

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Figures

Figure 1
Figure 1
Outline of the clinical trial protocol. Non-small cell lung cancer patients stages I-IIIa undergo curative surgery to prepare an autologous vaccine. The autologous tumor cells are physically minced and digested by a triple enzyme solution. The vaccine is then irradiated at 10,000 rads, tested for sterility and cryopreserved. Immediately following the intradermal vaccination (biweekly, total of up to five vaccinations) GM-CSF is infused subcutaneously for 6 days at a rate of 50 μg/24 h. Prior to vaccination plus GM-CSF administration, lymphopenia is induced by a 3-day combination chemotherapy (cyclophosphamide 350 mg/m2 and fludarabine 20 mg/m2) followed by reconstitution with autologous peripheral blood mononuclear cells. Two additional leukaphereses are harvested pre- and post-vaccination for immunemonitoring purposes. (CTX: chemotherapy)
Figure 2
Figure 2
A. Intradermal application on the abdominal wall; B. Continuous infusion of GM-CSF at the vaccine site using a minipump. Vaccines are administered intradermally on alternating sides of the abdominal wall (A). Before clinical administration, the irradiated autologous tumor cells are thawed, washed extensively, and resuspended in 1 mL of sterile saline. Immediately following the vaccine application a catheter is placed at the vaccine site and GM-CSF is infused continuously at a rate of 50 μg/24 h for a total of 6 days using an osmotic minipump (Smiths Medical, Kirchseeon, Germany) (B).
Figure 3
Figure 3
Local vaccine site reaction and immunohistochemistry of punch biopsies. (A) Typical vaccine site reaction 48 h following the third vaccination. Clinically, the reaction was characterized by erythema, induration and pruritus. Note the catheter for local infusion of GM-CSF placed in the center of the vaccine application site. Punch biopsies were taken at the reaction site and assessed immunohistochemically. Histopathological assessment of punch biopsy specimens taken at the local vaccine site reaction 48 h following the third vaccine revealed a dense infiltrate consisting predominantly of macrophages, eosinophils, neutrophils and lymphocytes (B, ×100). In contrast to unrelated skin sites, CD1a-positive dendritic cells were present at the vaccine site (C, ×400). Further immunohistochemical staining showed that most of the CD3-positive lymphocytes (D, ×400) in the patient with the strongest vaccine site reaction were CD4-positive cells (E, ×100).
Figure 4
Figure 4
Baseline chest x-ray (left) and MRI scan (right) of patient 1. Note the large tumor originating from the upper right lobe of the lung (arrows). The patient was resected with an upper bilobectomy (pT2 pN2, IIIA) and systematic lymphadenectomy and the diagnosis of NSCLC (adenocarcinoma) was confirmed.
Figure 5
Figure 5
Analysis of absolute white blood cell count (WBC), neutrophil count and flow-cytometric assessment of lymphocytes, T cell subsets and natural killer cells in patient 1 during the treatment phase. After preparative chemotherapy (CTX) (cyclophosphamide 350 mg/m2 and fludarabine 20 mg/m2) the patient was reconstituted with 0.9 × 1010 autologous PBMC (5.05 × 107 CD3-positive cells/kg) followed by 5 cycles of the autologous tumor cell vaccine. All of the determined CD3-positive cell subsets (CD4, CD8, CD4CD25) were affected by cyclophosphomide and fludarabine. As seen in all patients, neutrophil counts recovered to pre-chemotherapy levels within 30 days. Recovery of different T cell subsets (CD4, CD8, CD4CD25) was slower than normalization of neutrophil counts in all patients but varied inter-individually. The post-chemotherapy increase in CD4 numbers followed the same kinetics as the other subsets with no extended depression as one might expect following fludarabine treatment. Patient 1 experienced an extended depression of CD19-positive B lymphocytes, whereas NK cells (CD3-CD16+CD56+) recovered within 30 days (as observed in all patients). Additional leukaphereses were harvested prior to preparative chemotherapy and after the vaccination phase.
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