A pilot study with a therapeutic vaccine based on hydroxyapatite ceramic particles and self-antigens in cancer patients

Abstract

We describe an approach to produce an autologous therapeutic antitumor vaccine using hydroxyapatite (HA) for vaccinating cancer patients. The novel approach involved (1) the purification of part of the self-tumor antigens/ adjuvants using column chromatography with HA, (2) the employ of HA as a medium to attract antigen-presenting cells (APCs) to the vaccination site, and (3) the use of HA as a vector to present in vivo the tumor antigens and adjuvants to the patient's APCs. The vaccine was prepared using and combining HA particles, with at least 3 heat shock proteins (gp96 was one of them possibly with chaperoned proteins/peptides as shown in the slot blots) and with proteins from the cell membrane system (including Hsp70, Hsp27, and membrane proteins). The timing of HA degradation was tested in rats; the HA particles administered under the skin attracted macrophages and were degraded into smaller particles, and they were totally phagocytized within 1 week. In patients (n = 20), the vaccine was then administered weekly and showed very low toxicity, causing minor and tolerable local inflammation (erythema, papule, or local pain); only 1 patient who received a larger dose presented hot flashes, and there were no systemic manifestations of toxicity or autoimmune diseases attributed to the vaccine. Our study suggests that this therapeutic vaccine has shown some efficacy producing a positive response in certain patients. Stable disease was noted in 25% of the patients (renal carcinoma, breast carcinoma, and astrocytoma), and a partial response was noted in 15% of the patients (breast carcinoma and astrocytoma). The most encouraging results were seen in patients with recurrent disease; 4 patients in these conditions (20%) are disease free following the vaccine administration. However, we do not want to overstate the clinical efficacy in this small number of patients. The therapeutic vaccine tested in our study is working by activating the T-cell response as was shown in the comparative histological and immunohistochemical study performed in the pre- and postvaccine biopsy taken from a patient with inflammatory breast carcinoma. However, we cannot ruled out that the vaccine could also be producing an antibody(ies)-mediated response. In conclusion, this therapeutic vaccine based on HA ceramic particles and self-antigens can be safely administered and is showing some encouraging clinical results in cancer patients.

Fig 1.

Effect of the HA administration in rats. (A) The skin was separated from the muscle of the limb to show the congested injection area (arrows) 7 days after HA administration. (B) Photomicrograph showing large deposits of HA (brown) 2 hours after HA administration. Note the beginning of an inflammatory response around the HA. (C) This photomicrograph shows the increased inflammatory response noted 2 days after HA administration. (D) For days after HA administration, showing the mononuclear cell infiltration. (E) For days after HA administration, a large deposit of HA and degranulation of HA can be seen. The arrows point to macrophages engulfing the small HA granules. (F) Same as above but immunostained to reveal CD68+ macrophages (arrows). Photomicrographs taken from tissue sections stained with hematoxylin and eosin (B–D), stained with hematoxylin alone (E), and after immunohistochemistry (F). Bar = 45 μm (B–D); bar = 28 μm (E–F)

Fig 2.

Purification of the vaccine components. (A) Slot blot to reveal gp96 after HA column chromatography. Lane 1: positive control; lane 2: negative control; lane 3: flow-through after column equilibration; lanes 4–6: fractions collected after column washing with 50 mM NaCl; lanes 7–9: fractions collected after column washing with 100 mM NaCl; lanes 10, 11 and 14: fractions collected after column washing with 200 mM NaCl; lanes 15–17: fractions collected after column washing with 300 mM NaCl; lanes 18–20: fractions collected after column washing with 400 mM NaCl; lanes 21, 22: fractions collected after column washing with 500 mM NaCl. (B) SDS-PAGE and silver staining of the fractions collected from the HA chromatography column. Note that the fractions collected with 200 mM of NaCl contained the highest amount of gp96 (arrow), which appeared as a doublet (lanes 4–6). The other lanes were loaded with fractions obtained washing with lower and higher mM of NaCl concentrations (as shown in the slot blot). (C) Western blot showing gp96 as a doublet (arrow) after HA purification. (D) Slot blots of the gp96 purification fractions to show examples of the identified proteins. lane 1: β-catenin; lane 2: Her-2/neu; lane 3: P-cadherin; lane 4: survivin. (E) Western blot to reveal Hsp70 after membrane purification with sucrose gradient centrifugation. Lanes 1 and 2: loaded with 20 and 10 μg of proteins obtained from a patient with a parotid adenocarcinoma; lanes 3 and 4: loaded with 20 and 10 μg of proteins obtained from a patient with a renal clear cell carcinoma. Note that lane 3, loaded with a higher protein concentration, shows 2 Hsp70 bands: the upper corresponds to the constitutive form and the lower corresponds to the inducible form of Hsp70. (F) Slot blots of the purified membranes to show examples of the identified proteins. Lane 1: Her2/neu; lane 2: Hsp27; lane 3: P-cadherin; lane 4: β-catenin

Fig 3.

Patient with renal carcinoma (Table 2, patient 1), effect of the vaccine on a large bone metastasis in the humerus (X-rays). (A) Prevaccine. (B) and (C) 5 months after vaccine administration. One year after the last vaccine administration this large lesion (4.5 cm) showed again progressive disease, but the minor size bone lesions (<1 cm) disappeared or presented stable disease

Fig 4.

Patient with inflammatory breast carcinoma (Table 2, patient 3), effect of the vaccine on the skin lesions. (A) Prevaccine. The arrow points to the area where the biopsy was taken to generate the vaccine. (B) 15 days after the beginning of vaccine administration. (C) 30 days after the beginning of vaccine administration. (D) 60 days after the beginning of vaccine administration. (E) 90 days after the beginning of vaccine administration. (F) 180 days after the beginning of vaccine administration

Fig 5.

Immunohistochemical evaluation of the lymphoid cells in the pre- and postvaccine (180 days) biopsies taken from a patient with inflammatory breast carcinoma (Table 2, patient 3; Fig. 4). (A) and (B) Hematoxylin and eosin staining to show the tumor cells infiltrating the skin. (C) and (D) T cells evaluated by CD43. (E) and (F) T cells evaluated by CD45Ro. (G) and (H) NK cells evaluated by CD57. (I) and (J) Macrophages evaluated by CD68. (K) and (L) B cells evaluated by CD20. (M) and (O) Leukocytes evaluated by CD15. Bar = 60 μm (A–F); bar = 100 μm (G–O)

Fig 6.

Effect of the vaccine treatment on 2 grade III recurrent astrocytomas. Patient 13, Table 2 (A–D); patient 12, Table 2 (E–H). MRI with gadolinium for tumor visualization. (A) The arrows show the tumor in this prevaccine image. (B) 30 days after the beginning of the vaccine. (C) 180 days after the beginning of the vaccine. (D) 330 days after the beginning of the vaccine. (E) The arrows show the tumor in this prevaccine image. (F) 30 days after the beginning of the vaccine. (G) 180 days after the beginning of the vaccine. (H) 270 days after the beginning of the vaccine. In both cases note the reduction of the tumor mass, a minor gadolinium uptake in the final MRIs, and the enlargement of the ventricle (retraction like a scar)