Laura J. Peterson

A non-activating humanized anti-CD3 monoclonal antibody retains immunosuppressive properties in vivo

OKT3, a mouse anti-human CD3 mAb, is a potent immunosuppressive agent used in clinical transplantation to prevent or treat allograft rejection. Associated with this therapy is the systemic release of several cytokines that result in a series of adverse side effects. This release of cytokines is dependent on the cross-linking mediated by OKT3 between T cells and the FcγR-bearing cells. To generate an anti-human CD3 mAb with reduced activating properties as compared with OKT3, we have transferred the complementary determining regions of OKT3 onto human IgG frameworks and then performed point mutations that reduce the affinity of the “humanized” anti-CD3 mAbs for FcγRs. Initial, in vitro, studies showed that whereas OKT3 and the parental humanized anti-CD3 mAbs activated T cells similarly, a humanized Fc variant failed to do so. Both the Fc variant and the activating anti-CD3 mAbs induced comparable modulation of the TCR and suppression of cytolytic T cell activity, in vitro. In the current study, we exploited an experimental model in which human splenocytes from cadaveric organ donors were inoculated into severe combined immunodeficient mice (hu-SPL-SCID mice) to test the activating and immunosuppressive properties of these anti-human CD3 mAbs in vivo. Unlike injection of OKT3 or of the parental humanized mAb, administration of the Fc variant did not result in T cell activation in vivo, as evidenced by the lack of induction of surface markers of activation, and of systemic human cytokines, including IL-2. Importantly, similar prolongation of human allograft survival was achieved with all anti-CD3 mAbs, indicating that the nonactivating anti-CD3 mAbs retained significant immunosuppressive properties in vivo. Thus, the use of an Fc variant in clinical transplantation should result in fewer side effects than observed with OKT3, while maintaining its clinical efficacy.

Methylguanine methyltransferase–mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model

Clinical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase-mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34+ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.

Direct comparison of steady-state marrow, primed marrow, and mobilized peripheral blood for transduction of hematopoietic stem cells in dogs.

The optimal stem cell source for stem cell gene therapy has not been defined. Most gene transfer studies have used peripheral blood or marrow repopulating cells collected after administration of granulocyte colony-stimulating factor and stem cell factor (G-CSF/SCF). For clinical applications, however, growth factor administration may not be feasible. Thus, in the current study we used a competitive repopulation assay in the dog to directly compare transduction efficiency of steady-state marrow, G-CSF/SCF-primed marrow, and G-CSF/SCF-mobilized peripheral blood. Cells from all three sources were transduced, cryopreserved, and thawed together before infusion into myeloablated dogs. Gene marking in hematopoietic repopulating cells was assessed by polymerase chain reaction. While primed marrow resulted in the highest long-term marking levels, steady-state marrow was transduced at least as efficiently as mobilized peripheral blood in all three dogs. These results suggest that steady-state marrow may be an appropriate source for genetic modification of hematopoietic stem cells.

Foamy and Lentiviral Vectors Transduce Canine Long-Term Repopulating Cells at Similar Efficiency

Foamy viral vectors and lentiviral vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy because they both efficiently transduce stem cells using rapid ex vivo transduction protocols designed to maintain engraftment potential. Here we directly compared the ability of foamy and lentiviral vectors to transduce long-term hematopoietic repopulating cells in the dog model, using a competitive repopulation assay with vectors that express enhanced yellow or green fluorescent proteins (EY/GFP). Mobilized canine peripheral blood CD34(+) cells were divided into two fractions and exposed to either foamy (EGFP) or lentiviral (EYFP) vectors at a multiplicity of infection of 5 in an 18-hr transduction protocol and then reinfused after conditioning with 920 cGy of total body irradiation. Both dogs studied had rapid neutrophil engraftment and multilineage engraftment of transduced cells. Marking was similar for both vectors, particularly at later time points, indicating that both vector types transduce long-term repopulating cells at similar frequencies.

Antagonistic and agonistic anti-canine CD28 monoclonal antibodies: tools for allogeneic transplantation

Background. It has been presumed that antibody-mediated selective costimulatory molecule blockade of CD28 is superior to cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig. This is based on the premise that specifically blocking CD28 allows inhibitory signals through CTLA-4 to proceed, which furthermore suppresses T-cell function. Methods. The extracelluar domain of canine (ca)CD28 was cloned from dog peripheral blood mononuclear cells. Mice were immunized with a caCD28/murine IgG2a fusion protein. Hybridomas were produced by fusing splenocytes with mouse NSO cells and screened for caCD28 binding by ELISA. Agonistic and antagonistic activities of the monoclonal antibodies (mAb) were tested in mixed leukocyte reactions. Canine regulatory T cells were expanded using plate-bound anti-CD3 and an anti-CD28 agonist mAb. Results. One agonistic and seven antagonistic mAbs to canine (ca)CD28 were cloned. Binding studies indicated that an agonistic (5B8) and an antagonistic (1C6) mAb bound equally well to a caCD28/caIgG1 fusion protein and to CD28 expressed on CD4+ and CD8+ peripheral blood T cells. Antagonistic antibody blocked mixed lymphocyte reactions (MLR) in a dose-dependent manner similar to CTLA4-Ig, whereas the agonistic antibody to caCD28 enhanced MLR. The 5B8 was superior to 1C6 when either was combined with anti-caCD3 to stimulate lymphocyte proliferation. Furthermore, the agonistic mAb, 5B8, together with anti-CD3 mAb induced 100-fold proliferation of canine regulatory T cells. Relative to untreated control cells, anti-caCD28 (1C6) and CTLA4-Ig equivalently inhibited cytotoxic T lymphocyte-mediated killing of alloreactive target cells. Conclusion. These studies demonstrated that mouse anti-caCD28 mAbs can be generated with agonistic or antagonistic function.

Establishment of Long-term Tolerance to SRBC in Dogs by Recombinant Canine CTLA4-Ig

Background. Blockade of the CD28 costimulatory molecule by recombinant human cytotoxic T lymphocyte (CTL)-associated antigen (CTLA4)-Ig or CD40-CD154 interaction with the monoclonal antibody 5C8 together with donor-specific transfusion led to enhanced engraftment in the canine model of dog leukocyte antigen (DLA)-identical marrow transplantation after 1 Gy total body irradiation. To reduce or eliminate total body irradiation conditioning regimens, we have sought to develop canine specific reagents. Methods. We have created a fusion protein of the extracellular domain of canine (c) CTLA-4 linked to the hinge-CH2-CH3 domains of canine IgG1 in a pcDNA3.1+ vector. Chinese hamster ovarian cells were cotransfected with CTLA4-Ig vector and a dihydrofolate reductase-containing vector. Stable, high producing clones were generated. Results. Cell binding and mixed leukocyte reactions indicated no significant differences in activity between cCTLA4-Ig and human CTLA4-Ig. Mixed leukocyte reaction data indicated that combinations of cCTLA4-Ig and the monoclonal antibody 5C8 were superior in blocking 3H-thymidine uptake compared to either reagent alone. In dogs, the circulating half-life of cCTLA4-Ig was approximately 7 days with no immune response against the fusion protein. Finally, two injections of cCTLA4-Ig effectively tolerized two dogs against eight consecutive challenges with sheep red blood cells, given over 330 days as indicated by a complete block of IgG antibody production. Tolerance was broken in one of the two dogs when a ninth injection of sheep red blood cell was given subcutaneously in incomplete Freund’s adjuvant. Conclusion. cCTLA4-Ig is an effective nonimmunogenic blocking reagent of the CD28 costimulatory pathway in dogs and is a promising reagent for studies of tolerance induction in hematopoietic cell transplantation in the canine model.

Ex vivo selection for oncoretrovirally transduced green fluorescent protein-expressing CD34-enriched cells increases short-term engraftment of transduced cells in baboons

In an effort to improve hematopoietic stem cell gene transfer rates using gibbon ape leukemia virus (GALV)-pseudotype retroviral vectors in baboons, we have studied preselection of transduced green fluorescent protein (GFP)-expressing CD34-enriched marrow cells. Three animals were transplanted with GFP-selected (GS) CD34-enriched marrow. To ensure engraftment, preselected GFP-positive cells were infused together with unselected neo-transduced cells. After transduction on fibronectin, cells were cultured for an additional 2 days to allow for expression of GFP. GFP-expressing cells were enriched by fluorescence-activated cell sorting and infused together with cells from the unselected fractions after myeloablative irradiation of the recipient. Three other animals were transplanted with GFP-transduced CD34-enriched cells without prior GFP selection (GU). At 4 weeks after transplant, the percentage of GFP-expressing white blood cells was significantly higher in the GS group (6.6%) than in the GU group (1.3%) (p < 0.002). The higher gene transfer levels in the animals transplanted with GS cells gradually declined, and by day 100 after transplant, gene transfer levels were similar in both groups. PCR analysis performed on genomic DNA isolated from peripheral blood cells demonstrated that the decline in GFP-positive cells was due to the loss of gene-marked cells and not due to loss of expression. These results show that transplantation of CD34-positive marrow cells selected for GFP-positive cells after transduction provides high levels of transduced granulocytes in the short term. However, using this experimental design with concomitant infusion of unselected cells and the use of oncoretroviral vectors, preenrichment of vector-expressing, transduced CD34-enriched cells does not improve long-term persistence and expression.

Delaying DLA-Haploidentical Hematopoietic Cell Transplantation after Total Body Irradiation

Exposure to accidental or deliberate radiation poses a threat to public health, proving lethal at higher doses in large part because of deleterious effects on marrow. In those cases, allogeneic hematopoietic cell transplantation (HCT) might be required to restore marrow function. Most radiation accident victims will have HLA-haploidentical relatives who could serve as HCT donors. Here, we assessed in a canine HCT model the total body irradiation (TBI) doses after which transplants might be required and successful engraftment would be possible. In an attempt at mimicking the logistical problems likely to exist after radiation accidents, 4-, 8- or 10-day intervals were placed between TBI and HCT. To keep the experimental readout simple, no graft-versus-host disease (GVHD) prevention was administered. All dogs transplanted after a 4-day delay following 700 or 920 cGy TBI successfully engrafted, whereas virtually all those given 450 or 600 cGy rejected their grafts. Transplant delays of 8 and 10 days following 920 cGy TBI also resulted in successful engraftment in most dogs, whereas a delay of 8 days after 700 cGy resulted in virtually uniform graft failure. The time courses of acute GVHD (aGVHD) and rates of granulocyte recovery in engrafting dogs were comparable among dogs regardless of the lengths of delay. In other studies, we showed that most dogs not given HCT survived 700 cGy TBI with intensive supportive care, whereas those given 800 cGy TBI and higher died with marrow aplasia. Thus, DLA-haploidentical HCT was successful even when carried out 4, 8, or 10 days after TBI at or above radiation exposures where dogs survived with intensive care alone.

Transmission and expansion of HOXB4-induced leukemia in two immunosuppressed dogs: implications for a new canine leukemia model.

OBJECTIVE There are currently no large animal models to study the biology of leukemia and development of novel antileukemia therapies. We have previously shown that dogs transplanted with homeobox B4 (HOXB4)-transduced autologous CD34(+) cells developed myeloid leukemia associated with HOXB4 overexpression. Here we describe the transmission, engraftment, and expansion of these canine leukemia cells into two genetically unrelated, immunosuppressed dogs. MATERIALS AND METHODS Two dogs immunosuppressed after major histocompatibility complex-haploidentical hematopoietic cell transplantation and exhibiting mixed donor-host chimerism were accidentally infused trace amounts of HOXB4-overexpressing leukemia cells from a third-party dog. RESULTS Six weeks after infusion of HOXB4-overexpressing leukemia cells, these two dogs rapidly developed myeloid leukemia consisting of marrow and organ infiltration, circulating blasts, and, in one dog, chloromatous masses. Despite neither of these dogs sharing any dog leukocyte antigen haplotypes with the sentinel case, the HOXB4-transduced clones engrafted and proliferated without difficulty in the presence of immunosuppression. Chimerism studies in both dogs confirmed that donor and, in one case, host hematopoietic cell engraftment was lost and replaced by third-party HOXB4 cells. CONCLUSIONS The engraftment and expansion of these leukemia cells in dogs will allow studies into the biology of leukemia and development and evaluation of novel antileukemia therapies in a clinically relevant large animal model.

Improved short‐term engraftment of lentivirally versus gammaretrovirally transduced allogeneic canine repopulating cells

Gammaretroviral vectors require cell division for efficient transduction. Thus, extended cell culture times are necessary for efficient transduction with gammaretroviral vectors, which in turn can lead to stem cell loss and impaired engraftment. Lentiviral vectors transduce nondividing cells and are therefore able to transduce stem cells in short transduction protocols. Here, we compared the short‐term engraftment of lentivirally and gammaretrovirally transduced canine allogeneic DLA‐matched littermate cells. A reduced conditioning regimen of 400 cGy total body irradiation was used in preparation for clinical studies. Two dogs received a graft of gammaretrovirally transduced CD34‐selected cells. CD34+ cells were prestimulated for 30 h and then exposed twice to concentrated RD114 pseudotype vector. Three dogs received lentivirally transduced CD34‐selected cells. Cells were transduced overnight with concentrated VSV‐G pseudotype lentiviral vector. The animals in the lentiviral group showed a significantly faster granulocyte recovery. VNTR analysis 40–50 days after transplantation revealed higher donor chimerism for the lentiviral group compared to the retroviral group. These data suggest that short lentiviral transduction protocols may be superior to extended gammaretroviral transduction protocols with respect to engraftment potential of transduced CD34+ hematopoietic repopulating cells. Copyright