J Immunother  Volume 27, Number 6, November/December 2004
`
`Abstracts
`
`cells(APC). SV-BR1 cells had been previously incubated in 100 u/ml
`interferon-gamma to upregulate cell-surface antigens and stored frozen over
`liquid nitrogen. Immediately prior to use the cells are thawed, washed and
`irradiated to 200cGy. Inoculation was repeated q2weeks 3 3, then monthly
`until progression. Patients received low-dose cyclophosphamide, 300 mg/m2,
`2–3days prior to inoculation and also subcutaneous GMCSF, 125 mcg,
`immediately prior to inoculation and then daily 3 8 to enhance APC function.
`Fourteen patients have been entered on study. In 44 treatments, the median
`dose was 14 3 106 cells (range 9–26 3 106), median viability 93% (range 73–
`97%). The median age was 53.5 years (range 39–70), HER2/neu positive 6 of
`11, median previous chemotherapy regimens: 3 (range 1–5). Of 13 patients
`treated two are still alive at 48 and 129 weeks; overall median survival 48.7
`weeks (range 5.4–139 weeks); Five patients survived .52 weeks. There were
`no objective responses in 11 evaluable patients but 4 of 6 patients developed
`significant increases in anti-SV-BR1 antibody by ELISA., and DTH responses
`to SV-BR1 developed in 3 of 7. Toxicity was mild and consisted principally of
`erythema and pruritus at injection sites. One patient developed transient atrial
`fibrillation and one patient, after 3 inoculations, declined further treatment for
`psychological reasons. While selection bias cannot be excluded, the survival
`data compares favorably with other third-line studies. Given the safety and
`feasibility of this preliminary trial, SV-BR1 has been transfected with the
`pcDNA3.1/GS/GM-CSF plasmid (Invitrogen) using Zeocin selection.
`GMCSF production by ELISA in 3 representative lots was 115, 129, and
`110 ng/106 cells/24 hours. Biological activity was validated by culture with the
`GMCSF-dependent MUTZ-2 leukemic cell line. A clinical trial is planned
`pending final regulatory review.
`
`Dendritic Cell Vaccines in Patients with Non Small Cell Lung
`Cancer
`John Yannelli, Jamie Sturgill, Edward Hirschowitz. Microbiology, Immunol-
`ogy and Molecular Genetics;Pulmonary Medicine and The Markey Cancer
`Center, University of Kentucky, Lexington, KY.
`Non small cell lung cancer (NSCLC) is a leading killer of men and women
`throughout the world. We have developed an immunotherapy for this disease
`using dendritic cells (DCs) pulsed with apoptotic bodies derived from an
`allogeneic NSCLC cell line called 1650TC. The 1650TC line, (HLA-A2; B15;
`C7), expressed the NSCLC associated antigens Her-2neu, CEA, WT1,
`Survivin and Mage-2. The DCs were generated from leukapheresis products
`obtained from 16 patients with disease stages ranging from 1A to 3B. CD14+
`monocytes were purified from the leukapheresis products using Miltenyi
`magnetic bead separation technique. The CD14+ cells which were greater that
`95% pure were cultured in XVIVO 15 serum free medium containing 10%
`human allogeneic AB serum in the presence of 20 ng/ml GMCSF and IL-4 for
`7 days. The CD14+ cells were cultured in either 6 well cell culture plates or
`T175cm2 flasks at 1.0 3 106 CD14+ cells/ml. At day 7, 1650 TC was
`subjected to uvB light and irradiation. The resultant % of apoptotic cells after
`this treatment ranged from 35–40%. The apoptotic cells were added to the
`DCs at a ratio of 1:1. After 18hr culture,
`the DCs were harvested for
`intradermal injection in the patients. All 16 patients received an average of
`100 million antigen pulsed DCs in both a prime and a boost dose separated
`by 30 days. The DCs used for injection appeared to be immature based on the
`low levels of expression of CD80 and low levels of secretion of IL-12p70.
`There were no adverse events associated with the DC injections. Peripheral
`blood was obtained from each patient post injection and lymphocytes moni-
`tored for anti-tumor reactivity by ELISPOT technique. Results of ELISPOT
`analysis examining gamma interferon release showed that 5 of 16 patients had
`no clear anti-tumor immunologic response (no response); 5 of 16 patients had
`lymphocytes which showed a tumor antigen independent response(recognized
`autologous DCs alone) and 6 of 16 patients had lymphocytes showing an
`anti tumor immunologic response (recognized DCs following ingestion of
`1650-apoptotic bodies). Immunologic responses were independent of prior
`therapy or stage of the disease. Both favorable and unfavorable clinical
`outcomes were independent of measured immunologic outcomes. In con-
`clusion, vaccines were well tolerated and induced biologic activity in a number
`of patients. Studies are continuing in our lab to determine the antigens
`recognized by the anti-tumor reactive lymphocytes and also examine the
`nature of the reactivity against DCs alone. The clinical trial is also continuing
`and includes a randomized trial using DCs and the COX-2 inhibitor Celebrex
`in NSCLC patients.
`
`HEMATOLOGIC MALIGNANCIES
`
`A Phase I/II Study of the Oral mTOR Inhibitor RAD001 in
`Patients with Advanced Hematologic Malignancies
`Karen Yee1, Susan O’Brien1, William Wierda1, Deborah Thomas1, Razelle
`Kurzrock1, Luis Fayad1, Fredrick Hagemeister1, Andre Goy1, Jorge Cortes1,
`Judith Prestifilippo2, Ted Szatrowski2, Hagop Kantarjian1, Francis Giles1..
`1Leukemia, UT MD Anderson Cancer Center, Houston, TX; 2Novartis
`Pharmaceuticals Corporation, East Hanover, NJ.
`RAD001 (everolimus, Novartis)
`is an orally bioavailable derivative of
`rapamycin with demonstrated anti-proliferative activity against a broad panel
`of tumor cell lines and antitumor activity in experimental animal models of
`human cancer. RAD001 inhibits the mammalian target of rapamycin (mTOR)
`signaling pathway, which is involved in regulating many aspects of cell growth
`and cell cycle progression. Several lines of evidence implicate the importance
`of the PI3K/Akt/mTOR pathway in hematological malignancies. As there has
`been extensive experience with this agent in the solid organ transplantation
`setting, two dose levels (5 and 10 mg/day) were evaluated in the Phase I
`portion of this study to determine the maximum tolerated dose (MTD) to be
`used in the Phase II portion. RAD001 was administered orally once a day at
`the starting dose level of 5 mg. Seventeen patients have been enrolled, of
`which 16 (4 B-CLL, 4 MDS, 1 MF, 1 NK/T cell lymphoma/leukemia, 2 MCL,
`3 AML, 1 T-PLL) were evaluable for safety and toxicity. Median age was 65
`years (range, 51 to 76 years). Fourteen patients (87.5%) had received prior
`therapy (median, 2 regimens; range, 0 to 6 regimens). Median time on study
`was 39 days (range, 5 to 119 days). No dose-limiting toxicities were observed
`in the 6 patients enrolled in the Phase I portion of the study (cohorts of 3
`patients per dose level). The most common adverse events were grade # 2 and
`consisted of hyperglycemia, hyperlipidemia, hypocalcemia, hypomagnesemia,
`hypophosphatemia and hypokalemia, transaminitis, diarrhea or constipation,
`dermatitis, mucositis, anorexia, and asthenia. Grade 3 toxicities consisted of
`asymptomatic hypophosphatemia (2), hyperglycemia (4), asthenia (1),
`anorexia (1), and bone pain (1). No patient experienced grade 4 toxicities
`or death from RAD001. Of the 16 evaluable patients, 2 patients with B-CLL
`had a 33% and 65% reduction in the size of lymph nodes, respectively, and 1
`patient with MDS had a minor increase in the platelet count. At the time of
`analysis, 2 patients had discontinued therapy due to disease progression and 1
`patient had died from bleeding secondary to AV malformations in the
`gastrointestinal
`tract (unrelated to RAD001 therapy). These preliminary
`findings indicate that RAD001 is well tolerated at a daily dose of 10 mg/day
`and may have clinical activity in patients with hematologic malignancies.
`Enrollment is ongoing.
`
`CPG Oligodeoxynucleotides Enhance Immunogenicity In Vitro in
`All Cytogenetic Subgroups of B-Cell Chronic Lymphocytic
`Leukemia (B-CLL), but Preferentially Augment Apoptosis in
`B-CLL with Good Prognosis Cytogenetics
`Bernd Jahrsdoerfer1, Sue Blackwell1, James Wooldridge1, Christiana Taylor1,
`George Weiner1. 1Holden Comprehensive Cancer Center, University of Iowa,
`Iowa City, IA.
`Immunostimulatory CpG oligodeoxynucleotides (CpG ODN) are TLR9
`agonists that mediate a number of immunologic effects in normal and
`malignant B cells including upregulation of immunogenic molecules. They
`are therefore felt to be attractive as potential components of immunotherapy
`for B cell chronic lymphocytic leukemia (B-CLL). The cytogenetic status of
`B-CLL is known to be predictive of clinical prognosis, but little is known
`about how treatment of B-CLL cells correlates with cytogenetic status. The
`present study was designed to explore the impact cytogenetic status has on in
`vitro response to CpG ODN. B-CLL cytogenetic status was determined by
`interphase FISH. Immunophenotype and cell survival in the absence or
`presence of CpG ODN was determined in 23 samples. CpG ODN decreased in
`vitro survival of B-CLL cells with good prognosis cytogenetics, but had little
`effect on cells with poor prognosis cytogenetics. In contrast, CpG ODN
`upregulated costimulatory and antigen-presenting molecules and enhanced
`allogeneic T cell response in samples with either good or poor prognosis
`cytogenetics (Figure 1). We conclude CpG ODN induce changes in B-CLL
`consistent with enhanced immunogenicity in all samples studied, but induce
`apoptosis most effectively in the subset of B-CLL cells with good prognosis
`
`q 2004 Lippincott Williams & Wilkins
`
`S35
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`Roxane Labs., Inc.
`Exhibit 1022
`Page 001
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`

`
`Abstracts
`
`J Immunother  Volume 27, Number 6, November/December 2004
`
`suggest CpG ODN may be useful as
`cytogenetics. These studies
`immunotherapeutic agents for B-CLL irrespective of cytogenetic status
`because of their potential effects on immunogenicity.
`
`Notable Phenomena of the 1960s Recapitulated. I. Graft-Versus-
`Leukemia Reaction and Natural Hybridoma Formation in Mice
`Joseph G Sinkovics. St. Joseph’s Hospital Cancer Institute, University of
`South Florida, Tampa, FL.
`In 1962–3 at MD Anderson Hospital viral leukemia-susceptible Balb/c mice
`were rendered to be chronically runted with allogeneic disease due to cellular
`chimerism by injections of lymphoid cells from leukemia virus-resistant
`C57Bl mice. In these Balb/c mice leukemia viruses replicated to very low titers
`and induced no or low incidence of leukemia (AACR 1963; TX Rep Biol Med
`1965; 7th Internat Symposium: Autologous Marrow and Blood Trans-
`plantation, Arlington, TX 1994). Later 20% of Balb/c mice were cured of
`virally induced leukemias by lethal irradiation followed by inoculations of
`Balb/c lympho- and hematopoietic cells deriving from donors that were
`actively immunized with a photodynamically inactivated leukemia virus
`vaccine (CR 1965; MD Anderson 20th Symposium: Carcinogenesis: A Broad
`Critique, WW 1967). A virally induced but cell-passaged murine lymphoma
`(#620) presented with the starry sky histological picture (J Inf Dis 1968;
`1969), induced cell- and antibody-mediated immune reactions in its host and it
`could be rejected. Immune modulators either accelerated or decelerated its
`course (Science 1967; J Retic Soc 1970). The diploid lymphoma cells
`expressed budding retroviral particles and could fuse with immune plasma
`cells secreting leukemia virus-neutralizing antibodies. The fusion product
`tetraploid cells (#818) contained both leukemia virus antigens and IgG2b
`immunoglobulins; grew in suspension cultures and as lethal ascites tumors,
`while continuously secreting the specific antibody for over 10 years (1968–
`1980) (Lancet 1970; MD Anderson 14th Clinical Conference: Leukemia-
`Lymphoma, Yearbook, Chicago 1970; CR 1970; 1981). Antibody-coated
`lymphoma cells can be phagocytized by macrophages or dendritic cells (hence
`the starry sky feature of Burkitt lymphoma as the EBV-positive tumor cells are
`antibody-coated) (Critic Rev Immun 1991) to present tumor antigens to
`
`immune T cells. In human lymphomas two-directional FasL to Fas reactions
`occur between lymphoma cells and immune T cells (Inter J Oncol 2001). A
`human lymphoma cell line (T1 or #778) established in 1968 (JM Trujillo and
`the author) replicated unidentified retroviral particles. Lymphoma cells fused
`with plasma cells of the patient. During the RAG-induced V(D)J somatic
`hypermutations silent resident retrotransposons may acquire env sequences
`and emerge as endogenous retroviruses (AACR 2001; ASCO 2002). When
`these lymphoid cells generate autoantibody production, they may fuse with the
`antibody-producer plasma cells. Natural hybridoma formation in human
`lymphomas may be of decisive prognostic significance (Wainwright: Persp
`Biol Med 1992). This presentation will be illustrated with genuine original
`data and microphotographs from the 1960s to 1970s.
`
`IMAGING
`
`Homing of Tumor-Specific T Cells in the B16-OVA/OT-I Model
`System - towards T Cells as Carriers of Cytotoxic Substances in
`Therapy of Cancer
`Mikkel S Petersen1, Charlotte C Fleischer2, Søren B Hansen3, Hans Stødkilde-
`Jørgensen4, Ulrik Skands1, Tom E Andersen2, Egil F Hulgaard1, Jan T
`Jørgensen1, Jørgen Marqversen5, Hans J Gundersen6, Marianne Hokland2,
`Ralf Agger2. 1t-cellic A/S, Hørsholm, Denmark; 2Medical Microbiology and
`Immunology, Aarhus University, Aarhus, Denmark; 3PET Centre, Aarhus
`University Hospital, Aarhus, Denmark; 4MR Research Centre, Aarhus
`University Hospital, Aarhus, Denmark; 5Nuclear Medicine, Aarhus University
`Hospital, Aarhus, Denmark; 6Stereological Research Laboratory, Aarhus
`University, Denmark.
`Background: The use of adoptively transferred, tumor-specific T cells as
`carriers of cytotoxic substances to tumor tissue represents a possible new
`therapeutic modality for the treatment of cancer. In this approach, optimizing
`homing of adoptively transferred T cells to tumor tissue is critical. We have
`studied the ability of radioactively labeled, adoptively transferred tumor-
`specific T cells in the B16-OVA/OT-I model system to home to tumor sites
`using combined PET and MR imaging.
`Methods: OVA-specific CD8+ T lymphocytes labeled with 124-Iodine-
`conjugated deoxyuridine (124-IdU) were injected into C57BL/6J mice
`carrying subcutaneous tumors of the ovalbumin (OVA)-expressing malignant
`melanoma cell line B16-OVA
`Five days after adoptive transfer of the labeled cells, the mice were killed and
`subjected to PET- and MR imaging. Using a newly developed method for
`co-registration of the two image modalities, the anatomical localization of the
`transferred cells could be visualized and the amount of radioactivity in various
`anatomical locations accurately determined.
`Results: The experiments showed a clear accumulation of the transferred cells
`in the tumors. In two independent experiments comprising 12 and 13 mice,
`respectively, we found a statistically significant difference in the mean activity
`between the tumor regions and the control regions (p ¼ 0.002 and p ¼ 0.011,
`respectively) in the corresponding contra-lateral control volumes).
`Conclusions: These studies show that tumor-specific T lymphocytes home to
`subcutaneous tumors in substantial numbers, and that the cells can act as car-
`riers for radioactive imaging substances. We suggest that such migrating T cells
`could be employed in a future therapy of cancer as carriers of toxic substances
`to tumors. This form of therapy would rely solely on the ability of the
`adoptively transferred cells to home effectively to tumor sites, and would not
`depend on any inherent capacity of the T cells to elicit an anti-tumor response.
`
`Quantification and Visualization of Peptide/Major Histocompat-
`ability Complex Antigens on Live Cells Using High-Affinity
`Soluble T Cell Receptors
`Marco Purbhoo1. 1Avidex Ltd., Abingdon, Oxfordshire, United Kingdom.
`Like monoclonal antibodies, soluble T-cell receptors (TCRs), have evident
`uses in the study and treatment of cancer, autoimmunity, and infection.
`However, the use of soluble TCRs for analytical and therapeutical applications
`is hampered by both the difficulty of producing stable, soluble TCRs and the
`natural low affinity of TCRs for their ligands.
`We have overcome these limitations by introducing an inter-chain disulphide
`bond to stabilize the abTCR, and subsequent phage-display based affinity
`
`S36
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`q 2004 Lippincott Williams & Wilkins
`
`Roxane Labs., Inc.
`Exhibit 1022
`Page 002