Characterization of a Trispecific PD-L1 Blocking Antibody That Exhibits EGFR-Conditional 4-1BB Agonist Activity.

Immune checkpoint blockade has changed the treatment paradigm for advanced solid tumors, but the overall response rates are still limited. The combination of checkpoint blockade with anti-4-1BB antibodies to stimulate tumor-infiltrating T cells has shown anti-tumor activity in human trials. However, the further clinical development of these antibodies has been hampered by significant off-tumor toxicities. Here, we generated an anti-4-1BB/EGFR/PD-L1 trispecific antibody consisting of a triple-targeting tandem trimerbody (TT) fused to an engineered silent Fc region. This antibody (IgTT-4E1-S) was designed to combine the blockade of the PD-L1/PD-1 axis with conditional 4-1BB costimulation specifically confined to the tumor microenvironment (TME). The antibody demonstrated simultaneous binding to purified EGFR, PD-L1, and 4-1BB in solution, effective blockade of the PD-L1/PD1 interaction, and potent 4-1BB-mediated costimulation, but only in the presence of EGFR-expressing cells. These results demonstrate the feasibility of IgTT-4E1-S specifically blocking the PD-L1/PD-1 axis and inducing EGFR-conditional 4-1BB agonist activity.

Targeting IL-6 trans-signalling by sgp130Fc attenuates severity in SARS-CoV-2 -infected mice and reduces endotheliopathy.

BACKGROUND: SARS-CoV-2 infection is considered as a relapsing inflammatory process with a dysregulation of IL-6 signalling. Classic IL-6 signalling is thought to represent a defence mechanism against pathogens. In contrast, IL-6 trans-signalling has pro-inflammatory effects. In severe COVID-19, therapeutic strategies have focused on global inhibition of IL-6, with controversial results. We hypothesized that specific blockade of IL-6 trans-signalling could inhibit inflammatory response preserving the host defence activity inherent to IL-6 classic signalling. METHODS: To test the role of the specific IL-6 trans-signalling inhibition by sgp130Fc in short- and long-term consequences of COVID-19, we used the established K18-hACE2 transgenic mouse model. Histological as well as immunohistochemical analysis, and pro-inflammatory marker profiling were performed. To investigate IL-6 trans-signalling in human cells we used primary lung microvascular endothelial cells and fibroblasts in the presence/absence of sgp130Fc. FINDINGS: We report that targeting IL-6 trans-signalling by sgp130Fc attenuated SARS-CoV-2-related clinical symptoms and mortality. In surviving mice, the treatment caused a significant decrease in lung damage. In vitro, IL-6 trans-signalling induced strong and persisting JAK1/STAT3 activation in endothelial cells and lung fibroblasts with proinflammatory effects, which were attenuated by sgp130Fc. Our data also suggest that in those cells with scant amounts of IL-6R, the induction of gp130 and IL-6 by IL-6:sIL-6R complex sustains IL-6 trans-signalling. INTERPRETATION: IL-6 trans-signalling fosters progression of COVID-19, and suggests that specific blockade of this signalling mode could offer a promising alternative to mitigate both short- and long-term consequences without affecting the beneficial effects of IL-6 classic signalling. These results have implications for the development of new therapies of lung injury and endotheliopathy in COVID-19. FUNDING: The project was supported by ISCIII, Spain (COV-20/00792 to MB, PI23/01351 to MARH) and the European Commission-Next generation EU (European Union) (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global, SGL2103029 to MB). PID2019-110587RB-I00 (MB) supported by MICIN/AEI/10.13039/501100011033/and PID2022-143034OB-I00 (MB) by MICIN/AEI/10.13039/501100011033/FEDER. MAR-H acknowledges support from ISCIII, Spain and the European Commission-Next generation EU (European Union), through CSIC's Global Health PTI.

Self-Amplifying RNA: A Second Revolution of mRNA Vaccines against COVID-19.

SARS-CoV-2 virus, the causative agent of COVID-19, has produced the largest pandemic in the 21st century, becoming a very serious health problem worldwide. To prevent COVID-19 disease and infection, a large number of vaccines have been developed and approved in record time, including new vaccines based on mRNA encapsulated in lipid nanoparticles. While mRNA-based vaccines have proven to be safe and effective, they are more expensive to produce compared to conventional vaccines. A special type of mRNA vaccine is based on self-amplifying RNA (saRNA) derived from the genome of RNA viruses, mainly alphaviruses. These saRNAs encode a viral replicase in addition to the antigen, usually the SARS-CoV-2 spike protein. The replicase can amplify the saRNA in transfected cells, potentially reducing the amount of RNA needed for vaccination and promoting interferon I responses that can enhance adaptive immunity. Preclinical studies with saRNA-based COVID-19 vaccines in diverse animal models have demonstrated the induction of robust protective immune responses, similar to conventional mRNA but at lower doses. Initial clinical trials have confirmed the safety and immunogenicity of saRNA-based vaccines in individuals that had previously received authorized COVID-19 vaccines. These findings have led to the recent approval of two of these vaccines by the national drug agencies of India and Japan, underscoring the promising potential of this technology.

Oleuropein-driven reprogramming of the myeloid cell compartment to sensitise tumours to PD-1/PD-L1 blockade strategies.

BACKGROUND: Previous studies have shown that functional systemic immunity is required for the efficacy of PD-1/PD-L1 blockade immunotherapies in cancer. Hence, systemic reprogramming of immunosuppressive dysfunctional myeloid cells could overcome resistance to cancer immunotherapy. METHODS: Reprogramming of tumour-associated myeloid cells with oleuropein was studied by quantitative differential proteomics, phenotypic and functional assays in mice and lung cancer patients. Combinations of oleuropein and two different delivery methods of anti-PD-1 antibodies were tested in colorectal cancer tumour models and in immunotherapy-resistant lung cancer models. RESULTS: Oleuropein treatment reprogrammed monocytic and granulocytic myeloid-derived suppressor cells, and tumour-associated macrophages towards differentiation of immunostimulatory subsets. Oleuropein regulated major differentiation programmes associated to immune modulation in myeloid cells, which potentiated T cell responses and PD-1 blockade. PD-1 antibodies were delivered by two different strategies, either systemically or expressed within tumours using a self-amplifying RNA vector. Combination anti-PD-1 therapies with oleuropein increased tumour infiltration by immunostimulatory dendritic cells in draining lymph nodes, leading to systemic antitumour T cell responses. Potent therapeutic activities were achieved in colon cancer and lung cancer models resistant to immunotherapies, even leading to complete tumour regression. DISCUSSION: Oleuropein significantly improves the outcome of PD-1/PD-L1 blockade immunotherapy strategies by reprogramming myeloid cells.

AG5 is a potent non-steroidal anti-inflammatory and immune regulator that preserves innate immunity.

An archetypal anti-inflammatory compound against cytokine storm would inhibit it without suppressing the innate immune response. AG5, an anti-inflammatory compound, has been developed as synthetic derivative of andrographolide, which is highly absorbable and presents low toxicity. We found that the mechanism of action of AG5 is through the inhibition of caspase-1. Interestingly, we show with in vitro generated human monocyte derived dendritic cells that AG5 preserves innate immune response. AG5 minimizes inflammatory response in a mouse model of lipopolysaccharide (LPS)-induced lung injury and exhibits in vivo anti-inflammatory efficacy in the SARS-CoV-2-infected mouse model. AG5 opens up a new class of anti-inflammatories, since contrary to NSAIDs, AG5 is able to inhibit the cytokine storm, like dexamethasone, but, unlike corticosteroids, preserves adequately the innate immunity. This is critical at the early stages of any naïve infection, but particularly in SARS-CoV-2 infections. Furthermore, AG5 showed interesting antiviral activity against SARS-CoV-2 in humanized mice.

Checkpoint blockade meets gene therapy: Opportunities to improve response and reduce toxicity.

Immune checkpoint inhibitors (ICIs) based on monoclonal antibodies represent a breakthrough for the treatment of cancer. However, their efficacy varies among tumor types and patients, and they can lead to adverse effects due to on-target/off-tumor activity, since they are administered systemically at high doses. An alternative and attractive approach for the delivery of ICIs is the use of gene therapy vectors able to express them in vivo. This review focuses on the most recent studies using viral vectors able to express ICIs locally or systemically in preclinical models of cancer. These vectors include non-replicating viruses, oncolytic viruses able to propagate specifically in tumor cells and destroy them, and self-amplifying RNA vectors, armed with different formats of antibodies against immune checkpoints. Non-replicating vectors usually lead to long-term ICI expression, potentially eliminating the need for repeated administration. Vectors with replication capacity, although they have a shorter window of expression, can induce inflammation which enhances the antitumor effect. Finally, these engineered vectors can be used in combination with other immunostimulatory molecules or with CAR-T cells, further boosting the antitumor immune responses.

Local Delivery of Immunomodulatory Antibodies for Gastrointestinal Tumors.

Cancer therapy has experienced a breakthrough with the use of immune checkpoint inhibitors (ICIs) based on monoclonal antibodies (mAbs), which are able to unleash immune responses against tumors refractory to other therapies. Despite the great advancement that ICIs represent, most patients with gastrointestinal tumors have not benefited from this therapy. In addition, ICIs often induce adverse effects that are related to their systemic use. Local administration of ICIs in tumors could concentrate their effect in the malignant tissue and provide a higher safety profile. A new and attractive approach for local delivery of ICIs is the use of gene therapy vectors to express these blocking antibodies in tumor cells. Several vectors have been evaluated in preclinical models of gastrointestinal tumors to express ICIs against PD-1, PD-L1, and CTLA-4, among other immune checkpoints, with promising results. Vectors used in these settings include oncolytic viruses, self-replicating RNA vectors, and non-replicative viral and non-viral vectors. The use of viral vectors, especially when they have replication capacity, provides an additional adjuvant effect that has been shown to enhance antitumor responses. This review covers the most recent studies involving the use of gene therapy vectors to deliver ICIs to gastrointestinal tumors.

JNK3 Overexpression in the Entorhinal Cortex Impacts on the Hippocampus and Induces Cognitive Deficiencies and Tau Misfolding.

c-Jun N-terminal kinases (JNKs) are a family of protein kinases activated by a myriad of stimuli consequently modulating a vast range of biological processes. In human postmortem brain samples affected with Alzheimer's disease (AD), JNK overactivation has been described; however, its role in AD onset and progression is still under debate. One of the earliest affected areas in the pathology is the entorhinal cortex (EC). Noteworthy, the deterioration of the projection from EC to hippocampus (Hp) point toward the idea that the connection between EC and Hp is lost in AD. Thus, the main objective of the present work is to address if JNK3 overexpression in the EC could impact on the hippocampus, inducing cognitive deficits. Data obtained in the present work suggest that JNK3 overexpression in the EC influences the Hp leading to cognitive impairment. Moreover, proinflammatory cytokine expression and Tau immunoreactivity were increased both in the EC and in the Hp. Therefore, activation of inflammatory signaling and induction of Tau aberrant misfolding caused by JNK3 could be responsible for the observed cognitive impairment. Altogether, JNK3 overexpression in the EC may impact on the Hp inducing cognitive dysfunction and underlie the alterations observed in AD.

Local delivery of optimized nanobodies targeting the PD-1/PD-L1 axis with a self-amplifying RNA viral vector induces potent antitumor responses.

Despite the success of immune checkpoint blockade for cancer therapy, many patients do not respond adequately. We aimed to improve this therapy by optimizing both the antibodies and their delivery route, using small monodomain antibodies (nanobodies) delivered locally with a self-amplifying RNA (saRNA) vector based on Semliki Forest virus (SFV). We generated nanobodies against PD-1 and PD-L1 able to inhibit both human and mouse interactions. Incorporation of a dimerization domain reduced PD-1/PD-L1 IC50 by 8- and 40-fold for anti-PD-L1 and anti-PD-1 nanobodies, respectively. SFV viral particles expressing dimeric nanobodies showed a potent antitumor response in the MC38 model, resulting in >50% complete regressions, and showed better therapeutic efficacy compared to vectors expressing conventional antibodies. These effects were also observed in the B16 melanoma model. Although a short-term expression of nanobodies was observed due to the cytopathic nature of the saRNA vector, it was enough to generate a strong proinflammatory response in tumors, increasing infiltration of NK and CD8+ T cells. Delivery of the SFV vector expressing dimeric nanobodies by local plasmid electroporation, which could be more easily translated to the clinic, also showed a potent antitumor effect.

JNK Activation in Alzheimer's Disease Is Driven by Amyloid ß and Is Associated with Tau Pathology.

c-Jun N-terminal kinase 3 (JNK3) is suggested to play a key role in neurodegenerative disorders, especially in Alzheimer's disease (AD). However, it remains unclear whether JNK or amyloid ß (Aß) appears first in the disease onset. Postmortem brain tissues from four dementia subtypes of patients (frontotemporal dementia, Lewy body dementia, vascular dementia, and AD) were used to measure activated JNK (pJNK) and Aß levels. pJNK expression is significantly increased in AD; however, similar pJNK expression was found in other dementias. Furthermore, there was a significant correlation, co-localization, and direct interaction between pJNK expression and Aß levels in AD. Significant increased levels of pJNK were also found in Tg2576 mice, a model of AD. In this line, Aß42 intracerebroventricular injection in wild-type mice was able to induce a significant elevation of pJNK levels. JNK3 overexpression, achieved by intrahippocampal injection of an adeno-associated viral vector expressing this protein, was enough to induce cognitive deficiencies and precipitate Tau aberrant misfolding in Tg2576 mice without accelerating amyloid pathology. JNK3 overexpression may therefore be triggered by increased Aß. The latter, together with subsequent involvement of Tau pathology, may be underlying cognitive alterations in early stages of AD.

Photodynamic nasal SARS-CoV-2 decolonization shortens infectivity and influences specific T-Cell responses.

Background: The main objective was to evaluate the efficacy of intranasal photodynamic therapy (PDT) in SARS-CoV-2 mildly symptomatic carriers on decreasing the infectivity period. SARS-CoV-2-specific immune-stimulating effects and safety were also analysed. Methods: We performed a randomized, placebo-controlled, clinical trial in a tertiary hospital (NCT05184205). Patients with a positive SARS-CoV-2 PCR in the last 48 hours were recruited and aleatorily assigned to PDT or placebo. Patients with pneumonia were excluded. Participants and investigators were masked to group assignment. The primary outcome was the reduction in in vitro infectivity of nasopharyngeal samples at days 3 and 7. Additional outcomes included safety assessment and quantification of humoral and T-cell immune-responses. Findings: Patients were recruited between December 2021 and February 2022. Most were previously healthy adults vaccinated against COVID-19 and most carried Omicron variant. 38 patients were assigned to placebo and 37 to PDT. Intranasal PDT reduced infectivity at day 3 post-treatment when compared to placebo with a ß-coefficient of -812.2 (CI95%= -478660 - -1.3, p<0.05) infectivity arbitrary units. The probability of becoming PCR negative (ct>34) at day 7 was higher on the PDT-group, with an OR of 0.15 (CI95%=0.04-0.58). There was a decay in anti-Spike titre and specific SARS-CoV-2 T cell immunity in the placebo group 10 and 20 weeks after infection, but not in the PDT-group. No serious adverse events were reported. Interpretation: Intranasal-PDT is safe in pauci-symptomatic COVID-19 patients, it reduces SARS-CoV-2 infectivity and decelerates the decline SARS-CoV-2 specific immune-responses.

Treatment with the senolytics dasatinib/quercetin reduces SARS-CoV-2-related mortality in mice.

The enormous societal impact of the ongoing COVID-19 pandemic has been particularly harsh for some social groups, such as the elderly. Recently, it has been suggested that senescent cells could play a central role in pathogenesis by exacerbating the pro-inflammatory immune response against SARS-CoV-2. Therefore, the selective clearance of senescent cells by senolytic drugs may be useful as a therapy to ameliorate the symptoms of COVID-19 in some cases. Using the established COVID-19 murine model K18-hACE2, we demonstrated that a combination of the senolytics dasatinib and quercetin (D/Q) significantly reduced SARS-CoV-2-related mortality, delayed its onset, and reduced the number of other clinical symptoms. The increase in senescent markers that we detected in the lungs in response to SARS-CoV-2 may be related to the post-COVID-19 sequelae described to date. These results place senescent cells as central targets for the treatment of COVID-19, and make D/Q a new and promising therapeutic tool.

Identification of HLA class I-restricted immunogenic neoantigens in triple negative breast cancer.

Immune checkpoint inhibitor (ICI)-based immunotherapy in triple negative breast cancer (TNBC) is achieving limited therapeutic results, requiring the development of more potent strategies. Combination of ICI with vaccination strategies would enhance antitumor immunity and response rates to ICI in patients having poorly infiltrated tumors. In heavily mutated tumors, neoantigens (neoAgs) resulting from tumor mutations have induced potent responses when used as vaccines. Thus, our aim was the identification of immunogenic neoAgs suitable as vaccines in TNBC patients. By using whole exome sequencing, RNAseq and HLA binding algorithms of tumor samples from a cohort of eight TNBC patients, we identified a median of 60 mutations/patient, which originated a putative median number of 98 HLA class I-restricted neoAgs. Considering a group of 27 predicted neoAgs presented by HLA-A*02:01 allele in two patients, peptide binding to HLA was experimentally confirmed in 63% of them, whereas 55% were immunogenic in vivo in HLA-A*02:01+ transgenic mice, inducing T-cells against the mutated but not the wild-type peptide sequence. Vaccination with peptide pools or DNA plasmids expressing these neoAgs induced polyepitopic T-cell responses, which recognized neoAg-expressing tumor cells. These results suggest that TNBC tumors harbor neoAgs potentially useful in therapeutic vaccines, opening the way for new combined immunotherapies.

Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma.

The outcomes of metastatic and nonresponder pediatric osteosarcoma patients are very poor and have not improved in the last 30 years. These tumors harbor a highly immunosuppressive environment, making existing immunotherapies ineffective. Here, we evaluated the use of Semliki Forest virus (SFV) vectors expressing galectin-3 (Gal3) inhibitors as therapeutic tools, since both the inhibition of Gal3, which is involved in immunosuppression and metastasis, and virotherapy based on SFV have been demonstrated to reduce tumor progression in different tumor models. In vitro, inhibitors based on the Gal3 amino-terminal domain alone (Gal3-N) or fused to a Gal3 peptide inhibitor (Gal3-N-C12) were able to block the binding of Gal3 to the surface of activated T cells. In vivo, SFV expressing Gal3-N-C12 induced strong antitumor responses in orthotopic K7M2 and MOS-J osteosarcoma tumors, leading to complete regressions in 47% and 30% of mice, respectively. Pulmonary metastases were also reduced in K7M2 tumor-bearing mice after treatment with SFV-Gal3-N-C12. Both the antitumor and antimetastatic responses were dependent on modulation of the immune system, primarily including an increase in tumor-infiltrating lymphocytes and a reduction in the immunosuppressive environment inside tumors. Our results demonstrated that SFV-Gal3-N-C12 could constitute a potential therapeutic agent for osteosarcoma patients expressing Gal3.

Rapid SARS-CoV-2 disinfection on distant surfaces with UV-C: The inactivation is affected by the type of material.

SARS-CoV-2 is responsible for the COVID-19 pandemic, which has caused almost 570 million infections and over six million deaths worldwide. To help curb its spread, solutions using ultraviolet light (UV) for quick virus inactivation inside buildings without human intervention could be very useful to reduce chances of contagion. The UV dose must be sufficient to inactivate the virus considering the different materials in the room, but it should not be too high, not to degrade the environment. In the present study, we have analyzed the ability of a 254 nm wavelength UV-C lamp to inactivate dried samples of SARS-CoV-2 exposed at a distance of two meters, simulating a full-scale scenario. Our results showed that virus inactivation was extremely efficient in most tested materials, which included plastic, metal, wood, and textile, with a UV-C exposure of only 42 s (equivalent to 10 mJ/cm2). However, porous materials like medium density fibreboard, were hard to decontaminate, indicating that they should be avoided in hospital rooms and public places.

Intratumoral electroporation of a self-amplifying RNA expressing IL-12 induces antitumor effects in mouse models of cancer.

Alphavirus vectors based on self-amplifying RNA (saRNA) generate high and transient levels of transgene expression and induce innate immune responses, making them an interesting tool for antitumor therapy. These vectors are usually delivered as viral particles, but it is also possible to administer them as RNA. We evaluated this possibility by in vivo electroporation of Semliki Forest virus (SFV) saRNA for local treatment of murine colorectal MC38 subcutaneous tumors. Optimization of saRNA electroporation conditions in tumors was performed using an SFV vector coding for luciferase. Then we evaluated the therapeutic potential of this approach using an SFV saRNA coding for interleukin-12 (SFV-IL-12), a proinflammatory cytokine with potent antitumor effects. Delivery of SFV-IL-12 saRNA by electroporation led to improvement in tumor control and higher survival compared with mice treated with electroporation or with SFV-IL-12 saRNA alone. The antitumor efficacy of SFV-IL-12 saRNA electroporation increased by combination with systemic PD-1 blockade. This therapy, which was also validated in a hepatocellular carcinoma tumor model, suggests that local delivery of saRNA by electroporation could be an attractive strategy for cancer immunotherapy. This approach could have easy translation to the clinical practice, especially for percutaneously accessible tumors.

Gene Therapy for Acquired and Genetic Cholestasis.

Cholestatic diseases can be caused by the dysfunction of transporters involved in hepatobiliary circulation. Although pharmacological treatments constitute the current standard of care for these diseases, none are curative, with liver transplantation being the only long-term solution for severe cholestasis, albeit with many disadvantages. Liver-directed gene therapy has shown promising results in clinical trials for genetic diseases, and it could constitute a potential new therapeutic approach for cholestatic diseases. Many preclinical gene therapy studies have shown positive results in animal models of both acquired and genetic cholestasis. The delivery of genes that reduce apoptosis or fibrosis or improve bile flow has shown therapeutic effects in rodents in which cholestasis was induced by drugs or bile duct ligation. Most studies targeting inherited cholestasis, such as progressive familial intrahepatic cholestasis (PFIC), have focused on supplementing a correct version of a mutated gene to the liver using viral or non-viral vectors in order to achieve expression of the therapeutic protein. These strategies have generated promising results in treating PFIC3 in mouse models of the disease. However, important challenges remain in translating this therapy to the clinic, as well as in developing gene therapy strategies for other types of acquired and genetic cholestasis.

A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector.

BACKGROUND: Bile acid (BA) homeostasis is mainly regulated by bile salt excretory pump (BSEP), a hepatocyte transporter that transfers BAs to the bile. BSEP expression is regulated by BA levels through activation of farnesoid X receptor transcription factor, which binds to the inverted repeat (IR-1) element in the BSEP promoter. Gene therapy of cholestatic diseases could benefit from using vectors carrying endogenous promoters physiologically regulated by BAs, however their large size limits this approach, especially when using adeno-associated viral vector (AAV) vectors. RESULTS: We evaluated the functionality and BA-mediated regulation of minimal versions of human and mouse BSEP promoters containing IR-1 using AAV vectors expressing luciferase. Unexpectedly, a minimal mouse BSEP promoter (imPr) showed higher BA-mediated expression and inducibility than a minimal human promoter (ihPr) or than full-length BSEP promoters in human hepatic cells. In addition, in mice receiving an AAV8 vector carrying imPr promoter-driven luciferase expression was efficiently regulated by administration of a BA-enriched diet. Interestingly, this vector also expressed significantly higher luciferase levels in Abcb4-/- mice, which have high levels of BAs, compared to wild type mice, or to mice receiving a vector containing the luciferase gene downstream of the constitutive alpha-1 antitrypsin promoter. In contrast, the AAV vector containing ihPr showed very low luciferase expression with no inducibility. Finally, we optimized imPr by adding three IR-1 repeats at its 5' end. This new promoter provided higher levels of luciferase than imPr both in vitro and in vivo. CONCLUSIONS: The imPr could represent a useful tool for gene therapy approaches in which physiological BA regulation is desired.

Idiotype vaccines produced with a non-cytopathic alphavirus self-amplifying RNA vector induce antitumor responses in a murine model of B-cell lymphoma.

A promising therapy for patients with B-cell lymphoma is based on vaccination with idiotype monoclonal antibodies (mAbs). Since idiotypes are different in each tumor, a personalized vaccine has to be produced for each patient. Expression of immunoglobulins with appropriate post-translational modifications for human use often requires the use of stable mammalian cells that can be scaled-up to reach the desired level of production. We have used a noncytopathic self-amplifying RNA vector derived from Semliki Forest virus (ncSFV) to generate BHK cell lines expressing murine follicular lymphoma-derived idiotype A20 mAb. ncSFV/BHK cell lines expressed approximately 2 mg/L/24 h of A20 mAb with proper quaternary structure and a glycosylation pattern similar to that of A20 mAb produced by hybridoma cells. A20 mAb purified from the supernatant of a ncSFV cell line, or from the hybridoma, was conjugated to keyhole limpet hemocyanin and used to immunize Balb/c mice by administration of four weekly doses of 25 µg of mAb. Both idiotype mAbs were able to induce a similar antitumor protection and longer survival compared to non-immunized mice. These results indicate that the ncSFV RNA vector could represent a quick and efficient system to produce patient-specific idiotypes with potential application as lymphoma vaccines.