T cells can recognize tumor cells specifically by their TCR and the transfer of TCR-engineered T cells is a promising novel tool in anticancer therapies. We isolated and characterized four allorestricted TCRs with specificity for the HER2/neu-derived peptide 369 (HER2369) demonstrating high peptide specificity. PBMCs transduced with especially one TCR, HER2-1, mediated specific tumor reactivity after TCR optimization suggesting that this TCR represents a potential candidate for targeting HER2 by TCR-transduced effector cells. Another TCR showed high-peptide specificity without tumor reactivity. However, the TCRα-chain of this TCR specifically recognized HER2369 not only in combination with the original β-chain but also with four other β-chains of the same variable family deriving from TCRs with diverse specificities. Pairing with one β-chain derived from another HER2369-specific TCR potentiated the chimeric TCRs in regard to functional avidity, CD8 independency, and tumor reactivity. Although the frequency of such TCR single chains with dominant peptide recognition is currently unknown, they may represent interesting tools for TCR optimization resulting in enhanced functionality when paired to novel partner chains. However, undirected mispairing with novel partner chains may also result in enhanced cross-reactivity and self-reactivity. These results may have an important impact on the further design of strategies for adoptive transfer using TCR-transduced T cells.

Adoptive transfer of TCR-transduced effector cells represents an interesting novel strategy to treat a variety of viral and malignant diseases by specific targeting. This therapy has already been applied in the clinic, demonstrating feasibility of this approach (1, 2). However, the outcome of the first trial (1) showed limited antimelanoma reactivity when compared with the application of tumor-infiltrating lymphocytes potentially because of the intermediate avidity of the TCRs used for transfer. TCRs with higher avidity for a tumor-associated self-Ag might be rarely isolated from autologous T cells, but rather be selected from T cells that recognize their target peptide in the context of a foreign MHC molecule or recognize allogeneic or xenogeneic peptides (36). In fact, in the second clinical trial (2) two TCRs with specificity for MART1 and GP100 exhibiting higher avidity were transduced into PBMCs. This trial demonstrated improved response rates compared with the first trial. However, unexpected oculotoxicity and ototoxicity occurred, likely originating from specific targeting of MART1 and GP100 in these tissues. Recognition of low amounts of peptide on normal tissue therefore represents a major concern and these results emphasize the delicate choice of target Ag to be selected for this approach. Moreover, additional mechanisms may be involved in unexpected self-reactivity using TCR-transduced T cells. Introduced TCRα- and TCRβ-chains can pair with endogenous TCR chains potentially resulting in self-reactive T cells (3, 7). In addition, potential intrinsic cross-reactivity, alloreactivity, and xenoreactivity of each TCR candidate must be carefully evaluated, as these TCRs have not been negatively selected for the MHC environment of the novel host. Thus, TCRs with multiple specificities for diverse classes of tumor-associated Ags need to be investigated to further explore mechanisms involved in target specificity and potential self-reactivity to better evaluate the potential of this therapeutic approach for a broader application.

We previously identified an allorestricted T cell clone with specificity for the FMNL1-derived peptide PP2 (8). This clone recognizes the FMNL1-PP2 peptide with high specificity in the context of HLA-A2, but also demonstrates a defined cross-reactivity against an unknown peptide presented in the context of HLA-A*3303. We similarly targeted the well-described tumor-associated Ag, HER2/neu (HER2), which is overexpressed in diverse malignancies (9, 10). We isolated and characterized several allo-HLA-A2–restricted T cell clones and TCRs with specificity for the HER2-derived peptide 369 (HER2369). PBMCs transduced with these TCRs demonstrated high peptide-specificity and some tumor reactivity. One TCR specifically recognized endogenously processed HER2 representing a potential candidate for further evaluation for clinical studies. Moreover, investigation of mixed chain chimeras revealed one α-chain derived from a HER2369-specific TCR, recognizing the specific peptide in combination with β-chains derived from TCRs with diverse specificities. Importantly, one novel combination of TCRαβ-chains derived from two HER2369-specific TCRs primarily lacking tumor reactivity resulted in enhanced functional avidity, CD8 independency, and tumor-target recognition. These data may have a significant impact on the further development of adoptive T cell therapies using TCR-transgenic T cells.

PBMCs from healthy donors were collected with donors’ informed consent according to the requirements of the local ethical board and the principles expressed in the Helsinki Declaration. PBMC subpopulations from healthy donors were isolated by negative or positive magnetic bead depletion (Invitrogen, Karlsruhe, Germany), and high purity was confirmed by flow cytometric analysis. The T2 cell line that is a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (ATCC CRL-1992, Manassas, VA) has been reported to be defective in TAP molecules and to be deficient in peptide presentation (11). However, T2 cells have been demonstrated to express HLA-A2 at some level and to present a limited set of endogenous peptides (12). Peptide-pulsed T2 cells were used for priming and restimulation of HLA-A2–negative T cells (8). The TCR-deficient T cell line Jurkat76 (J76) (13) and J76 transduced with CD8α (J76CD8) cells were used for TCR-transfer experiments. The following malignant cell lines were used as targets to test tumor reactivity and cross-reactivity: HLA-A2–positive breast carcinoma cell lines MCF-7 (ATCC HTB-22) and MDA-MB 231 (CLS, Eppelheim, Germany), the HLA-A2–negative ovarian cancer cell lines SKOV3 and SKOV3 transfected with HLA-A2 (SKOV3tA2) (kindly provided by H. Bernhard, Munich, Germany) (14), the HLA-A2–positive melanoma cell lines SK-Mel 29, 624.38MEL (kindly provided by E. Noessner, Munich, Germany), wild-type (WT) K562 (ATCC CCL-243), HLA-A2–positive 143 TK lung fibroblasts (kindly provided by R. Mocikat, Munich, Germany), MRC-5 lung fibroblasts (CCL-171), and the human B cell lines C1R untransfected and transfected with HLA-A*0201 (kindly provided by S. Stevanovic, Tübingen, Germany) (15). CIR cells transfected with HLA-A*0201 and HER2, as well as fetal cardiomyocytes, were kindly provided by J. Charo (Berlin, Germany) (16).

We used the following peptides for pulsing of APCs: the HLA-A2–restricted HER2 369–377 (KIFGSLAFL) (17), single amino acid analog peptides of HER2 369–377 substituting all amino acids at all positions by either alanine or threonine, the HER1-derived peptide 364–372 (SISGDLHIL) (14), the HER3-derived peptide 356–364 (KILGNLDFL) (14), the HER4-derived peptide 361–369 (KINGNLIFL) (14), the HLA-A2–restricted influenza matrix peptide MP58 (GILGFVFTL) (18), the HLA-A2–restricted tyrosinase-derived peptide 369–377 (YMNGTMSQV) (19), the Formin-related protein in leukocytes (FMNL1)-derived HLA-A2–binding peptide PP2 (RLPERMTTL) (8), the HDAC6-derived peptide (RLAERMTTR) (8), the HLA-A2–restricted GP100-derived peptide 209–217 (ITDQVPFSV) (20), and the CMV-phosphoprotein (pp) 65-derived HLA-A2–restricted peptide 495–503 (NLVPMVATV) (21). Peptides were synthesized by standard fluorenylmethoxycarbonyl (Fmoc) synthesis (Biosyntan, Berlin, Germany). Purity was above 90% as determined by reverse phase HPLC and verified by mass spectrometry.

MHC-peptide tetramers and streptamers (multimers) detecting T cells with specificities for HLA-A2–HER2369, HLA-A2–Flu (MP58), and HLA-A2–GP100209 were synthesized as previously reported and used for detection and sorting of specific TCRs (22, 23). Streptamers were only used once for sorting of HER2369-specific T cells before T cell cloning, resulting in isolation of HER2-3. For selecting HER2369-specific T cells, multimer staining assays were performed essentially as previously described (24). The following Abs were used to characterize PBMC-derived cells, primary tumor cells, and malignant cell lines: anti-CD3–FITC (UCHT1, BD Diagnostic Systems, Heidelberg, Germany), anti-CD4–FITC (RPA-T4, BD, Heidelberg, Germany), anti-CD8–FITC (V5T-HIT8a, BD Diagnostic Systems), anti-CD8–PE and –APC (RPA-T8, BD), anti-IFN-γ–FITC (25723.11, BD Diagnostic Systems), anti-CD19–FITC and –PE (HIB19, BD Diagnostic Systems), anti-CD14–PE (M5E2, BD Diagnostic Systems), anti-CD56–PE (B159, BD Diagnostic Systems), anti-HLA-A2–FITC (BB7.2, ATCC), anti-αβ-TCR–FITC (T10B9.1A-31, BD Diagnostic Systems), anti-TCR gene variable β-chain (TRBV)12 Ab (IM1233, Beckman Coulter, Krefeld, Germany), and anti-TRBV27 Ab (CAS1.1.3, Beckman Coulter).

CTLs were generated from PBMCs using peptide-pulsed T2 cells for specific stimulation. T2 cells were pulsed with specific peptides (10−5 M and 10−7 M) and used for CTL priming at stimulator/effector cell ratios of 1:10 and for restimulation at stimulator/effector cell ratios of 1:100. Cytokines were added as follows: IL-2 (50 U/ml) (Chiron Vaccines International, Marburg, Germany), IL-7 (10 ng/ml) (Peprotech, London, U.K.), and IL-15 (10 ng/ml) (Peprotech). Peptide-specific T cells were detected by flow cytometry using PE-conjugated peptide-presenting HLA-A2 multimers and sorted by a high-performance cell sorter (MoFlo, Dako, Hamburg, Germany). Sorted cells were cloned by limiting dilution and nonspecifically restimulated every 2 wk using pooled allogeneic irradiated PBMCs, together with anti-CD3 Ab (OKT3), IL-2, IL-7, and IL-15.

Primary clones were tested for their specificity by standard 51Cr-release assay as previously described (8). TCR-transduced PBMCs were analyzed for their specificity by cytokine release. For stimulation assays, effector and target cells were incubated for 24 h at E:T ratio = 5:1 using 50,000:10,000 cells if not otherwise indicated. Supernatants were collected and the presence of IFN-γ was analyzed by ELISA (BD Diagnostics Systems), according to the recommendations of the manufacturer. In addition, the specificity of IFN-γ–producing TCR-transduced CD8+ T cells was determined by surface multimer and β-chain staining in combination with intracellular staining of IFN-γ. Effector and target cells were cocultivated at E:T ratio = 1:1 for 6 h in the presence of brefeldin A (BD Diagnostics Systems). Subsequently, TCR-transduced CD8+ T cells were at first stained with surface Ab or multimers as indicated, followed by fixation with 1% paraformaldehyde A (Sigma-Aldrich, Munich, Germany) and permeabilization with serum-supplemented PBS containing Saponin (Sigma-Aldrich). Intracellular staining of IFN-γ was performed using the anti-human IFN-γ–FITC (25723.11, BD Diagnostics Systems) Ab. Stained samples were analyzed using the LSRII flow cytometer (BD Diagnostics Systems).

TCR-PCR analysis of HER2369-specific T cell clones was performed as previously described (25). Total RNA from T cell clones and lines was extracted according to the manufacturer`s recommendation (Trizol reagent, Invitrogen). cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) and oligo(dT) primers. Subfamily-specific TCR-PCR was performed using 34 Vα and 37 Vβ primers, followed by gel isolation (NucleoSpin, Macherey-Nagel, Düren, Germany) and direct DNA sequencing of the amplified products. The TCR nomenclature was used according to International Immuno-Genetics Database (26). Sequences of isolated HER2369-specific and selected control TCR (Table II) have been deposited in Genbank, National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov/Genbank/).

Table II.
Variable and joining region family affiliation and CDR3 sequences of HER2369-specific and control TCR
CloneSpecificityTRAVTRAJCDR3NCBITRBVTRBJTRBDCDR3NCBI
HER2-1 HER2 (369) 19*01 24*02 CALYTTDSWGKLQF FJ795357 12-3*01 2-3*01  CASSFVLGDTQYF FJ795358 
HER2-2 HER2 (369) 27*01 20*01 CAGVPSNDYKLSF FJ795359 12-3*01 2-7*01 2*02 CASSPPLGSGIYEQYF FJ795360 
HER2-3 HER2 (369) 38-1*01 28*01 CAFIDSGAGSYQLTF FJ795361 7-8*01 2-7*01 2*01 CASSLAADEQYF FJ795362 
HER2-4 HER2 (369) 21*01 20*01 CAVRPQNDYKLSF FJ795363 12-3*01 2-1*01 2*01 CASSSWTSGDEQFF FJ795364 
R6C12 (33)a GP100(209) 41*01 54*01 CAASLIQGAQKLVF — 12-3*01 2-1*01 2*01 CASSPGGNEQFF — 
JG-9 (32)a pp65(495) 35*02 50*01 CAGPMKTSYDKVIF FJ795368 12-4*01 1-2*01 1*01 CASSSANYGYTF FJ795367 
SK22 (8)a FMNL1-PP2 38-2/DV8*01 41*01 CAYENSGYALNF FJ795365 27*01 2-5*01 2*01 CASSFLGETQYF FJ795366 
CloneSpecificityTRAVTRAJCDR3NCBITRBVTRBJTRBDCDR3NCBI
HER2-1 HER2 (369) 19*01 24*02 CALYTTDSWGKLQF FJ795357 12-3*01 2-3*01  CASSFVLGDTQYF FJ795358 
HER2-2 HER2 (369) 27*01 20*01 CAGVPSNDYKLSF FJ795359 12-3*01 2-7*01 2*02 CASSPPLGSGIYEQYF FJ795360 
HER2-3 HER2 (369) 38-1*01 28*01 CAFIDSGAGSYQLTF FJ795361 7-8*01 2-7*01 2*01 CASSLAADEQYF FJ795362 
HER2-4 HER2 (369) 21*01 20*01 CAVRPQNDYKLSF FJ795363 12-3*01 2-1*01 2*01 CASSSWTSGDEQFF FJ795364 
R6C12 (33)a GP100(209) 41*01 54*01 CAASLIQGAQKLVF — 12-3*01 2-1*01 2*01 CASSPGGNEQFF — 
JG-9 (32)a pp65(495) 35*02 50*01 CAGPMKTSYDKVIF FJ795368 12-4*01 1-2*01 1*01 CASSSANYGYTF FJ795367 
SK22 (8)a FMNL1-PP2 38-2/DV8*01 41*01 CAYENSGYALNF FJ795365 27*01 2-5*01 2*01 CASSFLGETQYF FJ795366 
a

These TCRs have been previously described as indicated.

NCBI, National Center for Biotechnology Information.

TCR cloning was performed as previously described (27). Shortly, the specific TCRα- and β-chain coding cDNA of the allo-HLA-A2–restricted T cell clones were amplified from isolated T cell clones using variable chain-specific oligonucleotides containing a NotI restriction site (Supplemental Table I). In addition, constant chain-specific primers containing an EcoRI restriction site were selected (Supplemental Table I). The TCR genes were first cloned separately as single TCR genes into the retroviral vector pMP71-PRE. Murinization of the constant chains (28) and codon optimization of the whole murinized TCRs (Geneart, Regensburg, Germany) (29, 30) were performed as indicated. In addition, bicistronic constructs with TCR single-chain genes separated by the picorna virus-derived peptide element P2A were cloned as previously described (31). A GFP-encoding MP71 vector was used as a mock control. All TCR cassettes used in this study were verified by sequence analysis (Eurofins, Ebersberg, Germany). The following TCRs were used as control TCRs: The FMNL1-specific TCR SK22 (8), the CMV-specific TCR JG-9 with specificity for the CMVpp65-derived HLA-A2–restricted peptide NLVPMVATV (32) and the previously described GP100209-specific TCR derived from clone R6C12 (33).

The TCR-containing retroviral vector plasmids pMP71-TCR-PRE were cotransfected with plasmids harboring retroviral genes for gag/pol derived from Moloney murine leukemia virus (pcDNA3.1-Mo-MLV) and env (pALF-10A1) into 293 T cells by calcium phosphate precipitation to generate amphotropic vector particles (31). PBMCs were activated for 2–3 d with IL-2 (50 U/ml) and OKT3 (50 ng/ml), whereas sorted CD8+ and CD4+ cells were stimulated with IL-2 (50 U/ml), plate-coated OKT3 (5 μg/ml), and CD28 mAb (clone CD28.2, BD, 1 μg/ml). Activated cells were transduced twice with retrovirus-containing supernatant in 24-well nontissue culture plates coated with RetroNectin (Takara, Apen, Germany) containing protamine sulfate (4 μg/ml) and IL-2 (100 U/ml). After addition of retroviral supernatant, the plates were spinoculated with 800 × g for 1.5 h at 32°C. Medium was replaced by fresh medium 24 h after second transduction. Transduced PBMCs were cultured with low-dose IL-2 (50 U/ml) every 3 d and analyzed for multimer staining and surface markers, as well as functional assays at different time points after transduction as indicated. PBMCs transduced with a GFP-containing MP71 vector were used as mock control. For analysis of CD8-depleted cell populations after TCR-transfer, cells were stained with anti-CD8 and then sorted by the high-performance cell sorter (MoFlo; Dako) by negative selection. TCR-transduced PBMCs were depleted from CD8+ cells by flow cytometry sorting where indicated and restimulated with OKT3 (30 ng/ml), IL-2 (50 U/ml), IL-7 (5 ng/ml), and IL-15 (5 ng/ml) as well as allogeneic PBMCs pooled from three different donors. Cells were used for further analysis 2 wk after sorting (4 wk after transduction). In addition, TCR-transduced T cells were cloned by limiting dilution 12 d after transduction and phenotypically, as well as functionally, analyzed.

Allo–HLA-A2–restricted T cells specific for HER2369 were isolated by MHC-peptide multimers after stimulation of HLA-A2–CD8+ T cells with peptide-pulsed T2 cells (22). A total of 0.05–0.8% of cells stained positive for the specific HER2369 multimer after stimulation and before sorting (data not shown). HER2369 multimer+ cells were then enriched by flow cytometric sorting and cloned by limiting dilution. A total of 33 different clones with peptide specificity for HER2369 were generated in three independent stimulation experiments using T cells derived from two different healthy donors. Most clones could not be cultured over a longer period preventing further extensive functional testing. However, RNA from four clones with high HER2369 specificity but low background reactivity against irrelevant peptides (Table I) was harvested and the TCR repertoire of these selected clones was determined (Table II). Interestingly, three of these four clones (HER2-1, HER2-2, and HER2-4) used a TCRβ-chain of the TRBV12-3 family (Table II).

Table I.
Peptide recognition and tumor reactivity of allorestricted HER2369-specific T-cell clones
DonorCloneCytotoxicity (%)
T2 + HER2369T2 + FluSK-Mel29
HER2-1 65 ± 6.2 8 ± 7.6 21 ± 8.2 
HER2-2 60 ± 4.7 0 ± 0.4 1 ± 0.9 
HER2-3 64 ± 6.5 6 ± 0.9 29 ± 1.3 
HER2-4 79 ± 4.2 2 ± 2.1 1 ± 0.0 
DonorCloneCytotoxicity (%)
T2 + HER2369T2 + FluSK-Mel29
HER2-1 65 ± 6.2 8 ± 7.6 21 ± 8.2 
HER2-2 60 ± 4.7 0 ± 0.4 1 ± 0.9 
HER2-3 64 ± 6.5 6 ± 0.9 29 ± 1.3 
HER2-4 79 ± 4.2 2 ± 2.1 1 ± 0.0 

The TCRα- and β-chain genes derived from these four different clones demonstrating peptide-specificity were used for cloning of TCR chain genes for transfer studies (Table II). In addition, we used the GP100209-specific TCR derived from clone R6C12 (33), a CMV-pp65495–specific TCR (JG-9) (32), and the FMNL1-PP2–specific TCR SK22 (8) as control TCR with defined specificities for other Ags than HER2 (Table II). Retroviral TCR gene transfer with unmodified TCRα- and β-chain genes using single TCR chain vectors into TCR knockout J76CD8 cells or PBMCs resulted in cells positive for the specific MHC-peptide multimer but negative for control multimer (Fig. 1A, 1B). TCR HER2-1 was the only HER2369-specific TCR demonstrating a significant percentage of multimer+ cells in the CD8 population (Fig. 1B) corresponding to multimer positivity of HER2-1–transduced J76 lacking CD8 (data not shown). Although the percentage of multimer+ cells was overall low, we observed high expression of the specific TRBV12 chain in CD4+ and CD8+ populations indicating efficient transduction and suggesting that either transduced TCRs are not well enough assembled to be detected by the specific multimer or that mispairing with endogeneous α-chains occurs. PBMCs transduced with unmodified TCR chain pairs from HER2369-specific T cell clones, as well as the control TCR R6C12, exerted reactivity toward the specific peptide in a dose-dependent manner (Fig. 2A, 2B). All transduced TCR demonstrated high-peptide specificity and did not respond to a panel of irrelevant peptides (Fig. 2C). Analyzing the tumor reactivity of PBMCs transduced with diverse HER2369-specific TCRs, we observed mainly tumor reactivity of HER2-3 against diverse tumor cell lines as MCF-7 and MDA-MB 231, as well as marginal tumor reactivity of HER2-1 against SK-Mel 29 (Fig. 2D).

FIGURE 1.

HER2369-specific TCRs are expressed after retroviral gene transfer. A, TCRα- and β-chain genes of the HER2369-specific TCR HER2-1, HER2-2, HER2-3, and HER2-4 as well as the control TCR R6C12 with specificity for GP100209 were retrovirally transduced into J76CD8 and analyzed by flow cytometry 4 d after transduction. Transduced cells were analyzed for specific TCR expression by staining with the specific multimer (thick line) as well as control multimers (thin line). B, Similarly, single TCR chains of HER2369-specific TCR and control TCR were retrovirally transduced into PBMCs and stained with the specific multimer (first panel), the control multimer (middle panel), as well as the specific TRBV Ab (as available) 4 d after transduction. Nontransduced PBMC were used as control.

FIGURE 1.

HER2369-specific TCRs are expressed after retroviral gene transfer. A, TCRα- and β-chain genes of the HER2369-specific TCR HER2-1, HER2-2, HER2-3, and HER2-4 as well as the control TCR R6C12 with specificity for GP100209 were retrovirally transduced into J76CD8 and analyzed by flow cytometry 4 d after transduction. Transduced cells were analyzed for specific TCR expression by staining with the specific multimer (thick line) as well as control multimers (thin line). B, Similarly, single TCR chains of HER2369-specific TCR and control TCR were retrovirally transduced into PBMCs and stained with the specific multimer (first panel), the control multimer (middle panel), as well as the specific TRBV Ab (as available) 4 d after transduction. Nontransduced PBMC were used as control.

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FIGURE 2.

HER2369-specific TCRs show peptide-specific function after retroviral gene transfer. PBMCs transduced with the HER2369-specific TCR as well as the control TCR R6C12 were incubated 11 d after transduction for 24 h with T2 cells pulsed with a range of titrated concentrations of HER2369 (A), GP100209 (B), or a panel of control peptides at 10 μM (C). Selected tumor cell lines were used as target cells for TCR-transduced PBMCs (D). Tumor target cells were treated with IFN-γ (100 U/ml) 48 h prior to the stimulation assay. Supernatants were analyzed by IFN-γ ELISA. The numbers in brackets indicate the percentage of cells stained positive with the specific multimer. SDs of triplicates are shown.

FIGURE 2.

HER2369-specific TCRs show peptide-specific function after retroviral gene transfer. PBMCs transduced with the HER2369-specific TCR as well as the control TCR R6C12 were incubated 11 d after transduction for 24 h with T2 cells pulsed with a range of titrated concentrations of HER2369 (A), GP100209 (B), or a panel of control peptides at 10 μM (C). Selected tumor cell lines were used as target cells for TCR-transduced PBMCs (D). Tumor target cells were treated with IFN-γ (100 U/ml) 48 h prior to the stimulation assay. Supernatants were analyzed by IFN-γ ELISA. The numbers in brackets indicate the percentage of cells stained positive with the specific multimer. SDs of triplicates are shown.

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To improve expression and reactivity of the three TCRs with the highest HER2369 reactivity, we introduced modifications into TCR constructs of HER2-1, HER2-2, and HER2-3. We first murinized constant chains as previously described (28). We additionally performed codon optimization and cloned both TCRβ- and α-chain genes in one vector separated by the picorna virus-derived peptide element P2A as previously described (29, 31). These modifications improved multimer staining and reduced expression of the specific Vβ chain indicating that these modifications resulted in a reduction of mispairing (Table III). Moreover, these modifications improved peptide-specific function (Fig. 3A, 3B) of TCR HER2-1 and HER2-2. Functional avidity of PBMCs transduced with HER2-1 was increased by these modifications when compared with transduction of unmodified chain genes (Fig. 3A). In addition, these modifications improved tumor reactivity of HER2-1, but not HER2-2 and HER2-3 (Fig. 3C). Modification of TCR HER2-3 revealed only improvement of multimer staining, but not HER2369-specific function and tumor reactivity (Fig. 3A–C). HLA-A2 and HLA-A2+ PBMCs transduced with this TCR showed reduced proliferation (data not shown) indicating a more general alloreactive potential of this TCR.

Table III.
Percentage of specific multimer+ and TRBV12+PBMCs after retroviral transduction with vector constructs containing either WT or modified TCR chain genes
Day 4
Day 11
TCRMultimer+ (%)TRBV12+ (%)GFP+ (%)Multimer+ (%)
HER2-1 WT 4.8 30.4  6.1 
Mod 14.1 19.9  15.6 
HER2-2 WT 5.6 29.3  6.0 
Mod 8.8 17.3  16.2 
HER2-3 WT 5.3 ND  9.6 
Mod 7.3 ND  20.3 
HER2-4 WT 2.7 27.9  3.7 
Mod ND ND  ND 
R6C12 WT 6.7 49.5  8.8 
Mod 9.1 21.5  13.8 
Nontransduced  0.2 4.5  0.3 
Mock  ND ND 31.3 ND 
Day 4
Day 11
TCRMultimer+ (%)TRBV12+ (%)GFP+ (%)Multimer+ (%)
HER2-1 WT 4.8 30.4  6.1 
Mod 14.1 19.9  15.6 
HER2-2 WT 5.6 29.3  6.0 
Mod 8.8 17.3  16.2 
HER2-3 WT 5.3 ND  9.6 
Mod 7.3 ND  20.3 
HER2-4 WT 2.7 27.9  3.7 
Mod ND ND  ND 
R6C12 WT 6.7 49.5  8.8 
Mod 9.1 21.5  13.8 
Nontransduced  0.2 4.5  0.3 
Mock  ND ND 31.3 ND 

WT, Wild type usage of vector constructs containing unmodified TCR chain genes; Mod, Modified usage of bicistronic vector constructs containing TCRα- and β-chain genes with murinized constant chains and codon optimization.

FIGURE 3.

PBMCs transduced with modified TCR constructs show enhanced functions with preserved peptide specificity. PBMCs transduced with either single TCR chains (WT) or modified constructs (modified) were stimulated 11 d after transduction for 24 h with target cells at E:T ratios of 5:1. Supernatants were then harvested and analyzed by IFN-γ ELISA. The percentage of multimer+ cells in the effector cell population is shown in Table III. Nontransduced PBMCs as well as mock-transduced PBMCs were used as controls. SDs of triplicates are shown. A, TCR-transduced PBMCs were tested against T2 cells pulsed with a range of titrated concentrations of specific peptide. HER2369 was used for TCR HER2-1, HER2-2, and HER2-3; GP100209 was used for TCR R6C12. B, TCR-transduced PBMCs were tested against T2 cells pulsed with a set of alternative peptides at a concentration of 10−5 M. C, TCR-transduced PBMCs were tested against selected tumor cell lines. Tumor target cells were treated with IFN-γ (100 U/ml) 48 h prior to the stimulation assay.

FIGURE 3.

PBMCs transduced with modified TCR constructs show enhanced functions with preserved peptide specificity. PBMCs transduced with either single TCR chains (WT) or modified constructs (modified) were stimulated 11 d after transduction for 24 h with target cells at E:T ratios of 5:1. Supernatants were then harvested and analyzed by IFN-γ ELISA. The percentage of multimer+ cells in the effector cell population is shown in Table III. Nontransduced PBMCs as well as mock-transduced PBMCs were used as controls. SDs of triplicates are shown. A, TCR-transduced PBMCs were tested against T2 cells pulsed with a range of titrated concentrations of specific peptide. HER2369 was used for TCR HER2-1, HER2-2, and HER2-3; GP100209 was used for TCR R6C12. B, TCR-transduced PBMCs were tested against T2 cells pulsed with a set of alternative peptides at a concentration of 10−5 M. C, TCR-transduced PBMCs were tested against selected tumor cell lines. Tumor target cells were treated with IFN-γ (100 U/ml) 48 h prior to the stimulation assay.

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As the optimized TCR HER2-1 demonstrated the most specific reactivity pattern, including evidence of multimer+ cells in the CD4+ and CD8+ T cell population as well as potent antitumor reactivity, we analyzed T cell populations transduced with the optimized TCR HER2-1 to further test specificity and tumor reactivity of this TCR (Figs. 4, 5). In contrast to a previous report about a HER2369-specific allorestricted T cell clone (14), TCR HER2-1 specifically recognized T2 cells pulsed with HER2369 but not HER1364, HER3356, or HER4361 (Fig. 4). We additionally used single-peptide analoga substituting all amino acids of HER2369 by either alanine or threonine. Although exchange of most central amino acids by threonine mainly abolished recognition by this TCR, several exchanges by alanine were tolerated. However, several peptide amino acid positions as 3 aa (phenylalanine), 6 aa (leucine), and 8 aa (phenylalanine) were essential for target recognition (Fig. 4). To analyze the specificity for endogenously presented HER2, we analyzed the reactivity against C1R cells transfected with HLA-A2 and HER2. CD8+ T cells transduced with HER2-1 showed specific IFN-γ–secretion in response to HLA-A2+ C1R cells transfected with HER2 (Fig. 5A). Moreover, HER2-multimer+ T cell clones generated from HER2-1–transduced T cell lines preferentially recognized C1R cells transfected with HLA-A2 and HER2 (Fig. 5B). Specificity of IFN-γ expression in bulk cultures was further analyzed by investigation of the presence of intracellular IFN-γ in combination with the expression of the specific TCR or β-chain indicating preferential recognition of C1R cells transfected with HLA-A2 and HER2 (Fig. 5C). Moreover, MCF-7 cells were specifically recognized by multimer+ cells. Limited recognition of HLA-A2+ C1R cells by TCR-transduced CD8+ cells as investigated by intracellular IFN-γ staining may indicate alloreactive recognition of HLA-A2+. We therefore investigated the recognition of a panel of nontransformed HLA-A2+ targets as lung fibroblasts, fetal cardiomyocytes, PBMC subpopulations, and activated PBMCs demonstrating no major recognition by CD4+ or CD8+ populations transduced with TCR HER2-1 as analyzed by IFN-γ ELISA (Fig. 5D).

FIGURE 4.

Detailed peptide-specificity of PBMC populations transduced with the modified construct of TCR HER2-1. Sorted CD4+ and CD8+ cell populations transduced with TCR HER2-1 were tested for their reactivity against T2 cells pulsed with the following peptides at concentrations of 10−5 M: HER2369 as well as single amino acid analogs of HER2369 using alanine and threonine for substitution, HER1364, HER3356, HER4361, and Flu (E:T = 5:1). Supernatants were harvested after 24 h and analyzed by IFN-γ ELISA. SDs of triplicates are shown. Nontransduced PBMCs (data not shown) as well as mock-transduced PBMCs were used as controls. The number in brackets indicate the percentage of multimer+ cells in the given cell population. Transduction efficiencies were determined 4 d after transduction by multimer staining, TRBV12 staining, and GFP (mock control) as shown in Supplemental Table II.

FIGURE 4.

Detailed peptide-specificity of PBMC populations transduced with the modified construct of TCR HER2-1. Sorted CD4+ and CD8+ cell populations transduced with TCR HER2-1 were tested for their reactivity against T2 cells pulsed with the following peptides at concentrations of 10−5 M: HER2369 as well as single amino acid analogs of HER2369 using alanine and threonine for substitution, HER1364, HER3356, HER4361, and Flu (E:T = 5:1). Supernatants were harvested after 24 h and analyzed by IFN-γ ELISA. SDs of triplicates are shown. Nontransduced PBMCs (data not shown) as well as mock-transduced PBMCs were used as controls. The number in brackets indicate the percentage of multimer+ cells in the given cell population. Transduction efficiencies were determined 4 d after transduction by multimer staining, TRBV12 staining, and GFP (mock control) as shown in Supplemental Table II.

Close modal
FIGURE 5.

Specified target recognition of PBMC populations transduced with the modified construct of TCR HER2-1. A, HER2-1–transduced CD4+ and CD8+ cells were stimulated 11 d after transduction for 24 h with WT C1R cells, C1R cells transfected with HLA-A2 as well as HLA-A2 and HER2. Standard deviations of triplicates are shown (E:T = 5:1). B, CD8+ cells transduced with HER2-1 were cloned by limited dilution and growing T cell clones were analyzed with flow cytometry for specific multimer binding. The mean fluorescence intensity is shown for selected clones. Selected clones were analyzed for their HER2369-specificity with specified target cells at E:T ratios of 1:5. SDs of duplicates are shown. C, HER2-1–transduced CD8+ cells were cocultivated 12 d after transduction with indicated target cells (E:T = 1:1). Cells were harvested after 6 h and stained with CD8, TCRBV12-specific Abs as well as specific and control multimers. Cells were then fixed and permeabilized, followed by treatment with anti-human IFN-γ Ab and analysis by flow cytometry. IFN-γ secretion of CD8-gated cells is shown. D, HER2-1–transduced CD4+ and CD8+ cells were stimulated 11 d after transduction with HER2369-pulsed T2 cells as positive control, lung fibroblasts, fetal cardiomyocytes, CD4+ and CD8+ sorted PBMCs as well as PBMCs stimulated with OKT3 and IL-2 (E:T = 5:1). SDs of duplicates are shown. For IFN-γ ELISA analysis, supernatants were harvested after 24 h and then analyzed. Nontransduced PBMCs as well as mock-transduced PBMCs were used as controls. The number in brackets indicate the percentage of multimer+ cells in the given cell population. Transduction efficiencies were determined at day 4 after transduction by multimer staining, TRBV12 staining, and GFP (mock control) and are shown in Supplemental Table II.

FIGURE 5.

Specified target recognition of PBMC populations transduced with the modified construct of TCR HER2-1. A, HER2-1–transduced CD4+ and CD8+ cells were stimulated 11 d after transduction for 24 h with WT C1R cells, C1R cells transfected with HLA-A2 as well as HLA-A2 and HER2. Standard deviations of triplicates are shown (E:T = 5:1). B, CD8+ cells transduced with HER2-1 were cloned by limited dilution and growing T cell clones were analyzed with flow cytometry for specific multimer binding. The mean fluorescence intensity is shown for selected clones. Selected clones were analyzed for their HER2369-specificity with specified target cells at E:T ratios of 1:5. SDs of duplicates are shown. C, HER2-1–transduced CD8+ cells were cocultivated 12 d after transduction with indicated target cells (E:T = 1:1). Cells were harvested after 6 h and stained with CD8, TCRBV12-specific Abs as well as specific and control multimers. Cells were then fixed and permeabilized, followed by treatment with anti-human IFN-γ Ab and analysis by flow cytometry. IFN-γ secretion of CD8-gated cells is shown. D, HER2-1–transduced CD4+ and CD8+ cells were stimulated 11 d after transduction with HER2369-pulsed T2 cells as positive control, lung fibroblasts, fetal cardiomyocytes, CD4+ and CD8+ sorted PBMCs as well as PBMCs stimulated with OKT3 and IL-2 (E:T = 5:1). SDs of duplicates are shown. For IFN-γ ELISA analysis, supernatants were harvested after 24 h and then analyzed. Nontransduced PBMCs as well as mock-transduced PBMCs were used as controls. The number in brackets indicate the percentage of multimer+ cells in the given cell population. Transduction efficiencies were determined at day 4 after transduction by multimer staining, TRBV12 staining, and GFP (mock control) and are shown in Supplemental Table II.

Close modal

By investigating the TCR repertoire of selected T cell clones with specificity for HER2369, we detected a preferential usage of β-chains of the TRBV12 family in these clones (Table II and data not shown). Restricted usage of variable chains involved in detection of a defined Ag has been previously described (34, 35). Moreover, it has been previously reported that the usage of mixed TCR chain combinations derived from diverse TCR with a defined Ag-specificity resulted again in the defined Ag-specificity (13). We therefore analyzed the capability of chimeric chain combinations to form TCRs with specificity for HER2369. We transduced J76CD8 and PBMCs with random combinations of unmodified TCR single chains derived from diverse TCR with specificity for HER2 but also alternative specificities and investigated these cells for TCR expression and specific multimer binding. All combinations showed surface expression of TCR or CD3 in transduced J76CD8 indicating that the TCR single chains easily mispair with novel partner TCR chains (data not shown). Interestingly, combinations of HER2-2α with diverse β-chains belonging to the TRBV12 family resulted in HER2369-multimer+ cells, whereas this was not observed using combinations with β-chains of other TRBV families (Table IV and Fig. 6A). Two of the β-chains belonging to the TRBV12 family were derived from receptors with specificities other than HER2369, namely, GP100209 (R6C12) and CMV-pp65495 (JG-9). PBMCs transduced with HER2-2α in combination with these TCRβ-chains did not stain positive with multimers specific for the original TCRs, HLA-A2–GP100209 or HLA-A2–CMV-pp65495, respectively (Fig. 6A). Moreover, transduction of single TCR HER2-2α-chain in PBMCs resulted in a slight increase of HER2369-multimer+ cells (Fig. 6A, 6B). These HER2369-multimer+ cells stained mainly concomitantly positive for anti-TRBV12 (Fig. 6B). Interestingly, combinations of HER2-2α with HER2-4β as well as R6C12β resulted in HER2369-multimer binding of TCR-chain–transduced CD8- PBMCs suggesting that specific multimer staining of these chimeric TCRs was less dependent on CD8 (Fig. 6A). Murinization of TCR constant chains, cloning of HER2-2α and HER2-1β or R6C12β in a bicistronic vector and codon optimization of chimeric TCR chain genes was performed on selected chain genes of chimeric TCRs and resulted in efficient HER2369-multimer binding of surface expressed chimeric TCRs after transduction in PBMCs.

Table IV.
Specific HER2369-multimer staining after transduction of chimeric TCR combinations into J76CD8 cells
HER2369 Multimer+HER2-1 TRBV 12-3*01HER2-2 TRBV 12-3*01HER2-3 TRBV 7-8*01HER2-4 TRBV 12-3*01R6C12 TRBV 12-3*01JG-9 TRBV 12-4*01SK22 TRBV 27*01
HER2-1 TRAV19*01 − − ND − ND ND 
HER2-2 TRAV27*01 − − 
R6C12 TRAV41*01 − − ND ND − ND ND 
HER2369 Multimer+HER2-1 TRBV 12-3*01HER2-2 TRBV 12-3*01HER2-3 TRBV 7-8*01HER2-4 TRBV 12-3*01R6C12 TRBV 12-3*01JG-9 TRBV 12-4*01SK22 TRBV 27*01
HER2-1 TRAV19*01 − − ND − ND ND 
HER2-2 TRAV27*01 − − 
R6C12 TRAV41*01 − − ND ND − ND ND 

(+) indicates specific HER2369-multimer recognition.

(−) indicates no HER2369-multimer recognition.

FIGURE 6.

Transduction of PBMCs with HER2-2α alone or in combination with β-chains derived from the TRBV12 family with diverse specificities results in significant HER2369-specific multimer binding. A, PBMCs transduced with TCR chain genes of HER2-2α alone or in combination with β-chains derived from six different receptors with diverse specificities were stained 10 d after transduction with HER2369-specific multimer and control multimer (left and right data sets). The specific multimers used are indicated in brackets above the graphs. B, PBMCs transduced with HER2-2α alone, HER2-2α/HER2-2β, and nontransduced PBMCs were stained with the HER2369-multimer and anti-TRBV12 Ab at day 4 and day 10 after transduction and analyzed by flow cytometry.

FIGURE 6.

Transduction of PBMCs with HER2-2α alone or in combination with β-chains derived from the TRBV12 family with diverse specificities results in significant HER2369-specific multimer binding. A, PBMCs transduced with TCR chain genes of HER2-2α alone or in combination with β-chains derived from six different receptors with diverse specificities were stained 10 d after transduction with HER2369-specific multimer and control multimer (left and right data sets). The specific multimers used are indicated in brackets above the graphs. B, PBMCs transduced with HER2-2α alone, HER2-2α/HER2-2β, and nontransduced PBMCs were stained with the HER2369-multimer and anti-TRBV12 Ab at day 4 and day 10 after transduction and analyzed by flow cytometry.

Close modal

In parallel to the specific multimer binding, combinations of HER2-2α with different β-chains of the TRBV12 family displayed specific reactivity for T2 cells pulsed with HER2369 in a dose-dependent manner (Fig. 7A, Table V). Whereas chimeric TCR combinations of HER2-2α with HER2-1β or JG-9β resulted in only marginal recognition of T2 cells pulsed with high concentrations of HER2369 (Table V), combinations of HER2-2α with R6C12β but especially HER2-4β resulted in an increased functional avidity in peptide titration experiments when compared with the original chain combination (Fig. 7A). Moreover, the combination of HER2-2α and HER2-4β resulted in reactivity against diverse tumor cell lines (Fig. 7B, Table V), although the original TCR chain combinations HER2-2αβ and HER2-4αβ showed no tumor reactivity. The combination of HER2-2α and HER2-4β demonstrated high-peptide specificity for HER2369 but also revealed increased reactivity in response to T2 cells pulsed with alternative peptides or unloaded T2 cells (Table V).

FIGURE 7.

Enhanced peptide-specific functional avidity as well as recognition of natural targets by PBMC populations transduced with novel chimeric TCR chain combinations of HER2-2α with HER2-4β and R6C12β. A, PBMCs transduced with TCR chain genes of HER2-2α in combination with β-chains derived from HER2-4β and R6C12β, as well as the native TCR combinations of HER2-2αβ and HER2-4αβ, were stimulated with T2 cells pulsed with a range of titrated concentrations of HER2369 at E:T ratios of 5:1. Supernatants were harvested after 24 h and analyzed by IFN-γ ELISA. The percentage of maximum IFN-γ release and the SD50 are shown. B and C, PBMCs (B) and CD8-depleted populations (C) transduced with TCR chain genes of HER2-2α in combination with β-chains derived from HER2-4β and R6C12β were stimulated 11 d (B) and 25 d (C) after TCR-transduction with diverse target cells at E:T ratios of 5:1. The percentage of multimer+ cells in the effector cell population is indicated. Nontransduced PBMCs as well as mock-transduced PBMCs were used as controls. SDs of triplicates are shown. *Usage of optimized constructs. D, Proliferation of total cell counts (upper panel) and multimer+ cells (lower panel) of CD4+ and CD8+ cells of HLA-A2 (left four panels) and HLA-A2+ (right four panels) donors are shown after transduction with the chimeric TCR combination HER2-2α/HER2-4β as well as HER2-1. Mock- and nontransduced cells were used as controls. Transduction efficiencies were primarily determined at day 4 after transduction by multimer staining, TRBV12 staining, and GFP (mock control) and are shown in Supplemental Table III.

FIGURE 7.

Enhanced peptide-specific functional avidity as well as recognition of natural targets by PBMC populations transduced with novel chimeric TCR chain combinations of HER2-2α with HER2-4β and R6C12β. A, PBMCs transduced with TCR chain genes of HER2-2α in combination with β-chains derived from HER2-4β and R6C12β, as well as the native TCR combinations of HER2-2αβ and HER2-4αβ, were stimulated with T2 cells pulsed with a range of titrated concentrations of HER2369 at E:T ratios of 5:1. Supernatants were harvested after 24 h and analyzed by IFN-γ ELISA. The percentage of maximum IFN-γ release and the SD50 are shown. B and C, PBMCs (B) and CD8-depleted populations (C) transduced with TCR chain genes of HER2-2α in combination with β-chains derived from HER2-4β and R6C12β were stimulated 11 d (B) and 25 d (C) after TCR-transduction with diverse target cells at E:T ratios of 5:1. The percentage of multimer+ cells in the effector cell population is indicated. Nontransduced PBMCs as well as mock-transduced PBMCs were used as controls. SDs of triplicates are shown. *Usage of optimized constructs. D, Proliferation of total cell counts (upper panel) and multimer+ cells (lower panel) of CD4+ and CD8+ cells of HLA-A2 (left four panels) and HLA-A2+ (right four panels) donors are shown after transduction with the chimeric TCR combination HER2-2α/HER2-4β as well as HER2-1. Mock- and nontransduced cells were used as controls. Transduction efficiencies were primarily determined at day 4 after transduction by multimer staining, TRBV12 staining, and GFP (mock control) and are shown in Supplemental Table III.

Close modal
Table V.
Peptide and tumor recognition of PBMCs transduced with HER2-2α in combination with different TCR β-chains
α-Chain
HER2-2 AV27
β-Chain (HER2369- Multimer+%) HER2-1 BV12 (10.5)a HER2-2 BV12 (16.2)a HER2-3 BV7 (0.2)a HER2-4 BV12 (10.8) R6C12 BV12 (13.3)a JG-9 BV12 (5.2) Mock Nontransduced 
T2 + 10−5 M HER2369 99 (2)b 2697 (123) 74 (0) 3624 (108) 2899 (132) 111 (8) 48 (3) 33 (5) 
T2 + 10−6 M HER2369 86 (4) 2094 (93) 57 (3) 3924 (81) 2202 (63) 68 (3) 42 (9) 39 (8) 
T2 + 10−7 M HER2369 53 (8) 1114 (40) 52 (3) 3261 (51) 1436 (4) 62 (17) 41 (4) 37 (3) 
T2 + 10−8 M HER2369 60 (10) 276 (2) 45 (2) 2129 (21) 573 (16) 43 (1) 61 (5) 30 (12) 
T2 + 10−9 M HER2369 35 (5) 175 (0) 53 (1) 1208 (42) 265 (19) 52 (18) 47 (8) 20 (3) 
T2 + 10−10 M HER2369 36 (10) 158 (19) 38 (1) 826 (7) 208 (4) 49 (8) 28 (1) 28 (3) 
T2 + 10−11 M HER2369 70 (7) 147 (7) 42 (6) 754 (5) 194 (11) 42 (4) 31 (3) 24 (7) 
T2 unpulsed 32 (2) 183 (123) 44 (4) 820 (13) 236 (6) 51 (3) 35 (5) 25 (5) 
T2 + GP100209 48 (1) 163 (4) 49 (2) 911 (70) 228 (3) 61 (2) 41 (7) 31 (8) 
T2 + pp65495 38 (9) 155 (6) 62 (8) 650 (50) 182 (7) 53 (4) 35 (15) 31 (8) 
T2 + Flu58 45 (2) 168 (32) 50 (3) 813 (44) 207 (0) 61 (0) 40 (2) 89 (12) 
T2 + FMNL1-PP2 42 (1) 140 (6) 46 (4) 870 (131) 225 (1) 57 (2) 33 (7) 92 (20) 
T2 + HDAC6862 56 (21) 142 (24) 40 (3) 721 (73) 221 (6) 48 (11) 36 (2) 65 (0) 
T2 + Tyrosinase369 66 (4) 139 (2) 45 (2) 951 (74) 181 (1) 49 (9) 37 (3) 51 (7) 
MCF-7 (A2+, HER2+66 (9) 112 (9) 82 (4) 1094 (13) 134 (0) 67 (10) 168 (10) 95 (11) 
MDA-MB-231 (A2+, HER2+34 (4) 29 (2) 35 (2) 2412 (33) 56 (0) 38 (0) 60 (0) 84 (3) 
SKOV3 tA2 (A2+, HER2+40 (1) 51 (4) 67 (4) 413 (17) 85 (7) 37 (4) 92 (4) 98 (9) 
SKOV3 (A2, HER2+31 (4) 64 (3) 54 (0) 82 (0) 63 (5) 47 (0) 19 (5) 32 (6) 
SK-Mel 29 (A2+, HER2+91 (0) 100 (4) 133 (1) 2716 (238) 169 (2) 94 (18) 132 (3) 114 (6) 
624.38 MEL (A2+, HER2+61 (0) 61 (4) 37 (1) 709 (10) 102 (4) 95 (4) 69 (6) 32 (7) 
K562 WT (A2, HER226 (0) 14 (2) 8 (23) 10 (4) 9 (4) 15 (2) 14 (1) 22 (1) 
Medium 21 (2) 24 (1) 25 (5) 21 (5) 10 (7) 18 (4) 21 (5) 4 (0) 
α-Chain
HER2-2 AV27
β-Chain (HER2369- Multimer+%) HER2-1 BV12 (10.5)a HER2-2 BV12 (16.2)a HER2-3 BV7 (0.2)a HER2-4 BV12 (10.8) R6C12 BV12 (13.3)a JG-9 BV12 (5.2) Mock Nontransduced 
T2 + 10−5 M HER2369 99 (2)b 2697 (123) 74 (0) 3624 (108) 2899 (132) 111 (8) 48 (3) 33 (5) 
T2 + 10−6 M HER2369 86 (4) 2094 (93) 57 (3) 3924 (81) 2202 (63) 68 (3) 42 (9) 39 (8) 
T2 + 10−7 M HER2369 53 (8) 1114 (40) 52 (3) 3261 (51) 1436 (4) 62 (17) 41 (4) 37 (3) 
T2 + 10−8 M HER2369 60 (10) 276 (2) 45 (2) 2129 (21) 573 (16) 43 (1) 61 (5) 30 (12) 
T2 + 10−9 M HER2369 35 (5) 175 (0) 53 (1) 1208 (42) 265 (19) 52 (18) 47 (8) 20 (3) 
T2 + 10−10 M HER2369 36 (10) 158 (19) 38 (1) 826 (7) 208 (4) 49 (8) 28 (1) 28 (3) 
T2 + 10−11 M HER2369 70 (7) 147 (7) 42 (6) 754 (5) 194 (11) 42 (4) 31 (3) 24 (7) 
T2 unpulsed 32 (2) 183 (123) 44 (4) 820 (13) 236 (6) 51 (3) 35 (5) 25 (5) 
T2 + GP100209 48 (1) 163 (4) 49 (2) 911 (70) 228 (3) 61 (2) 41 (7) 31 (8) 
T2 + pp65495 38 (9) 155 (6) 62 (8) 650 (50) 182 (7) 53 (4) 35 (15) 31 (8) 
T2 + Flu58 45 (2) 168 (32) 50 (3) 813 (44) 207 (0) 61 (0) 40 (2) 89 (12) 
T2 + FMNL1-PP2 42 (1) 140 (6) 46 (4) 870 (131) 225 (1) 57 (2) 33 (7) 92 (20) 
T2 + HDAC6862 56 (21) 142 (24) 40 (3) 721 (73) 221 (6) 48 (11) 36 (2) 65 (0) 
T2 + Tyrosinase369 66 (4) 139 (2) 45 (2) 951 (74) 181 (1) 49 (9) 37 (3) 51 (7) 
MCF-7 (A2+, HER2+66 (9) 112 (9) 82 (4) 1094 (13) 134 (0) 67 (10) 168 (10) 95 (11) 
MDA-MB-231 (A2+, HER2+34 (4) 29 (2) 35 (2) 2412 (33) 56 (0) 38 (0) 60 (0) 84 (3) 
SKOV3 tA2 (A2+, HER2+40 (1) 51 (4) 67 (4) 413 (17) 85 (7) 37 (4) 92 (4) 98 (9) 
SKOV3 (A2, HER2+31 (4) 64 (3) 54 (0) 82 (0) 63 (5) 47 (0) 19 (5) 32 (6) 
SK-Mel 29 (A2+, HER2+91 (0) 100 (4) 133 (1) 2716 (238) 169 (2) 94 (18) 132 (3) 114 (6) 
624.38 MEL (A2+, HER2+61 (0) 61 (4) 37 (1) 709 (10) 102 (4) 95 (4) 69 (6) 32 (7) 
K562 WT (A2, HER226 (0) 14 (2) 8 (23) 10 (4) 9 (4) 15 (2) 14 (1) 22 (1) 
Medium 21 (2) 24 (1) 25 (5) 21 (5) 10 (7) 18 (4) 21 (5) 4 (0) 
a

Indicates usage of TCR constructs modified by codon optimization, usage of murinized constant chains, and bicistronic vectors.

b

SDs of triplicates are shown in parentheses.

In parallel to the CD8-independent multimer staining of chimeric TCR combinations, mixed combinations of HER2-2α with HER2-4β or R6C12β expressed in CD8 cells sorted after TCR-transfer by flow cytometry resulted in CD8-independent peptide recognition in a dose-dependent manner demonstrating again a high-functional avidity of these chimeric chain combinations (Table VI). Sorted CD8 cells transduced with HER2-2α in combination with HER2-4β also demonstrated high-peptide specificity, although background reactivity against T2 cells pulsed with alternative peptides was again noticed (Table VI). In addition, reactivity against selected tumor cell lines was observed (Fig. 7C, Table VI). In contrast to HER2-1, T cells transduced with HER2-2α/HER2-4β showed similar recognition of HLA-A2+ C1R cells either transfected with HER2 or not (data not shown). As autoreactivity against PBMCs might be a major risk of transgenic T cells targeting overexpressed self-Ags, we analyzed proliferation of HLA-A2 and HLA-A2+ cells after transduction of TCR HER2-1 as well as HER2-2α/HER2-4β (Fig. 7D). HLA-A2 PBMCs transduced with either HER2-1 or HER2-2α/HER2-4β showed similar proliferation as mock-transduced or nontransduced PBMCs although proliferation of multimer+ cells was reduced in the CD8+ but not CD4+ cell population transduced with HER2-2α/HER2-4β. Using HLA-A2+ PBMC populations, we observed a slightly decelerated proliferation of cells transduced with HER2-1. In contrast, proliferation of CD4 and CD8 PBMC populations was completely abrogated when transduced with HER2-2α/HER2-4β (Fig. 7D).

Table VI.
Peptide and tumor recognition of PBMCs transduced with HER2-2α in combination with different TCR β-chains followed by depletion of CD8+ cells
α-Chain
HER2-2 AV27
β-Chain (CD8,HER2369 multimer+%) HER2-4 BV12 (14.1) R6C12 BV12 (2.3)a Mock Nontransduced 
T2 + 10−5 M HER2369 4287 (105)b 369 (20) 27 (1) 30 (1) 
T2 + 10−6 M HER2369 4347 (55) 315 (20) 24 (4) 19 (2) 
T2 + 10−7 M HER2369 4059 (174) 185 (8) 16 (0) 22 (5) 
T2 + 10−8 M HER2369 3827 (44) 126 (5) 14 (1) 16 (3) 
T2 + 10−9 M HER2369 1806 (25) 18 (3) 11 (1) 31 (4) 
T2 + 10−10 M HER2369 750 (9) 14 (3) 11 (2) 26 (1) 
T2 unpulsed 602 (35) 18 (2) 17 (1) 15 (4) 
T2 + GP100209 472 (6) 23 (1) 19 (5) 17 (2) 
T2 + pp65495 484 (17) 14 (2) 14 (1) 14 (1) 
T2 + Flu58 762 (129) 16 (2) 16 (4) 21 (0) 
T2 + FMNL1-PP2 606 (16) 18 (3) 19 (1) 14 (3) 
T2 + HDAC6862 593 (28) 21 (2) 15 (2) 16 (2) 
T2 + Tyrosinase369 644 (24) 28 (5) 20 (2) 18 (2) 
MCF-7 (A2+, HER2+715 (3) 7 (2) 19 (2) 16 (1) 
MDA-MB-231 (A2+, HER2+511 (47) 1 (1) 12 (2) 11 (4) 
SKOV3 tA2 (A2+, HER2+95 (1) 17 (2) 13 (3) 9 (3) 
SKOV3 (A2, HER2+47 (2) 22 (1) 12 (2) 10 (0) 
SK-Mel 29 (A2+, HER2+271 (0) 16 (0) 21 (2) 50 (19) 
624.38 MEL (A2+, HER2+271 (19) 25 (2) 11 (2) 13 (5) 
K562 WT (A2, HER229 (2) 8 (1) 7 (3) 16 (7) 
Medium 22 (2) 11 (3) 9 (2) 5 (1) 
α-Chain
HER2-2 AV27
β-Chain (CD8,HER2369 multimer+%) HER2-4 BV12 (14.1) R6C12 BV12 (2.3)a Mock Nontransduced 
T2 + 10−5 M HER2369 4287 (105)b 369 (20) 27 (1) 30 (1) 
T2 + 10−6 M HER2369 4347 (55) 315 (20) 24 (4) 19 (2) 
T2 + 10−7 M HER2369 4059 (174) 185 (8) 16 (0) 22 (5) 
T2 + 10−8 M HER2369 3827 (44) 126 (5) 14 (1) 16 (3) 
T2 + 10−9 M HER2369 1806 (25) 18 (3) 11 (1) 31 (4) 
T2 + 10−10 M HER2369 750 (9) 14 (3) 11 (2) 26 (1) 
T2 unpulsed 602 (35) 18 (2) 17 (1) 15 (4) 
T2 + GP100209 472 (6) 23 (1) 19 (5) 17 (2) 
T2 + pp65495 484 (17) 14 (2) 14 (1) 14 (1) 
T2 + Flu58 762 (129) 16 (2) 16 (4) 21 (0) 
T2 + FMNL1-PP2 606 (16) 18 (3) 19 (1) 14 (3) 
T2 + HDAC6862 593 (28) 21 (2) 15 (2) 16 (2) 
T2 + Tyrosinase369 644 (24) 28 (5) 20 (2) 18 (2) 
MCF-7 (A2+, HER2+715 (3) 7 (2) 19 (2) 16 (1) 
MDA-MB-231 (A2+, HER2+511 (47) 1 (1) 12 (2) 11 (4) 
SKOV3 tA2 (A2+, HER2+95 (1) 17 (2) 13 (3) 9 (3) 
SKOV3 (A2, HER2+47 (2) 22 (1) 12 (2) 10 (0) 
SK-Mel 29 (A2+, HER2+271 (0) 16 (0) 21 (2) 50 (19) 
624.38 MEL (A2+, HER2+271 (19) 25 (2) 11 (2) 13 (5) 
K562 WT (A2, HER229 (2) 8 (1) 7 (3) 16 (7) 
Medium 22 (2) 11 (3) 9 (2) 5 (1) 
a

Indicates usage of TCR constructs modified by codon optimization, usage of murinized constant chains and bicistronic vectors.

b

SDs of triplicates are shown in parentheses.

Transfer of Ag-specific TCR chains might be a therapeutic option for the treatment of cancer and has been previously shown to be a feasible and effective approach (1, 2). However, for broader application and further assessment of potential benefits and side effects, a broad range of TCRs with specificity for diverse classes of tumor-associated Ags need to be characterized. HER2 is a tumor-associated Ag overexpressed in diverse malignancies and has been previously demonstrated to be a preferable target for anticancer therapies using Abs (36). In this study, we investigated the function of HER2369-specific T cell clones that were generated using the allorestricted approach (4), and their specific TCR after transfer into diverse recipient cells.

Transfer of HER2369-specific allorestricted TCRs into PBMCs resulted in high-peptide specificity, whereas tumor reactivity was primarily low. Peptide specificity and tumor reactivity of especially one TCR, HER2-1, could be significantly enhanced after codon optimization, usage of a bicistronic vector construct, and murinization of constant chain genes as previously described (28, 29, 31). Moreover, these modifications also reduced mispairing with endogenous TCR chains. Murine sequences may induce immune responses. However, recent clinical data show effectivity and in vivo persistence of effector cells transduced with a murine TCR (2) suggesting that TCRs containing murine sequences should not be prematurely deleted from further investigation. However, other modifications reducing the risk for mispairing might be applied for HER2-1 (3739). High specificity of HER2-1 was proven by recognition of endogenously presented HER2369 as well as lack of cross-reactivity against HER1364, HER3356, or HER4361. We additionally tested recognition of single amino acid peptide analoga of HER2369 presented in the context of HLA-A2 using threonine and alanine for substitution. Although several single substitutions with the inert amino acid alanine were still recognized, exchange by threonine was less accepted by this TCR potentially because of the polarity as well as a longer side chain of this amino acid. Limited cross-reactivity for analog peptides has been previously reported for other TCRs with specificity for viral Ags (40) and this might reflect the general cross-reactive potential reported for TCRs (41). Of note, exchange of the HLA-A2 anchor residues in positions 2 and 9 by alanine or threonine was tolerated at high-peptide concentrations of 10 μM. However, melanoma epitopes have been previously described containing alanine or threonine on position 2 and alanine on position 9 (42). In addition, it has been previously published that position 3 may represent a secondary anchor and that aromatic residues are frequent in this position (43, 44). Substitution of phenylalanine at position 3 with either alanine or threonine completely abolished recognition by HER2-1 potentially pointing to a role of the aromatic residue at this position in peptide binding of HER2369 to HLA-A2 as well as recognition by TCR HER2-1. Importantly, PBMCs transduced with HER2-1 did not show reactivity against nonmalignant HLA-A2+ targets as fibroblast cell lines, cardiomyocytes, and unstimulated or stimulated PBMCs suggesting that peptide-independent HLA-A2 recognition or cross-reactivity against a peptide broadly presented by HLA-A2 may not play a role in target recognition. However, C1R cells transfected with HLA-A2 induced limited intracellular IFN-γ production in HER2-1–transduced CD8+ cells and transduction of this TCR in HLA-A2+ PBMCs resulted in slightly decelerated proliferation after longer in vitro culture suggesting that proliferating cells of hematopoietic origin may represent a target of HER2-1–transduced PBMCs. This is in common with previously published data demonstrating that mature hematopoietic cells express low levels of HER2 and increase HER2 mRNA and protein expression after mitogenic stimulation (45). Transient leukopenia may be tolerated during therapy. However, to further assess the value of HER2-specific TCRs and especially TCR HER2-1 for clinical application, it will be necessary to investigate the reactivity against a broad panel of HLA-A2+ healthy tissues in vitro and in vivo. Moreover, possibilities for selective deletion of TCR-transduced PBMCs (46) may be considered to avoid prolonged undesired side effects when targeting this Ag. It is interesting in this regard that the most promising TCR candidate (HER2-1) showed only an intermediate functional avidity, although this TCR has been selected in an allogeneic environment. This may rely on several factors (47). First, binding of HER2369 to HLA-A2 has been reported to have a relative short t1/2 (48) that may have an impact on the intermediate functional avidities observed. Second, the discriminative HER2369 epitope density between tumor cells and nontransformed cells might be marginal for HER2369. In fact, HER2369 peptide has been previously shown to be presented only at low levels on tumor cells (49). Moreover, overexpression of HER2 seems to result in proteasomal dysfunction and therefore reduced peptide presentation on tumor cells overexpressing this protein (50). It might be therefore possible that TCRs with higher avidity have not been selected on the T cell clone level because of enhanced cross-reactivity. TCRs with high avidity might be therefore problematic when targeting overexpressed self-Ags and an individual optimal functional TCR avidity potentially needs to be determined for any specific MHC-peptide complex.

The other selected TCRs seemed at first less interesting as they demonstrated either limited peptide specificity (HER2-3) or lack of tumor reactivity (HER2-2 and HER2-4). However, as we observed a preferential usage of β-chains of the TRBV12 family in the isolated HER2-specific TCR repertoire, we investigated random combinations of isolated single TCRα- and β-chains of these TCRs. We thereby identified a TCRα-chain (HER2-2α) with specific HER2369 peptide recognition in combination with diverse β-chains, all belonging to the TRBV12 family. Combinations with β-chains of two other TRBV families did not result in HER2369-multimer+ cells or peptide-specific target recognition. Importantly, all different TRBV12-derived β-chains contributing to HER2369-specific multimer staining in combination with HER2-2α possessed different CDR3 regions and were not only derived from allorestricted HER2369-specific TCRs from diverse HLA-A2 donors, but also from TCRs with alternative specificities as GP100209 and CMVpp65495. These latter TCRs were selected in HLA-A2–expressing individuals. Moreover, HER2369-multimer+ PBMCs transduced with HER2-2α alone stained mainly positive for anti-TRBV12 further emphasizing that HER2-2α is dominantly responsible for HLA-A2–restricted HER2369-specific recognition in combination with diverse TCRβ-chains of the TRBV12 family. A prevalent role of the TCRα-chain in the selection of the preimmune TCR repertoire specific for melan-A has been previously reported (34, 35). Heemskerk et al. described novel chimeric TCR chain combinations containing single TCR chains derived from diverse HA-2–specific TCR resulting again in HA-2 specificity (13). Moreover, Yokosuka et al. reported a murine HIV-specific TCRα-chain in TCR-transgenic mice responsible for recognition of HIVgp160 peptide/H-2Dd complexes in combination with a variety of TCRβ-chains (51). In these experiments, one third of randomly picked β-chains of the same TRBV family could reconstitute for specific peptide recognition in combination with the HIVgp160 peptide-specific TCRα-chain. In this study, we report that such TCR single chains with dominant peptide recognition in combination with TCRβ-chains derived from TCRs with diverse specificities are present in the circulating T cell pool of humans. MHC-peptide recognition by random combinations of TCRα- and β-chains has been previously suggested to be related to an intrinsic affinity of the TCRs toward MHC (52) and a genetic bias toward MHC recognition has been proposed (53, 54). More recently, germline interaction codons have been structurally defined for TCRα- and β-chains contacting specific MHC molecules (55, 56). Our data can be well explained by the existence of such germline restrictions of TCR–MHC-peptide complexes and favor for a preferential germline restriction of β-chains of the TRBV12 family toward HLA-A2 when paired to HER2-2α. However, the CDR3 regions of the matching β-chains still play an important role in HER2369 peptide recognition of HER2-2α and determined the dimensions of functional avidity, CD8 dependency, and tumor reactivity. Two β-chains of the TRBV12 family (HER2-1β and JG-9β) resulted in only low percentages of HER2369-multimer+ cells lacking specific functions. This might potentially result from low interchain affinity of the novel chimeric TCR chain combination. In addition, the β-chain of JG-9 represented another subtype (TRBV12-4). In contrast, two other TCRβ-chains potentiated peptide-specific Ag recognition (HER2-4β and R6C12β). These chimeric TCR chain combinations resulted in increased peptide-specific avidity and CD8 independent function. Combination of HER2-2α and HER2-4β additionally resulted in potent tumor reactivity. Both TCR single chains derived from different HER2369-specific TCRs and, when dimerized with their native partners, primarily exhibited low-functional avidities for peptide-pulsed T2 cells and a lack of tumor reactivity. Currently, the frequency of such TCR single chains with dominant peptide recognition in combination with diverse β-chains is unknown and it is not clear whether TCRβ-chains may have similar properties. It will be interesting to further evaluate the docking pattern on MHC-peptide complexes of these TCRs. Apart from that, the existence of these TCR single chains with dominant peptide recognition may have an important impact on the further development of TCR transfer strategies. Chimeric TCR combinations involving such TCR single chains with dominant peptide recognition may be a source for TCRs with improved properties as enhanced functional avidity for tumor-associated Ags and preferential recognition of natural tumor targets. Otherwise, effector T cells transduced with the chimeric TCR HER2-2α/HER2-4β also showed increased background reactivity against T2 cells pulsed with alternative peptides as well as strong reactivity against HLA-A2+ PBMCs. This reactivity might result from enhanced germline MHC interactions but also from recognition of low level HER2369 on these target cells. TCRs with enhanced affinity because of mutations within the CDR3 region have been previously demonstrated to be cross-reactive for self-peptides and might therefore harbor risks when used for clinical approaches with TCR-transgenic T cells (57, 58). Thus, the presence of such single TCR chains in the circulating T cell pool displaying dominant peptide recognition with different affinities in combination with diverse partner chains derived from introduced TCR may represent a risk for TCR transfer in PBMCs.

In conclusion, we isolated and characterized diverse TCRs with specificity for the HER2369 as potential candidates for TCR transfer. One TCR showed preferential characteristics for further investigation regarding clinical application. We additionally identified a single TCRα-chain with dominant HER2369 peptide recognition in combination with β-chains of the TRBV12 family derived from TCRs with diverse specificities demonstrating that pairing with novel partner chains can result in an increase of peptide-specific avidity as well as CD8 independency. Although the frequency of such TCRs is currently unknown, they may represent interesting tools for TCR optimization resulting in enhanced functionality when paired to novel partner chains. However, mispairing with novel partner chains may also result in enhanced cross-reactivity or self-reactivity, and these data further indicate that enhanced efforts for improved chain pairing (3739), as well as selective deletion of TCR-transduced PBMCs (46), may be important.

We are highly thankful to Richard Morgan and Steve Rosenberg for providing cDNA of the TCR R6C12. We also thank Helga Bernhard, Jehad Charo, Heinke Conrad, Elfriede Noessner, Ralph Mocikat, and Stefan Stevanovic for providing cell lines.

Disclosures X. L., L. W., and A. K. have a patent application for the described HER2-specific TCR sequences.

This work was supported by grants from the Life Science-Stiftung and Deutsche Forschungsgemeinschaft (KR2305/2-1) to A.M.K., Deutsche Forschungsgemeinschaft SPP1230 to W.U., and Deutsche Forschungsgemeinschaft SFB/TR36 (projects A4 [to A.M.], A8 [to A.M.K.], B3 [to D.H.B.], and Z1 [to W.U.]).

The online version of the article contains supplemental material.

Abbreviations used in this paper:

     
  • HER2

    HER2/neu

  •  
  • J76

    Jurkat 76

  •  
  • J76CD8

    J76 transduced with CD8α

  •  
  • Mod

    modified

  •  
  • NCBI

    National Center for Biotechnology Information

  •  
  • TRBV

    TCR gene variable β-chain

  •  
  • WT

    wild type.

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