© 1999 Nature America Inc. • http://biotech.nature.com RESEARCH Genetic fusion of chemokines to a self tumor antigen induces protective, T-cell dependent antitumor immunity Arya Biragyn, Kenji Tani1, Michael C. Grimm1, Steven Weeks, and Larry W. Kwak2* Science Application International Corporation, National Cancer Institute, Frederick, MD 21702. 1Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, MD 21702. 2Department of Experimental Transplantation and Immunology, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD 20892. *Corresponding author (e-mail: [email protected]). Received 24 September 1998; accepted 29 December 1998 We converted a model, syngeneic, nonimmunogenic tumor antigen into a vaccine by fusing it with a proinflammatory chemokine. Two chemokines, interferon inducible protein 10 and monocyte chemotac- tic protein 3, were fused to lymphoma Ig variable regions (sFv). The sFv–chemokine fusion proteins elicit- ed chemotactic responses in vitro and induced inflammatory responses in vivo. Furthermore, in two inde- pendent models, vaccination with DNA constructs encoding the corresponding fusions generated supe- rior protection against a large tumor challenge (20 times the minimum lethal dose), as compared with the best available protein vaccines. Immunity was not elicited by controls, including fusions with irrelevant sFv; fusions with a truncated chemokine that lacked receptor binding and chemotactic activity; mixtures of free chemokine and sFv proteins; or naked DNA plasmid vaccines encoding unlinked sFv and chemokine. The requirement for linkage of conformationally intact sFv and functionally active chemokine strongly suggested that the mechanism underlying these effects was the novel targeting of antigen pre- http://biotech.nature.com senting cells (APC) for chemokine receptor-mediated uptake of antigen, rather than the simple recruit- • ment of APC to tumor by the chemokine. Finally, in addition to superior potency, these fusions were dis- tinguished from lymphoma Ig fusions with granulocyte-macrophage colony-stimulating factor or other cytokines by their induction of critical effector T cells. Keywords: interferon inducible protein 10, monocyte chemotactic protein 3, chemokine fusion, antigen presenting cell targeting, idiotypic vaccine Chemokines are a group of small (7–15 kDa) secreted proteins that We explore a novel strategy for induction of antitumor immunity, 1999 Nature America Inc. induce inflammatory response by orchestrating the selective migra- in which APCs are specifically targeted in vivo by a fusion protein con- © tion, diapedesis, and activation of blood-borne leukocytes1–3. sisting of chemokine and tumor antigen. We demonstrate that genetic Chemokines act by binding to a specific cell-surface heptahelical G- fusion of chemokine moieties to lymphoma-derived sFv, a model non- protein–coupled receptor, which is internalized after binding with the immunogenic antigen, converted this “self” tumor antigen into a ligand4–6. Moreover, chemokines may have potent effects in promoting potent immunogen. In both murine lymphoma models tested, 38C-13 tumor immunity7–9. For example, interferon inducible protein 10 (IP- (ref. 18) and A20 (ref. 19), immunization with either chemokine–sFv 10) can generate a T-lymphocyte–dependent antitumor response by protein or naked DNA vaccines encoding the fusion elicited a T-cell recruiting other lymphocytes, neutrophils, and monocytes1–3,7–9. In dependent, protective antitumor immunity. addition, monocyte chemotactic protein 3 (MCP-3) is a potent chemoattractant for monocytes and dendritic cells, T lymphocytes, Results basophils, and eosinophils1–3,10. Importantly, most antigen presenting Chemokine-fused sFv proteins retain conformation of native lym- cells (APCs), particularly dendritic cells, express CCR1, CCR2, and phoma-derived Ig and functional properties of chemokine. Ig variable CCR3 chemokine receptors, which bind MCP-3 (refs. 1, 11, and 12). region fragments were cloned by reverse transcription PCR from two Clonal B-cell malignancies express variable region sequences (idio- different B-cell lymphomas, 38C-13 (ref. 19) and A20 (ref. 20). The types) on the Ig receptor, which can serve as potential tumor-specific fragments were arranged as sFv and produced as recombinant fusion antigens13. However, a major obstacle is that syngeneic lymphoma Ig proteins with either IP-10, MCP-3, control viral epitopes DomA (a protein is nonimmunogenic and therefore requires conjugation to 40–amino acid fragment containing major B- and T-cell epitopes of S immunogenic, exogenous carriers (e.g., keyhole-limpet hemocyanin antigen), or PreS2 of hepatitis B surface antigen (HBsAg) (Fig. 1). [KLH])14,15. Even then, Ig–KLH conjugates elicit primarily antibody Recombinant fusion proteins were purified from Escherichia coli20 and responses, with little evidence of T-cell responses against the idiotype. fully characterized. The proper folding of sFv proteins was demonstrat- Recent advances in antibody engineering make it possible to produce a ed by their ability to inhibit polyclonal or monoclonal anti-idiotypic refined lymphoma-specific single-chain Ig (sFv), consisting solely of antibodies binding to 38C-13–derived native Ig protein (Ig38) (Fig. 2). linked VH and VL domains that retain the conformation of the native In contrast, control fusions lacking a spacer (IP10dsFv38), fusions with Ig16,17. However, KLH crosslinked to sFv failed to induce the anti-idiotyp- sFv derived from the irrelevant lymphoma, or fusions with viral epi- ic antibody response usually observed with native Ig–KLH conjugates, topes DomA and PreS2 failed to inhibit binding to native 38C-13, sug- due to disruption of the fragile conformation of sFv (data not shown). gesting that they did not retain proper folding of the antigen. Folding NATURE BIOTECHNOLOGY VOL 17 MARCH 1999 http://biotech.nature.com 253 © 1999 Nature America Inc. • http://biotech.nature.com RESEARCH was not affected by the arrangement of sFv in VH–VL or inverse VL–VH orientation (INV). Next, binding experiments were conducted using confocal microscopy analysis of purified T cells and unfractionated murine splenocytes. IP-10 and MCP-3 sFv fusions (Fig. 3A, panels C and E), but not control viral epitope fusions (DomAsFv38; Fig 3A, panel A), were found to bind their respec- tive chemokine receptors. Binding was specific, as it was inhibited by native ligands IP-10 or MCP-3, respectively (Fig. 3A, panels D and F). Moreover, no binding was detected with the truncated IP-10 sFv fusion, IP10TsFv38, which contained a 9–amino acid. deletion of the amino-terminal portion of IP- 10 (Fig. 3A, panel B). Similarly, binding of 125I- labeled free chemokine was specifically inhibited by chemokine fusion proteins (data not shown). We further tested the ability of the samples to induce in vitro and in vivo chemotaxis. Both chemokine-sFv proteins (IP10sFv38 and MCP3sFv38) but not control PreS2sFv38, induced in vitro chemotaxis of murine lympho- cytes in a dose-dependent manner, with a maxi- Figure 1. Lymphoma fusion protein and DNA genetic constructs. Expression vectors both mum activity at 100 ng/ml (Fig. 3B). These sam- for E. coli-derived proteins and naked DNA vaccines for 38C-13 (ref. 18) and A20 (ref. 19) ples also induced in vivo chemotaxis in syngeneic lymphomas are shown. DNA fragments encoding IP-10, MCP-3, or control viral epitopes mice. Histologic evaluation of the site of subcuta- DomA (a 40–amino acid fragment containing major B- and T-cell epitopes of S antigen) or PreS2 of HBsAg, were fused in frame with DNA encoding sFv. IPI0TsFv38 and IP10dsFv38 neous injection of 10 µg protein showed that are identical to IPI0sFv38, except for truncation of the 9 N-terminal residues of IP10 and lack injection of IP10sFv38 or MCP3sFv38, but not of a spacer, respectively. Protein vaccines were expressed in modified pet11d vector PreS2sFv38, was associated with substantial num- (Stratagene, La Jolla, CA), and naked DNA plasmid vaccines were cloned in pCMVE/AB. bers of infiltrating mononuclear cells, and fewer http://biotech.nature.com • polymorphonuclear cells, in the dermis and subcutaneous layers (Table 1). Taken together, these results suggest that IP-10 and MCP- 3 fused sFv proteins retained both the conformation of native anti- gen and functional properties of their respective chemokine moi- eties. Chemokine fusion renders sFv immunogenic and induces antitu- mor immunity. C3H/HeN mice were immunized twice biweekly with 100 µg IP10sFv38 or MCP3sFv38 fusion proteins in phosphate-buffered saline (PBS). Their sera were then assayed for anti-idiotypic antibody 1999 Nature America Inc. response using Ig38. Both IP-10 and MCP-3 sFv fusions elicited signifi- © cant anti-idiotypic antibody responses (mean ± standard deviation, 77 ± 41 and 13 ± 3 µg/ml, respectively; Fig. 4A). This result was in distinct contrast to the lack of any anti-idiotypic antibody response observed in mice injected with a truncated chemokine sFv fusion that did not retain chemokine activity (IP10TsFv38), sFv38 alone, or fusions that did not retain the native antigen conformation (IP10dsFv38 or Pres2sFv38). The highest antibody levels (286 ± 78 µg/ml) were induced by immu- nization with chemically conjugated Ig38–KLH. Figure 2. Effect of chemokine fusions on folding of sFv antigen. sFv- chemokine fusion proteins were tested for their ability to inhibit Ten mice per group were immunized with either IP10sFv38 or with binding of anti-idiotypic