Identification of fibroblast growth factor-5 as an overexpressed antigen in multiple human adenocarcinomas.

Methodology for identifying tumor-associated antigens recognized by T cells has been successfully used to clone antigens from melanoma cells. Similar efforts for nonmelanoma tumors have had limited success with few antigens identified. To identify potentially relevant tumor-associated antigens expressed in renal cell carcinoma cell lines, a tumor-specific CTL clone was established from tumor-infiltrating lymphocytes from a regressing pulmonary lesion. This CTL recognized nonmutated fibroblast growth factor-5 (FGF-5). Quantitative real-time reverse transcription PCR revealed that FGF-5 was overexpressed in the majority of renal cell carcinomas, as well as in some prostate carcinoma and breast carcinoma lines. FGF-5 expression by quantitative real-time reverse transcription PCR in normal tissues was below the recognition threshold for this CTL. As a normal protein with significant overexpression by multiple adenocarcinomas and little normal tissue expression, FGF-5 represents an immunotherapy target with potential utility against a broad array of nonmelanoma cancers.

The envelope protein of an endogenous murine retrovirus is a tumor-associated T-cell antigen for multiple murine tumors.

Recently, significant progress has been made in identifying specific tumor-associated antigens recognized by T cells and defining the specific peptide epitopes within these proteins that are processed and presented on class I major histocompatibility antigens. Most of these antigens have been identified in human melanoma, where many of them appear to be tissue-specific, nonmutated proteins expressed by melanoma and normal melanocytes but not by other tissues. There has been much less progress in identifying the tumor antigens on murine tumors that are recognized by T cells, and this has restricted the development of preclinical animal models for immunotherapy. The authors previously described a method for generating tumor-reactive T cells from murine tumors (tumor infiltrating lymphocytes) that are CD8+ T cells recognizing autologous tumor and that can inhibit established tumor on adoptive transfer. Here the authors show that the envelope protein of an endogenous murine retrovirus of the AKV family, found in the germline of the C57BL/6 mouse, is recognized by tumor-infiltrating lymphocytes from two histologically different tumors syngeneic to that mouse strain. Furthermore, the authors identify the specific 9-amino acid peptide from the p15E transmembrane component of this envelope protein that is recognized in the context of major histocompatibility complex Kb, show that it is naturally presented and recognized on several other H-2b tumors, and that cytotoxic T lymphocytes specific for this epitope are therapeutic for these antigen-expressing tumors on adoptive transfer.

High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy.

A majority of the human tumor-associated Ags characterized to date are derived from nonmutated "self"-proteins. Little is currently understood about the nature of the self-reactive lymphocytes that recognize these Ags. We recently characterized two nonmutated tumor-associated Ags for the B16 murine melanoma: tyrosinase-related protein-2 (TRP-2) and the endogenous retroviral envelope protein, p15E. We previously reported that both TRP-2 and p15E reactive CTL could be detected in the spleens of naive animals after a single in vitro stimulation using 10(-5)-10(-6) M of the appropriate Kb-binding 9-amino acid epitope. In this report we show that the CTL found in naive animals are low avidity lymphocytes, that respond only to high concentrations of peptide in vitro. We demonstrate that titration of in vitro-stimulating peptide to limiting concentrations distinguishes qualitative differences in the lymphocyte reactivity to these two Ags between vaccinated and unvaccinated animals. We further demonstrate that in vitro expansion of CTL in either high or low concentrations of stimulating peptide generated CTL cultures with different avidities for the relevant epitopes. CTL expanded in low concentrations demonstrated higher avidity for peptide-pulsed targets and better tumor recognition, when compared to CTL generated in the presence of high concentrations of Ag. More importantly, high avidity CTL demonstrated superior in vivo antitumor activity. These results demonstrate that qualitative differences in the CTL that recognize these two self-Ags are critically important to their in vitro and in vivo anti-tumor efficacy.

Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma.

Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinase-related protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.

The use of interleukin-6 to generate tumor-infiltrating lymphocytes with enhanced in vivo antitumor activity.

Tumor-infiltrating lymphocytes (TIL) are cytotoxic T cells isolated from solid tumors and expanded in vitro in recombinant interleukin-2 (rIL-2). TIL have antitumor effects in murine models and in some patients with melanoma. In an effort to generate murine TIL with enhanced in vivo therapeutic efficacy, viable tumor cells were coinjected with a collagen matrix plus recombinant human IL-6 (rIL-6) subcutaneously into syngeneic mice to achieve sustained local concentrations of rIL-6 at the tumor site from which TIL were derived. In five separate experiments, single cell suspensions of tumors were admixed with either (a) Hanks' balanced salt solution (HBSS), (b) 2% (20 mg/ml) collagen matrix only, (c) 250 micrograms rIL-6 only, or (d) 250 micrograms rIL-6 in a 2% collagen matrix (prolonged release) before subcutaneous inoculation. These tumors were subsequently resected and TIL were isolated and expanded in vitro. TIL generated from tumors admixed with matrix plus rIL-6 were significantly more effective than TIL expanded from tumors admixed with HBSS (four of five experiments), TIL from tumors admixed with matrix only (five of five experiments), and TIL from tumors admixed with rIL-6 only (three of four experiments) in an established tumor treatment model. In no experiment was any other TIL culture superior to TIL grown from tumors augmented with collagen matrix plus rIL-6. These results suggest that strategies designed to increase the local concentrations of cytokines at tumor sites may lead to the generation of more potent TIL for clinical administration.

Mechanisms of adoptive immunotherapy: improved methods for in vivo tracking of tumor-infiltrating lymphocytes and lymphokine-activated killer cells.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and lymphokine-activated killer cells has been demonstrated to mediate regression of tumors in murine models and in selected patients with advanced cancer. Improved methods for monitoring immune cell traffic, particularly to sites of tumor, are needed to elucidate mechanisms of antitumor activity and optimize treatment protocols. Traditional cell tracking methods such as fluorescent protein labeling and radiolabeling using 111In, 125I, or 51Cr are limited by isotope half-life, leakage or transfer of label from immune cells, and toxicity or altered cell function caused by the labeling process. Labeling with genetic markers allows long-term cell tracking but is laborious to perform and difficult to quantitate. We have used two recently described lipophilic cell tracking compounds (PKH26 and 125I-PKH95) which stably partition into lipid regions of the cell membrane to track immune cells in vivo. Concentrations of each tracking compound which had no adverse effects were determined for a variety of murine TIL and lymphokine-activated killer cell functions. Viability was unimpaired at labeling concentrations of up to 5 microM for PKH95 and 20 microM for PKH26. TIL proliferation was unaltered by labeling with up to 5 microM PKH95, 20 microM PKH26, or a combination of 15 microM PKH26 and 5 microM PKH95. In vivo cytotoxic effector function and in vivo therapeutic efficacy of lymphokine-activated killer cells and TIL were also unimpaired by labeling with 20 microM PKH26 or 1 microM 125I-PKH95. Subsequent studies in an adoptive transfer immunotherapy model used 125I-PKH95 to track the biodistribution of TIL in tumor and in non-tumor-bearing animals and PKH26 fluorescence to monitor microdistribution within tissues and distinguish TIL from host T-cells. The results suggest that differential accumulation, selective retention, or proliferation at the tumor site cannot account for the observed pattern of therapeutic efficacy. We hypothesize that a minimum number of TIL must reach the tumor site in order to achieve a demonstrable therapeutic effect.

A tumor-elaborated supernatant factor chemotactic for IL-2 expanded tumor infiltrating T-lymphocytes.

Very little is known about factors influencing the migration of highly activated T-lymphocytes. One such lymphocyte population is the IL-2 expanded population of T cells infiltrating tumors. These tumor-infiltrating lymphocytes (TIL) can cause tumor regression in patients with metastatic cancer and in murine tumor models when given in adoptive transfer. In patients with melanoma, these TIL have been shown to migrate to sites of tumor and this may be a critical factor in their antitumor activity. In this study, a 48-well microchemotaxis chamber and a 5 microns pore nitrocellulose filter membrane system was utilized to study the motility of murine TIL. A chemotactic response was observed to supernatants from freshly explanted, autologous, and nonautologous tumor cultured for 24 h. Serially passaged autologous and nonautologous tumors also produced supernatants with chemotactic activity. Supernatants from single cell suspensions of normal tissues prepared and cultured identically did not elicit chemotaxis. Chemotactic activity for TIL was not removed by dialysis (2000 MW exclusion limit), its activity was undiminished by heat treatment at 60 degrees C for up to 60 min, and it was trypsin sensitive. Tumor supernatants were also chemotactic for two IL-2-dependent specifically alloreactive CTL lines (CTL-TIM and OE-4), but not two helper T cell lines (D-10 and D-1.5) or normal resting lymphocytes. This is the first demonstration of a chemotactic effect on IL-2-dependent, activated T cells. Characterization and purification of factors from tumor responsible for this directed migration are in progress.

Murine studies using polyethylene glycol-modified recombinant human interleukin 2 (PEG-IL-2): antitumor effects of PEG-IL2 alone and in combination with adoptive cellular transfer.

A polyethylene glycol-modified form of recombinant human IL-2 (PEG-IL-2) was tested for murine antitumor effects in vitro and in vivo. This PEG-IL-2 was demonstrated to retain the in vitro ability to support T cell proliferation, enhance a mixed lymphocyte reaction, and generate lymphokine-activated killer (LAK) cells. It was found to have a circulating half-life in mice 25 times longer than unmodified recombinant IL-2 (RIL-2). Serum levels were detected up to 60 h after a single intravenous injection. When given as a single, intravenous administration the antitumor effect of this material was similar to multiple, repeated bolus doses of RIL-2. PEG-IL-2 was also found to support the in vivo efficacy of adoptively transferred LAK cells and tumor infiltrating lymphocytes (TIL). Using a congenic TIL (Thy 1.1), persistence of adoptively transferred TIL was found to be prolonged with PEG-IL-2 compared to repeated boluses of RIL-2. Four days after transfer, twice as many Thy 1.1 TIL were recoverable from the lungs of mice given PEG-IL-2. These studies show that PEG-IL-2 is a modified lymphokine with significant antitumor activity in murine systems and is superior to bolus RIL-2 in enhancing the survival of adoptively transferred TIL.

An improved method for growing murine tumor-infiltrating lymphocytes with in vivo antitumor activity.

Tumor-infiltrating lymphocytes (TILs) are host T cells that can be grown from fresh murine and human tumors using interleukin-2 (IL-2) in bulk cultures. These activated T cells have been shown to have significant antitumor activity both in vitro and in vivo. A technique is described for the separation of Thy-1.2-positive TILs from fresh murine tumors using antibody-coated magnetic beads, permitting the examination of growth conditions for these cells. TILs with increased therapeutic efficacy are obtained from the immunogenic MCA 38 and MCA 105 tumors when culture conditions employing low levels of IL-2 (10 vs. 1,000 U/ml) and irradiated autologous tumor restimulation are used. TILs grown under these conditions can mediate a 93% reduction of 3-day-old established pulmonary metastases when as few as 2.5 x 10(5) cells are adoptively transferred with systemic IL-2. These culture conditions are utilized to grow TILs from the nonimmunogenic MCA 102 tumor for which bulk TIL culture methods are unsuccessful. MCA 102 TILs grown in this fashion demonstrate in vivo therapeutic efficacy against established autologous pulmonary metastases.

Distinct immunologic specificity of tumor regression mediated by effector cells isolated from immunized and tumor-bearing mice.

Sensitized T lymphocytes can mediate potent antitumor effects when transferred to tumor-bearing animals. Employing the MCA 105 and MCA 106 sarcomas, we were able to generate antitumor effector cells by immunization of syngeneic mice with tumor cells admixed with Corynebacterium parvum. These immune splenocytes could be further sensitized and expanded in culture by the in vitro sensitization (IVS) method utilizing tumor stimulator cells and IL-2. Adoptive immunotherapy of pulmonary metastases mediated by noncultured splenocytes from immunized mice or immune IVS cells showed exquisite specificity between the two sarcomas. These results demonstrate the presence of tumor-specific antigens on MCA 105 and MCA 106 tumor cells which can serve as target molecules for immunotherapy. Recently, we have generated therapeutic T lymphocytes from mice bearing progressively growing tumors by the IVS method. However, IVS cells from tumor-bearing mice showed cross-reactivity between the MCA 105 and 106 sarcomas in adoptive immunotherapy experiments. Since these IVS cells did not affect other control tumors, the limited cross-reactivity suggests the presence of common tumor-associated antigens on MCA 105 and MCA 106 tumor cells which can also serve as the target for tumor rejection. Therefore, immune responses to progressive tumor growth and to immunization are distinct with respect to antigen recognition by T lymphocytes.

Development of antitumor reactivity in regional draining lymph nodes from tumor-immunized and tumor-bearing murine hosts.

With use of the weakly immunogenic MCA 105 tumor of the C57BL/6 mouse, the antitumor reactivity of lymphoid cells derived from the regional draining lymph nodes (RLN) of tumor-immunized and tumor-bearing mice was examined. Mice were immunized with an inoculation of viable tumor admixed with Corynebacterium parvum. Excision of tumor immunization sites within 4 days abrogated the development of systemic immunity to reject a tumor challenge. However, excision of the immunization site on day 6 did not interfere with the development of systemic antitumor immunity. In subsequent experiments, tumor immunization sites were excised on day 6 in all mice and the RLN either left intact or excised on day 6 or day 14. The development of systemic tumor immunity was severely impaired if RLN were excised on day 6, indicating the pivotal role of the RLN. Excision of the RLN on day 14 had no impact on the development of systemic immunity, thus indicating that the requirement for the RLN was time dependent. In mice bearing progressively growing tumors, lymphoid cells derived from RLN were examined for therapeutic efficacy in adoptive immunotherapy experiments. Although fresh RLN cells harvested 6 and 14 days after tumor inoculation did not demonstrate inherent therapeutic efficacy, after in vitro sensitization with irradiated tumor cells and interleukin-2, these RLN cells acquired significant antitumor activity in adoptive immunotherapy experiments. These data indicate that RLN are essential in the development of tumor immunity and may be used as a source of therapeutic effector cells for adoptive immunotherapy.

Cellular basis of immunologic interactions in adoptive T cell therapy of established metastases from a syngeneic murine sarcoma.

The adoptive transfer of specifically sensitized T lymphocytes can effectively mediate the regression of established local and metastatic tumors. Previous experiments using the weakly immunogenic MCA 105 sarcoma indicated that cellular interactions between transferred L3T4+ helper and Lyt-2+ cytotoxic immune T cells were necessary for mediating tumor regression. In this study, the kinetics of T-T cell interactions were analyzed by in vivo depletion of T cell subsets with mAb. The anti-tumor efficacy of transferred immune cells was abrogated by in vivo administration of either L3T4 or Lyt-2 mAb on the day of cellular therapy. However, if mAb were given 3 days after the transfer of immune cells, depletion of Lyt-2+ but not L3T4+ cells abrogated anti-tumor efficacy. T cell depletion on day 6 after transfer of immune cells had no adverse effect on tumor regression, indicating the period required for T cell reactivity in vivo. Furthermore, depletion of Ia+ cells by in vivo mAb treatment abrogated the anti-tumor efficacy of immune cells. It is thus hypothesized that there are two distinct but sequential phases of in vivo T cell interactions leading to the regression of established tumors after adoptive immunotherapy. An initial "helper/inducer" phase apparently requires the interaction of L3T4+ immune cells and the tumor Ag involving Ia+ cells. The inducement of L3T4+ cell activation is to provide helper function via the secretion of IL-2. The second phase designated as an "effector phase" involves differentiation of immune Lyt-2+ cells under the influence of IL-2 secreted during the helper/inducer phase for generation of mature Lyt-2+ effector cells. To further support the hypothesis of a two-phase process we have examined the phenotype and kinetics of tumor regression mediated by effector cells generated by secondary in vitro sensitization (IVS). Although the IVS cells were generated from fresh MCA 105 immune spleen cells, their anti-tumor efficacy was mediated solely by Lyt-2+ lymphocytes. Kinetic studies revealed that the in vivo requirement of IVS Lyt-2+ effector cells to mediate tumor regression was less than 3 days, and the anti-tumor reactivity of these cells was not affected by in vivo depletion of Ia+ cells. Thus, the IVS reaction is likely representative of the in vivo counterpart of the helper/inducer phase leading to the generation of mature Lyt-2+ immune effector cells. Tumor regression after transfer of Lyt-2+ cells generated in IVS therefore required a relatively shorter period of time than that required after the transfer of fresh noncultured MCA 105 immune spleen cells.