CD4+ T CELL CLONES SPECIFIC FOR THE HUMAN ...

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melanoma-antigen p97 (melanotransferrin) deduced from the mRNA. 18. Furakawa, K. S., K. Furakawa, F. X. Real, L. J. Old, and K. 0. sequence. Proc. Natl.
0022-1767/91/1469-3235S02.00/0 THEJOURNAL OF fMMUNOLOCY Copyrlght Q 1991 by The Amerlcan Assoclatlonof Immunologists

Vol. 146.3235-3241. No. 9. May 1. 1991 Prlnted in U.S.A.

CD4+ T CELL CLONES SPECIFICFOR THE HUMAN p97 MELANOMA-ASSOCIATED ANTIGEN CAN ERADICATE PULMONARY METASTASES FROM A MURINE TUMOR EXPRESSING THE p97 ANTIGEN' MARIAKAHN,2*HIROYUKI SUGAWARA,+PATRICKMcGOWAN,* KIYOTAKAOKUNO,' SATOSHI NAGOYA,'KARLERIK HELLSTROM,"INGEGERDHELLSTROM,* AND PHILIP GREENBERG' From *Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle. WA 98121; and 'Departmentsof Medicine and Immunology. University of Washington. andFred Hutchinson Cancer Research Center, Seattle,WA 98195

p97 is a human tumor-associated Ag present on most melanomacells that represents a possible target for immunologicattack. To evaluate the capacity of T cells reactive with this protein to promote elimination of melanoma cells expressing p97, a murine model was developed by transfecting a C3H/ HeN melanoma withthe p97 cDNA, generating p97specific CD4+ T cells by in vivo immunization of C3H/HeN mice with a vaccinia/p97 recombinant virus followed by in vitro cloning with soluble p97 protein, and determining whether these CD4+T cells could mediate rejection of pulmonary metastases. Characterization of the T cell clones demonstrated the presence of both &Akand I-Ek-restrictedclones, although the majority of clones recognized p97 in the context of I-Ek. Analysis of clonal specificity using truncated p97 proteins revealed that atleast three epitopes were immunogenic,and further studies with overlapping 15-aminoacid peptides from a region of the p97 molecule defined by these truncated proteins identified an immunodominant epitope responsible for the majority of the I-Ek response. The T cell clones were not capable of directly recognizing the p97-expressingmelanoma cells but responded to the tumor if syngeneic APC were present to process the tumor-derived p97Ag. The therapeutic efficacyof these CD4+ T cell clones was evaluated in an adoptive therapy model in which mice bearing metastatic pulmonary lesions were treated by i.v. administration of the p97-specific cells. Despitethe inability of the CD4+ clones to directly respond to or lyse the tumor cells, the clones were effective in promoting tumor eradication. In vitro studies demonstrated that this may have reflected secretion of lymphokines that activated macrophages to lyse the tumor. The results suggest that noncytolytic p97-specific CD4+ T cell clones can be effective in therapy of pulmonary melanoma metastases. Moreover, if human T cells Received for publication November 19, 1990. Accepted for publlcation February 5, 1991. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked aduertisement in accordance with 1 8 U.S.C. Section 1 7 3 4 solely to indicate this fact. 'Thisstudywassupported by National CancerInstituteGrant CA33084. American Cancer Society Grant IM304. and by Bristol-Myers Squibb Pharmaceutical Research Institute. Address correspondenceand requestsfor reprints to Dr. Maria Kahn. Bristol-Myers Squibb pharmaceutical Research Institute.3005 First Avenue. Seattle,WA 9 8 1 2 1 .

reactive with the p97 protein could be generated, the expression of this tumor-associated Ag in melanoma cells might be adequate for such T cells to mediate a therapeutic antitumor response. Cell-mediated rejection of tumors requires that the tumor express an Ag recognizable by T cells and that an effector response capable of lysing tumor cells be induced. The ideal tumor Ag would be a n immunogenic protein uniquely expressedby a neoplastic cell. Although there are examples of such Ag among animal tumors, such as the SV-40 T Ag or the F-MuLV gag and envelope Ag (1, Z ) , most human tumor Ag that have been characterized are tumor-associated rather than tumor-specific. i.e., present athigh levels in tumor cells and at low levels in some normal tissues(2). There are many factors that influence the potential for suchAg to serve as the focus of a tumor rejection response. Studies in transgenic mice evaluating responses to transgenic products expressed under control of different tissue-specific promoters have demonstratedthat T cell toleranceresultsfromboth clonaldeletion and clonalanergy (3-8).and there is abundant evidence inthese models that, despite apparent in vivo tolerance, T cells specific for some self Ag expressed only in peripheral tissues can be activated in vitro [7- 12). Some self-Ag (or particular epitopes derived from the Ag) may fail to induce tolerance but still not induce an autoimmune response due to expression in very limited amounts or presentation in cells with very low levels of MHC molecules (13, 14). Thus. it may be possible to make a non-unique tumor-associated Ag immunogenic by modifying its presentation, such as by immunizing the host with large amounts of purified fragments of the protein or by introducing the Ag into a highly immunogenic vehicle s u c h a s a recombinant infectious virus (14. 15).It is hard to predict, however, whether a n immune response induced in this fashionwill be able to destroy tumor cells expressing large amounts of the Ag and/or will cause irreversible damage to normal cells that express small amountsof the Ag. One of the best characterized human tumor-associated Ag is p97, a glycoprotein present at high levels in most human melanomas and in trace amounts innormal human tissues(16. 17).Antibodies to a variant of p97 have been detected in a patient with melanoma (18), and a n tibodies to p97 have been generated by sensitization of human lymphocytes in vitro ( 1 9). which implies that the

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3236

ELIMINATION OF PULMONARY METASTASES BY p97-SPECIFIC CD4' CLONES

posterior draining lymph nodes p97 protein can be presented ina n immunogenic context ml). applied twice 20 days apart. The were removed, and cell suspensions were cultured in lMDM (GIBCO) and recognized by the human immune system. Therefore, with 10%FCS and 5 x 2-ME (Eastman Kodak, Rochester, NY). we have been interested in determining whether T cell 4 x 10" lymphocytes were seededin 16-mm wells (Costar,Camresponses can be elicited to thisAg, and if such responses bridge, MA) together with soluble p97 Ag and 2 x 10" syngeneic irradiated (3300 rad) spleen cellsas a source of APC in a final volume can be manipulatedfor a therapeuticbenefit. Before of 2 ml. Some lymphocytes were stimulated withUV-irradiated cl2A evaluating this in humans with melanoma, in whom melanoma cells at 1 x lo5 cells/well as a source of Ag plus APC. responses to p97could cause autoimmune injury as well Forty-eight hours later, 50U/ml of rIL-2 (Cetus Corporation, EmeryT cell lines were as antitumor activity, it is necessary to establish in a n ville. CA) was added to thecultures,andthe restimulated a s described above at 10- to 14-day intervals. The T animal model whether T cell reactivity to this Ag can cell lines were cloned by limiting dilution48 h after stimulation with accomplish the desired effect, i.e., eradication of a tumor Ag. and then maintainedas above. Proliferation assays. Ten to 14 days after the last stimulation expressing p97. For this purpose, the human p97 gene with Ag. T cells were seeded into 96-well plates (2 x 1O4 cells/well) h a s been transfected intoa murine melanoma for useas together with 5 x lo5syngeneic irradiated (3000 rad)spleen cells (as a target (20). and a recombinant vaccinia virus contain- APC) and Ag in the form of purified p97, OVA, or irradiated tumor ing the p97 gene, v-p97 NY, has been constructed for use cells. For analysis of the immunodominant regions of p97. cells were with truncated p97 molecules. After incubation for 4 8 to as a n immunogen to induce both cell-mediated and hu- stimulated 72 hours, theT cells were pulsed for 16 to 18h with 1 pCi tritiated moral p97-specific antitumor immunityin mice (2 1, 22). thymidine (New England Nuclear, Boston. MA), harvested. and In this study,we have evaluated ina murine model the counted in a liquid scintillation counter. Phenotypic andfunctional characterization of T cell clones. T generation of CD4+ T cells in response to the p97 molecell clones were tested for the expression of Lyt-2, L3T4 and Thycule, characterizedby clonal analysisof the finespecific- 1.2 markersby using fluoresceinated mAb 53.6, GK1.5. and 30H12, ity of the CD4' T cell response to peptides from p97, andrespectively, kindly provided by J. A. Ledbetter (Oncogene, Seattle, determined whether p97-specific CD4+ T cells can me- WA). After labeling, the cells were analyzed on a n Epics-C fluorescell sorter (Coulter, Hialeah,FL). diate a therapeutic effect against metastatic p97' mela- cence-activated To analyze theMHC restriction of the T cell clones, two anti-MHC noma in syngeneic(C3H)mice. CD4+T cells were selected class I1 mAb, 26-7-11s (anti-I-A') and 14-4-4s (anti-LEk).derived as the desired effector population for study, because such from hybridomas obtained from the American Type Culture Collection (Rockville, MD), were used. The mAb were added to wells a t a cells can be readily generated in vitro in situations in final concentration of 50 pglml before the addition of Ag at the which a tumor-associated Ag can be identified and puri- initiation of the proliferation assays. The requirement for Ag procfied, and since CD4+ Tcellshavebeenshownto be essing byAPC was evaluated by adding chloroquine (Sigma) at a M/well before the addition of Ag for capable of mediating complete eradication of tumors such concentration of 5 x proliferation assays. as disseminated leukemia in the absence of any contriGeneration of truncated proteins. Two truncated proteins were bution of CD8+ T cells (23). Our results demonstrate that generated by deletion of genes encoding the C-terminal region of CD4+ T cells can be generated in response to p97, that p97. E8wasgenerated when pSV2p97 was digested with SaiI; with exonuclease 11, nuclease S1, and klenow (Boehringer there is at least one immunodominant epitope for this treated Manneheim Diagnostics. Houston, TX) according to manufacturers' response, and that the p97-specific CD4+T cells can cure instructions: digested with BamHl; and gel-excised and ligated to a mice bearing p97+ melanoma cells already metastatic toBamHI-restricted pSV2p97 vector. Because the BamHI site a t 1599 was missing in this gene preparation. insertion into the vector was the lungs.

accomplished with blunt-endligation. This plasmid was transfected into DH5n bacteria. The gene encodingE8, as confirmed by sequencMATERIALS AND METHODS ing, was 1422 bp with a stop codon 49 bp downstream. Thesecond protein, generated by using restriction endonuleases Eco471II and Animals. Six- to eight-week-old female C3H/HeN and BALB/c mice were purchased from Charles River Breeding Laboratories (Wil- NruI and ligating the blunt ends, was 368 bplarger than E8 with a stop codon 30 bp downstream. These constructs were co-transfected mington. MA). with PMCl/NEO plasmid (Stratagene, San Diego,CA) into K1735 Tumor cell lines. Cells from the M2 clone of the K1735 mouse cells by the Capo4method and selected with 500 pglml G418. Stable melanoma were obtained from Dr. 1. J. Fidler (24). The par.3which does not express human p97, was transfectedwith the human p97 transformants were obtained and the supernatants were analyzed gene, and a series of cl expressing the human p97 Ag were estab- for the presence of p97 by a double-determinant immunoassay (26). The size of the truncated proteins was confirmed by Western blot lished (20).For our studies, cl 2A and cl 62, which express approxianalysis of the supernatants. mately 10" and 5 X lo5 molecules of p97 per cell, respectively Synthesis of peptides. Peptides were synthesized by using a (unpublished observations). were used. peptide synthesizer (430A: Applied Biosystems. Foster City, CA) and Tumor uaccine. A tumorvaccine,v-p97 NY, was prepared by analyzed by HPLC. inserting the p97 gene into the NY strain of vaccinia. This recomIn uiuo tumor therapy. Six- to eight-week-old female C3H/HeN binant vaccinia virus results in expression of the p97 protein in mice were injected i.v. on day 0 with 1.6 to 2 X IO6 cl62 or parental infected cells (21). melanoma cells. One, 2, or 6 days later, mice were treated with 3.3 Soluble p97Ag. Cells from the mouse melanoma cl 2A (20)were to 5 x 1O6 p97-specific cloned Th cells by i.v. administration. Control grown in Cell Factories (Nunc, Roskilde. Denmark) in DMEM with mice received the same number of lymphocytes from an OVA-speHEPES containing 10%FCS (GIBCO. Grand Island, NY) for a period of 7 days. The supernatants were then removed, and p97 was cific T cell clone. Mice were either followed for survival for 80 days or killed 18 days after injection of tumor cells for analysis of pulpurified on a n affinity column prepared by coupling the anti-p97 mAb 96.5 (16) onto Affi-Gel Hz Immunoaffinity Support (Bio-Rad. monary metastases. Lungs were removed and stained as previously described (22), and the number of lung metastases counted. The Richmond, CA). The column was eluted with 0.1 M KH2P04adjusted counting of lung metastases was performed on coded material. and of the final to pH 4.0 with citric acid, and the p97 preparation purity was monitored bygel permeationchromatography and by SDS- the histopathologic evaluation was done by a surgical pathologist. Analysis of production of macrophage-actiuatingfactor by T PAGE. Each p97 preparation waschecked for the absenceof mycocell clones. To determine whether the CD4+ T cell clones effective plasma. in therapy of melanoma metastases could activate macrophages to Chicken egg ovalbumin (Sigma Chemical Co., St. Louis, MO) was a tumoricidal state, the2D9 and 4F4clones were cultured at 5 X lo6 used a s a control Ag. cells/well with 5 x 10" irradiated spleen cells and p97 (5pg/ml) in 1 Generation of p97-specific Th cell clones. The Th cell clones ml for 24 h. and the supernatant was harvested. 5 X lo4c l 6 2 cells. were established by using methods described by Matis et al. (25) labeled for 16 h with I3H]uridine. were seeded into 96-well flatwith some modifications. C3H/HeN mice were immunized by tail bottomed microplates with 2 x lo5thioglycollate-induced peritoneal scarification with 10 p1 of v-p97 NY (1 X lo9 plaque-forming units/ macrophages in 0.1 ml and cultured with 0.1 ml of the supernatant Abbreviations used inthis paper: par. parental tumor clone: cl. clone from a T cell clone a t 37°C. Cultured cells were harvested after 24h on a glass filter, and cytolysis was determined by measuring the from tumor line; v-p97 NY, recombinant vaccinia virus expressing the amount of radioactivity remaining in the tumor cells. p97 gene.

ELIMINATION OF PULMONARY METASTASES

3237

BY 1197-SPECIFIC CD4' CLONES

macrophagestoefficientlystimulate a response. Although the majority of clones (>75%)recognized p97 Ag Specificity of CD4+T cell clones for p97. T cell clones presented in the context of LE", as revealed by inhibition were generated from the draining lymph nodesof C3H/ of the response if monoclonal d-E" was added at the HeN mice previously immunized in vivo with v-p97 NY initiation of culture, occasional clones were found to be and were cultured for a n initial period with 10 pg/ml of restricted to the I-A" molecule. purified p97 asdescribed inMaterials and Methods. The Activation of p97-specific CD4+ T cells by tumor cells clones, obtained by limiting dilution, were maintained expressing p97. The CD4+ T cell clones specific for p97 and expanded by cyclical stimulation with soluble p97 were generated by immunization of the host with v-p97 and autologousirradiatedspleencells as APC inthe NY and in vitro stimulation with the purified protein. presence of exogenous rIL-2. Data for the dose response However, for such p97-specific T cells to have therapeutic to stimulation with p97Ag for a representative groupof activity, tumor cells expressing the protein must also 14 clones derived from the lymph nodes of four separate induce a response. Therefore, T cell clones were stimumice are presented in TableI. The dose of p97 necessary lated with the p97' cl 2A tumor cell line or the p97toinduceproliferationvaried,withseveralclonesreparental cell line, and the response was compared to T sponding at dosesas low as 0.33 pg/ml, but stimulation cells directly stimulated with the p97 protein. The rewith 1 0 pg/ml always induced a larger proliferative responses of five representative clones are presented in sponse thanlower concentrations. The phenotype of the Table 111. All tested clones responded, in the presence of T cell clones was analyzedon a cell sorter with fluores- syngeneicspleencells as APC. to thep97-expressing ceinated antibodies, and all clones examined exhibited tumor cell line. Despite the expression by immunofluothe predicted Thy- 1.2+, CD4'. CD8- phenotype (data not rescence of low levels of class I1 Ag on the p97' cl 2A shown). tumorcells(datanotshown),none of the clonesreThe role of class 11' APC in this response was assessed, sponded tothis tumor in the absence of syngeneic spleen and the results with representative clones are presented cells. The APC appeared to be necessary to process p97 in Table 11. All tested clones required biochemical procAg derived from the tumor,as the 2A tumor did not elicit essing by APC in a n acid-sensitive compartment,as dem- a response if the APC were treated with chloroquine. onstrated by the failureof Ag-pulsed chloroquine-treated Stimulation with the p97- parental tumorfailed to elicit a response. In general, the maximal response achievable TABLE I with soluble protein was somewhat greater than that Proliferative response of T cell clones to varying doses of p97" resulting from stimulation with the 2A cells, but this may f'H]Thymidine Incorporation (cpm) after Responding have merely reflected a limitation by the in vitro culture Stimulationb with p97 (pg/ml) T Cell Media conditions for the numberof irradiated stimulator tumor Clone 10 3.3 1 0.33 cells that could be added to each well. Thus, p97-specific 2D9 220 48.386 18.239 1.798 145 T cell clones cannot directly recognize p97+ tumor cells, 4C12 363 152,005 108.663 28.245 1,249 but do respond if syngeneic class II+ APC are available to 195 53,273 29.604 5,915 207 4F4 5,322 635 5A7 362 54,197 24,176 process and present tumor-derived p97. RESULTS

FB5 143 FB7 203 2A7 485 2A4 40,003 87,777 243 2D1 83 384 178 3D11 143 4H5 262 5A3 193 5H4 400

24.581 114.243 32.894

166,199 109.717 82.155 121.305 94,625 34,672 51,823 70.043 125.127

12,645 49,717 17.500

494 6.275 1,926 10.044 763 66,999 36,873

28.800 59.781 32,244 93.079

16.385 1,642 56,687

121 203 121 754

13,257 246 3,105 206 10,357

"CD4+ T cell clones were harvested 10 to 14 days after the previous stimulation and placed in 96-well plates with fresh syngeneic irradiated spleen cells a s APC and the indicated amountof Ag. Wells were pulsed with 1 pCi of [3H]TdRfor the final 18 h of a 72-h culture.

Analysis for immunodominant epitopes on the p97 protein. The p97 protein is translated from a 2214-bp coding sequence as a 728-amino-acid precursormolecule containing a 19-amino-acid N-terminal signal sequence and 25-amino-acidC-terminalhydrophobicsequence (17). To identify regions containing possible immunodominant epitopes, sequential C-terminal truncations were made and expressed. The results with eight representative clones using two revealing truncated proteins are presented in Table IV. The long protein, denotedEco47IIIl NruI, was derived from a coding sequence containing the

TABLE I1 Requirementf o r Ag processing by APC to trigger responses by class11-restricted p97-specific CD4' T cell clones [3H]Thymidine Incorporation (cpm] after Stimulation' with Resuondine T Cell Cloneb .

3837 3A22 5A 1 5810 1Al1 1A14

Y

p97c APC Alone

88 46 16,487 48 329 89 94 48

APCd Alone

45 1 166 190 225 354

+ P97

23.477 168.855 111.095 110.734 122.230

APCe + chloroquine + P97

APd+ p97 + ai-A

5,000 3.505 29,745 24.988 280 7.930

22.456 19.199 101.398 128,983 12.311 20.550

APCIt p97 + al-E

79 1 123 2,883 2,232 115.848 161,483

Thymidine uptake was measuredby pulsing wells with 1 WCi of [3H]TdRfor the final 1 8 h of a 72-h stimulation. CD4+T cell clones. generated and maintaineda s described in Materials and Methods. were tested 10 to 14 days after previous stimulation. p97 was addeda t 5 @g/ml. Irradiated C3H/HeN spleen cells were added a s a source of APC. 'APC were cultured with 5 x M chloroquine before pulsingwith Ag. '01-A and 01-E mAb were added to wells a t 50 pglml.

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ELIMINATION OF PULMONARY METASTASES BY p97-SPECIFICCD4+

CLONES

TABLE 111 Requirement f o r Ag processing by APC in the responseof CD4' T cells to a p97+ tumor [3H]Thymidine incorporation (cpm) after Stimulationa with

Responding T Cell 'loneb

Media

APC' Alone

2D9 4F4 2D1 5A3 5H4

1.895 5.485 3,867 3.150 2.700

1.135 4.461 2.663 2.999 6.252

~ 9 7 p9 ~p79+7A+P C + Alone APC Chloroquinee

2it' Alone

2A+APC

2 A + A P C + p a r +p a r ' Chloroquine Alone

APC

1.459 105.318 2.097 3.796 57,908 2.193 2.080 1.941 3,709 50,199 4,311 4,889 37,905 4,559 3,485 4.181 5.412 1.275 4.381 2.787 15,377 3,345 25.202 5,632 2,596 1,768 104.607 4.232 35,121 3.032 2,923 4.259 4,627 1,356 148,977 2.001 101,718 2,659 6,641 1,703 "Thymidine uptake was measuredby pulsing wells with 1 pCi of [3H]TdRduring the finalI 8 h of a 72-h stimulation. bCD4+T cell clones were generateda s described (TableI). lrradiated C3H/HeN spleen cells were added a s a source of APC. p97 was added at a final concentrationof 5 pg/ml. 'APC were treated with5 x M chloroquine before the additionof either soluble p97 or p97' 2.4 tumor cells. 'p97+ 2A or p97- par tumor cells wereadded as a source of Ag.

TABLE IV Proliferafiue response ofCD4' T cells to truncated p97 molecules

defines a n immunodominant epitope on p97 presented in the context of I-E", but there are at least two other 13H]Thymidineincorporation (cpm) after Stimulation" epitopes on this molecule that can be recognized by T with cells respondingto immunization with the whole protein. T Cell Clone Media Soluble Adoptive therapy of pulmonary metastases with p97E8 (Eco47 1 1 /Nrui) p9P specific T cell clones. The ability of these CD4+ p972D9 66.887 115 264 144,557 specific Tcell clones to promote eradication of pulmonary 4C12 109 15.139 107 89.533 4F4 134 29.605 melanomamicrometastaseswasexamined. C3H mice 69 46.151 2A7 118 20,963 212 28,514 were injected intravenously with 2 X 10' cl 62 (p97+) 1All 88 63.050 84,890 72,071 melanoma cells on day 0 and were left untreated: or, 1 1,414 195 ND 31.691 43.359 3B37 ND 2.750 3.194 14,941 day later, after establishment of pulmonary micrometas5A 1 473 93.536 1.206 18.886 tases, were treated by i.v. adoptive transfer of cloned "Thymidine incorporation was measured and T cell clones were genCD4+ T cells. Thetwo clones evaluated in adoptive thererated a s previously described (TableI). apy were demonstrated by bioassay with CTLL cells in Soluble p97, or the truncatedp97 proteins E8 or (Eco471 l/Nrul) were added to wells at a final concentrationof 10 rg/ml. vitro tomake IL-2 in response to stimulation by p97 (data not shown). Untreatedmice had a median survival of 1 5 days (Fig. l , A and B), and postmortem examination refirst 1417 bp from the ATG start site, and the short protein, denoted E8, was derived froma coding sequence vealed massive pulmonary metastases with consolidated containing the first 1097 Only bp. twoof the eight clones lungs in allmice. Therapy with eitherclone 2D9 (Fig. 1A) or 4F4 (Fig. 1B)resulted in long-term survival of most respondedto the short protein. Althoughmostclones tested proliferated in responseto the long protein, oneof mice. The efficacy of the CD4+ clones was dependent on clone the clones described in TableIV, 3B37, failed to respond recognition of thep97protein,becauseneither of the to the long protein, which suggests that it recognized a exhibited any therapeutic activity in treatment the tumor was more C-terminal sequence.Of a total of 1 9 clones exam- parental p97- melanoma from which p97' derived (Fig. 2). ined that were derived from four separate immunized The efficacy of T cell clones in treating established donor mice, 17 responded to the long protein, including pulmonary metastases was also evaluated by killing mice three that also recognized the short protein, and two after adoptive therapy and counting the number of metwhich failed to recognize either protein. The described clones that recognized the short protein were I-A"-re- astatic colonies in the lung. Mice were inoculated with 0, stricted, whereas all other tested clonesrecognizing the 1.7 X lo5 par (p97-j or c l 6 2 (p97+) tumor cells on day treated on day 2 or day 6 with 1.5 X 10' p97-specific moreC-terminalregions of the proteinwere I-E'-reclone 2D9 T cells or a n OVA-specific CD4' T cell clone, stricted (Table 11: data not shown). These results suggested that an immunodominant ep-and killed on day 18. Untreated mice or mice treated with itope presented in the contextof I-E" might be located in a n OVA-specific T cell clone contained >200 metastatic the region between the terminationsof the short and long colonies per lung [Fig. 3 , A and B). Therapy on day 2 or proteins. Therefore,a sequential seriesof 15-amino-acid- day 6 with a p97-specific clone significantly reducedthe length peptides, overlapping by six amino acidscovering number of pulmonary metastases detected. By contrast, this entire coding region, were constructed. The results administration of p97-specific cloned T cells had no effect on the development of metastases from the par (p97-) with seven representative clones and multiple peptides CD4' p97-specific T are shown in TableV. The five clones that responded to tumor. Thus, adoptive therapy with cell clones, even if delayed to permit a longer time for the long but not the short protein responded to peptide the establishment andgrowth of pulmonary metastases, can MK-05 sequence. Neither the two I-A"-restricted clones that responded to the short protein (1All and 1A14) norresult in a significant Ag-specific antitumor effect and the clone that responded to a n epitope C-terminal to the has the potential to cure many metastatic pulmonary long protein (3837)responded to any of the peptides. Of lesions. Activation of tumoricidal macrophages by CD4+ T cell the 14 clones analyzed that were predicted by the truncated proteins to respond to this region, all 1 4 responded clones. The therapeutic efficacy of non-cytolytic CD4' T to peptide MK-05, and noneresponded toany of the other cells is dependent on the activation of other cytolytic peptides encompassing this region. Thus, this peptide effectormechanisms,andpreviousstudiesfromour

ELIMINATION OF PULMONARY METASTASES

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BY p9i"SPECIFIC CD4' CLONES

TABLE V Response of CD4+ T cell clones to epitopes defined by representative 15-amino-acid peptide stretchesfrom p97 [3HJThymidineIncorporation (cpm] Clone M e dM i aKM - 1K3M - 1K2M -P97 0K9M - 0K8M - 0K5M - 0K4M - 0K3 - 0 2

2D9 178.013 447 330 351 41281,605 333360 387 3327 485 21253.487326 4F4 271 228 727 348 469 4C12 34 241 71,193 311 1 56,742 283 272 230 442 62.734 243 171 674 47,72047 5 A 1 258 152 144 160 l 474 A l l76.327 174 351 402 449 268 1A14 32645.028191 3B37552 741 573 a79 792 713 604 379 72827,508

385

271

295

311

a Thymidine incorporationwas measured and CD4+T cell clones were generatedas previously described. bSoluble p97 protein or 15-amino-acid length peptides produced on a peptide synthesizer based on the known p97 sequence were added to wells a t a final concentrationof 10 rgiml.

100

-

I

80

80

-

1.OxlO'

4F4

1

3.3~10~ 4F4

-

60

1

60

.-Q>>

-

L

a

U,

40

s

40-

20

20

-

Tumor alone i

0

I

"

"

20

0

l

"

I

"

i

40

80

60

Day Figure 1 . Therapy of mice bearing metastatic pulmonary lesions from a P97+ melanoma (cl 62) with p97-specific CD4' T cell clones. C3H/HeN mice were inoculated i.v.with 2 x 10' tumor cells (cl 62) on day 0. and 24 h later, on day 1, were injected i.v. with either HBSS (tumor alone). alow of CD4+T cell clones specific for p97. cell dose (3.3X IO6). or a high cell dose (10') of either 2D9 [ A ] or 4F4 [B)

e

II

1

--c

4

par alone

3.3~10~ 2D9 1.0~10' 2 ~ 9 3.3~10' 4F4

VI). Supernatants obtained 24 h after stimulation of the CD4+ T cell clones with p97 had no direct lytic activity for the tumor. However, addition of the same supernatants to a macrophage monolayer resulted in the generation of macrophages that lysed the tumor. Thus, activation of macrophages by lymphokines secreted by the p97-specific CD4+ T cell clones may contribute to or be responsible for the observed in vivo efficacy of these clones. DISCUSSION

1

II

Human tumors express a variety of tumor-associated

Ag (2). Although most of these Ag have been defined by

mAb, some have been demonstrated to induce cell-mediated andlor humoral immune responses in some cancer 0 10 20 30 40 50 patients (2, 28-30).In view of the fundamental role of T Day cell responses in the killing and in vivo rejection of tumor Figure 2. Therapy of mice bearing metastatic pulmonary lesionsfrom cells, the identity of tumor Ag capable of inducing cellular the P97- par tumor with p97-specific CD4+ T cell clones. C3H/HeN mice immunity may be of the greatest interest for generating were inoculated i.v. with 2 x 10' tumor (par) cells on day 0, and 24 h later, on day 1. were inoculated i.v. with either HBSS (par alone)or p97therapeutic tumor immunity. However, it is a complex spectfic CD4+T cell clones 2D9 or 4F4 in dosesof 3.3 X IO6 or lo7 cells. task to define which human tumor Ag might represent appropriate immunogens for generatingclinically benegroup have demonstrated that tumoricidal macrophages ficial antitumor responses. One approach has been to may be responsible for this in vivo antitumor activity probe for T cell responses in patients bearing a tumor (27). Therefore, we examined whether the CD4+ T cell and then attempt to identifythe antigenic targets of these clones,demonstratedaboveto beeffectivein vivo in antitumor reactions (29, 30). An alternative approach, a treatment of pulmonary metastases, were capable of se- which is beingexplored by our groups, is to select protein that exhibits high expression in tumor cells and cretinglymphokinesincludingmacrophage-activating factors thatcould render macrophages tumoricidal (Tablea limited expression in normal tissues, and determine 2o

0 :

I

I

I

I

1

3240

1

not entirely tumor-specific, normal tissues generallyexpress two logs less p97 than melanoma cells (16, 26). 450 Recent studies have demonstrated thatlow-level expresf 400 sion of some proteins in peripheral tissue may not induce tolerance (13, 14) but still do not cause autoimmunity due to quantitatively insufficient presentation totrigger a T cell response. This may be particularly true for Ag Par expressed in peripheral tissues, such as musclecells, cl62 that expressonly small amounts of MHC molecules (31). Thus, Ag such as p97 may fail to induce tolerance, rendering it possible to generate immune response if the Ag ispresentedinanappropriatecontext.Thepotential immunogenicity of p97 is supported by the demonstraPBS T ova 2D9 tion that antibodies atovariant of p97 have been detected Treatment in the serumfrom one patient with melanoma 8). (1 In the present study, mice were immunized to p97 by B. vaccination with v-p97NY, a recombinant vaccinia virus 500 containing the p97 gene. Such a vaccinia vector offers 450 a n immunogenic matrix for enhancing the response ato 400 protein that cannot otherwise be readily accomplished $ 350 without the use of adjuvants. After priming with v-p97 f 300 NY, CD4+ T cell clones specific for p97 were generated M 250 by repetitive stimulation in vitro with purified p97 procl tein and syngeneic APC. The CD4+ T cell clones were “0 200 0 Par cl62 I-A” and I-EkMHC molecules found to be restricted to both * 150 M and responded to p97+-expressing tumor cells, provided 4 100 syngeneic APC were available to processand present the 50 p97 protein in the context of a class I1 molecule. 0 Our data show that such cloned murine CD4+ T cells PBS T ova 2D9 can mediate a therapeutic effect against metastatic synTreatment geneic melanoma, curing many tumors mice. in Although Figure 3. Delayed T cell therapy of mice bearing metastatic pulmonary previous studies have demonstrated that CD4+ T cell lesions. C3H/HeN mice were inoculated i.v. on day0 with 1.7 x lo5 p97+ cl 62 or p97- par. On day 2 (A] or day 6 (€3). mice were inoculated i.v. clones can promote the complete eradication of dissemiwith PBS or 1.5 X lo6 T cells of the CD4+ p97-specific 2D9 clone or a n nated leukemia ina murine model (23). the present findOVA-specific T cell clone (T ova]. Mice were killed 18 days later, and the ings represent the first demonstration that such clones number of metastases present in the lungs counted. can be effective in therapyof metastatic melanoma. The TABLE VI fact that CD4+ T cells can activate tumoricidal effectors Tumoricidal actiuityof macrophages lM01 actiuated by supernatants such as macrophages (27, 32), which lyse transformed from p97-specific CD4+clones cells independent of expression of the immunizing Ag, Tumor Cultured with: Residual may have contributed to the cures,as the inflammatory [3HIUdR Cytolysis T Cell Source after response promoted by a CD4+ T cell response to p97+ I%)* EffectoP of SupernaCulture tumor cells may be able to eliminate variant tumor cells IcomP tantsb present within a metastatic focus that express little or 17,947 0 None undetectable levels of p97. 15,541 0 MP) None 2D9 19,074 0 Analysis of the fine specificity of the CD4+ T cell re17,738 0 None 4F4 sponse to p97 demonstrated that there are at least three 10,098 35 M0 2D9 7.406 4F4 M@ immunogenic epitopes, but that the majority of T cell Peritoneal exudate cellsa s a source of MQ)were induced inC3H mice clones generated from several separate immunizations by injection of thioglycollate and were seededat a density of 2 X lo5cells/ recognized a n immunodominant epitope contained well into 96-well plates. within the same 15-amino-acid stretch. The relevance of CD4+ T cell clones were stimulated with irradiated spleen cells and 5 pg p97 and the supernatant harvested after 24 h. this epitope to Tcell responses elicited in other strainsof p97+ cl 62 tumor cells were labeled for 16 h with I3H]UdRand added mice or, more importantly, in humans, will require furto wells containing no cells, M 0 effectors, or MP)effectors plus supernather analysis. However, analysis of the CD4+ T cell recells were harvested on tant from a T cell clone. After 24 h, the cultured a glass filterformeasurement of viable tumor cells as reflected by sponse to HIV envelope has demonstrated that an imintracellular label. munodominant epitope revealed by studying the murine Cytolysis was calculatedas thetotal label recovered from tumorcells cultured with macrophages alone minus the label remaining in tumor 160 predicted a n immunodominant epitope response to gp cells cultured with MP) and supernatant divided by the label from tumor T cell response (33). Presumably, this for the human cells cultured withMP)alone. reflects the outcome of biochemical processing of the whether it can elicit specific T cell immunity and serve proteinwithsimilarenzymesinthetwospeciesand as a target for Tcell therapy in an animal model. Ag that preferential preservationof the epitope and/or a desirable satisfy these preclinical criteria can then be more exten- three-dimensional structure and physiochemical affinity of the peptide for relatively preserved regions of the class sively evaluated as potential immunogens in humans. I1 Ag-binding cleft. Preliminary studies in primates have We have chosen to examine the melanoma-associated p97 glycoprotein Ag with this approach. Although p97 isdemonstrated that, despite expression of trace levels of

A*

500

pll

52

ELIMINATION O F PULMONARY METASTASES BY p97-SPECIFIC CD4’ CLONES

ELIMINATION OF PULMONARY METASTASES

p97 in monkey cells, v-p97NY can elicit proliferative T cell responses (22) and should permit further analysis of the potential role of this epitope. The identification of a potentially immunodominant epitope offers thepossibility of constructing vaccines that present this epitope in a more immunogenic fashion, suchas in tandem repeats or linked to B cell epitopes, that might provide a means for priming hosts for secondary responses to the native as well as v-p97NY, may be worth protein. Such vaccines, exploring in patients with melanomaas potential immunogens for the inductionof T cell responses that canbe subsequently expanded to mediate tumor destruction. Acknowtedgrnents. The authors wish to thank Kent Slaven, Sandy Emery, and Ulrike Stevenson for their expert technical assistance, and Joanne Factor, Anita Rogers and Joe Smith for preparation of the manuscript. REFERENCES 1. Klarnet. J. P., D. E. Kern, K. Okuno, C . Holt. F. Lilly. and P. D. Greenberg. 1989. FBL-reactive CD8+ cytotoxic and CD4+ helper T lymphocytes recognize distinct Friend murine leukemia virus-encoded antigens. J . Exp. Med. 169:457. 2. Hellstrom, K. E.. and I. Hellstrom. 1989.Oncogene-associated tumor antigens as targets for immunotherapy. FASEB J . 3: 1715 . 3. Kisielow, P.. H. Bluthmann, U. D. Staerz, M. Steinmetz, and H. von Boehmer. 1988. Tolerance in cell T receptor transgenicmice involves deletion of nonmature CD4+CD8+ thymocytes. Nature 333:742. 4. Sha, W. C., C. A. Nelson, R. D. Newberry, D. M. Kranz, J. H. Russell, and D. Y.Loh. 1988. Selective expression of a n antigen receptor on CD8-bearing T lymphocytes in transgenic mice. Nature 335271. 5. Sha, W. C.. C. A. Nelson, R. D. Newberry. D. M. Kranz. J. H. Russell, and D. Y. Loh. 1988. Positive and negative selection of an antigen receptor on T cells in transgenic mice. Nature 336:73. 6. Teh, H. S . , P. Kisielow, B. Scott, H. Kishi, Y. Uematsu. H. Bluthmann, andH. von Boehmer. 1988. Thymicmajor histocompatibility complex antigen and the ol(j T cell receptor determine theCD4/CD8 phenotype of T cells. Nature 335229. 7. Burkly, L.C.. D. Lo, 0. Kanagawa, R. L. Brinster. and R. A. Flavell. 1989. T cell tolerance by clonal anergr in transgenic mice with nonlymphoid expression ofMHC class I1 I-E. Nature 342:564. 8. Miller, J. F. A. P.. G. Morahan, and J. Allison. 1989. Extrathymic acquisition of tolerance by T lymphocytes. Cold Spring Harbor Symp. Quant. Biol. LlV:807. 9. Morahan. G., J. Allison, and J. F. A. P. Miller. 1989. Tolerance of class I histocompatibility antigens expressed extrathymically. Nature 339522. 10. Morahan, G.,F.E. Brennan, P. S . Bhathal. J. Allison, K. 0.Cox, and J. F. A. P. Miller. 1989. Expression in transgenic mice of class I histocompatibility antigens controlled by the metallothionein promoter. Proc. Natl. Acad. Sci. USA 86:3782. 11. Miller, J.. L. Daitch. s. Rath, and E. Selsing. 1990. Tissue-specific expression of allogeneic class I1 MHC molecules inducesneither tissue rejection nor clonalinactivation of alloreactiveT cells. J . lmmunol. 144:334. 12. Bohme, J.. K. Haskins, P. Stecha. W. van Ewijk. M. LeMeur, P. Gerlinger, C. Benoist. and D. Mathis. 1989. Science 244:1179. 13. Schild, H., 0. Rotzschke, H. Kalbacher, and H.-G. Rammensee. 1990. Limit of T cell tolerance to self proteins by peptide presentation. Science 247:1587. 14. Gammon, G.. and E. Sercarz. 1989. How some T cells escape tolerance induction. Nature 342:183. 15. Moss, B.. and C. Flexner. 1987. Vaccinia virus expression vectors. Annu. Rev. Irnrnunol. 5305.

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