ALBERT

Description: INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (alloHSCT) provides the best chance for disease control in acute myeloid leukemia (AML). The known anti-leukemia benefit of alloHSCT is attributable to pre-transplant conditioning with chemotherapy and/or radiation and the graft-versus-leukemia (GvL) effect. However, the utility of alloHSCT is limited by significant treatment-related morbidity and mortality resulting from conditioning regimen-related toxicities and graft versus host disease (GvHD). AML is primarily diagnosed in elderly patients, for whom alloHSCT may be contraindicated due to an inability to tolerate these adverse effects. Therefore, an unmet clinical need exists for novel alloHSCT conditioning regimens that minimize toxicity without sacrificing therapeutic efficacy. Previously, we and others (Palchaudhuri et al. (2016), Nat Biotech; Persaud et al. (2018), Blood) have shown that antibody-drug conjugates (ADCs) composed of a CD45.2-specific antibody linked to the ribosome-inactivating protein saporin (CD45-SAP) permitted stable, high-level engraftment in murine syngeneic HSCT. We hypothesize that such targeted, immunotherapeutic strategies can be applied to the setting of alloHSCT, enhancing its safety and applicability to the treatment of hematologic diseases. METHODS: For engraftment studies, recipient mice were conditioned with 3 mg/kg CD45-SAP 7 days prior to infusion of 10-20 x 106 whole bone marrow cells. The following alloHSCT models were used: MHC-matched (BALB/c→DBA/2), F1-to-parent (CB6F1→B6), and F1-to-F1 (B6CBAF1→CB6F1). In some experiments, CD45-SAP treatment was combined with CD4+ and CD8+ T cell depletion and/or the Janus kinase (JAK) inhibitor, baricitinib. For GvHD studies, a parent-to-F1 (B6→CB6F1) model was used in which recipients were infused with 25 x 106 donor splenocytes after CD45-SAP treatment or sublethal irradiation. For studies with human CD45-SAP, MOLM13 AML cell line viability was measured with XTT assays, and cord-blood CD34+cell colony formation was assessed using methylcellulose assays. RESULTS: CD45-SAP combined with CD4+ and CD8+ T cell depletion allowed high-level engraftment in MHC-matched and F1-to-parent alloHSCT models (Figure 1A-B). Baricitinib, previously shown by our lab to prevent GvHD while preserving the GvL effect (Choi et al. (2018), Leukemia), permitted short-term HSC engraftment in F1-to-parent alloHSCT when mice were dosed daily with the drug during the first 21 days post-HSCT (Figure 1C). Graft stability in baricitinib-treated mice was improved by either combining daily baricitinib treatment with pre-transplant T cell depletion (Figure 1D), or by delivering baricitinib via continuous infusion (Figure 1E). In parent-to-F1 GvHD models, CD45-SAP conditioned mice showed minimal morbidity or mortality when infused with allogeneic splenocytes, whereas mice conditioned with sublethal irradiation succumbed within 3 weeks to severe GvHD (Figure 1F). That alloreactive T cells did not cause GvHD in CD45-SAP treated mice prompted us to use donor lymphocyte infusions to enhance engraftment in a fully haploidentical (F1-to-F1) HSCT model. Our results suggest that addition of donor T cells to the bone marrow graft promotes donor engraftment without eliciting clinically apparent GvHD (Figure 1G). Finally, anti-human CD45 (clone BC8), when conjugated to saporin, inhibited MOLM13 cell growth and cord blood CD34+ cell colony formation in vitro (Figure 1H-I), consistent with both an anti-stem cell and anti-leukemic effect. CONCLUSIONS: Combining CD45-SAP with T cell depletion and/or pharmacologic immunomodulation with the JAK inhibitor baricitinib effectively conditions mice for engraftment of MHC-matched and haploidentical HSCs without causing significant toxicity. Strikingly, mice conditioned with CD45-SAP, unlike mice conditioned with irradiation, did not develop GvHD when infused with allogeneic splenocytes. Finally, we have demonstrated that human CD45-SAP is cytotoxic to both the MOLM13 AML cell line and cord-blood derived stem cells. Taken together, these studies provide key proof-of-principle evidence that targeted immunotherapeutics (CD45-SAP plus JAK inhibitors) can accomplish the goals of alloHSCT - creating marrow space for donor HSCs, overcoming immune barriers to engraftment, and eliminating leukemia cells - with limited toxicity or GvHD. Disclosures Cooper: Wugen: Consultancy, Equity Ownership, Patents & Royalties. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. DiPersio:Incyte: Consultancy, Research Funding; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Macrogenics: Research Funding, Speakers Bureau; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Magenta Therapeutics: Equity Ownership; Karyopharm Therapeutics: Consultancy; Bioline Rx: Research Funding, Speakers Bureau.

Description: Background: Maintaining T cell function and survival after ex vivo manipulation remains a major challenge in adoptive immunotherapy. We previously demonstrated that murine T cells activated and expanded ex-vivo with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads) exhibit reduced GVHD-inducing potential compared to naïve (unmanipulated) T cells in a murine allogeneic BMT model. Blood diagnostics are commonly used to help identify patterns of biomarkers in multiple disease states. Objective: Determine if plasma profiles of 59 different analytes are altered in murine allogeneic BMT recipients who receive ex-vivo activated, suicide gene transduced and selected (Td) T cells compared with animals that received naïve suicide gene expressing T cells. Methods: Murine T cells were Td with a chimeric CD34-thymidine kinase (CD34-TK) fusion suicide gene. High efficiency (70%) gene transfer of CD34-TK to C57BL/6 (B6) murine T cells was accomplished 24 h after CD3/CD28 bead activation and gene-modified cells were purified to 〉 96% by CD34 immunomagnetic selection 2 days post-infection. Naïve B6 CD34-TK-expressing T cells were purified from the spleens of CD34-TK transgenic mice the day of BMT. To induce GVHD, lethally irradiated BALB/c allogeneic recipients were given T cell depleted (TCD) B6 BM supplemented with or without B6 CD34-TK purified naïve or Td T cells. Animals were bled 1 week after BMT and EDTA plasma samples were prepared and frozen. Quantitative measurements of 59 analytes were obtained using a rodent multi-analyte profile test (MAP test; Charles River Lab) on the plasma of 2 mice that received naïve T cells and died from GVHD on days 16 and 21 after BMT, 2 mice that received Td T cells and died from GVHD on day 34 after BMT, and 2 mice that received TCD BM only and survived 〉 100 days. Results: As before, we found that ex vivo activation of the donor T cells before BMT significantly prolonged the survival of mice transplanted with the Td T cells compared with mice receiving naïve T cells (p = 0.0028). Eighteen analytes, including IFN-γ, IL-2, IL-3, IL-4, IL-7, IL-11, and TNF-α were below the detection limits of the rodent MAP test for all samples analyzed. Twenty-six analytes, including VEGF, SCF, MIP-2, MIP-1α, MIP-3β, MCP-5, IL-1β, IL-18 and GM-CSF were detectable but not significantly different than the BM only controls for either the naïve or Td T cell groups. Nine analytes, including eotaxin, MIP-1γ, MCP-3, MCP-1, and IL-10 were similarly increased 2- to 3-fold in both the Td and naïve T cell groups compared to the BM only control. Interestingly, recipients of naive T cells exhibited increased plasma levels of growth hormone (≥14-fold), MIP-1β (4.6-fold), IP-10 (2.3-fold), and lymphotactin (2-fold), as well as decreased levels of leptin (≥14-fold), compared to both the BM only and Td T cell groups. This extreme modulation of growth hormone and leptin levels is particularly interesting given the fact that leptin influences local growth hormone secretion from lymphocytes and that both of these analytes have multiple biologic effects on T cells. Finally, only a single analyte, monocyte derived chemokine (MDC), was increased (4-fold) in mice that received Td T cells compared to the naïve T cell recipients. Conclusion: These results indicate that several plasma analytes with important immunological functions are altered when donor T cells are manipulated ex-vivo. These alterations may account for the decreased GVHD-inducing potential of ex vivo manipulated T cells after allogeneic BMT.

Description: Background: Mobilization of hematopoietic stem cells (HSCs) in mice and man can be induced using G-CSF (G) or by the use of chemokines and chemokine receptor antagonists. Recent preclinical and clinical data using the bicyclam AMD3100 (A) suggests that the combination of G+A results in significantly improved yields of HSCs compared to G alone in both mice and man (Blood.2005;106:1867). In humans, optimal mobilization of HSCs occurs 6–9 h after subcutaneous (s.c.) A. These mobilization kinetics necessitate the somewhat undesirable administration of A the evening before the first day of apheresis. Methods: We assessed the kinetics of HSC mobilization in mice and man after IV or s.c. administration of A. Human donors were initially mobilized with increasing doses of IV A (80, 160, 240 or 320 μg/kg). After 4 days of drug clearance, the same donors were then mobilized with a single s.c. dose of A (240 μg/kg) and collected cells were used as a source of HSCs for HLA-matched sibling recipients undergoing single dose (550 cGy) TBI and cytoxan allogeneic HSC transplantation (Devine et. al. Blood. 2006;108:53). Results: Peak mobilization of mouse CFU-GM occurred 3 h after s.c A (5 mg/kg; 15–18-fold; n=5) and 0.5–1 h after IV A (1–3 μg/kg; 10–12-fold; n=3). Identical CFU-GM kinetics were observed after mice were first given G (250 μg/kg) for 4 days then administered either s.c. A (250–500 fold in 3 h; n=3) or IV A (170–350 fold n=3). To date, 6 normal HLA-matched sib donors have been treated with 80 μg/kg (n=3) or 160 μg/kg (n=3) IV A over 30 minutes followed 4 days later by 240 μg/kg s.c. A. Peak mobilization of CD34+ cells occurred 1–4 h after 80 μg/kg IV A, 4–6 h after 160 μg/kg IV A, and 9 h after s.c. A. The more rapid kinetics of CD34 mobilization after IV A is in contrast to the kinetics of s.c. A in autologous transplant patients receiving both G and A (J Clin. Oncol.2004;22:1095) and in normal volunteers receiving A alone (∼9 hours; Blood. 2003;102:2728). This data suggests that IV A not only mobilizes HSCs from mouse and man more quickly than s.c. A, but also, that the peak of mobilization may be enhanced (6.4-fold at 160 uμg/kg IV vs. 5.2-fold for 240 μg/kg s.c.) and prolonged (5.7 fold at 160 μg/kg IV vs. 3.8 fold for 240 μg/kg s.c. at 6 h post-A) after IV A. IV A also induced: (i.) a more rapid and greater peak of CD19+ B cell mobilization (6-fold at 1 h and 6.7-fold at 4 h) than s.c. A (2.2-fold at 6 h), (ii.) a modest increase in CD4+ and CD8+ CD3+ T cell mobilization (3.2-fold vs. 1.1-fold for sc A at 6h) and (iii.) no significant changes in Treg, NK, CMV-specific CD8, and γδ T cells. To identify genes that are differentially expressed following G or A mobilization, we performed RNA profiling analyses using Affymatrix U133+2 arrays and RNA isolated from purified (〉95%) CD34+ HSCs obtained from 5 individual normal donors mobilized sequentially with A and G. Of note, CXCR4, CCR9, ALCAM, ICAM, and CEECAM1 were expressed more abundantly in all A mobilized CD34+ cells while mucin, Integrin α 6, Integrin α 2b, and CEACAM1 were more abundantly expressed in all G-mobilized CD34+ cells. Conclusions: IV A results in more rapid and prolonged mobilization of mouse HSC and human CD34+ cells compared to s.c. A. IV A was not associated with any adverse events in the 6 donors mobilized at the first two IV dose levels. The enhanced and more rapid mobilization of CD34+ cells by IV A may have significant applications for the optimal collection of peripheral blood HSC products.

Description: Herpes simplex virus thymidine kinase (TK) gene-modified T cells are currently being evaluated in gene therapy clinical trials for the control of graft-versus-host disease (GVHD) after allogeneic BMT. Unfortunately, these trials have been limited by a consistent failure of the ex-vivo manipulated T cells to survive and function properly in vivo. We recently developed a technique for retrovirally transducing and selecting murine T cells with a novel chimeric CD34-TK fusion suicide gene that preserves their alloreactivity after allogeneic BMT. In this study, we assessed the trafficking, survival, and GVHD-inducing potential of ex vivo manipulated murine T-cells in fully allogeneic transplant recipients by in vivo bioluminescence imaging (BLI) with two novel reporter vectors. The first vector encodes a fusion protein comprised of click beetle red (CBR) luciferase and EGFP (CBR/EGFP). In the second vector, we inserted a click beetle green (CBG) luciferase between CD34 and TK in our chimeric suicide gene (CD34/CBG/TK). Murine T cells, stimulated 24 h with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads), were transduced with Phoenix-Eco-derived CBR/EGFP or CD34/CBG/TK retrovirus and purified to 〉85% using a MoFlo cell sorter or CD34 immunomagnetic selection 48 h post-infection. To induce GVHD, lethally irradiated BALB/c allogeneic recipients were given T cell depleted C57BL/6 (B6) bone marrow supplemented with either 1e6 CBR/EGFP or CD34/CBG/TK purified B6 T cells. The CBR/EGFP BLI signal was significantly increased over background at 24 h post-injection, with the allogeneic T cells localizing primarily to the spleen and secondary lymph nodes. Over the next 2–3 days the CBR/EGFP+ cells migrated to the entire intestinal area followed rapidly by infiltration of the skin. Overall, the CBR/EGFP BLI signal increased nearly 3 orders of magnitude between days 1 and 8 post-BMT, remained steady for a week, and then only gradually declined over the next month (only a 3-fold decrease between days 14 to 42 post-BMT). Consistent with GVHD, these mice lost 〉20% of their pretransplant body weight and exhibited impaired lymphoid reconstitution. We observed similar trafficking and GVHD-inducing potential when CD34/CBG/TK gene-modified T cells were injected into BALB/c recipients. However, the maximum BLI signal intensity from the CD34/CBG/TK T cells was decreased nearly 2 orders of magnitude compared to the CBR/EGFP-modified T cells. Nevertheless, we were still able to demonstrate a significant reduction in BLI signal intensity when recipients of CD34/CBG/TK-modified allogeneic T cells were treated with ganciclovir (GCV) from days 1 to 7 post-BMT. This observation is consistent with in vitro cell sensitivity assays, which demonstrated that cells modified with the CD34/CBG/TK reporter gene retain TK activity similar to CD34-TK modified cells. In summary, this study demonstrated by in vivo BLI that allogeneic murine T cells activated and expanded ex vivo with CD3/CD28 beads retain significant GVHD-inducing potential and can be eliminated by HSV-TK/GCV suicide gene therapy.

Description: Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and interact with stroma cells and extracellular matrix. CXCR4/SDF-1 axis regulates the trafficking of HSC to and from the BM. We utilized a PML-RARα knock-in mouse model of human acute promyelocytic leukemia (APL) to study APL interaction with the normal BM. We have previously shown there is a rapid mobilization of APL cells from the BM into peripheral blood (PB) after administration of AMD3100, a competitive inhibitor of CXCR4. We hypothesize that we can sensitize these tumor cells to chemotherapy by interrupting the interaction between APL and the BM stroma. We transduced banked APL cells with a dual function reporter gene that encodes a fusion protein comprised of Click Beetle Red luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (Luc/EGFP). Upon iv injection into genetically compatible recipients (F1 129/B6 mice), APL rapidly migrated to the BM with increased BLI signal in the femurs, spine, ribs, and skull, at 4 days after injection, followed by spleen infiltration and death due to leukostasis by day 15. 129/B6 F1 mice (n=28) were injected iv with 106 APL cells. By day 12 all mice had ±5% APL cells in PB. 8 mice received AraC (500mg/kg/sq) on days 12 and 13, and another 8 mice received AraC+AMD (5mg/kg/sq) 1 hour before and 3 hours after each AraC injection. 6 mice received only AMD and 6 control mice were observed. Total body BLI signal, WBC, and blasts per μl of blood on days 19 and 23 were higher in AraC versus AraC+AMD (p

Description: Maintaining T cell function after ex vivo manipulation remains a major challenge in adoptive immunotherapy. We previously showed that murine T cells activated ex vivo with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads) retain greater GVHD-inducing potential than cells activated with either soluble anti-CD3 antibody alone or plate bound anti-CD3 and anti-CD28 antibodies after allogeneic BMT. However, CD3/CD28 bead activated T cells still exhibit reduced GVHD-inducing potential compared to naïve T cells. In this study, we used CFSE and congenic mice to monitor the proliferative kinetics of naïve and CD3/CD28 bead activated, transduced, and selected T cells in the same mouse after allogeneic and syngeneic BMT. High efficiency (〉50%) gene transfer of our chimeric suicide gene, CD34/TK, into C57BL/6 (B6) murine T cells was achieved 24 h after CD3/CD28 bead activation and gene-modified cells were purified to 〉98% by CD34 immunomagnetic selection 48 h later. CD34/TK+ T cells were then rested for 3 days in medium containing 10 U/mL IL-2. Purified CD34/TK+ (CD45.1+) and naïve (CD45.2+) T cells from B6 mice were then labeled with CFSE, mixed 1/1 (3e6 total T cells), and injected, along with T cell depleted B6 (CD45.1+) BM, into lethally irradiated allogeneic (BALB/c, CD45.2+) or syngeneic (B6, CD45.2+) recipients. Mice were sacrificed daily up to 6 days after BMT to assess donor T cell engraftment, division, and phenotype by five color flow cytometry. The CD34/TK+ donor T cells (both CD4+ and CD8+ T cells) underwent 1–2 rounds of division within 24 h after infusion. In contrast,

Description: GVHD is the major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. We developed a consistent and sensitive model of human GVHD by retroorbitally (ro) injecting huT cells into sublethally irradiated NOD/SCID-β2mnull mice (β2). Using this model, 59% (20/34) mice conditioned with 250cGy and injected ro with 107 huT cells developed lethal XGVHD and exhibited huT cell engraftment, weight loss, and infiltration in multiple target tissues. β2 mice injected with huT through the lateral tail vein (iv) had only small and transient huT cell engraftment and failed to develop lethal XGVHD. To evaluate why the ro versus iv route affected huT cell engraftment and the development of lethal XGVHD, we performed in vivo BLI and sequential [18F]FHBG microPET imaging studies. We used huT cells transduced with a click beetle red luciferase/enhanced green fluorescent protein (CBRluc/egfp) BLI reporter gene (huT*) and/or a chimeric CD34-thymidine kinase fusion suicide/PET gene. We injected 107 huT* cells either iv or ro into 250cGy irradiated β2 mice. Mice injected iv (n=5) failed to develop lethal XGVHD, whereas 60% of the mice injected ro (n=5) developed lethal XGVHD. During the first week, mice injected ro presented early expansion of the huT cells in the retroorbital cavity and local regional draining lymph nodes followed by widespread dissemination throughout the whole body, with increased percentage of huT in the blood (p

Description: Nonmyeloablative conditioning regimens followed by allogeneic hematopoietic cell transplantation (HSCT) rely on pre and post transplant immunosuppression to overcome host-versus-graft reactions and control graft-versus-host-disease (GVHD). Patients with less than 50% of donor T cells by day 14 after transplant have a higher risk of graft rejection and relapse than those with more than 50% donor chimerism (Blood2004; 104(8):2254). However, with increasing donor T-cell chimerism the risk of acute GVHD increases. Immunosuppression may inhibit the beneficial graft-versus-infection and graft-versus-leukemia effects mediated by mature donor T cells. Genetically modifying T cells with a drug resistance gene is one way to confer protection from the effects of the immunosuppressive agents, retain donor T cell function, and increase donor T cell chimerism by day 14. Mycophenolate mofetil (MMF) has been used in the post transplant setting to decrease the severity of GVHD and reduce the risk of host vs. graft reaction leading to graft failure. The active metabolite of MMF, mycophenolic acid (MPA), inhibits the enzyme inosine monophosphate dehydrogenase (IMPDH). The type II isoform of IMPDH is the rate-limiting enzyme in de novo guanosine synthesis and is preferentially expressed in activated T and B cells which lack the salvage pathway for guanosine synthesis. A number of investigators have identified IMPDH II mutants that have altered MPA binding capacity and normal guanosine synthetic activity ex vivo (BBA2002;1594:27, JBC.1997;272:961, BBA.1994;1217:156). We have generated six mutants of IMPDH II based on in vitro data and x-ray crystallography of the MPA binding site and transduced them into a human T cell line (Jurkat), a canine large granular lymphocyte cell line (CLGL-90) and a fibroblast cell line (NIH3T3) using oncoretroviral transduction. The mutants include: S276A (Amut), S276F (Fmut), T333I + S351Y (IYmut), L30F + Q277R (FRmut), Q277R + A462T (RTmut), and S276Y (Ymut). We generated bicistronic (IRES containing) retroviral vectors that expressed either HA-tagged wild type human IMPDH II (WT) or an HA-tagged IMPDH mutant and EGFP. We show that forced expression of WT will increase the MPA IC50 3 fold (IC50=3uM) compared to non-transduced cells. Forced expression of only the IY mutant in all cell lines tested was able to transfer resistance to MPA in excess of WT (IC50〉50uM). This is clinically relevant since MPA concentrations measured after transplant in patients on MMF are around 8uM (Blood.2005;106(13):4381). To show that resistance was related to the expression of the mutant enzyme we correlated GFP by FACS with HA-IMPDH II expression by western blot analysis, and correlated this with resistance to MPA in vitro. This was true regardless of selection in MPA or transduction and cell sorting for 〉90% GFP positive cells. Analysis of IMPDH activity in lysates of transduced cells is currently underway. This is the first report where forced expression only of the IY mutant provides a clear enhanced resistance to MPA in vitro compared to cells over expressing wild type IMPDH II. Currently, we are generating bicistronic maloney based oncoretroviral vectors which carry a suicide gene (CD34-TK) and the IMPDH-HA IY mutant which will allow us to pharmacologically modulate the level of donor T cells after allogeneic stem cell transplantation.

Description: Background: Hematopoietic stem cell (HSC) transplant is an essential treatment for a variety of blood disorders and malignancies. A key step in this procedure is the mobilization of donor stem cells. The most commonly used regimen for donor mobilization is a 5-day course of G-CSF. The length of this regimen coupled with the associated side effects emphasizes a need for superior alternatives. In recent years, there has been a growing understanding of mechanisms governing stem cell retention within the bone marrow niche. This has led to the development of new mobilization drugs that specifically target these processes. Two examples of previously described drugs that target mechanisms of stem cell retention are Plerixafor (a CXCR4 inhibitor already in clinical use), and truncated Gro-Beta (tGroβ; a CXCR2 agonist). Another potential target for inducing mobilization is disruption of the interaction between the VLA-4 integrin and its ligand VCAM-1. In this study, we evaluate the efficacy of novel VLA-4 inhibitors (VLA4i) alone and in combination with Plerixafor and/or tGroβ for the purposes of hematopoietic stem cell mobilization. Methods: We synthesized over 15 novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. The 5 inhibitors determined to be most potent were then tested in vivo in DBA mice for their ability to mobilize HSCs alone and in combination with tGroβ and/or Plerixafor (n=5). HSC mobilization was measured in wild-type and splenectomized mice via flow cytometry to quantify the proportion of LSK (Lineage- Sca+ cKit+) cells as well as via Colony Forming Unit (CFU) assays. For competitive transplant, mobilized CD45.1+ BALB/c mouse blood (10 uL) was injected into lethally irradiated CD45.2+ BALB/c recipients alongside 2.5x105 CD45.2+ BALB/c bone marrow cells (n=10 / cohort). HSC engraftment was monitored monthly via flow cytometry for ratio of 45.1+ vs. 45.2+ cells in peripheral blood. Results: Firetagrast and BIO5192 are previously characterized VLA4i that have been administered to humans for indications unrelated to HSC mobilization. Our best VLA4i to date, LGB-2019, exhibited similar potency as BIO5192 in preventing the binding of sVCAM-1 to VLA-4 (IC50: 1.7nM) and was 〉200-fold more potent than firategrast. LGB-2019 showed increased aqueous solubility and mobilized 1.5-fold more murine LSK cells for a longer time period (peak HSC mobilization maintained for 4 hours) than BIO5192 when administered alone. Simultaneous injection of C57BL/6 mice with LGB-2019 (VLA4i), Plerixafor (CXCR4i) and tGro-β (CXCR2a) resulted in a synergistic increase in circulating CFUs (Fig. 1A; 9.8 x 103 CFUs/mL) and LSKs (Fig. 1B; 12.8 LSKs/uL) at 4 hours post-injection. In contrast, 5 days of G-CSF treatment mobilized approximately 3-fold and 8-fold less CFUs and LSKs, respectively (Fig. 1A-B). We saw no significant difference in mobilization for splenectomized vs. wildtype mice (23.4 x 103 CFUs/mL vs. 23.0 x 103 CFUs/mL) when mobilizing DBA/2 mice via VLA4i+CXCR4i+CXCR2a. Three months after competitive transplantation, blood obtained from BALB/c mice mobilized with the triple combination engrafted significantly better than blood obtained from mice treated with G-CSF or the dual combinations (Fig. 1C). Summary: New insights about the stem cell niche have allowed for the development of targeted drugs for the purposes of mobilization. Here, we show that a novel VLA-4 receptor inhibitor in combination with two other known mobilizers induces mobilization of hematopoietic stem and progenitor cells (CFU/LSK) at levels superior to the standard of care G-CSF and in a dramatically shortened time frame. Mouse transplant data also show superior engraftment in lethally irradiated recipients when using the triple cocktail regimen compared to the G-CSF mobilized graft. Secondary transplants are ongoing and will provide a more complete picture of primitive HSC mobilization and serial engraftment properties of the cells. Disclosures Rettig: WashU: Patents & Royalties: Patent Application 16/401,950. Karpova:WashU: Patents & Royalties: Patent Application 16/401,950. Ruminski:WahU: Patents & Royalties: Patent Application 16/401,950. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. DiPersio:Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Equity Ownership; Incyte: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Karyopharm Therapeutics: Consultancy; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; NeoImmune Tech: Research Funding.

Description: The therapeutic benefits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) are primarily derived from graft-versus-leukemia (GvL) that is mediated by mature T cells in donor grafts. Unfortunately, graft-versus-host disease (GvHD) results from immune reactions of these same donor T cells against host tissues and organs after allo-HSCT. Due to this strong association between GvHD and the beneficial GvL, our lab and others have been working on developing effective therapeutic strategies to selectively prevent and treat GvHD without abrogating GvL. Recently we have reported that pharmacologic inhibition of Janus kinases 1/2 (JAK1/JAK2) prevents GvHD by blocking IFNGR and IL6R signaling. Although baricitinib (BARI) and ruxolitinib (RUX) are both JAK1/JAK2 inhibitors with similar potency, BARI was found to be superior to RUX in prevention of GvHD in our mouse models of allo-HSCT. One of our proposed mechanistic pathways by which BARI prevents GvHD is via a robust increase in regulatory T cells (Tregs) which play a pivotal role in controlling GvHD. Since the proliferation and survival of Tregs are mediated through the IL2-JAK1/JAK3-STAT5 signaling pathway and that RUX is a more JAK3 inhibitor, we hypothesized that sparing JAK3 by BARI is critical for this increase of Tregs. To test this hypothesis, we administered BARI to allo-HSCT recipient mice with or without JAK3-specific inhibitor and monitored them for GvHD signs. The mice treated with BARI and iJAK3 had significantly lower Tregs and overall survival rates compared to the mice treated with BARI alone. This data suggests that BARI prevents GvHD by retaining JAK3, thereby increasing Tregs. We have also previously reported that BARI treats established GvHD. However, the mechanisms by which BARI reverses ongoing GvHD remain unclear. Consequently, we hypothesized that BARI not only modulates immune cell functions as we previously reported, but also promotes restoration of GvHD damaged tissues. To test our hypothesis, we delayed the administration of BARI until mice developed clinically apparent GvHD and treated them starting on days 10, 17, or 24 after allo-HSCT. Despite this latency, the day 10 group showed significantly better overall survival (p