ALBERT

Description: Background: Progressive Multifocal Encephalopathy (PML) is a subacute infection of the central nervous system (CNS) mediated by John Cunningham (JC) polyomavirus (PyV). The disease is frequently fatal, unless adaptive immunity to JC virus is restored. The disease typically occurs in immunosuppressed patients (e.g. HIV/AIDS). In the modern era, patients with hematological malignancies treated with rituximab and/or fludarabine or following immunosuppressive therapy for transplant or autoimmunity (e.g. multiple sclerosis (MS) or Crohn's disease) are also at risk. JC-PML can also occur in patients with genetic defects of immunity. Specific treatments do not exist and immunocompromised patients with history of cancer or genetic defects of immunity have no realistic chance for rapid recovery of their ability to fight infections. In subjects with Rituximab-related PML the case-fatality ratio is 〉90%. Survivors can be left with severe neurological disabilities. Methods: Based on our previously established procedure for generation of multi-virus virus-specific T cells using overlapping peptide libraries (pepmixes) as immunogens, we have developed dedicated polyomavirus-specific T cells (PyVST) targeting polyomavirus BK large T (LT) and Viral Protein 1 (VP1) antigens and highly cross-reactive with the structurally-homologous JC LT and VP1 proteins (see Figure). We hypothesized that adoptive transfer of donor-derived PyVSTs could be safely used for therapy of patients with refractory JC-PML. We have developed a pilot study to test the feasibility and safety of adoptive immunotherapy strategy using PyVSTs from the partially matched healthy 1st degree relative donors (sibling, parent or offspring; NIH study 16-N-0072). Adults diagnosed with PML who have no other treatment options were eligible. Patients with MS or HIV were excluded. PyVSTs were generated from donor blood leukocytes cultured for 14 days in G-rex flasks upon stimulation with BK LT and VP1 pepmixes. Subjects underwent baseline physical examination, MRI and lumbar puncture. Upon enrollment subjects received an intravenous infusion of a fixed dose of 1x10e6 (+/-10%) PyVST cells/kg. Safety monitoring period was 28 days after each infusion. Subjects were eligible for up to two additional infusions (dose 2x10e6 PyVSTs/kg) a minimum of 28 days apart if no toxicities were observed. Serial MRI and lumbar punctures were performed to monitor response. Subjects were followed for 12 months after the last infusion. Results: Nine subjects have been enrolled and treated with at least one infusion of PyVSTs under this protocol and the trial is still accruing subjects. No immediate infusion reactions or adverse events have been observed within the safety monitoring period. No CNS immune system reconstitution syndrome (IRIS) has occurred. One subject received a single infusion and withdrew from the study following Day 14 visit due to unwillingness to travel. One subject received two doses of PyVSTs with stabilization of the disease, not requiring the 3rd infusion. This subject was withdrawn from the study just prior to the 1 year follow up visit, as he was diagnosed with the stage IV lung cancer and entered hospice. Seven subjects received 3 infusions of PyVSTs. 3 of these 7 patients died of refractory PML, all 〉30 days after the 3rd dose of T cells. At the time of this report one subject completed the study at one year with stable PML. The remaining subjects are in the follow-up phase of the protocol. Conclusions: We report that partially matched T cells targeting PyVs generated from healthy related donors can be safely used for adoptive immunotherapy of severely immunocompromised patients with PML. Multiple infusions of T cells are very well tolerated at the doses of up to 2x10e6 PyVST cells/kg, displaying excellent safety profile without adverse events. Furthermore, our data suggest possible efficacy of this strategy as a life-saving therapy for patients who otherwise face a dismal prognosis. Disclosures No relevant conflicts of interest to declare.

Description: Background A widely accepted in vivo model for studying leukemia and its treatment is the highly immune-deficient mice NOD/SCID (b2M-/- or rag-/-). While this model is powerful and recapitulates the phenotypes of blood malignancies in vivo. it is costly and complex, requiring 1-2 months to establish engraftment and the mice are susceptible to spontaneous neoplasms. For these and other reasons the testing of new drugs on leukemia is primarily performed in vitro. The development of antileukemia therapies could be facilitated by a rapid and cost-effective in vivo system for evaluating human leukemia growth and its response to new drugs. Additionally, the treatment of relapsed or refractory disease could be individually tailored by this rapid and cost-effective in vivo system by evaluating patient's cells response to new agents. Turkey embryos are inexpensive, require no maintenance, are larger than chicks are more easily manipulated and have a more robust engraftment (Grinberg I, et al, Leuk Res, 2009; 33:1417-26). We recently described this new in-vivo system for studying multiple myeloma in the pre-immune turkey embryo (Farnoushi, Y., et al.,Br J Cancer, 2011; 105:1708-18). We now demonstrate application of this rapid alternative xenograft system for the preclinical assessment of leukemia growth and therapy. Methods BCR/Abl+ human leukemia lines K562 or LAMA-84 c-Kit+ CHRF 4288 and fresh patient cells were injected into turkey egg chorioallantoic membrane (CAM) veins. Cell injections were performed on day embryonic day E11as previously optimized (Farnoushi, Y., et al.,as above). To determine the engraftment of human AML cells on E19-23, in hematopoietic tissue, the engraftment of human AML cells in the BM was detected in BM by flow cytometry (FC) using anti-human CD71 for LAMA and K562, anti- human CD33 for CHRF and fresh leukemia samples. Engraftment in bone marrow (BM) and other organs was also monitored using Quantitive real time PCR (Q-PCR) comparing the amount of genomic human to the amount of avian DNA and number of human cells / avian cells in BM. Drug response was tested by IV injection of therapeutic range doses of Imatinib (Glivec ®) and Doxorubicin, 48H after cell grafting, at drug levels precalibrated to be non-toxic to the developing embryo by LD50 and BM cell viability compared to control. Six days later (E19) the embryos were sacrificed and the BM collected for FC and hematopoietic and non-hematopoietic tissues for molecular analysis. Results The optimal treatment and readout times were resolved by injecting cells on E11 and determining the kinetics of leukemia cell engraftment in the BM on E15, E18, and E23 in BM and liver. The highest engraftment level in the BM bone marrow (BM) and liver of lines tested was detected at E18 by Q-PCR, and FC in more than 90% of the injected embryos. The average engraftment (±s.d.) in the BM after one week was 4.6%+0.75 K562, 5.16%+2.15 LAMA-84, 7.65%+1.15 CHRF-4288 ( n=7-12 per group) and 2.5% fresh leukemia cells was detected by FC. Q-PCR results were similar to those of FC. Imatinib toxicity testing revealed 100% survival of embryos with no BM toxicity on embryos treated on E13 with doses similar to a human therapeutic dose, up to 0.75 mg/egg. Treatment of embryos with 100 ug Doxorubicin was previously shown to be not toxic to the embryos (Taizi M et al. Exp Hematol 2006; 34:1698–708). A single dose of 0.75 mg Imatinib/embryo dramatically reduced engraftment in BM and several other organs of all 3 AML cell lines or fresh patient leukemia cells. A similar effect was also obtained by a single dose Rx 100ug Doxorubicin. Treatment of a single dose of 0.75 Imatinib mg/embryo 48H after injecting ARH-77 (multiple myeloma) had no effect on cell engraftment. Treatment with a single non toxic dose of Revlimid as previously described (Farnoushi, Y., et al. as above) eliminated engraftment of ARH77 cells, clearly demonstrating the specificity of the drug treatments. Conclusions The results presented demonstrate the potential utility of a practical avian embryo model for testing drug activity in vivo. With further work the turkey embryo may provide a new xenograft in vivo method for studying the biology of leukemia engraftment, and for rapidly and affordably testing leukemia therapies. This system may provide a new platform for developing individualized patient screening for response or resistance to particular therapeutic agents. Disclosures: No relevant conflicts of interest to declare.

Description: While umbilical cord blood provides an important source of hematopoietic stem/progenitor cells (HSPC) for allogeneic transplantation in children, its use in adults is limited by the inadequate number of primitive stem cells and megakaryocyte progenitors (Mk-p) in single or even double CB units resulting in prolonged thrombocytopenia. Thrombopoietin treatment is not effective in these patients due to the paucity of target progenitors and patients require multiple platelet transfusions until the long-term engrafting cells can support thrombopoiesis, thus new modalities to increase progenitor cell dose are needed. A new transplantation strategy could involve the infusion of ex vivo-generated Mk-p together with unmanipulated single or double CB units. While CB CD34+ cells can be expanded by several reported methods, these rare cells cannot be sacrificed from the CB units due to their limited number. We propose a novel ex-vivo strategy to facilitate HSPC and Mk-p expansion from mononuclear cells (MNC) of a small aliquot of CB using conditions that mimic the hematopoietic niche, in short term cultures. Fibronectin (FN) was considered to be a prime candidate to support proliferation because it is a major extracellular matrix (ECM) component of all bone marrow hematopoietic microenvironments which is known to enhance viability and proliferation of HSPC. Other growth stimulators added were thrombopoietin (r-hu-TPO), the major physiological stimulator of MK and the synthetic hematopoietic stress peptide ARP derived from acetylcholinesterase, shown to increase transplantable Mk-p and produce human platelets in NOD/SCID mice (Pick et al, Blood 2006, Grisaru et al, J Imm 2006). High definition flow cytometry enabled assessing expansion of the SSClow/CD34high HSPC, and the SSClow/CD45dim/neg/CD41high Mk-p, and their subpopulations on day 0 and 10 of culture. True MK expansion was assessed by gating out of granulocyte and monocytes, which acquire CD41+ adherent platelets in culture. FN alone increased viability and expansion of HSPC by 6.9 fold and MK-p by 4-fold, while r-hu-TPO alone enhanced Mk-p proliferation with an average expansion of 8.3-fold in agreement with its known activity. Combining FN with r-hu-TPO produced a 25-fold increase in the number of MK-p while adding ARP to FN and r-hu-TPO was even more powerful, doubling the number of cells with a highly significant average expansion of 59-fold (p 〈 0.001). To define the progenitor subpopulations that contributed to Mk-p proliferation with FN, r-hu-TPO and ARP, we further analyzed the resulting subsets of MK-p cells, which also expressed either CD34, or the early myeloid marker CD33. The CD41high/CD34high population was increased by 4 fold, while the CD41high/CD33+ Mk-p, a subset with properties similar to clonogenic GEMM progenitors that could provide both myeloid and megakaryocytic cells post-transplant, were stimulated 30–50 fold. This notion is confirmed by the stimulation of CFU-MK and CFU-GEMM obtained under these conditions. Considering that expansion of MK-p requires proliferation of the HSPC precursor, we examined the proliferation of CD34+ progenitor cells and their subpopulations; CD34high/CD33+ or CD34high/CD41low uncommitted HSPC and CD41 high committed Mk subpopulations. The addition of FN alone stimulated CD34+ HSPC expansion by 6.9-fold (p 〈 0.05). All cultures that contained the ARP peptide maintained a high proliferation capacity, confirming that ARP protects and drives CD34+HSPC and early myeloid cell proliferation (Deutsch et al Exp Hem 2002). The addition of r-hu-TPO and ARP to FN produced a synergistic proliferative effect on the CD34+/CD41low HSPC stimulating a dramatic 440 fold increase of these uncommitted cells. These data support the notion that FN is protective and plays an essential role in enabling HSPC and MK-p expansion driven by r-hu-TPO and ARP. These conditions also supported MK maturation, as measured by increased high ploidy cells and elevated expression of GPIIb/IIIa detected by quantitative real time PCR. We demonstrate that expansion of both very early myeloid and Mk-p from a small fraction of the CB unit in short term cultures under conditions that mimic the hematopoietic niche is feasible, easy to perform and can comply with GTP requirements. This approach may lead to the development of more effective cell therapy modalities to facilitate myelopoiesis and platelet production following CBT.