Autologous lysate-pulsed dendritic cell vaccination followed by adoptive transfer of vaccine-primed ex vivo co-stimulated T cells in recurrent ovarian cancer

Abstract

Novel strategies for the therapy of recurrent ovarian cancer are warranted. We report a study of a combinatorial approach encompassing dendritic cell (DC)-based autologous whole tumor vaccination and anti-angiogenesis therapy, followed by the adoptive transfer of autologous vaccine-primed CD3/CD28-co-stimulated lymphocytes. Recurrent ovarian cancer patients for whom tumor lysate was available from prior cytoreductive surgery underwent conditioning with intravenous bevacizumab and oral metronomic cyclophosphamide, sequentially followed by (1) bevacizumab plus vaccination with DCs pulsed with autologous tumor cell lysate supernatants, (2) lymphodepletion and (3) transfer of 5 × 10⁹ autologous vaccine-primed T-cells in combination with the vaccine. Feasibility, safety as well as immunological and clinical efficacy were evaluated. Six subjects received this vaccination. Therapy was feasible, well tolerated, and elicited antitumor immune responses in four subjects, who also experienced clinical benefits. Of these, three patients with residual measurable disease received outpatient lymphodepletion and adoptive T-cell transfer, which was well tolerated and resulted in a durable reduction of circulating regulatory T cells and increased CD8⁺ lymphocyte counts. The vaccine-induced restoration of antitumor immunity was achieved in two subjects, who also demonstrated clinical benefits, including one complete response. Our findings indicate that combinatorial cellular immunotherapy for the treatment of recurrent ovarian cancer is well tolerated and warrants further investigation. Several modifications of this approach can be envisioned to optimize immunological and clinical outcomes.

Keywords: DC vaccines; adoptive T-cell transfer; autologous immunotherapy.

Figure 1. Radiological response in UPCC-11807. (A) Radiological assessments were performed by CT (CT) at enrollment or baseline (BL), and at the end of study (EOS). Two subjects also had evaluation after completing bevacizumab and metronomic cyclophosphamide and before starting vaccine (pre-vac). Volumes of all individual tumor metastases are shown. Estimated tumor volumes were calculated based on the formula V = ½ S * ½ S * L, where L is the longest and S is the shortest tumor diameter. Tumors were measured on the CT section that had the biggest tumor dimensions. Subject (S)-01 and -02 achieved partial response. S-02 demonstrated disease progression on bevacizumab and metronomic cyclophosphamide, although her lesions regressed after vaccine administration. S-03 had overall bulky disease, while S-04 had stable no evidence of disease. Subjects S-05 and S-06 exhibited disease progression. (B) Example of a left para-aortic lymph node regressing in S-01 post-vaccination CT (right) relative to pre-vaccination CT (left). (C) S-04 experienced remission inversion with progression-free survival (PFS3) being twice as long as PFS2.

Figure 2. Lymphocyte changes during UPCC-11807 and UPCC-10808. (A) CD4⁺CD25⁺FOXP3⁺ regulatory T cell (Treg) frequency during therapy. No Treg reduction was seen following bevacizumab and metronominc cyclophosphamide. A mild (statistically significant) reduction in Treg frequency was seen after vaccine plus bevacizumab relative to pre-vaccine. Two out of three subjects exhibited marked and sustained reduction in circulating Tregs after T -cell transfer. (B) Circulating CD4:CD8 T-cell ratio as measured at baseline and at the end of the study (EOS). Longitudinal assessment of tumor-reactive T cells by interferon γ (IFNγ) ELISpot in S-01, S-02 and S-03 during UPCC-10808.

Figure 3. Immune responses in UPCC-11807. (A,B) Thawed peripheral blood mononuclear cells (PBMCs) obtained at baseline (BL) and at the end of study (EOS) were co-incubated, in the absence of additional stimulation, with autologous DCs pulsed with autologous tumor-cell lysates or control DCs for ~18 h and evaluated by interferon γ (IFNγ) ELISpot. (A) Representative data of T-cell responses. Tumor-specific responses are not detectable among PBMCs collected prior to therapy or after two doses of bevacizumab and metronomic cyclophosphamide, demonstrating the requirement for vaccine to elicit antitumor immunity. S-02 was HLA-A0201, allowing for the detection of immune responses to HER2 (H2N)- or hTERT-derived epitopes. A response is seen against HER2_689–697. (B) Summary of vaccine-induced T cell responses. A positive response was detected in S-01, -02, -03 and -04. (C) Humoral responses induced by the vaccine. Sera from S-01 and S-02 were profiled on ProtoArray^® Human Protein Microarrays v. 4.1, containing more than 8,000 human proteins. Increased IgG and IgM seropositivity is detected against human proteins post-vaccine as compared with pre-vaccine. Y-axes indicate the number of proteins recognized by serum antibodies. (D) CD3⁺ tumor-infiltrating T-cells (top) and MHC Class I expression levels (bottom) in tumors at BL. (E) Table depiciting the scoring system.

Figure 4. Hematopoietic reconstitution in UPCC-10808. (A) Lymphocytes purified from peripheral blood mononuclear cells (PBMCs) by elutriation were stimulated and expanded for ~11 d using Dynal microbeads coated with anti-CD3 and anti-CD28 antibodies. Expansion of three subjects and two normal donors is shown. A population doubling level (PDL) average of 5.14 is seen for three subjects with ovarian cancer. (B) Expanded T cells from S-02 were tested by interferon γ (IFNγ) ELISpot for cross-reactivity against non-transduced or HLA-A2-transduced SKOV3 cells, T2 cells pulsed with HER2/neu 369 or 689 peptides or unpulsed T2 cells, autologous dendritic cells (DCs) pulsed with SKOV3-cell lysates or unpulsed DCs, or media (T cells only). Prior to assessments, T cells were incubated for 7 d with autologous DCs pulsed with SKOV3-cell lysates prepared either by freeze-thawing (FTL) or UV B (UVB) irradiation. Results are means of the number of IFNγ spots per 10 T cells ± SEM of triplicates (p < 0.001; paired Student’s t-test) (C) Absolute lymphocyte count (ALC), white blood cells (WBC) and absolute neutrophil count (ANC) in subjects receiving adoptive T-cell transfer. S-02 (black) and S-03 (gray) received pegfilgrastim on the day of T-cell transfer.

Figure 5. (A,B) Clinical design of UPCC-11807 (A) and UPCC-10808 (B).