Shark VNAR phage display libraries: An alternative source for therapeutic and diagnostic recombinant antibody fragments.

The development of recombinant antibody fragments as promising alternatives to full-length immunoglobulins offers vast opportunities for biomedicine. Antibody fragments have important advantages compared with conventional monoclonal antibodies that make them attractive for the biotech industry: superior stability and solubility, reduced immunogenicity, higher specificity and affinity, capacity to target the hidden epitope and cross the blood-brain barrier, the ability to refold after heat denaturation and inexpensive and easy large-scale production. Different antibody formats such as antigen-binding fragments (Fab), single-chain fragment variable (scFv) consisting of the antigen-binding domains of Ig heavy (VH) and light (VL) chain regions connected by a flexible peptide linker, single-domain antibody fragments (sdAbs) like camelid heavy-chain variable domains (VHHs) and shark variable new antigen receptor (VNARs), and bispecific antibodies (bsAbs) are currently being evaluated as diagnostics or therapeutics in preclinical studies and clinical trials. In the present review, we summarize and discuss studies on VNARs, the smallest recombinant antibody fragment, obtained after the screening of different types of phage display antibody libraries. Results published until March 2023 are discussed.

Generation of multiepitope cancer vaccines based on large combinatorial libraries of survivin-derived mutant epitopes.

Immune tolerance is the main challenge in the field of cancer vaccines, so modified peptide sequences or naturally occurring mutated versions of cancer-related wild-type (WT) antigens represent a promising pathway. However, the low immunogenicity of mutation-induced neoantigens and, particularly, their incapacity to activate CD8+ T cells are generating doubts on the success of neoantigen-based cancer vaccines in clinical trials. We developed a novel vaccine approach based on a new class of vaccine immunogens, called variable epitope libraries (VELs). We used three regions of survivin (SVN), composed of 40, 49 and 51 amino acids, along with the complete SVN protein to generate the VELs as multiepitope vaccines. BALB/c mice, challenged with the aggressive and highly metastatic 4T1 cell line, were vaccinated in a therapeutic setting. We showed significant tumor growth inhibition and, most importantly, strong suppression of lung metastasis after a single immunization using VEL vaccines. We demonstrated vaccine-induced broad cellular immune responses concomitant with extensive tumor infiltration of T cells, the activation of CD107a+  IFN-γ+ T cells in the spleen and a significant increase in the number of CD3+  CD8+  Ly6C+ effector T cells. In addition, we observed the presence of interferon-γ-, granzyme B- and perforin-producing lymphocytes along with modifications in the amount of CD11b+  Ly6Cint/low  Ly6G+ granulocytic myeloid-derived suppressor cells and CD4+  CD25+  FoxP3+ regulatory T cells in the lungs and tumors of mice. In summary, we showed that the VELs represent a potent new class of cancer immunotherapy and propose the application of the VEL vaccine concept as a true alternative to currently available vaccine platforms.

Immunogenic properties of immunoglobulin superfamily members within complex biological networks.

Antibodies, T cell receptors and major histocompatibility complex molecules are members of the immunoglobulin superfamily and have pivotal roles in the immune system. The fine interrelation between them regulates several immune functions. Here, we describe lesser-known functions ascribed to these molecules in generating and maintaining immune response. Particularly, we outline the contribution of antibody- and T cell receptor-derived complementarity-determining region neoantigens, antigenized antibodies, as well as major histocompatibility complex class I molecules-derived epitopes to the induction of protective/therapeutic immune responses against pathogens and cancer. We discuss findings of our own and other studies describing protective mechanisms, based on immunogenic properties of immunoglobulin superfamily members, and evaluate the perspectives of application of this class of immunogens in molecular vaccines design.

Are we getting closer to a successful neoantigen cancer vaccine?

Although significant advances in immunotherapy have revolutionized the treatment of many cancer types over the past decade, the field of vaccine therapy, an important component of cancer immunotherapy, despite decades-long intense efforts, is still transmitting signals of promises and awaiting strong data on efficacy to proceed with regulatory approval. The field of cancer vaccines faces standard challenges, such as tumor-induced immunosuppression, immune response in inhibitory tumor microenvironment (TME), intratumor heterogeneity (ITH), permanently evolving cancer mutational landscape leading to neoantigens, and less known obstacles: neoantigen gain/loss upon immunotherapy, the timing and speed of appearance of neoantigens and responding T cell clonotypes and possible involvement of immune interference/heterologous immunity, in the complex interplay between evolving tumor epitopes and the immune system. In this review, we discuss some key issues related to challenges hampering the development of cancer vaccines, along with the current approaches focusing on neoantigens. We summarize currently well-known ideas/rationales, thus revealing the need for alternative vaccine approaches. Such a discussion should stimulate vaccine researchers to apply out-of-box, unconventional thinking in search of new avenues to deal with critical, often yet unaddressed challenges on the road to a new generation of therapeutics and vaccines.

Recombinant Antibody Fragments for Immunotherapy of Parkinson's Disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. Multiple genetic and environmental factors leading to progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SN) and consequent depletion of dopamine were described. Current clinical approaches, such as dopamine replacement or deep brain stimulation using surgically implanted probes, provide symptomatic relief but cannot modify disease progression. Therefore, disease-modifying therapeutic tools are urgently needed. Immunotherapy approaches, including passive transfer of protective antibodies and their fragments, have shown therapeutic efficacy in several animal models of neurodegenerative diseases, including PD. Recombinant antibody fragments are promising alternatives to conventional full-length antibodies. Modern computational approaches and molecular biology tools, directed evolution methodology, and the design of tissue-penetrating fusion peptides allowed for the development of recombinant antibody fragments with superior specificity and affinity, reduced immunogenicity, the capacity to target hidden epitopes and cross the blood-brain barrier (BBB), higher solubility and stability, the ability to refold after heat denaturation, and inexpensive large-scale production. In addition, antibody fragments do not induce microglia Fcγ receptor (FcγR)-mediated proinflammatory response and tissue damage in the central nervous system (CNS), because they lack the Fc portion of the immunoglobulin molecule. In the present review, we summarized data on recombinant antibody fragments evaluated as immunotherapeutics in preclinical models of PD and discussed their potential for developing therapeutic and preventive protocols for patients with PD.

Recombinant Antibody Fragments for Neurological Disorders: An Update.

Recombinant antibody fragments are promising alternatives to full-length immunoglobulins, creating big opportunities for the pharmaceutical industry. Nowadays, antibody fragments such as antigen-binding fragments (Fab), single-chain fragment variable (scFv), single-domain antibodies (sdAbs), and bispecific antibodies (bsAbs) are being evaluated as diagnostics or therapeutics in pre-clinical models and in clinical trials. Immunotherapy approaches, including passive transfer of protective antibodies, have shown therapeutic efficacy in several animal models of Alzheimer ́s disease(AD), Parkinson ́s disease (PD), frontotemporal dementia (FTD), Huntington ́s disease (HD), transmissible spongiform encephalopathies (TSEs) and multiple sclerosis (MS). There are various antibodies approved by the Food and Drug Administration (FDA) for treating multiple sclerosis and two amyloid beta-specific humanized antibodies, Aducanumab and Lecanemab, for AD. Our previous review summarized data on recombinant antibodies evaluated in pre-clinical models for immunotherapy of neurodegenerative diseases. Here, we explore recent studies in this fascinating research field, give an update on new preventive and therapeutic applications of recombinant antibody fragments for neurological disorders and discuss the potential of antibody fragments for developing novel approaches for crossing the blood-brain barrier (BBB) and targeting cells and molecules of interest in the brain.

Tumor antigen-unbiased variable epitope library contains mimotopes with antitumor effect in a mouse model of breast cancer.

Breast cancer is one of the leading causes of death that affects the female population worldwide. Despite advances in treatments and a greater understanding of the disease, there are still difficulties in successfully treating patients. Currently, the main challenge in the field of cancer vaccines is antigenic variability which can reduce antigen-specific T- cell response efficacy. The search for and validation of immunogenic antigen targets increased dramatically over the past few decades and, with the advent of modern sequencing techniques, permitting the fast and accurate identification of the neoantigen landscape of tumor cells, will undoubtedly continue to grow exponentially for years to come. We have previously implemented Variable Epitope Libraries (VEL) as an unconventional vaccine strategy in preclinical models and for identifying and selecting mutant epitope variants. Here, we used an alanine-based sequence to generate a 9-mer VEL-like combinatorial mimotope library G3d as a new class of vaccine immunogen. An in silico analysis of the 16,000 G3d-derived sequences revealed potential MHC-I binders and immunogenic mimotopes. We demonstrated the antitumor effect of treatment with G3d in the 4T1 murine model of breast cancer. Moreover, two different T cell proliferation screening assays against a panel of randomly selected G3d-derived mimotopes allowed the isolation of both stimulatory and inhibitory mimotopes showing differential therapeutic vaccine efficacy. Thus, the mimotope library is a promising vaccine immunogen and a reliable source for isolating molecular cancer vaccine components.

Antitumor efficacy of MUC1-derived variable epitope library treatments in a mouse model of breast cancer.

The identification of novel targets for cancer immunotherapy and the development of new vaccine immunogens are subjects of permanent interest. MUC1 is an overexpressed antigen found in most tumors, and its overexpression correlates with poor prognosis. Many attempts to direct the immune response against MUC1 in tumor cells have failed, including several clinical trials. We have previously developed an innovative Variable Epitope Library (VEL) vaccine platform that carries massively substituted mutant variants of defined epitopes or epitope regions as an alternative to using wild-type peptide sequences-based immunogens. Here, two murine MUC1-derived epitopes equivalent to the previously tested in cancer immunotherapy human MUC1 regions were used to generate VELs. We observed that vaccination with the 23L VEL immunogens, encompassing the entire signal peptide region of MUC1, reduces the tumor area compared to the wild-type sequence treatment. Contrastingly, vaccination with the MUC1 signal peptide-derived predicted CD8++ T cell epitope-based VEL, 9MUC1spL, showed similar tumor area reduction as the wild-type treatment; however, a decrease in lung metastasis after 9MUC1spL treatment was observed. In addition, vaccination induced a large pool of CD8+ T cells which recognized most variant epitopes from 9MUC1spL. Also, we generated MUC1 variable number tandem repeat (VNTR)-based VELs that reduced the metastatic burden when dendritic cells and M13 recombinant bacteriophages were used as vaccine carriers. Collectively, our data demonstrate the immunogenic and antitumor properties of MUC1 signal peptide- and VNTR-derived VEL immunogens.

Neoantigen Cancer Vaccines: Real Opportunity or Another Illusion?

In this communication, we will analyze some important factors and immunological phenomena related to neoantigen cancer vaccines, with particular emphasis on recently published Phase I clinical trials. Several obstacles and issues are addressed that challenge the current paradigm and inquire if neoantigens, which are essentially single-use vaccine candidates, are legitimate targets to induce protective immune responses with regard to the evolving mutational landscape. We also share insights into the striking similarities between cancer and antigenically variable pathogens and suggest that any successful vaccine against either should demonstrate a similar property: efficient induction of a diverse pool of immune cells equipped to prevent immune escape. Hence, to confront antigenic variability directly, we have employed our innovative vaccine concept, Variable Epitope Libraries, composed of large combinatorial libraries of heavily mutated epitopes, as a "universal" vaccine platform. Collectively, we offer critical analyses on key issues, which ultimately reflect on the prospective clinical relevance of personalized neoantigen vaccines which is still undefined.

Generation of cancer vaccine immunogens derived from Oncofetal antigen (OFA/iLRP) using variable epitope libraries tested in an aggressive breast cancer model.

After decades of cancer vaccine efforts, there is an imperious necessity for novel ideas that may result in better tumor control in patients. We have proposed the use of a novel Variable Epitope Library (VEL) vaccine strategy, which incorporates an unprecedented number of mutated epitopes to target antigenic variability and break tolerance against tumor-associated antigens. Here, we used an oncofetal antigen/immature laminin receptor protein-derived sequence to generate 9-mer and 43-mer VEL immunogens. 4T1 tumor-bearing mice developed epitope-specific CD8+IFN-γ+ and CD4+IFN-γ+ T cell responses after treatment. Tumor and lung analysis demonstrated that VELs could increase the number of tumor-infiltrating lymphocytes with diverse effector functions while reducing the number of immunosuppressive myeloid-derived suppressor and regulatory T cells. Most importantly, VEL immunogens inhibited tumor growth and metastasis after a single dose. The results presented here are consistent with our previous studies and provide evidence for VEL immunogens' feasibility as promising cancer immunotherapy.

The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction.

It is generally accepted that carbohydrate binding modules (CBMs) recognize their carbohydrate ligands by hydrophobic and CH-π interactions. Point mutations of one CBM26 of the Lactobacillus amylovorus α-amylase starch-binding domain (LaCBM26) showed that conserved non-aromatic residue are essential in the starch recognition function of the domain, as the mutation of a single glutamine (Q68L) eliminates binding to starch and ß-cyclodextrin, even in the presence of aromatic amino acids necessary for ligand binding. The secondary structure of mutated proteins was verified and showed no differences from the wild-type domain. However, random mutations of five residues involved in binding (Y18, Y20, Q68, E74, and F77) did cause change in the secondary structure of the protein, which also causes loss of function. Much of the diversity introduced in the LaCBM26 was probably incompatible with the appropriate folding of these proteins, suggesting that the domain has little tolerance to change.

Amyloid-beta peptide binds to microtubule-associated protein 1B (MAP1B).

Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer's disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer's disease.

Generation of cancer vaccine immunogens derived from major histocompatibility complex (MHC) class I molecules using variable epitope libraries.

Although various immune checkpoint inhibitors (ICIs), used for the treatment of advanced cancer, showed remarkably durable tumor regression in a subset of patients, there are important limitations in a large group of non-responders, and the generation of novel immunogens capable of inducing protective cellular immune responses is a priority in cancer immunotherapy field. During the last decades, several types of vaccine immunogens have been used in numerous preclinical studies and clinical trials. However, although immunity to tumor Ags can be elicited by most vaccines tested, their clinical efficacy remains modest. Recently, we have developed an innovative vaccine concept, called Variable Epitope Libraries (VELs), with the purpose to exploit the high antigenic variability of many important pathogens and tumor cells as starting points for the construction of a new class of vaccine immunogens capable of inducing the largest possible repertoire of both B and T cells. In the present study, we decided to generate VEL immunogens derived from both classical and non-classical major histocompatibility complex (MHC) class I molecules. The MHC molecules, responsible for antigen presentation and subsequent activation of T lymphocytes, undergo multiple modifications that directly affect their proper function, resulting in immune escape of tumor cells. Two large VELs derived from multi-epitope region of H2-Kd and Qa-2 sequences (46 and 34 amino acids long, respectively), along with their wild type counterparts have been generated as synthetic peptides and tested in an aggressive 4T1 mouse model of breast cancer. Significant inhibition of tumor growth and the reduction of metastatic lesions in the lungs of immunized mice were observed. This study demonstrated for the first time the successful application of VELs carrying combinatorial libraries of epitope variants derived from MHC class I molecules as novel vaccine immunogens.

Data concerning secondary structure and alpha-glucans-binding capacity of the LaCBM26.

Carbohydrate-binding modules (CBMs) are auxiliary domains into glycoside-hydrolases that allow the interaction between the insoluble substrate and the solubilized enzyme, through hydrophobic, CH-π interactions and hydrogen bonds. Here, we present the data article related to the interaction of one LaCBM26 and some mutated proteins with soluble α-glucans determined by enzyme-linked carbohydrate-binding assay, isothermal titration calorimetry (ITC), and affinity gel electrophoresis (AGE). The data of the behavior of proteins in presence and absence of substrate analyzed by circular dichroism CD and thermofluor are also presented. These results are complementary to the research article "The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction" (Armenta et al., 2019).

Recombinant Antibody Fragments for Neurodegenerative Diseases.

BACKGROUND: Recombinant antibody fragments are promising alternatives to full-length immunoglobulins and offer important advantages compared with conventional monoclonal antibodies: extreme specificity, higher affinity, superior stability and solubility, reduced immunogenicity as well as easy and inexpensive large-scale production. OBJECTIVE: In this article we will review and discuss recombinant antibodies that are being evaluated for neurodegenerative diseases in pre-clinical models and in clinical studies and will summarize new strategies that are being developed to optimize their stability, specificity and potency for advancing their use. METHODS: Articles describing recombinant antibody fragments used for neurological diseases were selected (PubMed) and evaluated for their significance. RESULTS: Different antibody formats such as single-chain fragment variable (scFv), single-domain antibody fragments (VHHs or sdAbs), bispecific antibodies (bsAbs), intrabodies and nanobodies, are currently being studied in pre-clinical models of cancer as well as infectious and autoimmune diseases and many of them are being tested as therapeutics in clinical trials. Immunotherapy approaches have shown therapeutic efficacy in several animal models of Alzheimer´s disease (AD), Parkinson disease (PD), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), Huntington disease (HD), transmissible spongiform encephalopathies (TSEs) and multiple sclerosis (MS). It has been demonstrated that recombinant antibody fragments may neutralize toxic extra- and intracellular misfolded proteins involved in the pathogenesis of AD, PD, DLB, FTD, HD or TSEs and may target toxic immune cells participating in the pathogenesis of MS. CONCLUSION: Recombinant antibody fragments represent a promising tool for the development of antibody-based immunotherapeutics for neurodegenerative diseases.

Identification of amyloid-beta 1-42 binding protein fragments by screening of a human brain cDNA library.

Extracellular and intraneuronal formation of amyloid-beta (Abeta) deposits have been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of Abeta neurotoxicity is not completely understood. Previous studies suggest that binding of Abeta with a number of targets have deleterious effects on cellular functions. It has been shown that Abeta directly interacted with intracellular protein ERAB (endoplasmic reticulum amyloid beta-peptide-binding protein) also known as ABAD (Abeta-binding alcohol dehydrogenase) resulting in mitochondrial dysfunction and cell death. In the present study we have identified another mitochondrial enzyme, ND3 of the human complex I, that binds to Abeta1-42 by the screening of a human brain cDNA library expressed on M13 phage. Our results indicated a strong interaction between Abeta and a phage-displayed 25 amino acid long peptide TTNLPLMVMSSLLLIIILALSLAYE corresponding to C-terminal peptide domain of NADH dehydrogenase, subunit 3 (MTND3) encoded by mitochondrial DNA (mtDNA). This interaction may explain, in part, the inhibition of complex I activity in astrocytes and neurons in the presence of Abeta, described recently. To our knowledge, the present study is the first demonstration of interaction between Abeta and one of the subunits of the human complex I.

Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants.

Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed.

Mimotopes of conformational epitopes in fibrillar beta-amyloid.

In Alzheimer's disease (AD) beta-amyloid peptide accumulates in the brain in different forms including fibrils. Amyloid fibrils could be recognized as foreign by the mature immune system since they are not present during its development. Thus, using mouse antisera raised against the fibrillar form of Abeta42, we have screened two phage peptide libraries for the presence of foreign conformational mimotopes of Abeta. Antisera from wild type animals recognized predominately peptides with the EFRH motif from Abeta42 sequence, whereas amyloid precursor protein (APP) transgenic mice recognized mainly phage clones that mimic epitopes (mimotopes) within the fibrillar Abeta42 but lack sequence homology with this peptide.

Antigenic properties of phage displayed peptides comprising disulfide-bonded loop of the immunodominant region of HIV-1 gp41.

The HIV-1 envelope glycoprotein gp41 contains Cys(X)5Cys motif, which has been shown to elicit a strong antibody response in almost all HIV-1 infected individuals. This disulfide-bonded loop region is conserved in most retroviruses suggesting the existence of an essential function in virus life cycle. In this study, we displayed the peptides comprising 12 amino acids of the immunodominant loop of gp41 on the surface of M13 phage as N-terminal fusions to the minor coat protein pIII and major coat protein pVIII of the phage and demonstrated that cysteine loop containing peptide expressed on phage recognized 62 out of 63 (98.4%) HIV-1 positive samples but not control negative sera while phage bearing linear peptides detected 4-30% of HIV-1-positive sera. The main advantage of phage-based ELISA or other antibody detection-based diagnostic tests of HIV-infection to be used for massive screening in developing countries is the reproducible, simple, rapid and low-cost production of recombinant antigens.

New approaches to improve a peptide vaccine against porcine Taenia solium cysticercosis.

Cysticercosis caused by Taenia solium frequently affects human health and rustic porciculture. Cysticerci may localize in the central nervous system of humans causing neurocysticercosis, a major health problem in undeveloped countries. Prevalence and intensity of this disease in pigs and humans are related to social factors (poor personal hygiene, low sanitary conditions, rustic rearing of pigs, open fecalism) and possibly to biological factors such as immunity, genetic background, and gender. The indispensable role of pigs as an obligatory intermediate host in the life cycle offers the possibility of interfering with transmission through vaccination of pigs. An effective vaccine based on three synthetic peptides against pig cysticercosis has been successfully developed and proved effective in experimental and field conditions. The well-defined peptides that constitute the cysticercosis vaccine offer the possibility to explore alternative forms of antigen production and delivery systems that may improve the cost/benefit of this and other vaccines. Encouraging results were obtained in attempts to produce large amounts of these peptides and increased its immunogenicity by expression in recombinant filamentous phage (M13), in transgenic plants (carrots and papaya), and associated to bacterial immunogenic carrier proteins.