Tumor-specific Ab-mediated targeting of MHC-peptide complexes induces regression of human tumor xenografts in vivo

Abstract

A cancer immunotherapy strategy is described herein that combines the advantage of the well established tumor targeting capabilities of high-affinity recombinant fragments of Abs with the known efficient, specific, and potent killing ability of CD8 T lymphocytes directed against highly antigenic MHC-peptide complexes. Structurally, it consists of a previously uncharacterized class of recombinant chimerical molecules created by the genetic fusion of single-chain (sc) Fv Ab fragments, specific for tumor cell surface antigens, to monomeric scHLA-A2 complexes containing immunodominant tumor- or viral-specific peptides. The fusion protein can induce very efficiently tumor cell lysis, regardless of the expression of self peptide-MHC complexes. Moreover, these molecules exhibited very potent antitumor activity in vivo in nude mice bearing preestablished human tumor xenografts. These in vitro and in vivo results suggest that recombinant scFv-MHC-peptide fusion molecules could represent an approach to immunotherapy, bridging Ab and T lymphocyte attack on cancer cells.

Fig. 1.

Design, expression, and purification of scHLA-A2/scFv fusion molecules. (A) Binding of in vitro refolded scHLA-A2-peptide complexes to CTLs. Melanoma differentiation antigen gp100-specific CTL clones R6C12 and R1E2 or the EBV-specific CTL line DZ/EBV were reacted with in vitro refolded purified scHLA-A2-peptide tetramers containing the G9-209M epitope recognized by R6C12, the G9-280V peptide recognized by R1E2, or the EBV-derived BMLF1 protein epitope GLC_280-288 recognized by DZ/EBV CTLs. CTLs were stained with FITC-anti-CD8 (a and d), with phycoerythrin-labeled scHLA-A2-G9-209M tetramers (b, f, and h), with scHLA A2-G9-280V tetramers (c and e), or with scHLA-A2-EBV tetramers (g). R6C12, R1E2, and DZ/EBV CTLs were stained with the specific G9-209M, G9-280V, or EBV tetramers, respectively, but not with the control tetramer. (B) Design of scHLA-A2/scFv fusion. (C) SDS/PAGE analysis of inclusion bodies of scHLA-A2/aTac(scFv) (a) or scHLA A2/SS1(scFv) (b). (D) SDS/PAGE analysis of scHLA-A2/aTac(scFv) (a) or scHLA-A2/SS1(scFv) (b) folded around the G9-209M peptide after ion-exchange purification.

Fig. 2.

Binding of scHLA-A2/scFv fusion molecules to target antigens. (A and B) scHLA-A2/aTac(scFv) or scHLA-A2/SS1(scFv) folded around the G9-209M or EBV peptide, respectively, was tested for dose-dependent binding to recombinant purified p55 (Tac, IL-2 receptor α-subunit) (A) or mesothelin (B). Binding was monitored with anti-HLA mAb W6/32. (C) Flow cytometry analysis of the binding of scHLA-A2/aTac(scFv) folded around the G9-209M peptide to antigen-positive HLA-A2-negative cells. The binding of human anti-Tac mAb to A431 (a), ATAC4 (c), and HUT102W leukemic (e) cells monitored the expression of p55 (Tac). ATAC4 are A431 cells stably transfected with the Tac/p55 IL-2 receptor α-subunit. Control cells with secondary Ab are shown; HLA-A2 expression on these cells was monitored before and after incubation with the scHLA-A2/aTac(scFv) fusion (b, d, and f). The conversion of these targets from HLA-A2 negative to positive due to the binding of the fusion molecule is shown. In g, mesothelin-expressing A431/K5 cells were tested for binding of BB7.2 before and after incubation with the scHLA-A2/SS1(scFv) fusion refolded around the EBV peptide. Two versions of the fusion with or without a 5-aa connector between the scHLA-A2 and the scFv gene (see Fig. 1B) are shown. The scHLA-A2/SS1(scFv) with the connector was slightly but significantly better in binding and thus was selected for further analysis. (D) Confocal microscopy detection of the binding of scHLA-A2/aTac(scFv) fusion folded around the G9-209M peptide to p55-positive ATAC4 but not to Tac-negative A431 cells. Detection was with anti-HLA-A2 mAb BB7.2 and FITC-labeled secondary Ab.

Fig. 3.

Potentiation of CTL-mediated lysis of HLA-A2-negative tumor cells by the scHLA-A2/scFv fusion molecule. CD25-transfected ATAC4 or CD25-positive leukemic HLA-A2-negative cells coated or not coated with the scHLA-A2/aTac(scFv) fusion molecule folded around the G9-209M (A, C, and E) or G9-280V (B, D, and F) gp100-derived peptides were incubated with melanoma-reactive gp100-peptide-specific CTL clones R6C12 or JR1E2, respectively, in a [³⁵S]methionine release assay. Mesothelin-expressing A431/K5 cells were coated or not coated with a scHLA-A2/SS1(scFv) fusion molecule folded around the gp100-derived G9-209M peptide (G) or EBV peptide (H) and incubated with the R6C12 G9-209M-specific CTL clone or EBV-specific line, respectively. A431/K5 cells incubated with scHLA-A2/SS1(scFv) folded around the EBV (G) or G9-209M (H) peptides were not killed by R6C12 G9-209M-specific CTLs or DZ/EBV EBV-specific CTLs, respectively. Insensitivity of scHLA-A2/SS1(scFv)-coated A431 cells is also shown as the control. (I) Dose-dependent activity of the scHLA-A2/SS1(scFv) molecule folded around the EBV peptide on A431/K5 cells using EBV-specific CTLs.

Fig. 4.

Antitumor activity of scHLA-A2/scFv fusion molecules in nude mice model of human tumor xenografts. (A and B) ATAC4 cells (3 × 10⁶) were injected s.c. into nude mice, and 4-7 days after injection ≈40- to 50-mm³ tumors were generated. Mice were injected i.v. three times every other day with 100 μg of purified scHLA A2/aTac(scFv) folded around the gp100-derived G9-209M peptide. Five to 6 h after fusion molecule injection, gp100 G9-209M R6C12-specific CTLs (2-3 × 10⁶) were injected i.v. or i.t. In B, the mean tumor size of treated and control groups is shown. (C and D) A431/K5 cells (3 × 10⁶) were injected as above. Mice were injected i.v. three times every other day with 100 μg of purified scHLA-A2/SS1(scFv) folded around the EBV-derived peptide. Five to 6 h after fusion molecule injection, EBV-specific CTLs (2-3 × 10⁶) were injected i.t. (C) or i.v. (D). Tumor size was measured every 2 days, and tumor volume was calculated. The days of treatment are indicated.