First-in-human Study With LIS1, a Next-generation Porcine Low Immunogenicity Antilymphocyte Immunoglobulin in Kidney Transplantation.

BACKGROUND: Polyclonal rabbit antithymocyte globulins (ATGs) are commonly used in organ transplantation as induction. Anti- N -glycolylneuraminic acid carbohydrate antibodies which develop in response to rabbit carbohydrate antigens might lead to unwanted systemic inflammation. LIS1, the first new generation of antilymphocyte globulins (ALGs) derived from double knockout swine, lacking carbohydrate xenoantigens was already tested in nonhuman primates and rodent models. METHODS: This open-label, single-site, dose escalation, first-in-human, phase 1 study evaluated the safety, T cell depletion, pharmacokinetics, and pharmacodynamics of LIS1. In an ascending dose cohort (n = 5), a primary kidney transplant recipient at low immunologic risk (panel reactive antibody [PRA] < 20%), received LIS1 for 5 d at either 0.6, 1, 3, 6, or 8 mg/kg. After each patient completed treatment, the data safety monitoring board approved respective dose escalation. In the therapeutic dose cohort (n = 5) in patients with PRA <50% without donor specific antibodies, 2 patients received 8 mg/kg and 3 patients 10 mg/kg. RESULTS: CD3 + T cell depletion <100/mm 3 at day 2 was observed in all patients who received 6, 8, and 10 mg/kg of LIS1. The terminal half-life of LIS1 was 33.7 d with linearity in its disposition. Lymphocyte repopulation was fast and pretransplant lymphocyte subpopulation counts recovered within 2-4 wk. LIS1 was well tolerated, neither cytokine release syndrome nor severe thrombocytopenia or leukopenia were noticed. Antibodies to LIS1 were not detected. CONCLUSIONS: In this first-in-human trial, genome-edited swine-derived polyclonal LIS1 ALG was well tolerated, did not elicit antidrug antibodies, and caused time-limited T cell depletion in low- and medium-risk kidney transplant recipients.

First-in-human Study With LIS1, a Next-generation Porcine Low Immunogenicity Antilymphocyte Immunoglobulin in Kidney Transplantation.

BACKGROUND: Polyclonal rabbit antithymocyte globulins (ATGs) are commonly used in organ transplantation as induction. Anti- N -glycolylneuraminic acid carbohydrate antibodies which develop in response to rabbit carbohydrate antigens might lead to unwanted systemic inflammation. LIS1, the first new generation of antilymphocyte globulins (ALGs) derived from double knockout swine, lacking carbohydrate xenoantigens was already tested in nonhuman primates and rodent models. METHODS: This open-label, single-site, dose escalation, first-in-human, phase 1 study evaluated the safety, T cell depletion, pharmacokinetics, and pharmacodynamics of LIS1. In an ascending dose cohort (n = 5), a primary kidney transplant recipient at low immunologic risk (panel reactive antibody [PRA] < 20%), received LIS1 for 5 d at either 0.6, 1, 3, 6, or 8 mg/kg. After each patient completed treatment, the data safety monitoring board approved respective dose escalation. In the therapeutic dose cohort (n = 5) in patients with PRA <50% without donor specific antibodies, 2 patients received 8 mg/kg and 3 patients 10 mg/kg. RESULTS: CD3 + T cell depletion <100/mm 3 at day 2 was observed in all patients who received 6, 8, and 10 mg/kg of LIS1. The terminal half-life of LIS1 was 33.7 d with linearity in its disposition. Lymphocyte repopulation was fast and pretransplant lymphocyte subpopulation counts recovered within 2-4 wk. LIS1 was well tolerated, neither cytokine release syndrome nor severe thrombocytopenia or leukopenia were noticed. Antibodies to LIS1 were not detected. CONCLUSIONS: In this first-in-human trial, genome-edited swine-derived polyclonal LIS1 ALG was well tolerated, did not elicit antidrug antibodies, and caused time-limited T cell depletion in low- and medium-risk kidney transplant recipients.

Polyclonal antibodies selectively inhibit tumor growth and invasion and synergize with immune checkpoint inhibitors.

Heterologous polyclonal antibodies (pAb) were shown to possess oncolytic properties a century ago with reported clinical responses. More recent preclinical models confirmed pAb efficacy, though their ability to tackle complex target antigens reduces susceptibility to tumor escape. Owing to the recent availability of glyco-humanized pAb (GH-pAb) with acceptable clinical toxicology profile, we revisited use of pAb in oncology and highlighted their therapeutic potential against multiple cancer types. Murine antitumor pAb were generated after repeated immunization of rabbits with murine tumor cell lines from hepatocarcinoma, melanoma, and colorectal cancers. Antitumor pAb recognized and showed cytotoxicity against their targets without cross-reactivity with healthy tissues. In vivo, pAb are effective alone; moreover, these pAb synergize with immune checkpoint inhibitors like anti-PD-L1 in several cancer models. They elicited an antitumor host immune response and prevented metastases. The anticancer activity of pAb was also confirmed in xenografted NMRI nude mice using GH-pAb produced by repeated immunization of pigs with human tumor cell lines. In conclusion, the availability of bioengineered GH-pAb allows for revisiting of passive immunotherapy with oncolytic pAb to fight against solid tumor and cancer metastasis.

LIS1, a glyco-humanized swine polyclonal anti-lymphocyte globulin, as a novel induction treatment in solid organ transplantation.

Anti-thymocyte or anti-lymphocyte globulins (ATGs/ALGs) are immunosuppressive drugs used in induction therapies to prevent acute rejection in solid organ transplantation. Because animal-derived, ATGs/ALGs contain highly immunogenic carbohydrate xenoantigens eliciting antibodies that are associated with subclinical inflammatory events, possibly impacting long-term graft survival. Their strong and long-lasting lymphodepleting activity also increases the risk for infections. We investigated here the in vitro and in vivo activity of LIS1, a glyco-humanized ALG (GH-ALG) produced in pigs knocked out for the two major xeno-antigens αGal and Neu5Gc. It differs from other ATGs/ALGs by its mechanism of action excluding antibody-dependent cell-mediated cytotoxicity and being restricted to complement-mediated cytotoxicity, phagocyte-mediated cytotoxicity, apoptosis and antigen masking, resulting in profound inhibition of T-cell alloreactivity in mixed leucocyte reactions. Preclinical evaluation in non-human primates showed that GH-ALG dramatically reduced CD4+ (p=0.0005,***), CD8+ effector T cells (p=0.0002,***) or myeloid cells (p=0.0007,***) but not T-reg (p=0.65, ns) or B cells (p=0.65, ns). Compared with rabbit ATG, GH-ALG induced transient depletion (less than one week) of target T cells in the peripheral blood (<100 lymphocytes/L) but was equivalent in preventing allograft rejection in a skin allograft model. The novel therapeutic modality of GH-ALG might present advantages in induction treatment during organ transplantation by shortening the T-cell depletion period while maintaining adequate immunosuppression and reducing immunogenicity.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID-19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrates, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3-galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate-N-acetylneuraminic acid hydroxylase and α1,3-galactosyl-transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain to produce glyco-humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3-galactose epitopes. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized spike/angiotensin converting enzyme-2 interaction at a concentration <1 µg/mL, and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. We also found that pig GH-pAb Fc domains fail to interact with human Fc receptors, thereby avoiding macrophage-dependent exacerbated inflammatory responses and a possible antibody-dependent enhancement. These data and the accumulating safety advantages of using GH-pAbs in humans warrant clinical assessment of XAV-19 against COVID-19.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal α1,3-galactose (αGal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and α1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and α-Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1µg/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.

Quantitative and qualitative changes in anti-Neu5Gc antibody response following rabbit anti-thymocyte IgG induction in kidney allograft recipients.

Antibodies of non-human mammals are glycosylated with carbohydrate antigens, such as galactose-α-1-3-galactose (α-Gal) and N-glycolylneuraminic acid (Neu5Gc). These non-human carbohydrate antigens are highly immunogenic in humans due to loss-of-function mutations of the key genes involved in their synthesis. Such immunogenic carbohydrates are expressed on therapeutic polyclonal rabbit anti-human T-cell IgGs (anti-thymocyte globulin; ATG), the most popular induction treatment in allograft recipients. To decipher the quantitative and qualitative response against these antigens in immunosuppressed patients, particularly against Neu5Gc, which may induce endothelial inflammation in both the graft and the host. We report a prospective study of the antibody response against α-Gal and Neu5Gc-containing glycans following rabbit ATG induction compared to controls. We show a drop in the overall levels of anti-Neu5Gc antibodies at 6 and 12 months post-graft compared to the pre-existing levels due to the major early immunosuppression. However, in contrast, in a cross-sectional study there was a highly significant increase in anti-Neu5Gc IgGs levels at 6 months post-graft in the ATG-treated compared to non-treated patients(P = 0.007), with a clear hierarchy favouring anti-Neu5Gc over anti-Gal response. A sialoglycan microarray analysis revealed that the increased anti-Neu5Gc IgG response was still highly diverse against multiple different Neu5Gc-containing glycans. Furthermore, some of the ATG-treated patients developed a shift in their anti-Neu5Gc IgG repertoire compared with the baseline, recognizing different patterns of Neu5Gc-glycans. In contrast to Gal, Neu5Gc epitopes remain antigenic in severely immunosuppressed patients, who also develop an anti-Neu5Gc repertoire shift. The clinical implications of these observations are discussed.

Anti-Gal and Anti-Neu5Gc Responses in Nonimmunosuppressed Patients After Treatment With Rabbit Antithymocyte Polyclonal IgGs.

BACKGROUND: Polyclonal antihuman thymocyte rabbit IgGs (antithymocyte globulin [ATG]) are popular immunosuppressive drugs used to prevent or treat organ or bone-marrow allograft rejection, graft versus host disease, and autoimmune diseases. However, animal-derived glycoproteins are also strongly immunogenic and rabbit ATG induces serum sickness disease in almost all patients without additional immunosuppressive drugs, as seen in the Study of Thymoglobulin to arrest Type 1 Diabetes (START) trial of ATG therapy in new-onset type 1 diabetes. METHODS: Using enzyme-linked immunosorbent assay, we analyzed serial sera from the START study to decipher the various anti-ATG specificities developed by the patients in this study: antitotal ATG, but also antigalactose-α1-3-galactose (Gal) and anti-Neu5Gc antibodies, 2 xenocarbohydrate epitopes present on rabbit IgG glycans and lacking in humans. RESULTS: We show that diabetic patients have substantial levels of preexisting antibodies of the 3 specificities, before infusion, but of similar levels as healthy individuals. ATG treatment resulted in highly significant increases of both IgM (for anti-ATG and anti-Neu5Gc) and IgG (for anti-ATG, -Gal, and -Neu5Gc), peaking at 1 month and still detectable 1 year postinfusion. CONCLUSIONS: Treatment with rabbit polyclonal IgGs in the absence of additional immunosuppression results in a vigorous response against Gal and Neu5Gc epitopes, contributing to an inflammatory environment that may compromise the efficacy of ATG therapy. The results also suggest using IgGs lacking these major xenoantigens may improve safety and efficacy of ATG treatment.

Rabbit antithymocyte globulin-induced serum sickness disease and human kidney graft survival.

BACKGROUND: Rabbit-generated antithymocyte globulins (ATGs), which target human T cells, are widely used as immunosuppressive agents during treatment of kidney allograft recipients. However, ATGs can induce immune complex diseases, including serum sickness disease (SSD). Rabbit and human IgGs have various antigenic differences, including expression of the sialic acid Neu5Gc and α-1-3-Gal (Gal), which are not synthesized by human beings. Moreover, anti-Neu5Gc antibodies have been shown to preexist and be elicited by immunization in human subjects. This study aimed to assess the effect of SSD on long-term kidney allograft outcome and to compare the immunization status of grafted patients presenting with SSD following ATG induction treatment. METHODS: We analyzed data from a cohort of 889 first kidney graft recipients with ATG induction (86 with SSD [SSD(+)] and 803 without SSD [SSD(-)]) from the Données Informatisées et Validées en Transplantation data bank. Two subgroups of SSD(+) and SSD(-) patients that had received ATG induction treatment were then assessed for total anti-ATG, anti-Neu5Gc, and anti-Gal antibodies using ELISA assays on sera before and after transplantation. RESULTS: SSD was significantly associated with long-term graft loss (>10 years, P = 0.02). Moreover, SSD(+) patients exhibited significantly elevated titers of anti-ATG (P = 0.043) and anti-Neu5Gc (P = 0.007) IgGs in late post-graft samples compared with SSD(-) recipients. CONCLUSION: In conclusion, our data indicate that SSD is a major contributing factor of late graft loss following ATG induction and that anti-Neu5Gc antibodies increase over time in SSD(+) patients. FUNDING: This study was funded by Société d'Accélération du Transfert de Technologies Ouest Valorisation, the European FP7 "Translink" research program, the French National Agency of Research, Labex Transplantex, the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation for Innovation.