Neoantigen and tumor antigen-specific immunity transferred from immunized donors is detectable early after allogeneic transplantation in myeloma patients

Abstract

To enhance the therapeutic index of allogeneic hematopoietic SCT (HSCT), we immunized 10 HLA-matched sibling donors before stem cell collection with recipient-derived clonal myeloma Ig, idiotype (Id), as a tumor antigen, conjugated with keyhole limpet hemocyanin (KLH). Vaccinations were safe in donors and recipients. Donor-derived KLH- and Id-specific humoral and central and effector memory T-cell responses were detectable by day 30 after HSCT and were boosted by post-transplant vaccinations at 3 months in most recipients. One patient died before booster vaccinations. Specifically, after completing treatment, 8/9 myeloma recipients had persistent Id-specific immune responses and 5/9 had improvement in disease status. Although regulatory T cells increased after vaccination, they did not impact immune responses. At a median potential follow-up period of 74 months, 6 patients are alive, the 10 patients have a median PFS of 28.5 months and median OS has not been reached. Our results provide proof of principle that neoantigen and tumor antigen-specific humoral and cellular immunity could be safely induced in HSCT donors and passively transferred to recipients. This general strategy may be used to reduce relapse of malignancies and augment protection against infections after allogeneic HSCT.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that there are no competing financial interests.

Figures

Figure 1. Clinical trial schema

Multiple myeloma (MM) patients underwent plasmapheresis after enrollment to isolate myeloma idiotype (Id) from plasma and were treated with three to five cycles of etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and fludarabine (EPOCH-F) induction chemotherapy every 21 days. While MM patients were receiving EPOCH-F, their HLA-matched sibling donors were immunized with Id-KLH+GM-CSF vaccine three times at the indicated time points and peripheral blood stem cells were harvested 3–4 weeks after third vaccination. Patients received reduced-intensity preparative chemotherapy with fludarabine and cyclophosphamide (Flu/CTX) prior to stem cell transfer. MM Patients received three post-transplant immunizations with Id-KLH+GM-CSF vaccine at the indicated time points.

Figure 2. KLH and Id-specific antibody responses were induced in the donors and transferred to recipients

Prevaccine (pre-V) or pre-hematopoietic stem cell transplant (pre-SCT) and postvaccine (post-V) or post-SCT serum samples from the indicated time points in the donors (A, B, E, G) and recipients (C, D, F, H) were tested in parallel for KLH (A–D) and Id- (E–H) specific antibody responses by ELISA as described in the Materials and Methods. Post-SCT samples at 4 mo, 6 mo, 7 mo and 9 mo were obtained one month after the 1st, two months after the 2nd, and one and three months after the 3rd post-SCT vaccination, respectively. Vaccination time points are indicated by arrows as V1, V2, and V3 in the donors (A) and recipients (C). (A–F) Sample optical density (OD) measurements at a serum dilution of 1:32 are shown. Horizontal bars indicate median for each group. Significant increase (P < 0.05) in antibody titers in postvaccine or post-SCT groups compared with prevaccine or pre-SCT groups respectively is indicated by an asterisk. Significant anti-Id antibody responses in individual patients are indicated by #. P values were calculated by Wilcoxon signed-rank test. Antibody responses against KLH were either IgM (A, C) or IgG (B, D) subtype. (G, H) Representative anti-Id antibody titration curves in donor 8 and recipient 8 are shown. Serum samples from the indicated time points were tested at various dilutions for reactivity against vaccinated Id or isotype-matched Id proteins of irrelevant specificity (Irr Id).

Figure 3. Cellular responses against KLH

Cryopreserved pre and postvaccine or pre and post-hematopoietic stem cell transplant (pre- and post-SCT) PBMC samples from the indicated time points in the donors (D1–D10) (A) and recipients (R2–R10) (B) were tested in parallel for reactivity against KLH in a cytokine induction assay as described in the Materials and Methods. Post-SCT samples at 4 mo, 6 mo, 7 mo and 9 mo were obtained one month after the 1st, two months after the 2nd, and one and three months after the 3rd post-SCT vaccinations, respectively. Vaccination time points are indicated as V1, V2, and V3 in the donors (A) and recipients (B). KLH-specific cytokine production was calculated by subtracting cytokines produced by PBMC in the absence of antigen from that in the presence of KLH at each time point. KLH-specific cytokine production is presented as a heat map according to the scale shown.

Figure 3. Cellular responses against KLH

Cryopreserved pre and postvaccine or pre and post-hematopoietic stem cell transplant (pre- and post-SCT) PBMC samples from the indicated time points in the donors (D1–D10) (A) and recipients (R2–R10) (B) were tested in parallel for reactivity against KLH in a cytokine induction assay as described in the Materials and Methods. Post-SCT samples at 4 mo, 6 mo, 7 mo and 9 mo were obtained one month after the 1st, two months after the 2nd, and one and three months after the 3rd post-SCT vaccinations, respectively. Vaccination time points are indicated as V1, V2, and V3 in the donors (A) and recipients (B). KLH-specific cytokine production was calculated by subtracting cytokines produced by PBMC in the absence of antigen from that in the presence of KLH at each time point. KLH-specific cytokine production is presented as a heat map according to the scale shown.

Figure 4. Cellular responses against Id

Fresh (A, B) or cryopreserved (C, D) pre and postvaccine or pre and post-hematopoietic stem cell transplant (pre- and post-SCT) PBMC samples from the indicated time points in Donor 2 (A) and recipients R2, R3, and R5) (B–D) were tested for reactivity against Id or irrelevant Id (Irrel. Id) in a cytokine induction assay as described in the Materials and Methods. Post-SCT samples at 4 mo, 6 mo, 7 mo and 9 mo were obtained one month after the 1st, two months after the 2nd, and one and three months after the 3rd post-SCT vaccinations, respectively. Vaccination time points for the recipient are indicated as V1, V2, and V3 (C). Cytokine production in each well is shown (A, B). Id-specific cytokine production was calculated by subtracting cytokines produced by PBMC in the absence of antigen from that in the presence of Id at each time point (C, D).

Figure 4. Cellular responses against Id

Fresh (A, B) or cryopreserved (C, D) pre and postvaccine or pre and post-hematopoietic stem cell transplant (pre- and post-SCT) PBMC samples from the indicated time points in Donor 2 (A) and recipients R2, R3, and R5) (B–D) were tested for reactivity against Id or irrelevant Id (Irrel. Id) in a cytokine induction assay as described in the Materials and Methods. Post-SCT samples at 4 mo, 6 mo, 7 mo and 9 mo were obtained one month after the 1st, two months after the 2nd, and one and three months after the 3rd post-SCT vaccinations, respectively. Vaccination time points for the recipient are indicated as V1, V2, and V3 (C). Cytokine production in each well is shown (A, B). Id-specific cytokine production was calculated by subtracting cytokines produced by PBMC in the absence of antigen from that in the presence of Id at each time point (C, D).

Figure 5. Frequency of KLH-specific T cells in donors and recipients

Cryopreserved PBMC samples from various time points in donors (A, B) and recipients (C, D) were cultured in medium alone, KLH, or bovine serum albumin (BSA) for 24 h with Brefeldin A added for the last 14 h. Production of TNF-α (A, C) and IL-2 (B, D) was assessed by intracellular cytokine staining as described in Materials and Methods. Vaccination time points are indicated by arrows as V1, V2, and V3 in the donors (A) and recipients (C). Frequency of KLH-specific CD4+ T cells was calculated by subtracting cytokine-producing CD4+ T cells in the absence of KLH from that in the presence of KLH at each time point. Significant increase (P < 0.05) in KLH-specific CD4+ T cells in donor postvaccine or recipient post-SCT groups compared with donor prevaccine group is indicated by an asterisk. P values were calculated by Wilcoxon signed-rank test. Post-HSCT samples at 4 mo, 6 mo, and 7 mo were obtained one month after the 1st, two months after the 2nd, and one month after the 3rd post-SCT vaccination, respectively. KLH-specific CD4+ T cells producing TNF-α or IL-2 were detected in 7 of 8 evaluable recipients at 90 days post-SCT and in 8 of 8 following post-transplant immunizations.

Figure 6. Regulatory T cells (Tregs) in donors and recipients

(A, B) The percentage of Foxp3+ T cells in the peripheral blood CD4+ T cells and PBMC was determined by flow cytometry and methylation status of the Foxp3 gene, respectively in donors (A) and recipients (B). The absolute number of Foxp3+ T cells in the peripheral blood was calculated as described in Materials and Methods. P values were calculated by paired t-test for donors D2–D10. A significant increase in the percentage and absolute number of Foxp3+ cells was observed at postvaccine 1 time point in 8 of 10 donors as compared with prevaccine time point (D2–D10: p<0.01, paired T-test). (C). Cryopreserved PBMC from 7 mo post-HSCT time point from two recipients were cultured for 24 h in medium alone, KLH, or phorbol-12-myristate-13-acetate (PMA) and ionomycin. Intracellular staining was performed to determine the production of TNF-α and IL-2 in CD4+Foxp3+ and CD4+Foxp3− T cells. Representative data from recipient 7 is shown.