Clin. exp. Immunol. (1990) 82, 200-207

A novel strategy for targeting CD4 + PPD-reactive T cells against tumour cells using PPD monoclonal antibody heteroconjugates A. M. P. MONTGOMERY, M. G. WING & P. J. LACHMANN Molecular Immunopathologi' Unit, MRC Centre, Cambridge, England

(Acceptedfor publication 26 April 1990)

SUMMARY We have constructed PPD monoclonal antibody heteroconjugates specific for a tumour-associated antigen of C57BL/6 melanomas or for human complement component C3d fixed de novo to murine fibrosarcoma cells (MC6A). The ability of our heteroconjugates to target CD4+ PPD-reactive T cells against the appropriate tumour targets was then determined in vitro. Heteroconjugate-treated B16Fl 0 and MC6A tumour targets were both able to present PPD to the specific T cells, resulting in activation and concomitant lymphokine secretion. Secreted lymphokines were then demonstrated to cause significant tumour cytolysis and cytostasis in vitro. Preliminary experiments in vivo suggest that this targeting system may provide the basis for a future immunotherapeutic strategy. Keywords PPD T helper clones monoclonal antibody heteroconjugates immunotherapy

INTRODUCTION

tumour cytostasis

of the tumour cells. The rationale for this work is to focus

a

delayed-type hypersensitivity (DTH) response against selected Despite uniformity in the expression of CD5 (Ly-1) and CD4 (L3T4) surface markers, it is now apparent that there is considerable functional heterogeneity within the murine class 11-restricted T helper population. Two subsets have been defined on the basis of lymphokine secretion and concomitant function, and these have been variously named Thl/T inflammatory and Th2/T regulatory (Mosmann et al., 1986; Bottomly, 1988). While this functional dichotomy may prove to be oversimplistic with further heterogeneity within these subsets (Wong et al., 1988), it has been consistently demonstrated that Th 1, but not Th2 cells are able to secrete both tumour necrosis factor (TNF) alpha and beta and interferon (IFN) gamma, and consequently can mediate significant tumouricidal activity. Here we describe a novel system designed to focus class IIrestricted PPD-reactive Th I cells onto selected tumour targets coated with PPD. We have previously demonstrated (Vyakarnam, Lachmann & Sia, 1988) that murine Th I clones specific for PPD are able to lyse syngeneic tumour targets treated with a Con A-PPD heteroconjugate. Lysis was demonstrated to be PPD-specific and class II-restricted. The clones displayed bystander lysis indicative of a lymphokine-mediated mechanism. We have extended this system to specific tumour targeting using PPD monoclonal antibody heteroconjugates specific for a tumour-associated antigen of C57BL/6 melanomas or for the human complement component C3d fixed de novo to the surface Correspondence: A. M. P. Montgomery, Molecular Immunopathology Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.

tumour targets in vivo with a view to developing a new

immunotherapy that can be exploited in a BCG immune population. The use of heteroconjugates directed to complement components should allow the activation of T cells at sites of complement fixation resulting from a tumour-induced pathway or as a result of conventional monoclonal antibody

(MoAb) therapy.

MATERIALS AND METHODS

Antigens Tuberculin PPD was obtained from the Central Veterinary Laboratories, Weybridge, Surrey. Control antigens included streptokinase and ovalbumin (Sigma, Poole, UK.) Culture media

Complete medium (CM) consisting of RPMI 1640 (GIBCO, Paisley, UK), supplemented with 10% heat-inactivated FCS; 1 U/100 ml penicillin/streptomycin antibiotic mixture (GIBCO); 2 mM L-glutamine, and 5 x 105 M 2-mercaptoethanol was used. Mice

C57BI/6 (B6), C57BI/10 (B10) and recombinant B10.A 4R, 5R and 2R mouse strains were obtained from Olac, Bicester, UK.

Tumour cell lines A B16-FIO melanoma cell line (H-2b) and a MC6A methylcholanthrene induced fibrosarcoma (H-2b) of B6 and BO origin,

200

PPD monoclonal antibody conjugates respectively, were used. The B16-F1O melanoma cell line was kindly provided by Dr R. C. Rees, Department of Virology, Medical School, Royal Hallamshire Hospital, Sheffield, UK. Both tumour lines were maintained in RPMI 1640 supplemented with 10% FCS. Monoclonal antibodies Hybridoma cells producing murine MoAb MM2-9B6 (IgG2b) specific for B16 melanomas were a kind gift of Dr A. Eisenthal of the National Cancer Institute, Bethesda, MD. The MoAb is described by Leong et al. (1988). It was purified from culture supernatant using protein A. Rat MoAb clone 3, specific for human C3d (Lachmann et al., 1980), was purified from ascites using saturated ammonium sulphate (SAS) precipitation. Culture supernatant containing rat MoAb which recognizes the antigens Thy- I (YTS 154.7. 1), L3T4 (YTS 191.1.2), and Lyt 2 were generous gifts of Dr S. Cobbold, Department of Immunology, University of Cambridge, UK. Murine complement-fixing MoAb 166B7 specific for MC6A was purified from culture supernatant using protein A. A rat MoAb (M51 14) specific for a monomorphic determinant on murine I-A and I-E was purified using SAS precipitation. Establishment of PPD-reactit'e clones

C57BL/ 10 mice were immunized subcutaneously on both flanks

with one human dose of BCG (Glaxo Laboratories, Greenford, UK). A single cell suspension was prepared from draining inguinal lymph nodes 21 days post-immunization. Cells thus obtained (2 x 107) were restimulated with 20 pg/ml PPD in 10 ml of CM. After 5 days of culture, viable blast cells were isolated over ficoll (Pharmacia, Milton Keynes, UK), washed, and plated out in round-bottomed microtitre plates at I and 0-3 cells per well by limiting dilution. Plated cells were co-cultured with irradiated (40 Gy) syngeneic spleen cells (5 x 105/well) in CM supplemented with 20 pg/ml PPD and 10° rat conditioned media (RCM) as a source of interleukin-2 (IL-2). Positive wells were harvested 14 days later and expanded in 24-well Linbro plates in the presence of irradiated spleen cells (5 x 106/well) in CM supplemented with PPD (20 pg/ml) and 10°/., RCM. Characterization of PPD-reactii'e T cell clones The antigen specificity and class II restriction of the PPDreactive clones was determined by using a standard proliferation assay. Cloned T cells (1 x 105) were added to each well of a round-bottomed, 96-well microtitre plate and were stimulated with 5 x l05 irradiated (40 Gy) spleen cells of BO0 or BIOA.4R, 2R or 5R origin in 0 2 ml of CM containing PPD (10 pg/ml) or control antigens (20 pg/ml). After 48 h, the co-cultures were pulsed with I pCi/well of 3H-thymidine (Amersham International, Amersham, UK) for 18 h. Cells were harvested on a glass fibre filter and the radioactivity measured by liquid scintillation counting. Results are expressed as mean ct/min of triplicate cultures. The surface phenotype of PPD-reactive clones was determined using FACS analysis. Cloned T cells were incubated with MoAb specific for Thy 1.2, CD4 or CD8 surface antigens, followed by fluorescinated goat anti-rat immunoglobulin (Sigma).

Conjugation of MoAbs clone 3 and MM2-9B6 to PPD PPD (10000 MW) was covalently linked via a disulphide bond

201

to the MoAbs using the heterobifunctional cross-linking reagent N-succinimidyl 3-(2 pyridyldithio) propionate (SPDP). The protocol used was based on the recommendations of the manufacturers (Pharmacia). Ten milligrams of the purified MoAbs were dialysed into the SPDP coupling buffer (0-1 M NaCl, 0-1 M P04-; pH 7-5) and were treated with a 5-mol excess of SPDP at 20 mm in absolute ethanol for 20 min at 233C. This resulted in a substitution of 2-5: 1 (mol 2-pyridyl disulphide: mol MoAb). The degree of substitution with 2-pyridyl disulphide was determined by assaying the concentration of pyridine-2thione released after reduction with 10 mm dithiothrietol (Calbiochem-Behring, La Jolla, CA). Thiolated MoAb was separated from free SPDP using a Sephadex G-25 column. Eight milligrams of PPD in distilled water were spikelabelled with '251-PPD to a specific activity of 120 CPS/pg and treated with a 1 4-mol excess of SPDP at 20 mm in absolute ethanol for 20 min at 23-C. This resulted in a substitution of 0 8:1 (mol pyridyl disulphide:mol PPD). Thiolated PPD was separated from free SPDP using a sephadex G-25 column. Thiolated PPD was reduced with dithiothrietol (DTT) at a final concentration of 10 mm for 30 min at 23; C. Free DTT was removed using a sephadex G-25 column run with deoxygenated SPDP coupling buffer, gassed with N2, and containing 10 mM EDTA to prevent re-oxidation. A 2 5-mol excess of the reactive thiolated PPD was immediately mixed with the thiolated MoAb and the conjugation reaction allowed to continue for 16 h at 4 C. Monomeric MoAB-PPD heteroconjugate containing 1 52 mol PPD: 1 mol MoAb was separated from other reaction products using a Sephracryl-300 superfine column (Pharmacia) run with a 10 mM PO4 , 05 M NaCl buffer (pH 7-2). On the basis of the elution profiles obtained, and SDS gel electrophoresis of eluted fractions, pooled samples were judged to be free from significant competing levels of unconjugated MoAb. Pooled fractions were extensively dialysed against PBS.

Characterization of heteroconiugates Quantitative analysis of '251-PPD-MoAb binding to specific tumour targets was determined using standard radioimmunoassay techniques. Various concentrations of the iodinated clone 3-PPD were offered in a final volume of lO00 pl of PBS to 1 x I07 MC6A tumour cells coated with C3. Non-specific binding was determined by offering equivalent concentrations to uncoated MC6A. lodinated MM2-9B6-PPD was offered at various concentrations to 1 x 107 B16-FIO tumour cells. Non-specific binding was determined by offering equivalent concentrations to B16-FIO cells in the presence of excess cold MM2-9B6 MoAb. Pulsing of tumour cell targets i'th heteroconjugates Prior to incorporation into cytotoxicity/cytostasis assays, 1 x 106 tumour cells were pulsed for 45 min with saturating levels of the MoAb-PPD heteroconjugates containing 0-8 pg of conjugated PPD in 100 pl of PBS. Control cells were pulsed under identical conditions with an equivalent concentration of unconjugated PPD or MoAb. To enhance class II expression, the tumour cells were routinely pretreated overnight with 10% RCM (Jack & McVeigh, 1987; Vyakarham et al., 1988). MC6A tumour cells were coated with C3 via the classical pathway prior to being pulsed with the clone 3-PPD heteroconjugate. In brief, 1 x 106 MC6A cells were incubated with 5 pg of MoAb 1 6B7 in 100 p1 of

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A. M. P. Montgomery, M. G. Wing & P. J. Lachmann

PBS at 4°C prior to treatment with human serum for 20 min at 37TC. Cells were washed prior to incubation with clone 3-PPD.

Cytotoxicity assays Standard 5"Cr release assays were performed as previously described (Ishikawa et al., 1982). Five-million tumour cell targets were labelled with 100 yCi of 5"Cr at 37 C for 45 min, in 0 1 ml of PBS. Cells were washed and pulsed with the heteroconjugates or unconjugated components as described. Following incubation the cells were washed and plated out in triplicate at 5 x 103 cells/well in U-bottomed microtitre plates in 100 p1 of CM supplemented with 10% RCM. Cloned T cells or BCG immune spleen cells were added to the targets at different E: T ratios in a final volume of200 pl/well. Maximum killing was determined by incubating cells with 4-5% Brij; l00-pl samples of supernatant were collected after 16 h and their radioactivity measured on a gamma counter. Specific cytotoxicity (%) was calculated as mean experimental release -mean background release/mean maximum release -mean background release x 100. The results are expressed as a mean of triplicate determinations. To demonstrate class II restriction in the cytotoxicity observed, anti-murine class II MoAb M5114 was added to wells during the 16-h cytotoxicity assays; 20 pg/ml were added to each well containing MM2-9B6-PPD conjugate pre-pulsed B16 cells and PPD-MW3 cells. Cytotoxicity was then determined as described above.

Cytostasis assays Tumour targets were pulsed with relevant heteroconjugates and the unconjugated components as described. Pretreated tumour cells (5 x 104) were then co-cultured in duplicate with 5 x 105 cloned T cells in 24-well Linbro plates in CM containing 10% RCM at a final volume of 1 ml. After 72 h the cells were trypsinized for counting in the presence of trypan blue. Results are expressed as % cytostasis of the relevant control consisting of identically pretreated tumour cells cultured in the absence of T cells. Cytostasis and cytotoxicity assays using culture supernatant Tumour targets were pulsed with the relevant heteroconjugates and the unconjugated components as described. Pretreated tumour cells (5 x 104) were then co-cultured with 5 x 105 cloned T cells in 24-well Linbro plates in CM containing 10% RCM at a final volume of 1 ml. For cytostasis assays, culture supernatant was harvested 72 h later, dialysed into RPMI 1640 and 100-pl aliquots added in triplicate to a 96-well plate containing 2 x 103 tumour cells per well in 100 p1 of RPMI 1640 supplemented with 10% FCS. After 48 h the sub-confluent wells were pulsed with 1 pCi/well 3Hthymidine (Amersham) for 3 h. Cells were harvested on glass filters and their radioactivity measured on a liquid scintillation counter. Results are expressed as mean % specific cytostasis of triplicate determinations. For cytotoxicity assays, culture supernatant was harvested at 72 h, dialysed into RPMI 1640 and 100-pl aliquots added in triplicate to a 96-well plate containing 2 x 103 5'Cr-labelled tumour cells per well in 100 pl of RPMI 1640. Per cent specific cytotoxicity after 16 h was then determined using the standard release assay described above.

Induction of lymphokine secretion by heteroconjugate-treated tumours B16-F1O cells were pulsed with the MM2-9B6-PPD conjugate or the unconjugated components as described. Pretreated or untreated tumour cells (5 x 104) were then co-cultured with 5 x 105 cloned T cells in 24-well Linbro plates in CM containing 10% RCM at a final volume of 1 ml. TNF-a/# activity in 24, 48 and 72 h culture supernatant was assayed by measuring its cytostatic activity towards the TNF-sensitive murine fibroblast cell line L929. Logarithmically growing L929 cells were detached using trypsin and 2 x 103 cells/ well were incubated in triplicate in a 96-well microtitre plate with a 1-in-2 dilution of the control and test supernatant at a final volume of 200 pl. After 48 h the sub-confluent wells were pulsed with 1 pCi/well 3H-thymidine (Amersham) for 3 h. Cells were harvested on glass filters and their radioactivity measured on a liquid scintillation counter. Results are expressed as mean % specific cytostasis of triplicate determinations. The concentration of TNF in the test supernatant was determined by comparison with a human recombinant TNF-o standard curve. Assessment of tumour growth in vivo Thirty female C57BL/6 mice aged 6-8 weeks were injected intradermally in each hind limb with one human dose of BCG. Eighteen days post-immunization groups of 10 BCG-primed or untreated mice were injected subcutaneously into each hind limb with 1 x 105 B16-FIO tumour cells prepulsed as described with either MM2-9B6-PPD or with the unconjugated components. All animals produced tumours which were excised on day 11. Results are expressed as mean wet weight + 1 s.d. for each treatment group of 10 animals. Significance levels were determined using the Mann-Whitney U-test.

RESULTS

Characterization of PPD-reactive clones Four PPD-reactive T cell clones were established following cloning by limited dilution. The surface phenotype of three of these clones was determined by flow microfluorimetry to be Thy 1 -2+, CD4+ (L3T4+) and CD8- (Lyt 2 ). This is consistent with the helper/inducer subpopulation of T cells. One of these clones, designated PPD-MW3 was selected for further studies. The specificity and class II restriction of clone PPD-MW3 was investigated by in vitro proliferative responses. Figure 1 demonstrates that a marked proliferative response could be induced in the presence of PPD and syngeneic feeders, but not in the presence of the irrelevant antigens streptokinase and ovalbumin. To determine MHC restriction, the ability of the clones to proliferate to spleen cells from H-2 recombinant mice was examined. Figure 1 demonstrates that proliferation in the presence of PPD could only be induced by B1O.A (5R), indicating restriction by the I-Ab gene product. All proliferative responses observed could be inhibited by anti-class II MoAb M5114 (data not shown). Further studies on clone PPD-MW3 (Wing et al., 1990) have demonstrated that the clone secretes significant levels of both TNF-a/,B and IFN-y, consistent with the Thb subset.

203

PPD monoclonal antibody conjugates C57BL/10 + ovalbumin

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Fig. 2. Binding of 251I-PPD-MoAb heteroconjugates to tumour targets; (a) binding of clone 3-PPD heteroconjugate to C3-coated MC6A cells; (b) binding of MM2-9B6-PPD heteroconjugate to B16-FlOcells. Heteroconjugates were offered in a final volume of 100p1 in PBS. Non-specific binding was determined by offering equivalent concentrations to uncoated MC6A or B 16-FO cells in the presence of excess cold antibody.

Characterization of heteroconjugates MM2-9B6-PPD and clone 3-PPD were found to bind specifically to B16-F10 cells and C3d-coated MC6A cells, respectively. At saturating levels 3-5 x 105 molecules of conjugated 125-PPD were bound per B16-FO0 cell and 8 x 105 molecules to MC6A cells (Fig 2).

Cytolysis of heteroconjugate-treated tumour cells by cloned T cells The ability of clone PPD-MW3 to lyse pulsed tumour targets was determined in a 16-h 51Cr release assay. A 16-h time course had previously been established to be optimal for this system (Vyakarnam et al., 1988).

204

A. M. P. Montgomery, M. G. Wing & P. J. Lachmann 10

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Fig. 3. T cell-mediated cytolysis of heteroconjugate treated tumour cells. 51Cr-labelled tumour cells pretreated with heteroconjugate or the unconjugated components were co-cultured (5 x 103/well) with T cell effectors at various E: T ratios for 16 h. (a) T cell clone PPDMW3 mediated lysis of B16-FI0 tumour cells treated with MM2-9B6-PPD heteroconjugate; (b) T cell clone PPD-MW3 mediated lysis of C3-coated MC6A tumour cells treated with clone 3-PPD heteroconjugate; (c) BCG immune spleen cell mediated lysis of C3-coated MC6A tumour cells treated with clone 3-PPD heteroconjugate; (d) effect of anti-class II MoAb (M5114) on cytolysis of B16-FIO tumour cells by T cell clone PPD-MW3. Results are expressed as the mean 0X) specific lysis of triplicate determinations; s.d. using the T cell clone and immune spleen cells was < 100 and < 40%, respectively.

Marginal but significant lysis of MM2-9B6-PPD-treated B16-FlO cells and clone 3-PPD-treated MC6A cells was observed (Fig. 3a, b). No significant lysis of MoAb- or PPDpulsed cells was observed. MC6A cells not coated with C3 were also resistant to lysis despite pre-incubation with clone 3-PPD. Further to establishing the ability of the cloned T cells to lyse conjugate-treated targets we also wished to establish the cytotoxic potential of BCG immune spleen cells. Due to a low frequency of PPD-reactive effectors we were unable to demonstrate specific cytotoxicity with fresh immune spleen cells. However, expansion of PPD-reactive cells by re-stimulation with PPD for 5 days in vitro produced significant PPD-specific cytotoxicity at E: T ratios of 100: against clone 3-PPD-treated MC6A cells (Fig. 3c). Figure 3d demonstrates that the cytotoxicity observed can effectively be blocked by the addition of an anti-class II MoAb, suggesting a central role for class II-restricted presentation of PPD in initiating the cytolytic process. We have previously demonstrated with Con A-PPD heteroconjugate (Vyakarnam et al., 1988) that cytotoxicity is dependent on syngeneic class II expression on the tumour target.

Cytostasis of heteroconjugate-treated tumour cells In 72-h co-culture experiments we observed significantly increased levels of cytostasis in both tumour lines following pretreatment with the appropriate heteroconjugates (Table 1). We have demonstrated that B16-FlO cytostasis is extensively due to the effect of IFN-y, while TNF-a//3 and IFN-y act synergistically in the cytostasis of MC6A cells (Wing et al., 1989). Weak, I-A-restricted autoreactivity in clone PPD-MW3, resulting in low-level lymphokine secretion upon co-culture with syngeneic tumour cells may in part be responsible for the background levels of cytostasis observed in Table 1. The further increase in cytostasis observed following treatment with the heteroconjugate is, however, associated with

a more

than

10-

fold increase in the level of TNF secretion. Table I also demonstrates the cytolytic/static effect of supernatant derived from the T cell clone stimulated either with syngeneic feeder cells and PPD or with heteroconjugate pulsed tumour cells. From these data it is clear that while both tumour cell lines are relatively resistant to the cytolytic action of mediators within the supernatant, they are, however, highly susceptible to their cytostatic effects. In this respect the BI6-F10

PPD monoclonal antibody conjugates

205

Table 1. Heteroconjugate mediated cytostasis of tumour targets

Supernatantt Co-culture

Stimulator

Treatment

Target

BLl0 spleen BLl0 spleen

PPD PPD Clone 3-PPD Clone 3 PPD MM2-9B6-PPD MM2-9B6 PPD

MC6A B16-F10 MC6A

MC6A

B16-F10

cytostasis (%/,)*

47 + 5 26 + 3

27+3 B16-F10

60+6 30+ 3

31 + 1

Cytostasis (0)

Lysis (%)

30+2 70+4 8 +1 0 0 25 + 3 3 +1 4+2

3-6+2 2+0 4 0 0

T cell clone PPD-MW3-mediated cytostasis of heteroconjugate-treated tumour cells. * Tumour cells pretreated with heteroconjugate or the unconjugated components were cocultured (5 x 104/well) with PPD-MW3 cells (5 x 105/well). After 72 h, tumour cells were trypsinized for counting. Results are expressed as % cytostasis of the relevant control (i.e. identically pretreated B16-FIO cells cultured in the absence of PPD-MW3 cells). t Culture supernatant was derived from co-cultures of syngeneic spleen cells (I x 1 06/ml) and clone PPD-MW3 (1 x 105/ml) in the presence or absence of PPD or from co-cultures of PPDMW3 cells (5 x 105/well) and tumour cells (5 x 104/well) prepulsed with relevant heteroconjugates or unconjugated components. Supernatant diluted l-in-2 was then added to tumour targets in a standard 51Cr release assay or in a 3-day cytostasis assay.

tumour cell line is clearly more susceptible than the MC6A cells (Wing et al., 1989). The resistance of the tumour targets to cytolysis by secreted lymphokines may be an important factor in explaining the marginal cytotoxicity observed in Fig. 3. The high levels of cytostasis achieved using syngeneic spleen cells as stimulators could not be achieved using the heteroconjugate-pulsed tumour cells; this may reflect the relative efficiencies with which the tumour targets and 'professional' antigenpresenting cells (APC) within the feeder population are able to present the PPD and elicit optimal T cell activation. In addition, the tumour cells used as stimulators are themselves likely to absorb out a significant proportion of the lymphokines produced (Shiohara et al., 1987b). The levels of cytostasis and cytolysis achievable following transfer of supernatant was consistently lower than that seen when both cell types were present as a co-culture. Apart from a dilution effect, this may highlight the importance of the micro-environment between the opposing membranes of the effector cell and its target, which may allow the localized concentration of cytotoxic Iymphokines preferentially secreted towards this point of cell-cell contact (Shiohara et al., 1987b).

Induction of lymphokine secretion by heteroconjugate-treated tumour cell lines The ability of conjugate-treated tumour targets to activate MW3-PPD cells was further determined by assaying for TNF secretion. Figure 4 demonstrates that clone PPD-MW3 is stimulated to produce a level of TNF equivalent to 25 U/ml of recombinant TNF-a after a 72-h co-culture with B16-FO0 cells pretreated with MM2-9B6-PPD. Although this level may be an under-estimate due to absorption ofTNF by the tumour targets, it is significantly below the levels achievable when spleen cells are used as a source of APC (Wing et al., 1989). This again suggests that antigen presentation by the tumour cells and concomitant T cell activation is suboptimal.

30 25 20. 0o'

o0

15

0

10

5

24

36

48

60

72

Time (h) Fig. 4. Induction of TNF secretion by heteroconjugate-treated B16-F10 tumour cells. B 16-F 10 cells were pre-pulsed with MM2-9B6-PPD or the unconjugated components and co-cultured (5 x 104/well) with the T cell clone PPD-MW3 (5 x 105/well). Supernatant was harvested at 24, 48, and 72 h and added in triplicate at a 1/2 dilution to TNF-sensitive L929 cells (2 x 103/well). After 72 h, the wells were pulsed with 3H-thymidine. Results are expressed as mean % specific cytostasis of triplicate determinations. 0, MoAb-PPD; 0, PPD; A, MoAb.

A lack of demonstrable lymphokine activity in supernatant for up to 24 h following the initiation of co-culture has been reported under similar experimental conditions (Shiohara et al., 1987b), and may reflect the initial absorption of TNF production immediately following T cell activation. In vivo cytostasis of MM2-9B6-PPD conjugate-treated B16-F10 cells Table 2 demonstrates the levels of cytostasis that can be achieved following the administration of B16-FIO cells pre-

A. M. P. Montgomery, M. G. Wing & P. J. Lachmann

206

Table 2. Effect of MM2-9B6-PPD on B16-FIO tumour burden

Treatment

Non-immune

BCG immune

PPD MoAb MoAb-PPD

28+07 27+08

22+06 1 7+09 0.9+0 8*

1 5+1

Groups of 10 BCG-immunized or untreated B6 mice were injected subcutaneously with 1 x 105 B16FlO cells in each hind limb. B16 cells were prepulsed with MM2-9B6-PPD or the unconjugated components. Tumours were excised on day 11. Results are expressed as mean wet weight + 1 s.d. * Significantly different from control groups (P < 0 05).

pulsed with MM2-9B6-PPD. Relative to control groups there is a statistically significant decrease in tumour burden equivalent to 40% cytostasis. Whether this reduction in tumour burden is ultimately consequent upon a CD4+ T cell-mediated antitumour DTH response is currently been investigated. The ability of CD4+ T cells to mediate significant in vivo tumour cytostasis has, however, being documented (Shiohara et al., 1987a). It is interesting to observe that BCG immunization alone also has a significant cytostatic effect, possibly as a consequence of non-specific macrophage and NK cell activation. DISCUSSION We have demonstrated that two tumour cell lines which are not Iprofessional' APC and which have minimal class II expression (Vyakarnam et al., 1988; Jack & McVeigh, 1987) are able to present antigen to an antigen-specific helper T cell clone causing activation and concomitant lymphokine secretion. We have shown that the lymphokines secreted, including TNF and IFN-y, are then responsible for the tumour cytostasis observed (Wing et al., 1989). Effective presentation is dependent on the linkage of antigen onto molecules expressed on the surface of the tumour cells. In this respect, we have demonstrated that both a melanoma tumour-associated antigen expressed as a consequence of B-tropic ecotropic murine retrovirus replication (Leong et al., 1988) and a complement component fixed de novo to the cell surface act as suitable structures for heteroconjugate targeting. In a recent study by Lanzavecchia et al. (1988), the presentation of xenogenic antibody to specific T cells by human PBMC was shown to be markedly enhanced when the antibody offered bound to specific surface structures on the APC. These structures included surface immunoglobulin, class II and class I molecules, the transferrin receptor, and a membrane activation antigen (4F2C 13). These observations are encouraging since a wide range of surface molecules seem to fulfil the requirements of successful antigen targeting, which may include internalization for subsequent processing, and class II-restricted presentation. In the context of this paper, however, it is a matter of debate as to whether PPD needs to be processed for presentation. PPD is prepared from culture supernatant of Mycobacterium tuber-

culosis by ultrafiltration, heating to 100 C and precipitation of protein with trichloroacetic acid. In this sense, PPD is already partially processed, and may therefore contain polypeptide chains which have sufficient flexibility to associate with the class II binding site, as has been described recently for fibrinogen (Chain, Kaye & Shaw, 1988). At present, reports on PPD processing requirements are conflicting (Guidos, Wong & Lee, 1984) and may be dependent on the APC used (Kim, Solvay & Thomas, 1985) and the responding T cell clone. Using chloroquine as a lysosomotropic agent we have, however, been able to demonstrate that the PPD-reactive clone used in this study (PPD-MW3) requires to recognize processed PPD when presentation is mediated by peritoneal macrophages (data not shown). The system described has a number of advantages over current T cell targeting systems involving the use of heteroantibody duplexes or hybrid monoclonal antibodies specific for T cell-associated epitopes and target cell determinants. Clearly, rapid modulation of heteroconjugate does not present a problem provided a proportion of the conjugate is internalized for processing. MoAb MM2-9B6, for example, has been shown to modulate rapidly and can be detected in the cytoplasm within 15 min (Leong et al., 1988). Furthermore, once internalized and processed, the immunogenic peptides formed are believed to form a relatively stable interaction with the class II binding sites and are therefore available for recognition for an extended period. Ochi et al. (1987) demonstrated that a B lymphoma cell line pretreated with an anti-idiotypic MoAb-KLH heteroconjugate remained susceptible to cytolysis by a KLH-specific T cell clone for at least 24 h following pulsing with the heteroconjugate. Another important advantage of the targeting system described is bystander cytotoxicity consequent upon the nonspecific action and range of secreted lymphokines. We have previously demonstrated, for example, that untreated MC6A cells are destroyed when co-cultured with Con A-PPD-treated cells and a PPD-reactive clone (Vyakarnam et al., 1988). This bystander lysis could theoretically be exploited for the destruction of antigen-negative variants which often arise during conventional MoAb therapy. More specifically, the targeting system described has the advantage that PPD may safely be used within a BCG-immunized population. There are, however, a number of obvious restrictions that need to be recognized. Clearly, the system will be restricted to the treatment of class II-positive tumour targets that have a processing capacity and are susceptible to the action of lymphokines such as TNF and IFN. It is worth noting, however, that these lymphokines may also kill resistant targets through the intermediary of activated macrophages (Wing et al., 1989). However, the main barrier to effective immunotherapy may be the need to target a large amount of antigen onto tumour cells which are not professional APC. We have previously demonstrated, for example, that 1-2 x 106 molecules of PPD/cell are optimal for the Con A-PPD conjugate targeting system (Vyakarnam et al., 1988). In our experience with MoAb-PPD conjugates, less than 3 x 105 molecules of PPD per MC6A cell fail to produce successful targeting. Presumably, enough antigen must be internalized to allow effective competition for binding sites on a limited number ofclass II molecules. From the cytostasis data presented, it is evident that the limiting factor is the ability of the target tumours to present the PPD efficiently to the specific T cells, rather than the tumourstatic capability of T cells once effectively stimulated. With this in mind, the system

PPD monoclonal antibody conjugates

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may ultimately be optimized in treating malignancy of the professional 'APC' such as the B cell lymphomas. The rationale for targeting to complement components is to overcome the restrictions imposed by targeting tumour-specific or associated antigens. It is also hoped that such a targeting system can be usefully employed following conventional treatment with complement-fixing MoAbs. To this end we are currently testing heteroconjugates specific for C3 neoantigens only expressed when fixed to the cell surface. On the basis of the preliminary in vivo results we are currently testing the ability of targeted Thl to recruit and activate tumouricidal macrophages and ultimately to induce a targeted DTH response.

KIM, K.L., SOLVAY, M.J. & THOMAS, D.W. (1985) Cellular requirements for antigen presenting cells: evidence for different pathways in forming the same antigenic determinants. Cell Immunol. 96, 267.

ACKNOWLEDGMENTS

OCHI, A., WORTON, K.S., WOODS, G., GRAVELLE, M. & KITAGAMI, K. (1987) A novel strategy for immunotherapy using antibody coupled

This work was supported by the Medical Research Council and by a grant from the Cancer Research Campaign. We are grateful for the excellent technical assistance of Mrs C. Harley.

SHIOHARA, T., MOELLMANN, G.E., JACOBSON, K., KUKLINSKA, E.,

LACHMANN, P.J., OLDROYD, R.G., MILSTEIN, C. & WRIGHT, B.W. (1980)

Three rat monoclonal antibodies to human C3. Immunology, 41, 503. LANZAVECCHIA, A., ABRINGNANI, S., SCHEIDEGGER, D., OBRIST, R.,

DORKEN, B. & MOLDENHAUER, G. (1988) Antibodies as antigens. The use of mouse monoclonal antibodies to focus human T-cells against selected targets. J. exp. Med. 167, 345. LEONG, S.P.L., MULLER, J., YETTER, R.A., GORELIK, E., TAKAMI, T. &

HEARING, V.J. (1988) Expression and modulation of a retrovirusassociated antigen by murine melanoma cells. Cancer Res. 48, 4954. MOSMANN, T.R., CHERWINSKI, H., BOND, M.W., GIEDLIN, M.A. &

COFFMAN, R.L. (1986) Two types of murine helper T-cell clone. J. Immunol. 136, 2348. carriers to focus cytotoxic T-helper cells. Eur. J. Immunol. 17, 1645. RUDDLE, N.H. & LERNER, A.B. (1987a) Anti-tumour activity of class II antigen-restricted cloned autoreactive T-cells. II. Destruction of

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