Immunotherapy and Gene Therapy of Cancer

Steven A. Rosenberg Cancer Res 1991;51:5074s-5079s.

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[CANCER RESEARCH (SUPPL.) 51. 5074s-5079s. September 15. 1991)

Immunotherapy and Gene Therapy of Cancer1

Steven A. Rosenberg
National Cancer Institute, Bethesda, Maryland 20892

Abstract
In the past decade, immunothrapieshave been developed that are capable of causing prolonged cancer regressions in selected patients with advanced metastatic disease. In the past year, attempts at the gene therapy of cancer have begun. These experimental cancer treatments deserve vigorous exploration.

The last decade has witnessed the rise of new biological treatments for cancer based on stimulating natural immune reactions against the disease. These treatments, collectively referred to as biological therapy, are beginning to join surgery, radiation therapy, and chemotherapy as effective means for treating cancer (1, 2). Biological therapy can be denned as cancer treatments that act primarily through natural host de fense mechanisms or by the administration of natural mam malian substances. An increased understanding of the cellular immune system as well as new developments in biotechnology have made new approaches to biological therapy feasible. Using recombinant DNA technology, many biological compounds have become available in large amounts for the first time. One approach to the development of biological therapies for the treatment of cancer has involved attempts to identify im mune cells in tumor-bearing animals and patients that can distinguish cancer from normal cells and can be used in adoptive immunotherapy. Adoptive immunotherapy can be defined as the transfer to the tumor-bearing host of immunological re agents such as immune cells that have antitumor reactivity and can mediate, either directly or indirectly, antitumor effects. The major obstacle to the development of effective adoptive immu nothrapieshas been the inability to identify cells with specific antitumor reactivity that could be generated in large enough numbers for use in cancer treatment. In the past decade, however, several cell types have been identified that can cause the regression of cancer in some patients. We began our work in this field with the description of LAK cells which were non-MHC-restricted killer cells ca pable of lysing fresh tumor but not fresh normal cells in culture. These studies led us to the description of more specific antitumor cells called TIL that were capable of recognizing unique antigens on tumor cells. More recently we have begun genetic modification of these TIL in an attempt to increase their antitumor reactivity. Development of Interleukin Cancer 2-based Immunothrapies for

hormone, originally called T-cell growth factor, which was capable of causing the growth in culture of T-lymphocytes activated either by specific antigen or by lectins. This growth factor, subsequently renamed IL-2, opened new possibilities for expanding T-lymphocytes with antitumor activity for use in adoptive immunotherapy. In studies attempting to isolate Tcells from growing tumors we noted that lymphocytes exposed to IL-2 developed the ability to kill fresh tumor cells but not fresh normal cells in 4-h chromium release assays (3,4). Further studies revealed that lymphocytes from tumor-bearing as well as normal hosts in both the mouse and the human developed this lymphokine-activated killing activity for fresh cancer cells. In vitro these LAK cells showed ubiquitous non-MHC-re stricted killing of target cells altered either by malignant trans formation or by growth in culture. Shortly after the description of this in vitro activity we began extensive studies of the antitumor effects of the administration of IL-2 alone or IL-2 plus LAK cells (5-7). A summary of the findings in experimental animals using either IL-2 alone or LAK cells plus IL-2 is shown in Tables 1 and 2. These experi mental studies guided the development of the clinical protocols that followed. In these murine studies we demonstrated that treatment with either high dose IL-2 alone or LAK cells plus IL-2 could mediate the regression of established liver and lung mtastasesin a variety of tumor models. Each of these effects was dose related and using each dose of IL-2 more extensive antitumor effects were seen with the combined administration of LAK cells. The mechanism of action of these immunotherapy treatments was shown to depend on the activation of endoge nous CD8+ cytolytic T-cells and LAK cells, as well as the expansion in vivo of adoptively transferred lymphocytes. Thus a basic principle of adoptive immunotherapy was established in which IL-2-dependent lymphocytes with antitumor reactivity were used to treat the tumor-bearing host. Continued adminis tration of IL-2 led to the in vivo expansion of these lymphocytes which persisted as long as IL-2 administration continued. Fol lowing the discontinuance of IL-2 administration these IL-2dependent antitumor cells died in vivo. Our first studies of the in vivo administration of IL-2-dependent cells were performed in three patients with advanced sarcomas who received cells labeled with either MCr or '"In to monitor their in vivo distribution (4). It was found that these adoptively transferred IL-2-dependent cells distributed first to the lung followed by slow clearance to the liver and spleen. Although murine studies demonstrated that the administration of both IL-2 and LAK cells was necessary for maximal antitu mor effects, substantial practical difficulties existed in the ap plication of these findings to humans with advanced cancer. The very tiny amounts of IL-2 that were available precluded the administration of significant amounts to humans and also precluded the generation of LAK cells in sufficiently large amounts for use in human treatment. We thus began a series of Phase I studies in which naturally derived IL-2 from a high producer Jurkat cell line was administered to patients (8, 9). We also generated killer cells using exposure to phytohemagglutinin and administered these cells to patients in Phase I studies (10).

The difficulty in growing T-lymphocytes in culture for im munological studies represented a major impediment to the development of immunothrapies for the treatment of cancer. This difficulty was largely overcome by the description of a
1Presented at the Symposium. "Discoveries and Opportunities in Cancer Research: A Celebration of the 50th Anniversary of the Journal Cancer Research," May 15, 1991. during the 82nd Annual Meeting of the American Association for Cancer Research. Houston, TX. 'The abbreviations used are: LAK, lymphokine-activated killer; MHC, major histocompatibility complex; TIL, tumor-infiltrating lymphocytes; IL-2. interleukin 2; TNF, tumor necrosis factor.

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Table 1 Immunotherapy of murine tumors with IL-2 alone 1. Liver and lung micrometastases (3-day) from a variety of immunogenic and nonimmunogenic sarcomas, melanomas, and adenocarcinomas can be inhib ited by IL-2 administration. 2. Lung macrometastases (10-day) from two immunogenic sarcomas (but not from two nonimmunogenic sarcomas) can be inhibited by IL-2 administra tion. 3. A direct relationship exists between the dosage of IL-2 and therapeutic effect. 4. High dose IL-2 administration leads to in vivo lymphoid proliferation in visceral organs. These cells have LAK activity in vitro. 5. The immunotherapeutic effect of IL-2 on 3-day micrometastases is mediated by asialo-GMi-positive LAK cells. In immunogenic tumors, Lyt-2-positive cells also participate. 6. The immunotherapeutic effect of IL-2 on 10-day macrometastases is me diated by Lyt-2-positive cells. 7. Immunosuppression with irradiation or cyclophosphamide can inhibit IL-2 therapy against 3-day mtastases but can enhance the effects of IL-2 on 10day macrometastases. 8. The sensitivity of macrometastases to therapy involving IL-2 appears to be directly related to the expression of MHC antigens (Class I) on the tumor. 9. The administration of IL-2 can enhance the therapeutic effect of concomitantly administered LAK cells, TIL, and specifically sensitized T-lymphocytes.

Table 2 Immunotherapy of murine tumors with LAK cells plus IL-2 1. Liver and lung micrometastases (3-day) from a variety of immunogenic and nonimmunogenic sarcomas, melanomas, and adenocarcinomas can be inhibited by treatment with LAK cells plus IL-2. 2. A direct relationship exists between therapeutic effect and the dosage of IL2 and the dose of LAK cells. 3. The precursor of the LAK cell effective in vivo is Thy-1 negative, immunoglobulin negative, asialo-ASGMi positive. 4. Three-day incubation of splenocytes is optimal for the generation of LAK cells effective in vivo. 5. Immunotherapy of micrometastases with LAK cells and IL-2 is effective in hosts suppressed by total body irradiation or treatment with cyclophos phamide. Therapy is also effective in "B" mice (thymectomized, lethally irradiated, reconstituted with T-cell-depleted bone marrow). 6. Immunotherapy of micrometastases with allogeneic LAK cells plus IL-2 is effective. 7. LAK cells effective in immunotherapy can be generated from the spleno cytes of tumor-bearing mice. 8. Mtastases that persist after in vivo therapy with LAK cells plus IL-2 are sensitive to LAK cell lysis both in vitro and in subsequent in vivo experi ments. We have been unable to generate LAK-resistant tumor cells. 9. Administration of IL-2 leads to in vivo proliferation of transferred LAK cells. 10. Diffuse i.p. carcinomatosis can be successfully treated with i.p. LAK cells plus IL-2. 11. LAK cells can mediate antibody-dependent cellular cytotoxicity; thus ad ministration of IL-2 alone or LAK cells plus IL-2 can enhance the in vivo therapeutic efficacy of monoclonal antibodies with antitumor reac tivity.

The cloning of the gene for IL-2, its insertion into bacteria, and the availability of very large amounts of purified recombi nant IL-2, however, opened many new possibilities for the treatment of patients with these approaches. Phase I studies were conducted using either natural or recombinant IL-2 alone in 39 patients and using either phytohemagglutinin-activated killer cells or LAK cells alone in 27 patients with advanced cancer to determine the safe tolerable doses of each of these reagents (8-10). These Phase I studies have been reported in detail and served as the basis for the use of combinations of IL2 and LAK cells in patients with advanced cancer. None of the 66 patients with advanced cancer treated in these Phase I trials responded to treatment. In late 1984, we began studies combining treatment with LAK cells plus IL-2 in patients with advanced cancer and it was with this treatment that we observed our first antitumor responses in humans (11-14). In accord with lessons learned in animal models, LAK cells incubated for 3 or 4 days in IL-2 were administered in patients along with IL-2 given by bolus administration every 8 h. An update of the results of 180 patients treated as of 1990 with IL-2 plus LAK cells is shown in Table 3. Antitumor responses were seen in patients with advanced melanoma, renal cell cancer, colon cancer, and nonHodgkin's lymphoma. Approximately 10% of patients with advanced melanoma and renal cell cancer experienced a com plete remission of their cancer. As experience accumulated with the use of high dose IL-2 we began to see responses with IL-2 administration alone and these results are summarized in Table 3. These studies demonstrated that it was indeed possible to use strictly immune manipulations to mediate the regression of advanced cancer in humans. Prior studies with a-interferon had demonstrated tumor regression in selected patients with renal cell cancer although the direct antiproliferative effect of ainterferon made it unclear as to whether the antitumor effects were seen because of an alteration in host immune reactivity or the direct impact of a-interferon on the growing tumor (1, 2). IL-2 itself has no direct antitumor activity in any in vitro model and thus treatment with IL-2 or IL-2 plus LAK cells represented a true result of biological therapy in that it mediated its antitumor effects through natural host defense mechanisms. Tumor-infiltrating Lymphocytes

Although LAK cells could mediate antitumor effects, both in vitro and in vivo, we continued our search for more potent

Table 3 Immunotherapy of patients with advanced cancer using IL-2 alone or with LAK cells No. of patients IL-2 alone Diagnosis Evaluable" CR* CR + PR PR Evaluable' IL-2 + LAK cells CR + PR CR PR

RenalMelanomaColorectalNon-Hodgkin's lymphomaBreastSarcomaLungOtherTotal604112113111"1365000000056100000001618240000001572483071659'17884110000141763300002935211357000024

" Includes all treated patients except four who died of therapy. * CR, complete response; PR, partial response. ' Includes all treated patients except one lost to follow-up and one who died of therapy. Two patients each with hepatoma and brain cancer; one each with ovary, pancreas, and uterine cancer. ' One patient each with cancer of brain, esophagus, ovary, testes, thyroid, gastrinoma. unknown primary and two patients with Hodgkin's lymphoma.

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lymphocytes that might be used in adoptive immunotherapy. Our specific interest was the identification of T-cells that could recognize specific tumor antigens in a MHC-restricted fashion. Techniques were developed for isolating TIL from resected tumors and these cells provided evidence of specific tumor antigen recognition by lymphocytes obtained from a tumorbearing host (15-18). These cells were isolated by culturing single cell suspensions from tumors in IL-2. Infiltrating lym phocytes that bore IL-2 receptors, presumably because of their reactivity to tumor, grew in the presence of IL-2 and destroyed tumor cells simultaneously growing in the culture. Thus, 2 to 3 weeks after initiating cultures pure populations of TIL could be established without the presence of contaminating tumor cells. We established techniques for isolating TIL from both mouse and human tumors and extensively studied these cells in vitro as well as in tumor models in mice. In the mouse, TIL cells were exclusively CD8+ although in the human both CD4+ and CD8+ T-cells grew in culture. In the mouse, specific recognition of tumor antigens could be identified on the basis of specific lysis of target cells as well as by specific cytokine secretion when TIL were coincubated with the type of tumor cells from which they were derived (19-21). These reactivities were MHC restricted and could be inhibited by monoclonal antibodies against Class I antigens. Extensive studies were conducted in animal models of the antitumor effects of tumor-infiltrating lymphocytes. These studies are summarized in Table 4. Titration of TIL in mice with established lung and liver mtastasesindicated that these cells were from 50 to 100 times more potent than LAK cells in reducing lung mtastases (15). In advanced tumor models, TIL plus IL-2, but not LAK cells plus IL-2 in conjunction with cyclophosphamide, was effective in eliminating established m tastases from the lung and liver. Following these early studies with TIL cells, attempts were made to find ways to improve their antitumor efficacy in mice. Because of our finding that TIL might not be effective against tumors that expressed very low levels of Class I antigens we transfected genes coding for Class I antigens into tumors and showed that TIL raised from these tumors could mediate their regression as well as the regression of the parental non-Class I expressing tumor after treatment of the mouse with -y-interferon. Gamma interferon was shown to up-regulate Class I MHC antigens in vivo and thus the vital role played by MHC expression in these tumor immunothrapies was established (22-26). We further showed that local tumor irradiation could
Table 4 Immunotherapy of murine tumors with TIL and IL-2 1. The administration of TIL + IL-2 is 50-100 times more effective than LAK cells + IL-2 in reducing established (day 3) lung micrometastases. 2. The administration of TIL + IL-2 + cyclophosphamide is effective in treat ing mice with advanced lung (day 14) or liver (day 8) mtastases. All three agents are required for effective treatment. 3. The phenotype of TIL effective in vivo is CD3+, CD4-, CD8+. 4. TIL raised from tumors that express Class 1 antigens only after transfection with genes coding for Class I can mediate the regression of micrometastases from the parental tumor after treatment of the mouse with -y-interferon. 5. Local tumor irradiation synergizes with TIL and IL-2 administration in mediating the regression of established mtastases. Local irradiation can substitute for cyclophosphamide. 6. TIL with improved antitumor activity in vivo can be generated from tumor suspensions by using immunomagnetic beads to isolate lymphocytes followed by incubation of lymphocytes in low dose IL-2. 7. In mice cured of lung micrometastases by administration of TIL. live TIL can be identified in vivo 3 months after injection. 8. The specific secretion of f-interferon by TIL when cultured with tumor is the best in vitro correlate of the in vivo antitumor effectiveness of TIL. Nonlytic TIL that specifically secrete >-interferon can effectively treat established lung micrometastases.

Table 5 TIL treatment of patients with melanoma" Objective response (PR + CR) PR + CR (all)

NR"

(number of patients) NoIL-2IL-2 previous CYIL-2 + CY)Previous (No IL-2IL-2 CYrIL-2 + (no CY)177341143111/284/113/61/539365020 " Excludes patients with brain mtastases at start of treatment (all NR). * NR, no response; PR, partial response; CR, complete response; CY, cyclo phosphamide. ' Excludes two patients who received IL-2 at 30,000/kg; both were NR.

substitute for cyclophosphamide in synergizing with TIL and IL-2 (27). The reason for the need for this local tumor treat ment, either with cyclophosphamide or irradiation is not clear. It is possible that these treatments result in elimination of suppressor cells or increase the traffic of immune cells to the tumor site. The cytoreductive effect of these treatments may also play a role in their effectiveness. More recently, improved techniques for generating TIL with antitumor activity in vivo have been established. The use of low dose IL-2 (60 to 120 lU/ml) generates cells with more specific cytolytic activity and more effectiveness in vivo than the use of high dose IL-2 (6000 lU/ml) (21). The long term persistence of TIL in mice cured by the in vivo injection of TIL has been demonstrated using congenie Thy-1.1 TIL in Thy-1.2 mice bearing syngeneic tumors (28). Extensive efforts to identify the characteristics of TIL in mice that correlate with antitumor reactivity have shown that specific cytokine secretion rather than specific lysis is most critical (20). Thus these studies of the in vivo antitumor activity of TIL in murine models have played an important role in guiding the design and conduct of human trials with TIL in patients with advanced cancer. Based on these animal models, pilot studies have begun the treatment of patients with advanced metastatic melanoma using tumor-infiltrating lymphocytes (29). The results of our first 50 patients with advanced melanoma treated with TIL are shown in Table 5. Thirty-eight % of patients have responded to treat ment. Interestingly, similar response rates were seen in patients previously failing high dose IL-2-based therapy compared to those that had previously not been treated with IL-2. TIL have been valuable for studying the nature of the immune response of humans to their cancers. From approximately onethird of patients with melanoma, TIL with specific cytolytic activity for the autologous tumor and not autologous normal tissues can be isolated (16-18). More recently we have shown that specific cytokine secretion can also identify immune re sponses in patients with growing malignancy. Specific cytokine secretion has identified immune cells in patients with melanoma and breast cancer. TIL have been valuable in studying the nature of shared antigens among patients with cancers of similar histology. Extensive studies of TIL from patients with melanoma tested against a panel of allogeneic melanomas with known HLA subtypes have demonstrated that melanomas from different individuals appear to share common tumor antigens that are restricted by a specific HLA specificity (30). The HLA-A2 genotype appears to be particularly common as a restriction element in the recognition of melanoma antigens. To further demonstrate the nature of the shared antigens in patients with melanoma, we have transfected the gene for HLA-A2 into

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melanomas and have shown that 6 of 6 different melanomas transfected with the gene for HLA-A2 are recognized by a single HLA-A2-restricted TIL (31). It thus appears that anti gens shared among melanomas can be recognized by TIL and possibilities for the use of these antigens for immunization of patients against cancer appear feasible. Using TIL as a recog nition system we are currently in the process of attempting to clone the gene that codes for tumor antigens in both the mouse and human by transfection of genes from sensitive to resistant cell lines. Should such genes be cloned they could be inserted into vaccinia or other viruses for use in the immunization of patients against cancer antigens. Finally studies with TIL have demonstrated that these cells recirculate and specifically localize in cancer deposits. Up to 0.015% of all injected TIL localized per g of tumor based on in vivo studies in humans using TIL labeled with '"In (32, 33). Thus TIL cells have proved to be a valuable resource not only for the study of the immune reaction of patients against their tumors but also for use in immunotherapy. Extensive efforts under way to improve upon immunotherapy with TIL are listed in Table 6 including attempts to enhance the in vivo effective ness of existing TIL or to generate more effective TIL either by growing more effective cells, by genetically modifying TIL, or by raising TIL from genetically modified tumor. In addition studies are under way to study these IL-2-based immunothra pies in conjunction with other treatments such as chemotherapy and radiation therapy. New recombinant cytokines are being tested for antitumor reactivity. We have recently published our results showing that IL-6 has antitumor activity when admin istered to tumor-bearing mice (34) and clinical trials with IL-6 will be undertaken shortly. Gene Therapy of Cancer The ability to introduce and express foreign genes in eukaryotic cells has opened new possibilities for the therapy of cancer. Our first attempts at the gene therapy of cancer involved modification of TIL by the insertion of marker genes (35, 36). More recently genes coding for cytokines have been introduced into TIL. In the first phase of these studies we inserted into TIL a bacterial gene coding for neomycin phosphotransferase which could induce resistance to the antibiotic neomycin and enable us to differentiate adoptively transferred TIL from endogenous host lymphocytes (35, 36). The goal of these studies was to demonstrate the feasibility and safety of using retroviral me diated gene transfer to introduce genes into humans and to study the long term distribution and survival of autologous TIL. Because of the practical, safety, and ethical issues involved with human gene transfer and the possible consequences of using a modified retrovirus derived from the Moloney murine leukemia retrovirus randomly integrating a new gene into the human genome, our clinical studies utilizing human TIL trans duced with the gene coding for neomycin resistance (NeoR) were preceded by extensive in vitro studies and were extensively reviewed by a variety of review groups composed of clinicians, scientists, ethicists, and lay people. In these studies we dem onstrated that the gene could be successfully inserted into human TIL, that the properties of human TIL were not altered, that the gene was expressed in TIL, and that these procedures could be performed with little risk to the patient and no risk to health care personnel and the public. Retroviral mediated gene transduction of TIL was selected because of the high efficiency

Table 6 Experimental leads for improving immunotherapy \. Enhancing in vivo effectiveness of TIL a. Combination with other cytokines b. Combination with local radiation therapy 2. Generating more effective TIL a. Lymphocyte subpopulations or clones b. Repeated in vitro stimulation c. Low-dose IL-2 d. Culture in IL-2 + IL-4 e. Modify TIL by gene transfer 3. Immunize with gene-modified tumor a. Cytokine genes b. MHC antigen genes 4. Application of new cytokines a. IL-6 b. Cytokine combinations 5. Monoclonal antibodies a. + IL-2 + LAK cells (LAK cells mediate antibody-dependent b. + macrophage-colony-stimulating factor 6. Synergy of chemotherapy 4- IL-2

of gene transfer using this technique (37, 38). Studies performed on TIL from multiple cancer patients as well as the 10 patients who have now received NeoR genemodified TIL demonstrated that the NeoR gene could be in serted into human TIL, that it was satisfactorily expressed, and that the general properties of the TIL were not changed (35, 36). Semiquantitative Southern blots indicated that approxi mately one viral genome copy was present in the gene-trans duced TIL. The phenotype and cytotoxicity of the transduced and nontransduced cells were similar. Analyses of T-cell recep tor gene rearrangements revealed the oligoclonal nature of the TIL population and no major changes in the DNA re arrangement patterns or the level of mRNA expression of the and 7 chains following transduction and selection of TIL in the neomycin analogue G418 (35). Similarly, cytokine mRNA expression was not significantly altered following the transduc tion of TIL. Transduced TIL consistently showed resistance to G418, a neomycin analogue lethal to all eukaryotic cells. The polymerase chain reaction technique was utilized to detect the NeoR genome. Approximately one transduced cell in 10s normal cells could be consistently detected (36). Safety considerations were paramount in our consideration of the use of this gene transduction technique for introducing genes into TIL for use in patient therapy. Extensive safety studies were performed on all transduced TIL including tests for aerobic and anaerobic bacteria, fungi, and Mycoplasma and tests using sarcoma-positive, leukemia-negative assays for ecotropic, xenotropic, and amphotropic infectious viruses. Ampli fication tests using NIH 3T3 cells were performed and were shown to be capable of detecting a single viral particle per ml of solution. All safety tests performed on TIL in preclinical studies as well as on TIL from the patients who received the gene-modified cells were negative and no safety problems of any kind were detected (36). Gene-modified cells could be detected in the circulation up to 189 days and in tumor deposits up to 64 days after the infusion of gene-modified TIL (36). Laboratory and clinical studies using TIL transduced with the gene for neomycin resistance demonstrated that retroviral mediated gene transduction was a feasible and safe means for introducing genes into humans. Because of the accumulation of TIL at tumor deposits we have conducted studies attempting to modify human TIL with genes that can improve the antitumor effectiveness of these cells. The first gene that we have selected for these studies is the gene for TNF. Extensive animal research in the Surgery Branch, National Cancer Institute, and in many other groups have demonstrated

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that the injection of recombinant TNF can mediate the necrosis and regression of a variety of established murine cancers. How ever, tumor-bearing mice can tolerate up to 400 Mg/kg TNF and these doses are required to mediate tumor regression; the administration of less TNF is far less effective. In contrast, the maximum tolerated dose of TNF in humans in both Surgery Branch, National Cancer Institute, and other studies is approx imately 8 /g/kg day O)- Thus when given i.v. injections humans can tolerate only 2% of the TNF dose required to mediate antitumor effects in the mouse and this appears to explain why antitumor effects have not been seen when TNF was adminis tered systemically to humans. We have thus sought means to selectively increase the local concentration of TNF at the tumor site. Because TIL can traffic directly to tumor deposits and concentrate at those sites (32, 33) we have hypothesized that TIL that are genetically modified to produce large amounts of TNF may generate high TNF concentrations in the local tumor microenvironment and thus exhibit an increased antitumor effect compared to normal TIL. The retroviral vector construct selected for insertion of the TNF gene into human TIL contains the TNF gene promoted by the murine long terminal repeat and the neomycin resistance gene promoted by the SV40 early promoter region. The twogene retroviral construct was introduced into the PA317 pro ducer cell line and the supernatant from this line was used to transduce the TIL from patients with advanced metastatic melanoma. Although it has been relatively easy to express cytokine genes in tumor cells and demonstrate production of large amounts of TNF, difficulties exist in the expression of cytokine genes in lymphocytes. It has been difficult to achieve consistently high levels of cytokine production in lymphocytes using these retroviral vectors probably because regulatory mechanisms exist for cytokine transcription, translation, and/or secretion in lympho cytes that do not exist in other cell types. We have, however, been able to achieve the expression of TNF genes in lympho cytes from selected patients and have achieved cytokine secre tion in excess of 100 pg/106 cells/24 h. We have begun clinical studies using these TNF gene-modi fied TIL in patients with advanced cancer. Increasing numbers of TNF-transduced TIL are being administered, first in the absence of IL-2 and then when safely tolerated doses are achieved, in conjunction with IL-2. To the present time four patients with advanced melanoma have been treated with the TNF gene-modified TIL. No side effects have been seen at cell doses which have ranged up to 10" cells in a single infusion. A large series of patients treated with gene-modified TIL plus IL2 will be required to know whether or not this approach gives improved results compared to the use of unmodified TIL. We currently have permission to treat up to 50 patients with TNF gene-modified TIL utilizing this approach. Additional genes being studied for insertion into TIL to improve their antitumor activity include 7-interferon, IL-2, IL6, and genes for chimeric T-cell receptors. More recently we have begun studies investigating the use of genetic modification of tumor cells to increase their immunogenicity. We and others have demonstrated that insertion of cytokine genes can increase the immune recognition of tumor cells and can lead to the production by the host of cytolytic cells that are not produced in response to the parental nonmodified tumor (39-43). These experiments have been con ducted by a variety of groups and include insertion of genes for IL-4, IL-2, tumor necrosis factor, 7-interferon, and granulo-

cyte-macrophage-colony-stimulating factor. In related experi ments we have shown that insertion of the gene for Class I major histocompatibility antigens can also increase the immunogenicity of tumors and lead to the generation of TIL that cannot be produced from low MHC-expressing tumors (26). This approach to gene therapy, by genetically modifying tumors for use in immunization, holds promise not only for active immunotherapy but also for the development of immune cells that might be used in adoptive immunotherapy. In the last decade biological therapy has been shown to be capable of mediating the regression of established cancers in some patients with advanced malignancy. The continued devel opment of these biological approaches offers the hope that they can lead to the development of effective, safe, and practical treatments for patients with cancer. References
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31. Kawakami. Y.. Zakut. R., Topalian, S. L., Stotter. H.. and Rosenberg, S. A. Shared human melanoma antigens: recognition by tumor infiltrating lym phocytes in HLA-A2.1 transfected melanomas, submitted. 1991. 32. Fisher. B., Packard. B. S., Read, E. J., Carrasquillo, J. A., Carter. C. S., Topalian, S., Yang, J. C., Volles, P., Larson, S. M., and Rosenberg, S. A. Tumor localization of adoptively transferred indium-111 labeled tumor infil trating lymphocytes in patients with metastatic melanoma. J. Clin. Oncol., 7:250-261, 1989. 33. Griffith. K. D.. Read, E. J., Carrasquillo. J. A.. Carter, C. S., Yang, J. C., Fisher, B., Aebersold. P.. Packard. B. S., Yu, M. V.. and Rosenberg, S. A. In vivo distribution of adoptively transferred indium-Ill labeled tumor infiltrating lymphocytes and peripheral blood lymphocytes in patients with metastatic melanoma. J. Nail. Cancer Inst., 81: 1709-1717, 1989. 34. Mul. J. J.. Mclntosh. J. K.. Jablons. D. M., and Rosenberg. S. A. Antitumor activity of recombinant nterleukin-6 in mice. J. Exp. Med., 171: 629-636. 1990. 35. Kasid, A., Morecki. S.. Aebersold. P.. Cornetta, K.. Culver. K., Freeman, S., Director, E., Lotze, M. T., Blaese, R. M., Anderson. W. F.. and Rosenberg, S. A. Human gene transfer: characterization of human tumor infiltrating lymphocytes as vehicles for retroviral mediated gene transfer in man. Proc. Nati. Acad. Sci. USA. 87: 473-477. 1990. 36. Rosenberg, S. A., Aebersold, P., Cornetta, K., Kasid. A.. Morgan, R. A., Moen, R., Karson, E. M., Lotze. M. T., Yang. J. C., Topalian, S. L., Merino, M. J., Culver, K., Miller, A. D. E., Blaese, R. M., and Anderson. W. F. Gene transfer into humans: immunotherapy of patients with advanced melanoma using tumor-infiltrating lymphocytes modified by retrieval gene transduction. N. Engl. J. Med., 8: 323:570-578, 1990. 37. Bender, M. A., Palmer. T. D.. Gelinas, R. E., and Miller, A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J. Viro!.. 61: 1639-1646, 1987. 38. Armentano, D., Yu, S. F., Kantoff. P. S., von Rden, T., and Anderson, W. F. Effect of internal viral sequences on the utility of retroviral vectors. J. Virol., 61: 1647-1650. 1987. 39. Fearon, E. R.. Pardoll. D. M., Itaya, T.. Golumbek. P., Levitsky, H. I.. Simons, J. W., Karasuyama, H., Vogelstein, B., and Frost, P. Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response. Cell. 60: 397-403. 1990. 40. Gansbacher. B.. Zier, K.. Daniels, B., Cronin, K., Bannerji. R., and Gilboa. E. Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity. J. Exp. Med., 172: 1217-1224, 1990. 41. Gansbacher, B., Bannerji. R.. Daniels, B.. Zier, K., Cronin, K., and Gilboa, E. Retroviral vector-mediated -y-interferon gene transfer into tumor cells generates potent and long lasting antitumor immunity. Cancer Res., SO: 7820-7825, 1990. 42. Asher, A. L., Mul, J. J., Kasid, A., Restifo, N. P., Salo, J. C., Reichert, C. M., Jaffe, G., Fendly, B., Kriegler, M., and Rosenberg, S. A. Murine tumor cells transduced with the gene for tumor necrosis factor-. Evidence for paracrine immune effects of tumor necrosis factor against tumors. J. Immu nol., 146: 3227-3234, 1991. 43. Colombo, M. P., Ferrari, G., Stoppacciaro, A., Parenza, M., Rodolfo, M., Mavilio, F., and Parmiani. G. Granulocyte colony-stimulating factor gene transfer suppresses tumorigenicity Exp. Med.. 173: 889-897. 1991. " of a murine adenocarcinoma in vivo. J.

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