Conversion of tumors into autologous vaccines by intratumoral injection of alpha-Gal glycolipids that induce anti-Gal/alpha-Gal epitope interaction

Anti-Gal is the most abundant antibody in humans, constituting 1% of immunoglobulins. Anti-Gal binds specifically alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). Immunogenicity of autologous tumor associated antigens (TAA) is greatly increased by manipulating tumor cells to express alpha-gal epitopes and bind anti-Gal. Glycolipids with alphagal epitopes (alpha-gal glycolipids) injected into tumors insert into the tumor cell membrane. Anti-Gal binding to the multiple alpha-gal epitopes de novo presented on the tumor cells results in targeting of these cells to APC via the interaction between the Fc portion of the bound anti-Gal and Fcgamma; receptors on APC. The APC process and present immunogenic TAA peptides and thus, effectively activate tumor specific CD4+ helper T cells and CD8+ cytotoxic T cells which destroy tumor cells in micrometastases. The induced immune response is potent enough to overcome immunosuppression by Treg cells. A phase I clinical trial indicated that alpha-gal glycolipid treatment has no adverse effects. In addition to achieving destruction of micrometastases in cancer patients with advance disease, alpha-gal glycolipid treatment may be effective as neo-adjuvant immunotherapy. Injection of alpha-gal glycolipids into primary tumors few weeks prior to resection can induce a protective immune response capable of destroying micrometastases expressing autologous TAA, long after primary tumor resection

Antibody production and tolerance to the α-gal epitope as models for understanding and preventing the immune response to incompatible ABO carbohydrate antigens and for α-gal therapies

This review describes the significance of the α-gal epitope (Galα-3Galβ1-4GlcNAc-R) as the core of human blood-group A and B antigens (A and B antigens), determines in mouse models the principles underlying the immune response to these antigens, and suggests future strategies for the induction of immune tolerance to incompatible A and B antigens in human allografts. Carbohydrate antigens, such as ABO antigens and the α-gal epitope, differ from protein antigens in that they do not interact with T cells, but B cells interacting with them require T-cell help for their activation. The α-gal epitope is the core of both A and B antigens and is the ligand of the natural anti-Gal antibody, which is abundant in all humans. In A and O individuals, anti-Gal clones (called anti-Gal/B) comprise >85% of the so-called anti-B activity and bind to the B antigen in facets that do not include fucose-linked α1–2 to the core α-gal. As many as 1% of B cells are anti-Gal B cells. Activation of quiescent anti-Gal B cells upon exposure to α-gal epitopes on xenografts and some protozoa can increase the titer of anti-Gal by 100-fold. α1,3-Galactosyltransferase knockout (GT-KO) mice lack α-gal epitopes and can produce anti-Gal. These mice simulate human recipients of ABO-incompatible human allografts. Exposure for 2–4 weeks of naïve and memory mouse anti-Gal B cells to α-gal epitopes in the heterotopically grafted wild-type (WT) mouse heart results in the elimination of these cells and immune tolerance to this epitope. Shorter exposures of 7 days of anti-Gal B cells to α-gal epitopes in the WT heart result in the production of accommodating anti-Gal antibodies that bind to α-gal epitopes but do not lyse cells or reject the graft. Tolerance to α-gal epitopes due to the elimination of naïve and memory anti-Gal B cells can be further induced by 2 weeks in vivo exposure to WT lymphocytes or autologous lymphocytes engineered to present α-gal epitopes by transduction of the α1,3-galactosyltransferase gene. These mouse studies suggest that autologous human lymphocytes similarly engineered to present the A or B antigen may induce corresponding tolerance in recipients of ABO-incompatible allografts. The review further summarizes experimental works demonstrating the efficacy of α-gal therapies in amplifying anti-viral and anti-tumor immune-protection and regeneration of injured tissues

Induced Remodeling of Porcine Tendons to Human Anterior Cruciate Ligaments by alpha-GAL Epitope Removal and Partial Cross-Linking

This review describes a novel method developed for processing porcine tendon and other ligament implants that enables in situ remodeling into autologous ligaments in humans. The method differs from methods using extracellular matrices (ECMs) that provide postoperative orthobiological support (i.e., augmentation grafts) for healing of injured ligaments, in that the porcine bone-patellar-tendon-bone itself serves as the graft replacing ruptured anterior cruciate ligament (ACL). The method allows for gradual remodeling of porcine tendon into autologous human ACL while maintaining the biomechanical integrity. The method was first evaluated in a preclinical model of monkeys and subsequently in patients. The method overcomes detrimental effects of the natural anti-Gal antibody and harnesses anti-non-gal antibodies for the remodeling process in two steps: Step 1. Elimination of alpha-gal epitopes-this epitope that is abundant in pigs (as in other nonprimate mammals) binds the natural anti-Gal antibody, which is the most abundant natural antibody in humans. This interaction, which can induce fast resorption of the porcine implant, is avoided by enzymatic elimination of alpha-gal epitopes from the implant with recombinant alpha-galactosidase. Step 2. Partial cross-linking of porcine tendon with glutaraldehyde-this cross-linking generates covalent bonds in the ECM, which slow infiltration of macrophages into the implant. Anti-non-gal antibodies are produced in recipients against the multiple porcine antigenic proteins and proteoglycans because of sequence differences between human and porcine homologous proteins. Anti-non-gal antibodies bind to the implant ECM, recruit macrophages, and induce the implant destruction by directing proteolytic activity of macrophages. Partial cross-linking of the tendon ECM decreases the extent of macrophage infiltration and degradation of the implant and enables concomitant infiltration of fibroblasts that follow the infiltrating macrophages. These fibroblasts align with the implant collagen fibers and secrete their own collagen and other ECM proteins, which gradually remodel the porcine tendon into human ACL. This ligamentization process lasts approximately 2 years and the biomechanical integrity of the graft is maintained throughout the whole period. These studies are the first, and so far the only, to demonstrate remodeling of porcine tendon implants into permanently functional autologous ACL in humans

alpha-Gal Nanoparticles in Wound and Burn Healing Acceleration

Significance: Rapid recruitment and activation of macrophages may accelerate wound healing. Such accelerated healing was observed in wounds and burns of experimental animals treated with alpha-gal nanoparticles. Recent Advances: alpha-Gal nanoparticles present multiple alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). alpha-Gal nanoparticles applied to wounds bind anti-Gal (the most abundant antibody in humans) and generate chemotactic complement peptides, which rapidly recruit macrophages. Fc/Fc receptor interaction between anti-Gal coating the alpha-gal nanoparticles and recruited macrophages activates macrophages to produce cytokines that accelerate healing. alpha-Gal nanoparticles applied to burns and wounds in mice and pigs producing anti-Gal, decreased healing time by 40-60%. In mice, this accelerated healing avoided scar formation. alpha-Gal nanoparticle-treated wounds, in diabetic mice producing anti-Gal, healed within 12 days, whereas saline-treated wounds became chronic wounds. alpha-Gal nanoparticles are stable for years and may be applied dried, in suspension, aerosol, ointments, or within biodegradable materials. Critical Issues: alpha-Gal nanoparticle therapy can be evaluated only in mammalian models producing anti-Gal, including alpha1,3-galactosyltransferase knockout mice and pigs or Old World primates. Traditional experimental animal models synthesize alpha-gal epitopes and lack anti-Gal. Future Directions: Since anti-Gal is naturally produced in all humans, it is of interest to determine safety and efficacy of alpha-gal nanoparticles in accelerating wound and burn healing in healthy individuals and in patients with impaired wound healing such as diabetic patients and elderly individuals. In addition, efficacy of alpha-gal nanoparticle therapy should be studied in healing and regeneration of internal injuries such as surgical incisions, ischemic myocardium following myocardial infarction, and injured nerves

Conversion of Tumors into Autologous Vaccines by Intratumoral Injection of α-Gal Glycolipids that Induce Anti-Gal/α-Gal Epitope Interaction

Anti-Gal is the most abundant antibody in humans, constituting 1% of immunoglobulins. Anti-Gal binds specifically α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R). Immunogenicity of autologous tumor associated antigens (TAA) is greatly increased by manipulating tumor cells to express α-gal epitopes and bind anti-Gal. Glycolipids with αgal epitopes (α-gal glycolipids) injected into tumors insert into the tumor cell membrane. Anti-Gal binding to the multiple α-gal epitopes de novo presented on the tumor cells results in targeting of these cells to APC via the interaction between the Fc portion of the bound anti-Gal and Fcγ; receptors on APC. The APC process and present immunogenic TAA peptides and thus, effectively activate tumor specific CD4+ helper T cells and CD8+ cytotoxic T cells which destroy tumor cells in micrometastases. The induced immune response is potent enough to overcome immunosuppression by Treg cells. A phase I clinical trial indicated that α-gal glycolipid treatment has no adverse effects. In addition to achieving destruction of micrometastases in cancer patients with advance disease, α-gal glycolipid treatment may be effective as neo-adjuvant immunotherapy. Injection of α-gal glycolipids into primary tumors few weeks prior to resection can induce a protective immune response capable of destroying micrometastases expressing autologous TAA, long after primary tumor resection

In Situ “Humanization” of Porcine Bioprostheses: Demonstration of Tendon Bioprostheses Conversion into Human ACL and Possible Implications for Heart Valve Bioprostheses

This review describes the first studies on successful conversion of porcine soft-tissue bioprostheses into viable permanently functional tissue in humans. This process includes gradual degradation of the porcine tissue, with concomitant neo-vascularization and reconstruction of the implanted bioprosthesis with human cells and extracellular matrix. Such a reconstruction process is referred to in this review as “humanization”. Humanization was achieved with porcine bone-patellar-tendon-bone (BTB), replacing torn anterior-cruciate-ligament (ACL) in patients. In addition to its possible use in orthopedic surgery, it is suggested that this humanization method should be studied as a possible mechanism for converting implanted porcine bioprosthetic heart-valves (BHV) into viable tissue valves in young patients. Presently, these patients are only implanted with mechanical heart-valves, which require constant anticoagulation therapy. The processing of porcine bioprostheses, which enables humanization, includes elimination of α-gal epitopes and partial (incomplete) crosslinking with glutaraldehyde. Studies on implantation of porcine BTB bioprostheses indicated that enzymatic elimination of α-gal epitopes prevents subsequent accelerated destruction of implanted tissues by the natural anti-Gal antibody, whereas the partial crosslinking by glutaraldehyde molecules results in their function as “speed bumps” that slow the infiltration of macrophages. Anti-non gal antibodies produced against porcine antigens in implanted bioprostheses recruit macrophages, which infiltrate at a pace that enables slow degradation of the porcine tissue, neo-vascularization, and infiltration of fibroblasts. These fibroblasts align with the porcine collagen-fibers scaffold, secrete their collagen-fibers and other extracellular-matrix (ECM) components, and gradually replace porcine tissues degraded by macrophages with autologous functional viable tissue. Porcine BTB implanted in patients completes humanization into autologous ACL within ~2 years. The similarities in cells and ECM comprising heart-valves and tendons, raises the possibility that porcine BHV undergoing a similar processing, may also undergo humanization, resulting in formation of an autologous, viable, permanently functional, non-calcifying heart-valves

Acceleration of Wound Healing by α-gal Nanoparticles Interacting with the Natural Anti-Gal Antibody

Application of α-gal nanoparticles to wounds and burns induces accelerated healing by harnessing the natural anti-Gal antibody which constitutes ~1% of human immunoglobulins. α-gal nanoparticles present multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R), the carbohydrate ligand of anti-Gal. Studied α-gal nanoparticles were comprised of glycolipids with α-gal epitopes, phospholipids, and cholesterol. Binding of anti-Gal to α-gal nanoparticles in wounds activates the complement cascade, resulting in formation of chemotactic complement cleavage peptides that induce rapid recruitment of many macrophages. The Fc/Fcγ receptors interaction between anti-Gal coating α-gal nanoparticles and the recruited macrophages activates macrophages to produce cytokines/growth factors that promote wound healing and recruit stem cells. Studies of wound healing by α-gal nanoparticles were feasible in α1,3galactosyltransferase knockout mice and pigs. In contrast to other nonprimate mammals, these mice and pigs lack the α-gal epitope, and thus they are not immunotolerant to it and produce anti-Gal. Treatment of skin wounds and burns with α-gal nanoparticles resulted in 40–60% decrease in healing time in comparison with control wounds treated with saline. This accelerated healing is associated with increased recruitment of macrophages and extensive angiogenesis in wounds, faster regrowth of epidermis, and regeneration of the dermis. The accelerated healing further decreases and may completely eliminate fibrosis and scar formation in wounds. Since healing of internal injuries is mediated by mechanisms similar to those in external wound healing, it is suggested that α-gal nanoparticles treatment may also improve regeneration and restoration of biological function following internal injuries such as surgical incisions, myocardial ischemia following infarction, and nerve injuries

Discovery of the natural anti-Gal antibody and its past and future relevance to medicine

This is a personal account of the discovery of the natural anti-Gal antibody, the most abundant natural antibody in humans, the reciprocal distribution of this antibody and its ligand the alpha-gal epitope in mammals and the immunological barrier this antibody has formed in porcine to human xenotransplantation. This barrier has been overcome in the recent decade with the generation of alpha1,3-galactosyltransferase gene-knockout pigs. However, anti-Gal continues to be relevant in medicine as it can be harnessed for various therapeutic effects. Anti-Gal converts tumor lesions injected with alpha-gal glycolipids into vaccines that elicit a protective anti-tumor immune response by in situ targeting of tumor cells for uptake by antigen-presenting cells. This antibody further accelerates wound and burn healing by interaction with alpha-gal nanoparticles applied to injured areas and induction of rapid recruitment and activation of macrophages. Anti-Gal/alpha-gal nanoparticle immune complexes may further induce rapid recruitment and activation of macrophages in ischemic myocardium and injured nerves, thereby inducing tissue regeneration and prevention of fibrosis

Anti-Gal: An abundant human natural antibody of multiple pathogeneses and clinical benefits

Anti-Gal is the most abundant natural antibody in humans constituting ~1% of immunoglobulins. Anti-Gal is naturally produced also in apes and Old World monkeys. The ligand of anti-Gal is a carbohydrate antigen called the alpha-gal epitope with the structure Galalpha1-3Galbeta1-4GlcNAc-R. The alpha-gal epitope is present as a major carbohydrate antigen in nonprimate mammals, prosimians and New World monkeys. Anti-Gal can contribute to several immunological pathogeneses. Anti-Gal IgE produced in some individuals causes allergies to meat and to the therapeutic monoclonal antibody cetuximab, all presenting alpha-gal epitopes. Aberrant expression of the alpha-gal epitope or of antigens mimicking it in humans may result in autoimmune processes, as in Graves\u27 Disease. alpha-Gal epitopes produced by Trypanosoma cruzi interact with anti-Gal and induce autoimmune like inflammatory reactions in Chagas Disease. Anti-Gal IgM and IgG further mediate rejection of xenografts expressing alpha-gal epitopes. Because of its abundance, anti-Gal may be exploited for various clinical uses. It increases immunogenicity of microbial vaccines (e.g., flu vaccine) presenting alpha-gal epitopes by targeting them for effective uptake by APC. Tumor lesions are converted into vaccines against autologous tumor associated antigens by intratumoral injection of alpha-gal glycolipids which insert into tumor cell membranes. Anti-Gal binding to alpha-gal epitopes on tumor cells targets them for uptake by APC. Accelerated wound healing is achieved by application of alpha-gal nanoparticles which bind anti-Gal, activate complement, recruit and activate macrophages that induce tissue regeneration. This therapy may be of further significance in regeneration of internally injured tissues such as ischemic myocardium and injured nerves. This article is protected by copyright. All rights reserved