Assessment of two main therapeutic regimens of chronic lymphocytic leukemia in a major referral center in Syria

Introduction: Due to the high cost of targeted therapy, chemoimmunotherapy regimens remain the standard therapy for chronic lymphocytic leukemia in many developing countries. In this study, we compare the treatment outcomes of the two main chemoimmunotherapeutic regimens. Material and methods: Data was obtained from the oncology department archives at Tishreen University Hospital between 2016 and 2020. We enrolled previously untreated, fit patients with chronic lymphocytic leukemia who were treated with one of two regimens: either a fludarabine, cyclophosphamide, and rituximab regimen, or a bendamustine and rituximab regimen. Results: 78 patients were enrolled in the study. 56.8% of the fludarabine, cyclophosphamide, and rituximab group achieved complete response versus 73.5% of the bendamustine and rituximab group. Progression-free survival was slightly shorter for fludarabine, cyclophosphamide, and rituximab than for bendamustine and rituximab [median 15.1 months [95% confidence interval {CI} 12.4–17.8] vs. 17.7 months (95% CI 15.4–20.1)] without statistical significance. In elderly patients (>65 years) median progression-free survival (PFS) was significantly (p = 0.046) longer with the bendamustine and rituximab treatment [median 19.9 months (95% CI 17.2–22.5)] than with the fludarabine, cyclophosphamide, and rituximab [median 11.6 months (6–17.2)]. Regarding overall survival, no significant difference between the two groups was documented. Delay and deletion of cycles, neutropenia and anemia were more frequent with the fludarabine, cyclophosphamide, and rituximab group. Furthermore, we found that elevated lactate dehydrogenase, positive expression of ZAP-70, stage C, and splenomegaly are all indicators of poor prognosis in correlation with PFS. Conclusions: Our study found that the bendamustine and rituximab regimen is safer than, and has comparable efficacy to, the standard therapy of fludarabine, cyclophosphamide, and rituximab for previously untreated, fit patients with chronic lymphocytic leukemia

Patient-Derived Organoids for Precision Cancer Immunotherapy

Cancer immunotherapy has revolutionized the way tumors are treated. Nevertheless, efficient and robust testing platforms are still missing, including clinically relevant human ex vivo tumor assays that allow pretreatment testing of cancer therapies and selection of the most efficient and safe therapy for a specific patient. In the case of immunotherapy, this testing platform would require not only cancer cells, but also the tumor microenvironment, including immune cells. Here, we discuss the applications of patient-derived tumor organoid cultures and the possibilities in using complex immune-organoid cultures to provide preclinical testing platforms for precision cancer immunotherapy.Peer reviewe

A novel cancer vaccine for melanoma based on an approved vaccine against measles, mumps, and rubella

Common vaccines for infectious diseases have been repurposed as cancer immunotherapies. The intratumoral administration of these repurposed vaccines can induce immune cell infiltra-tion into the treated tumor. Here, we have used an approved trivalent live attenuated measles, mumps, and rubella (MMR) vaccine in our previously developed PeptiENV cancer vaccine platform. The intratumoral administration of this novel MMR-containing PeptiENV cancer vaccine significantly increased both intratumoral as well as systemic tumor-specific T cell responses. In addition, PeptiENV therapy, in combination with immune checkpoint inhibitor therapy, improved tumor growth control and survival as well as increased the number of mice responsive to immune checkpoint inhibitor therapy. Importantly, mice pre-vaccinated with the MMR vaccine responded equally well, if not better, to the PeptiENV therapy, indicating that pre-existing immunity against the MMR vaccine viruses does not compromise the use of this novel cancer vaccine platform.Peer reviewe

Peptides-Coated Oncolytic Vaccines for Cancer Personalized Medicine

Publisher Copyright: Copyright © 2022 Feola, Russo, Martins, Lopes, Vandermeulen, Fluhler, De Giorgi, Fusciello, Pesonen, Ylösmäki, Antignani, Chiaro, Hamdan, Feodoroff, Grönholm and Cerullo.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.Peer reviewe

Characterization of Expanded Gamma Delta T Cells from Atypical X-SCID Patient Reveals Preserved Function and IL2RG-Mediated Signaling

Abnormally high gamma delta T cell numbers among individuals with atypical SCID have been reported but detailed immunopheno typing and functional characterization of these expanded gamma delta T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG (NM_000206.3) c.172C > T;p.(Pro58Ser) variant. Here, we have further investigated the index patient's abnormally large gamma delta T cell population in terms of function and phenotype by studying IL2RG cell surface expression, STAT tyrosine phosphorylation and blast formation in response to interleukin stimulation, immunophenotyping, TCRv gamma sequencing, and target cell killing. In contrast to his alpha beta T cells, the patient's gamma delta T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. V delta 2 + population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's gamma delta T cells showed enhanced cytotoxicity towards A549 cancer cells. His TCRv gamma repertoire was versatile but sequencing of IL2RG revealed a novel c.534C > A; p.(Phe178Leu) somatic missense variant restricted to gamma delta T cells. Over time this variant became predominant in gamma delta T cells, though initially present only in part of them. IL2RG-Pro58Ser/Phe178Leu variant showed higher cell surface expression compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline p.(Pro58Ser) variant. In conclusion, our report indicates that expansion of gamma delta T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells.Peer reviewe

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-gamma 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.Peer reviewe

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

Novel oncolytic adenovirus expressing enhanced cross-hybrid IgGA Fc PD-L1 inhibitor activates multiple immune effector populations leading to enhanced tumor killing in vitro, in vivo and with patient-derived tumor organoids

Background Despite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to arm such antibodies with Fc-mediated effector mechanisms. However, these effector mechanisms are inhibited or reduced due to toxicity issues since PD-L1 is not confined to the tumor and also expressed on healthy cells. To increase efficacy while minimizing toxicity, we designed an oncolytic adenovirus that secretes a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit effector mechanisms of an IgG1 and also IgA1 consequently activating neutrophils, a population neglected by IgG1, in order to combine multiple effector mechanisms. Methods The cross-hybrid Fc-fusion peptide comprises of an Fc with the constant domains of an IgA1 and IgG1 which is connected to a PD-1 ectodomain via a GGGS linker and was cloned into an oncolytic adenovirus. We demonstrated that the oncolytic adenovirus was able to secrete the cross-hybrid Fc-fusion peptide able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity in various cancer cell lines, in vivo and ex vivo renal-cell carcinoma patient-derived organoids. Results Using various techniques to measure cytotoxicity, the cross-hybrid Fc-fusion peptide expressed by the oncolytic adenovirus was shown to activate Fc-effector mechanisms of an IgA1 (neutrophil activation) as well as of an IgG1 (natural killer and complement activation). The activation of multiple effector mechanism simultaneously led to significantly increased tumor killing compared with FDA-approved PD-L1 checkpoint inhibitor (Atezolizumab), IgG1-PDL1 and IgA-PDL1 in various in vitro cell lines, in vivo models and ex vivo renal cell carcinoma organoids. Moreover, in vivo data demonstrated that Ad-Cab did not require CD8+ T cells, unlike conventional checkpoint inhibitors, since it was able to activate other effector populations. Conclusion Arming PD-L1 checkpoint inhibitors with Fc-effector mechanisms of both an IgA1 and an IgG1 can increase efficacy while maintaining safety by limiting expression to the tumor using oncolytic adenovirus. The increase in tumor killing is mostly attributed to the activation of multiple effector populations rather than activating a single effector population leading to significantly higher tumor killing.Peer reviewe

Artificially Cloaked Viral Nanovaccine for Cancer Immunotherapy

Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer.Peer reviewe

Novel chimerized IgA CD20 antibodies : Improving neutrophil activation against CD20-positive malignancies

ABSTRACT Current combination therapies elicit high response rates in B cell malignancies, often using CD20 antibodies as the backbone of therapy. However, many patients eventually relapse or develop progressive disease. Therefore, novel CD20 antibodies combining multiple effector mechanisms were generated. To study whether neutrophil-mediated destruction of B cell malignancies can be added to the arsenal of effector mechanisms, we chimerized a panel of five previously described murine CD20 antibodies to the human IgG1, IgA1 and IgA2 isotype. Of this panel, we assessed in vitro antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and direct cell death induction capacity and studied the efficacy in two different in vivo mouse models. IgA antibodies outperformed IgG1 antibodies in neutrophil-mediated killing in vitro, both against CD20-expressing cell lines and primary patient material. In these assays, we observed loss of CD19 with both IgA and IgG antibodies. Therefore, we established a novel method to improve the assessment of B-cell depletion by CD20 antibodies by including CD24 as a stable cell marker. Subsequently, we demonstrated that only IgA antibodies were able to reduce B cell numbers in this context. Additionally, IgA antibodies showed efficacy in both an intraperitoneal tumor model with EL4 cells expressing huCD20 and in an adoptive transfer model with huCD20-expressing B cells. Taken together, we show that IgA, like IgG, can induce ADCC and CDC, but additionally triggers neutrophils to kill (malignant) B cells. We conclude that antibodies of the IgA isotype offer an attractive repertoire of effector mechanisms for the treatment of CD20-expressing malignancies.Peer reviewe