key: cord-0292697-cqb6115s
authors: Ylösmäki, Erkko; Fusciello, Manlio; Uoti, Arttu; Feola, Sara; Martins, Beatriz; Aalto, Karri; Hamdan, Firas; Ciaro, Jacopo; Viitala, Tapani; Cerullo, Vincenzo
title: A personalized anti-cancer vaccine for melanoma based on an approved vaccine against measles, mumps, and rubella
date: 2021-06-08
journal: bioRxiv
DOI: 10.1101/2021.06.07.447462
sha: ab238dcc27d72b367e90a839845d437a6552509c
doc_id: 292697
cord_uid: cqb6115s

Common vaccines for infectious diseases have been repurposed as cancer immunotherapies. Intratumoural administration of these repurposed vaccines can induce immune cell infiltration into the treated tumour. Here, we have used an approved trivalent live attenuated measles, mumps, and rubella (MMR) vaccine in our previously developed PeptiENV anti-cancer vaccine platform. Intratumoural administration of this novel MMR-containing PeptiENV anti-cancer vaccine significantly increased both intratumoural as well as systemic tumour-specific T cell responses. In addition, PeptiENV therapy, in combination with immune checkpoint inhibitor therapy, significantly improved tumour growth control and survival as well as increased the number of mice responsive to immune checkpoint inhibitor therapy.

The measles, mumps, and rubella (MMR) vaccine is a trivalent vaccine that contains live attenuated 30 strains of measles, mumps, and rubella viruses. MMR vaccines are indicated for the routine 31 immunization of children for the prevention of measles, mumps, and rubella. Recently, common 32 vaccines for infectious diseases, such as a seasonal influenza vaccine, rotavirus vaccines and a 33 vaccine against yellow fever, have been repurposed as intratumoural immunotherapies for the 34 modulation of the tumour microenvironment (TME) 1-3 . By intratumoural administration of these 35 viral vaccines, they were able to elicit immunostimulatory effects, such as enhancement of infiltration 36 of cytotoxic T cells (CTLs), natural killer (NK) cells and CD4 + Th1 T helper cells, reduction of T 37 regulatory cells (Tregs) and in some cases, exert oncolytic properties 1-5 . Immune checkpoint 38 inhibitors (ICIs), a novel class of therapeutic antibodies targeting immune checkpoint molecules, such 39 as programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte-40 associated antigen 4 (CTLA-4), block the negative feedback systems within the tumour 41 microenvironment to activate pre-existing anti-tumour immune responses 6 . ICIs have demonstrated 42 induction of long-term tumour regression and durable responses in some cancer patients, with 43 response rates of 10-25% in the majority of cancers 7 . The common feature of the patients responding 44

to ICI therapy seems to be that they have an existing anti-tumour immunity and immune cell 45 infiltration in the tumour tissue already prior to ICI therapy 8, 9 . This pre-exiting anti-tumour immunity 46 can then be enhanced and rendered functional by the ICI therapy. However, the remaining 75%-90% 47 were measured by calliper (largest tumour diameter and perpendicular tumour diameter) every second 129 day, starting on the day tumours were first treated. All injections and tumour measurements were 130 performed under isoflurane anaesthesia. 131

For the vaccination experiment using naïve mice, 8-to 9-week-old immunocompetent female 132 C57BL/6JOlaHsd mice were subcutaneously treated on days 0, 2 and 15 with one vaccination dose 133 of Priorix alone, one vaccination dose of Priorix together with OVA control peptide, one vaccination 134 dose of PeptiENV-OVA Priorix, or phosphate buffered saline (PBS) as a mock-treated group. 135

Animals were sacrificed at day 20 and spleens were harvested for immunological analysis. For the 136 B16.OVA melanoma experiment, 8-to 9-week-old immunocompetent female C57BL/6JOlaHsd 137 mice were injected in the right flank with 350.000 B16.OVA melanoma cells, and were treated 6-, 8-138

and 20-days post tumour implantation with one vaccination dose of Priorix alone, one vaccination 139 dose of PeptiENV-OVA Priorix, peptides alone or PBS as a mock-treated group. Animals were 140 sacrificed at day 24 and tumours, spleens and tumour-draining lymph nodes were harvested for 141 immunological analysis. For the B16F10.9/K1 melanoma experiment, 8-to 9-week-old immuno-142 competent female C57BL/6JOlaHsd mice were injected in the right flank with 600.000 B16F10.9/K1 143 cells together with a 3:2 ratio (v:v) of Matrigel Basement Membrane Matrix High Concentration 144 (Corning, USA), and were treated 7-, 9-, and 21-days post tumour implantation with one vaccination 145 dose of Priorix alone, one vaccination dose of PeptiENV-Trp2 Priorix, or left untreated (Mock). 146

Groups receiving anti-PD-1 (InVivoMab, USA, clone RMP1-14) were injected intraperitoneally 147 three times per week with 100 µg/dose starting at day 12 post tumour implantation. Tumour growth 148 and survival was monitored until all mice in the control group had died or sacrificed (day 35 post-149 tumour implantation). For the assessment of induction of systemic anti-tumour T cell responses, 150 surviving mice were rechallenged with 1.2x10 6 B16F10.9/K1 cells in the left flank and followed for 151 secondary tumour development/growth. 152

The amount of SIINFEKL (OVA257-264)-specific activated, interferon-γ secreting T cells were 155 measured by ELISPOT assay (CTL, Ohio USA) according to the manufacturer's instructions. Briefly, 156 2 µg of SIINFEKL peptide was used to stimulate the antigen presenting cells. After 3 days of 157 stimulation, plates where stained and sent to CTL-Europe GmbH for counting of the spots. For data analysis, one-way or two-way ANOVA were used. All results are expressed as the mean ± 171 SEM. presented by the DCs, no DC activation was seen. In contrast to the peptide administration, when Priorix was administered alone, robust DC activation, but not SIINFEKL presentation was seen. 203

When PeptiENV-OVA or Priorix mixed with OVA control peptide was administered, efficient 204 presentation and DC activation was seen with both groups, indicating again that PeptiENV-OVA 205

Priorix can act as a potent adjuvant for the delivered peptides. To study the immunostimulatory potential and anti-tumour effects of the PeptiENV platform with 225

Priorix, we used a well-established syngeneic mouse melanoma model B16 expressing chicken 226 ovalbumin (OVA) as a model antigen 13 . When mice bearing B16.OVA tumours were treated 227 intratumourally with OVA-targeting PeptiENV (PeptiENV-OVA Priorix), Priorix, peptides alone or 228 vehicle (mock), we observed only a moderate increase in tumour growth control in the PeptiENV-229 OVA Priorix group as compared to other treatment groups ( Figure 4A ). We set a tumour size 230 threshold of 500 mm 3 for defining the responders in each treatment group. Treating mice with the 231 CPP-containing SIINFEKL peptide alone did not have an effect on tumour growth, with 2 mice 232 defined as responders to the therapy in this group. Similarly, in Priorix-and mock-treated groups 233 there were two responders in each group. In contrast, PeptiENV-OVA Priorix treatment had a minor 234 effect on tumour growth with 5 mice defined as responders for the therapy. We went on to analyse 235 whether there were any differences in immunological responses against the OVA antigen between 236 the treatment groups, and we first assessed whether there were any differences in the infiltration of 237 immune cells into the tumour microenvironment (TME). We did not see any differences in the 238 number of tumour-infiltrating lymphocytes or in the number of cytotoxic CD8 + T cells infiltrated into 239 the tumours between the different treatment groups. However, PeptiENV-OVA Priorix-treated mice 240 had significantly enhanced infiltration of tumour-specific CD8 + T cells into the TME as compared to 241 the tumours of Priorix-, peptide alone-or mock-treated mice ( Figure 4B ). In addition, we saw an 242 increase of tumour-specific CD8 + T cells in tumour draining lymph nodes of PeptiENV-OVA-treated 243 animals as compared to other groups ( Figure 4C ). In striking contrast to Priorix-, peptide alone-and 244 mock-treated mice, a significant induction of a systemic OVA-specific T cell response was also seen 245

in PeptiENV-OVA Priorix-treated mice ( Figure 4D) . and anti-PD-1 therapy was given intraperitoneally. Here, with the more immunogenic tumour model, 266

we set the tumour size threshold of 250 mm 3 for defining the responders in each treatment group. In 267 contrast to mock-treated animals, Priorix-and anti-PD-1-treated groups showed modest tumour 268 growth control with response rates of 10% and 18%, respectively. Priorix in combination with anti-269 PD-1-treated animals showed moderate tumour growth control with a 45% response rate. PeptiENV-270

Trp2 Priorix-treated animals showed efficient tumour growth control with a response rate of 60%. 271

Remarkably, PeptiENV-Trp2 Priorix in combination with anti-PD-1-treated animals showed the most 272 efficient tumour growth control, with a 91% response rate; increasing the response rate for anti-PD-273 1 therapy from 18% to 91% ( Figure 5A ). All treatment groups increased the survival of the animals 274 as compared to mock group (Figure 5B) , however, treatment with PeptiENV-Trp2 Priorix-or animals, and by the time all mice in the mock group had died, 100% of the PeptiENV-Trp2 Priorix 277 in combination with anti-PD-1-treated mice were still alive. In order to study systemic anti-tumour 278 immunity elicited by the different treatments, surviving mice were rechallenged with a very high 279 amount (1.2x10 6 , 2x the initial dose) of the same tumour cells into the contralateral flank. In this study, we have exploited the immunostimulatory characteristics of a safe and widely used 291 FDA and EMA-approved vaccine against measles, mumps and rubella, the MMR vaccine trade 292 named Priorix. One vaccine dose of Priorix contains very low amounts of highly attenuated strains 293 of measles, mumps, and rubella viruses. As all these vaccine viruses are enveloped, we set up to test 294 the suitability of Priorix vaccine in our recently developed peptide-based cancer vaccine platform for 295 enveloped viruses PeptiENV 11 . By using surface plasmon resonance (SPR) analysis, we showed that 296 cell penetrating peptide (CPP) sequence derived from HIV Tat protein can efficiently act as an 297 attachment moiety when fused to the N-terminus of tumour antigens. Based on our earlier study, 298 addition of the CPP sequence to the therapeutic peptides does not affect the presentation of tumour 299 epitopes from these CPP-containing peptides 11 . By using an immunodominant epitope from chicken 300 ovalbumin (OVA) as a model antigen, we were able to study the characteristics of antigen presentation and concomitant dendritic cell activation by the PeptiENV platform. PeptiENV-OVA 302

Priorix was able to efficiently deliver tumour antigens into dendritic cells and induce their robust 303 activation. Similar characteristics was also seen with Priorix mixed with OVA control peptide lacking 304 the CPP attachment moiety. These in vitro results are expected since pulsing dendritic cell monolayer 305 in a cell culture dish does not take into account the differences in diffusion kinetics of peptides and antigen-specific T cells as well as increased number of OVA antigen-specific T cells in tumour-322 draining lymph nodes. These results prompted us to test the PeptiENV Priorix platform in 323 combination with immune checkpoint inhibitors as these therapies can benefit from the increased 324 immune cell infiltration into the TME 9 . To test the effects of the PeptiENV Priorix platform in 325 combination with anti-PD-1 ICI on tumour growth and tumour-specific T cell induction, we used a syngeneic mouse model of B16.F10.9/K1 melanoma together with a more relevant tumour epitope 327 from endogenous tumour-associated antigen Trp2(180-188). Interestingly, both Priorix and anti-PD-1 328 monotherapies had a very minimal effect on tumour growth control, but the combination therapy 329 (Priorix + ICI) had an enhanced effect on tumour growth control. However, already as a monotherapy, 330

PeptiENV-Trp2 Priorix had a robust effect on tumour growth control, and when combined with anti-331 PD-1 therapy, the antitumoural effects were further enhanced. 332

Vaccine strains of measles and mumps have been extensively tested for oncolytic cancer virotherapy 333 15, 16 . However, the number of viral particles given intratumourally have typically been at the level of 334 10 6 infectious viral particles per dose, which is 100 to 1000 times more than what is used in this study 335 (one vaccine dose of Priorix contains approximately 10 3.0 CCID50 and 10 3.7 CCID50 of measles and 336 mumps, respectively). Indeed, we wanted to use the same FDA/EMA-approved dose that has safely 337 been used in vaccination programmes worldwide for vaccinating against measles, mumps, and 338 rubella. With the dose used, we did not see any oncolysis-driven anti-tumoural effects by the Priorix 339 vaccine, however, when combined with the PeptiENV platform, Priorix acted as an outstandingly 340 potent biological adjuvant for the attached tumour antigen peptides and was able to induce anti-341 tumoural effects through the stimulation of tumour antigen-specific T cell immunity. 342

Recently, common live attenuated vaccines for various infectious diseases, have been repurposed as 343 intratumoural immunotherapy for the modulation of the tumour microenvironment (TME) 1, 3 . As an 

Repurposing the yellow fever vaccine for intratumoral 398 immunotherapy

Intratumoral injection of the seasonal flu shot converts immunologically cold tumors to 407 hot and serves as an immunotherapy for cancer

Repurposing rotavirus vaccines for intratumoral immunotherapy can overcome resistance to 412 immune checkpoint blockade

Repurposing infectious disease vaccines for intratumoral 415 immunotherapy

Overcoming 417 cancer therapeutic bottleneck by drug repurposing

CTLA-4 and PD-1/PD-L1 blockade: new 419 immunotherapeutic modalities with durable clinical benefit in melanoma patients

Acquired Resistance to Immune Checkpoint 422 Inhibitors

Single-institution experience with 425 ipilimumab in advanced melanoma patients in the compassionate use setting: lymphocyte count 426 after 2 doses correlates with survival

Integrated NY-ESO-1 antibody 430 and CD8+ T-cell responses correlate with clinical benefit in advanced melanoma patients treated 431 with ipilimumab

Mechanisms of resistance to immune 433 checkpoint inhibitors

Personalized Cancer Vaccine Platform for Clinically Relevant Oncolytic Enveloped Viruses

Enveloped viruses

H-2Kb transfection of B16 melanoma cells 442 results in reduced tumourigenicity and metastatic competence

Attenuated oncolytic measles 445 virus strains as cancer therapeutics

Combination of Vaccine-Strain Measles and Mumps Viruses 448 Enhances Oncolytic Activity against Human Solid Malignancies

Vaccine repurposing approach for 451 preventing COVID 19: can MMR vaccines reduce morbidity and mortality?

In silico comparative study of SARS-CoV-2 455 proteins and antigenic proteins in BCG, OPV, MMR and other vaccines: evidence of a possible 456 putative protective effect

Homology May Contribute to Cross-Reactivity or to Complement Activation Protection

Protective 462 heterologous T cell immunity in COVID-19 induced by MMR and Tdap vaccine antigens

Tumour antigens can readily be 470 attached to the viral envelopes of measles, mumps, and rubella (MMR) vaccine viruses by using a cell penetrating peptide 471 (CPP) sequence as an anchoring moiety. Anchor-modified peptides are complexed for 15 minutes with the MMR vaccine 472 for allowing efficient attachment. Various different peptides, including MHC class I and II epitopes, can be delivered by 473 the PeptiENV-platform for potent activation of antigen-presenting cells and increased antigen-specific immunological 474 responses