T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation.

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity.

Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue.

The use of animal organs could potentially alleviate the critical worldwide shortage of donor organs for clinical transplantation. Because of the strong immune response to xenografts, success will probably depend upon new strategies of immune suppression and induction of tolerance. Here we report our initial results using alpha-1,3-galactosyltransferase knockout (GalT-KO) donors and a tolerance induction approach. We have achieved life-supporting pig-to-baboon renal xenograft survivals of up to 83 d with normal creatinine levels.

Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience.

Hearts from alpha1,3-galactosyltransferase knockout pigs (GalT-KO, n = 8) were transplanted heterotopically into baboons using an anti-CD154 monoclonal antibody-based regimen. The elimination of the galactose-alpha1,3-galactose epitope prevented hyperacute rejection and extended survival of pig hearts in baboons for 2-6 months (median, 78 d); the predominant lesion associated with graft failure was a thrombotic microangiopathy, with resulting ischemic injury. There were no infectious complications directly related to the immunosuppressive regimen. The transplantation of hearts from GalT-KO pigs increased graft survival over previous studies.

How current understanding of clearance mechanisms and pharmacodynamics of therapeutic proteins can be applied for evaluation of their drug-drug interaction potential.

Increasing use of therapeutic proteins (TPs) in polypharmacy settings calls for more in-depth understanding of the biological interactions that can lead to increased toxicity or loss of pharmacological effect. Factors such as patient population, medications that are likely to be coadministered in that population, clearance mechanisms of a TP, and concomitant drugs have to be taken into account to determine the potential for drug-drug interactions (DDIs). The most well documented TP DDI mechanism involves cytokine-mediated changes in drug-metabolizing enzymes. Because of the limitations of the current preclinical models for addressing this type of DDI, clinical evaluation is currently the most reliable approach. Other DDI mechanisms need to be addressed on a case-by-case basis. These include altered clearance of TPs resulting from the changes in the target protein levels by the concomitant medication, displacement of TPs from binding proteins, modulation of Fcγ receptor expression, and others. The purpose of this review is to introduce the approach used by Pfizer scientists for evaluation of the DDI potential of novel TP products during drug discovery and development.

Complex pharmacokinetics of a humanized antibody against human amyloid beta peptide, anti-abeta Ab2, in nonclinical species.

PURPOSE: Anti-Aß Ab2 (Ab2) is a humanized monoclonal antibody against amino acids 3-6 of primate (but not rodent) amyloid ß (Aß) and is being evaluated for the treatment of Alzheimer's disease (AD). This study was conducted to predict the human pharmacokinetics of Ab2. METHODS: In vivo PK profile of Ab2 in preclinical species and in vitro mechanistic studies in preclinical and human systems were used for pharmacokinetic predictions. RESULTS: In Tg2576 and PSAPP mice that have ~100-fold higher circulating levels of human Aß compared to humans, elimination of Ab2 was target-mediated, such that exposure was 5-10 fold lower compared to wild-type rodents or to PDAPP mice that have human Aß concentrations in plasma similar to humans. In cynomolgus monkeys, the t(1/2) of Ab2 was faster (<2.5 days) compared to that of the control antibody (~13 days). The fast elimination of Ab2 in cynomolgus monkeys was linked to off-target binding to cynomolgus monkey fibrinogen that was also causing incomplete recovery of Ab2 in cynomolgus monkey serum in blood partitioning experiments. Ab2 had significantly weaker to undetectable binding to human (and mouse) fibrinogen and had good recovery in human serum in blood partitioning experiments. CONCLUSIONS: These data predict that elimination of Ab2 in healthy or AD humans is expected to be slow, with t(1/2) similar to that observed for other humanized antibodies.

Investigation of potential carbohydrate antigen targets for human and baboon antibodies.

BACKGROUND: The continued presence of a primate antibody-mediated response to cells and organs from alpha1,3-galactosyltransferase gene-knockout (GTKO) pigs indicates that there may be antigens other than Gal alpha 1,3Gal (alpha Gal) against which primates have xenoreactive antibodies. Human and baboon sera were tested for reactivity against a panel of saccharides that might be potential antigen targets for natural anti-non-alpha Gal antibodies. METHODS: Human sera (n = 16) and baboon sera (n = 15) of all ABO blood types were tested using an enzyme-linked immunoadsorbent assay for binding of IgM and IgG to a panel of synthetic polyacrylamide-linked saccharides (n = 15). Human sera were also tested after adsorption on alpha Gal immunoaffinity beads. Sera from healthy wild-type (WT, n = 6) and GTKO (n = 6) pigs and from baboons (n = 4) sensitized to GTKO pig organ or artery transplants (of blood type O) were also tested. Forssman antigen expression on baboon and pig tissues was investigated by immunohistochemistry. RESULTS: Both human and baboon sera showed high IgM and IgG binding to alpha Gal saccharides, alpha-lactosamine, and Forssman disaccharide. Human sera also demonstrated modest binding to N-glycolylneuraminic acid (Neu5Gc). When human sera were adsorbed on alpha Gal oligosaccharides, there was a reduction in binding to alpha Gal and alpha-lactosamine, but not to Forssman. WT and GTKO pig sera showed high binding to Forssman, and GTKO pig sera showed high binding to alpha Gal saccharides. Baboon sera sensitized to GTKO pigs showed no significant increased binding to any specific saccharide. Staining for Forssman was negative on baboon and pig tissues. CONCLUSIONS: We were unable to identify definitively any saccharides from the selected panel that may be targets for primate anti-non-alpha Gal antibodies. The high level of anti-Forssman antibodies in humans, baboons, and pigs, and the absence of Forssman expression on pig tissues, suggest that the Forssman antigen does not play a role in the primate immune response to pigs.

Alpha1,3-galactosyltransferase gene-knockout pigs for xenotransplantation: where do we go from here?

The ability to genetically engineer pigs that no longer express the Galalpha1,3Gal (Gal) oligosaccharide has been a significant step toward the clinical applicability of xenotransplantation. Using a chronic immunosuppressive regimen based on costimulatory blockade, hearts from these pigs have survived from 2 to 6 months in baboons. Graft failure was predominantly from the development of a thrombotic microangiopathy. Potential contributing factors include the presence of preformed anti-nonGal antibodies or the development of low levels of elicited antibodies to nonGal antigens, natural killer (NK) cell or macrophage activity, and inherent coagulation dysregulation between pigs and primates. The breeding of pigs transgenic for an "anticoagulant" gene, such as human tissue factor pathway inhibitor, hirudin, or CD39, or lacking the gene for the prothrombinase, fibrinogen-like protein-2, is anticipated to inhibit the change in the endothelium to a procoagulant state that takes place in the pig organ after transplantation. The identification of the targets for anti-nonGal antibodies and/or human macrophages might allow further genetic modification of the pig, and xenogeneic NK cell recognition and activation may be inhibited by the transgenic expression of human leukocyte antigen molecules and/or by blocking the function of activating NK receptors. The ultimate goal of induction of T-cell tolerance may be possible only if these hurdles in the coagulation system and innate immunity can be overcome.

Characterization of anti-Gal antibody-producing cells of baboons and humans.

BACKGROUND: Anti-Gal antibodies cause hyperacute and delayed xenograft rejection in pig-to-primate transplantation. The cell populations producing anti-Gal and other natural antibodies in primates are unknown. METHODS: Cells from different lymphoid compartments of naïve or sensitized baboons were examined for anti-Gal and total Ig production by ELISPOT. B and plasma cells from humans and baboons were purified by FACS sorting and characterized for anti-Gal and total Ig production and cytology. RESULTS: In naïve baboons, the spleen was the major source of anti-Gal IgM-secreting cells. Two months after sensitization with porcine tissues, high frequencies of anti-Gal IgM- and IgG-secreting cells were detected in the spleen, lymph nodes, and bone marrow. Six months after antigen exposure, anti-Gal IgM- and IgG-secreting cells were preferentially localized in the bone marrow. Cells from human spleen, bone marrow, and blood were also analyzed and anti-Gal IgM-secreting cells were detected mainly in the spleen. Sorting of baboon and human cells showed that anti-Gal IgM-secreting cells were mainly splenic B cells (CD20+, CD138-, and Ig+). Although low in percentage, sorted CD20-CD138+ plasma cells in spleen and bone marrow secreted large quantities of anti-Gal IgM. Most anti-Gal IgG-secreting cells were plasma cells (CD138+) at both early (Ig+) and late (Ig-) stages of differentiation. CONCLUSIONS: Similar to Gal knockout mice, natural anti-Gal IgM antibodies in primates are produced mainly by splenic B cells. After antigen exposure, anti-Gal IgM and IgG were secreted by both B and plasma cells. These results suggest strategies to remove xenoreactive antibody-secreting cells prior to transplantation.

alpha1,3-Galactosyltransferase gene-knockout pig heart transplantation in baboons with survival approaching 6 months.

BACKGROUND: The recent generation of alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pigs has allowed investigation of the survival of GalT-KO pig organs in nonhuman primates. METHODS: Heterotopic heart transplantation from GalT-KO pigs was carried out in baboons (n=8) using a human antihuman CD154 monoclonal antibody-based immunosuppressive regimen. RESULTS: In six of the eight cases, graft survival extended to between approximately 2 and 6 months. All grafts developed thrombotic microangiopathy (TM). In particular, the clinical course of one baboon in which the graft functioned for 179 days is summarized. This baboon received aspirin (40 mg on alternate days) from day 4 in addition to heparin, which may have been a factor in the delay of onset and progression of TM and in prolonged graft survival. Maintenance therapy with anti-CD154 mAb, mycophenolate mofetil, and methylprednisolone was associated with persistently low numbers of CD3CD4 and CD3CD8 cells. Despite persisting depletion of these cells, no infectious complications occurred. CONCLUSIONS: It remains to be established whether TM is related to a very low level of natural preformed or T-cell-induced antibody deposition on the graft, inducing endothelial activation and injury, or to molecular incompatibilities in the coagulation mechanisms between pig and baboon, or to both. However, function of a pig organ in a baboon for a period approaching six months, which has not been reported previously, lends encouragement that the barriers to xenotransplantation will eventually be overcome.

Vascularized thymic lobe transplantation in a pig-to-baboon model: a novel strategy for xenogeneic tolerance induction and T-cell reconstitution.

BACKGROUND: This laboratory has previously demonstrated the induction of allogeneic tolerance by vascularized thymic lobe (VTL) transplantation in miniature swine. We report here our initial attempt to induce tolerance by VTL transplantation in the clinically relevant, discordant, pig-to-baboon model of xenotransplantation. METHODS: Six baboons received xenografts of hDAF VTLs. Four of these baboons also received omental thymic tissue implants. All recipients were treated with an immunosuppressive conditioning regimen that included thymectomy, splenectomy, extracorporeal immunoadsorption of anti-alpha Gal antibodies, and T-cell depletion. Two control baboons received sham operations, of which one also received 5x10 hDAF porcine thymocytes/kg intravenously. RESULTS: Transplanted VTL grafts supported early thymopoiesis of recipient-type immature thymocytes, and facilitated engraftment of nonvascularized thymic omental implants. Recipients of the VTL grafts demonstrated donor-specific unresponsiveness in MLR assays, development of peripheral CD45RAhigh/CD4 double positive (DP) cells, and positive cytokeratin staining of thymic stroma in the grafts for 2 months following xenotransplantation. The control baboons did not show these markers of thymic reconstitution. The eventual return of Gal natural antibodies led to the destruction of graft epithelial cells and the rejection of all VTL grafts by 3 months posttransplantation. CONCLUSIONS: VTL transplantation from hDAF swine to baboons induced early thymopoiesis in the recipients and donor-specific cellular unresponsiveness in vitro. When coupled with additional strategies aimed at silencing humoral rejection, VTL transplantation may significantly prolong xenograft survival and result in long-term tolerance.

Activation of human endothelial cells by mobilized porcine leukocytes in vitro: implications for mixed chimerism in xenotransplantation.

BACKGROUND: The induction of immunologic tolerance to pig antigens in primates may facilitate the development of successful clinical xenotransplantation protocols. The infusion of mobilized porcine peripheral blood leukocytes (PBPC, consisting of approximately 2% peripheral blood progenitor cells) into preconditioned baboons, intended to induce mixed hematopoietic cell chimerism, however, results in a severe thrombotic microangiopathy (TM) that includes vascular injury, microvascular thrombosis, and pronounced thrombocytopenia. Because the mechanisms responsible for TM are unclear, we have explored the effects of PBPC on human umbilical vein endothelial cell (HUVEC) activation. METHODS: Confluent HUVEC monolayers were established in 96-well cell culture clusters. PBPC were mobilized from miniature swine with porcine interleukin 3 (pIL-3), porcine stem cell factor (pSCF), and human granulocyte-colony stimulating factor (hG-CSF) and were collected by leukapheresis. PBPC were added to HUVEC (0-1x10(7) PBPC/well) for 3- to 24-hr periods and, with cell-based ELISA techniques, surface levels of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) were measured. In some cases, peripheral blood leukocytes (PBL) were collected from pigs that did not receive pIL-3, pSCF, or hG-CSF and were added to HUVEC. PBPC were also sorted into subsets of CD2- cells, CD2+ cells, and cellular debris, each of which were added separately to HUVEC. Transwell permeable membrane inserts were placed over HUVEC to prevent direct cell-cell contact with PBPC in some instances. RESULTS: PBPC from different pigs (n=6) induced an increase in the expression of E-selectin, VCAM-1, and ICAM-1 to levels 5, 4, and 2 times greater than baseline, respectively. ICAM-1 expression reached maximum levels after the addition of 6x10(5) PBPC/well. Expression of E-selectin and VCAM-1 increased further with the addition of greater numbers of PBPC, reaching maximum levels after the addition of 1x10(7) PBPC/well. PBPC-induced up-regulation of E-selectin, VCAM-1, and ICAM-1 had a maximum effect after approximately 6 hr, 12 hr, and 6 to 9 hr, respectively (n=3). The effects of fresh and frozen PBPC on HUVEC were similar (n=2). Compared to PBPC, PBL induced higher levels of E-selectin, VCAM-1, and ICAM-1 on HUVEC (n=2). The addition of CD2- cells to HUVEC induced an increase in E-selectin and VCAM-1 to levels 4 times greater than baseline, whereas the addition of CD2+ cells or debris did not elicit a substantial effect (n=2). Transwell permeable membranes prevented PBPC-induced up-regulation of E-selectin, VCAM-1, and ICAM-1 on HUVEC (n=2), suggesting that the mechanism of activation requires direct cell-cell contact. CONCLUSIONS: Porcine PBPC activate HUVEC, as suggested by an increase in surface E-selectin, VCAM-1, and ICAM-1 levels, and have a maximum effect after 9 hr. Freezing of PBPC does not affect PBPC-induced activation of HUVEC. PBL induce greater activation of HUVEC than do PBPC. CD2- cells are primarily responsible for PBPC-induced activation of HUVEC and direct cell-cell contact is required. Removal of CD2- cells before the administration of PBPC or the use of agents that interrupt PBPC-endothelial cell interactions may prevent or treat TM in baboons.

Acute vascular rejection of xenografts: roles of natural and elicited xenoreactive antibodies in activation of vascular endothelial cells and induction of procoagulant activity.

BACKGROUND: Hyperacute rejection of vascularized discordant xenografts can now be effectively managed. However, acute vascular rejection (AVR) then ensues, resulting in graft destruction, coagulopathy, or both within weeks. The aim of this study was to determine associations between humoral responses to the xenograft and the induction of AVR, coagulopathy, or both. METHODS: In vitro, heat-inactivated, naive or sensitized baboon sera containing xenoreactive natural or elicited antibodies were used to activate porcine aortic endothelial cells (PAEC) in vitro. Tissue factor expression on PAEC was determined as an index of heightened procoagulant activity. In vivo, porcine renal xenografts were transplanted into immunosuppressed baboons, and at the time of rejection or the development of a consumptive coagulopathy, biopsy specimens were obtained for studies of xenoreactive antibody binding and tissue factor expression. RESULTS: In vitro, incubation of PAEC with naive baboon sera containing natural anti-Galalpha1,3Gal (Gal) antibodies resulted in minimal tissue factor induction; the addition of complement boosted procoagulant responses. Elicited xenoreactive antibodies, and to non-Gal epitopes alone, induced high amounts of procoagulant activity on PAEC; the addition of complement resulted in overt cytotoxicity. In vivo, AVR was associated with xenoreactive antibody deposition in the graft. When vascular endothelial binding of xenoreactive antibody was combined with the expression of tissue factor, consumptive coagulopathy developed irrespective of histopathologic features of AVR. CONCLUSIONS: Our in vitro results indicate that elicited antibodies, potentially to non-Gal epitopes, induce endothelial cell activation and tissue factor expression; in vivo, a consumptive coagulopathy occurred when there was xenoreactive antibody deposition and increase of tissue factor.

Xenogeneic thymus transplantation in a pig-to-baboon model.

BACKGROUND: We have tested whether fetal porcine thymic tissue transplantation can lead to tolerance across a discordant (pig-to-baboon) xenogeneic barrier. METHODS: Six baboons underwent a conditioning regimen with thymectomy, splenectomy, and anti-monkey CD3 antibody conjugated to a diphtheria toxin binding site mutant (FN18-CRM9). Porcine fetal or neonatal thymic tissue was transplanted into three baboons. Three control baboons received either no transplanted pig tissue (n=1) or adult pig lymph node (n=2). Cellular responses and skin xenografts were used to test for tolerance. RESULTS: After T-cell depletion and thymic transplantation, recovery of thymus-dependent naïve-type CD4 cells (CD4/CD45RA ) and in vitro xenogeneic hyporesponsiveness were observed. No sensitization of alpha-galactosyl antibody responses was observed. The thymic grafts survived up to 48 days. Porcine skin xenografts were performed in two of these animals with survival of 22 and 24 days. Only two of these animals were completely T-cell depleted, and both failed to recover thymus-dependent T cells (CD4/CD45RA ). In one animal, general in vitro hyporesponsiveness was observed, with subsequent death from infection. The second animal demonstrated delayed recovery of T cells and prolonged general hyporesponsiveness in vitro. Neither animal demonstrated prolongation of porcine skin grafts compared with allografts (both rejected by day 13). CONCLUSIONS: Porcine thymic tissue is able to induce xenogeneic hyporesponsiveness. More efficient thymic engraftment may allow this approach to induce xenograft tolerance.

Xenogeneic thymokidney and thymic tissue transplantation in a pig-to-baboon model: I. Evidence for pig-specific T-cell unresponsiveness.

BACKGROUND: The potential of xenotransplantation for clinical application will require overcoming barriers of humoral and cellular rejection, through strategies using immune suppression or tolerance induction. This laboratory has previously reported the induction of tolerance in the discordant xenogeneic model of pig-to-rodent thymic transplantation. We also have described a miniature swine model of fully mismatched allogeneic composite vascularized thymokidney transplantation that induced transplantation tolerance. We tested a combination of these approaches in a clinically relevant pig-to-primate model of xenotransplantation. METHODS: Composite thymokidney grafts were prepared 40 to 80 days before transplantation by the autologous implantation of thymic tissue under the renal capsule of human decay-accelerating factor transgenic swine. Baboons received xenotransplants of both human decay-accelerating factor composite thymokidneys and omental implants of thymic tissue. Recipients were treated with an immunosuppressive-conditioning regimen including thymectomy or thymic irradiation, extracorporeal immunoadsorption of anti-alphaGal antibodies and T-cell depletion. Recipients were followed for indicators of xenograft rejection, T-cell depletion and reconstitution, anti-alphaGal antibody levels, and mixed lymphocyte responses. Immunologic responses were studied in those animals that survived for more than 3 weeks. RESULTS: Thymokidney xenografts survived for up to 30 days, with evidence of viable thymic epithelium and Hassall's corpuscles under the renal capsule and in the omental implants, and with evidence of few host lymphocytes. Three animals demonstrated donor-specific unresponsiveness, while maintaining normal alloresponses, in mixed-lymphocyte-response assays performed after immunosuppression had been stopped. Rejected grafts demonstrated humoral damage without evidence of cellular infiltrates. After graftectomy, one animal maintained donor-specific cellular unresponsiveness and stable anti-alphaGal antibody levels for more than 2 months. CONCLUSIONS: We concluded that composite thymokidney and thymic-tissue xenotransplantation from swine to baboons can induce donor-specific cellular unresponsiveness and stable anti-alphaGal antibody levels, suggesting avoidance of sensitization after xenotransplantation. The presence of viable donor-swine thymic epithelium could have a role in the development of donor-specific T-cell tolerance. Further strategies to address humoral rejection could prolong graft survival and result in long-term tolerance to xenografts.

alpha1,3-Galactosyltransferase gene-knockout miniature swine produce natural cytotoxic anti-Gal antibodies.

BACKGROUND: The expression of galactose alpha 1,3 galactose (Gal) in pigs has proved a barrier to xenotransplantation. Miniature swine lacking Gal (Gal pigs) have been produced by nuclear transfer/embryo transfer. METHODS: The tissues of five Gal pigs of SLA dd haplotype (SLA) were tested for the presence of Gal epitopes by staining with the Griffonia simplicifolia IB4 lectin. Their sera were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from wild-type (Gal) SLA-matched pigs; serum cytotoxicity was also assessed. The cellular responses of PBMC from Gal swine toward Gal SLA-matched PBMC were tested by mixed leukocyte reaction and cell-mediated lympholysis assays. RESULTS: None of the tissues tested showed Gal expression. Sera from all five Gal pigs manifested IgM binding to Gal pig PBMC, and sera from three showed IgG binding. In all five cases, cytotoxicity to Gal cells could be demonstrated, which was lost after treatment of the sera with dithiothreitol, indicating IgM antibody-mediated cytotoxicity. PBMC from Gal swine had no proliferative or cytolytic T-cell response toward Gal SLA-matched PBMC. CONCLUSIONS: Gal pigs do not express Gal epitopes and develop anti-Gal antibodies that are cytotoxic to Gal pig cells. The absence of an in vitro cellular immune response between Gal and Gal pigs is related to their identical SLA haplotype and indicates the absence of immunogenicity of Gal in T-cell responses. The model of Gal organ transplantation into a Gal SLA-matched recipient would be a valuable large animal model in the study of accommodation or B-cell tolerance.

Porcine cytomegalovirus and coagulopathy in pig-to-primate xenotransplantation.

BACKGROUND: A rapidly progressive disorder termed consumptive coagulopathy (CC) has been observed frequently in pig-to-baboon renal xenotransplantation. CC may be initiated by endothelial activation and induction of procoagulant factors after immunologic injury or infection, or by molecular incompatibilities between porcine coagulation proteins and primate clotting factors. The activation of porcine (P) cytomegalovirus (PCMV) and baboon (B) CMV infections has been documented in pig-to-primate xenotransplantation. The purpose of this study was to determine the contribution of PCMV and BCMV to CC. METHODS: Endothelial activation was assessed by means of measurement of porcine tissue factor (pTF) in a functional assay in primary porcine aortic endothelial cells (PAEC) in vitro. Renal xenografts and native kidneys were studied by immunohistochemistry in immunosuppressed swine and baboons. BCMV and PCMV DNA was measured by quantitative molecular assays using real-time polymerase chain reaction. RESULTS: In vitro, infection of PAEC with PCMV resulted in a significant increase of pTF expression. In vivo, pTF increase occurred without the activation of PCMV in two xenografts, and in four grafts no pTF was detected despite PCMV activation. All animals with graft pTF increase developed CC. BCMV activation in the baboon xenograft recipients did not correlate with CC or pTF increase. Control pigs and baboons had activation of PCMV and BCMV, respectively, but without coagulation abnormalities. CONCLUSIONS: PCMV induces endothelial cell activation in vitro with procoagulant expression. However, in vivo, CC and pTF induction has an uncertain relationship to increased replication of PCMV within a xenograft. Although the data do not exclude a contributory role of PCMV in CC, other mechanisms are also likely to contribute to coagulopathies observed in pig-to-primate xenotransplantation.

Cryopreservation and mycophenolate therapy are detrimental to hematopoietic progenitor cells.

BACKGROUND: The aim of the present study was to determine whether certain components of nonmyeloablative regimens for hematopoietic cell transplantation might compromise the growth of hematopoietic progenitors. METHODS: Porcine peripheral blood progenitor cells (PBPC) were cytokine-mobilized, collected by leukapheresis, and cryopreserved using 5% dimethyl sulfoxide and 6% hydroxyethyl starch. The influence of cryopreservation on PBPC was tested in vitro by enumeration of colony-forming units (CFUs) in methylcellulose and cobblestone area-forming cell (CAFC) subsets in stromal-associated long-term cultures on fresh and frozen PBPC. The effects of mycophenolate mofetil (MMF) on porcine PBPC and baboon and human bone marrow (BM) were tested in vitro by adding varying doses of MMF to the CFU assays. One baboon was treated with increasing doses of MMF (100-500 mg/kg per day continuously intravenous), and sequential BM aspirations were tested for CFU content. RESULTS: Fresh cytokine-mobilized PBPC had similar frequencies of progenitor cells when compared with porcine BM. Freezing-thawing of PBPC had no effect on porcine CFUs but reduced the recovery of CAFCs by more than 90%. In vitro, MMF completely inhibited the development of porcine and human CFUs at a concentration of 1 microg/mL and of baboon CFUs at levels between 10 and 100 microg/mL. Plasma-free mycophenolic acid levels of 10 to 30 microg/mL were associated with decreased CFUs in the BM. CONCLUSIONS: Cryopreservation and MMF potentially prevent engraftment of porcine PBPC by reducing the content or development of progenitor cells. These results indicate that the use of fresh PBPC might improve the induction of mixed hematopoietic chimerism and raise the possibility that use of high doses of MMF in the poststem cell transplant may compromise engraftment.

An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics.

Concern around the lack of monitoring of proteinaceous subvisible particulates in the 0.1-10 microm range has been heightened (Carpenter et al., 2009, J Pharm Sci 98: 1202-1205), primarily due to uncertainty around the potential immunogenicity risk from these particles. This article, representing the opinions of a number of industry scientists, aims to further the discussion by developing a common understanding around the technical capabilities, limitations, as well as utility of monitoring this size range; reiterating that the link between aggregation and clinical immunogenicity has not been unequivocally established; and emphasizing that such particles are present in marketed products which remain safe and efficacious despite the lack of monitoring. Measurement of subvisible particulates in the <10 microm size range has value as an aid in product development and characterization. Limitations in measurement technologies, variability from container/closure, concentration, viscosity, history, and inherent batch heterogeneity, make these measurements unsuitable as specification for release and stability or for comparability, at the present time. Such particles constitute microgram levels of protein with currently monitored sizes >or=10 microm representing the largest fraction. These levels are well below what is detected or reported for other product quality attributes. Subvisible particles remain a product quality attribute that is also qualified in clinical trials.

Prolonged survival of porcine hepatocytes in cynomolgus monkeys.

BACKGROUND & AIMS: Management of patients with liver failure can be a significant medical challenge, and transplantation of the liver is the only definitive therapy. Whole liver allotransplantation is limited by a shortage of human donors and the risks of the surgery in those most ill. Transplants consisting of xenogeneic hepatocytes might overcome these problems, and work in rodents indicates that such transplants can correct some metabolic deficiencies and can prevent the complications and mortality associated with hepatic failure. As a prelude to clinical application, we tested the feasibility of hepatocyte xenotransplantation in nonhuman primates. METHODS: One to 2 billion hepatocytes from outbred swine were transplanted into the spleens of cynomolgus monkeys using conventional immunosuppression to control rejection. Duration of graft function was determined based on assay for porcine albumin. RESULTS: Following a single infusion, xenogeneic hepatocytes functioned for more than 80 days and, following re-transplantation, for more than 253 days. Engraftment in the spleen was confirmed 40 days after transplantation by asialoglycoprotein receptor-directed nuclear scanning. The humoral immune response to the transplanted porcine cells had no discernible impact on the survival of the grafts. CONCLUSIONS: Xenotransplantation of hepatocytes should be explored as a readily available, minimally invasive form of therapy for hepatic failure.

Selected physiologic compatibilities and incompatibilities between human and porcine organ systems.

The shortage of donor organs is a major barrier to clinical organ transplantation. Although xenotransplantation is considered one of the alternatives to human organ transplantation, there are immunologic and physiologic incompatibilities between humans and pigs. With the exception of coagulation, the major potential physiologic incompatibilities relating to function of the kidney, heart, liver, lungs, pancreatic islets, and hormones are reviewed. Some of these physiologic differences can be overcome by producing genetically altered pigs to improve compatibility with humans. The possibility of producing such pigs for organ transplantation is considered.