Tyrosinemia

IMMUNOTHERAPY USING HERCEPTIN(R)-TARGETED, ACTIVATED AUTOLOGOUS T CELLS

Women with high risk, locally advanced (stage II-III with [>, double =] 4 positive lymph nodes) breast cancer or metastatic (stage IV) breast cancer, and men with hormone refractory prostate cancer (HRPC) could benefit from new approaches to improve lengths of remissions and overall survival, to prolong clinical responses, and to improve quality of life. Even minor responses or relief from bone pain may help those with bone metastases. Many patients with these diseases share an overexpression of Her2/neu on their cancer cell membranes. Overexpressed Her2/neu is an excellent target for monoclonal antibody therapy with Herceptin(R) (anti-Her2/neu).1

Immunotherapy with autologous T cells has been studied both in vitro and in vivo for over 25 years. T cells obtained by leukapheresis can be expanded ex vivo and activated with the monoclonal antibody OKT3 (anti-CD3). Activated T cells (ATC) are efficient killers of tumor cell lines in vitro and show promise in vivo in women with stage IV breast cancer (reducing relapse rates) after massive cytoreduction from high dose chemotherapy and stem cell transplantation. In vitro studies show that ATC may be made even more efficient killers by "arming" them with a "bispecific" antibody produced by the chemical heteroconjugation of OKT3 (anti-CD3) and Herceptin, (anti-Her2/neu) monoclonal antibodies, yielding a product we call Her2Bi.2

When armed with nanogram amounts of Her2Bi, ATC aggregate with Her2/neu positive breast and prostate cancer cell lines and are highly cytotoxic to these cell lines at extraordinarily low effector to tumor cell (E:T) target ratios (e.g., 5:1). Armed ATC from 10 normal subjects and 6 cancer patients displayed mean (+ or -SD) cytotoxicity 59+ or -11% and 32+ or -9%, respectively, above that seen for ATC alone at E:T of 20:1.3 Tumoricidal cytokines (IFN[gamma], TNF[alpha], and GM-CSF) as well as chemokines (RANTES and MIP-1[alpha]) were secreted when armed ATC bound to tumor targets. Armed ATC were able to kill tumor targets 4 times consecutively and to divide 3 times in 13 days while continuing to bear Her2Bi on their surface.4 In SCID/Beige mice, prostate tumors were prevented in 100% of the mice by co-injections of armed ATC with tumor cells. Established prostate tumors could be ablated in 40% of the mice given twice weekly intra-tumoral injections of armed ATC.5 These preclinical studies support the rationale for clinical trials.

The FDA has granted us approval to expand anti-CD3 ATC ex vivo, to produce the bispecific antibody Her2Bi, and to arm ATC with Her2Bi in order to evaluate a unique non-toxic clinical treatment strategy for diseases that overexpress Her2/neu. This approach combines the cytotoxic capacity of ATC with the specific targeting ability of Herceptin antibody to improve tumor lysis. Immunotherapy with ATC armed with Her2Bi is being used to treat patients with breast, prostate, and pancreatic cancer in FDA approved Phase I/II clinical trials (listed at www.cancer.gov/clinica_trials). Phase I studies assess side effects of armed ATC during dose escalation in sequential cohorts of patients. Phase II studies assess clinical response rates of individual diseases to armed ATC.

To date, 5 patients with breast cancer, 4 patients with HRPC, and 1 patient with metastatic pancreatic cancer have been treated at beginning dose levels without any life-threatening reactions. Striking to us at this low dose level is the observation that 4 patients have had more than a 50% reduction in their pain medication requirement. We expect that these studies will provide proof-of-concept that high numbers of Her2Bi armed, activated T cells will provide a valuable anti-tumor effect without undue toxicities to patients.

AUTOLOGOUS ADULT STEM CELLS AND TISSUE REPAIR

Recently, our understanding of adult marrow stem cell biology dramatically changed. Marrow hematopoietic stem cells have the capacity to produce nonhematopoietic cells in diseased or injured tissues. Mice transplanted with lethal genetic tyrosinemia have had massive repopulation of liver cells with purified marrow stem cells and a number have been cured.6 In a similar vein, Orlic and coworkers7 have studied mice with ischemic cardiac lesions. Such mice were either infused with purified marrow stem cells or had endogenous marrow stem cells mobilized. These stem cell approaches resulted in the production of cardiac myocytes from marrow and a decrease in mortality of the mice. Similar work has shown that marrow cells can produce epithelial cells in lung,8 gut, and skin,9-11 neural cells in brain,12 bone cells13 and skeletal muscle cells.14 Critical aspects of these studies have been the method of tissue injury and the details of the transplantation. When cardiotoxin has been injected into the anterior tibialis muscle and the muscle has been irradiated, infusing syngeneic marrow cells or mobilizing endogenous stem cells has resulted in very significant conversion of marrow cells to skeletal muscle cells. Similarly, after irradiation and wound production, stem cells produce skin appendages. Not all methods of tissue injury, however, respond to stem cell "therapy."

These results are now being applied to treatment of human diseases. Marrow cells have been infused after angioplasty in cardiac patients in phase I/II studies in Germany and Italy with promising results as well as safety profile. Studies carried out by Drs. Evangelos Badiavas and Vincent Falanga at Roger Williams Medical Center (RWMC) have shown dramatic effects of healing of chronic refractory skin wounds by local application of autologous bone marrow cells.15 In these studies, patients with non-healing wounds (over 1 year) had fresh marrow collected and a portion was applied to the skin wound and injected in areas around the wound. The other portion of the marrow sample was cultured in vitro and subsequently applied to the wound in a similar manner. Three patients with chronic refractory wounds were treated in this manner, with healing of the wounds in all cases. Although the number of patients treated is small, we believe that this is the first clear-cut demonstration of adult marrow stem cells effectively treating a non-marrow disorder.

There has been a good deal of confusion of the roles of embryonic stem cells and adult marrow stem cells. The former have raised ethical and religious issues since the cells are derived from blastulas from developing embryos. These cells have been studied in mice and man and found to have great potential for indefinite growth in tissue culture and have the ability to produce many different cell types. With the establishment of approaches to culture human embryonic stem cells, many investigators have envisioned great therapeutic potential. Unfortunately, these cells are hard to regulate and, at least in murine transplant systems, ordinarily produce tumors, many of which become malignant. This has not been tested in human transplants. Adult marrow stem cells, on the other hand, have been used for years in marrow transplantation. Their clinical use has been established, as has their safety profile. There is little risk of tumor formation, and with autologous (same person) transplants, virtually no side effects of the marrow infusion. With allogeneic marrow or stem cell transplantation there is the risk of graft-versus-host disease, which has been extensively studied and can be treated. The discovery that adult marrow stem cells can produce cells in a variety of nonhematopoietic organs has opened the possibilities for a variety of therapeutic approaches to replace damaged or diseased tissues. Ischemic cardiac disease, serious lung or liver disease, chronic wounds or burns all may be appropriate candidates for stem cell therapies. Chronic neurologic disorders such as Parkinson's, Alzheimer's disease, and stroke are also candidates for therapy, as is muscular dystrophy. The critical issues are whether or not enough marrow stem cell to tissue cell conversion could occur to restore organ function. As noted, mouse models have shown that this is possible for both cardiac and liver disease, and our recent wound healing studies indicate that marrow treatment may be effective in human disease. Current studies in the Center for Stem Cell Biology at RWMC are focusing on details that may improve such conversions; these include the number and functional state of the infused cells, additional organ or tissue injuries that may enhance the conversion events and details of the actual transplantation regimens.

The work in the Stem Cell Center at RWMC and the pioneering clinical studies of Drs. Falanga and Badiavas have led to the formation of The Center for Stem Cell Tissue Restoration at RWMC. This Center will focus initially on the treatment of chronic refractory skin wounds in patients with peripheral vascular disease, diabetes, and other chronic diseases. Recent progress in basic work in the Center, showing very high rates of cellular replacement in skeletal muscle and lung tissues, has provided the basis for the development of clinical protocols for the treatment of muscular dystrophy and serious lung disorders. We anticipate that such experimental therapy will become available over the next 1-2 years.

ACKNOWLEDGMENTS

Supported by grants R01 CA092344 from the National Cancer Institutes, National Institutes of Health, DHHS, #DAMB 17-01-0618 from the Department of Defense, and funds from the Adele R. Decof Cancer Center.

REFERENCES

1. Cuitis MA. New monoclonal antibodies for hematologic malignancies and breast cancer. M&H/RI 2003;86.

2. Lum LG, Sen M. ActivatedT-cell and bispecific antibody immunotherapy for high-risk breast cancer. Acta Haematol 2001; 105: 130-6.

3. Sen M, Wankowski DM, Garlie NK, et al. Use of anti-CD3 x anti-HER2/neu bispecific antibody for redirecting cytotoxiciry of activated T cells toward HER2/neu Tumors. J Hematother Stem Cell Res 2001;10: 247-60.

4. Grabert RC, Smith J, Tiggs J, et al. Anti-CD3 Activated T Cells Armed with OKT3 x Herceptin Bispecific Antibody, Survive and Divide, and Secrete Cytokines and Chemokines after Multiple Cycles of Killing Directed at Her2/neu+ Tumor Targets. Proceedings Amer Assoc Cancer Res 2003:44:656.

5. Smith JA, Davol PA, Kouttab N, et al. Activated T cells armed with anti-CD3 x anti-HER2/neu Bispecific antibody I: Anti-tumor activity and survival in SCID/beige mice. Blodd 2002;100:673a.

6. Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229.

7. Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infracted heart, improving function and survival. Proc Natl Acad Sci 2001;28;98:10344-9.

8. Kotton DN, Ma BY, Cardoso WV, et al. Bone marrow-derived cells as progenitors of king alveolar epithelium. Development 2001;128:5181-8.

9. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, and Sharkis SJ: Multi-organ, multi-lineage engraftment by a single bone marrow derived stem cell. Cell 2001;105:369-77.

10. Abedi M, Badiavas E, Lambert JF, et al. Trafficking and Transdifferentiation of bone marrow cells in a skin injury model. Abstracts/Experimental Hematol 2002;30:47.

11. Badiavas E, Abedi M, Butmarc J, et al. Participation of Bone Marrow Derived Cells in Cutaneous Wound Mealing. J Cell Physiol 2002;9999:1-6.

12. Brazelton TR, Rossi FMV, Keshet GI, Blau HM: From marrow to brain: Expression of neuronal phenotypes in adult mice. Science 2000;290: 1775-9.

13. Nilsson SK, Dooner MS, Weier HU, et al. Cells capable of bone production engraft from whole bone marrow transplants in nonablated mice. J Exp Med 1999; 189:729-34.

14. Ferrari G, Cusella-DeAngelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;227:1528-30.

15. Badiavas EV, Falanga V. Treatment of chronic wounds with bone marrow derived cells. Arch Dermatol in press 2003.

Lawrence G. Lum, MD, Ritesh Rathore, MD, Peter J. Quesenberry, MD, and Gerald J. Elfenbein, MD

Lawrence G. Lum, MD, is Chief of Basic Research, Director of Immunotherapy, and Director of Stem Cell Laboratory, Adele R. Decof Cancer Center, Roger Williams Medical Center, and Professor of Medicine, Boston University School of Mediane.

Peter J. Quesenberry, MD, is Director, Department of Research; Director, Center for Stem Cell Biology, and Professor of Medicine, Boston University School of Medicine.

CORRESPONDENCE

Lawrence G. Lum, MD

Adele R. Decof Cancer Center

Roger Williams Medical Center

825 Chalkstone Avenue

Providence, RI 02908

phone: (401) 456-2672

fax: (401) 456-2398

e-mail: llum@rwmc.org

Copyright Rhode Island Medical Society Aug 2003
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