Active Antiviral T-Lymphocyte Response Can Be Redirected against

Cancer Therapy: Preclinical

Active Antiviral T-Lymphocyte Response Can Be Redirected against Tumor Cells by Antitumor Antibody MHC/Viral Peptide Conjugates Vale¤rie Cesson,1 Kathrin Stirnemann,1 Bruno Robert,3,4,5 Immanuel Luescher,2 Thomas Filleron,5,6 Giampietro Corradin,1 Jean-Pierre Mach,1 and Alena Donda1

Abstract

Purpose:To redirect an ongoing antiviralT-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides. Experimental Design: First, lymphochoriomeningitis virus (LCMV) ^ infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA) ^ transfected MC38 colon carcinoma cells precoated with anti-CEA H-2Db/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab¶)2 fragments. Second, influenza virus ^ infected mice were injected i.v., to induce lung metastases, with HER2transfected B16F10 cells, coated with either anti-HER2 H-2Db/NP366 influenza peptide conjugates, or anti-HER2 F(ab¶)2 fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA H-2Db conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA+ cells, s.c. grafted in LCMV-infected mice. Results: In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38CEA+ cells did not develop into tumors, whereas all grafts with F(ab¶)2-precoated MC38-CEA+ cells did so (P = 0.0022). In influenza virus ^ infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA H-2Db/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016). Conclusion: The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody MHC/viral peptide conjugates.

In recent years, a major effort has been dedicated to the development of active vaccination protocols for immunotherapy of cancer patients, using newly discovered tumor-specific or differentiation antigens (1 – 3). Successful induction of specific T-cell responses and tumor infiltration by T lymphocytes were well documented, but the number of tumor remissions remained low (4, 5). It is hard to understand the reasons for Authors’ Affiliations: 1Department of Biochemistry and 2Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; 3 Institut National de la Sante et de la Recherche Medicale, EMI 0227, Centre de Recherche en Cance¤rologie de Montpellier; 4Universite¤ de Montpellier I, 5CRLC Val d’Aurelle-Paul Lamarque, and 6Biostatistiques, Montpellier, France Received 7/27/06; revised 9/7/06; accepted 9/21/06. Grant support: Cancer Research Institute New York (A. Donda), OncoSuisse Foundation (V. Cesson and K. Stirnemann), and Novartis Foundation (V. Cesson). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Alena Donda, Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland. Phone: 41-21-692-57-47; Fax: 41-21692-57-05; E-mail: alena.donda@ unil.ch. F 2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-1862

Clin Cancer Res 2006;12(24) December 15, 2006

the relatively poor antitumor activity of the induced specific T lymphocytes, given that such effector cells are known to be so efficient in the elimination of virus-infected cells and in allograft rejection. Three major explanations have been given for the inefficiency of the T-cell antitumor response: the poor antigenicity of autologous tumor antigens, the low expression or absence of MHC molecules on the tumor cell surface, or some functional defects in their antigen-processing machinery (6, 7). Here, we present a new immunotherapeutic strategy that has the potential to overcome these obstacles. We coupled an antitumor antibody fragment to an MHC molecule loaded with an immunodominant viral peptide and showed that tumor cells coated with the conjugate are rejected by mice immunized against the relevant virus, whereas growth of the same tumor cells coated with unconjugated antibody fragment alone was not affected. Indeed, the proposed strategy allows one to both increase the number of MHC molecules on the surface of tumor cells and to redirect the ongoing high-affinity T-cell response against the MHC/viral peptide complex coated on the tumor cells. Overall, this strategy takes advantage of the efficient tumor-targeting properties of selected monoclonal antibodies (mAb) directed against tumor-associated antigens (TAA), such as carcinoem-

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TumorTargeting of Antibody-MHC/Viral Peptides

bryonic antigen (CEA; refs. 8, 9) and HER2 (10, 11) combined with the strong cytotoxic activity of CD8+ T cells against viral antigens (12). In fact, the bifunctional conjugates represent a way of bridging the antibody and T-cell attack on cancer because the antibody arm allows a specific binding to a cell surface TAA, whereas the MHC/peptide complex is recognized by the T-cell receptor. The first partial demonstration of this new immunotherapeutic strategy was based on in vitro experiments, using biotinylated MHC complexes tetramerized with streptavidin (13, 14). Then it was shown that monomeric MHC complexes coupled to anti-TAA Fab fragments were sufficient to induce killing by CD8+ T cells through the oligomerization of the MHC on tumor cells (15). The first entirely in vivo demonstration of the feasibility of the study was done in C57BL/6 mice using anti-OVA peptide T cells derived from OT-1 mice and anti-CEA H-2Kb/ova-peptide conjugates (16). Single-chain MHC molecules genetically fused to scFv antibody were subsequently shown to inhibit the growth of human tumor xenografts in nude mice injected with large amounts of cloned human T cells (17). Here, we show that T cells, induced by infection with two different viruses in immunocompetent mice, can be redirected against tumor cells by the use of antibodyMHC/viral peptide conjugates. To test this strategy, we used two viral models [lymphochoriomeningitis virus (LCMV) and influenza virus] and tested the retargeting of antiviral CTLs to two types of syngeneic tumor (either a s.c. grafted CEAexpressing colon carcinoma or lung metastases induced by i.v. injection of HER2-expressing B16 melanoma cells). To prevent or to treat these tumors, two conjugates were synthesized and used to precoat tumor cells: anti-CEA Fab H-2Db, loaded with the LCMV immunodominant peptide GP33-41 (18, 19) or anti-HER2 Fab H-2Kb, loaded with the influenza virus immunodominant peptide NP366-374 (20). Furthermore, to test the efficacy of the approach against previously grafted, uncoated tumor cells, we synthesized an anti-CEA MHC conjugate in which the H-2Db molecule was covalently crosslinked to the GP33 peptide and examined the antitumor effect of systemic injection of this conjugate in the LCMV model.

Materials and Methods Anti-TAA Fab class I MHC/peptide conjugates. Bifunctional conjugates were prepared as previously described by Robert et al. (15) and Donda et al. (16). Briefly, the recombinant soluble class I H-2Db heavy chain was mutagenized to express an extra cysteine at the COOHterminal end (position 903). The modified H-2Db molecule and the h2microglobulin were expressed in bacteria and purified from inclusion bodies before being refolded together by dialysis in the presence of either the LCMV peptide GP33-41 (KAVYNFATA) or the influenza peptide NP366-374 (ASNENMETM). For the LCMV GP33-41 peptide, the COOH-terminal cysteine was replaced by an alanine to avoid reactivity with the dimaleimide coupling reagent (Sigma-Aldrich, St. Louis, MO; ref. 21). In parallel, F(ab¶)2 fragments of murine anti-CEA mAb 35A7 (22) and of humanized anti-HER2 mAb Herceptin (trastuzumab; Genentech, South San Francisco, CA/Roche, Indianapolis, IN; ref. 23) were prepared by pepsin digestion (Sigma Chemical Co., St. Louis, MO) followed by reduction with 5 mmol/L h-mercaptoethanol (Fluka, Buchs, Switzerland) to obtain F(ab¶) fragments. Soluble H-2Db/GP33 monomers with cross-linked peptide. Photoreactive GP33-41 peptide [F(N3)AVYNFATA] was synthesized by solidphase chemistry, by replacing the NH2-terminal lysine with an azidophenylalanine, using Fmoc for transient NH2-terminal protection


[Fmoc-4-azidophenylalanine F(N3)-OH; Bachem AG, Bubendorf, Switzerland]. Covalent H-2Db/F(N3)AVYNFATA complexes were obtained by UV irradiation for 40 s at 4jC with a 90-W fluorescence UV lamp emitting at 312 F 40 nm. Cross-linked MHC/peptide monomers were purified from non – cross-linked complexes by incubation at 37jC for 4 h, followed by size-exclusion chromatography on a Superdex S200 column (24). Tetramers with LCMV and influenza immunodominant peptides. To obtain the different tetramers, purified H-2Db/GP33 and H-2Db/NP366 monomers and H-2Db/GP33 cross-linked monomers were biotinylated by the reaction of biotin maleimide (EZ-link BM, Pierce, Rockford, IL) with the COOH-terminal cysteine (25), followed by tetramerization by stepwise addition of extravidin phycoerythrin (Sigma-Aldrich) at a ratio of 4:1. Tumor cell lines. The murine chemically induced colon carcinoma cell line MC38, transfected with the CEA (clone C15.4.3.AP) and subsequently referred to as MC38-CEA+, was provided by J. Primus (Department of Pathology, Vanderbilt University, Nashville, TN; ref. 26). B16F10 melanoma cells were transfected with the human HER2 gene, provided by Y. Yarden (The Weizman Institute of Science, Israel; ref. 27), sorted, and cloned. B16F10-HER2+ cells, subsequently referred to as B16-HER2+, were maintained in complete DMEM supplemented with 1.2 mg/mL G418 (Calbiochem, San Diego, CA). The CEA-expressing LoVo human colon carcinoma cell line and HER2expressing SKBR3 human breast carcinoma cell line were obtained from the American Type Culture Collection (Rockville, MD) and maintained as recommended. Flow cytometry analysis. CEA expression on MC38-CEA+ cells and HER2 expression on B16-HER2+ cells were assessed by a two-step incubation with anti-CEA or anti-HER2 (Herceptin) mAb, respectively, followed by goat anti-mouse IgG labeled with Alexa 480, or a FITClabeled goat anti-human IgG (BD PharMingen, San Jose, CA). Specific coating of the anti-CEA H-2Db/GP33 conjugates on LoVo cells and of the anti-HER2 H-2Db/NP366 conjugates on SKBR3 cells was assessed by incubation with the relevant target cells at 10 Ag/mL for 45 min at 4jC in 50 AL of PBS containing 5% FCS, 0.02% azide. The conjugates were revealed by a two-step incubation: first with an antiH-2Db conformation-sensitive mAb (BD PharMingen; clone 28-14-8) then followed by a FITC-labeled goat anti-mouse IgG (Sigma). Samples were analyzed on a FACScan, and data were analyzed using CellQuest software (both from Becton Dickinson, Mountain View, CA). Animals and viral infections. All experiments on mice were done according to the Swiss guidelines for experimental animal studies (authorization 839.6). C57BL/6 mice were purchased from Harlan Laboratories (Indianapolis, IN) and housed in an animal facility with P2 level of biosafety. CEA-transgenic mice (C57BL/6J) were initially provided by F.J. Primus (26). LCMV-WE virus was obtained from Ch. Mueller (University of Bern, Switzerland; ref. 28) and R. Zinkernagel (University Hospital, Zurich, Switzerland; ref. 29). Mice were infected by i.v. injection of virus (200 plaque-forming units in 200 AL of PBS) in the tail vein. The murine strain of influenza virus PR8 (H1N1) was provided by M. Kopf (ETH Zurich; ref. 30); 25 plaque-forming units of virus in 2 50 AL PBS were used for intranasal infection under mild anesthesia with isofluran. Chromium release cytotoxicity assay. MC38-CEA+ or B16-HER2+ target cells were pulsed for 1 h at 37jC with 1 Amol/L of the relevant peptide (GP33-41 and NP366-374, respectively) or the irrelevant peptide (NP366-374 and GP33-41, respectively) to saturate endogenous MHC class I. Pulsed cells were then labeled with 51Cr for 45 min at 37jC, washed, and plated in 96-well plates (2 103 per well). Conjugates were added at 10 Ag/mL to target cells pulsed with the irrelevant peptide. Effector T cells were obtained either from splenocytes of a day 8 LCMV-infected mouse, restimulated by a 5-day in vitro culture with 1 Amol/L GP33-41 peptide in DMEM 10% FCS, or from bronchoalveolar lavage lymphocytes of a day 15 influenza-infected mouse, after a similar 5-day in vitro restimulation with 1 Amol/L NP366-374 peptide.

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Cancer Therapy: Preclinical Growth inhibition of tumor cells precoated with conjugates. On day 8 after LCMV infection, a group of six B6 mice were grafted on the right flank with 1 106 MC38-CEA+ cells precoated with anti-CEA H-2Db/ GP33 conjugate and on the other flank with 1 106 MC38-CEA+ cells precoated with anti-CEA F(ab¶)2 fragments alone. Precoating was done by in vitro incubation (1 h at 4jC) of 1 106 cells with 10 Ag/mL of conjugates or F(ab¶)2 fragments diluted in 200 AL PBS. Tumor growth was monitored every 2 days by measuring the three orthogonal diameters with a caliper and using the formula (length width thickness) / 2. Mean tumor size and SD of each tumor were plotted for each time point. On day 13 after influenza infection, 18 B6 mice were injected i.v. with 0.7 106 B16-HER2+ cells to induce lung metastases. One group of six mice were injected with B16-HER2+ cells precoated in vitro with anti-HER2 H-2Db/NP366 conjugate; a second group of six mice received B16-HER2+ cells precoated with Herceptin F(ab¶)2; and a third group of six mice received intact Herceptin-precoated B16-HER2+ cells. After 15 days, mice were sacrificed, and entire lungs were excised, perfused with PBS, and fixed with 4% paraformaldehyde for microscopic analyses. Tumor therapy with systemic conjugate injection. On day 8 after LCMV infection, 10 CEA-transgenic mice were grafted on the right flank with 0.7 106 MC38-CEA+ cells. Twenty-four hours later, a group of five mice were injected i.p. every 2 days with 70 Ag of conjugate, whereas the remaining five mice were injected i.p. with 70 Ag of antiCEA F(ab¶)2 fragments. Tumor growth was measured as mentioned previously. Statistical analysis. For continuous variables, the means F SD and range were computed. To investigate the association between trial features, exact Wilcoxon tests were done for statistical analysis using the SAS procedure. All reported Ps are two sided. For all statistical tests, differences were considered as significant at the 5% level. Statistical analyses were done on an IBM PC-compatible personal computer using the SAS 8.0 software (SAS Institute, Inc., Cary, NC).

Results Synthesis and characterization of anti-CEA Fab H-2Db/ GP33 and anti-HER2 Fab H-2Db/NP366 conjugates. The synthesis of our conjugates is based on the chemical coupling of a soluble recombinant class I MHC/peptide complex with a F(ab¶) fragment from a monoclonal anti-TAA antibody (15, 16). An ortho-phenylene dimaleimide linker is used to form stable thioether bonds between free SH groups on the cysteine residues from F(ab¶) fragments and an SH group of an engineered cysteine at the COOH terminus of soluble MHC class I molecules. A schematic representation of the monomeric and bifunctional conjugate is shown in Fig. 1A.

To produce an anti-CEA Fab H-2Db/GP33 conjugate, antiCEA F(ab¶) fragment, derived from the high-affinity murine anti-CEA mAb 35 (22), was first derivatized by the dimaleimide linker and then coupled to an H-2Db molecule, refolded in presence of the GP33-41 LCMV peptide. To produce the antiHER2 Fab H-2Db/NP366 conjugate, the H-2Db molecule refolded with NP366-374 influenza virus peptide was first derivatized by the dimaleimide linker and then coupled to antiHER2 F(ab¶) fragment, derived from the humanized anti-HER2 mAb Herceptin (31). Monomeric F(ab¶) MHC/peptide conjugates were purified by filtration on a S200 Superdex column. The purified conjugates eluted as a single symmetrical peak with the expected apparent molecular weight of 95 kDa (Fig. 1B). The molecular weight of the conjugate was confirmed by SDSPAGE, which showed one major band at 83 kDa under nonreducing conditions, corresponding to the conjugate minus the dissociated h2-microglobulin of 12 kDa (outside the gel). The thioether bond, made between the maleimide on the cysteine of the F(ab¶) fragment and the COOH-terminal cysteine residue engineered on the MHC molecules, is resistant to reducing conditions, as shown by the presence of a 58-kDa band in the reduced SDS-PAGE gel, corresponding to the antibody heavy chain linked to the class I MHC. The other band of 25 kDa corresponds to the antibody light chains (Fig. 1C). Antitumor Fab MHC conjugates can selectively coat tumor cell surface with MHC/viral peptide complexes. The capacity of the two anti-TAA Fab MHC/peptide conjugates to coat tumor cells expressing the relevant TAA with class I MHC/peptide complexes was tested by flow cytometry on four different tumor cell lines, expressing either the CEA or the HER2 antigen (Fig. 2). The expression of CEA by the murine MC38-CEA+ and by the human LoVo colon carcinoma cell lines and the presence of HER2 on the surface of the murine B16-HER2+ melanoma and on the breast carcinoma line SKBR3 were first confirmed by using the anti-CEA mAb 35A7 and the humanized anti-HER2 mAb Herceptin, respectively (Fig. 2A-D). The coating of the MHC/peptide by our conjugate was then shown on the two human carcinoma lines (LoVo and SKBR3) that did not express H-2Db. When preincubated with the anti-CEA Fab H-2Db/ GP33 or the anti-HER2 Fab H-2Db/NP366 conjugate, respectively, the two human carcinoma cell lines became positively stained by a conformation-dependent anti-H-2Db

Fig. 1. Characterization of antitumor Fab MHC/peptide conjugates. A, schematic representation of antibody Fab fragment coupled to MHC/peptide complexes via the ortho-phenylene dimaleimide linker. B, FPLC profile of purified anti-CEA Fab H-2Db/GP33 conjugates eluted as a single peak with an apparent molecular weight of 95 kDa on a Superdex S200 column. C, 10% SDS-PAGE analysis of an FPLC-purified anti-HER2 Fab H-2Db/ NP366 conjugate, under nonreducing (NR) and reducing (R) conditions. Calculated apparent molecular weights are indicated in kDa.


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Fig. 2. A to D, antigen expression on tumor cell lines. The expression of CEA on the murine MC38-CEA+ colon carcinoma and on the human colon carcinoma LoVo cell lines is shown by the use of the anti-CEA mAb 35A7 (open histograms, A and B). The presence of HER2 on the B16-HER2+ melanoma and on the human breast carcinoma SKBR3 cell lines is confirmed by the use of the anti-HER2 mAb Herceptin (open histograms, C and D). E to H, specific coating of Fab MHC/peptide conjugates on tumor cell lines. Binding of anti-CEA H-2Db/GP33 conjugate on MC38-CEA+ cells and of anti-HER2 H-2Db/NP366 conjugate on B16HER2+ is demonstrated by an anti-Fab-specific second antibody (open histograms, E and G, respectively). Coating of anti-CEA H-2Db/GP33 conjugate to LoVo cells and of anti-HER2 H-2Db/NP366 conjugate on SKBR3 cells is revealed by a conformation-sensitive anti-H-2Db antibody (open histograms, F and H, respectively). Controls with conjugates containing Fab with anti-TAA specificity absent from target cells gave negative histograms (gray lines) similar to the filled histograms obtained with untreated tumor cells.

antibody (Fig. 2F and H). The coating specificity was shown by the negative results obtained with control conjugates. The results also indicate that the MHC/peptide complexes coated on tumor cells have a correct conformation. The specific coating of anti-TAA Fab H-2Db/peptide conjugates on the murine tumor cell lines MC38-CEA+ and B16-HER2+, which do express H-2Db, was shown by using a second antibody directed against the Fab fragment of the conjugate (Fig. 2E and G). Sensitization of conjugate-coated tumor cells to lysis by virusspecific CTLs. Preincubation of tumor cells with the appropriate anti-TAA Fab MHC/viral peptide conjugate induced their lysis by viral peptide-specific CTLs. The anti-CEA Fab H-2Db/ GP33 conjugate induced efficient lysis of the MC38-CEA+ tumor cells (Fig. 3A) by in vitro stimulated LCMV-specific CTLs, whereas uncoated MC38-CEA+ tumor cells were not lysed by the same CTLs. The anti-HER2 H-2Db/NP366 conjugates similarly induced efficient lysis of the B16-HER2+ tumor cells (Fig. 3B) by in vitro stimulated, influenza-specific CTLs, whereas uncoated B16HER2+ tumor cells were not sensitive to influenza-specific CTLs. Antitumor activity of the conjugates precoated on tumor cells grafted in mice infected with LCMV or influenza virus. The ability of antitumor Fab MHC/viral peptide conjugates to redirect active antiviral T-cell responses against the tumor cells and inhibit their growth was first tested with tumor cells, which were precoated in vitro with the appropriate conjugate before tumor grafting in virus-infected B6 mice.


In the first viral model, B6 mice were infected i.v. with 200 plaque-forming units LCMV-WE, which was shown to induce 50% to 60% of the CTL activity against the GP33-41 peptide epitope (18). The kinetics of expansion of specific H-2Db/GP33 CTLs, followed by tetramer and CD8 fluorescence-activated cell sorting staining on peripheral blood mononuclear cells, showed that maximum frequency of specific CTLs was reached by day 8 with 15% to 20% tetramer-CD8 double-positive cells. At the peak of the primary immune response, MC38-CEA+ tumor cells, precoated with the anti-CEA Fab H-2Db/GP33 conjugate or with the control anti-CEA F(ab¶)2 fragments, were grafted on the right and left flank of each mouse, respectively, so that each animal carried its own control tumor. Equal saturation of tumor cells with conjugate or F(ab¶)2 was confirmed by flow cytometry (Fig. 4B). Figure 4A shows the kinetics of the mean tumor growth, in the same six mice, of cells precoated with either anti-CEA Fab H-2Db/GP33 or only with the anti-CEA F(ab¶)2. All tumor grafts precoated with F(ab¶)2 progressed into growing tumors, whereas only one of the six conjugate-precoated tumor grafts produced a tumor that started to grow a week later than F(ab¶)2-coated cells. This efficient and statistically significant (P = 0.0022) tumor graft inhibition was reproduced in three independent experiments. The results show that the H-2Db/GP33 – specific CTLs, generated by the LCMV infection, were able to reach the s.c. tumor cells and recognize in vivo the MHC/viral peptide complexes bound to the tumor cells, leading to their activation

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Fig. 3. Specific lysis of conjugate-coated tumor cells by viral-specific CTLs. A, MC38-CEA+ tumor cells coated with anti-CEA H-2Db/GP33 conjugates (.) are lysed by H-2Db/GP33 ^ specific splenocytes from an LCMV-infected mouse. Control targets are uncoated tumor cells (o). Effector cells were splenocytes taken from a mouse at day 8 after LCMV infection and stimulated 5 d in vitro with GP33-41peptide. B, B16-HER2+ tumor target cells are lysed by CTLs obtained from bronchoalveolar lavages of an influenza-infected mouse only when coated with anti-HER2 H-2Db/ NP366 conjugate (.), whereas they are not lysed when not coated (o). Effector cells were CTLs from bronchoalveolar lavages stimulated 5 d in vitro with NP366-374 peptide.

and destruction of the coated tumor cells. The very few mice (one of six mice per experiment) in which a conjugate-coated tumor escaped from the conjugate-induced killing had similar frequencies of virus-specific CTLs, and immunohistochemistry showed that the CEA was still expressed on those tumors. Thus, we think that the very rare cases of tumor escape of conjugateprecoated cells are not due to a lack of CTL or to a subpopulation of tumor cells with low CEA expression but may be due to a too rapid dissociation of the conjugate from the tumor cells, or of the peptide from the MHC molecule. A second viral and syngeneic tumor model was developed, in which B6 mice were infected intranasally with the influenza virus PR8 (30) and challenged by an i.v. injection of B16HER2+ melanoma cells to induce artificial lung metastases (32). Because after nasal infection the immune response is mostly localized in the lungs, the lung metastasis model was preferred to the above-described s.c. tumor graft. The antiviral response was followed by flow cytometry of the T cells obtained by bronchoalveolar lavages, using an H-2Db/NP366 tetramer synthesized as described in Materials and Methods, and an anti-CD8 mAb (20, 33). Tumor challenge was done when a frequency of H-2Db/NP366 – specific CTL of 10% to 15% could be detected in the bronchoalveolar lavages. A group of six mice were injected with tumor cells precoated in vitro with antiHER2 Fab H-2Db/NP366 conjugate. As controls, a group of six mice received F(ab¶)2 precoated tumor cells, whereas a third group of another six mice received tumor cells precoated with the intact anti-HER2 antibody, allowing a comparison of the effect of our conjugate with that of the clinically used Herceptin antibody on tumor metastases development. The equal and complete saturation of the precoated B16-HER2+ tumor cells with the anti-HER2 Fab H-2Db/NP366 conjugate, or with the anti-HER2 F(ab¶)2, was verified by the fact that a further incubation of the precoated tumor cells with the intact Herceptin mAb did not lead to any additional binding of the mAb detectable by cytofluorometry (Fig. 4D). To evaluate the antitumor activity of the conjugate, compared with that of F(ab¶)2 or intact Herceptin mAb, mice were sacrificed 2 weeks after tumor cell injection to assess the number of metastatic nodules in the lungs. After perfusion and fixation of the lungs with 4% paraformaldehyde, pictures of each lung side were taken for counting of metastatic nodules.


As shown in Fig. 4C, all mice from conjugate-precoated B16HER2 group had only few metastatic nodules, with a mean of 15 metastases per lung side, compared with mice from the F(ab¶)2 and Herceptin-precoated groups, which had a statistically significant higher number of nodules, averaging 110 and 120 metastases, respectively (P = 0.022 and P = 0.013). In addition, larger and deeper metastases were observed in the control groups compared with the conjugate-treated group, as shown by representative lung photographs (Fig. 4C). These results show that the coating of HER2-expressing tumor cells with anti-HER2 Fab H-2Db/NP366 conjugates produces a dramatic reduction in the number of metastases developing in the mice, with some mice exhibiting a complete inhibition of metastases development. Photo-crosslinking of the GP33 peptide to the H-2Db MHC molecule. To prevent peptide dissociation from the MHCbinding groove in vitro and in vivo, covalent H-2Db/GP33 peptide MHC complexes were prepared by replacing the Lys33 of the GP33-41 peptide (KAVYNFATA), by a photoreactive 4-azidophenylalanine [F(N3)-AVYNFATA, thereafter referred as cross-linked peptide]. The purified refolded complexes were then UV irradiated at 312 nm for 40 s to photo-crosslink the peptide in the MHC-binding groove (34). To test the recognition of the photo-crosslinked complexes by H-2Db/ GP33 – specific CD8+ T cells, H-2Db/GP33 tetramers were made with the cross-linked peptide and compared with wild-type peptide tetramers for the staining of peripheral blood mononuclear cells from LCMV-infected mice. As shown in Fig. 5A and B, similar fluorescence intensities were obtained with the two tetramers, indicating that both tetramers bind with the same avidity to the T-cell receptor from the viral peptidespecific CTLs. In support of this finding, tetramer titration, done on peripheral blood mononuclear cells from B6 mice at day 25 after LCMV infection, showed that both stainings reached saturation at the same tetramer concentration (f15 Ag/mL; Fig. 5C). However, the frequency of H-2Db/ GP33 – specific CD8+ T cells detected in the spleen of mice at day 8 after LCMV infection was 10% as detected by H-2Db/ GP33 cross-linked tetramers, whereas 15% were detected by H-2Db/GP33 wild-type tetramers (Fig. 5A and B), suggesting that the NH2-terminal peptide modification precludes its recognition by f30% of H-2Db/GP33 – specific CD8+ T-cell clones.

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Specific T-cell recognition of the cross-linked GP33 peptide was further tested in ex vivo cytotoxicity experiments. Splenocytes from LCMV-infected mice were incubated at different ratios with MC38-CEA+ tumor cells coated with conjugates containing the H-2Db cross-linked or bound to GP33 peptide. Both conjugates efficiently induced MC38-CEA+ target cell lysis by ex vivo H2Db/GP33 – specific CTLs (Fig. 5D). Antitumor effect of the systemic injection of conjugates containing H-2Db with cross-linked viral peptide. The antitumor activity of anti-CEA H2Db/GP33 conjugates with crosslinked peptide was tested in the previously described model of B6 CEA-transgenic mice infected with LCMV and s.c. transplanted with MC38-CEA+ colon carcinoma cells. Ten B6 mice were infected i.v. with 200 plaque-forming units of LCMV virus and s.c. grafted 8 days later with 7 105 MC38-CEA+ tumor cells. Conjugate treatment (70 Ag per injection) was started 1 day after the graft, at the peak of T-cell response. Injections were done alternately i.v. and i.p. every 2 days during 2 weeks. Complete tumor growth inhibition was obtained in four of the five conjugate-treated mice, whereas all control mice

receiving anti-CEA F(ab¶)2 fragments alone developed fastgrowing tumors (P = 0.016; Fig. 6). The only conjugate-treated mouse that escaped the treatment had a barely palpable tumor that appeared 7 days after the end of treatment and was removed to verify its histology and its possible infiltration by T lymphocytes. Histology confirmed the presence of tumor cells, but the tumor specimen was too small to allow proper analysis of the T-cell infiltration. This tumor inhibition experiment was reproduced twice with similar results. Interestingly, the CEA transgenic mice, known to express CEA on cells from their normal colonic mucosa (26), tolerated well the repeated injections of anti-CEA H-2Db/GP33 conjugates, without symptoms of autoimmune disease, such as weight loss or diarrhea.

Discussion The results show that it is possible to redirect a physiologic antiviral T-cell response against syngeneic tumor cells in vivo, through an antibody-mediated coating of the tumor cells with

Fig. 4. Antitumor activity of anti-TAA MHC/viral peptide conjugates in vivo. A, tumor growth was followed in the same six LCMV-infected mice, s.c. grafted on the right flank with 0.7 106 MC38-CEA+ cells precoated with anti-CEA Fab H-2Db/GP33 conjugates (.) and on the left flank with 0.7 106 MC38-CEA+ cells precoated with anti-CEA F(ab¶)2 fragments (o) Vertical bars, 2 SD. B, evidence that the MC38-CEA+ tumor cells were precoated with similar amounts of conjugate, or F(ab¶)2 fragments, as shown by flow cytometry, using an FITC-labeled anti-mouse Fab antibody. C, inhibition of lung metastases development, counted 14 d after tumor i.v. injections in three groups of influenza-infected mice. Mice received either B16-HER2+ tumors precoated with anti-HER2 H-2Db/NP366 conjugates (group I), control anti-HER2 F(ab¶)2 (group II), or intact anti-HER2 mAb (group III). The number of lung metastases detected per mouse (., E, n, x, and ); average number of metastases per group (large horizontal bar). D, saturation of B16-HER2+ tumor cells precoated with conjugate or F(ab¶)2 fragments is shown by the absence of staining by Herceptin, followed by FITC-labeled Fc-specific anti-human IgG (open columns). Saturation of Herceptin-precoated B16-HER2+ tumor cells is demonstrated by positive staining when incubated with the FITC-labeled Fc-specific anti-human IgG, which is not markedly increased when excess of Herceptin is added.


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an optimal T-cell antigen consisting of autologous MHC molecules loaded with an immunodominant viral peptide. The bifunctional conjugate can play the role of an adaptor molecule, which replaces the cell surface TAA (recognized by an antibody-binding site) by a selected immunodominant viral peptide, associated with an MHC molecule (recognized by the T-cell receptor). In view of the positive in vivo results presented here, obtained in immunocompetent mice, we will now consider how this new strategy has developed in recent years, what are its intrinsic advantages, and how it could be integrated with the use of active T-cell vaccination and mAb treatment, in the overall field of cancer immunotherapy. The concept of coating tumor cells with a highly antigenic viral T-cell antigen in the context of autologous MHC molecules is very attractive. As soon as soluble recombinant MHC class I molecules were available (35), preliminary attempts were made to bind the MHC complexes, loaded with specific peptides to tumor cells (by using an anti-h2-microglobulin antibody chemically coupled to the cell surface), to verify if these molecules could activate T cells and induce lysis of the target cells (36). Then, by taking advantage of the biotinylation and tetramerization of MHC complexes on streptavidin, it was shown that the antibody-mediated binding of MHC tetramers on tumor cells could induce their in vitro lysis by specific CTL (13, 14). Soon afterwards, however, we showed that the streptavidin was not necessary, and that the oligomerization on the tumor cell surface of anti-TAA antibody fragments coupled to monomeric MHC/peptide complexes was sufficient to induce efficient in vitro and in vivo tumor cell lysis (15, 16). More recently, progress was made by the development of a recombinant fusion protein among h2-microglobulin, MHC

heavy chain, and a single-chain Fv antibody fragment, which, when loaded with an antigenic peptide, was also able to induce lysis of tumor cells in vitro and in vivo (17). Thus far, however, most results were obtained in immunodeficient animals xenografted with human tumor cell lines and injected with human T-cell clones (17, 37). In contrast, we have shown the feasibility of this immunotherapy strategy in immunocompetent animals grafted with syngeneic tumors, first in the OT-1 transgenic model, using H-2Kb/ova peptide complex (16) and here by the use of two different clinically more relevant models of viral infection. The present results obtained in B6 mice previously infected with viruses show that the induction phase of the T-cell response can be produced by viral immunization, whereas the effector function can be redirected against tumor cells by the targeting of antibody-MHC/viral peptide conjugates. The results suggest that the need to increase efficiency of antigen presentation (e.g., using adjuvants) is not a concern in the present form of immunotherapy, contrary to active tumor antigen vaccination, because the live virus infection, or boost, can produce sufficient memory T cells, which can be redirected against tumor cells coated by antibody-MHC/viral peptide conjugates. A second potential advantage of this new tumor immunotherapy strategy, compared with active T-cell immunization against autologous tumor antigens, is that it allows the targeting on the tumor cell surface of multiple copies of autologous MHC complexes loaded with the same viral antigenic peptide. In contrast, peptides derived from autologous tumor or differentiation antigens are often not sufficiently expressed on tumor cells because they have to compete against numerous normal cellular peptides for transport to the

Fig. 5. H-2Db/GP33 cross-linked monomers are efficiently recognized by H-2Db/GP33 ^ specific CD8+ Tcells. A and B, comparison of the ability of H-2Db tetramers wild-type GP33 peptide (A) or cross-linked GP33 peptide (B) to stain murine CD8+ spleen cells obtained 8 d after LCMV infection. Frequencies (top right) are expressed as percentage of tetramer-specific cells from the CD8 population. C, titration of the two types of tetramer with wild-type peptide (o) and cross-linked peptide (.) for the staining of peripheral blood mononuclear cells from day 25 LCMV-infected mice. Staining with both cross-linked and wild-type tetramers is concentration dependent and reaches saturation around 15 Ag/mL. D, cytotoxicity of ex vivo anti-LCMV CTLs on MC38-CEA+ tumor cells uncoated (o), coated with anti-CEA Fab H-2Db/Gp33 with either wild-type peptide (n) or cross-linked peptide (E), tested in a chromium release assay.


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Fig. 6. MC38-CEA+ tumor growth inhibition induced by systemic injection of anti-CEA H-2Db/GP33 cross-linked conjugate, in LCMV-immune mice. Points, mean tumor growth curves in each group; bars, 2 SD. Mice were treated every 2 d by systemic injection of 70 Ag of conjugate from days 1to 17 after tumor graft. LCMV infection was made 8 d before tumor grafting. Control mice received anti-CEA F(ab¶)2 fragments alone.

endoplasmic reticulum and presentation through binding to MHC molecules, and functional defects in the antigen processing delivery of some tumors have been recently reported (7). A third advantage of this immunotherapy, compared with other related strategies, such as bispecific antibodies (38, 39), is that the MHC class I/peptide complexes oligomerized by antibody-mediated targeting on the surface of the tumor cells will provide multiple binding site for specific CTL, not only via the T-cell receptor but also via the CD8 molecule, which has been shown to play an essential role in the interaction with MHC class I/peptide complexes (40, 41). In the present study, to minimize the known risk of peptide dissociation (42, 43) after systemic injection, we have introduced the covalent cross-linking of the viral peptide to the MHC molecule by photoactivation, whereas all previous in vivo studies were made with non – covalently bound peptide (16, 17, 37). The peptide cross-linked to MHC was well recognized by specific T cells in vitro and was active in preventing tumor growth in vivo after systemic injection. Alternatively, the peptide can be genetically fused directly to the MHC heavy chain or through the h2-microglobulin (44, 45). One limitation of the present study is that we showed tumor growth inhibition by early systemic injection of our conjugates but not yet regression of growing tumors. The difficulty in obtaining regression of larger tumors might be due to the malignant phenotype and the poor vascularization of the syngeneic colon carcinoma cell line used. Indeed, we have previously shown that, when radiolabeled, such conjugates were specifically but

not abundantly localized in this tumor (16). One way to improve this strategy, would be to use a conjugate with two antibody-binding sites to increase its residence time on the tumor cell surface. If we now consider the potential of this type of immunotherapy for clinical use, it should not be difficult to identify, in the majority of cancer patients to be treated, a robust T-cell memory against an endemic virus, such as EBV, CMV, or influenza, for which immunodominant viral peptides have been identified, as shown by a very recent study of B-chronic lymphocytic leukemia leukemia, in which the patients’ antiCMV T cells were redirected in vitro against leukemic cells by the use of a streptavidin-fused anti-CD20 ScFv antibody fragment in combination with biotinylated MHC class I molecules (46). The TAA most expressed on the tumor cells, such as HER2, CEA, CA125 (47), or PSMA (ref. 48; for review, see ref. 9), as well as the patient’s MHC class I phenotype, will be identified. Furthermore, before tumor treatment by injection of the anti – TAA-MHC/viral peptide conjugate, the T-cell antiviral response could be boosted. The advantages of the MHC/viral peptide tumor targeting strategy suggest that it should be particularly useful in conjunction with active T-cell vaccination or monoclonal anticancer antibody treatment. In the first case, when patients undergoing active T-cell immunotherapy do not show tumor regression despite induction of a significant T-cell response against the immunizing antigen, the injection of Fab-MHC conjugates loaded with viral peptide should mobilize antiviral CTL to react against tumor cells. This local induction of CTLmediated tumor cell lysis may trigger the recruitment, crosspriming, and full activation of the T cells induced by active vaccination and additionally kill the resistant clones with low MHC expression. Indeed, it has been shown that soluble MHC class I, loaded with specific peptides, can act as powerful antigens (49). In the second case, when treatment with the classic antitumor mAbs, such as anti-HER2, anti – epidermal growth factor receptor, or anti-CD20, have induced significant tumor remission, but tumor cells are still present, an additional injection with the antibody-MHC conjugate loaded with viral peptide may add the necessary local CTL targeting to eliminate the tumor cells resistant to the action of antibody alone. The antibody-mediated tumor targeting of antigenic MHC complexes described here may also open the way to similar approaches, such as the antibody-targeting of MHC class I – related MICA molecules, which we have shown by in vitro experiments to induce the activation of natural killer cells, leading to specific target tumor cells lysis (50).

Acknowledgments We thank Richard Pink for reviewing the article, Karine Fournier for excellent technical help, and Pedro Romero for advice and suggestions.

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