Vol. 3, 1789-1 797. October 1997 Clinical Cancer Research 1789 Successful Treatment of Canine Malignant Histiocytosis with the Human Major Histocompatibility Complex Nonrestricted Cytotoxic T-Cell Line TALL-1041 Sophie Visonneau, Alessandra Cesano, effectors and their therapeutic potential even in the most Thuy Tran, K. Ann Jeglum, and Daniela Santoli2 aggressive forms of the disease. The Wistar Institute, Philadelphia, Pennsylvania 19104 [S. V., A. C.. T. T., D. S.], and Veterinary Oncology Services and Research Center, INTRODUCTION West Chester, Pennsylvania 19382 [K. A. J.] MH3 in dogs, first reported in 1978 ( I ), is a tumor char- actenized by neoplastic proliferation of invasive atypical eryth- nophagocytic histiocytes in various tissues. The disease fre- ABSTRACT quently becomes manifest in the middle-age years and has been The human MHC nonrestricted cytotoxic T-cell line observed more frequently in males than in females (2). Bernese TALL-104 exerts potent antitumor effects in animal models mountain dogs are genetically prone to this type ofcancer (3, 4), with both induced and spontaneous cancers. The present but other breeds are also sporadically affected (5). Clinical report documents the ability of systemically delivered findings commonly include fever, generalized lymphoadenopa- TALL-104 cells to induce durable clinical remissions in four thy, and hepatosplenomegaly as well as concomitant anemia, of four dogs with malignant histiocytosis (MH). The animals leukopenia, and thrombocytopenia (I). Neoplastic histiocytes received multiple i.v injections oflethally irradiated (40 Gy) mainly infiltrate the spleen, liver, lungs, lymph nodes, bone TALL-iN cells at a dose of 108 cells/kg, with (two dogs) or marrow, and skin. The visceral form of this disease is rapidly without (two dogs) cyclosporin A, followed by monthly progressive, and the prognosis is poor (1). The diagnosis of MH boosts. No significant clinical or laboratory toxicities devel- presents a challenge to both clinicians and pathologists because oped during cell therapy; interestingly, a strong correlation the disease lies within a spectrum of histiocytic and hematolym- was found between the dogs’ clinical and immunological phoid disorders and may be confused with granulomatous in- responses. One dog with advanced disease (intrathoracic flammation. Recently, phenotypic similarities between malig- involvement) refractory to chemotherapy achieved a corn- nant fibrous histiocytoma and malignant histiocytosis have been plete remission (CR) within 2 months of the first TALL-104 reported (6). A diagnosis of MH is appropriate once the malig- cell infusion. This dog died 14 months later of unrelated nant nature and histiocytic differentiation of the process have causes: histological analysis of its organs postmortem re- been established. vealed no evidence of neoplasia, thus confirming the achieve- We have developed a new cell therapy approach based on ment of CR also at the pathological level. The other three the use of the human leukemic T-cell line TALL-104 (CD3/ dogs with MH that at diagnosis had multiple s.c. and cuta- TCRaf3, CD8, CDS6, and CDI6), which is endowed with neous lesions and lyrnphadenopathy, but no visceral involve- potent MHC nonrestricted tumoricidal activity, without lysing ment, were treated with TALL-104 cells as single agent (no cells from normal tissues (7, 8). Unlike patient-derived lympho- chemotherapy was administered). Two of these dogs kine-activated killer cells, TALL-l04 cells provide an unlimited achieved a CR soon after cell therapy, and the third dog had and reliable source of clonal effecton cells with stable cytotoxic two long-lasting partial responses; CR in this dog was later activity that is ideal for cell therapy approaches. Although achieved by combined administration of chemotherapy and dependent on recombinant human IL-2 for expression of cyto- cell therapy. None of the three dogs that received cell ther- toxicity and long-term survival in vitro, TALL-104 cells can apy at diagnosis developed visceral disease in the -9-22 exert antitumon effects in vivo without the concomitant admin- months of follow-up. The clinical responses experienced by istration of IL-2, thus eliminating the potential toxicity of this all four MH cases to TALL-104 cell therapy suggest the high cytokine. TALL-104 cells induce necrotic tumor cell death responsiveness of this canine tumor to these xenogeneic through secretory pathways involving perform and senine ester- ases and/or kill targets through the release of cytostatic/cyto- toxic mediators such as TNF-a, TNF-3, IFN-y, or transforming growth factor 3 (7, 9). Despite the fact that TALL-104 cells Received 1 1/8/96; revised 5/24/97; accepted 7/1/97. would be rejected by a tumor-bearing MHC-incompatible host, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with I 8 U.S.C. Section 1734 solely to indicate this fact. I Supported in part by American Cancer Society Grant RD391, the Connelly Foundation, the Parker Hughes Trust, and the Butler Family 3 The abbreviations used are: MH, malignant histiocytosis; CsA, cyclos- Fund. porn A: PBMC, peripheral blood mononuclear cell: FACS. fluores- 2 To whom requests for reprints should be addressed, at The Wistar cence-activated cell-sorting: TNF, tumor necrosis factor: GM-CSF, Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: (215) granulocyte macrophage-colony stimulating factor: CR, complete 898-3978: Fax: (215) 573-7919. response: PR, partial response: IL, interleukin: NK, natural killer. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 1997 American Association for Cancer Research. 1790 Cell Therapy of Canine Malignant Histiocytosis Table 1 Charactenis tics of the MH dogs treated with TALL- 104 cell therapy Canine patient Sexlage (yr)fbreed Previous therapy Clinical stage at diagnosis Dog I M/8/Scottish Terrier Five cycles of chemotherapy with Multiple cutaneous and s.c. lesions, doxorubicin, cyclophosphamide. metastatic lymphadenopathy, vincnistine, dacarbazine, and intrathoracic involvement L-asparaginase Dog 2 M/7IWest Highland Terrier None Multiple cutaneous and s.c. lesions, metastatic lymphadenopathy Dog 3 F/3/Bernese Mountain Dog None Cutaneous lesions on the muzzle and right axilla, metastatic lymphadenopathy Dog 4 F/l/Golden Retriever None Cutaneous and s.c. lesions on the muzzle; metastatic lymphadenopathy we have used -1’-irradiation (40 Gy) as a precautionary measure three dogs were not given chemotherapy and were treated with to irreversibly arrest the growth of this leukemic cell line in the TALL-104 cells as the single agent. host tissues ( I 0). This treatment did not impair the antitumor Histological and Immunohistochemical Analysis. 5cc- effects of TALL-l04 cells in immunodeficient mice engrafted tions (6 jim) of paraffin blocks from the dogs’ tumor lesions with human neoplasms ( 1 1, 1 2) and in immunocompetent mice were stained with H&E for histological analysis. Immunohisto- bearing syngeneic leukemia ( I 3), suggesting the feasibility of chemical staining was done using the avidin-biotin-peroxidase using -i-irradiated (nonproliferating) TALL-l04 cells in cancer complex method (iS): sections were deparaffinized, dehydrated therapy. through graded alcohol, and washed in 0.01 M PBS (pH 7.4). To To determine the potential safety of TALL-l04 cells as an block nonspecific binding, sections were incubated with normal anticancer agent in a clinically relevant setting, we recently horse serum (1 :75; Vector Laboratories, Burlingame, CA) for 20 conducted a Phase I trial in pet dogs with spontaneous end-stage mm, followed by overnight incubation at 4#{176}Cwith primary tumors (14). Results of that study clearly showed not only the antibodies antilysozyme ( 1 :200), anti-a-i-trypsin (1:300; absence of significant adverse reactions in terminally ill canine DAKO, Carpinteria, CA), and anticathepsin B (1:300; ICN patients but also the manifestation of various levels of clinical Biochemicals, Costa Mesa, CA). Normal mouse serum was used responses including one CR and one PR in two dogs with MH. as control antibody. Sections were incubated in biotinylated The present report extends the follow-up for these two dogs and horse antimouse IgG (1 :200; Vector Laboratories) for 45 mm. describes the successful application of TALL-104 cell therapy All incubations were done in a humidified chamber at room to two more dogs with less-advanced MH. Results indicated temperature. 3-Amino-9-ethylcarbazole (Sigma Chemical Co., that: (a) this potentially fatal canine disease is extremely sensi- St. Louis, MO) was used as chromogen, and sections were tive to treatment with xenogeneic TALL-l04 cells; (b) durable counterstained with Mayer’s hematoxylin. CRs could be achieved even in dogs with advanced tumors, Large-Scale Expansion of TALL-104 Cells for Therapy. refractory to chemotherapy; and (c) TALL-l04 cells were very TALL-104 cells were grown in endotoxin-free Iscove’s modi- effective when used as a single agent in newly diagnosed cases fled Duibecco’s medium (Life Technologies, Inc., Grand Island, with systemic disease but no visceral involvement. NY) supplemented with 10% heat-inactivated fetal bovine se- rum (Atlanta Biologicals, Norcross, GA) and 100 units/ml re- MATERIALS AND METHODS combinant human IL-2 (Chiron Therapeutics, Emenyville, CA) Canine Patients. Four dogs diagnosed with MH were in a humidified