How integrin phosphorylations regulate cell adhesion and signaling.

Cell adhesion is essential for the formation of organs, cellular migration, and interaction with target cells and the extracellular matrix. Integrins are large protein α/ß-chain heterodimers and form a major family of cell adhesion molecules. Recent research has dramatically increased our knowledge of how integrin phosphorylations regulate integrin activity. Phosphorylations determine the signaling complexes formed on the cytoplasmic tails, regulating downstream signaling. α-Chain phosphorylation is necessary for inducing ß-chain phosphorylation in LFA-1, and the crosstalk from one integrin to another activating or inactivating its function is in part mediated by phosphorylation of ß-chains. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus receptor angiotensin-converting enzyme 2 (ACE2) and possible integrin coreceptors may crosstalk and induce a phosphorylation switch and autophagy.

Regulation of cell adhesion: a collaborative effort of integrins, their ligands, cytoplasmic actors, and phosphorylation.

Integrins are large heterodimeric type 1 membrane proteins expressed in all nucleated mammalian cells. Eighteen α-chains and eight ß-chains can combine to form 24 different integrins. They are cell adhesion proteins, which bind to a large variety of cellular and extracellular ligands. Integrins are required for cell migration, hemostasis, translocation of cells out from the blood stream and further movement into tissues, but also for the immune response and tissue morphogenesis. Importantly, integrins are not usually active as such, but need activation to become adhesive. Integrins are activated by outside-in activation through integrin ligand binding, or by inside-out activation through intracellular signaling. An important question is how integrin activity is regulated, and this topic has recently drawn much attention. Changes in integrin affinity for ligand binding are due to allosteric structural alterations, but equally important are avidity changes due to integrin clustering in the plane of the plasma membrane. Recent studies have partially solved how integrin cell surface structures change during activation. The integrin cytoplasmic domains are relatively short, but by interacting with a variety of cytoplasmic proteins in a regulated manner, the integrins acquire a number of properties important not only for cell adhesion and movement, but also for cellular signaling. Recent work has shown that specific integrin phosphorylations play pivotal roles in the regulation of integrin activity. Our purpose in this review is to integrate the present knowledge to enable an understanding of how cell adhesion is dynamically regulated.

Phosphorylation of the α-chain in the integrin LFA-1 enables ß2-chain phosphorylation and α-actinin binding required for cell adhesion.

The integrin leukocyte function-associated antigen-1 (LFA-1) plays a pivotal role in leukocyte adhesion and migration, but the mechanism(s) by which this integrin is regulated has remained incompletely understood. LFA-1 integrin activity requires phosphorylation of its ß2-chain and interactions of its cytoplasmic tail with various cellular proteins. The α-chain is constitutively phosphorylated and necessary for cellular adhesion, but how the α-chain regulates adhesion has remained enigmatic. We now show that substitution of the α-chain phosphorylation site (S1140A) in T cells inhibits the phosphorylation of the functionally important Thr-758 in the ß2-chain, binding of α-actinin and 14-3-3 protein, and expression of an integrin-activating epitope after treatment with the stromal cell-derived factor-1α. The presence of this substitution resulted in a loss of cell adhesion and directional cell migration. Moreover, LFA-1 activation through the T-cell receptor in cells expressing the S1140A LFA-1 variant resulted in less Thr-758 phosphorylation, α-actinin and talin binding, and cell adhesion. The finding that the LFA-1 α-chain regulates adhesion through the ß-chain via specific phosphorylation at Ser-1140 in the α-chain has not been previously reported and emphasizes that both chains are involved in the regulation of LFA-1 integrin activity.

A nebulin super-repeat panel reveals stronger actin binding toward the ends of the super-repeat region.

INTRODUCTION: Nebulin is a giant actin-binding protein in the thin filament of the skeletal muscle sarcomere. Studies of nebulin interactions are limited by the size, complexity, and poor solubility of the protein. We divided the nebulin super-repeat region into a super-repeat panel, and studied nebulin/actin interactions. METHODS: Actin binding was studied using a co-sedimentation assay with filamentous actin and 26 different nebulin super-repeats. RESULTS: The panel revealed notable differences in actin binding between the super-repeats. Both ends of the super-repeat region bound actin significantly more strongly, whereas the central part of the protein bound actin weakly. Thus, the binding between nebulin and actin formed a location-dependent pattern of strong vs. weak binding. DISCUSSION: The nebulin super-repeat panel allowed us to study the actin binding of each super-repeat individually. The panel will be a powerful tool in elucidating nebulin function in health and disease. Muscle Nerve 59:116-121, 2019.

LFA-1 integrin antibodies inhibit leukocyte α4ß1-mediated adhesion by intracellular signaling.

Binding of intercellular adhesion molecule-1 to the ß2-integrin leukocyte function associated antigen-1 (LFA-1) is known to induce cross-talk to the α4ß1 integrin. Using different LFA-1 monoclonal antibodies, we have been able to study the requirement and mechanism of action for the cross-talk in considerable detail. LFA-1-activating antibodies and those inhibitory antibodies that signal to α4ß1 induce phosphorylation of Thr-758 on the ß2-chain, which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the ß1-chain of α4ß1 and loss of binding to its ligand vascular cell adhesion molecule-1. The results show that with LFA-1 antibodies, we can activate LFA-1 and inhibit α4ß1, inhibit both LFA-1 and α4ß1, inhibit LFA-1 but not α4ß1, or not affect LFA-1 or α4ß1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications.

Specific phosphorylations transmit signals from leukocyte ß2 to ß1 integrins and regulate adhesion.

The regulation of integrins expressed on leukocytes must be controlled precisely, and members of different integrin subfamilies have to act in concert to ensure the proper traffic of immune cells to sites of inflammation. The activation of ß2 family integrins through the T cell receptor or by chemokines leads to the inactivation of very late antigen 4. The mechanism(s) of this cross-talk has not been known. We have now elucidated in detail how the signals are transmitted from leukocyte function-associated antigen 1 and show that, after its activation, the signaling involves specific phosphorylations of ß2 integrin followed by interactions with cytoplasmic signaling proteins. This results in loss of ß1 phosphorylation and a decrease in very late antigen 4 binding to its ligand vascular cell adhesion molecule 1. Our results show how a member of one integrin family regulates the activity of another integrin. This is important for the understanding of integrin-mediated processes.

Mutation update and genotype-phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies.

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core-rod myopathy, congenital fiber-type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal-dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca(2+) sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non-hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin-actin association or tropomyosin head-to-tail binding.

K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity.

Mutations in the TPM2 gene, which encodes ß-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of ß-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-ß-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-ß-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.

Regulation of integrin activity by phosphorylation.

Integrins are heterodimeric complex type I membrane proteins involved in cellular adhesion and signaling. They exist as inactive molecules in resting cells, and need activation to become adhesive. Although much is known about their structure, and a large number of interacting molecules have been described, we still only partially understand how their activities are regulated. In this review we focus on the leukocyte-specific ß2-integrins and, specifically, on the role of integrin phosphorylation in the regulation of activity. Phosphorylation reactions can be fast and reversible, thus enabling strictly directed regulatory activities both time-wise and locally in specific regions of the plasma membrane in different leukocytes.

Low-dose decitabine enhances the efficacy of viral cancer vaccines for immunotherapy.

Cancer immunotherapy requires a specific antitumor CD8+ T cell-driven immune response; however, upon genetic and epigenetic alterations of the antigen processing and presenting components, cancer cells escape the CD8+ T cell recognition. As a result, poorly immunogenic tumors are refractory to conventional immunotherapy. In this context, the use of viral cancer vaccines in combination with hypomethylating agents represents a promising strategy to prevent cancer from escaping immune system recognition. In this study, we evaluated the sensitivity of melanoma (B16-expressing ovalbumin) and metastatic triple-negative breast cancer (4T1) cell lines to FDA-approved low-dose decitabine in combination with PeptiCRAd, an adenoviral anticancer vaccine. The two models showed different sensitivity to decitabine in vitro and in vivo when combined with PeptiCRAd. In particular, mice bearing syngeneic 4T1 cancer showed higher tumor growth control when receiving the combinatorial treatment compared to single controls in association with a higher expression of MHC class I on cancer cells and reduction in Tregs within the tumor microenvironment. Furthermore, remodeling of the CD8+ T cell infiltration and cytotoxic activity toward cancer cells confirmed the effect of decitabine in enhancing anticancer vaccines in immunotherapy regimens.

Novel peptide-based oncolytic vaccine for enhancement of adaptive antitumor immune response via co-engagement of innate Fcγ and Fcα receptors.

BACKGROUND: Cancer immunotherapy relies on using the immune system to recognize and eradicate cancer cells. Adaptive immunity, which consists of mainly antigen-specific cytotoxic T cells, plays a pivotal role in controlling cancer progression. However, innate immunity is a necessary component of the cancer immune response to support an immunomodulatory state, enabling T-cell immunosurveillance. METHODS: Here, we elucidated and exploited innate immune cells to sustain the generation of antigen-specific T cells on the use of our cancer vaccine platform. We explored a previously developed oncolytic adenovirus (AdCab) encoding for a PD-L1 (Programmed-Death Ligand 1) checkpoint inhibitor, which consists of a PD-1 (Programmed Cell Death Protein 1) ectodomain fused to an IgG/A cross-hybrid Fc. We coated AdCab with major histocompatibility complex (MHC-I)-restricted tumor peptides, generating a vaccine platform (named PeptiCab); the latter takes advantage of viral immunogenicity, peptide cancer specificity to prime T-cell responses, and antibody-mediated effector functions. RESULTS: As proof of concept, PeptiCab was used in murine models of melanoma and colon cancer, resulting in tumor growth control and generation of systemic T-cell-mediated antitumor responses. In specific, PeptiCab was able to generate antitumor T effector memory cells able to secrete various inflammatory cytokines. Moreover, PeptiCab was able to polarize neutrophils to attain an antigen-presenting phenotype by upregulating MHC-II, CD80 and CD86 resulting in an enhanced T-cell expansion. CONCLUSION: Our data suggest that exploiting innate immunity activates T-cell antitumor responses, enhancing the efficiency of a vaccine platform based on oncolytic adenovirus coated with MHC-I-restricted tumor peptides.

TCR-induced activation of LFA-1 involves signaling through Tiam1.

Adhesion is pivotal for most leukocyte functions, and the ß(2) integrin family of adhesion molecules plays a central role. The integrins need activation to become functional, but the molecular events resulting in adhesion have remained incompletely understood. In human T cells, activation through the TCR results in specific phosphorylation of the T758 on the ß(2) chain of LFA-1. We now show that this phosphorylation leads to downstream binding of 14-3-3 proteins, followed by engagement of the guanine nucleotide exchange factor protein Tiam1 and Rac1 activation. Downregulation of the signaling molecules inhibits LFA-1 activity. Activation by the chemokine stromal cell-derived factor-1α also results in T758 phosphorylation and integrin activation. Thus, TCR and chemokine activation converges on LFA-1 phosphorylation, followed by similar downstream events affecting adhesion.

Abnormal actin binding of aberrant ß-tropomyosins is a molecular cause of muscle weakness in TPM2-related nemaline and cap myopathy.

NM (nemaline myopathy) is a rare genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding ß-Tm (tropomyosin). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium-sensitive manner. In the present study, we have investigated the pathogenetic mechanisms underlying five disease-causing mutations in Tm. We show that four of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, whereas two show defective Ca2+ activation of contractility. We have also mapped the amino acids altered by the mutation to regions important for actin binding and note that two of the mutations cause altered protein conformation, which could account for impaired actin affinity.

Controlled release of enhanced cross-hybrid IgGA Fc PD-L1 inhibitors using oncolytic adenoviruses.

Immune checkpoint inhibitors have clinical success in prolonging the life of many cancer patients. However, only a minority of patients benefit from such therapy, calling for further improvements. Currently, most PD-L1 checkpoint inhibitors in the clinic do not elicit Fc effector mechanisms that would substantially increase their efficacy. To gain potency and circumvent off-target effects, we previously designed an oncolytic adenovirus (Ad-Cab) expressing an Fc fusion peptide against PD-L1 on a cross-hybrid immunoglobulin GA (IgGA) Fc. Ad-Cab elicited antibody effector mechanisms of IgG1 and IgA, which led to higher tumor killing compared with each isotype alone and with clinically approved PD-L1 checkpoint inhibitors. In this study, we further improved the therapy to increase the IgG1 Fc effector mechanisms of the IgGA Fc fusion peptide (Ad-Cab FT) by adding four somatic mutations that increase natural killer (NK) cell activation. Ad-Cab FT was shown to work better at lower concentrations compared with Ad-Cab in vitro and in vivo and to have better tumor- and myeloid-derived suppressor cell killing, likely because of higher NK cell activation. Additionally, the biodistribution of the Fc fusion peptide demonstrated targeted release in the tumor microenvironment with minimal or no leakage to the peripheral blood and organs in mice. These data demonstrate effective and safe use of Ad-Cab FT, bidding for further clinical investigation.

Development of mesothelioma-specific oncolytic immunotherapy enabled by immunopeptidomics of murine and human mesothelioma tumors.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. As the available therapeutic options show a lack of efficacy, novel therapeutic strategies are urgently needed. Given its T-cell infiltration, we hypothesized that MPM is a suitable target for therapeutic cancer vaccination. To date, research on mesothelioma has focused on the identification of molecular signatures to better classify and characterize the disease, and little is known about therapeutic targets that engage cytotoxic (CD8+) T cells. In this study we investigate the immunopeptidomic antigen-presented landscape of MPM in both murine (AB12 cell line) and human cell lines (H28, MSTO-211H, H2452, and JL1), as well as in patients' primary tumors. Applying state-of-the-art immuno-affinity purification methodologies, we identify MHC I-restricted peptides presented on the surface of malignant cells. We characterize in vitro the immunogenicity profile of the eluted peptides using T cells from human healthy donors and cancer patients. Furthermore, we use the most promising peptides to formulate an oncolytic virus-based precision immunotherapy (PeptiCRAd) and test its efficacy in a mouse model of mesothelioma in female mice. Overall, we demonstrate that the use of immunopeptidomic analysis in combination with oncolytic immunotherapy represents a feasible and effective strategy to tackle untreatable tumors.

Distinct overlapping sequences at the carboxy-terminus of merlin regulate its tumour suppressor and morphogenic activity.

The Neurofibromatosis 2 (NF2) gene product merlin is a tumour suppressor, which in addition to inhibiting cell proliferation regulates cell morphology. The morphogenic properties of merlin may play a role in tumour suppression, as patient-derived tumour cells demonstrate cytoskeletal abnormalities. However, it is still unclear how these functions are linked. The N-terminal FERM-domain of merlin is highly homologous to the oncogenic protein ezrin, while the C-termini are less conserved, suggesting that the opposite effect of the proteins on proliferation could be mediated by their distinct C-terminal regions. In this study we characterize the role of the most C-terminal residues of merlin in the regulation of proliferation, cytoskeletal organization, phosphorylation and intramolecular associations. In addition to the two full-length merlin isoforms and truncating mutations found in patients, we focused on the evolutionally conserved C-terminal residues 545-547, also harbouring disease-causing mutations. We demonstrate that merlin induces cell extensions, which result from impaired retraction of protrusions rather than from increased formation of filopodia. The residues 538-568 were found particularly important for this morphogenic activity. The results further show that both merlin isoforms are able to equally inhibit proliferation, whereas C-terminal mutants affecting residues 545-547 are less effective in growth suppression. This study demonstrates that the C-terminus contains distinct but overlapping functional domains important for regulation of the morphogenic activity, intramolecular associations and cell proliferation.

Platelet-derived microvesicles: multitalented participants in intercellular communication.

Platelets can release a heterogeneous pool of vesicles which include plasma membrane-derived microparticles (PMPs) and multivesicular body-derived exosomes. As both vesicle types are generated upon activation and their distinction is complicated due to an overlap in their molecular properties and sizes, they are best discussed as an entity, the platelet-derived microvesicles (PMVs). PMPs can be formed through several induction pathways, which determine their different molecular profiles and facilitate tailor-made participation in intercellular communication. This dynamic variability may lie behind the multifaceted and sometimes very different observations of the PMPs in physiological and pathological settings. Currently, little is known of platelet-derived exosomes. In all, PMVs not only participate in several homeostatic multicellular processes, such as hemostasis, maintenance of vascular health, and immunity, but they also play a role in thrombotic and inflammatory diseases and cancer progression. In the past few years, the number of original articles and reviews on microvesicles has dramatically increased, but the data simultaneously raise further questions, the answers to which depend on forthcoming analytical improvements. In this article, the differential activation pathways and the molecular and functional properties of PMVs are reviewed in context with their sometimes paradoxical role in health and in disease. Also, the methodological issues of PMV detection and analysis are discussed in the light of recent advances within the field.

Regulation of Dynamic Cell Adhesion by Integrin-Integrin Crosstalk.

Most cells express several integrins. The integrins are able to respond to various cellular functions and needs by modifying their own activation state, but in addition by their ability to regulate each other by activation or inhibition. This crosstalk or transdominant regulation is strictly controlled. The mechanisms resulting in integrin crosstalk are incompletely understood, but they often involve intracellular signalling routes also used by other cell surface receptors. Several studies show that the integrin cytoplasmic tails bind to a number of cytoskeletal and adaptor molecules in a regulated manner. Recent work has shown that phosphorylations of integrins and key intracellular molecules are of pivotal importance in integrin-cytoplasmic interactions, and these in turn affect integrin activity and crosstalk. The integrin ß-chains play a central role in regulating crosstalk. In addition to Integrin-integrin crosstalk, crosstalk may also occur between integrins and related receptors, including other adhesion receptors, growth factor and SARS-CoV-2 receptors.

Peptides-Coated Oncolytic Vaccines for Cancer Personalized Medicine.

Oncolytic Viruses (OVs) work through two main mechanisms of action: the direct lysis of the virus-infected cancer cells and the release of tumor antigens as a result of the viral burst. In this sc.enario, the OVs act as in situ cancer vaccines, since the immunogenicity of the virus is combined with tumor antigens, that direct the specificity of the anti-tumor adaptive immune response. However, this mechanism in some cases fails in eliciting a strong specific T cell response. One way to overcome this problem and enhance the priming efficiency is the production of genetically modified oncolytic viruses encoding one or more tumor antigens. To avoid the long and expensive process related to the engineering of the OVs, we have exploited an approach based on coating OVs (adenovirus and vaccinia virus) with tumor antigens. In this work, oncolytic viruses encoding tumor antigens and tumor antigen decorated adenoviral platform (PeptiCRAd) have been used as cancer vaccines and evaluated both for their prophylactic and therapeutic efficacy. We have first tested the oncolytic vaccines by exploiting the OVA model, moving then to TRP2, a more clinically relevant tumor antigen. Finally, both approaches have been investigated in tumor neo-antigens settings. Interestingly, both genetically modified oncolytic adenovirus and PeptiCRAd elicited T cells-specific anti-tumor responses. However, in vitro cross-representation experiments, showed an advantage of PeptiCRAd as regards the fast presentation of the model epitope SIINFEKL from OVA in an immunogenic rather than tolerogenic fashion. Here two approaches used as cancer oncolytic vaccines have been explored and characterized for their efficacy. Although the generation of specific anti-tumor T cells was elicited in both approaches, PeptiCRAd retains the advantage of being rapidly adaptable by coating the adenovirus with a different set of tumor antigens, which is crucial in personalized cancer vaccines clinical setting.

A novel immunopeptidomic-based pipeline for the generation of personalized oncolytic cancer vaccines.

Besides the isolation and identification of major histocompatibility complex I-restricted peptides from the surface of cancer cells, one of the challenges is eliciting an effective antitumor CD8+ T-cell-mediated response as part of therapeutic cancer vaccine. Therefore, the establishment of a solid pipeline for the downstream selection of clinically relevant peptides and the subsequent creation of therapeutic cancer vaccines are of utmost importance. Indeed, the use of peptides for eliciting specific antitumor adaptive immunity is hindered by two main limitations: the efficient selection of the most optimal candidate peptides and the use of a highly immunogenic platform to combine with the peptides to induce effective tumor-specific adaptive immune responses. Here, we describe for the first time a streamlined pipeline for the generation of personalized cancer vaccines starting from the isolation and selection of the most immunogenic peptide candidates expressed on the tumor cells and ending in the generation of efficient therapeutic oncolytic cancer vaccines. This immunopeptidomics-based pipeline was carefully validated in a murine colon tumor model CT26. Specifically, we used state-of-the-art immunoprecipitation and mass spectrometric methodologies to isolate >8000 peptide targets from the CT26 tumor cell line. The selection of the target candidates was then based on two separate approaches: RNAseq analysis and HEX software. The latter is a tool previously developed by Jacopo, 2020, able to identify tumor antigens similar to pathogen antigens in order to exploit molecular mimicry and tumor pathogen cross-reactive T cells in cancer vaccine development. The generated list of candidates (26 in total) was further tested in a functional characterization assay using interferon-γ enzyme-linked immunospot (ELISpot), reducing the number of candidates to six. These peptides were then tested in our previously described oncolytic cancer vaccine platform PeptiCRAd, a vaccine platform that combines an immunogenic oncolytic adenovirus (OAd) coated with tumor antigen peptides. In our work, PeptiCRAd was successfully used for the treatment of mice bearing CT26, controlling the primary malignant lesion and most importantly a secondary, nontreated, cancer lesion. These results confirmed the feasibility of applying the described pipeline for the selection of peptide candidates and generation of therapeutic oncolytic cancer vaccine, filling a gap in the field of cancer immunotherapy, and paving the way to translate our pipeline into human therapeutic approach.