Therapeutic immunization against cancer antigens using genetically engineered cells

Immunotechnology

3 (1997) 161-172

Review article

Therapeutic

immunization against cancer antigens using genetically engineered cells Hans Olov Sjiigren

Tumor Immunology Unit, Department of Cell and Molecular Biology, Wallengerg Laboratory, S-220 07 Lund, Sweden

University qf Lund,

Received 19 June 1997; accepted 26 June 1997

1. Introduction A cornerstone in the field of tumor immunology is the full identification of tumor derived peptides, proven to be target antigens for cytolytic T-cell effector cells (CTL) of certain tumor patients [1,2]. A generally accepted implication is that such tumor peptides make a rational approach to the development of clinically useful therapeutic immunizations possible. The basis for this is that such tumor derived peptides may be exploited to generate more tumor selective immunity and possibly also stronger antitumor immune responses. Facing the weak and/or misdirected anti-tumor immune response of the cancer patient at start of therapy, the task of increasing the strength of the immune response up to the level leading to dramatic regressions even of fairly large tumors has to be given top priority. Viable tumor cells genetically engineered to produce or express certain immunological key molecules are important tools in developing the principle ways of achieving this goal. Development of sufficiently powerful procedures is conceptually so important that certain

anticipated side effects of those initially designed procedures should not necessarily detract from their importance. The risk of, e.g. inducing autoimmune reactivity by an immunization procedure involving viable tumor cells, due to presence of a variety of tissue selective antigens shared by tumor cells and some normal cells of the organ of tumor origin, can be dealt with at a later stage when the requirements for induction of tumor regression have been defined in detail. At that stage, if not before, the replacement of genetically engineered tumor cells with defined tumor derived peptides and appropriately activated antigen-presenting cells (APC) and other normal cells engineered to produce or express certain key molecules can be foreseen.

2. Rationale for therapeutic immunization against cancer For a long time the main issue in tumor immunology was whether or not tumor specific antigens exist in spontaneous tumors. Failure of inducing rejection or elimination of growth of a

1380-2933/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PUS1380-2933(97)00015-S

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certain tumor was taken as evidence that tumor antigens did not exist. The present realization that cells of many tumors do indeed display peptides on their MHC class I, which are tumor antigens in the sense that the tumor bearer has expanded T-cell clones with receptors recognizing the peptides, has altered the focus of interest to the mechanisms involved in allowing these tumor cells to grow despite tumor antigen recognition. One has been forced to accept that also established tumors displaying such recognizable peptides are often difficult to eliminate by various measures to augment the antitumor immune responses. Preimmunization with irradiated tumor cells can often induce a relative immunity to subsequent isografting of the same tumor, although this immunity can in the majority of cases be overcome by increasing the dose of tumor cells used for challenge. In very few experimental models has it been possible to demonstrate an immunity rejecting for example 10 x lo6 cells. In the therapeutic situation, we request the treatment to cause an eradication of established tumors containing much more than 10 x lo6 cells. The therapy must induce rejection of tumor cells that are already part of a well established tumor tissue, when it is initiated. This should have allowed ample time for previous selection of low immunogenicity in the tumor cell population and the possible mounting of a noncytolytic response, which counteracts the induction of immune responses that could eliminate the tumor. 2.1. Provision of tumor-derived peptides in an immunogenic form

The quantity of tumor peptides displayed on the MHC class I of tumor cells as the direct target antigen, may for a variety of reasons be very limited. This may be due to production of only a small amount of the protein involved or a deficiency of any of the molecules required for peptide formation, transport and presentation on MHC class I at the cell surface. Though this amount may be quite sufficient for the cell to be sensitive to already generated effector cells, it may be too low to induce a strong T-cell response. Since the immunogenicity is highly dependent on

3 (1997) 161~ 172

the adequate presentation of appropriate peptides on MHC class I and II of professional APC capable of providing costimulation [3], deficient availability of such cells may cause the most serious limitation of T-cell activation (Fig. 1). The presentation can be improved by augmenting the MHC class I expression by gene transduction, exposure of the cells to IFN-y, or replacing tumor cells with tumor derived heat shock protein (HSP) with bound tumor peptides [4] or by activated dendritic cells preincubated with ‘peptide preparations’ eluted from tumor cells [5]. This can greatly enhance the T-cell responses to tumor antigens. Another way of mobilizing dendritic cells and macrophages at the site of immunization is to use tumor cells transduced with GM-CSF. Such cells have been shown to induce an infiltrate mostly of dendritic cells and macrophages and a few T-cells [3]. In humans the dendritic differentiation is enhanced by TNF-c( as a cofactor [6], whereas IL-4 appears to have this role in mice [7]. Mature dendritic cells express high levels of MHC class I and II, costimulator molecule B7 and cell adhesion molecules such as ICAM-1. The presentation of these molecules and the production of key cytokines make them to the most potent antigenpresenting cells. It is important to remember that although these various procedures to increase the immunogenicity results in an augmented generation of effector cells, the parental tumor that is to be inhibited has to have at least some expression of antigenic peptide displayed on its surface MHC class I in order to be sensitive to the induced effector cells. However, the level of expression required is known to be much lower than that required to induce the immunity. 2.2. Provision of improved costimulatory signals or adhesive contacts between tumor cells and APC and/or responsive lymphocytes

One possible reason for the inefficiency of the cytolytic response is lack of costimulating signals. The T-cells recognizing tumor peptides will as a consequence become anergic rather than activated [S]. The costimulatory signals may be lacking or are insufficient because the tumor cells, as normal

H.O. SjGgren /Immunotechnology

IFN-)I TNF 1

I IL-2

TNF-a \

3 (1997) 161- 172

GM-CSF IL-1 I

163

IL-4 / ILL2R FasL

CD40L

CD40

CTLA-4 CD2

J

IL-l 2R

0CTL FcR signal

TGF-P PGEQ C3b signal

\

IL-10

Glucocorticoids

Fig. 1. Key molecules in the interaction between T-lymphocytes and professional antigen presenting cells (APC), particularly dendritic cells (DCs). Blood monocytes are derived from bone marrow progenitors and may differentiate into macrophages under influence of M-CSF, or into DCs when exposed to GM-CSF and TNF-r and/or IL-4. Immature DCs have high capacity to capture antigen and process it to peptides presented on their MHC, both on class II and class I. Phagocytic processes and MHC expression are upregulated by IFN-7. DCs mature as an effect of exposure either to TNF-x, IL-l, LPS, or to CD40L of activated T-cells. Upon maturation DCs upregulate the costimulatory molecule B7 and adhesion molecule ICAM- 1 and if also receiving a signal from their CD48158 adhesion molecules (by interaction with CD2 of activated T-cells), they start production of IL-12. T-cells get a necessary costimulatory signal from the B7 interacting with their CD28 and CTLA-4 receptors allowing them to get activated. If exposed to IL-12 and/or IL-18 and IFN-), they differentiate into Thl cells producing IL-2, IFN-y and TNF, which help activate those CTL effector cells which have encountered their peptide antigen presented on MHC class I. The function of DC and particularly their IL-12 production, is suppressed by several different factors, including TGF-P, prostaglandin-E-2 (PGE-2) IL-IO, glucocorticoids and signals via C3b complement receptors (CD46) or Fc-receptors binding immune complexes.

cells expressing tissue-specific peptides, may not be able to activate the resting APC to provide the costimulatory signals [9]. The necessary costimulatory signals can be provided at the site of immunization in different ways. The genes of the most important molecules, B7-1 and/or B7-2, can be transduced into the tumor cells or these molecules can be induced in the local dendritic cells or macrophages by e.g. signaling via CD40/ CD40L [lo], possibly by CD40L transduced cells.

2.3. Provision of stimulators of proliferative T-cell responses

A sufficiently high local concentration of T-cell growth factors, e.g. IL-2, IL-7 and IL-12, is mandatory in inducing a strong cytolytic immune response. This can be achieved by using cells producing these interleukins or by use of local treatment with recombinant interleukins. IL-2 is a main T-cell growth factor triggering several secondary events in the process of T-cell activation.

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Dendritic cells Dendritic cell

Cytolytic effector cells Fig. 2. Activation of cytolytic effector cells. Generation of CTL, NK and activated macrophages is dependent on production of sufficiently high concentrations of IFN-y, IL-2 and TNF. These molecules are produced by T helper-cells and NK-cells stimulated by IL-12, TNF-a and IL-7. Cytolytic responses are consequently inhibited by factors suppressing IL-12, IL-2 and IFN-7 production.

T-cells recognizing an MHC-presented peptide by its TCR and receiving adequate costimulatory signals will be triggered to express high affinity IL-2 receptors. The amount of IL-2 available to the cell is a most important factor determining the amplitude of the proliferative response. IL-7 is critically important for the maturation of both B and T lymphocytes [11,12]. After maturation only T-cells are responsive to IL-7, which then often acts synergistically with IL-2. Its receptor also shares a common signaling gamma chain with that of IL-2 [13]. IL-7 is stimulatory for both

CD4 + and CD8 + cells, often with preference for CD8 + [14,15]. As IL-2, it supports T-cell proliferation in vitro, but often for longer periods than IL-2 [16]. This might be due to its inhibitory effect on the production of the immunosuppressor TGF-P by macrophages, whereas IL-2 stimulates this production [ 171. IL-12 is produced by professional APCs (dendritic cells, macrophages and B-cells) and acts on preactivated T- and NK-cells, stimulating their proliferation [18], IFN-:/ production [19] and cytolytic activity [20]. It is the most potent factor

H.O. Sjigren / Immunotechnology 3 (1997) 161- 172

inducing the type 1 cytolytic T-cell differentiation and counteracting the Th2 generation [21] (Fig. 2). The initiation of IL-12 production is triggered by interaction between multiple molecules present on the APC and activated T-cells (Fig. 1). The production of IL-12 by the APC is dependent on their CD40 binding to the CD40 ligand (L) of an activated T-cell, the expression of which is induced as a result of interaction between the TCR and MHC-peptide.and is independent of costimulation signals from B7 [lo]. The responsiveness of activated T-cells to IL-12 is regulated via signaling from CD2 interacting with CD58 or CD48

[W. 2.4. Provision of antigenic stimulation combined with local cytokines forcing stimulation of the cytolytic type 1 T-cell response with or without simultaneous inhibition of the noncytolytic type 2 T-cell responses A major reason for a weak cytolytic T-cell response is likely to be that the response is diverted to a type 2 reactivity leading to support for antibody production but not for cytolytic functions, which are instead inhibited. IL-12 appears to be a dominating key molecule in determining whether or not a cytolytic response should develop. It has a variety of immunomodulatory effects on activated T- and NK-cells, stimulating the proliferative response [ 181, interferon-y (IFNy) production [19] and cytolytic activity [20]. It is a most powerful inducer of the T-helper cell type 1 (Thl) differentiation [21], characterized by the production of IFN-y, TNF-P and IL-2 and therefore of the total cytolytic response. IL-12 is produced by nonfollicular dendritic cells (DC) and by activated macrophages in response to CD40CD40 ligand interaction [23], which also has been reported to upregulate the expression of MHC class I and II, as well as the costimulators B7-1 and B7-2 [24]. IFN-), has a powerful, enhancing effect on IL-12 production by monocytes/ macrophages, most pronounced when preexposing the cells prior to induction via CD40/CD40L [25,26]. The IL-12 responsiveness of activated Tand NK-cells is critically dependent on the upregulation of the IL-12 receptor [27] but is

165

augmented also by IL-2 [19], IL-7 [28], costimulation via CD28-B7 [29] and via CD2-CD58 or CD2-CD48 [22]. IL-12 production is down-regulated by IL-10 and IL-4 in an additive manner [30], by TGF-/3 [31] and by PGE-2 [32]. The PGE-2 effect is partly mediated by a CAMP elevation, partly by an augmented IL-10 production [33]. IL-10 inhibits IL-12 production from APC and also the production of IFN-7 by NK cells [34]. One factor that would tend to shift the balance to primarily IL-10 production is lack of signaling through CD40/ CD40L [23], signaling through Fc-receptors of the APC by binding of immune complexes [34], exposure to p15E of endogenous retroviruses [35], or exposure to TGF-/I [31]. These mechanism would help to maintain the type 2 reactivity once initiated. Some molecules are known to drive the T-cell differentiation in the type 1 direction and can be supplied at the immunization site. These would include IL-12, IFN-y and probably IL-l& which all can be produced by transduced cells. Furthermore, cells expressing the CD40L and thereby capable of triggering the CD40/CD40L signal that induces IL-12 could be of interest. IL-18, formerly designated IGIF, is produced by monocytes/macrophages in response to exposure to certain bacterial materials [36], but also as a normal step in the immune response to other antigens [37]. It is a potent inducer of IFN-7 and GM-CSF [38] and although acting independently, it shares also several other properties with IL-12 and acts synergistically with it [36]. 2.5. Rescuing anergized T-cells to an activated state If T-cells are anergized due to deficient costimulation, they might be possible to rescue by exposure to relatively high doses of IL-2 and/or IL-12 [39,40]. 2.6. Counteraction of immunosuppressing molecules produced by the tumor cells Several tumors produce immunosuppressive molecules or factors which push the immune re-

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sponse towards type 2 reactivity. Examples of this are TGF-/3 [41,42], PGE-2 [43,44] and IL-10 [45], which all inhibit the cytolytic immune responses. It is particularly important to counteract any such inhibitor when cells are being used for immunization. This has been proven to be feasible by e.g. transducing an antisense TGF-P into cells used for immunization and an analogous technique could possibly be used also for IL-lo.

3. Evaluation of augmented immunogenicity Increased immunogenicity may be evaluated in several different ways representing different levels of immunogenicity (Table 1). One commonly used technique is to test for decreased tumorigenicity in immunocompetent animals, i.e. decreased capacity of the engineered cells to form a progressively growing tumor upon inoculation of various cell doses into nonimmunized, immunocompetent animals as compared to in vitro growth or growth in immunodeficient animals. The idea would be that highly immunogenic cells would be anticipated to induce a strong immune response capable of rejecting the cells. A problem with the use of a decreased capacity of the altered cells to grow as isografts is that peptides derived from the proteins corresponding to the transferred genes, including the resistance gene required for efficient selection of transfectants, will be presented on their MHC class I along with the peptides derived from tumor Table 1 Evaluation of augmented immunogenicity Decreased tumorigenicity in nonimmunized, immunocompetent recipients. Increased capacity to induce transplantation immunity to subsequent isografts of parental tumor. Capacity to induce regression of preexisting parental tumor growing at a realistic organ site. Dependence of the effects of immunization on continued presence of CD8+ and/or CD4+ T-cells and/or NK-cells. Enhanced capacity to induce T-cell responses to parental tumor cells as evaluated in vitro by proliferation assays (MLTC), cytolytic assays or antigen selective production of any of an array of T-cell related cytokines.

3 (1997) 161- 172

antigens of the original tumor. The response to these additional antigens may contribute to an inhibited growth capacity and can be misinterpreted to indicate a stronger response to the original tumor antigens. The acquisition of an increased capacity to induce rejection of a subsequent isograft of the parental, genetically unaltered tumor may be regarded as a minimum requirement for concluding that an enhanced relevant immunogenicity has been demonstrated. Indicative of a usefully enhanced immunogenicity is that the cells have acquired the capacity to induce regression of preexisting parental tumors. This can be complemented with the demonstration that the effect is dependent on the continued presence of CD8 + and/or CD4+ T-cells and is no longer observed after depletion of these subsets by specific antibodies. In vitro evidence of enhanced T-cell responses against parental tumor cells can be obtained in assays for proliferative responsiveness, production of various cytokines upon exposure to parental tumor cells or in cytolytic assays. 3.1. Results of therapeutic mod$ied

immunization

ex vivo to express immunological

with cells key

molecules

Various experimental tumor models have been used for analysis of the effect of immunization with tumor cells -genetically engineered to produce a variety of cytokines or expressing various other immunological key molecules. 3.1.1. IL-2 The first molecules that became available were different cytokines, and first of those the IL-2. After having been studied in recombinant form for systemic treatment intravenously or intralymphatically with some clinically significant responses in certain types of cancer [46,47], it became clear that the toxicity (hypotension and edema) at high doses limits its use in this form [46]. This might be true for most candidate cytokines since they are physiologically acting locally with only low concentrations seen in circulation. Therefore, IL-2 has been tried intralesionally [48] or in the form of transduced cells for local action

H.O. Sjtigren / Immunotechnology 3 (1997) 161-l 72

at the site of immunization, but without sufficient production to result in systemic spill over causing toxicity. Immunization with tumor cells expressing IL-2 has resulted in significant antitumor immune responses against several different murine tumors (Table 2), including fibrosarcomas, melanomas, mammary carcinomas, colon carcinomas and pancreas carcinomas [49-521 and also against rat prostate cancer [53]. The immunization was performed subcutaneously, intraperitoneally or intradermally, mostly with irradiated cells and was shown to induce systemic immunity demonstrated as capacity to reject subsequent isografts of the parental tumor, usually tested by S.C. challenge with low cell numbers. The growth of preexisting pulmonary micrometastasis can also be significantly inhibited by immunization started 3 days after the challenge [54]. This effect is most likely mediated by CTL effector cells rather than activated NK or LAK cells since it was shown that additional transfection of MHC class I to the tumor cells used for immunization greatly enhanced their immunizing effect. In the rat prostate experiments it was demonstrated that immunization initiated 3 days after challenge with the parental tumor caused significant regression, and very importantly, regressions were obtained also when the challenge tumor was given orthotopitally [53]. No general toxicities were recorded as a result of the immunizations. It is of considerable interest that when transduced murine melanoma B16 cells producing the highest levels of IL-2 were compared to those producing intermediary amounts, the former failed to induce any significant systemic immunity [55]. This might be consistent with the view that low concentrations of IL-2 preferentially bind to high affinity IL-2 receptors [56] and may therefore selectively stimulate antigen-specific CDS + CTL, whereas high concentrations of IL-2 allow binding to NK-cells and LAK-cells. This emphasizes the importance and the complexity of the quantitative aspect in optimizing this type of therapy. In the clinical application of this concept of therapeutic immunization a serious limitation is that many tumors may not be possible to culture well enough to allow the gene transduction. It is therefore of great importance that IL-2 has been

161

shown to be readily transducible to freshly explanted normal fibroblasts, which can always be obtained from any patient. Immunization with IL-2 transduced fibroblasts admixed to tumor cells have been demonstrated to induce immunity to subsequent parental tumor isografts and also to cause regression of preexisting tumors [51,57]. 3.1.2. IL-7 Immunization with IL-7 transduced murine fibrosarcomas and mammary adenocarcinomas protects against subsequent parental tumor challenge and inhibits the growth of preexisting pulmonary micrometastases of the parental tumor [58,59]. A heavy lymphocyte infiltration was induced at the immunization site and these infiltrating T-cells demonstrated a selective cytolytic effect against the parental tumor cells [60]. In the mammary tumor model double transfectants expressing both IL-7 and B7.1 showed a significantly increased immunogenicity. In a rat glioma model, subcutaneous immunization with rat IL-7 transduced cells caused a significant frequency of regressions and cures of preexistent intracerebral parental tumors [61]. 3.1.3. IFN-y Immunization with IFN-), transduced tumor cells of murine neuroblastoma, fibrosarcoma, plasmocytoma and mammary carcinoma origin has been shown to induce protection against subsequent parental tumor challenge [62-641. In other cases the transduced cells were reported to be efficiently rejected but failing to induce systemic immunity [65]. In a fibrosarcoma model in which cells transduced with IFN-9 alone failed to induce immunity, transduction with simultaneous expression of both IL-2 and IFN-1/ resulted in cells inducing protective immunity [66]. In a rat model, regression of preexisting intracerebral parental glioma isografts was induced by subcutaneous immunization with rat IFN-), transduced glioma cells [61]. 3.1.4. IL-12 IL-12 has been transduced into cells of three principally different types: tumor cells, normal fibroblasts and normal dendritic cells. Immuniza-

S.C. S.C.

S.C. S.C.

Murine Tumor cells

Murine tumors Rat glioma

tumors

Tumor

Murine

Rat glioma Rat ovary tumor

Murine Colon cancer

B7-1

B7-2

IFN-;

GM-CSF

TGF-B antisense

MCP/MIP

cells

Murine Tumor cells Fibroblasts Dendritic cells

IL-12

s.c

SC.

S.C.

S.C.

id. id. i.v.

i.p. S.C.

S.C.

Murine Fibrosarcoma Rat glioma

IL-7

s.c., 1.p.. i.d. S.C. id.

Immunization site

Murine Tumor cells

tu-

cell

IL-4

Transduced

Murine Tumor cells Fibroblasts Rat prostate mor

ex-

Immunization

IL-2

Molecule pressed

Table 2

Non-IR

IR IR

IR

IR IR

Non-IR

NowIR

IR Non-IR Non-IR

Non-IR IR

IR

IR Non-IR IR

Irradiated (IR)/ nonirradiated cells

+

+ +

+

+

+

+

+

+

+

+

immunogenicity

+

+ +

+

+ +

+

+ +

+

+ + +

+

+ + +

t-

+

+ + +

+

+ + +

Ectopic

Augmented CTL response in vitro

+ +

+

+

+

Orthotopic

Regression of preexisting parental tumor isograft

+

+

+ +

+

+

+ +

Rejection of subsequent parental tumor isograft

of enhanced

Decreased tumorigenicity

Evidence

H. 0. Sjigren / Immunotechnology 3 (1997) 161- 172

tion with transduced tumor cells or fibroblasts protected against subsequent, simultaneous and preexisting challenge with the parental tumor [67]. Inoculation of transduced fibroblasts peritumorally in a parental tumor isografted 7 days earlier resulted in complete regression and later rechallenge confirmed selective immunity [68]. The antitumor effect appears to be mostly dependent on CD8 + T-cells but also on CD4+ and NK-cells [69]. Dendritic cells cultured from bone marrow were transduced with IL-12 and exposed to peptides eluted at low pH from collagenase suspended parental tumor cells and inoculated intravenously into mice bearing established tumors after receiving parental tumor isografts 8 days earlier [70]. A significant, major tumor growth inhibition was observed, although not complete cures. 3.1.5. B7 It was first demonstrated that B7-1 transduced murine tumors regressed upon isografting to immunocompetent recipients and this resulted in a subsequent immunity also to the parental tumor mediated by CD8 + CTL [71]. These results could be reproduced with several tumors having a detectable level of original immunogenicity but not with entirely nonimmunogenic tumors [72,73]. 3.1.6. GM-CSF In a comparative study of a number of moderately or weakly immunogenic murine tumors, tumor cells transduced with each of several different cytokines were used for immunization and the immunity to a subsequent parental tumor isograft was evaluated [74]. The GM-CSF expressing cells were found to produce the most consistent and strongest systemic immunity. Systemic immunity was induced by GM-CSF transduced rat prostate cancer cells and growth inhibition was demonstrated also of preexisting parental tumors, but in this model the effect was weaker than that of IL-2 expressing cells [53]. 3.1.7. TGF-/3 antisense Decreased growth capacity in immunocompetent recipients by murine mesothelioma treated with TGF-B antisense RNA was reported to be associated with infiltration of CD8+ T-cells [75].

169

Immunization with TGF-P2 antisense transduced rat gliomas was then demonstrated to significantly inhibit growth of subsequent or preexistent intracerebral tumor parental isografts [76]. Analogously, TGF-P 1 antisense transduced murine mammary carcinoma cells with a confirmed decrease of their TGF-P production have been shown to have an inhibited growth capacity in immunocompetent recipients compared to the parental tumor [77]. In this case there has been no investigation of systemic antitumor immunity. 3.2. Combined immunization with two or more different, genetically engineered cells So far most efforts have been devoted to analysis of the rather simple concept of immunizing with one, or in a few cases two, type(s) of genetically modified cell(s) at a time. Although this is the natural way to initiate the analysis, it is not likely to be the way to obtain an optimal therapeutic effect. Rather one may anticipate that the prerequisite for induction of an exceptionally strong cytolytic response is the simultaneous optimization of several different features of the immune stimulation. An important task is to define which combinations give synergistic effects, which are additive and ~which are less than additive. This work has presently just started. A component that probably always should be one part of the combination is an optimized presentation of the tumor antigen. Shortcomings regarding this aspect may have many different reasons. One is that the MHC class I expression is insufficient. The results obtained indicate positive effect both by using tumor cells directly transduced with MHC class I or cells with an augmented MHC expression secondary to IFN-y transduction. The immunogenicity is obviously very much affected by the type, stage of activation and number of professional APC present at the site of immunization. This is determining the efficiency by which tumor peptides are presented to CD4+ helper T-cells, but it is conceivable that also tumor peptides presented on MHC class I of optimized APC provide for a much stronger activation of CD8+ CTL than the tumor cells can

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ever do. Capacity to process antigen and express it is a function of immature dendritic cells [78]. Their capacity to stimulate T-cells has to await maturation, but this is accompanied with a loss of antigen processing ability. Here one has the option to further increase the stimulation by preexposing the APC to tumor derived peptides, tumor derived HSP with bound tumor peptides [79], or synthesized peptides, in the cases where their sequences have been determined. It is also always required to provide for an optimal costimulation and the importance of this is well established. So far this has mostly been done by transducing B7-1 or B7-2 into tumor cells. However, it might be at least as important that the local APC have an optimal expression of B7. This can be achieved in different alternative ways, either by admixing a large number of APC, in vitro induced to express B7, or theoretically by transducing the tumor cells with e.g. CD40L, which can be anticipated to induce B7 in the local professional APC, besides ICAMand IL-l 2 that should also be beneficial. If B7 tumor transfectants are being used a synergistic effect is obtained by transducing also CD48 [80]. It is to be remembered that much of the effect of GM-CSF transduced tumor cells is likely to be due to an improved peptide presentation and costimulation. Though optimizing tumor peptide presentation and costimulation should often result in a strong response, this response could most likely become even stronger if one could provide for one or more additional growth factors, e.g. by using e.g. fibroblasts transduced with IL-2, IL-7 or IL-12. By themselves, cells transduced with each of these interleukins have proven to have considerable immunogenicity and the combination of IL-12 with either IL-2 or IL-7 synergizes the effect [31]. Besides optimizing the proliferative T-cell response either one of these combinations would also provide for a dominating cytolytic response and would be expected to rescue some anergic T-cells. An even stronger activation of the cytolytic response could possibly be obtained by adding cells releasing IL-18, which has a strongly synergistic effect with IL-12 [36], or IFN-y. All these cytokines could be transduced into fibroblasts [68] or possibly into cultured dendritic cells [81].

3 (1997) 161 -172

In view of the substantial evidence for the suppressive effects of TGF-/?, it would seem natural to test each tumor to be used for immunization for release of TGF-fi and if it produces higher amounts, either transduce antisense TGF-B of the right isotype, or possibly transduce it with IL-7, which has been shown to suppress TGF-P production [17]. Since several tumor types have been shown to produce IL-10 [43,44], which has been reported to be a potent inhibitor of cytolytic responses, an analogous procedure would be important for that cytokine. Finally, one could further enhance the response by adding an additional component to the immunization, facilitating the influx of leukocytes through the capillary walls into the immunizing site. Candidate molecules are MCP-1 and/or MIF-1, which have been transduced to tumor cells, augmenting the macrophage and neutrophilic infiltration [82]. As an alternative or complement to therapeutic immunization with genetically engineered cells, recombinant cytokines are available for systemic administration, or for local treatment when combined with an immunization procedure. Extensive efforts devoted to systemic treatment of various types of cancer with certain cytokines have met with some limited success but also considerable side effects due to the systemic administration [46]. This may indicate that in the majority of cases recombinant cytokine therapy should better be combined with other components which can focus the effect on a longer-term augmentation of the cytolytic anti-tumor response. A further major alternative mode of immunotherapy is based on the technology involved in proliferative growth of T-lymphocytes in vitro, making it possible to expand the number of patient lymphocytes, to modify their responsiveness by antigen stimulation and exposure to, e.g. various cytokines and to select certain subpopulations before readministering them into the patients. In principle, this form of therapy can of course be combined with therapeutic immunization, particularly at the early stages of immunization before full effect can be anticipated. Alternatively, local transfer of Tlymphocytes and/or activated dendritic cells at the site of immunization or perilesionally might have potential.

H.O. Sjiigren /Immunotechnology 3 (1997) 161-l 72

It is important to keep in mind that although all these aspects should theoretically be optimized simultaneously in order to achieve the strongest possible immunization effect, this has to be balanced against the complexity of the procedure. However, it would be of considerable importance to test in some model systems, whether it is possible in this way to design a procedure that is potent enough to cause regression even of big tumors, although perhaps to the price of certain serious side effects. If this would turn out to be possible, it would clearly demonstrate that the goal of curing patients by therapeutic immunizations is indeed realistic. The procedures can then stepwise be simplified and it is very likely that one can learn how to avoid some of the most serious side effects.

Acknowledgements

This work was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Society and the GAE Nilsson Foundation.

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