ALBERT

Description: The etiology of graft-versus-host disease (GVHD) is rooted in the alloreactive response of donor T cells present in the hematopoietic graft resulting in the destruction of the patient's tissues, particularly the gastrointestinal tract. Gut GVHD affects up to 50% of patients, is a leading cause of death, and has overlapping features with inflammatory bowel disease (IBD). Increased gut permeability, alterations of the gut microbiome and intestinal stem cell niche damage predispose the gut to the local and systemic effects of GVHD, and is exacerbated by the inability of the gut to adequately regenerate. Severe shifts in metabolism and reduced oxygen (O2) availability in the inflamed gut are major underlying factors in the pathogenesis of IBD. Two related transcription factors, hypoxia-inducible factor-1 (HIF-1) and HIF-2, originally discovered as master regulators of the adaptive response to hypoxia, have been recently shown to be gut protective and promote mucosal healing in response to injury. Using a MHC mismatched B10.BR→B6 bone marrow transplant (BMT) model, we previously found that loss of intestinal epithelial (IE) HIF-1α or HIF-2α worsened survival compared to wild-type mice and exhibited increased GVHD-induced-histopathology. Thus, we hypothesized that HIF-1 and HIF-2 protects and repairs the gut from conditioning and GVHD-related damage. HIF-1 and HIF-2 are heterodimers consisting of an O2-labile HIF-1α or HIF-2α subunit respectively and a constitutively expressed HIF-1β subunit. The recent discovery that iron-dependent prolyl hydroxylase enzymes (PHD1-3) can trigger the O2-dependent proteasomal degradation of the HIF-1α subunit has led to the development of pan-PHD inhibitors (PHDi) that activate HIF-1 and HIF-2. PHDi such as dimethyloxallyl glycine (DMOG) and AKB-4924 have been shown to attenuate colitis and radiation-induced gut toxicity in animal models. We thus sought to determine whether PHDi could also ameliorate gut GVHD in the B10.BR→B6 BMT model. B6 mice were lethally irradiated (10Gy, split dose) and transplanted with 5x106 T-cell depleted bone marrow (BM) cells from B10.BR donors with 2x106 enriched T cells from spleens and lymph nodes (BM+T). B6 mice transplanted with only T cell depleted BM cells served as our non-GVHD control group. B6 mice were treated with AKB-4924 (AKB, 5 mg/kg) or vehicle (β-cyclodextrin) via intraperitoneal delivery, beginning 1 day prior to BMT and for 6 consecutive days post-BMT. Treatment with AKB prevented significant weight loss, compared to vehicle-treated mice, 7 days post-BMT (n=6, p

Description: ADCT-301, currently in Phase I clinical trial, is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human IL-2R-α (CD25) conjugated through a cleavable linker to a PBD dimer warhead with a drug-antibody ratio of 2.3. In vitro and ex vivo, ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and targeted cytotoxicity against a panel of human lymphoma cell lines. On release, PBD dimers bind in the DNA minor groove and exert their cytotoxic action via the formation of DNA interstrand cross-links. In vivo, ADCT-301 demonstrates dose-dependent antitumor activity against subcutaneous and disseminated lymphoma models. For example, in the Karpas 299 xenograft model, 10/10 tumor-free survivors are observed following a single dose of 0.5 mg/kg, whereas Adcetris® gives only a modest delay in mean tumor growth at 0.5 mg/kg, despite this tumor expressing three-fold higher target antigen levels for this drug. The current study aimed to further define the mechanism of action of ADCT-301 and validate pharmacodynamic assays for clinical development. In Karpas 299 cells, evidence for internalization of ADCT-301 was shown by a reduction of CD25 molecules on the cell surface over the first three hours post-treatment followed by a return to pre-treatment levels by 16 hours. This is consistent with the documented rapid recycling of CD25 to the membrane after exposure to IL-2 (Hemar et al Journal of Cell Biology 1995). Furthermore, ADCT-301 on the cell surface declined by 〉70% over four hours. Following a two-hour exposure to ADCT-301, DNA interstrand cross-linking, measured using a modification of the single cell gel electrophoresis (comet) assay, reached a peak between 4 and 8 hours after which cross-links persisted up to 36 hours. In contrast, the peak of cross-link formation for an equimolar concentration of warhead was immediately following drug exposure and a non-targeted PBD-containing ADC did not produce crosslinks in these cells. A strong correlation (r = 0.97) between loss of viability and DNA cross-link formation provides support for this DNA damage being the critical initiating mechanism of cytotoxicity of ADCT-301. We have previously shown that PBD-induced DNA interstrand cross-links elicit a robust, but delayed γ-H2AX response (Wu et al Clinical Cancer Research 2013). In Karpas 299 cells phosphorylation of H2AX was observed 24 hours after a two-hour exposure to sub-GI50 concentrations of ADCT-301. In these cells continuous exposure to ADCT-301 resulted in a dose-dependent G2/M arrest, peaking at 48 hours, later than for the naked warhead. The peak of the early apoptosis marker annexin-V on the cell surface of Karpas 299 cells was observed between 60 and 72 hours and maximal loss of viability was at 96 hours. Significant bystander killing of CD25-negative human Burkitt's lymphoma-derived Ramos cells was demonstrated for ADCT-301 both by co-culture experiments with CD25-positive Karpas 299 cells, and by media transfer from Karpas 299 cells treated with ADCT-301. This is important as many lymphomas are heterogeneous in their CD25 expression profile (Strauchen et al American Journal of Pathology 1987). In SCID mice with Karpas 299 subcutaneous tumors a single dose of ADCT-301 was administered at 0.2 or 0.6 mg/kg. 24 hours after treatment, excised tumors showed a dose proportional increase in intensity of membrane and cytoplasmic staining by an anti-PBD payload antibody. Cross-linking was determined as 23% (0.2 mg/kg) vs 49% (0.6 mg/kg) (p ≤ 0.01) reduction in Tail Moment using the comet assay and dose-dependent γ-H2AX formation measured by immunohistochemistry was observed. No cross-linking was observed in matched lymphocyte samples. These data confirm the mechanism of cell killing of ADCT-301 and provide relevant pharmacodynamic assays for use in the clinical development of PBD-based ADCs. Disclosures Flynn: Spirogen/Medimmune: Employment. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics: Employment. Tyrer:Spirogen/Medimmune: Employment. Williams:Spirogen/Medimmune: Employment. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Interleukin-2 receptor-α (IL2R-α, CD25), one of a heterotrimer that makes up the IL2R, plays a key role in signal transduction pathways involved in the pathogenesis of autoimmunity and graft rejection (Burchill et al Immunol Lett 2007). In addition, the preponderance of CD25+ cells in hematological malignancies (Srivastava et al Leuk Lymphoma 1994) and the relationship between increased CD25 expression and poor prognosis (Yoshida et al PLoS One 2013) raise the possibility of using an anti-CD25 antibody to deliver a cytotoxin to these cells in patients. Clinical proof of concept for treatment of CD25-positive malignancies has previously been established using radio-immunoconjugates (Dancey et al Clin Cancer Res 2009) and immunotoxins (Kreitman et al J Clin Oncol 2000) utilising antibodies basiliximab and daclizumab. ADCT-301 is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human CD25 attached to a PBD warhead. The drug-antibody ratio is 2.3 ± 0.3. ADCT-301 was potently cytotoxic against CD25-expressing anaplastic large cell lymphoma lines SUDHL1 (341,000 CD25 copies/cell, GI50 0.7 ng/ml) and Karpas 299 (112,000 copies/cell, GI50 3.9 ng/ml) and Hodgkin's lymphoma line L540 (91,000 copies/cell, GI50 3.9 ng/ml). In contrast, CD25-negative Burkitt's lymphoma line, Daudi, gave a GI50 〉〉 1 mg/ml. The released PBD dimer warhead induces highly cytotoxic interstrand cross-links in the DNA minor groove. Unique to this class of ADCs, the single cell gel electrophoresis (comet) assay can therefore be used as a pharmacodynamic endpoint. For ADCT-301, cross-link formation was dose dependent and the peak of cross-linking occurred 16 to 24 hours after a 2 hour exposure of Karpas 299 cells. In vivo, ADCT-301 demonstrated dose-dependent antitumor activity against SUDHL1 and Karpas 299 xenograft and disseminated models. For example, ADCT-301 at a single dose of 0.2 mg/kg significantly delayed Karpas 299 tumor growth compared to vehicle-treated and isotype control ADC-treated mice, and at 0.4 and 0.6 mg/kg gave 3/10 and 10/10 tumor-free survivors, respectively (Figure A). 10/10 tumor-free survivors were also observed at a single dose of 0.5 mg/kg, whereas Adcetris gave only a modest delay in mean tumor growth at a single dose of 0.5 mg/kg despite this tumor expressing three times the level of Adcetris target CD30 antigen compared to CD25 (Figure B). ADCT-301 was well tolerated with no signs of toxicity at 6 mg/kg, currently the highest dose tested. Together, these data clearly demonstrate the potent antitumor activity of ADCT-301 against CD25-expressing hematological tumors and warrants the rapid development of this agent into the clinic. Figure 1A B Figure 1A B. Disclosures Flynn: Medimmune: Employment. van Berkel:ADC Therapeutics Sarl: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics Sarl: Employment. Levy:Medimmune: Employment. Tiberghien:Medimmune: Employment, Patents & Royalties. Masterson:Medimmune: Employment. D'Hooge:Medimmune: Employment. Adams:Medimmune: Employment. Williams:Medimmune: Employment. Howard:ADC Therapeutics Sarl: Equity Ownership, Patents & Royalties. Hartley:Medimmune: Employment, Equity Ownership, Patents & Royalties; ADC Therapeutics Sarl: Equity Ownership, Patents & Royalties.

Description: Thrombotic thrombocytopenic purpura (TTP) is an acute, life threatening disorder. The mainstay of treatment is plasma exchange (PEX) as a source of ADAMTS 13. In the UK, 20–25% of all plasma consumed is in patients with TTP. In our protocol (up until 31st December 2005) apheresis was initially with cryosupernatant (National Blood Service, UK) unless patients had a previous severe allergic reaction or refractory disease. Apheresis therefore continued with Solvent-Detergent Fresh Frozen Plasma (S/D FFP) Octaplas, (Octapharma, Vienna Austria) virally inactivated plasma, available throughout Europe. We reviewed 50 acute TTP episodes involving 32 patients. Thirteen episodes used cryosupernatant only and in 15 episodes, treatment started with cryosupernatant and changed to Octaplas. The reasons for changing were refractory disease in 2 episodes and major or recurrent allergic reactions to cryosupernatant in 13 cases. Once Octaplas had been used, it was continued on further admissions. In 22 episodes, Octaplas was used exclusively; in 4 cases as physicians choice and in the remaining due to previous reactions to cryosupernatant. The total volume of cryosupernatant used was 508250mls, 27.6% of all plasma; total volume of Octaplas was 1327600mls, 72.4% of all plasma. Citrate mediated reactions associated with symptomatic hypocalcaemia during apheresis were present in 11% of Octaplas and 20% of cryosupernatant. Acute or delayed urticarial or allergic reactions were noted in 5% of Octaplas and 10% cryosupernatant procedures. A particular complication of apheresis is central line infection. There were 21 line infections and in 43% of cases the infection was associated with a reduction in platelet count 〈 150 × 109/L. In all 50 episodes, the only documented thrombosis was a superficial non central vein in a patient who had received Octaplas. Prevention of venous thrombosis is by use of thromboembolic stockings, low dose aspirin and low molecular weight heparin in patients when platelet counts 〉50 × 109/L. In episodes receiving only cryosupernatant or Octaplas, there was no significant difference in the median number of PEX to remission, 7(3–14) and 8.5 (5–30) respectively. Baseline viral screen in all episodes was negative after discharge following an acute TTP episode. In conclusion: cryosupernatant and S/D FFP (Octaplas) appear equally efficacious. However, the risk of allergic/urticarial reactions was twice as common with cryosupernatant, as were citrate reactions. Milder allergic reactions to cryosupernatant are possibly higher, but may have been treated with antihistamines and data not recorded. There was no documented viral transmission with either product.