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The Journal of Immunology, 1999, 162: 6024-6028.
Copyright © 1999 by The American Association of Immunologists

Covalent Linkage to ß2-Microglobulin Enhances the MHC Stability and Antigenicity of Suboptimal CTL Epitopes1

Robert A. Uger, Steven M. Chan and Brian H. Barber2

Department of Immunology, Medical Sciences Building, University of Toronto, Toronto, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Many CTL epitopes of clinical importance, particularly those derived from tumor Ags, display relatively poor MHC binding affinity and stability. Because in vivo immunogenicity, and thus the efficacy of peptide-based vaccines, is thought to be determined by MHC/peptide complex stability, there is a need to develop a simple strategy for enhancing the binding of suboptimal epitopes. Toward this goal, the ability to enhance suboptimal peptides through covalent linkage to ß2-microglobulin (ß2m) was explored. Two suboptimal variants of a high-affinity Db-restricted influenza nucleoprotein peptide were covalently linked, via a polypeptide spacer, to the amino terminus of human ß2m and the recombinant fusion proteins expressed in Escherichia coli. When compared with their uncoupled counterparts, the ß2m-linked epitopes display enhanced MHC stabilization and antigenicity. Thus, tethering epitopes to ß2m provides a simple method for augmenting the biological activity of suboptimal peptides and could be useful in the design of peptide-based vaccines or immunotherapeutics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The identification of MHC class I epitopes of both viral and tumor origin, and the ability to routinely generate ex vivo dendritic cells, has increased the prospects of developing effective peptide-based vaccines and immunotherapies. The design of such strategies requires careful selection of peptide epitopes to maximize in vivo efficacy. Numerous experiments have demonstrated that the degree of immunogenicity of a peptide is related to its binding affinity for class I molecules (1, 2, 3). More specifically, the rate of dissociation of MHC/peptide complexes appears to be the most important binding parameter, with immunogenic peptides displaying slower off-rates (4, 5). Thus, rational peptide vaccine design should focus on class I epitopes that display a high MHC/peptide complex stability. Unfortunately, many epitopes that are clinically relevant, including HIV and melanoma-derived peptides, display relatively poor MHC binding and suboptimal immunogenicity (6, 7, 8, 9, 10, 11, 12). This is of particular concern in tumor immunology, because many tumor-specific CTL are directed against suboptimal self-epitopes due to the tolerance of CTLs to high-affinity self-peptides.

A potential solution to this problem is to convert suboptimal epitopes into more effective immunogens by enhancing their MHC binding and stability. Numerous groups have taken such an approach, synthesizing peptide analogues in which deleterious MHC binding residues are replaced with more favorable ones, while preserving TCR contact amino acids. This strategy has been employed successfully in mouse viral (13) and tumor models (14). In human systems, modifications made to an HIV epitope (15) and numerous melanoma peptides (12, 16, 17) have resulted in enhanced MHC binding and immunogenicity. Furthermore, Rosenberg et al. recently reported that immunization of melanoma patients with a modified, higher-affinity melanoma gp100 peptide analogue can result in a positive clinical outcome (18). Thus, engineering class I epitopes to have increased MHC binding capacity appears to be a promising approach for the in vivo induction of specific CTL responses. However, this technique can be laborious, in so far as it requires analyzing the binding contribution of individual residues within a given epitope and selection of appropriate high-affinity analogues.

In this report, we describe an alternative strategy for enhancing suboptimal class I peptides: tethering epitopes to human ß2-microglobulin (hß2m).3 Previously, we demonstrated that CTL target structures could be effectively generated by covalently linking optimal Db- or Kd-restricted influenza virus nucleoprotein (NP) peptides to hß2m (19). Interestingly, we observe that the ability of the optimal Kd-restricted epitope to sensitize target cells for CTL lysis is enhanced when linked to hß2m. To further examine the enhancing role of hß2m linkage, we generated bacterially expressed fusion proteins in which suboptimal variants of the Db-restricted NP (366–374) peptide are tethered to hß2m. Covalently linking these peptides results in greater MHC/peptide stability and increased antigenicity over uncoupled peptides. Therefore, this approach offers a simple method of enhancing the biological activity of suboptimal epitopes and could be useful for the design of peptide-based vaccines and immunotherapeutics.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell lines, Ab, and hß2m

EL4 (H-2b), P815 (H-2d), and TAP-defective RMA-S (H-2b) cells were grown in AIM-V serum-free medium (Life Technologies, Grand Island, NY). The anti-Db Ab B22.249.R1 (B22) has been previously described (20, 21). Purified hß2m was purchased from Fluka (Ronkonkoma, NY).

Construction and purification of peptide-hß2m fusion proteins

Peptide-hß2m fusion proteins have a 9-aa NP epitope (Kd- or Db-restricted) connected to hß2m via a 12-aa linker (GGGSGTGSGSGS, single letter amino acid code). The corresponding DNA constructs were generated by standard PCR reactions using a wild-type hß2m cDNA clone as a template and were inserted into the pCAL-n-EK bacterial expression vector (Stratagene, La Jolla, CA) immediately downstream of an N-terminal calmodulin binding protein tag and enterokinase (EK) cleavage site. Digestion with EK liberates peptide-hß2m fusion proteins without any additional amino acids. Vectors were transformed into BL21(DE3)plysS bacteria (Stratagene), isopropyl ß-D-thiogalactoside induced, and lysates prepared essentially as described previously (19). Refolded fusion proteins were purified over a calmodulin affinity column (Stratagene) eluting in 50 mM Tris, pH 8.0, 300 mM NaCl, and 2 mM EDTA. Eluted fusion protein was then exchanged into EK cleavage buffer (20 mM Tris, pH 8.0, 50 mM NaCl, and 2 mM CaCl2) using a G-25 Sephadex column (PD-10; Pharmacia, Uppsala, Sweden) and digested overnight at room temperature with EK. Enzyme and free calmodulin binding protein were removed by addition of soybean trypsin inhibitor-agarose and calmodulin affinity resin, and the digested protein was concentrated (Centriprep, Amicon, Beverly, MA) and exchanged into PBS.

MHC stability determination

The stability of folded Db complexes was determined using a cell panning enzyme immunoassay described previously (22). RMA-S cells were grown overnight in serum-free (AIM-V) medium at 26°C to favor the formation of "empty" class I molecules and were pulsed with 10 µM free peptide and 10 µM hß2m or 10 µM fusion protein for 1 h at 26°C in the presence of 5 µg/ml brefeldin A. Cells were washed in PBS, resuspended at 2 x 106 cells/ml in serum-free medium with brefeldin A, and incubated at 37°C. At various times during the 37°C incubation, 2 x 105 cells were removed and dispensed into a 96-well plate that had been coated with 10 µg/ml purified B22 Ab (2 h at 37°C) and blocked with 5% milk (2 h at 37°C). Cells were subsequently incubated on the plate for 1 h at room temperature, and the wells were washed gently six times with PBS. Bound cells were lysed and quantitated by adding 100 µl of lactate dehydrogenase (LDH) substrate (0.167 mg/ml p-iodonitrotetrazolium violet, 0.043 mg/ml phenazine methosulfate, 2.435 mg/ml lactic acid, 0.431 mg/ml ß-NAD, and 1% Triton X-100 in 0.2 M Tris, pH 8.2), which undergoes a yellow to red color change. The reaction was stopped by adding 100 µl of 3 M HCl, and absorbance at 492 nM was read. All samples were analyzed in triplicate, and the percentage stability was determined by normalizing the LDH values relative to the initial (no 37°C incubation) point.

CTL assays

Bulk CTL cultures were generated by immunizing C57BL/6 or BALB/c mice with influenza virus and restimulating spleen cells with 1 µM optimal peptide, essentially as described (23). EL4 (H-2b) or P815 (H-2d) target cells were labeled with 100 µCi of Na2[51Cr]O4 (Amersham, Arlington Heights, IL), washed, and pulsed with titrated amounts of purified fusion protein or equivalent amounts of free peptide and hß2m at a concentration of 105 cells/ml for 1 h at room temperature. A total of 104 targets were dispensed into 96-well plates, effectors were added, and the plates were incubated at 37°C for 4 h. Plates were centrifuged, and the supernatant was harvested and quantitated using a TopCount scintillation counter (Canberra Packard, Mississauga, Ontario). The percent specific lysis was calculated, using the mean of triplicate samples, as 100 x [(experimental cpm - spontaneous cpm)]/[(maximum cpm - spontaneous cpm)]. Spontaneous cpm values were determined by incubating target cells alone in medium, and maximum values were determined by lysis of targets in 1% Triton X-100 (v/v).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Covalently coupling a peptide epitope to its presenting MHC class I molecule, through either the heavy chain or ß2m subunit, has recently emerged as a potentially useful method of generating defined CTL target structures (19, 24, 25). The physical association between epitope and MHC could increase the stability of the MHC/peptide complex by restricting peptide diffusion and slowing dissociation, which can be critical for immunogenicity (4, 5). From the perspective of peptide vaccine design, the ß2m subunit appears ideally suited, because it is a small, soluble molecule that is known to promote peptide binding in vitro (26, 27, 28) and augment peptide-specific CTL responses in vivo (29). It was previously demonstrated that Db and Kd binding peptides could be genetically fused to hß2m, and CTL target structures could be formed by both an endogenous (expression in transfected cell lines) and exogenous pathway (pulsing target cells with fusion protein) (19). Interestingly, we observed that when NP(147–155), an optimal Kd-restricted epitope, is tethered to hß2m there is enhanced target cell sensitization compared with the free peptide mixed with hß2m (Fig. 1Go). To further explore this phenomenon, we have generated recombinant fusion proteins in which Db-binding class I peptides are tethered to hß2m via a flexible polypeptide linker (Fig. 2Go). The optimal peptide NP(366–374) and two variant peptides (designated R2 and D2) were chosen as a model system. These variant peptides have a single amino acid substitution at the P2 position (Ser changed to Arg and Asp, respectively). This peptide position, although not a dominant anchor, is buried within the MHC groove (30) and is thus expected to affect MHC binding but not TCR recognition. Previous measurements indicate that the R2 and D2 variant peptides show significantly reduced Db binding (31, 32), and we observe a dramatic (3 log) decrease in the ability of the mutant peptides to sensitize target cells for CTL lysis (see Fig. 4Go).



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  		FIGURE 1. Linking NP(147–155) peptide to hß2m enhances the formation of CTL target structures. 51Cr-labeled P815 cells (H-2d) were pulsed with 1 nM, 0.1 nM or 0.01 nM NP(147–155)-hß2m fusion protein (A) or equivalent amounts free NP(147–155) peptide and hß2m (B) and then incubated in a standard chromium release assay using NP(147–155)-specific CTLs. The NP(147–155)-hß2m fusion protein (19) has the NP(147–155) nonamer epitope connected to the amino terminus of hß2m via a 12-residue linker (GGGSGTGSGSGS, single letter amino acid code).

 


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  		FIGURE 2. Epitope-linked hß2m fusion proteins. The Db-restricted epitope NP(366–374) (ASNENMETM, single letter amino acid code) and two suboptimal variants (designated R2 and D2) were linked to the amino terminus of hß2m via a 12-residue linker (GGGSGTGSGSGS). The altered residues in the P2 position are underlined.

 


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  		FIGURE 4. Peptides linked to hß2m exhibit increased antigenicity over unlinked peptides. 51Cr-labeled EL4 (H-2b) cells were pulsed with titrated, equal amounts of free peptide and hß2m or peptide-hß2m fusion protein and incubated in a standard 4-h chromium release assay using NP(366–374)-specific CTLs at an E:T ratio of 40:1. A, Comparison of uncoupled NP(366–374) peptide and hß2m and the corresponding fusion protein, NP(366–374)-hß2m. B, Comparison of free R2 peptide and R2-hß2m fusion protein. C, Comparison of D2 peptide and D2-hß2m fusion protein.

 
Initially, we compared the ability of the purified peptide-hß2m fusion proteins and their corresponding free peptides to stabilize Db complexes on RMA-S cells. Incubation of RMA-S at low temperature (26°C) promotes the formation of thermolabile empty class I molecules, which can be stabilized with exogenous peptides (33). The rate of decay of these complexes at 37°C gives an indication of stability, which we quantified using a cell panning immunoassay (22). This assay demonstrates high sensitivity and specificity, and thus compares favorably with conventional flow cytometric analysis (data not shown). RMA-S cells were first grown at 26°C overnight, pulsed with 10 µM of fusion protein or equivalent amounts of free peptide and hß2m, washed, and transferred to 37°C for various time periods. The extent of class I stabilization was determined by exposing the cells to a plate coated with a conformation-sensitive Ab (B22) followed by the measurement of LDH activity to quantitate the number of cells bound. This cell capture was B22 specific, as plates coated with an isotype-matched control Ab failed to generate an LDH signal (data not shown). Note that brefeldin A was included in the assay to prevent the emergence of new class I molecules at the cell surface, increasing the likelihood that any observed loss of class I stabilization will reflect peptide off-rates. As shown in Fig. 3GoA, both free NP(366–374) peptide and the corresponding fusion protein, NP(366–374)-hß2m, stabilized Db molecules equally well. In contrast, when the suboptimal R2 variant peptide was tethered to hß2m, there was a marked increase in stability over the free peptide simply mixed with hß2m in equal amounts (Fig. 3GoB). Pulsing with uncoupled R2 peptide and hß2m results in complexes that are 50% dissociated by 30 min, while the tethered fusion protein extends this time to nearly 1.5 h. This effect was even more pronounced with the D2 variant (Fig. 3GoC). Like the R2 peptide, free D2 peptide and hß2m result in class I complexes that dissociate rapidly (50% loss by 30 min). However, the D2-hß2m fusion protein stabilized complexes such that the half life was extended beyond 1.5 h. Thus, tethering suboptimal peptides to hß2m results in a measurable increase in MHC complex stability over uncoupled peptide and hß2m alone.



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  		FIGURE 3. Suboptimal peptides tethered to hß2m have an enhanced ability to stabilize class I molecules. RMA-S cells cultured overnight at 26°C were pulsed with 10 µM fusion protein or 10 µM free peptide and 10 µM hß2m for 1 h at 26°C and then shifted to 37°C for the indicated time periods. The decay of Db complexes was measured using a cell panning assay as described in Materials and Methods. A, Comparison of uncoupled NP(366–374) peptide and hß2m and the corresponding fusion protein, NP(366–374)-hß2m. B, Comparison of free R2 peptide and R2-hß2m fusion protein. C, Comparison of D2 peptide and D2-hß2m fusion protein. The dotted lines indicate the time required for 50% decay.

 
To determine whether this increase in stability translates into an increase in antigenicity, free peptides and fusion proteins were compared for their ability to sensitize EL4 (H-2b) target cells for lysis by flu-specific CTLs in standard chromium release assays. As shown in Fig. 4GoA, the free optimal peptide and NP(366–374)-hß2m fusion protein are generally comparable in their ability to sensitize for CTL lysis, although a difference is observed at the 1-pM concentration. Note that tethering an optimal Kd peptide to hß2m actually augments antigenicity compared with the uncoupled counterparts (Fig. 1Go). This reason for this discrepancy is not clear, although it could relate to the relatively weak binding of ß2m to Db heavy chains (34). In the case of the R2 variant epitope, covalent coupling to hß2m dramatically increases antigenicity (Fig. 4GoB). Free R2 peptide and hß2m sensitize target cells for lysis out to 10-10 M, whereas the corresponding fusion protein titers out near 10-13 M, a ~1000-fold improvement. To a lesser extent, D2-hß2m also outperforms its uncoupled counterpart (Fig. 4GoC). While free D2 peptide and hß2m titer out at 10-10 M, the hß2m fusion protein is active at a log lower concentration. Thus, tethering an optimal peptide to hß2m does not significantly impair its ability to sensitize target cells for lysis, and most importantly, coupling suboptimal epitopes to hß2m improves antigenicity. It is curious that the D2-hß2m fusion protein, which induces slightly more Db stability than R2-hß2m, actually shows a smaller improvement in antigenicity. This may be due to suboptimal TCR recognition of the D2 variant epitope by CTLs restimulated on the optimal NP(366–374) peptide.

The ability to enhance the stability and antigenicity of suboptimal epitopes by covalent linkage to ß2m, as described above, may offer new approaches for the development of peptide-based vaccines and immunotherapies. It would allow the repertoire of class I epitopes to be expanded to include low-affinity peptides, which is of clinical relevance for viral and neoplastic disease treatments (7, 8, 9, 10, 11, 12). A ß2m-coupling approach does not require analyzing the contribution that individual amino acids make toward the binding of a particular peptide, and may therefore represent a simple, global strategy for converting suboptimal peptides into optimal epitopes. It could be amenable for use in protein immunizations, in an analogous fashion to the peptide immunization experiments conducted by Rosenberg et al. (18), as a DNA immunogen or used in combination with ex vivo-generated dendritic cells for adoptive immunotherapy. In considering the latter strategy, it is noteworthy that Berzofsky and colleagues have shown that the avidity and thus in vivo efficacy of CTLs are inversely related to the concentration of restimulating peptide (35, 36). Thus, maximally effective CTL responses may require exceedingly low amounts of high-affinity peptides, which may only be attainable by modifying suboptimal epitopes, such as by a ß2m-coupling approach. Furthermore, recent MHC tetramer technology has provided new opportunities to assess epitope-specific CD8+ T cell responses (37). Using an epitope-linked ß2m approach, the repertoire of MHC tetramer reagents could be increased to include low-affinity peptides that would otherwise form unstable complexes.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada (MT 6004). R.U. is a recipient of a Medical Research Council Studentship Award. Back

2 Address correspondence and reprint requests to Dr. Brian H. Barber, Department of Immunology, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Toronto, M5S 1A8 Canada. E-mail address: Back

3 Abbreviations used in this paper: hß2m, human ß2-microglobulin; NP, nucleoprotein; LDH, lactate dehydrogenase; EK, enterokinase. Back

Received for publication January 4, 1999. Accepted for publication February 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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