Preclinical evaluation of a MAGE-A3 vaccination utilizing the oncolytic Maraba virus currently in first-in-human trials

Abstract

Multiple immunotherapeutics have been approved for cancer patients, however advanced solid tumors are frequently refractory to treatment. We evaluated the safety and immunogenicity of a vaccination approach with multimodal oncolytic potential in non-human primates (NHP) (Macaca fascicularis). Primates received a replication-deficient adenoviral prime, boosted by the oncolytic Maraba MG1 rhabdovirus. Both vectors expressed the human MAGE-A3. No severe adverse events were observed. Boosting with MG1-MAGEA3 induced an expansion of hMAGE-A3-specific CD4+ and CD8+ T-cells with the latter peaking at remarkable levels and persisting for several months. T-cells reacting against epitopes fully conserved between simian and human MAGE-A3 were identified. Humoral immunity was demonstrated by the detection of circulating MAGE-A3 antibodies. These preclinical data establish the capacity for the Ad:MG1 vaccination to engage multiple effector immune cell populations without causing significant toxicity in outbred NHPs. Clinical investigations utilizing this program for the treatment of MAGE-A3-positive solid malignancies are underway (NCT02285816, NCT02879760).

Keywords: Cancer immunotherapy; MAGE-A3; MG1 Maraba; Oncolytic vaccination; endogenous immunity.

Figures

Figure 1.

Ad-MAGEA3 and MG1-MAGEA3 are well tolerated by non-human primates. (A) Study timeline in cynomolgus macaques. MG1-MAGEA3 was infused twice (3 days apart) at low (1 x 1010 PFU; ‘lo”) or high (1 x 1011 PFU; “hi”) doses, either as a standalone treatment (cohorts M-lo and M-hi; n = 4 each) or sequentially after receiving one intramuscular injection of 1 × 1010 PFU Ad-MAGEA3. For the latter setting, three intervals between Ad-MAGEA3 and MG1-MAGEA3 injections were tested: 2 weeks (cohorts AM2w-lo and AM2w-hi; n = 4 each), 4 weeks (cohort AM4w-hi; n = 4) or 6 weeks (cohorts AM6w-lo and AM6w-hi; n = 4 each). Blood samples were collected at the indicated time-points for immunogenicity assessments. Of note, the AM6w cohorts were dedicated to assessing the immunogenicity of the Ad-MAGEA3:MG1-MAGEA3 prime-boost and were not enrolled in the toxicology study. Body weight change relative to body weight at day 0 (cohorts M, AM2w and AM4w; n = 20) (B,C) and rectal temperatures (all macaques; n = 28) (D,E) by treatment arm (MG1-MAGEA3 alone versus Ad-MAGEA3:MG1-MAGEA3 regardless of the interval) or MG1-MAGEA3 dose (low dose versus high dose regardless of the treatment arm). Fever was classified as a rectal temperature ≥ 39.8°C (indicated by the horizontal dashed line). Body weight change is illustrated as mean ± SD and temperature as mean ± SEM. p-values (linear mixed models) are indicated and were considered significant when p < 0.05. MAGE-A3, human melanoma-associated antigen, family A, member 3; PFU, plaque-forming unit.

Figure 2.

Ad-MAGEA3 primed CD8+ T-cell population against hMAGE-A3. hMAGE-A3-specific CD8+ T-cell responses were detected by intracellular staining of IFNγ following ex vivo re-stimulation with pools of overlapping peptides covering the full length MAGE-A3 antigen. (A,B) Pre-immune and MG1-MAGEA3-mediated prime responses against hMAGE-A3 were measured before and 9 days after receiving MG1-MAGEA3. MG1-MAGEA3 was delivered systemically at two doses (3 days apart) of 1e10 PFU to the M-lo cohort (n = 4, A) or 1e11 PFU to the M-hi cohort (n = 4, B). (C,D) The Ad-MAGEA3 vaccine was administered intramuscularly at 1e10 PFU to a total of 20 macaques in the AM2w-lo, AM2w-hi, AM4w-hi, AM6w-lo and AM6w-hi cohorts. CD8+ T-cell responses against hMAGE-A3 were measured before and 14 days after Ad-MAGEA3 administration. Ad-MAGEA3-mediated prime response was also measured at day 28 in the AM4w-hi cohort (n = 4) and at day 42 in the AM6w-lo and AM6w-hi cohorts (n = 8). Mean hMAGE-A3-specific prime response is displayed both as the count (cells per µl of blood; C) and as the frequency (% among total circulating CD8+ T-cell population); D) of IFNγ-producing CD8+ T-lymphocytes. Histograms represent mean ± SD. p-value considered non significant (NS) when > 0.05; **p < 0.01 (Wilcoxon’s paired test for A and B; Dunn’s test for C and D). hMAGE-A3, human melanoma-associated antigen, family A, member 3; IFNγ, interferon gamma; PFU, plaque-forming unit.

Figure 3.

Ad-MAGEA3:MG1-MAGEA3 induced CD4+ T-cell reactivity against hMAGE-A3. hMAGE-A3-educated CD4+ T-cells were quantified by intracellular staining of IFNγ following ex vivo (re-)stimulation with pools of overlapping peptides covering the full length MAGE-A3 antigen. (A, B) Kinetics of CD4+ T-cell responses against hMAGE-A3 in the 20 macaques that received the Ad-MAGEA3:MG1-MAGEA3. Response is displayed both as the count (cells per µl of blood; A) and as the frequency (% among total CD4+ T-lymphocytes; B) of IFNγ-producing CD4+ T-lymphocytes. Histograms show mean ± SD. p-value (Dunn’s test) considered non significant (NS) when > 0.05; *p < 0.05, ***p < 0.001, ****p < 0.0001. (C, D) Peak CD4+ T-cell boost responses against hMAGE-A3 in each individual macaque, ranked according to their magnitude expressed as absolute count per µl of blood (C). Corresponding MG1-MAGEA3 boost responses expressed as the frequency (%) of reactive cells inside the total CD4+ T-cell population (D). Bars illustrate the number of IFNγ-producing CD4+ T-cells per µl blood, with the dashed line indicating pre-immune baseline (set to 0.1 reactive CD4+ T-cells/µl blood). Sub-responses to each of the nine peptide pools used for antigenic (re-)challenge are color-coded: pool 1–10 in red, pool 11–20 in white, pool 21–30 in pink, pool 31–40 in green, pool 41–50 in blue, pool 51–60 in yellow, pool 61–70 in purple, pool 71–80 in orange and pool 81–87 in grey. hMAGE-A3, human melanoma-associated antigen, family A, member 3; IFNγ, interferon gamma.

Figure 4.

Marked expansion of the hMAGE-A3-specific CD8+ T-cell population following MG1-MAGEA3 boost. (A,B) Kinetics of CD8+ T-cell responses against hMAGE-A3 in 20 macaques treated with Ad-MAGEA3:MG1-MAGEA3. Response is displayed both as the count (cells per µl of blood; A) and as the frequency (% among total CD8+ T-lymphocytes; B) of IFNγ-producing CD8+ T-lymphocytes. Histograms represent mean ± SD. p-value (Dunn’s test) considered non significant (NS) when > 0.05; *p < 0.05, **p < 0.01. (C-E) Illustrations of the three different kinetics of the boost response witnessed across the vaccinated macaques. (F, G) Peak CD8+ T-cell boost responses against hMAGE-A3 in each individual macaque, ranked according to their magnitude with bottom 10 and top 10 responders respectively displayed in the panels F and G. (H, I) Corresponding MG1-MAGEA3 boost responses expressed as the frequency (%) of reactive cells inside the total CD8+ T-cell population. (C-I) Sub-responses to each of the nine pools of hMAGE-A3 peptides used for antigenic re-stimulation are color-coded: pool of peptides 1–10 in red, 11–20 in white, 21–30 in pink, 31–40 in green, 41–50 in blue, 51–60 in yellow, 61–70 in purple, 71–80 in orange and 81–87 in grey. (J) Fold by which hMAGE-A3-reactive CD8+ T-cell population expanded between day 0 and the peak response day following MG1-MAGEA3 boost. In the absence of any detectable prime response in AM2w-lo 2, its boost fold was arbitrarily set to 200. Red dashed line illustrates boost fold of 1 (i.e. failure of MG1 boost). Green dotted line and dashed line illustrate median and mean boost folds, respectively. hMAGE-A3, human melanoma-associated antigen, family A, member 3; IFNγ, interferon gamma.

Figure 5.

Genotypic and biological traits impacting on the profile of Ad:MG1 vaccination. (A) Distribution of the CD8+ T-cell reactivity against the different regions of the hMAGE-A3 antigen following Ad-MAGEA3:MG1-MAGEA3 immunization. Each region was color-coded according to the peptide pool covering it, as follows: the N-terminal extremity was encompassed within the pool of peptides 1–10 in red, the inner regions were covered by the pools of peptides 11–20 in white, 21–30 in pink, 31–40 in green, 41–50 in blue, 51–60 in yellow, 61–70 in purple and 71–80 in orange while the C-terminal extremity was covered by the peptides 81–87 in grey. (B-D) Quality of vaccine immunogenicity was assessed by measuring the proportion of IFNγ+ reactive CD8+ T-cells that co-produced TNFα. (B) Flow cytometry gating strategy. CD8+ lymphocytes were first gated (top left plot – magenta frame). In the child gates (right), IFNγ TNFα double positive CD8+ lymphocytes were counted following each (re-)stimulation with one of the nine peptide pools from the hMAGE-A3 library (pool 1–10 to pool 81–87). Illustrated dot plots were recorded in the AM6w-lo 4 macaque who best responded to Ad-MAGEA3 prime:MG1-MAGEA3 boost. (C) Quality of the CD8+ T-lymphocyte reactivity illustrated as the percentage of IFNγ+ T cells co-producing TNFα upon ex vivo (re-)exposure to hMAGE-A3 peptides in each macaque immunized with Ad-MAGEA3:MG1-MAGEA3. (A, C) Primates are ranked according to the magnitude of the CD8+ T-cell boost response illustrated in the Figure 5E-F, from the weakest (on the left) to strongest (on the right). Green dashed line illustrates both mean (62%) and median (61%) TNFα positivity values. (D) Quality of vaccine immunogenicity according to the age of the macaque (3 year-old versus 4 to 6 year-old subjects). Histograms represent mean ± SD. p-value (Dunn’s test) considered non significant (NS) when > 0.05; *p < 0.05. hMAGE-A3, human melanoma-associated antigen, family A, member 3; IFNγ, interferon gamma; TNFα, tumor necrosis factor alpha.

Figure 6.

Ad-MAGEA3:MG1-MAGEA3 induces auto-reactive T-cells. 5x105 PBMCs were seeded and re-stimulated for 24 hours with one of the 87 overlapping peptides that cover the complete sequence of the hMAGE-A3 antigen. Reactivity against each peptide was detected by ELISpot. (A) Representative epitope mapping from the AM6w-hi4 macaque. Spots of T-lymphocytes secreting IFNγ following exposure to each peptide were counted and displayed in a bar graph. Cut-off was set to 10 spots (dashed line). Peptides with highly conserved sequences between human and simian MAGE-A3 were labeled as follows: ** = sequences identical or * = sequences diverging by one amino acid located at an extremity. (B) Reactivity against each of the highly conserved MAGE-A3 peptides was measured in 16 macaques at the indicated time-point post-MG1-MAGEA3 injection and reported as the number of spots detected. For each macaque, highlighting illustrates the relative immunogenicity of each peptide expressed as the percentage of the response measured against the immunodominant peptide. The immunodominant peptide was determined as the peptide leading to the highest number of IFNγ+ T-cell spots and consisted, except in AM6w-hi4, of a poorly conserved epitope not displayed in the table. For instance, in AM6w-hi4, as indicated by the red highlighting, the conserved peptide #42 turned out immunodominant with 263 spots detected (100% immunogenicity) while the conserved peptide #31 was subdominant with 128 spots detected (49% immunogenicity of the immunodominant peptide #42 illustrated by the yellow highlight). MAGE-A3, human melanoma-associated antigen, family A, member 3; IFNγ, interferon gamma; PBMCs, peripheral blood mononuclear cells.