Selective CD28 Blockade Research Articles

Selective CD28 blockade impacts T cell differentiation during homeostatic reconstitution following lymphodepletion.

Costimulation blockade targeting the CD28 pathway provides improved long-term renal allograft survival compared to calcineurin inhibitors but may be limited as CTLA-4-Ig (abatacept, belatacept) blocks both CD28 costimulation and CTLA-4 coinhibition. Directly targeting CD28 while leaving CTLA-4 intact may provide a mechanistic advantage. Fc-silent non-crosslinking CD28 antagonizing domain antibodies (dAb) are currently in clinical trials for renal transplantation. Given the current standard of care in renal transplantation at most US centers, it is likely that lymphodepletion via thymoglobulin induction therapy could be used in patients treated with CD28 antagonists. Thus, we investigated the impact of T cell depletion (TCD) on T cell phenotype following homeostatic reconstitution in a murine model of skin transplantation treated with anti-CD28dAb. Skin from BALB/cJ donors was grafted onto C56BL/6 recipients which were treated with or without 0.2mg anti-CD4 and 10μg anti-CD8 one day prior to transplant and with or without 100μg anti-CD28dAb on days 0, 2, 4, 6, and weekly thereafter. Mice were euthanized six weeks post-transplant and lymphoid cells were analyzed by flow cytometry. Anti-CD28dAb reversed lymphopenia-induced differentiation of memory CD4+ T cells in the spleen and lymph node compared to TCD alone. Mice treated with TCD+anti-CD28dAb exhibited significantly improved skin graft survival compared to anti-CD28dAb alone, which was also improved compared to no treatment. In addition, the expression of CD69 was reduced on CD4+ and CD8+ T cells in the spleen and lymph node from mice that received TCD+anti-CD28dAb compared to TCD alone. While a reduced frequency of CD4+FoxP3+ T cells was observed in anti-CD28dAb treated mice relative to untreated controls, this was balanced by an increased frequency of CD8+Foxp3+ T cells that was observed in the blood and kidney of mice given TCD+anti-CD28dAb compared to TCD alone. These data demonstrate that CD28 signaling impacts the differentiation of both CD4+ and CD8+ T cells during homeostatic reconstitution following lymphodepletion, resulting in a shift towards fewer activated memory T cells and more CD8+FoxP3+ T cells, a profile that may underpin the observed prolongation in allograft survival.

Superior inhibition of alloantibody responses with selective CD28 blockade is CTLA-4 dependent and T follicular helper cell specific.

Anti-donor antibodies cause immunologic injury in transplantation. CD28 blockade with CTLA-4-Ig has the ability to reduce the incidence of these donor-specific antibodies (DSA), but its mechanism is suboptimal for the inhibition of alloimmunity in that CTLA-4-Ig blocks both CD28 costimulation and CTLA-4 coinhibition. Thus selective CD28 blockade that spares CTLA-4 has potential to result in improved inhibition of humoral alloimmunity. To test this possibility, we utilized a full allogeneic mismatch murine transplant model and T follicular helper (Tfh):B cell co-culture system. We observed that selective blockade with an anti-CD28 domain antibody (dAb) compared to CTLA-4-Ig led to superior inhibition of Tfh cell, germinal center, and DSA responses in vivo and better control of B cell responses in vitro. CTLA-4 blockade enhanced the humoral alloresponse and, in combination with anti-CD28 dAb, abrogated the effects of selective blockade. This CTLA-4-dependent inhibition was Tfh cell specific in that CTLA-4 expression by Tfh cells was necessary and sufficient for the improved humoral inhibition observed with selective CD28 blockade. As CD28 blockade attracts interest for control of alloantibodies in the clinic, these data support selective CD28 blockade as a superior strategy to address DSA via the sparing of CTLA-4 and more potent targeting of Tfh cells.

Role of CD28 Signaling on T cell Phenotype following Lymphodepletion and Homeostatic Reconstitution

Calcineurin inhibitors, the current standard of care in immunosuppression for transplant patients, leads to nephrotoxicity and loss of graft function over time. Preclinical studies have shown selective CD28 blockade (CD28 dAb, Lulizumab) to be superior to calcineurin inhibitors in reducing toxicity and prolonging allograft survival. Emory University will soon commence a Phase 2 clinical trial testing the efficacy of a Lulizumab‐based immunosuppression regimen in kidney transplantation. Patients in the trial will be T cell depleted (TCD), rendering them lymphopenic at the time of transplant and will therefore undergo homeostatic reconstitution in the weeks following transplantation. Lulizumab prevents T cell activation by selectively targeting CD28 signaling, but it is presently unclear what effect blocking this pathway will have on T cell phenotypes following homeostatic reconstitution (HR) in these patients. Previous studies have shown that HR leads to increased differentiation of naïve T cells into memory cells and to premature senescence. We hypothesized that blocking CD28 signaling in T cells during this process could prevent memory differentiation and senescence. We investigated this question using a murine model of TCD and skin transplantation. Four groups of mice were established: no treatment control, TCD alone, CD28 dAb alone, and both TCD and CD28 dAb (n = 4). Mice were sacrificed at 6 weeks post‐depletion and transplantation, and blood and tissues were collected for flow cytometric analysis. Using both traditional flow cytometry analysis and an unsupervised machine‐learning approach, we found that TCD CD4+ T cells have significantly elevated expression of the memory markers CD27, CD103, and CD69 when compared to no treatment control CD4+ T cells, indicating HR‐induced differentiation into memory T cells. In addition, TCD CD4+ T cells also expressed significantly higher levels of PD‐1, TIGIT, and CD226 (DNAM‐1) when compared to no treatment control CD4+ T cells, indicating a higher degree of senescence. Finally, we found that treating TCD mice with CD28 dAb restored expression of both memory markers and costimulatory markers to baseline levels. As such, we believe CD28 dAb presents a viable method to control HR‐induced differentiation and senescence of CD4+ T cells. These results were consistent across the blood, lung, and spleen. Our future directions include using murine models to determine whether the selective CD28 blockade‐mediated rescue of the HR‐induced phenotypic changes significantly affects protective immunity to infection.Support or Funding InformationResearch reported here was supported by the National Institutes of Health under award number R25 DK 101390 ‐ Title: Summer Undergraduate Program in Renal Research (SUPERR).

CD28-Dependent CTLA-4 Expression Fine-Tunes the Activation of Human Th17 Cells.

SummaryPrevious work has demonstrated that Th17 memory cells but not Th1 cells are resistant to CD28/CTLA-4 blockade with CTLA-4 Ig, leading us to investigate the individual roles of the CD28 and CTLA-4 cosignaling pathways on Th1 versus Th17 cells. We found that selective CD28 blockade with a domain antibody (dAb) inhibited Th1 cells but surprisingly augmented Th17 responses. CD28 agonism resulted in a profound increase in CTLA-4 expression in Th17 cells as compared with Th1 cells. Consistent with these findings, inhibition of the CD28 signaling protein AKT revealed that CTLA-4 expression on Th17 cells was more significantly reduced by AKT inhibition relative to CTLA-4 expression on Th17 cells. Finally, we found that FOXO1 and FOXO3 overexpression restrained high expression of CTLA-4 on Th17 cells but not Th1 cells. This study demonstrates that the heterogeneity of the CD4+ T cell compartment has implications for the immunomodulation of pathologic T cell responses.

Impact of selective CD28 blockade on virus-specific immunity to a murine Epstein-Barr virus homolog.

CTLA-4Ig (belatacept) blocks the CD80/CD86 ligands for both CD28 and CTLA-4; thus, in addition to the intended effect of blocking CD28-mediated costimulation, belatacept also has the unintended effect of blocking CTLA-4-mediated coinhibition. Recently, anti-CD28 domain antibodies (dAb) that selectively target CD28 while leaving CTLA-4 intact were shown to more effectively inhibit alloimmune responses and prolong graft survival. However, the impact of selective CD28 blockade on protective immunity has not been extensively investigated. Here, we sought to compare the impact of CTLA-4Ig vs anti-CD28dAb on CD8+ T cell immunity to a transplant-relevant pathogen, a murine homolog of Epstein-Barr virus. Mice were infected with murine gammaherpesvirus-68 (MHV) and treated with vehicle, CTLA-4Ig, or anti-CD28dAb. Although anti-CD28dAb resulted in a decrease in virus-specific CD8+ T cell numbers as compared to CTLA-4Ig, cytolytic function and the expression of markers of high-quality effectors were not different from CTLA-4Ig treated animals. Importantly, MHV-68 viral load was not different between the treatment groups. These results suggest that preserved CTLA-4 coinhibition limits MHV-specific CD8+ T cell accumulation, but the population that remains retains cytolytic function and migratory capacity and is not inferior in its ability to control viral burden relative to T cell responses in CTLA-4Ig-treated animals.

Selective CD28 blockade attenuates CTLA-4-dependent CD8+ memory T cell effector function and prolongs graft survival.

Memory T cells pose a significant problem to successful therapeutic control of unwanted immune responses during autoimmunity and transplantation, as they are differentially controlled by cosignaling receptors such as CD28 and CTLA-4. Treatment with abatacept and belatacept impede CD28 signaling by binding to CD80 and CD86, but they also have the unintended consequence of blocking the ligands for CTLA-4, a process that may inadvertently boost effector responses. Here, we show that a potentially novel anti-CD28 domain antibody (dAb) that selectively blocks CD28 but preserves CTLA-4 coinhibition confers improved allograft survival in sensitized recipients as compared with CTLA-4 Ig. However, both CTLA-4 Ig and anti-CD28 dAb similarly and significantly reduced the accumulation of donor-reactive CD8+ memory T cells, demonstrating that regulation of the expansion of CD8+ memory T cell populations is controlled in part by CD28 signals and is not significantly impacted by CTLA-4. In contrast, selective CD28 blockade was superior to CTLA-4 Ig in inhibiting IFN-γ, TNF, and IL-2 production by CD8+ memory T cells, which in turn resulted in reduced recruitment of innate CD11b+ monocytes into allografts. Importantly, this superiority was CTLA-4 dependent, demonstrating that effector function of CD8+ memory T cells is regulated by the balance of CD28 and CTLA-4 signaling.

Selective blockade of CD28 on human T cells facilitates regulation of alloimmune responses.

T cells are central to the detrimental alloresponses that develop in autoimmunity and transplantation, with CD28 costimulatory signals being key to T cell activation and proliferation. CTLA4-Ig molecules that bind CD80/86 and inhibit CD28 costimulation offer an alternative immunosuppressive treatment, free from some of the chronic toxicities associated with calcineurin inhibition. However, CD80/86 blockade by CTLA4-Ig also results in the loss of coinhibitory CTLA4 signals that are critical to the regulation of T cell activation. Here, we show that a nonactivating monovalent anti-CD28 that spares CTLA4 signaling is an effective immunosuppressant in a clinically relevant humanized mouse transplant model. We demonstrate that selective CD28 blockade prolongs human skin allograft survival through a mechanism that includes a reduction in the cellular graft infiltrate. Critically, selective CD28 blockade promotes Treg function in vivo and synergizes with adoptive Treg therapy to promote transplant survival. In contrast to CTLA4-Ig treatment, selective CD28 blockade promotes regulation of alloimmune responses and facilitates Treg-based cellular therapy.

CD28 Blockade Ex Vivo Induces Alloantigen-Specific Immune Tolerance but Preserves T-Cell Pathogen Reactivity.

Donor T-cells contribute to reconstitution of protective immunity after allogeneic hematopoietic stem cell transplantation (HSCT) but must acquire specific tolerance against recipient alloantigens to avoid life-threatening graft-versus-host disease (GvHD). Systemic immunosuppressive drugs may abrogate severe GvHD, but this also impedes memory responses to invading pathogens. Here, we tested whether ex vivo blockade of CD28 co-stimulation can enable selective T-cell tolerization to alloantigens by facilitating CD80/86-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) signaling. Treatment of human allogeneic dendritic cell/T-cell co-cultures with a human CD28 blocking antibody fragment (α-huCD28) significantly abrogated subsequent allospecific immune responses, seen by decreased T-cell proliferation and of type 1 cytokine (IFN-γ and IL-2) expression. Allo-tolerization persisted after discontinuation of CD28 blockade and secondary alloantigen stimulation, as confirmed by enhanced CTLA-4 and PD-1 immune checkpoint signaling. However, T-cells retained reactivity to pathogens, supported by clonotyping of neo-primed and cross-reactive T-cells specific for Candida albicans or third-party antigens using deep sequencing analysis. In an MHC-mismatched murine model, we tolerized C57BL/6 T-cells by ex vivo exposure to a murine single chain Fv specific for CD28 (α-muCD28). Infusion of these cells, after α-muCD28 washout, into bone marrow-transplanted BALB/c mice caused allo-tolerance and did not induce GvHD-associated hepatic pathology. We conclude that selective CD28 blockade ex vivo can allow the generation of stably allo-tolerized T-cells that in turn do not induce graft-versus-host reactions while maintaining pathogen reactivity. Hence, CD28 co-stimulation blockade of donor T-cells may be a useful therapeutic approach to support the immune system after HSCT.

Selective CD28 Blockade Results in Superior Inhibition of Donor-Specific T Follicular Helper Cell and Antibody Responses Relative to CTLA4-Ig.

Donor-specific antibodies (DSAs) are a barrier to improved long-term outcomes after kidney transplantation. Costimulation blockade with CTLA4-Ig has shown promise as a potential therapeutic strategy to control DSAs. T follicular helper (Tfh) cells, a subset of CD4+ T cells required for optimal antibody production, are reliant on the CD28 costimulatory pathway. We have previously shown that selective CD28 blockade leads to superior allograft survival through improved control of CD8+ T cells relative to CTLA4-Ig, but the impact of CD28-specific blockade on CD4+ Tfh cells is unknown. Thus, we identified and characterized donor-reactive Tfh cells in a murine skin transplant model and then used this model to evaluate the impact of selective CD28 blockade with an anti-CD28 domain antibody (dAb) on the donor-specific Tfh cell-mediated immune response. We observed that the anti-CD28 dAb led to superior inhibition of donor-reactive CXCR5+ PD-1high Tfh cells, CD95+ GL7+ germinal center B cells and DSA formation compared with CTLA4-Ig. Interestingly, donor-reactive Tfh cells differentially upregulated CTLA4 expression, suggesting an important role for CTLA4 in mediating the superior inhibition observed with the anti-CD28 dAb. Therefore, selective CD28 blockade as a novel approach to control Tfh cell responses and prevent DSA after kidney transplantation warrants further study.

Prevention of lupus nephritis development in NZB/NZW mice by selective blockade of CD28.

Systemic lupus erythematosus (SLE) is a chronic systemic inflammatory disease. Autoantibodies (autoAbs) against double-stranded DNA (ds DNA), the hallmark of lupus, are produced and maintained by the interaction between auto-reactive B cells and CD4+ T cells. This interplay is controlled by the CD28/CD80-86/CTLA-4 axis. Here we investigated whether selective blockade of CD28-CD80/86 co-stimulatory interactions abrogates lupus nephritis development in a murine model of SLE. To this aim, NZB/NZW F1 mice were treated for 3 months, either with an anti-CD28 Fab' fragment or a control Fab'-IgG. The effect of CD28 blockade on lupus nephritis onset, survival, production of anti-ds DNA antibodies and costimulatory molecules was evaluated. CD28 blockade prevented the development of lupus nephritis and prolonged survival during the 3-month treatment and 12 weeks after. Furthermore, the production of anti-ds DNA autoAbs was decreased. Lastly, the protective effect of CD28 blockade was associated with increased intrarenal expression of the immunoregulatory molecule, Indoleamine 2, 3-dioxygenase, of the co-inhibitory receptor programmed cell-Death - 1 (PD-1) and of its ligand programmed death ligand - 1 (PDL-1).In conclusion, CD28 blockade prevented the development of lupus nephritis in NZB/NZW F1 mice. This immunomodulatory strategy is a promising candidate for SLE therapy in humans.

Costimulation Blockade in Autoimmunity and Transplantation: The CD28 Pathway.

T cell activation is a complex process that requires multiple cell signaling pathways, including a primary recognition signal and additional costimulatory signals. TCR signaling in the absence of costimulatory signals can lead to an abortive attempt at activation and subsequent anergy. One of the best-characterized costimulatory pathways includes the Ig superfamily members CD28 and CTLA-4 and their ligands CD80 and CD86. The development of the fusion protein CTLA-4-Ig as an experimental and subsequent therapeutic tool is one of the major success stories in modern immunology. Abatacept and belatacept are clinically approved agents for the treatment of rheumatoid arthritis and renal transplantation, respectively. Future interventions may include selective CD28 blockade to block the costimulatory potential of CD28 while exploiting the coinhibitory effects of CTLA-4.

Anti-CD28 Antibody and Belatacept Exert Differential Effects on Mechanisms of Renal Allograft Rejection.

Belatacept is a biologic that targets CD80/86 and prevents its interaction with CD28 and its alternative ligand, cytotoxic T lymphocyte antigen 4 (CTLA-4). Clinical experience in kidney transplantation has revealed a high incidence of rejection with belatacept, especially with intensive regimens, suggesting that blocking CTLA-4 is deleterious. We performed a head to head assessment of FR104 (n=5), a selective pegylated Fab' antibody fragment antagonist of CD28 that does not block the CTLA-4 pathway, and belatacept (n=5) in kidney allotransplantation in baboons. The biologics were supplemented with an initial 1-month treatment with low-dose tacrolimus. In cases of acute rejection, animals also received steroids. In the belatacept group, four of five recipients developed severe, steroid-resistant acute cellular rejection, whereas FR104-treated animals did not. Assessment of regulatory T cell-specific demethylated region methylation status in 1-month biopsy samples revealed a nonsignificant trend for higher regulatory T cell frequencies in FR104-treated animals. Transcriptional analysis did not reveal significant differences in Th17 cytokines but did reveal higher levels of IL-21, the main cytokine secreted by CD4 T follicular helper (Tfh) cells, in belatacept-treated animals. In vitro, FR104 controlled the proliferative response of human preexisting Tfh cells more efficiently than belatacept. In mice, selective CD28 blockade also controlled Tfh memory cell responses to KLH stimulation more efficiently than CD80/86 blockade. Our data reveal that selective CD28 blockade and belatacept exert different effects on mechanisms of renal allograft rejection, particularly at the level of Tfh cell stimulation.

Selective CD28 Antagonist Blunts Memory Immune Responses and Promotes Long-Term Control of Skin Inflammation in Nonhuman Primates.

Novel therapies that specifically target activation and expansion of pathogenic immune cell subsets responsible for autoimmune attacks are needed to confer long-term remission. Pathogenic cells in autoimmunity include memory T lymphocytes that are long-lived and present rapid recall effector functions with reduced activation requirements. Whereas the CD28 costimulation pathway predominantly controls priming of naive T cells and hence generation of adaptive memory cells, the roles of CD28 costimulation on established memory T lymphocytes and the recall of memory responses remain controversial. In contrast to CD80/86 antagonists (CTLA4-Ig), selective CD28 antagonists blunt T cell costimulation while sparing CTLA-4 and PD-L1-dependent coinhibitory signals. Using a new selective CD28 antagonist, we showed that Ag-specific reactivation of human memory T lymphocytes was prevented. Selective CD28 blockade controlled both cellular and humoral memory recall in nonhuman primates and induced long-term Ag-specific unresponsiveness in a memory T cell-mediated inflammatory skin model. No modification of memory T lymphocytes subsets or numbers was observed in the periphery, and importantly no significant reactivation of quiescent viruses was noticed. These findings indicate that pathogenic memory T cell responses are controlled by both CD28 and CTLA-4/PD-L1 cosignals in vivo and that selectively targeting CD28 would help to promote remission of autoimmune diseases and control chronic inflammation.

Inhibition of Donor-Reactive CD8+ T Cell Responses by Selective CD28 Blockade Is Independent of Reduced ICOS Expression.

Programmed T cell differentiation is critically influenced by the complement of costimulatory and coinhibitory signals transmitted during initial antigen encounter. We previously showed that selective CD28 blockade with novel domain antibodies that leave CTLA-4-mediated coinhibitory signaling intact resulted in more profound attenuation of donor-reactive T cell responses and improved graft survival in a murine transplant model. Selective CD28 blockade was also associated with decreased ICOS expression on donor-reactive CD8+ T cell responses as compared to CTLA-4 Ig, but the functional importance of this reduced ICOS expression was not known. In this study, we created retrogenic donor-reactive CD8+ T cells that overexpress ICOS in order to determine whether reduced ICOS expression mechanistically underlies the increased efficacy of selective CD28 blockade in controlling graft-specific T cell responses as compared to conventional costimulation blockade with CTLA-4 Ig. Results indicated that the ability of selective CD28 blockade to blunt donor-reactive CD8+ T cell expansion following transplantation was independent of its ability to inhibit ICOS expression. Furthermore, we have previously published that 2B4 coinhibitory signals are functionally important for controlling graft-specific CD8+ T cell responses in mice treated with CD28 blockade. Here we used a co-adoptive transfer approach to determine that 2B4 coinhibitory signals on antigen-specific CD8+ T cells function in a cell-intrinsic manner to limit ICOS expression in the setting of selective CD28 blockade.

Effect of selective CD28 blockade with FR104 alone or combined with CD154 blockade in monkey cardiac allograft transplantation. (TRAN2P.967)

Abstract Selective CD28 blockade, which preserves CTLA-4-dependent immune regulation, is a promising approach to induce tolerance. FR104, a novel CD28 antagonist, was evaluated in monkey heart transplant recipients that received αCD28 alone (n=3, FR104) or in combination with αCD154 (n=5, hu5C8), dosed to achieve receptor coverage for 84 days; results were compared to αCD154 alone (n=4). Acute (AR) and chronic (CAV) rejection scores were quantified histologically. Median graft survival time (MST) with FR104 (167d), hu5C8 (133d), and FR104+hu5C8 (173d) was not statistically different between groups. Rejection was seen in 1 of 3 animals with FR104 monotherapy despite CD28 receptor saturation, but not with additional CD154 blockade. CAV was decreased with FR104 (0.7±0.3) and FR104+hu5C8 (0.5±0.3) compared to hu5C8 (1.6±0.7 p<0.05). AR tended to be lower with FR104 (0.5±0.4, p=0.047) or combined therapy (0.8±0.4, p=0.07) on biopsy/explanted functional grafts relative to hu5C8 (1.4±0.6). Blood Tregs frequency on d7-21 was increased (fold change vs. baseline) with FR104 (1.5±0.2), hu5C8 (1.4±0.3), and FR104+hu5C8 (2.3±0.1). CD28 saturation became undetectable by d150 in blood and LN; subsequent graft failure was associated with alloantibodies. We conclude that selective non-activating CD28 blockade protects primate cardiac allografts from pathogenic alloimmunity, an effect enhanced by simultaneous CD154 blockade, supporting the hypothesis that CD28 blockade is potentially tolerogenic.

Selective Blockade of CD28-Mediated T Cell Costimulation Protects Rhesus Monkeys against Acute Fatal Experimental Autoimmune Encephalomyelitis

Costimulatory and coinhibitory receptor-ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28-CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4-CD80/86 interactions and the function of regulatory T (Treg) cells, abrogates the induction of experimental autoimmune encephalomyelitis (EAE) in rhesus monkeys. EAE was induced by intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA on day 0. FR104 is a monovalent, PEGylated-humanized Fab' Ab fragment against human CD28, cross-reactive with rhesus monkey CD28. FR104 or placebo was administered on days 0, 7, 14, and 21. FR104 levels remained high until the end of the study (day 42). Placebo-treated animals all developed clinical EAE between days 12 and 27. FR104-treated animals did not develop clinical EAE and were sacrificed at the end of the study resulting in a significantly prolonged survival. FR104 treatment diminished T and B cell responses against rhMOG, significantly reduced CNS inflammation and prevented demyelination. The inflammatory profile in the cerebrospinal fluid and brain material was also strongly reduced. Recrudescence of latent virus was investigated in blood, spleen, and brain. No differences between groups were observed for the β-herpesvirus CMV and the polyomaviruses SV40 and SA12. Cross-sectional measurement of lymphocryptovirus, the rhesus monkey EBV, demonstrated elevated levels in the blood of FR104-treated animals. Blocking rhesus monkey CD28 with FR104 mitigated autoreactive T and B cell activation and prevented CNS pathology in the rhMOG/CFA EAE model in rhesus monkeys.

Preventing Primate GvHD Using a Novel Antagonistic Anti-CD28 Antibody Plus Rapamycin: Downregulation of CD8 Activation and Preservation of the Naïve Cell Phenotype Predicts GvHD-Free Survival

▪Introduction: We have previously shown that blocking CD28:CD80/86 costimulation with CTLA4-Ig can be a successful strategy to inhibit GVHD. However, since CTLA4-Ig targets CD80/86, there remain concerns of off-target, immune activating effects, given that it can inhibit both positive signaling through CD28 and negative signaling through CTLA4. In order to specifically target CD28, we have developed a humanized anti-CD28 monovalent Fab antibody (FR104) and have tested its ability to protect against GVHD.Methods: NHP underwent MHC-mismatched transplantation after myeloablative radiation-based pre-transplant conditioning. They were transplanted with GCSF-mobilized PBSCs (4.2x10^8 +/- 1.1x10^8 TNC/kg and 1.9 x 10^7 +/- 0.5 x 10^7 CD3+ T cells/kg. GVHD prophylaxis consisted of either monotherapy with FR104 (5mg/kg given weekly) or with combination FR104 and rapamycin (rapamycin trough 5-15 ng/ml). These were compared to three control groups: no GVHD prophylaxis, rapamycin monotherapy and autologous HSCT. Clinical and histologic GVHD was monitored using our NHP grading scale, and longitudinal immunologic analysis was performed. Treatment was continued as tolerated with planned terminal analysis at 35 days in all survivors.Results: Untreated controls (n = 5) developed rapid, severe multi-organ GVHD (MST=7 days). Rapamycin monotherapy (n=6) partially protected against GVHD (MST =14 days). Monotherapy with FR104 showed a significant prolongation in survival compared to untreated animals (MST = 21 days, p=0.02 n = 3), with breakthrough GVHD (liver, skin predominant) ultimately occurring. In contrast to all other groups, combination therapy with FR104 and rapamycin (n=4) resulted in striking protection from GVHD with all animals reaching planned terminal analysis with minimal clinical disease (p=0.01 vs. untreated controls, p=0.01 vs. rapamycin monotherapy, Fig. 1).We have documented a major role for both T cell proliferation (Ki-67) and Granzyme B (Gran-B) overexpression in GVHD, with untreated controls demonstrating rampant CD8+ proliferation (81% + 5.4% Ki-67+ at day 7 vs. 4% pre-transplant) and Gran-B overexpression (81% + 2.7% Gran-Bvery high at day 7 vs. 0.5% + 0.5% pre-transplant). Monotherapy with either rapamycin or FR104 partially controlled proliferation (53% + 23% and 30% + 21.7%, respectively) and Gran-B overexpression (21% + 8.7% and 11% + 5%, respectively) on day 7. Combination therapy with FR104 + rapamycin resulted in synergistic control of both proliferation and Gran-B overexpression (0.6% + 0.2% Ki67+ and 2.3% + 1.3% Gran-B overexpression on Day 7), reverting the CD8+ activation phenotype to that observed in auto-HSCT controls (5.6% + 2.1% Ki67+, 9% + 3.2% Gran-B overexpression, Fig. 2). This reversion to the auto-HSCT phenotype was maintained until the terminal analysis, and the control of Day 7 proliferation and Gran-B overexpression correlated closely with GVHD-free survival.The control of CD28-mediated T cell activation in FR104-treated recipients resulted in significant preservation of the naïve CD8+ T cell (CD8 Tn) phenotype in these animals. Thus, in untreated controls and rapamycin monotherapy groups, the CD8 Tn population virtually disappeared post-transplant (2.1% + 0.7% and 6.6% + 1.5% at day 7, respectively compared to 33.6%+ 4.1% pre-transplant), consistent with widespread allo-stimulation and costimulation. In contrast, FR104 monotherapy resulted in partial preservation of CD8 Tn (24.3% + 2.8% at Day 7) and FR104 + rapamycin combination therapy promoted nearly complete preservation of CD8 Tn (48.3% + 0.9% at day +7), with maintenance of this naïve population for the duration of therapy (Fig. 3).Discussion: These results show, for the first time, that specific blockade of CD28 can inhibit NHP GVHD, and when coupled with rapamycin it can effectively control both the clinical and immunologic sequelae of this disease. CD28 blockade with FR104 + rapamycin significantly inhibited both CD8+ T cell proliferation and Granzyme B overexpression, and lead to robust preservation of the naïve T cell phenotype, consistent with efficient inhibition of allo-activation. Our results suggest that targeting the CD28:CD80/86 costimulation axis by selective CD28 blockade may be effective in inhibiting the T cell activation associated with GVHD, and is deserving of clinical evaluation for safety and efficacy in patients undergoing HCT. [Display omitted] [Display omitted] [Display omitted] DisclosuresVanhove:Effimune: Research Funding.

Selective CD28 Blockade Exhibits Superior Suppression of Donor-Specific Antibody Relative to CTLA4Ig.

Costimulation blockade in the form of CTLA4Ig is known to attenuate donor-specific antibody responses. However, CTLA4Ig indiscriminately blocks both the CD28 costimulatory and CTLA4 co-inhibitory pathways. While follicular helper T (TFH) cell development and germinal center (GC) formation are CD28-dependent, CTLA4 has been shown to play a critical role in suppressing the provision of T cell help for GC B cell function and immunoglobulin production. Thus, selective CD28 blockade has potential to inhibit GC development while garnering the inhibitory effects of intact CTLA4 signaling on T cell-dependent antibody responses. To test this possibility, we used a T cell receptor transgenic murine skin allograft model to compare a novel CD28-specific domain antibody (dAb) to CTLA4Ig. The selective CD28 dAb resulted in superior skin allograft survival in comparison to CTLA4Ig (MST >100d vs. 34d). Importantly, animals treated with the anti-CD28 dAb experienced a greater reduction in donor-specific IgG as compared to those treated with CTLA4Ig. Flow cytometric analysis of donor-reactive CD4+Thy1.1+ T cells from draining lymph nodes revealed increased expression of CTLA4 on TFH cells (CXCR5+GL7+/-) compared to naïve and non-TFH cells (CXCR5-GL7-), with the greatest level of CTLA4 expression on maximally polarized GC TFH cells (CXCR5+GL7+). Analysis of donor-specific CD4+Thy1.1+ T cells also revealed a greater reduction in the frequency and absolute number of TFH cells (CXCR5+GL7+/-) with selective CD28 blockade as compared to CTLA4Ig. Additionally, we observed a reciprocal increase in the frequency and absolute number of naïve and non-TFH cells (CXCR5-GL7-) that was greatest with CD28 dAb treatment. These findings suggest that selective CD28 blockade in the presence of intact CTLA4 signaling has a greater inhibitory effect on T cell dependent antibody responses than CTLA4Ig. DISCLOSURE:Suchard, S.: Employee, Bristol-Myers Squibb. Nadler, S.: Employee, Bristol-Myers Squibb.

Selective Blockade of CD28/B7/CTLA4 Pathway With Monovalent Anti-CD28 Versus Targeting of B7 With Belatacept, in Kidney Allograft in Non-Human Primate, After CNI Weaning.

Targeting the CD28/B7/CTLA-4 costimulation pathway with the fusion protein CTLA4-Ig (Belatacept) in association with mycofenolate mofetil (MMF), is a less toxic alternative to calcineurin inhibitors (CNI). However, B7-specific blocking strategies inhibit CTLA-4 signals crucial to the function of regulatory T cells (Tregs). We previously showed in a model of kidney allograft in non-human primate (NHP) that a monovalent antibody fragment against CD28, which selectively blocks CD28 without affecting CTLA4, increased intragraft and peripheral blood Tregs and delayed acute rejection as single therapy and synergized with CNI to prevent efficiently acute rejection. In the current study, we performed a comparison of FR104, a monovalent pegylated Fab'anti-CD28 antagonist antibody with B7 blockade in kidney allograft in the baboon. Biologics were used de novo together with an initial 1 month treatment of low dose tacrolimus, weaned thereafter between months 1 and 2. After 2 months, therefore, recipients were under monotherapy with their biologics. In case of acute rejection, animals were treated with bolus of steroids. In the Belatacept group (n=3), two recipients developed severe acute rejection before or during CNI weaning (day 15 and 43) and bolus of steroid were ineffective. Histological examination confirmed rejection (Banff classification IIB: i3t2v2 and i3t3v2). The third recipient was lost on day 70 for pyelonephritis. In FR104-treated baboons (n=2), one animal developed an acute rejection episode during the CNI weaning period, but steroids bolus was able to reverse this episode. These two first FR104-treated recipients still have a stable kidney function 6 and 8 months post-transplantation. Interestingly, on the 1 and 3-months protocol biopsies, both FR104-treated recipients displayed an interstitial infiltration. Immuno-phenotyping analyses, emphasizing on peripheral Treg count (CD4+CD127lowCD25+) by flow cytometry, did not reveal any difference between the two biologics. These results suggest that the selective blockade of CD28 was efficient to allow reducing immunosuppression and maintaining recipients under this single therapy. Additional experiments are necessary to confirm the data and to further understand the mechanisms of action, in particular Tregs quantification in the biopsy. DISCLOSURES:Poirier, N.: Employee, Effimune SAS. Vanhove, B.: Stockholder, Effimune SAS.

2B4 (CD244) Induced By Selective CD28 Blockade Functionally Regulates Allograft-Specific CD8+ T Cell Responses.

Mounting evidence in models of both autoimmunity and chronic viral infection suggests that the outcome of T cell activation is critically impacted by the constellation of costimulatory and coinhibitory receptors expressed on the cells' surface. Here, we identified a critical role for the coinhibitory SLAM family member 2B4 (CD244) in attenuating primary donor-reactive CD8+ T cell responses in the presence of immune modulation with selective CD28 blockade. Our results reveal a specific upregulation of 2B4 on donor-reactive CD8+ T cells in animals in which CD28 signaling was blocked. However, 2B4 upregulation was not observed in animals treated with CTLA-4 Ig (abatacept) or CD28 blockade in the presence of anti-CTLA-4 mAb, indicating that CTLA-4 coinhibitory signals are required for 2B4 upregulation. Skin graft experiments revealed a dramatic prolongation in survival in animals treated with CD28 dAbs, in which CTLA-4 coinhibitory signaling is maintained and 2B4 coinhibitory signaling is induced, as compared to CTLA-4 Ig in both major and minor histocompatibility models. Interestingly, the increased efficacy of CD28 dAbs in attenuating donor-reactive CD8+ T cell responses as compared to CTLA-4 Ig was not an effect of preserved PD-L1/CD80 coinhibitory signals, as blockade of PD-L1 did not abrogate the ability of CD28 dAb to inhibit donor-reactive CD8+ T cell responses or induce 2B4 upregulation. In order to directly assess the functional role of the 2B4 coinhibitory pathway in prolonging allograft survival, we assessed donor-reactive CD8+ T cell responses in 2B4-/- mice. 2B4 upregulation following CD28 blockade was functionally significant, as the inhibitory impact of CD28 blockade was diminished when donor-reactive CD8+ T cells were deficient in 2B4. In contrast, 2B4 deficiency had no effect on CD8+ T cell responses during unmodified rejection or in the presence of CTLA-4 Ig. We conclude that blockade of CD28 signals in the presence of preserved CTLA-4 signals results in the unique upregulation of 2B4 on primary CD8+ effectors, and that this 2B4 expression plays a critical functional role in controlling donor-reactive CD8+ T cell responses during transplantation. DISCLOSURES:Schneeweis, L.: Employee, Bristol Myers-Squibb. Stetsko, D.: Employee, Bristol Myers-Squibb. Suchard, S.: Employee, Bristol Myers-Squibb. Nadler, S.: Employee, Bristol Myers-Squibb.