Multivalent protein-drug conjugates - An emerging strategy for the upgraded precision and efficiency of drug delivery to cancer cells.

With almost 20 million new cases per year, cancer constitutes one of the most important challenges for public health systems. Unlike traditional chemotherapy, targeted anti-cancer strategies employ sophisticated therapeutics to precisely identify and attack cancer cells, limiting the impact of drugs on healthy cells and thereby minimizing the unwanted side effects of therapy. Protein drug conjugates (PDCs) are a rapidly growing group of targeted therapeutics, composed of a cancer-recognition factor covalently coupled to a cytotoxic drug. Several PDCs, mainly in the form of antibody-drug conjugates (ADCs) that employ monoclonal antibodies as cancer-recognition molecules, are used in the clinic and many PDCs are currently in clinical trials. Highly selective, strong and stable interaction of the PDC with the tumor marker, combined with efficient, rapid endocytosis of the receptor/PDC complex and its subsequent effective delivery to lysosomes, is critical for the efficacy of targeted cancer therapy with PDCs. However, the bivalent architecture of contemporary clinical PDCs is not optimal for tumor receptor recognition or PDCs internalization. In this review, we focus on multivalent PDCs, which represent a rapidly evolving and highly promising therapeutics that overcome most of the limitations of current bivalent PDCs, enhancing the precision and efficiency of drug delivery to cancer cells. We present an expanding set of protein scaffolds used to generate multivalent PDCs that, in addition to folding into well-defined multivalent molecular structures, enable site-specific conjugation of the cytotoxic drug to ensure PDC homogeneity. We provide an overview of the architectures of multivalent PDCs developed to date, emphasizing their efficacy in the targeted treatment of various cancers.

Human antigen R regulates autophagic flux by stabilizing autophagy-associated mRNA in calcific aortic valve disease.

AIMS: The incidence of calcific aortic valve disease (CAVD) has risen over the last decade and is expected to continue rising; however, pharmacological approaches have proven ineffective. In this study, we evaluated the role and underlying mechanisms of human antigen R (HuR)-mediated post-transcriptional regulation in CAVD. METHODS AND RESULTS: We found that HuR was significantly upregulated in human calcified aortic valves and primary aortic valvular interstitial cells (VICs) following osteogenic stimulation. Subsequent functional studies revealed that HuR silencing ameliorated calcification both in vitro and in vivo. For the first time, we demonstrated that HuR directly interacted with the transcript of phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A), which mediates phosphatidylinositol signalling, facilitates autophagy, and acts as an mRNA stabilizer. HuR positively modulated PIP4K2A expression at the post-transcriptional level and consequently influenced the AKT/mTOR/ATG13 pathway to regulate autophagy and CAVD progression. CONCLUSION: Our study provides new insights into the post-transcriptional regulatory role of HuR in modulating autophagy-positive factors to regulate the pathogenesis of CAVD. Our findings highlight the potential of HuR as an innovative therapeutic target in CAVD treatment.

Peptide Drug Conjugates and Their Role in Cancer Therapy.

Drug conjugates have become a significant focus of research in the field of targeted medicine for cancer treatments. Peptide-drug conjugates (PDCs), a subset of drug conjugates, are composed of carrier peptides ranging from 5 to 30 amino acid residues, toxic payloads, and linkers that connect the payload to the peptide. PDCs are further broken down into cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs), each having their own differences in the delivery of cytotoxic payloads. Generally, PDCs as compared to other drug conjugates-like antibody-drug conjugates (ADCs)-have advantages in tumor penetration, ease of synthesis and cost, and reduced off-target effects. Further, as compared to traditional cancer treatments (e.g., chemotherapy and radiation), PDCs have higher specificity for the target cancer with generally less toxic side effects in smaller doses. However, PDCs can have disadvantages such as poor stability and rapid renal clearance due to their smaller size and limited oral bioavailability due to digestion of its peptide structure. Some of these challenges can be overcome with modifications, and despite drawbacks, the intrinsic small size of PDCs with high target specificity still makes them an attractive area of research for cancer treatments.

Stimuli-Responsive Polymeric Nanomaterials for the Delivery of Immunotherapy Moieties: Antigens, Adjuvants and Agonists.

Immunotherapy has been investigated for decades, and it has provided promising results in preclinical studies. The most important issue that hinders researchers from advancing to clinical studies is the delivery system for immunotherapy agents, such as antigens, adjuvants and agonists, and the activation of these agents at the tumour site. Polymers are among the most versatile materials for a variety of treatments and diagnostics, and some polymers are reactive to either endogenous or exogenous stimuli. Utilizing this advantage, researchers have been developing novel and effective polymeric nanomaterials that can deliver immunotherapeutic moieties. In this review, we summarized recent works on stimuli-responsive polymeric nanomaterials that deliver antigens, adjuvants and agonists to tumours for immunotherapy purposes.

Engineered Attenuated Salmonella typhimurium Expressing Neoantigen Has Anticancer Effects.

Neoantigen vaccines are an immunotherapy strategy for treating cancer. The vaccine degrades quickly, so the strategy must include protection and precise targeting for immune cell stimulation. In this study, we engineered attenuated Salmonella typhimurium, which is highly infiltrative to tumors, to act as a carrier for Neoantigen peptide vaccine. Our system used a constitutive promoter vector, so that a single injection of Salmonella expressing Neoantigen could be used without requiring additional induction injections. In vivo experiments on bacteria-treated mice showed that Neoantigen expressed by the engineered carrier infiltrated tumors and resulted in suppressed tumor growth, higher survival rates and longer survival times, a relative increase of CD4 and CD8 T cells, and cytokine release. These results indicate that engineered Salmonella can be used as a carrier for Neoantigen immunotherapy.

Poly(hydrophobic amino acid)-Based Self-Adjuvanting Nanoparticles for Group A Streptococcus Vaccine Delivery.

Peptide antigens have been widely used in the development of vaccines, especially for those against autoimmunity-inducing pathogens and cancers. However, peptide-based vaccines require adjuvant and/or a delivery system to stimulate desired immune responses. Here, we explored the potential of self-adjuvanting poly(hydrophobic amino acids) (pHAAs) to deliver peptide-based vaccine against Group A Streptococcus (GAS). We designed and synthesized self-assembled nanoparticles with a variety of conjugates bearing a peptide antigen (J8-PADRE) and polymerized hydrophobic amino acids to evaluate the effects of structural arrangement and pHAAs properties on a system's ability to induce humoral immune responses. Immunogenicity of the developed conjugates was also compared to commercially available human adjuvants. We found that a linear conjugate bearing J8-PADRE and 15 copies of leucine induced equally effective, or greater, immune responses than commercial adjuvants. Our fully defined, adjuvant-free, single molecule-based vaccine induced the production of antibodies capable of killing GAS bacteria.

Experimental autoimmune prostatitis: different antigens induction and antigen-specific therapy.

PURPOSE: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has uncertain etiology and lacks effective treatment. Autoimmunity is an important pathogeny, and experimental autoimmune prostatitis (EAP) models have long been used for studying CP/CPPS. This review presents the detailed current knowledge of EAP models based on evaluation criteria aspects to provide a tool for model selection in pathogenesis studies and therapeutic drug screening. METHODS: We extensively searched the published literature on CP/CPPS and different antigen-induced EAP models focusing on the histopathology, clinical-related phenotypes, and biochemical indicators. We also cover the changes in the prostate function and other organs in EAP. Finally, we try to get some insights about antigen-based therapeutic approaches for CP/CPPS. RESULTS: Several inciting autoantigens were reported in EAP, including male accessory gland extracts, prostate extracts (PE), prostatic steroid-binding protein, prostatic spermine-binding protein (p25), prostatic acid phosphatase, seminal vesicle secretory protein 2, and T2 peptide. All of these models mimicked histological prostatitis, however only p25- and T2-induced models developed both pelvic pain and voiding behaviors. PE immunization is the most widely used method. Diminished fertility and mental health disorders can be found in PE model. Oral and intravenous T2 peptide have been studied for antigen-specific therapy and achieved preliminary progress in EAP models. CONCLUSIONS: PE-induced model is the most commonly used, while T2- or p25-model could serve as a promising CP/CPPS model. Antigen-specific therapy in CP/CPPS deserves further study.

Proton-driven transformable nanovaccine for cancer immunotherapy.

Cancer vaccines hold great promise for improved cancer treatment. However, endosomal trapping and low immunogenicity of tumour antigens usually limit the efficiency of vaccination strategies. Here, we present a proton-driven nanotransformer-based vaccine, comprising a polymer-peptide conjugate-based nanotransformer and loaded antigenic peptide. The nanotransformer-based vaccine induces a strong immune response without substantial systemic toxicity. In the acidic endosomal environment, the nanotransformer-based vaccine undergoes a dramatic morphological change from nanospheres (about 100 nanometres in diameter) into nanosheets (several micrometres in length or width), which mechanically disrupts the endosomal membrane and directly delivers the antigenic peptide into the cytoplasm. The re-assembled nanosheets also boost tumour immunity via activation of specific inflammation pathways. The nanotransformer-based vaccine effectively inhibits tumour growth in the B16F10-OVA and human papilloma virus-E6/E7 tumour models in mice. Moreover, combining the nanotransformer-based vaccine with anti-PD-L1 antibodies results in over 83 days of survival and in about half of the mice produces complete tumour regression in the B16F10 model. This proton-driven transformable nanovaccine offers a robust and safe strategy for cancer immunotherapy.

Stem cell antigen/Ly6a protects against bladder fibrosis in mice.

We have defined a population of stem cell antigen (Sca)-1+/CD34+/lin- mesenchymal stem cells in the mouse urinary bladder. These cells are reduced after partial bladder outlet obstruction (PO). To test the role of Sca-1 expressed by these cells, we analyzed bladders from Sca-1 knockout (KO) mice in both uninjured male mice and male mice subjected to PO. We found that loss of Sca-1 alone had little effect on bladder development or function but reduced the total number of mesenchymal stem cells by 30%. After PO, bladders from Sca-1-null KO male mice were larger, with more collagen and less muscle, than obstructed wild-type mice. Steady-state levels of caldesmon were significantly reduced and levels of fibroblast-specific protein 1 were significantly increased in Sca-1 KO mice compared with wild-type mice after PO. In investigating the effects of PO on cell proliferation, we found that loss of Sca-1 changed the timing of cell division in CD34+/lin-, collagen-producing, and smooth muscle cells. PO in combination with loss of Sca-1 drastically reduced the ability of CD34+/lin- cells to form colonies in vitro. Our findings therefore support the hypothesis that Sca-1 protects the bladder from fibrotic remodeling after obstruction, in part by influencing the proliferation of cells responding to the injury.

Effect of Oral T2 Antigen on Chronic Prostatitis/Chronic Pelvic Pain Syndrome in Mice Model.

The exact etiology and pathogenesis of chronic prostatitis (CP/CPPS) remain unclear. However, autoimmunity is a widely known theory. Precise treatment of CP/CPPS is not available. Here, we developed a new effective treatment method to prevent the occurrence of CP/CPPS. A total of 40 male C57BL/6 mice were randomly divided into four groups (n = 10): i.e., naive, model, high-dose (500 µg/ml), and low-dose (50 µg/ml) groups. High-dose and low-dose groups were orally given 0.4 ml of T2-containing soybean trypsin inhibitor (STI) at once after every 2 days for a total of 10 days. On day 10 and day 24 all the groups except naïve group were subcutaneously injected with 0.2 ml of T2 peptide along with CFA to make valid CP/CPPS models. Hematoxylin and eosin staining were used to evaluate the variation in CP/CPPS manifestation. Voiding behavior was recorded for the evaluation of urine frequencies. ELISA was used to measure the serum level of TNF-α in each group. The high- and low-dose groups of T2-containing STI displayed a reduction in urine frequencies, and inflammation, and there was a slight inflammatory infiltration as compared to the model group. In contrast, there was no difference observed in the TNF-α concentration of model as well as high- and low-dose groups compared to the naïve group. Our study demonstrates that oral T2-containing STI prevents CP/CPPS and provides an effective approach for the treatment of CP/CPPS.

Enhanced anti-tumor immunotherapy by dissolving microneedle patch loaded ovalbumin.

The skin is a very suitable organ for the induction of immune responses to vaccine antigens. Antigen delivery systems to the skin by needle and syringe directly deposit the antigen into the epidermal-dermal compartment, one of the most immunocompetent sites due to the presence of professional antigen-presenting cells aimed at the induction of antigen-specific T cells. In this study, we analyzed the amount of ovalbumin as an antigen delivered to the skin by a microneedle. When ovalbumin protein as an antigen was delivered to the skin of mice using a dissolving microneedle, it induced an immune response through the enhanced proliferation and cytokines production by the splenocytes and lymph nodes. Also, it effectively increased the ovalbumin-specific CD8+ T cell and CD4+ T cell population and induced an ovalbumin-specific CTL response against the graft of ovalbumin-expressing EG7 tumor cells in the immunized mice. Also, we identified the inhibition of tumor growth and prevention of tumor formation in the context of the therapeutic and prophylactic vaccine, respectively through EG-7 tumor mouse model. Finally, these data show the potential of patches as attractive antigen delivery vehicles.

Engineering Protein Delivery Depots for Cancer Immunotherapy.

Cancer immunotherapy that induces a tumor-specific immune response for cancer eradication has received tremendous attention. To enhance the immunotherapeutic effects, many drug delivery strategies have been developed to overcome the physiological barriers as well as to reduce toxicity. For example, intratumoral or peritumoral administration of injectable depot formulations can directly target tumor sites for immunotherapy. Compared with systemic administration of therapeutics, this strategy has minimal side effects. Such local treatment can also trigger a systemic immune response for inhibiting tumor metastasis. This Topical Review highlights the recent studies on depot-mediated delivery of protein/peptide therapeutics for cancer immunotherapy. Further opportunities and challenges in this field are also discussed.

How Similar Is Biosimilar? A Comparison of Infliximab Therapeutics in Regard to Charge Variant Profile and Antigen Binding Affinity.

Biosimilars are increasing in economic importance. Just how similar a biosimilar needs to be to gain market approval is currently still decided on a per case basis. The authors try to shed light on one often cited critical quality attribute of monoclonal antibodies, namely charge heterogeneity. Using high resolution electrophoretic and chromatographic methods, the authors are able to separate and quantify the charge variant content of infliximab originator and three biosimilars. Additionally the authors quantified and compared the antigen binding affinity in an SPR based binding assay and analyzed the glycosylation pattern of all four of these infliximab biosimilar products. Even though the analytical methods did not show full similarity between originator and some biosimilars, all of the biosimilars have gained approval based on their clinical comparability. The authors would therefore argue, that analytical comparison is not always a good predictor for clinical interchangeability. Any future regulatory framework for the approval of biosimilars should reflect that the parameters chosen for analytical comparability have to be chosen carefully.

Identifying neoantigens for use in immunotherapy.

This review focuses on the types of cancer antigens that can be recognised by the immune system and form due to alterations in the cancer genome, including cancer testis, overexpressed and neoantigens. Specifically, neoantigens can form when cancer cell-specific mutations occur that result in alterations of the protein from 'self'. This type of antigen can result in an immune response sufficient to clear tumour cells when activated. Furthermore, studies have reported that the likelihood of successful immunotherapeutic targeting of cancer by many different methods was reliant on immune response to neoantigens. The recent resurgence of interest in the immune response to tumour cells, in conjunction with technological advances, has resulted in a large increase in the predicted, identified and functionally confirmed neoantigens. This growth in identified neoantigen sequences has increased the contents of training sets for algorithms, which in turn improves the prediction of which genetic mutations may form neoantigens. Additionally, algorithms predicting how proteins will be processed into peptide epitopes by the proteasome and which peptides bind to the transporter complex are also improving with this research. Now that large screens of all the tumour-specific protein altering mutations are possible, the emerging data from assessment of the immunogenicity of neoantigens suggest that only a minority of variants will form targetable epitopes. The potential for immunotherapeutic targeting of neoantigens will therefore be greater in cancers with a higher frequency of protein altering somatic variants. There is considerable potential in the use of neoantigens to treat patients, either alone or in combination with other immunotherapies and with continued advancements, these potentials will be realised.

Caracterización biológica del marcador CD66c y su importancia clínica en la leucemia linfoide aguda / Biological characterization of the CD66c marker and its clinical importance in acute lymphoid leukemia

Las leucemias son un grupo heterogéneo de hemopatías con diversa etiología, patogenia, historia natural y pronóstico en las que se desencadena una proliferación clonal. La leucemia linfoblástica aguda (LLA) es el tipo de cáncer más común en los niños, se caracteriza por la infiltración de células neoplásicas del sistema hematopoyético a la médula ósea, sangre y otros tejidos. Fue considerada fatal hasta hace 30 años, hoy, la tasa de sobrevida a los 5 años supera el 70 por ciento, lo que implica que la mayoría de los pacientes puede curarse; sin embargo, la situación es diferente para la población infantil de los países en desarrollo. Se estima que la supervivencia al cáncer es entre 10 y 20 por ciento menor que para los infantes en países desarrollados. Uno de los marcadores de inmunofenotipo que ha cobrado mayor relevancia en los últimos años en el diagnóstico de LLA-B y seguimiento de la enfermedad mínima residual es el CD66c, este es una glicoproteína miembro de la familia del antígeno carcinoembrionario con función de adhesión celular y ampliamente utilizado como marcador tumoral que fue descubierto por SvenBerg a finales de los años 1970. Este antígeno se expresa en la superficie de los granulocitos y está implicado en varias funcionesbiológicas, que incluyen la adhesión celular, la migración, la transducción de señales y la regulación de la expresión génica. Este antígeno se presenta frecuentemente en varios tipos de cáncer y su sobrexpresión se asocia a menudo con pobre respuesta al tratamiento y disminución de la supervivencia de los pacientes. Diversos estudios evidencian que este marcador se relaciona con la presencia de diversas alteraciones cromosómicas como BCR-ABL, CRLF2, hiperdiploidía, que permiten orientar al pronóstico de la enfermedad(AU)

Leukemias are a heterogeneous group of blood diseases with a diverse etiology, pathogenesis, natural history and prognosis in which a clonal proliferation may be triggered. Acute lymphoid leukemia (ALL) is one of the most common types of cancer in children, it is characterized by the infiltration of neoplastic cells from the haematopoietic system to the bone marrow, blood and other tissues. Until 30 years ago, it was considered fatal; nowadays, the five-year survival rate exceeds 70 percent, which implies that most patients may heal. Nevertheless, in developing countries the situation might be different for pediatric population; it is estimated that cancer survival rate ranges between 10 and 20 percent less than those children living in developed countries. One of the immunophenotype markers that has been relevant in the last few years in the diagnosis of B-ALL and the follow-up of the minimal residual disease is CD66c. This is a member of the glycoprotein family from the carcinoembryonic antigen with a cellular adhesion function that has been widely used as a tumor marker as discovered by Sven Berg in the late 1970's. This antigen has been identified as a superficial protein expressed on the granulocytes and it is also involved in several biological functions, including cellular adhesion, migration, signal transduction and regulation of gene expression. This antigen is frequently presented in several types of cancer and its overexpression is often associated with a poor response to treatment and a decrease of survival rates for patients. Several studies have evidenced that this marker is relatedwith the presence of several chromosomal abnormalities, such as: BCR-ABL, CRLF2, and hyperdiploidy, which may help in the disease prognosis(AU)

"Minimalist" Nanovaccine Constituted from Near Whole Antigen for Cancer Immunotherapy.

One of the major challenges in vaccine design has been the over dependence on incorporation of abundant adjuvants, which in fact is in violation of the "minimalist" principle. In the present study, a compact nanovaccine derived from a near whole antigen (up to 97 wt %) was developed. The nanovaccine structure was stabilized by free cysteines within each antigen (ovalbumin, OVA), which were tempospatially exposed and heat-driven to form an extensive intermolecular disulfide network. This process enables the engineering of a nanovaccine upon integration of the danger signal (CpG-SH) into the network during the synthetic process. The 50 nm-sized nanovaccine was developed comprising approximately 500 antigen molecules per nanoparticle. The nanovaccine prophylactically protected 70% of mice from tumorigenesis (0% for the control group) in murine B16-OVA melanoma. Significant tumor inhibition was achieved by strongly nanovaccine-induced cytotoxic T lymphocytes. This strategy can be adapted for the future design of vaccine for a minimalist composition in clinical settings.

Photosensitizer-induced self-assembly of antigens as nanovaccines for cancer immunotherapy.

Herein, the photosensitizer indocyanine green (ICG) is used to induce the self-assembly of antigens to form nanovaccines. Under near-infrared (NIR) laser irradiation, reactive oxygen species can be generated by nanovaccines to disrupt the membranes of endo/lysosomes, which helps to release antigens into the cytosol efficiently, thereby enhancing antigen cross-presentation and anti-cancer immunity. To the best of our knowledge, this study represents the first example of ICG as a biocompatible adjuvant to improve cancer vaccine efficacy.

Bioconjugate Strategies for the Induction of Antigen-Specific Tolerance in Autoimmune Diseases.

Antigen-specific immunotherapy (ASI) holds great promise for the treatment of autoimmune diseases. In mice, administration of major histocompatibility complex (MHC) binding synthetic peptides which modulate T cell receptor (TCR) signaling under subimmunogenic conditions induces selective tolerance without suppressing the global immune responses. However, clinical translation has yielded limited success. It has become apparent that the TCR signaling pathway via synthetic peptide antigen alone is inadequate to induce an effective tolerogenic immunity in autoimmune diseases. Bioconjugate strategies combining additional immunomodulatory functions with TCR signaling can amplify the antigen-specific immune tolerance and possibly lead to the development of new treatments in autoimmune diseases. In this review, we provide a summary of recent advances in the development of bioconjugates to achieve antigen-specific immune tolerance in vivo, with the discussion focused on the underlying design principles and challenges that must be overcome to target these therapies to patients suffering from autoimmune diseases.

Pulmonary Administration of Soluble Antigen Arrays Is Superior to Antigen in Treatment of Experimental Autoimmune Encephalomyelitis.

Antigen-specific immunotherapy has been used to hyposensitize patients to allergens and offers an enticing approach for attenuating autoimmune diseases. Applying antigen-specific immunotherapy to mucosal surfaces such as the lungs may engage unique immune response pathways to improve efficacy. Pulmonary delivery of soluble antigen arrays (SAgAs) was explored in mice with experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model. SAgAs were designed to impede immune response to autoantigen epitopes and are composed of a hyaluronan backbone with peptides PLP139-151 (proteolipid protein) and LABL, a disease-causing proteolipid peptide epitope and an intracellular cell-adhesion molecule-1 ligand, respectively. Pulmonary instillation of SAgAs decreased disease score, improved weight gain, and decreased incidence of disease in EAE mice compared to pulmonary delivery of hyaluronic acid polymer, LABL, or PLP. Interestingly, treating with PLP alone also showed some improvement. Splenocytes from SAgA-treated animals showed increased interferon-gamma levels, and interleukin-6 (IL-6) and IL-17 were elevated in SAgA-treated animals compared to PLP treatments. IL-10, IL-2, and tumor necrosis factor-alpha levels showed no significant difference, yet trends across all cytokines suggested SAgAs induced a very different immune response compared to treatment with PLP alone. This work suggests that codelivery of peptide components is essential when treating EAE via pulmonary instillation, and the immune response may have shifted toward immune tolerance.