-imm.notker p
Immunology Today Vol. I 1, No. 6 1990
CANCERIMMUNOTHERAPYUPDATE Immunotherapy offers the tantalizing possibility of a fourth modality for the treatment of cancer, joining surgery, chemotherapy and radiotherapy. This has meant that great hopes, and considerable pressure, have been placed on immunotherapy programs, although these are still in the early stages of development. To assessthe current status of cancer immunotherapy, ImmunologyTodayhas brought together a short group of articles including (1) a progress update, in the form of a report from a recent international meeting, (2) a cautionary view of the difficulties being encountered in rationalizing immunotherapies and (3) an examination of the potential for new methods of lymphokine targetring. The outlook remains cautiously optimistic as the application of recent developments in immunology begins.
Recent
advancesin cellularimmunology: implicationsfor immunity to cancer
Basicimmunologists, animal experimentersand clinicianscame frv~th~r t ~ i ~ r
thi¢ v ~ r * tn rb'~'ml~ incinhP¢ intn ~nd ~nnli-
cations of, cellular immunity to cancer. Major questions critical to the further evaluation and exploitation of the cellular immune response to cancer were ~~plored. It was clear from this conference that there has been a move beyond phenomenology to reductionistic approaches to understanding immune events, raising hopes that the specificity of the T-cellresponse might be applied to the problem of human cancer.
Michael T. L01ze and Oiivera J. finn
T-cell recognitionof antigenand T-cellactivation An efficient cellular immune response against tumors requires that: (1) antigenic determinants are preferentially expressed on tumor cells; (2) these antigens must bind major histocompatibiiity complex (MHC) molecules; (3) peripheral T cells must recognize the presented antigens; it has been said that cancer has done more for the science (4) presented antigens must be able to stimulate both of immunology than immunology has ever done for helper T-cell and cytotoxic T-cell responses; and (5) effeccancer. Recent developments in immunology promise to tor cells must reach the tumor site and cause tumor change this perception. New insights regarding antigen destruction. presentation to T cells, T-cell recognition, activation, The keynote speaker, Philippa Marrack (Denver) exgrowth, traffic and interaction with endothelial cells tended earlier observations that a number of self antisuggest how tumor antigens might be recognized by the gens, such as MIs, in combination with products of the immure s/stem and what requirements must be met to murine major histocompatibility complex (MHC) stimulate dpply these principles to animal models and human tumor many T cells within an individual V~ subfamily. In the case immunology. of the staphylococcal enterotoxin 13, the response is largely due to V~3 and Ve8 T cells which are completely *TheUCLASymposiumon CellJlar=mmunityandthe Immunotherapyof Cancer deleted from the peripheral T-cell repertoire after injecwasheldin ParkC=ty,Utahon27January-3February1990. tion of this molecule in neonatal mice, resultir~g in tolerance. Tolerance is dominant in heterozygous individuals and, although the mouse genome can encode 21 or more MichaelLotzeisat the TumormmunologySection,SurgeryBranch,NCl, Vp subfamilies, not all of these are expressed in all aniBeOSesda,MD 20892, USAand OliveraFinn is at the Departmer,t of mals. Even mice of the same MHC type can differ greatly Microbiology and Immunology, Duke University Medical Center, in their T-cell repertoires. Interestingly, predominant use Durham, NC27710, USA. of particular T-cell receptors in human tumor immunity
190
(~) 1990, Elsevier Science Publishers Ltd, UK. 0167--4919/901502.00
Immunology Today, /ol. 1 I, No. 6 1990
(V~2 predominantly expressed by anti-melanoma T-cell clones and lines, V. Kumar, Pasadena), alloimmunity (oligoclonal Vp segment usage, S. Hand, Durham) and autoimmunity (preferential V~ 12.1 usage in multiple sclerosis plaques, R. Bell, Stanford) was reported. The important question of immunogenicity of antigens recognized by T cells was addressed by J. Berzofsky (Bethesda). The coupling of a helper T-cell site to a B-cell site can be used to produce a synthetic immunogen that elicits neutralizing antibodies, for example to HIV and in malaria, and the same strategy could be considered for the rational design of vaccines for anti-tumor immunity. The effective therapy of a disseminated Friend retrovirusinduced leukemia, FBL-3 (P. Greenberg, M. Cheever, St Louis) requires in vivo priming to tumor antigen. CD8 ÷ T cells could not be adequately primed in the absence of concurrent activation of CD4 ÷ T cells which suggests that, even if a tumor expresses a potential immunogen, such a molecule may be incapable of inducing both T-cell subsets. It is unclear how frequently antigens can stimulate both T-cell subsets. One such molecule, epithelial cell mucin, has been identified on pancreatic and breast adenocarcinomas and both CD8 ÷ and CD4 ÷ T cells reactive with a tandemly repeated peptide on the native molecule have been isolated and characterized (O. Finn, Durham). The question of what constitutes a tumor antigen was addressed in a number of presentations. Three genes encoding antigens that elicit anti-tumor responses were isolated and sequenced (T. Boon, Melbourne) using a highly transfectable P815 tumor and its in vivo mutagenized variants. Each mutation was identified as a unique codon change. Synthetic peptides encompassing the mutation bind to autologous H-2 molecules and stimulate cytotoxic T cells. Frequent single base point mutations at three unique sites on the ras oncogene are associated with malignant transformation and are found in a variety of animal and human tumors. Us:ng synthetic peptides encompassing the mutation at codon 12, M. Cheever derived murine CD4 ÷ T cells which proliferated specifically in an MHC-restricted fashion. Adenovirusinduced lymphomas in mice also produce immunologically recognizable E1A oncogene products. Adoptive transfer of E1A-specific T-cell clones and IL-2 can cure animals of this tumor (C. Melief, Amsterdam). More problematic and largely unaddressed at this conference are issues regarding so-called non-immunogenic tumors and apparent tolerance to presumed tumor antigens. H. Schreiber (Chicago) used a highly immunogenic MHC c!ass I antigen, K216, as a model tumor antigen and showed that mice bearing a K216positive tumor reject K216-positive normal tissue grafts but cannot reject the tumor even when immunity specific to the alloantigen is generated. One mechanism for enhancing anti-tumor immunity was presented by D. Pardoll (Baltimore) and S. Russell(London) and involves transfection of poorly immunogenic tumors with the IL-2 gene. An allograft model for tolerance was presented in which skin grafts expressing MHC disparities fail to be rejected (A. Rosenberg, Washington). The response of B6 mice to both Qa-1 and bin6 alloantigens is characterized by a deficiency of antigen-specific helper T cells. CD4÷ T cells specific for otl~er determinants on the same graft (such as H-Y) provide the requisite second signal for generation of effector cells. Exposure to Qa-1 in the absence of such
determinants leads to tolerance. Adhesion is actively regulated by the biochemical events that result from activation (S. Shaw, Bethesda). Expression of adhesion molecules is enhanced on memory cel!s, which may be responsible for their preferential localization in tissues and at epithelial surfaces. The accessory molecules, CD4 and CD8, appear to have a dua! role, one being adhesion which facilitates T-cell activation, and the other being an intimate association with the T-ceil receptor complex and the membrane bound tyrosine kinase p56 Ick (J. Parnes, Stanford). Attachment of T cells to endothelial cells and migration into tissues involves different classes of cell adhesion molecules (CAMs). These include integrins (for example lymphocyte function-associated antigen 1 (LFA-1)), the CD44 glycoproteins and selectins (for example MEL-14, LAM-1) (M. Gallatin, F. Hutchinson, Seattle). T-cell activation leads to decreased MEL-14 expression and tissue retention after encountering antigen. Unique endothelial activation antigens induced by interleukin 4 (IL-4) (W.M. Gallatin) and IL-1 and tumor necrosis factor (TNF) (M. Cohen, Philadelphia) suggest that cytokine release at the site of inflammation is in part responsible for migration of activated T cells to those sites. The binding of metastatic tumor cells and growth in various tissue sites may be related to endothelial-derived organ-specific paracrine growth factors (G. Nicholson, Houston).
Natural killer cells and non-MHGrestricted lysis Most lymphoid cells that mediate non-MHC-restricted tumor lysis exist in a population of CD3- large and small agranular lymphocytes (F. Bach, Minneapolis) that require two signals for optimal activation. The first signal can be delivered via interaction with the surface of cultured cell lines such as K562 or !ymphoblastoid cell lines and the second is provided by T-cell growth factors Three different IgG Fc receptors, I (CD64), II (CD32) and III (CD16) (L. Lanier, San Jose) have been identified on null cells and myetomonocytic cells. The CD16 molecule on granutocytes exists as a 30 kDa form and on natural killer (NK) cells as a 36-38 kDa form. Binding of immune complexes leads to increased intercellular calcium and IP3 generation, transcription of various lymphokine messages, and ultimately to cytotoxicity. Expression of the CD16 molecule in transfectants requires co-transfection with either the CD3 ~ chain or the homologous ~/chain of the FcR high-affinity receptor. The c: chain of CD3, which appears to be important in signal transduction in T cells and null cells, does not exist in granulocytes. At least one candidate target molecule recognized by null cells has been identified, cloned and sequenced, after the generation of an anti-idiotypic antibody to an antibody which binds to K562 and blocks lysls by NK cells (J. Ortaldo, Bethesda). The molecule exists as a single nonrearranged gene encoding a 116 amino acid protein, incorporates a cyclophilin-like domain and is expressed on the cell surface as a glycosylated 140 kDa monomer. The mouse and human genes are 80% homologous. A 27met antisense molecule inhibits lysisof K562 as well as the nonspecific lysis mediated by certain cyt.otoxic T lymphocytes (CTL). Adenoviruses 2 and 12 prevent transcription or expression of MHC class I molecules at the surface of targets; this is associated with increased NK cell s~nsitivity. Mouse MHC class I molecules and certain human 191
Immunology Today, Vol. 11, No. 6 1990
MHC clas~ I molecuies, such as HLA-A2, when transfected into NK-cell-sensitive targets do not provide protection from NK-cell-mediated lysis(J. Dawson, Durham). Certain residues on the al and a2 domains are implicated in providing resistance. Using a line deficient in transport of HI.A, it was suggested that a transport molecule exists which might i~eff confer resistance to lysis based on its ability to bind only certain MHC class i molecules. Monocytes mediate tumor lysis after priming with IFN-~/and triggering with mc!ecules such as LPS. Three different molecular species on activated monocytes c~n be defined by monoclonal antibodies (D. Paulnock, Madison) and appear to be import3nt in the mechanisms of adhesion and signal transduction.
T-cell growth and expansion of specificcells Much of the progress in cellular immunology over the last decade depended on the ability to expand T cells in v/tro and in v/vo using the T-cell growth factor IL-2 (Lotze, Bethesda). At least three T-cell growth factors.., IL-2, IL-4 and IL-7, have now been identified and a variety of other molecules including IL-1, IL-6, TNF and transforming growth factor ~ (TGC-~) are known to be important regulatory factors. In clinical studies, IL-2 response and response duration correlate with the lymphocytosis observed following completior~ of IL-2 therapy, and profound lymphoid infilt ~tion of resDonsive tumors can be observed. IL-4 also promotes I-cell growth and modulates iymphokine-activated killer (LAK) cell actiwty induced by 11.-2,inhibiting it when added concurrently with 11_-2,but promoting LAK cell proliferation and activation after prior exposure. IL-4 has been given to over 60 patients (Lotze) in phase I trials with a maximum daily dose of 60 I~g kg -~. Marked transient lyrnphopenia and the induction of a vascular leak syndrome reminiscent of that observed with ii.-2 has been identified. IL-7 clearly promotes growth of fetal thymocytes and in humans stimulates growth of both CD4÷ and CD8÷ cells. It increases 11.-2receptor (IL-2R) expression, 11_-2production and ClL generation in primary human mixed lymphocyte reaction (MLR) (M. Widmer, Seattle). TGF-13inhibits primary murine CTL development in vitro (M. Palladino, San Francisco)and LAK cell generation (J. Mule, A. Kasid, Bethesda). TNF abrogates this inhibition and these two c),tokines appear to have inverse regulatory roles. The production of TNF-a and TNF-13(C. Ware, Riverside) is tightly regulated with activation signals provided by lectins needing to be continuously present to maintain transcription of these molecules. IL-6 exists as a circulating hormone that is important in the initial immune response as a proinflammatory cytokine (P. Sehgal, New York). The administration of !1.-2, TNF and endotoxin to mice or humans leads to detectable circulating levelsof IL-6 in the serum suggesting that it exists as an immunological hormone. It ~nergizes with a varie~/of cyl;okir~es and in-creases the motility of certain carcinoma cells.
The curious role of heat shockproteins (HSPs) HSPswere initialh, identified in Eschedchia coil follow;ng increases in temperature and were subsequently identified as highly conserved families of molecules from prokaryotes to humans. A variety of different immunological stimuli and other stresses are known to induce some HSPswhile others are expressed constitutively. The HSP70 gene has been mapped to the human MHC locus
192
and HSPs may be involved in MHC class I assembly and peptide presentation. The damage-induced gene products in skin associated with recognition by dendritic epidermal ~/8T cells are postulated to be heat shock proteins (J. Allison, Berkeley). T-ceU clones reactive to the mycobacteria 65 kDa HSP can induce arthritis in rats (S. Strober, Stanford). Approximately half of the l-cell clones expanded from synovia of patients with rheumatoid arthritis express the -/8 T-cell receptor and proliferate to *he 55 kDa HSP molecule without HLA restriction. The role of HSPsin tumor-specific cellular immunity was suggested by studies of gp96. This molecule is expressed in certain mouse and human tumors. Immunization with soluble gp96 in such animals confers resistanceto tumor growth. The transfer of such T cells from immunized animals leadsto reg-ession ef methycholanthrene (MCA)iqduced sarcomas (P. Srivastava, New York). It was suggested that these molecules, which are 40% homologous with HSPs, might present unique peptides of each tumor.
Melanoma in mice and humans The incidence of melanoma in humans is increasing; 1 in 1500 individuals in 1935 rising to a projected 1 in 90 individuals in the year 2000. Animal models of melanoma induction using chemical carcinogens such as DMBA and UV radiation can lead to the development of immunologically cross-reactive melanoma in a high percentage of animals (M. Kripke, Houston). Similar cross-reactivity of human melanoma -ea~ive T ce',!~ with HLA-matched tumors have been identified (V. Kumar; T Darrow, Durham) as well as unique T-cell receptors prefere~,tially using the V,2 segment in T cells reactive to melanoma in many patients. Melanomas appear to be able to process and present exogenous antigen_ The ability to present tetanus antigens appears to decreasewith more advanced tumors (D. Guerry, Philadelphia) and it was suggested that altered MHC class II molecules may provide a mechanism of escape Transfection cf murine melanomas with human melanoma antigens identified by murine monoc!onal antibodies (S. Liang, D. Herlyn, Pittsburgh; P. Greenberg, St Louis) suggests novel ways of studyir,g recognition of such antigens in animal models. Furthermore, generation of anti-idiotypic antibodies that can induce so-called Ab3, which are capable of binding tumor antigens in the host, have been extensively studied in humans (R. Levy, Stanford; D. Herlyn, Pittsburgh; S. Ferrone, NY Medical College) and suggest alternative strategies for induction of tumor recognition in patients. Many melanomas appear to express members of the integrin superfamily including VLA1-4 and VLA6 (A. Anichini, Milan) that are potentially important molecules in T-cell recognition of such tumors. T-cell clones with restricted reactivity to melanoma have now been identified by many groups (M. Lotze; T. Boon, P. Hersey, Newcastle; K. Itoh, Houston). At least four different antigens can be defined by such clones(T. Boon). Attempts to characteriz the antigens are being undertaken. The administration of IL-2 to melanoma patients is associated with a 25-40% objective response rate which is believed to be related to induction of specific tumorreactive T cells (M. Lotze; M. Mitchell, Los Angeles; P. Sondel, Madison). This has been most clearly shown with
Immunology Today, Vol. i 1, No. 6 1990
°
i,i
the adoptive transfer of so-called tumor infiltrating lymphocytes (TILs), which can be expanded in tissue culture using gas-permeable bags or hollow fiber systems with over 100-fold expansion to cell numbels exceeding 10 ~ (S. Rosenberg, P. Aebersold, Bethesda; P. Simon, Wilmington). Such ,-~lls can be identified following transfer at tumor sites using radiolabels or using cells genetically marked with the neomycin phosphotransferase bacterial gene (S. Rosenberg). These gene-marked cells have been given to _fix patients and recovered from peripheral s:,tes up to 60 days after transfer. Use of adoptively transferred T cells as vehicles for gene therapy is now being actively pursued (M. Blaese, Bethesda). Although TILs can be derived from a variety of tumors, other means of generatincj such cells including in vitro stimulation (S. Slovin, Philadelphia; A. Moser, M. Wettendoff, Brussels) and following in vivo immunization (F. Chang, M;nneapolis) or directly from tumor explants (J. Kurnick, Boston) have been carried out.
Problems The last decade has witnessed a veritable exolosion in the investigational study and clinical use of immunotherapy for the treatment of cancer. Although this is an exalting development, the promise of cancer immunotherapy has not yet been fulfilled 1,2. Why is there an apparent discrepancy between the u~euly u~ cancer ~mmunomerepy ana the actual results from clinical studies? Michael Osband and Susan Ross suggest that there are several basic problems with the clinical study ar,~ therapeutic use of immunotherapy that must be overcome before it can be considered a viable treatment for a broad range of tt,mors. The purpose of this article is to desoibe some of the more important of these problems. .LL
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Summary It is now beyond question that T cells are central to the immune response to tumors in both mice and humans. Means to modulate the immune response to tumors in murine models and in human melanoma are now well established and studies to determine whether these findings can be generalized to a larger number of neoplasms are in hand. Attempts are also being made to define where immune recognition breaks down, focussing on the T-cell receptor, MHC molecules, cytokines and other accessory molecules involved in the response to tumor antigens. Thus tumor immunology is clearly entering the new decade on a far st:rer footing. Generous support for this conference was provided by the sponsors Cetus, Immunex,Triton and SterlingOncologyas well as by Armand H.~mmer, Genentech,GeneticTherapy Inc. and IDECpharmaceuticals.Carefulpreparationof this manuscriptby Ms Etta Owens is appreciated.
in the investigationalstudy and clinical use of cancer immunotherapy Michael E. Osbandand SusanRoss
r
The Tolstoyanmodelof immunopathophysiology 'All happy families are alike. Each unhappy family is unhappy in its own way.' Tne.~e are the opening lines of Leo Tolstoy's Anna Karemna and they can be paraphrased to create a useful model by which to understand human immunopathophysiology: 'All healthy immune systems are alike. Each sick patient is immunologically dysfunctional in his or her own way.' The human immune system is complex, multi-faceted and highly specific. It is, therefore, naive to assume that the immunobiology of the host-tumor relationship is identical, or even similar, in all patients with cancer. In fact, the variable clinical response seen with most cancer immunotherapy suggests that individual patients have very different immunological mechanisms involved in the
Joint Clinical Immunotherapy Program, Boston UniversitySchool of Medicine and New England Baptist Hospital, 125 ParkerHill Avenue, Boston, MA 02135, USA. ~) 1990, ElsevierScience PublishersLtd, UK. 0167--49191901502.00
n
etiology and pathophysiology of their specific malignancy3. These can include important differences in the existence, nature and distribution of tumor antigens on the malignant cells4, as weli as the specific immune response of the patient to the tumor 5. The entire premise of a well-designed and well-conducted clinical trial is to test the safety and efficacy of the same therapeutic agent when used in comparable patients. The unique nature of the host-tumor relationship in each individual patient makes the dev-=Icpment and clinical study of immunotherapy very difficult. These issues are even more pronounced with certain treatment approaches, such as adoptive immunotherapy6 or autologous tumor vaccines7. In these settings, both the actual therapeutic that is used and the patient who is treated are one-of-a-kind. This results in clinical studies that often consist of no more than a series of anecdotes 8-1°. Another result of the unique nature of the host-tumor relationship is the lack of useful animal models that can be applied to the development of immunotherapy. The response of animals to chemotherapy, radiation and surgery is generally predictive of their effect in human patients 11.12.This is not the case with immunotherapy ~3'14. Many immunotherapeutic agents are inactive in other species. In addition, owing to the extreme complexity of the host-tumor immunorelationship, animal models do not fully mimic the biology of human patients with cancer. Finally, the immune system is obviously different in
193
Immunology Today Vol. I 1, No. 6 1990
CANCERIMMUNOTHERAPYUPDATE Immunotherapy offers the tantalizing possibility of a fourth modality for the treatment of cancer, joining surgery, chemotherapy and radiotherapy. This has meant that great hopes, and considerable pressure, have been placed on immunotherapy programs, although these are still in the early stages of development. To assessthe current status of cancer immunotherapy, ImmunologyTodayhas brought together a short group of articles including (1) a progress update, in the form of a report from a recent international meeting, (2) a cautionary view of the difficulties being encountered in rationalizing immunotherapies and (3) an examination of the potential for new methods of lymphokine targetring. The outlook remains cautiously optimistic as the application of recent developments in immunology begins.
Recent
advancesin cellularimmunology: implicationsfor immunity to cancer
Basicimmunologists, animal experimentersand clinicianscame frv~th~r t ~ i ~ r
thi¢ v ~ r * tn rb'~'ml~ incinhP¢ intn ~nd ~nnli-
cations of, cellular immunity to cancer. Major questions critical to the further evaluation and exploitation of the cellular immune response to cancer were ~~plored. It was clear from this conference that there has been a move beyond phenomenology to reductionistic approaches to understanding immune events, raising hopes that the specificity of the T-cellresponse might be applied to the problem of human cancer.
Michael T. L01ze and Oiivera J. finn
T-cell recognitionof antigenand T-cellactivation An efficient cellular immune response against tumors requires that: (1) antigenic determinants are preferentially expressed on tumor cells; (2) these antigens must bind major histocompatibiiity complex (MHC) molecules; (3) peripheral T cells must recognize the presented antigens; it has been said that cancer has done more for the science (4) presented antigens must be able to stimulate both of immunology than immunology has ever done for helper T-cell and cytotoxic T-cell responses; and (5) effeccancer. Recent developments in immunology promise to tor cells must reach the tumor site and cause tumor change this perception. New insights regarding antigen destruction. presentation to T cells, T-cell recognition, activation, The keynote speaker, Philippa Marrack (Denver) exgrowth, traffic and interaction with endothelial cells tended earlier observations that a number of self antisuggest how tumor antigens might be recognized by the gens, such as MIs, in combination with products of the immure s/stem and what requirements must be met to murine major histocompatibility complex (MHC) stimulate dpply these principles to animal models and human tumor many T cells within an individual V~ subfamily. In the case immunology. of the staphylococcal enterotoxin 13, the response is largely due to V~3 and Ve8 T cells which are completely *TheUCLASymposiumon CellJlar=mmunityandthe Immunotherapyof Cancer deleted from the peripheral T-cell repertoire after injecwasheldin ParkC=ty,Utahon27January-3February1990. tion of this molecule in neonatal mice, resultir~g in tolerance. Tolerance is dominant in heterozygous individuals and, although the mouse genome can encode 21 or more MichaelLotzeisat the TumormmunologySection,SurgeryBranch,NCl, Vp subfamilies, not all of these are expressed in all aniBeOSesda,MD 20892, USAand OliveraFinn is at the Departmer,t of mals. Even mice of the same MHC type can differ greatly Microbiology and Immunology, Duke University Medical Center, in their T-cell repertoires. Interestingly, predominant use Durham, NC27710, USA. of particular T-cell receptors in human tumor immunity
190
(~) 1990, Elsevier Science Publishers Ltd, UK. 0167--4919/901502.00
Immunology Today, /ol. 1 I, No. 6 1990
(V~2 predominantly expressed by anti-melanoma T-cell clones and lines, V. Kumar, Pasadena), alloimmunity (oligoclonal Vp segment usage, S. Hand, Durham) and autoimmunity (preferential V~ 12.1 usage in multiple sclerosis plaques, R. Bell, Stanford) was reported. The important question of immunogenicity of antigens recognized by T cells was addressed by J. Berzofsky (Bethesda). The coupling of a helper T-cell site to a B-cell site can be used to produce a synthetic immunogen that elicits neutralizing antibodies, for example to HIV and in malaria, and the same strategy could be considered for the rational design of vaccines for anti-tumor immunity. The effective therapy of a disseminated Friend retrovirusinduced leukemia, FBL-3 (P. Greenberg, M. Cheever, St Louis) requires in vivo priming to tumor antigen. CD8 ÷ T cells could not be adequately primed in the absence of concurrent activation of CD4 ÷ T cells which suggests that, even if a tumor expresses a potential immunogen, such a molecule may be incapable of inducing both T-cell subsets. It is unclear how frequently antigens can stimulate both T-cell subsets. One such molecule, epithelial cell mucin, has been identified on pancreatic and breast adenocarcinomas and both CD8 ÷ and CD4 ÷ T cells reactive with a tandemly repeated peptide on the native molecule have been isolated and characterized (O. Finn, Durham). The question of what constitutes a tumor antigen was addressed in a number of presentations. Three genes encoding antigens that elicit anti-tumor responses were isolated and sequenced (T. Boon, Melbourne) using a highly transfectable P815 tumor and its in vivo mutagenized variants. Each mutation was identified as a unique codon change. Synthetic peptides encompassing the mutation bind to autologous H-2 molecules and stimulate cytotoxic T cells. Frequent single base point mutations at three unique sites on the ras oncogene are associated with malignant transformation and are found in a variety of animal and human tumors. Us:ng synthetic peptides encompassing the mutation at codon 12, M. Cheever derived murine CD4 ÷ T cells which proliferated specifically in an MHC-restricted fashion. Adenovirusinduced lymphomas in mice also produce immunologically recognizable E1A oncogene products. Adoptive transfer of E1A-specific T-cell clones and IL-2 can cure animals of this tumor (C. Melief, Amsterdam). More problematic and largely unaddressed at this conference are issues regarding so-called non-immunogenic tumors and apparent tolerance to presumed tumor antigens. H. Schreiber (Chicago) used a highly immunogenic MHC c!ass I antigen, K216, as a model tumor antigen and showed that mice bearing a K216positive tumor reject K216-positive normal tissue grafts but cannot reject the tumor even when immunity specific to the alloantigen is generated. One mechanism for enhancing anti-tumor immunity was presented by D. Pardoll (Baltimore) and S. Russell(London) and involves transfection of poorly immunogenic tumors with the IL-2 gene. An allograft model for tolerance was presented in which skin grafts expressing MHC disparities fail to be rejected (A. Rosenberg, Washington). The response of B6 mice to both Qa-1 and bin6 alloantigens is characterized by a deficiency of antigen-specific helper T cells. CD4÷ T cells specific for otl~er determinants on the same graft (such as H-Y) provide the requisite second signal for generation of effector cells. Exposure to Qa-1 in the absence of such
determinants leads to tolerance. Adhesion is actively regulated by the biochemical events that result from activation (S. Shaw, Bethesda). Expression of adhesion molecules is enhanced on memory cel!s, which may be responsible for their preferential localization in tissues and at epithelial surfaces. The accessory molecules, CD4 and CD8, appear to have a dua! role, one being adhesion which facilitates T-cell activation, and the other being an intimate association with the T-ceil receptor complex and the membrane bound tyrosine kinase p56 Ick (J. Parnes, Stanford). Attachment of T cells to endothelial cells and migration into tissues involves different classes of cell adhesion molecules (CAMs). These include integrins (for example lymphocyte function-associated antigen 1 (LFA-1)), the CD44 glycoproteins and selectins (for example MEL-14, LAM-1) (M. Gallatin, F. Hutchinson, Seattle). T-cell activation leads to decreased MEL-14 expression and tissue retention after encountering antigen. Unique endothelial activation antigens induced by interleukin 4 (IL-4) (W.M. Gallatin) and IL-1 and tumor necrosis factor (TNF) (M. Cohen, Philadelphia) suggest that cytokine release at the site of inflammation is in part responsible for migration of activated T cells to those sites. The binding of metastatic tumor cells and growth in various tissue sites may be related to endothelial-derived organ-specific paracrine growth factors (G. Nicholson, Houston).
Natural killer cells and non-MHGrestricted lysis Most lymphoid cells that mediate non-MHC-restricted tumor lysis exist in a population of CD3- large and small agranular lymphocytes (F. Bach, Minneapolis) that require two signals for optimal activation. The first signal can be delivered via interaction with the surface of cultured cell lines such as K562 or !ymphoblastoid cell lines and the second is provided by T-cell growth factors Three different IgG Fc receptors, I (CD64), II (CD32) and III (CD16) (L. Lanier, San Jose) have been identified on null cells and myetomonocytic cells. The CD16 molecule on granutocytes exists as a 30 kDa form and on natural killer (NK) cells as a 36-38 kDa form. Binding of immune complexes leads to increased intercellular calcium and IP3 generation, transcription of various lymphokine messages, and ultimately to cytotoxicity. Expression of the CD16 molecule in transfectants requires co-transfection with either the CD3 ~ chain or the homologous ~/chain of the FcR high-affinity receptor. The c: chain of CD3, which appears to be important in signal transduction in T cells and null cells, does not exist in granulocytes. At least one candidate target molecule recognized by null cells has been identified, cloned and sequenced, after the generation of an anti-idiotypic antibody to an antibody which binds to K562 and blocks lysls by NK cells (J. Ortaldo, Bethesda). The molecule exists as a single nonrearranged gene encoding a 116 amino acid protein, incorporates a cyclophilin-like domain and is expressed on the cell surface as a glycosylated 140 kDa monomer. The mouse and human genes are 80% homologous. A 27met antisense molecule inhibits lysisof K562 as well as the nonspecific lysis mediated by certain cyt.otoxic T lymphocytes (CTL). Adenoviruses 2 and 12 prevent transcription or expression of MHC class I molecules at the surface of targets; this is associated with increased NK cell s~nsitivity. Mouse MHC class I molecules and certain human 191
Immunology Today, Vol. 11, No. 6 1990
MHC clas~ I molecuies, such as HLA-A2, when transfected into NK-cell-sensitive targets do not provide protection from NK-cell-mediated lysis(J. Dawson, Durham). Certain residues on the al and a2 domains are implicated in providing resistance. Using a line deficient in transport of HI.A, it was suggested that a transport molecule exists which might i~eff confer resistance to lysis based on its ability to bind only certain MHC class i molecules. Monocytes mediate tumor lysis after priming with IFN-~/and triggering with mc!ecules such as LPS. Three different molecular species on activated monocytes c~n be defined by monoclonal antibodies (D. Paulnock, Madison) and appear to be import3nt in the mechanisms of adhesion and signal transduction.
T-cell growth and expansion of specificcells Much of the progress in cellular immunology over the last decade depended on the ability to expand T cells in v/tro and in v/vo using the T-cell growth factor IL-2 (Lotze, Bethesda). At least three T-cell growth factors.., IL-2, IL-4 and IL-7, have now been identified and a variety of other molecules including IL-1, IL-6, TNF and transforming growth factor ~ (TGC-~) are known to be important regulatory factors. In clinical studies, IL-2 response and response duration correlate with the lymphocytosis observed following completior~ of IL-2 therapy, and profound lymphoid infilt ~tion of resDonsive tumors can be observed. IL-4 also promotes I-cell growth and modulates iymphokine-activated killer (LAK) cell actiwty induced by 11.-2,inhibiting it when added concurrently with 11_-2,but promoting LAK cell proliferation and activation after prior exposure. IL-4 has been given to over 60 patients (Lotze) in phase I trials with a maximum daily dose of 60 I~g kg -~. Marked transient lyrnphopenia and the induction of a vascular leak syndrome reminiscent of that observed with ii.-2 has been identified. IL-7 clearly promotes growth of fetal thymocytes and in humans stimulates growth of both CD4÷ and CD8÷ cells. It increases 11.-2receptor (IL-2R) expression, 11_-2production and ClL generation in primary human mixed lymphocyte reaction (MLR) (M. Widmer, Seattle). TGF-13inhibits primary murine CTL development in vitro (M. Palladino, San Francisco)and LAK cell generation (J. Mule, A. Kasid, Bethesda). TNF abrogates this inhibition and these two c),tokines appear to have inverse regulatory roles. The production of TNF-a and TNF-13(C. Ware, Riverside) is tightly regulated with activation signals provided by lectins needing to be continuously present to maintain transcription of these molecules. IL-6 exists as a circulating hormone that is important in the initial immune response as a proinflammatory cytokine (P. Sehgal, New York). The administration of !1.-2, TNF and endotoxin to mice or humans leads to detectable circulating levelsof IL-6 in the serum suggesting that it exists as an immunological hormone. It ~nergizes with a varie~/of cyl;okir~es and in-creases the motility of certain carcinoma cells.
The curious role of heat shockproteins (HSPs) HSPswere initialh, identified in Eschedchia coil follow;ng increases in temperature and were subsequently identified as highly conserved families of molecules from prokaryotes to humans. A variety of different immunological stimuli and other stresses are known to induce some HSPswhile others are expressed constitutively. The HSP70 gene has been mapped to the human MHC locus
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and HSPs may be involved in MHC class I assembly and peptide presentation. The damage-induced gene products in skin associated with recognition by dendritic epidermal ~/8T cells are postulated to be heat shock proteins (J. Allison, Berkeley). T-ceU clones reactive to the mycobacteria 65 kDa HSP can induce arthritis in rats (S. Strober, Stanford). Approximately half of the l-cell clones expanded from synovia of patients with rheumatoid arthritis express the -/8 T-cell receptor and proliferate to *he 55 kDa HSP molecule without HLA restriction. The role of HSPsin tumor-specific cellular immunity was suggested by studies of gp96. This molecule is expressed in certain mouse and human tumors. Immunization with soluble gp96 in such animals confers resistanceto tumor growth. The transfer of such T cells from immunized animals leadsto reg-ession ef methycholanthrene (MCA)iqduced sarcomas (P. Srivastava, New York). It was suggested that these molecules, which are 40% homologous with HSPs, might present unique peptides of each tumor.
Melanoma in mice and humans The incidence of melanoma in humans is increasing; 1 in 1500 individuals in 1935 rising to a projected 1 in 90 individuals in the year 2000. Animal models of melanoma induction using chemical carcinogens such as DMBA and UV radiation can lead to the development of immunologically cross-reactive melanoma in a high percentage of animals (M. Kripke, Houston). Similar cross-reactivity of human melanoma -ea~ive T ce',!~ with HLA-matched tumors have been identified (V. Kumar; T Darrow, Durham) as well as unique T-cell receptors prefere~,tially using the V,2 segment in T cells reactive to melanoma in many patients. Melanomas appear to be able to process and present exogenous antigen_ The ability to present tetanus antigens appears to decreasewith more advanced tumors (D. Guerry, Philadelphia) and it was suggested that altered MHC class II molecules may provide a mechanism of escape Transfection cf murine melanomas with human melanoma antigens identified by murine monoc!onal antibodies (S. Liang, D. Herlyn, Pittsburgh; P. Greenberg, St Louis) suggests novel ways of studyir,g recognition of such antigens in animal models. Furthermore, generation of anti-idiotypic antibodies that can induce so-called Ab3, which are capable of binding tumor antigens in the host, have been extensively studied in humans (R. Levy, Stanford; D. Herlyn, Pittsburgh; S. Ferrone, NY Medical College) and suggest alternative strategies for induction of tumor recognition in patients. Many melanomas appear to express members of the integrin superfamily including VLA1-4 and VLA6 (A. Anichini, Milan) that are potentially important molecules in T-cell recognition of such tumors. T-cell clones with restricted reactivity to melanoma have now been identified by many groups (M. Lotze; T. Boon, P. Hersey, Newcastle; K. Itoh, Houston). At least four different antigens can be defined by such clones(T. Boon). Attempts to characteriz the antigens are being undertaken. The administration of IL-2 to melanoma patients is associated with a 25-40% objective response rate which is believed to be related to induction of specific tumorreactive T cells (M. Lotze; M. Mitchell, Los Angeles; P. Sondel, Madison). This has been most clearly shown with
Immunology Today, Vol. i 1, No. 6 1990
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the adoptive transfer of so-called tumor infiltrating lymphocytes (TILs), which can be expanded in tissue culture using gas-permeable bags or hollow fiber systems with over 100-fold expansion to cell numbels exceeding 10 ~ (S. Rosenberg, P. Aebersold, Bethesda; P. Simon, Wilmington). Such ,-~lls can be identified following transfer at tumor sites using radiolabels or using cells genetically marked with the neomycin phosphotransferase bacterial gene (S. Rosenberg). These gene-marked cells have been given to _fix patients and recovered from peripheral s:,tes up to 60 days after transfer. Use of adoptively transferred T cells as vehicles for gene therapy is now being actively pursued (M. Blaese, Bethesda). Although TILs can be derived from a variety of tumors, other means of generatincj such cells including in vitro stimulation (S. Slovin, Philadelphia; A. Moser, M. Wettendoff, Brussels) and following in vivo immunization (F. Chang, M;nneapolis) or directly from tumor explants (J. Kurnick, Boston) have been carried out.
Problems The last decade has witnessed a veritable exolosion in the investigational study and clinical use of immunotherapy for the treatment of cancer. Although this is an exalting development, the promise of cancer immunotherapy has not yet been fulfilled 1,2. Why is there an apparent discrepancy between the u~euly u~ cancer ~mmunomerepy ana the actual results from clinical studies? Michael Osband and Susan Ross suggest that there are several basic problems with the clinical study ar,~ therapeutic use of immunotherapy that must be overcome before it can be considered a viable treatment for a broad range of tt,mors. The purpose of this article is to desoibe some of the more important of these problems. .LL
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Summary It is now beyond question that T cells are central to the immune response to tumors in both mice and humans. Means to modulate the immune response to tumors in murine models and in human melanoma are now well established and studies to determine whether these findings can be generalized to a larger number of neoplasms are in hand. Attempts are also being made to define where immune recognition breaks down, focussing on the T-cell receptor, MHC molecules, cytokines and other accessory molecules involved in the response to tumor antigens. Thus tumor immunology is clearly entering the new decade on a far st:rer footing. Generous support for this conference was provided by the sponsors Cetus, Immunex,Triton and SterlingOncologyas well as by Armand H.~mmer, Genentech,GeneticTherapy Inc. and IDECpharmaceuticals.Carefulpreparationof this manuscriptby Ms Etta Owens is appreciated.
in the investigationalstudy and clinical use of cancer immunotherapy Michael E. Osbandand SusanRoss
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The Tolstoyanmodelof immunopathophysiology 'All happy families are alike. Each unhappy family is unhappy in its own way.' Tne.~e are the opening lines of Leo Tolstoy's Anna Karemna and they can be paraphrased to create a useful model by which to understand human immunopathophysiology: 'All healthy immune systems are alike. Each sick patient is immunologically dysfunctional in his or her own way.' The human immune system is complex, multi-faceted and highly specific. It is, therefore, naive to assume that the immunobiology of the host-tumor relationship is identical, or even similar, in all patients with cancer. In fact, the variable clinical response seen with most cancer immunotherapy suggests that individual patients have very different immunological mechanisms involved in the
Joint Clinical Immunotherapy Program, Boston UniversitySchool of Medicine and New England Baptist Hospital, 125 ParkerHill Avenue, Boston, MA 02135, USA. ~) 1990, ElsevierScience PublishersLtd, UK. 0167--49191901502.00
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etiology and pathophysiology of their specific malignancy3. These can include important differences in the existence, nature and distribution of tumor antigens on the malignant cells4, as weli as the specific immune response of the patient to the tumor 5. The entire premise of a well-designed and well-conducted clinical trial is to test the safety and efficacy of the same therapeutic agent when used in comparable patients. The unique nature of the host-tumor relationship in each individual patient makes the dev-=Icpment and clinical study of immunotherapy very difficult. These issues are even more pronounced with certain treatment approaches, such as adoptive immunotherapy6 or autologous tumor vaccines7. In these settings, both the actual therapeutic that is used and the patient who is treated are one-of-a-kind. This results in clinical studies that often consist of no more than a series of anecdotes 8-1°. Another result of the unique nature of the host-tumor relationship is the lack of useful animal models that can be applied to the development of immunotherapy. The response of animals to chemotherapy, radiation and surgery is generally predictive of their effect in human patients 11.12.This is not the case with immunotherapy ~3'14. Many immunotherapeutic agents are inactive in other species. In addition, owing to the extreme complexity of the host-tumor immunorelationship, animal models do not fully mimic the biology of human patients with cancer. Finally, the immune system is obviously different in
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