anti-Gal Ab Research Articles

Immunoglobulin M and Immunoglobulin G Subclass Distribution of Anti-galactose-Alpha-1,3-Galactose and Anti-N-Glycolylneuraminic Acid Antibodies in Healthy Korean Adults

BackgroundHuman preformed antibodies (Abs), anti-galactose-alpha-1,3-galactose (Gal) and anti-N-glycolylneuraminic acid (Neu5Gc), can react with porcine antigens of wild-type pigs. To provide basic population data of the Abs for potential application in clinical xenotransplantation, we developed enzyme-linked immunosorbent assay methods and investigated the serum titers of anti-Gal and anti-Neu5Gc Abs, including immunoglobulin (Ig) M and IgG along with its subclasses, in humans. MethodsAnti-Gal and anti-Neu5Gc Abs serum titers were measured in 380 healthy Korean adults using the in-house enzyme-linked immunosorbent assays. The frequency and median values of anti-Gal and anti-Neu5Gc were measured, and their class and subclass distribution were evaluated. ResultsThe detection frequencies of anti-Gal were 99.2%, 95.0%, 23.2%, 94.5%, 12.4%, and 3.4% for IgM, IgG, IgG1, IgG2, IgG3, and IgG4, respectively. The detection frequencies of anti-Neu5Gc Abs were 87.4%, 96.6%, 1.6%, 46.3%, 0.0%, and 0.0% for IgM, IgG, IgG1, IgG2, IgG3, and IgG4, respectively. The median values of anti-Gal IgM (1001.6 ng/mL) and IgG (1198.3 ng/mL) were significantly higher than those of anti-Neu5Gc Abs (IgM, 328.4 ng/mL; IgG, 194.7 ng/mL; P < .001). IgG2 titers of both anti-Gal and anti-Neu5Gc Abs correlated better with the IgG class than the titers of other IgG subclasses. ConclusionsThe titers of anti-Gal Abs were higher than those of anti-Neu5Gc Abs. IgG2 was the main IgG subclass in both anti-Gal and anti-Neu5Gc Abs. Variation in the titers of anti-Gal or anti-Neu5Gc Abs may partly explain the biological and immunologic changes that occur in recipients of xenotransplants.

The IgM and IgG subclass distribution of anti-Galactose-alpha-1,3-galactose and anti-N-glycolylneuraminic acid antibodies in healthy adults

Abstract Background The major xenoreactive antibodies (Abs), anti-galactose-alpha-1,3-galactose (Gal) and anti-N-glycolylneuraminic acid (Neu5Gc), exist naturally in humans and can contribute to several immunological pathogenesis in xenotransplantation. In this study, we investigated the serum titers of anti-Gal and anti-Neu5Gc Abs including the IgM, IgG, and IgG subclasses in humans to gather basic population data before launching clinical xenotransplantation. Methods Serum titers of anti-Gal and anti-Neu5Gc Abs were measured in 380 healthy adults using an in-house-developed enzyme-linked immunosorbent assay. The detection frequency and distribution of anti-Gal and anti-Neu5Gc IgM, IgG and IgG subclass were evaluated. Results The detection frequencies of Abs were as follows: for anti-Gal, IgG, 95.0%; IgM, 99.2%; IgG1, 23.2%; IgG2, 94.5%; IgG3, 12.4%; and IgG4, 3.4%, and for anti-Neu5Gc, IgG, 96.6%; IgM, 87.4%; IgG1, 1.6%; IgG2, 46.3%. The median titers of anti-Gal IgG (1,198 ng/ml, range &amp;lt;401 – 128,758 ng/ml) and IgM (996 ng/ml, range &amp;lt;113 – 24,698 ng/ml) were significantly higher than those of anti-Neu5Gc Abs (IgG, 194 ng/ml, range &amp;lt;30 – 12,415 ng/ml; IgM, 328 ng/ml, range &amp;lt;154 – 6,514 ng/ml; all P&amp;lt;0.001). IgG2 of both anti-Gal and anti-Neu5G Abs correlated better with the IgG class than other IgG subclasses (rs = 0.762, P &amp;lt; 0.001; rs = 0.606, P &amp;lt; 0.001). Conclusions Most healthy adults possess anti-Gal and anti-Neu5Gc IgG and IgM in their sera. Overall, the titers of anti-Gal Abs are higher than those of anti-Neu5Gc Abs. IgG2 seems to be the main IgG subclass in both anti-Gal and anti-Neu5Gc natural Abs. Changes in the titers of these Abs would be a clue to understand an immunological pathogenesis in the recipients of xenotransplantation.

A New Humanized Mouse Model Mimics Humans in Lacking α-Gal Epitopes and Secreting Anti-Gal Antibodies

Mice have been used as accepted tools for investigating complex human diseases and new drug therapies because of their shared genetics and anatomical characteristics with humans. However, the tissues in mice are different from humans in that human cells have a natural mutation in the α1,3 galactosyltransferase (α1,3GT) gene and lack α-Gal epitopes on glycosylated proteins, whereas mice and other nonprimate mammals express this epitope. The lack of α-Gal epitopes in humans results in the loss of immune tolerance to this epitope and production of abundant natural anti-Gal Abs. These natural anti-Gal Abs can be used as an adjuvant to enhance processing of vaccine epitopes to APCs. However, wild-type mice and all existing humanized mouse models cannot be used to test the efficacy of vaccines expressing α-Gal epitopes because they express α-Gal epitopes and lack anti-Gal Abs. Therefore, in an effort to bridge the gap between the mouse models and humans, we developed a new humanized mouse model that mimics humans in that it lacks α-Gal epitopes and secretes human anti-Gal Abs. The new humanized mouse model (Hu-NSG/α-Galnull) is designed to be used for preclinical evaluations of viral and tumor vaccines based on α-Gal epitopes, human-specific immune responses, xenotransplantation studies, and in vivo biomaterials evaluation. To our knowledge, our new Hu-NSG/α-Galnull is the first available humanized mouse model with such features.

Distribution of Bacterial α1,3-Galactosyltransferase Genes in the Human Gut Microbiome

Because of a loss-of-function mutation in the GGTA1 gene, humans are unable to synthetize α1,3-Galactose (Gal) decorated glycans and develop high levels of circulating anti-α1,3-Galactose antibodies (anti-Gal Abs). Anti-Gal Abs have been identified as a major obstacle of organ xenotransplantation and play a role in several host-pathogen relationships including potential susceptibility to infection. Anti-Gal Abs are supposed to stem from immunization against the gut microbiota, an assumption derived from the observation that some pathogens display α1,3-Gal and that antibiotic treatment decreases the level of anti-Gal. However, there is little information to date concerning the microorganisms producing α1,3-Gal in the human gut microbiome. Here, available α1,3-Galactosyltransferase (GT) gene sequences from gut bacteria were selectively quantified for the first time in the gut microbiome shotgun sequences of 163 adult individuals from three published population-based metagenomics analyses. We showed that most of the gut microbiome of adult individuals contained a small set of bacteria bearing α1,3-GT genes. These bacteria belong mainly to the Enterobacteriaceae family, including Escherichia coli, but also to Pasteurellaceae genera, Haemophilus influenza and Lactobacillus species. α1,3-Gal antigens and α1,3-GT activity were detected in healthy stools of individuals exhibiting α1,3-GT bacterial gene sequences in their shotgun data.

Development of α-Gal-Antibody Conjugates to Increase Immune Response by Recruiting Natural Antibodies.

Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α-gal-Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α-gal and anti-gal Abs. The potency of the α-gal-Ab conjugates depended on the amount of α-gal conjugated to the antibody: the larger the amount of α-gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α-gal dendrimer allowed the introduction of large amounts of α-gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re-development of Abs to improve their potency.

Abstract 732: A practical strategy to pancreatic cancer immunotherapy using resected human tumor lysate vaccines remodeled to express α-gal epitopes

Abstract Cancer immunotherapy is a potential treatment for pancreatic ductal adenocarcinoma (PDAC) patients. As the predominant natural Ab found in humans, anti-Gal accounts for approximately 1% of immunoglobulins. The ligand of anti-Gal, “α-gal epitope (Galα1-3Galβ1-4GlcNAc-R),” is a major carbohydrate antigen, expressed by non-primate mammals, New World monkeys. One useful application of anti-Gal abundance is the enhancement of the immunogenicity of tumor-associated antigens (TAAs) that promote effective uptake by antigen-presenting cells (APCs). To develop an effective vaccine-based immunotherapy for PDAC, we hypothesized that resected tumor tissue lysates from patients might be an attractive source of PDAC-associated TAAs vaccination. This study presents a novel immunotherapy expressing α-gal epitopes using freshly obtained human PDAC tumor tissue homogenates from patients. Tumor and normal pancreatic tissue specimens were obtained from 10 patients at the time of surgical exploration for primary PDAC. To synthesize α-gal epitopes on either tumor membranes or normal pancreatic tissue membranes, we employed recombinant α1,3 galactosyltransferase (α1,3GT). PDAC membranes or normal pancreatic tissues were homogenized, and were incubated with UDP-Gal and α1,3GT. α1,3GT KO mice were immunized with pig tissues to produce anti-Gal Ab. The high anti-Gal KO mice were vaccinated by i.p. injection with unprocessed or processed PDAC tumor lysate (α-gal(-) PDAC-ly or α-gal(+) PDAC-ly). The high anti-Gal KO mice were also vaccinated with unprocessed normal or processed normal pancreatic tissue lysate (α-gal(-) N-ly or α-gal(+) N-ly). Effective synthesis of α-gal epitopes was demonstrated after processing of PDAC tumor lysates in Western blot analysis. α-gal(+) PDAC-ly vaccines elicited significant antibody production against multiple TAAs, assessed by ELISPOT (anti-MUC1; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly=28.7 vs. 151.8 spots [P=0.0008], anti-mesothelin; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly=36.5 vs. 97.2 spots [P=0.029]) and activated multiple tumor-specific T cells, assessed by ELISPOT (MUC1; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly=146.0 vs. 828.0 spots [P=0.0009], mesothelin; α-gal(-) PDAC-ly vs. α-gal(+) PDAC-ly= 250.7 vs. 988.0 spots [P=0.021], α-gal(-) N-ly and α-gal(+) N-ly groups did not display significant spots.). To demonstrate in vivo effectiveness, splenocytes from vaccinated KO mice were prepared, and these isolated cells were transferred by i.p. injection into NOD/SCID mice. Then, live PANC1 cells were challenged with s.c. injection. The survival time of NOD/SCID mice received from α-gal(+) PDAC-ly groups was significantly extended (95.0 days) compared with α-gal(-) PDAC-ly groups (45 days, p&amp;lt;0.01). We conclude that α-gal(+) PDAC-ly vaccination may be a practical and effective new immunotherapeutic approach for treating PDAC. Citation Format: Masahiro Tanemura, Kenta Furukawa, Eiji Miyoshi, Hidetoshi Eguchi, Hiroaki Nagano, Katsuyoshi Matsunami, Satoshi Nagaoka, Manabu Mikamori, Kentaro Kishi, Hiroki Akamatsu, Masaki Mori, Yuichiro Doki. A practical strategy to pancreatic cancer immunotherapy using resected human tumor lysate vaccines remodeled to express α-gal epitopes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 732.

Abstract 2355: Strong immune responses to pancreatic cancer cells induced by human tumor lysate vaccine remodeled to express α-gal epitopes and their implication for a universal vaccine

Abstract Pancreatic cancer (PC) is a lethal disease that remains one of the most resistant to traditional therapies. We are in urgent need of other therapies. Immunotherapy designed to target tumor-associated antigens (TAAs) is a promising treatment approach for PC. But vaccination against a single TAA seems to be insufficient. In our previous study, we showed effectiveness vaccination by whole PC cells, engineered to express α-gal epitopes (Cancer Res, 2010). Subsequently we investigated strong immune response of tumor lysate vaccine engineered to express α-gal epitopes (Int J Oncol, 2015). In this study, for developing clinical application of this immunotherapy, we investigated the effects of vaccination with human PC tumor lysate obtained from PC patients, expressing α-gal epitopes. Tumor specimens were obtained from 11 PC patients at the time of surgical resection. To express α-gal epitopes, we cloned the α1,3galactosyltransferas (α1,3GT) from a New World Monkey and expressed it in a soluble form in the yeast expression system of Pichia pastoris. α1,3GT KO mice were immunized with pig tissue to generate anti-Gal Ab in their sera. These high anti-Gal KO mice were vaccinated intraperitoneally by either unsynthesized (control group; group C) or α-gal tumor lysate (α-gal group; group A). Vaccination with α-gal PC tumor lysate elicited strong immune responses of anti-PC cell IgG and anti-PC TAAs, including MUC1 and Mesothelin. Productions of anti-PC cell IgG in group A were 8∼32-fold higher than those of group C. Furthermore, productions of anti-MUC1 and of ant-Mesothelin Ab in group A were 4∼8-fold higher than those of group C. Expansion of TAAs (MUC1 and Mesothelin)-specific B cells was significantly higher [MUC1: spots of group A vs. C = 151 vs. 28 (p&amp;lt;0.001), Mesothelin: 97 vs. 36 (p = 0.03)]. The number of spots of IFN-γ secreting T cells in the presence of MUC1 and Mesothelin peptide stimulation was significantly higher in group A than in group C [MUC1: 828 vs. 146 (p&amp;lt;0.001), Mesothelin: 988 vs. 384 (p = 0.02)]. To demonstrate in vivo tumor destruction, an animal experiment was performed. Splenocytes from vaccinated KO mice were prepared, and then transferred intraperitoneally into NOD/SCID mice. Followed by transferring, mice were challenged with 1×107 of live PANC-1 cells. In mice from group A, regrowth of tumors was significantly prevented and survival period was significantly prolonged [group A vs. C = 95.0 vs. 45.0 days (p&amp;lt;0.001)]. In the immunohistochemical analysis, the tumor created in NOD/SCID mice from group A was shown severe central necrosis and strong infiltration of CD4+ and CD8+ T cells and macrophages into the periphery. In contrast, the tumor obtained from NOD/SCID mice of group C indicated no lymphocytic infiltration. We conclude that the use of human PC tumor lysate vaccine, remodeled to express α-gal epitopes can elicited strong immune responses to pancreatic cancer. Citation Format: Kenta Furukawa, Masahiro Tanemura, Eiji Miyoshi, Tomoki Hata, Kentaro Kishi, Hiroki Akamatsu, Hidetoshi Eguchi, Masaki Mori, Yuichiro Doki. Strong immune responses to pancreatic cancer cells induced by human tumor lysate vaccine remodeled to express α-gal epitopes and their implication for a universal vaccine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2355.

The Role of Liver Sinusoidal Endothelial Cells in Induction of Carbohydrate Reactive B Cells Tolerance Through the Programmed Death 1/Programmed Death Ligand 1 Pathway.

A spontaneous tolerance of B cells responding to blood group antigens frequently develops in ABO-incompatible pediatric liver transplantation (LT). Liver sinusoidal endothelial cells (LSECs), which exclusively express blood group antigens in the liver, possess a capacity to induce alloantigen-specific tolerance. In this study, we elucidated the role of LSECs in the tolerance induction of blood group antigen-reactive B cells after ABO-incompatible LT using mice that lack galactose-α(1,3)galactose (Gal) epitopes resembling blood group carbohydrate antigens. Using adoptive transfer of LSECs from wild type (WT) C57BL/6J mice to congenic α1,3-galactosyltransferase gene knockout (GalT) mice, we established orthotropic GalT → GalT LSEC chimera mice. Anti-Gal Ab (antibody) production was evaluated after immunization of GalT → GalT LSEC chimera mice with Gal rabbit RBC. Adoptive transfer of LSECs isolated from WT GalT mice via the portal vein resulted in persistent engraftment of Gal LSECs in congenic GalT mouse livers. Only when GalT mice were splenectomized before LSEC inoculation, the GalT → GalT LSEC chimera lost the ability to produce anti-Gal Abs. The administration of blocking monoclonal Abs (mAbs) against programmed death ligand 1 to the splenectomized GalT → GalT LSEC chimera resulted in the recovery of anti-Gal Ab production. These findings suggest that LSECs take a part in tolerization of immature but not mature B cells specifically for Gal. Furthermore, the programmed death 1/programmed death ligand 1 pathway likely plays a crucial role in the mechanisms underlying spontaneous tolerization of B cells responding to ABO-blood group antigens in LT.

Abstract 2512: Novel immunotherapy using a tumor lysate vaccine with α-gal epitopes against pancreatic cancer

Abstract Pancreatic cancer (PC) is a lethal disease that remains one of the most resistant to traditional therapies, so we are in urgent need of other therapies. Immunotherapies act differently than chemotherapy or radiotherapy and might be an alternative treatment modality to this deadly disease with significantly less toxicity. Immunotherapy designed to target tumor-associated antigens (TAAs) is a promising treatment approach for PC. But vaccination against a single TAA seems to be insufficient. In our previous study, we showed effectiveness vaccination by whole PC cells engineered to express α-gal epitopes (Cancer Res, 2010). Subsequently we showed effectiveness of tumor lysate vaccine engineered to α-gal epitopes (Int J Oncol, 2015). For clinical development of more effective immunotherapy, we proposed that human tumor lysate originated from resected PC is more suitable because it contains several TAAs that could elicit a marked anti-tumor response. The present study addresses the effectiveness of elicitation of both antibody production and in vivo destruction against PC by vaccination with human tumor lysate with α-gal epitopes enzymatically. Tumor specimens were obtained from 11 patients at the time of surgical resection. To express α-gal epitopes, we cloned the α1,3galactosyltransferas (α1,3GT) from a New World Monkey and expressed it in a soluble form in the yeast expression system of Pichia pastoris. α1,3GT KO mice were immunized with pig tissue to generate anti-Gal Ab in their sera. These mice were vaccinated intraperitoneally by either unsynthesized (control group; group C) or α-gal tumor lysate (α-gal group; group A). Production of anti-PC cell Ab in group A was 8∼16-fold higher than that of group C. Also production of anti-TAA Ab in group A was 8∼16-fold higher than that of group C. Expansion of TAAs-specific B cells was significantly higher [number of spots at 1×106 splenocytes: MUC1: group A vs. C; 151±33 vs. 28±13 (p&amp;lt;0.001), Mesothelin: 97±26 vs. 36±19 (p = 0.03)]. In group A, we detected 828±180 spots of IFN-γ secreting T cells in the presence of the MUC1, and 988±232 spots were detected in the presence of the Mesothelin. In group C, 146±59 spots by the MUC1 and 384±219 spots by the Mesothelin were detected. The number of spots in the presence of TAA stimulation was significantly higher in group A than in group C. To demonstrate in vivo tumor destruction, an animal experiment was performed. Splenocytes from vaccinated KO mice were prepared, and then transferred intraperitoneally (90×106 cells) into NOD/SCID mice. Followed by transferring, mice were challenged with 1×107 of live PANC-1 cells. In mice from group A, regrowth of tumors was significantly prevented and survival period was significantly prolonged [group A vs. C: 91.5 vs. 47.0 days (p&amp;lt;0.01)]. We conclude that immunotherapy with α-gal tumor lysate obtained from PC patients with enzymatic engineering could be more effective and practicable methods for PC treatment. Citation Format: Kenta Furukawa, Masahiro Tanemura, Eiji Miyoshi, Hiroaki Nagano, Masaki Wakasugi, Toru Masuzawa, Mitsuyoshi Tei, Kentaro Kishi, Hiroki Akamatsu, Hidetoshi Eguchi, Katsuyoshi Matsunami, Masaki Mori, Yuichiro Doki. Novel immunotherapy using a tumor lysate vaccine with α-gal epitopes against pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2512. doi:10.1158/1538-7445.AM2015-2512

Abstract 2884: Immune-based therapy with human tumor lysate, expressing α-gal epitopes induce significant B cell response and in vivo tumor destruction against pancreatic cancer

Abstract Current strategies in the treatment of advanced pancreatic cancer (PC) offer little hope for a cure. Now treatment modalities, including immunotherapy are warranted for PC, which easily metastasizes in many organs. Although MUC1 and Mesothelin are potential targets for the immunotherapy, vaccination against a single tumor-associated antigen (TAA) seems to be insufficient. In our previous study, we showed the in vitro and in vivo effectiveness of immunotherapy though vaccination with whole PC cells engineered to express α-gal epitopes (Cancer Res; 70(13); 5259-69, 2010). It was thought that the immunogenicity of well-known as well as unknown multiple TAAs in cancer cells was upregulated by addition of α-gal epitopes and these TAAs ultimately lead to polyclonal expansion of both B and T cells. For clinical development of more effective immunotherapy, we proposed that human tumor lysate originated from resected PC is more suitable because it contains several TAAs that could elicit a marked anti-tumor response. The present study addresses the effectiveness of elicitation of both antibody production and in vivo destruction against PC by vaccination with human tumor lysate from PC patients, expressing α-gal epitopes enzymatically. Tumor specimens were obtained from 11 patients at the time of surgical resection. To express α-gal epitopes, we cloned the α1,3galactosyltransferas (α1,3GT) from a New World Monkey and expressed it in a soluble form in the yeast expression system of Pichia pastoris. For synthesis of α-gal epitopes, tumor tissues were homogenized in MES buffer, and incubated with MnCl2, UDP-Gal and α1,3GT for 4 hours at 37°C. α1,3GT KO mice were immunized with pig tissue to generate anti-Gal Ab in their sera. These mice were vaccinated intraperitoneally by either unsynthesized (control group) or α-gal tumor lysate (α-gal group). Production of anti-PC cell Ab in α-gal group was 4∼8-fold higher than that of control group. Anti-TAA Ab production was also markedly increased in comparison with that in control group (anti-MUC1 2-fold, anti-Mesothelin 4-fold). Expansion of TAAs-specific B cells was significantly higher [number of spots at 1×106 splenocytes: MUC1: α-gal vs. control; 414±26 vs. 178±46 (p=0.015), Mesothelin: 401±56 vs. 209±19 (p=0.03)]. To demonstrate in vivo tumor destruction, an animal experiment was performed. Splenocytes from vaccinated KO mice were prepared, and then transferred intraperitoneally (90×106 cells) into NOD/SCID mice. Followed by transferring, mice were challenged with 1×107 of live PANC-1 cells. In mice from α-gal group, regrowth of tumors was significantly prevented and survival period was significantly prolonged [α-gal vs. control: 91.5 vs. 43.3 days (p=0.03)]. We conclude that immunotherapy with α-gal tumor lysate obtained from PC patients with enzymatic engineering could be more effective and practicable methods for PC treatment. Citation Format: Kenta Furukawa, Masahiro Tanemura, Eiji Miyoshi, Hiroaki Nagano, Hidetoshi Eguchi, Toshimitsu Irei, Masashi Inoue, Shinya Yamashita, Yoshito Tomimaru, Naoki Hama, Hiroshi Wada, Koichi Kawamoto, Shogo Kobayashi, Katsuyoshi Matsunami, Kiyomi Taniyama, Wataru Kamiike, Masaki Mori, Yuichiro Doki. Immune-based therapy with human tumor lysate, expressing α-gal epitopes induce significant B cell response and in vivo tumor destruction against pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2884. doi:10.1158/1538-7445.AM2014-2884

Abstract 2560: Strong cross-reactive responses to pancreatic cancer cells induced by α-gal epitopes-expressing tumor lysate and their implications for a universal cancer vaccine

Abstract We investigated the effects of whole cell vaccination with α-gal epitopes-expressing pancreatic cancer (PC) cells (Cancer Res; 70 (13); 5259-69, 2010) and examined the usefulness of this vaccine in the induction of specific anti-tumor-responses and improvement of survival. But, these effects were somewhat weak. We hypothesized that tumor lysate (t-ly), which contains many tumor-associated antigens (TAAs) of both PC and stromal cells is a suitable vaccination material and it can elicit a broadly protective anti-tumor responses. In this study, we investigate that vaccination with α-gal epitopes-expressing PC t-ly can induce cross-reactive protections to heterosubtypic PC cells. Human PC cell lines, BxPC3 (Bx), expressing Mesothelin, and MiaPaCa (Mia), expressing MUC1 were employed. PC cell lines were injected s.c. into NOD/SCID mice. The grown tumors were enucleated and homogenized for making t-ly. To synthesize α-gal epitopes on t-ly, we cloned the α1,3 galactosyltransferase (α1,3GT) cDNA from a New World monkey and isolated a soluble form of enzyme in the yeast expression system of Pichia pastoris. These t-lys were incubated with UDP-Gal and α1,3GT. α1,3GT KO mice were immunized with pig tissues to produce anti-Gal Ab. The high anti-Gal KO mice were vaccinated intraperitoneally by either parental (control group; Bx-t-ly or Mia-t-ly) or α-gal-t-ly (α-gal group; α-gal-Bx-t-ly or α-gal-Mia-t-ly). No expression of α-gal epitopes were observed in parental t-lys, whereas high expression levels were clearly detected on α-gal-t-lys. α-gal-Bx-t-ly vaccination elicited strong responses of anti-Bx IgG and anti-Mesothelin IgG, whereas Bx-t-ly vaccination did not induce such Ab responses. For Mia-t-ly vaccination, KO mice of α-gal group clearly induce anti-Mia IgG and anti-MUC1 IgG, but no Ab responses were observed in control group. Productions of either anti-Bx or anti-Mia IgG in α-gal group were 64-fold higher than those in control group. α-gal-Bx-t-ly vaccinated KO mice abundantly produced cross-reactive IgG Abs to Mia cells, whereas no cross-reactive humoral responses to Mia cells were induced by Bx-t-ly vaccine. Vaccinated mice sera from α-gal-Mia-t-ly strongly cross-react to Bx cells. To demonstrate in vivo tumor destruction, splenocytes from vaccinated KO mice were prepared, and these splenocytes were inoculated i.p. into NOD/SCID mice. Either live Bx or Mia cells were challenged with s.c. injection. Both Bx and Mia tumors in mice inoculated from parental group reached the size of 100 mm2 at approximately 22 days after injection. For tumors in mice transferred from α-gal group, no regrowth tumors were identified. We conclude that α-gal epitopes-expressing t-ly vaccine can elicit durable and broadly protective immune response to subtypic PC cells, and that such vaccination may provide implications for a universal cancer vaccine to cure for the patients with PC. Citation Format: Masahiro Tanemura, Eiji Miyoshi, Hiroaki Nagano, Masafumi Inoue, Toshimitsu Irei, Shinya Yamashita, Kenta Furukawa, Katsuyoshi Matsunami, Wataru Kamiike, Masaki Mori, Yuichiro Doki. Strong cross-reactive responses to pancreatic cancer cells induced by α-gal epitopes-expressing tumor lysate and their implications for a universal cancer vaccine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2560. doi:10.1158/1538-7445.AM2014-2560

Role of α‐gal epitope/anti‐Gal antibody reaction in immunotherapy and its clinical application in pancreatic cancer

Pancreatic cancer is one of the most common causes of death from cancer. Despite the availability of various treatment modalities, such as surgery, chemotherapy and radiotherapy, the 5-year survival remains poor. Although gemcitabine-based chemotherapy is typically offered as the standard care, most patients do not survive longer than 6 months. Therefore, new therapeutic approaches are needed. The α-gal epitope (Galα1-3Galβ1-4GlcNAc-R) is abundantly synthesized from glycoproteins and glycolipids in non-primate mammals and New World monkeys, but is absent in humans, apes and Old World monkeys. Instead, they produce anti-Gal antibody (Ab) (forming approximately 1% of circulating immunoglobulins), which specifically interacts with α-gal epitopes. Anti-Gal Ab can be exploited in cancer immunotherapy as vaccines that target antigen-presenting cells (APC) to increase their immunogenicity. Tumor cells or tumor cell membranes from pancreatic cancer are processed to express α-gal epitopes. Subsequent vaccination with such processed cell membranes results in in vivo opsonization by anti-Gal IgG in cancer patients. The interaction of the Fc portion of the vaccine-bound anti-Gal with Fcγ receptors of APC induces effective uptake of the vaccinating tumor cell membranes by the APC, followed by effective transport of the vaccinating tumor membranes to the regional lymph nodes, and processing and presentation of the tumor-associated antigens. Activation of tumor-specific B and T cells could elicit an immune response that in some patients is potent enough to eradicate the residual cancer cells that remain after completion of standard therapy. This review addresses these topics and new avenues of clinical importance related to this unique antigen/antibody system (α-gal epitope/anti-Gal Ab) and advances in immunotherapy in pancreatic cancer.

Rapid Recruitment and Activation of Macrophages by Anti-Gal/α-Gal Liposome Interaction Accelerates Wound Healing

Macrophages are pivotal in promoting wound healing. We hypothesized that topical application of liposomes with glycolipids that carry Galα1-3Galβ1-4GlcNAc-R epitopes (α-gal liposomes) on wounds may accelerate the healing process by rapid recruitment and activation of macrophages in wounds. Immune complexes of the natural anti-Gal Ab (constituting ∼1% of Ig in humans) bound to its ligand, the α-gal epitope on α-gal liposomes would induce local activation of complement and generation of complement chemotactic factors that rapidly recruit macrophages. Subsequent binding of the Fc portion of anti-Gal coating α-gal liposomes to FcγRs on recruited macrophages may activate macrophage genes encoding cytokines that mediate wound healing. We documented the efficacy of this treatment in α1,3galactosyltrasferase knockout mice. In contrast to wild-type mice, these knockout mice lack α-gal epitopes and can produce the anti-Gal Ab. The healing time of excisional skin wounds treated with α-gal liposomes in these mice is twice as fast as that of control wounds. Moreover, scar formation in α-gal liposome-treated wounds is much lower than in physiologic healing. Additional sonication of α-gal liposomes resulted in their conversion into submicroscopic α-gal nanoparticles. These α-gal nanoparticles diffused more efficiently in wounds and further increased the efficacy of the treatment, resulting in 95-100% regeneration of the epidermis in wounds within 6 d. The study suggests that α-gal liposome and α-gal nanoparticle treatment may enhance wound healing in the clinic because of the presence of high complement activity and high anti-Gal Ab titers in humans.

Antigen‐binding specificity of anti‐αGal reagents determined by solid‐phase glycolipid‐binding assays. A complete lack of αGal glycolipid reactivity in α1,3GalT‐KO pig small intestine

αGal-specific lectins, monoclonal and polyclonal antibodies (Abs) are widely used in xenotransplantation research. Immunological assays such as immunohistochemistry, flow cytometry, Western blot and thin layer chromatography are often the only applicable characterization procedures when limited amount of tissue is available and biochemical characterization is impossible. Hence, detailed knowledge of the Ab/lectin carbohydrate-binding specificity is essential. The binding specificity of human blood group AB serum, three different affinity-purified human polyclonal anti-Gal Ab batches, and two anti-Gal mAb clones (TH5 and 15.101) as well as Griffonia simplicifolia isolectin B4 and Marasmius oreades agglutinin were examined for reactivity with glycolipid fractions isolated from human and pig (wild-type and α1,3GalT-KO) tissues using thin layer chromatogram and microtiter well binding assays. All anti-Gal-specific reagents reacted with the pentaglycosylceramide Galα1,3nLc4, and several 6-12 sugar compounds in wild-type pig kidneys. However, their staining intensity with different αGal antigens varied considerably. Some, but not all, anti-Gal reagents cross-reacted with a pure iGb3 glycolipid reference compound. No reactivity with glycolipids isolated from α1,3GalT-KO pig small intestine or human tissues was found, confirming the specificity of the anti-Gal reagents in those tissues for α1,3Gal-epitopes produced by the α1,3GalT (GGTA1). Different anti-Gal reagents vary in their carbohydrate epitope specificity. Mono-/polyclonal Abs and lectins have different carbohydrate epitope fine specificity toward pig glycolipids as well as purified Galα1,3nLc4, and iGb3. Despite the difference in αGal specificity, all reagents were completely non-reactive with glycolipids isolated from α1,3GalT-KO pig small intestine.

An in vitro model of pig liver xenotransplantation—pig complement is associated with reduced lysis of wild‐type and genetically modified pig cells

After pig liver transplantation in humans, the graft will produce pig complement (C). We investigated in vitro the lysis of wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and CD46 transgenic (CD46) pig peripheral blood mononuclear cells (PBMC) caused by human anti-pig antibodies (Abs) + pig C. Human serum IgM/IgG binding to WT and GTKO PBMC was determined by flow cytometry, and lysis of pig PBMC by a C-dependent cytotoxicity assay using (i) human serum (human Abs + C), (ii) GTKO pig serum (anti-Gal Abs + pig C), (iii) heat-inactivated human serum (human Abs) + rabbit C, or (iv) human Abs + pig C (serum). Binding of human IgM and IgG to GTKO PBMC was less than to WT PBMC (P < 0.05). In the presence of human Abs, lysis of WT and GTKO PBMC by rabbit C was 87 and 13%, respectively (WT vs. GTKO, P < 0.01), but was only 37 and 0.4% in the presence of pig C (WT vs. GTKO, P < 0.05). Human/rabbit C-induced lysis was greater than pig C-induced lysis for both WT and GTKO PBMC. CD46 pig PBMC reduced rabbit/human C- and pig C-mediated lysis (P < 0.05). Pig livers, particularly from GTKO and CD46 pigs, are likely to have an immunologic advantage over other organs after transplantation into humans. In the absence of pig antibodies directed to human tissues, pig complement is unlikely to cause problems after liver xenotransplantation, especially if GTKO/CD46 pigs are used as the source of the livers.

Anticarbohydrate Antibody Repertoires in Patients Transplanted with Fetal Pig Islets Revealed by Glycan Arrays

Ten patients with type I diabetes were transplanted with porcine fetal islet-like cell clusters (ICC) between 1990 and 1993. A significant rise in the anti-α-Gal antibody titers was seen posttransplant, but also non-α-Gal-specific antibodies were detected in some patients. We have reanalyzed the carbohydrate specificity of antibodies in the sera from seven of these patients taken before transplantation, 1, 6 and 12 months posttransplantation using a glycan array with 200 structurally defined glycans. The main findings were: (i) prepig ICC transplantation patients had antibodies reactive with terminal α-GalNAc (e.g. the Forssman antigen, but not the blood group A determinant in blood group A patients), α-Gal (except blood group B determinants in B individuals), β3-linked Gal especially Galβ1,3GlcNAc even if terminally sulfated or sialylated, β-GlcNAc except if β1,3-linked and oligomannosyl compounds; (ii) the titers of all carbohydrate-specific antibodies detected before transplantation rose after transplantation; (iii) the kinetics of the antibody responses differed between patients; (iv) in some patients antibodies reacting with Galα1,3Lex and several structures terminated with Neu5Gc appeared after transplantation. In conclusion, anti-α-Gal antibodies are the predominant anticarbohydrate antibodies detected after porcine ICC transplantation, with some patients also developing Neu5Gc-specific antibodies. Their clinical significance needs to be established.

Glycolipid Studies in Small Intestine and Pancreas of α1,3-Galactosyltransferase Knockout Miniature Swine: α1,3GALT-KO Animals Lack αGAL Antigens and Contain Novel Blood Group H Compounds

Background To avoid hyperacute rejection of xeno-organs, α1,3-galactosyltransferase knock-out (GalT-KO) pigs have been produced. However, Galα1,3Gal (Gal) determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens that might interfere with the human immune response. Methods Glycolipids isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig were tested for immune reactivity with antibodies on thin-layer chromatograms after separation by high-performance liquid chromatography, and selected fractions were analysed by proton NMR spectroscopy. Results Immunostaining using purified human anti-Gal Abs revealed that tissues from WT animals express large amounts of Gal-antigens whereas GalT-KO tissues lacked these antigens. Proton NMR spectroscopy on small intestine fractions revealed both linear and branched nona- and decaglycosylceramides, respectively, with terminal Gal-epitopes. In corresponding GalT-KO fractions, Gal-epitopes seemed to be replaced by terminal α1,2fucoses. Two novel branched blood group H compounds was found in the GalT-KO intestine. Conclusions The structural complexity of αGal-terminating antigens in the WT organs is very high. Knockout of α1,3GalT by gene-targeting results in elimination of Gal-determinants. In addition structurally novel α1,2fucose-terminated blood group H compounds were identified in the GalT-KO tissue. These compounds are not expected to be recognized by the human immune system.

Normal human serum contains high levels of anti‐Galα1‐4GlcNAc antibodies

Natural xenoreactive antibodies (Abs) directed against the Bdi-epitope (Gal alpha 1-3Gal beta) on the cells of non-primate mammals take part in hyperacute rejection of xenotransplanted organs. We found that some Abs, which were one-step affinity purified on Bdi-Sepharose, cross-reacted with the disaccharide Gal alpha 1-4GlcNAc beta. The epitope Gal alpha 1-4GlcNAc has not been identified on mammals or bacterial polysaccharides yet. To isolate the antibodies of the corresponding specificity the disaccharide was immobilized on Sepharose and antibodies were affinity purified from pooled serum of blood group O individuals. These one-step purified Abs cross-reacted with Bdi, but after a prior absorption step on Bdi-Sepharose no cross-reactivity with Bdi was observed any longer. Surprisingly, the quantity of anti-Gal alpha 1-4GlcNAc isolated from the same serum pool, 4-7 microg/ml, was equal to that of anti-Bdi or more. Independently of ABO blood groups all the tested healthy donors had anti-Gal alpha 1-4GlcNAc Abs at a similar level. Monospecific anti-Gal alpha 1-4GlcNAc Abs were not cytotoxic towards porcine cells. 1. The actual concentration of monospecific, xenoreactive Gal alpha 1-3Gal beta Abs in blood may be considerably lower than the value referred to in the literature for 'anti-alpha Gal' or 'anti-Galili' antibodies. 2. Anti-Gal alpha 1-4GlcNAc Abs seem not to be important for xenotransplantation.

Intratumoral Injection of α-gal Glycolipids Induces Xenograft-Like Destruction and Conversion of Lesions into Endogenous Vaccines

This study describes a novel cancer immunotherapy treatment that exploits the natural anti-Gal Ab to destroy tumor lesions and convert them into an endogenous vaccine targeted to APC via FcgammaR. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). The binding of anti-Gal to alpha-gal epitopes on pig cells mediates xenograft rejection. The proposed method uses glycolipid micelles with multiple alpha-gal epitopes (alpha-gal glycolipids). These glycolipids are extracted from rabbit red cell membranes and are comprised of ceramides with carbohydrate chains containing 5-25 carbohydrates, all capped with alpha-gal epitopes. Efficacy of this treatment was demonstrated in alpha1,3-galactosyltransferase knockout mice producing anti-Gal and bearing B16 melanoma or B16/OVA producing OVA as a surrogate tumor Ag. These mice are unique among nonprimate mammals in that, similar to humans, they lack alpha-gal epitopes and can produce the anti-Gal Ab. Intratumoral injection of alpha-gal glycolipids results in local inflammation mediated by anti-Gal binding to the multiple alpha-gal epitopes and activation of complement. These glycolipids spontaneously insert into tumor cell membranes. The binding of anti-Gal to alpha-gal expressing tumor cells induces the destruction of treated lesions as in anti-Gal-mediated xenograft rejection. Anti-Gal further opsonizes tumor cells within the lesion and, thus, targets them for effective uptake by APC that transport the tumor Ags to draining lymph nodes. APC further cross-present immunogenic tumor Ag peptides and elicit a systemic anti-tumor immune response. Similar intratumoral injection of alpha-gal glycolipids in humans is likely to induce the destruction of treated lesions and elicit a protective immune response against micrometastases.

Human complement‐activating immunoglobulin (Ig)G3 antibodies are essential for porcine endothelial cell activation

Complement-activating naturally occurring anti-porcine endothelial cell antibodies (Abs) are responsible for hyperacute rejection in porcine-to-primate transplantation, whereas the role of complement in acute vascular rejection, characterized by type II endothelial cell activation, is less well understood. We previously demonstrated a correlation between porcine type II endothelial cell activation, as detected by E-selectin expression, and human immunoglobulin (Ig)G3 anti-Gal alpha1-3Gal (Gal) Abs, which was not seen for IgG1, IgG2 or IgG4. The present study was undertaken to investigate whether there is a causal relationship between human anti-porcine IgG3 Abs and porcine endothelial cell activation. IgG3 was isolated employing a Protein A column to 98.3% purity. Porcine endothelial cells were incubated with isolated human IgG3 or the combination of IgG1, IgG2 and IgG4. E-selectin expression and complement activation were investigated by flow cytometry and Western blotting, respectively. Purified IgG3, in contrast to the other IgG subclasses, induced a substantial increase in E-selectin expression. This activation was accompanied by complement activation as detected by C3 cleavage, and was abolished by heat inactivation or by adding the complement inhibitor FUT-175. Depletion of anti-Gal Abs reduced E-selectin expression by 60%, consistent with the presence of complement-activating anti-porcine non-Gal Abs of the IgG3 subclass. Collectively, these data strengthen the hypothesis that human anti-porcine endothelial cell Abs of the IgG3 subclass are essential for endothelial cell activation in porcine-to-human species grafts and demonstrate such activation to be partly independent of Gal epitopes.