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Clinical Cancer Research Vol. 10, 7043-7052, October 15, 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Heteroclitic CD33 Peptide With Enhanced Anti-Acute Myeloid Leukemic Immunogenicity

Jooeun Bae, Jeffrey A. Martinson and Hans G. Klingemann

Section of Bone Marrow Transplant and Cell Therapy, Rush University Medical Center, Chicago, Illinois


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The goal of these studies was to engineer a synthetic CD33 peptide with enhanced immunogenicity for the induction of acute myeloid leukemia (AML)-specific CTLs. Eight modified CD33 peptides YLISGDSPV,YIGSGDSPV,YIIIGDSPV,YIILGDSPV,YIISGISPV,YIISGDLPV,YIISGDSWV andYIISGDSPL were designed for increased HLA-A2.1 or T cell receptor affinity and compared with the native CD3365–73 peptide, AIISGDSPV, for enhanced immunogenicity. The YLISGDSPV peptide was found to be the most immunogenic epitope producing highly cytolytic CTLs against AML target cells. The CTLs generated withYLISGDSPV peptide showed CD33 peptide-specificity through targeting of both native (AIISGDSPV) and modified (YLISGDSPV) peptide presenting EBV-BLCL. The CTL cultures displayed a distinct phenotype consisting of a high percentage of activated memory (CD69+/CD45RO+)-CD8+and a low percentage of naïve (CD45RA+/CCR7+)-CD8+cells. In addition, T-cell clones specific to theYLISGDSPV peptide were isolated and characterized to target AML cells. The clones exhibited both HLA-A2.1-restricted and AML cell-specific cytotoxicity that was mediated through a granule-dependent pathway. More importantly, the CTL clones did not lyse or inhibit the proliferation of normal CD34+ progenitor cells. In conclusion, we report on the identification of a highly immunogenic heterocliticYLISGDSPV CD33 epitope that is a promising candidate for immunotherapy targeting AML.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Self-peptides presented by the MHC may play an important role in T-cell function by providing critical stimulatory signals to immune effector cells. Recent studies have shown that self-proteins expressed by both normal and malignant cells can be used as target antigens for developing cellular therapies to treat many types of diseases including carcinomas, lung cancer, colorectal cancer, breast cancer, melanoma, and myeloma (1, 2, 3, 4) . Although the role of thymic self-peptides in T-cell selection has not been conclusively defined, there is evidence that positive selection of a significant proportion of the T-cell repertoire is mediated by direct T-cell receptor (TCR)-self-peptide interaction (5, 6, 7, 8, 9) . Studies by Hu et al. (8) provide evidence that abundant self-peptides, purified from thymic epithelial cells, are specifically recognized during the positive selection of CD8+ T cells. This positive selection process generates a reserve of T cells that are self-reactive and explains how the recognition of a limited repertoire of thymic self-peptides can select a diverse population of T cells through its cross-reactivity. Such findings imply that self-peptides can be used to stimulate the peripheral T-cell population leading to effector cells with an intrinsic affinity for self-epitopes.

Identification and utilization of peptides against self-antigens offer an attractive approach to augment antigen-specific immunity against tumor cells lacking defined tumor-specific antigens. Native peptides against melanoma-associated self-antigens have shown limited success in treating the disease because of low peptide immunogenicity. However, investigators have shown that modifying critical amino acid residues within the native melanoma peptide sequence induced greater T-cell responses and increased the targeting of melanoma cells by CTLs (10 , 11) . Modification of native self-peptides in solid tumors, leukemia, or lymphomas could also lead to peptides with increased immunogenicity against the tumor cells, although only a few studies on peptide modification to target these diseases have been published.

CD33, a normal self-antigen that is a Mr 67,000 cell surface glycoprotein largely restricted to the myeloid/monocytic lineage, was selected as the target antigen because of its overexpression on >90% of acute myeloid leukemia (AML) blasts and its lack of expression on pluripotent stem cells (12, 13, 14) . We have previously identified a novel native CD3365–73 peptide, AIISGDSPV, for ex vivo generation of CTLs targeting primary HLA.A2.1+ AML blasts (15) . In addition, we have shown that a modified CD33 peptide, YIISGDSPV, containing tyrosine (Y) instead of alanine (A) at position 1 had increased HLA-A2.1–binding affinity/stability along with enhanced immunogenicity compared with the native CD3365–73 peptide (15) . The objective of this study was to develop more effective heteroclitic analogs of the CD33 peptide by conserving the tyrosine (Y) substitution at position 1 (P1) and examining the effects of additional amino acid substitutions on peptide immunogenicity against AML. Peptide modifications were made through substitution of key amino acid residues within the CD33 peptide sequence to increase HLA-A2.1 affinity or interaction with the TCR. The resulting modified peptides were tested for their ability to induce AML-specific CTLs ex vivo.

Of eight modified CD33 peptides tested, the YLISGDSPV epitope displayed the highest HLA-A2.1 affinity/stability, which correlated with the induction of highly cytotoxic CTLs response against AML cells. We report here on the identification and characterization of a heteroclitic peptide, YLISGDSPV, which induces both polyclonal CTLs and T-cell clones specific for CD33 antigen on AML cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
The CD33+/HLA-A2.1+ AML cell line, ML-2 (provided by Dr. Y. Matsuo, Fujisaki Cell Center, Okayama, Japan) was used as target cells in the cytotoxicity assays. The T2 cell line provided by Dr. J. Molldrem (University of Texas M. D. Anderson Cancer Center, Houston, TX) was used as antigen presenting cells. EBV-transformed B-cell lines (EBV-BLCL) were established by transformation of CD19+ lymphocytes with the B95–8 strain of EBV (Nazaruk et al., 1998). The NK-sensitive K562 cell line was obtained from American Tissue Culture Collection (Manassas, VA). All cell lines were cultured in RPMI 1640 (Life Technologies, Inc. Rockville, MD) supplemented with 10% FCS (BioWhittaker, Walkersville, MD), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.).

Reagents.
Mouse antihuman HLA-A2.1 monoclonal antibody was purified from the culture supernatant of the hybridoma BB7.2 cell line (gift from Dr. J. Molldrem). Recombinant human granulocyte-colony–stimulating factor (G-CSF), GM-CSF, interleukin (IL)-3, and stem cell factor were obtained from Immunex (Seattle, WA). Recombinant human IL-2 was purchased from R&D Systems (Minneapolis, MN). Mouse antihuman CCR7, CD3, CD4, CD8, CD33, CD45RA, CD45RO, CD56, and CD69 fluorescein isthiocyanate-, phycoerythrin- or perinidin chlorophyll protein-conjugated and unconjugated antihuman CD3 and Fas monoclonal antibodies were purchased from BD/PharMingen (San Diego, CA).

Synthetic Peptide.
Modified HLA-A2.1–specific CD33 peptides were designed by altering two amino acid residues within the native CD33 peptide (AIISGDSPV) sequence. The sequences of the modified CD33 peptides are as follows: YLISGDSPV (1Y2L), YIGSGDSPV (1Y3G), YIIIGDSPV (1Y4I), YIILGDSPV (1Y4L), YIISGISPV (1Y7S), YIISGDLPV (1Y7L), YIISGDSWV (1Y8W), and YIISGDSPL (1Y9L). Influenza virus protein matrix peptide58–66 (GILGFVFTL) and MAGE-3 peptide271–279 (FLWGPRALV) were used as HLA-A2.1–specific peptide controls. All peptides (Biosynthesis, Lewisville, TX) were synthesized by standard 9-fluorenylmethyl-oxycarbonyl chemistry, purified to >90% with reverse-phase chromatography and validated by mass-spectrometry for Mr.

T2 Peptide-Binding Assay.
The T2 cell line, a TAP-deficient human B- x T-lymphoblastoid hybrid expressing HLA-A2.1 molecules (16 , 17) , was used to evaluate the CD33 peptides for HLA-A2.1–specific binding. In the assay, T2 cells were washed three times and resuspended in serum-free AIM-V medium (Life Technologies, Inc.) to a final concentration of 1 x 106 cells/ml and transferred into a 24-well tissue culture plate. Cells were pulsed with a respective CD33 peptide (100 µg/ml) or influenza virus protein matrix peptide (30 µg/ml) plus 3 µg human ß2-microglobulin (Sigma, St. Louis, MO) and incubated at 37°C, 5% CO2 in humidified air. After overnight incubation, the cells were washed, stained with mouse antihuman HLA-A2.1 monoclonal antibody for 15 minutes at 4°C, washed and incubated with goat antimouse IgG (F(ab')2)-FITC (Sigma) for 15 minutes at 4°C. The cells were washed and then analyzed on a FACSort flow cytometer with CellQuest v2.1 software (Becton Dickinson, San Jose, CA). The fluorescence index (mean channel fluorescence of T2 cells pulsed with the peptide plus ß2 microglobulin ÷ mean channel fluorescence of T2 cells pulsed with ß2 macroglobulin) was calculated to determine the up-regulation of HLA-A2.1 expression on T2 cells caused by HLA-A2.1–specific peptide binding.

Isolation of Primary Human Cells.
Informed consent was obtained from all donors, and the protocol was approved by the RUSH University Medical School Institutional Review Board.

CD3+ T Cells.
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood of normal HLA-A2.1+ donors by standard density gradient centrifugation over Ficoll-Paque Plus (Amersham Pharmacia Biotech AB, Uppsala, Sweden). PBMCs were harvested from the interface, washed twice, and resuspended in PBS supplemented with 5 mmol/L EDTA and 0.5% human serum albumin. CD3+ T cells were isolated with the Pan T cell isolation kit from Miltenyi Biotec (Auburn, CA). In brief, T-cell enrichment was accomplished by depletion of B cells, NK cells, early erythroid cells, platelets, and basophils by labeling with a mixture of hapten-conjugated CD11b, CD16, CD19, CD36, and CD56 antibodies and magnetic-activated cell sorting microbeads coupled to an antihapten monoclonal antibody. The effluent (negative cell fraction) was collected from the column as enriched CD3+ T cells. Purity (mean ± SD) of the enriched CD3+ T cells was examined by flow cytometry and was found to be 94 ± 4%.

PBMCs from AML Patients or Chronic B-Lymphocytic Leukemia Patients.
PBMCs were isolated (as described above) from heparinized peripheral blood of HLA-A2.1+ AML, HLA-A2.1– AML, or HLA-A2.1+ CLL patients. Patient PBMCs were stored frozen in liquid nitrogen and thawed before use as target cells in the cytotoxicity assays. The isolated PBMCs from AML patients (AML-PBMCs) were composed of between 54 to 77% AML blasts based on differential cell counts. In addition, flow cytometric analysis showed that >70% of the isolated AML-PBMCs were positive for CD33 antigen expression (data not shown). PBMCs from the CLL patient (CLL-PBMCs) samples were negative for CD33 antigen expression.

PBMCs from Normal Donors.
PBMCs were isolated from heparinized peripheral blood of normal human donors as described above. The PBMCs were stored frozen in liquid nitrogen and thawed before use.

CD34+ Stem Cells.
CD34+ stem cells were provided by Jim Bender (Nexel, Irvine, CA). In brief, the Isolex 300i stem cell isolation technology (Baxter Healthcare, Deerfield, IL) was used to isolate CD34+ cells from G-CSF-mobilized apheresis products collected from consented normal HLA-A2.1+ human volunteers. The isolated CD34+ cells were stored frozen in liquid nitrogen and thawed immediately before use.

Generation of CD33 Peptide-Specific CTLs.
CD33 peptide-specific CTLs (CD33-CTLs) were generated ex vivo by repeated stimulation of CD3+ T lymphocytes obtained from normal HLA-A2.1+ donors with native or modified CD33 peptide-pulsed T2 cells (T2/peptide). In brief, T2 cells were washed and resuspended in serum-free AIM-V medium and pulsed overnight at 37°C with 100 µg/ml of the appropriate CD33 peptide. Peptide-pulsed T2 cells were washed, counted, irradiated at 10 Gy and used to prime CD3+ T cells at a 1:20 T2/peptide (stimulator)-to-CD3+ T-cell (responder) ratio in AIM-V medium supplemented with 10% human AB serum (BioWhittaker). The T-cell cultures were restimulated every 7 days with irradiated T2/peptide for a total of 4 cycles. IL-2 (100 units/ml) was added to the cultures 3 days after the second stimulation. Control T-cell cultures (unstimulated with peptide) were maintained in AIM-V medium supplemented with 10% human AB serum containing 100 units/ml IL-2.

Isolation of CD33 Peptide-Specific T-Cell Clones.
T-cell clones were established by limiting dilution at 0.3, 1, and 3 cells/well in 96-well U-bottomed microtiter plates as described previously by Brucker et al. (18) . The clones were isolated and expanded in complete medium (RPMI 1640 + 10% Human AB serum) in the presence of 100 units/ml IL-2, 30 ng/ml anti-CD3 purified monoclonal antibody and pooled irradiated allogeneic PBMCs from three normal donors as feeder cells.

Chromium Release Cytotoxicity Assay.
The cytotoxic activity of the CD33-CTLs or CD33 peptide-specific T-cell Clones (CD33-T–cell clones) was measured in a standard 51Cr-release assay. Effector cells were seeded with 51Cr-labeled target cells (5 x 103 cells/well) at various E:T cell ratios in 96-well U-bottomed microtiter plates, and the plates were incubated for 6 hours at 37°C, 5% CO2. 51Cr-release was measured in 100 µL supernatant with a Beckman LS6500 liquid scintillation counter (Beckman Coulter, Brea, CA). Maximum release was obtained from detergent-released target cell counts and spontaneous release from target cell counts in the absence of effector cells. The percentage of specific cell lysis was calculated as [(experimental release – spontaneous release) ÷ (maximum release – spontaneous release)]. In the assay to examine the role of perforin or Fas in the CD33-T–cell clone-mediated cytotoxicity, effector cells were pretreated for 2 hours with either concanamycin A (CMA, Sigma) at 500 nmol/L per well or Fas antibody at 1 µg/ml per well. The treated or untreated effector cells were then incubated with target cells in a standard 51Cr-release assay.

Phenotypic Analysis of the CD33-CTLs or CD33-T–Cell Clones.
The CD33-CTLs were stained with CD8-Percp/CCR7-PE/CD45RA-FITC or CD8-Percp/CD45RO-PE/CD69-FITC mouse antihuman monoclonal antibodies for flow cytometric analysis. The CD33-T cell clones were stained with CD3-PerCP/CD4-PE/CD56-FITC or CD3-PerCP/CD8-PE/CD56-FITC mouse antihuman monoclonal antibodies. After staining with antibodies for 15 minutes at 4°C, the cells were washed and analyzed using a FACSort flow cytometer with CellQuest 228 v2.1 software.

Hematopoietic Progenitor Cell Proliferation Inhibition Assay.
Inhibition of HLA-A2.1+ normal donor CD34+ cells or HLA-A2.1+ AML-PBMCs proliferation was evaluated in the presence or absence of CD33-CTL clones. Irradiated (10 Gy) clones (1 x 105, 0.5 x 105, 0.25 x 105, or 0.125 x 105) were incubated with 2 x 104 HLA-A2.1+ CD34+ cells or HLA-A2.1+ AML-PBMCs per well in 96-well U-bottomed microtiter plates. The cells were cultured in MyeloCult5100 medium (StemCell Technologies Inc., Vancouver, Canada) supplemented with 100 ng/ml G-CSF, 100 ng/ml GM-CSF, 25 ng/ml IL-3, and 50 ng/ml stem cell factor, which support the proliferation and differentiation of early progenitor cells. After 6 days of culture, the wells were pulsed with 1 µCi [3H]thymidine for 18 hours and harvested to measure the proliferation of the hematopoietic progenitor cells or the AML blasts. Irradiated effector cells alone were used to determine the background [3H]thymidine incorporation. The Ph.D. cell harvester was used to harvest cells onto filter discs using, cells were resuspended in liquid scintillation fluid, and a Beckman LS6500 scintillation counter was used to evaluate 3H-cpm. The percentage growth inhibition (19) was determined by the following formula: % proliferation inhibition = [1 – (mean cpm wells with CTLs ÷ mean cpm wells without CTLs)] x 100%.

Cold Target Inhibition Assay.
Antigen-specific cell lysis was evaluated in a "cold" target inhibition assay with unlabeled HLA-A2.1+ AML-PBMCs or K562 cells as cold inhibitors to block lysis of 51Cr-labeled "hot" HLA-A2.1+ AML-PBMCs. The effector cells were incubated with an equal number of unlabeled cold inhibitors for 1 hour at 37°C, 5% CO2 before the addition of 51Cr-labeled HLA-A2.1+ AML-PBMCs as target cells. After 6 hours of incubation, the supernatants were harvested, and the specific 51Cr-release was measured as described previously. The inhibition of AML cell-specific lysis was measured by comparing the specific lysis percentage of the effector cells incubated with or without the unlabeled cold target cells.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Modified CD33 Peptides Display Improved Affinity/Stability to HLA-A2.1 Molecules.
Modified CD33 peptides were tested to determine whether alteration of key amino acid (AA) residues affects overall peptide affinity/stability to HLA-A2.1 molecules. In previous studies, we have shown that alteration of the secondary anchor motif (residue position 1) of the native AIISGDSPV peptide from alanine (A) to an aromatic amino acid tyrosine (Y) resulted in a heteroclitic YIISGSPV peptide with enhanced HLA-A2.1–binding affinity and stability (15) . In these studies, the YIISGSPV peptide was further modified by altering a residue at position 2 or position 9 for improvement of HLA-A2.1 affinity. Alternatively, a residue at position 3, 4, 6, 7, or 8 was changed to increase peptide interaction with the TCR. The T2 peptide-binding assay was used to evaluate the modified CD33 peptides for HLA-A2.1 affinity/stability.

Peptide binding to HLA-A2.1 molecules on T2 cells is shown as the Fluorescence Index (FI). The FI value >1 indicates the up-regulation of HLA-A2.1 attributable to peptide-specific binding of the molecules on the cells. Peptide stability to the HLA-A2.1 molecules is expressed as the dissociation complex50, which is defined as the time required for the loss of 50% of peptide/HLA complexes stabilized at time 0.

Table 1Citation summarizes the HLA-A2.1 affinity and stability of the native and eight modified CD33 peptides. The FI values for all of the modified CD33 peptides were >1.1- to 1.7-folds the native peptide, implying that tyrosine (Y) at position 1 consistently enhance the HLA-A2.1 affinity of the peptides. The stability of modified 1Y2L (YLISGDSPV) and 1Y6I (YIISGISPV) peptide binding to HLA-A2.1 molecule was increased to >18 hours. The 1Y7L (YIISGDLPV) and 1Y9L (YIISGDSPL) peptides also displayed increased stability (>4 hours) whereas the stability of the remaining modified and native CD33 peptides was 2 hours. Overall, the 1Y2L and 1Y6I peptides exhibited the highest peptide binding (FI, >5.60) and stability (dissociation complex50, >18 hours) that was similar to the influenza virus protein matrix peptide58–66 (FI, 5.72 ± 0.82; dissociation complex50, >18 hours) used as a control HLA-A2.1–binding peptide.


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  		Table 1 HLA-A2.1 affinity and stability of the native and modified CD33 peptides

 
Modified 1Y2L CD33 Peptide-Specific CTLs Display the Greatest Cytotoxic Activity against AML Target Cells.
A critical issue in these studies was to evaluate the modified CD33 peptide-specific CTL’s directed lysis of AML cells. Figure 1Citation summarizes the cytotoxic activity of the modified CD33 peptide-specific CTLs against HLA-A2.1+ AML-PBMCs or the HLA-A2.1+ ML-2 AML cell line. The modified 1Y2L CD33 peptide, which exhibited the highest HLA-A2.1 affinity/stability, generated the most effective CTLs targeting either primary AML (50.6 ± 1.1%) or the ML-2 (54.8 ± 4.7%) cell line. The remaining modified CD33 peptides generated CTLs displaying equivalent cytotoxic activity against AML targets from native CD33 peptide. Control T cells (IL-2 only stimulated) did not lyse either primary AML or ML-2 target cells (data not shown).



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  		Fig. 1. Cytotoxicity induced by native or modified CD33 peptides. These data display 51Cr-release assay results done 1 week after the fourth stimulation of CTLs at E:T ratio of 10:1. The results show the cytotoxic activity of the native or modified CD33 peptide-specific CTLs, which were generated from T cells obtained from a single donor, against (A) HLA-A2.1+ AML-PBMCs or (B) HLA-A2.1+ ML-2 AML cell line. The highest cytotoxicity was observed in CTLs induced with modified 1Y2L CD33 peptide against both target cells. The percentage of specific lysis is expressed as the mean ± SE from three independent experiments.

 
Cytotoxic Activity of 1Y2L-CTLs Is Peptide-Specific against Autologous EBV-BLCL Target Cells Presenting Either Native CD33 or Modified CD33 1Y2L Peptide.
The 1Y2L CD33 peptide-specific CTLs (1Y2L-CTLs) were tested for their ability to recognize and lyse native CD3365–73 peptide-presenting cells. In the cytotoxicity assay, autologous EBV-BLCL pulsed with native CD33 (AIISGDSPV), modified CD33 (YLISGDSPV), irrelevant MAGE-3 (FLWGPRALV), or no peptides were used as the target cells. Figure 2Citation shows the cytotoxicity of the 1Y2L-CTLs against EBV-BLCL presenting 1Y2L CD33 peptide. More importantly, the 1Y2L-CTLs displayed equivalent cytotoxic activity against target cells presenting native CD33 peptide. However, the 1Y2L-CTLs did not show a significant lysis (<15%) of MAGE-3 peptide-presenting EBV-BLCL or EBV-BLCL itself, verifying that the cytotoxicity of the CTLs was CD33 peptide-specific. These results confirm that the recognition of native CD33 peptide by the 1Y2L-CTLs was not altered by the peptide modification.



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  		Fig. 2. Cytotoxicity of 1Y2L-CTLs against native or modified CD33 peptide presenting EBV-BLCL. The cytotoxicity of the 1Y2L-CTLs was measured in a 51Cr-release assay against native CD33 peptide ({blacksquare}), modified 1Y2L CD33 peptide ({blacktriangleup}), irrelevant HLA-A2.1–binding MAGE-3 peptide ({diamondsuit}), or no peptide (•) pulsed with autologous EBV-BLCL target cells. The 1Y2L-CTL cytotoxicity is peptide-specific against either native or modified 1Y2L CD33 peptide presenting autologous EBV-BLCL but not against MAGE-3 peptide presenting EBV-BLCL or EBV-BLCL alone. The percentage of specific lysis is expressed as the mean ± SE from three independent experiments.

 
Cytotoxic Activity of 1Y2L-CTLs Is Enhanced against Native CD33 Peptide.
Figure 3Citation compares the cytotoxic activity of native CD33 peptide-specific CTLs or modified CD33 1Y2L-CTLs with autologous EBV-BLCL pulsed with native CD3365–73 peptide. The 1Y2L-CTLs showed increased cytotoxicity (32–75%) against native CD33 peptide-presenting EBV-BLCL compared with native CD33 peptide CTLs (21–43%) at all of the E:T cell ratios tested. Therefore, the 1Y2L peptide shows a higher potential as an immunogen than the native CD33 peptide to generate the CD33 antigen-specific CTLs for targeting AML cells.



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  		Fig. 3. Cytotoxicity of native CD33 peptide-specific CTLs or 1Y2L-CTLs against native peptide presenting EBV-BLCL. Native CD33 peptide-specific CTLs ({blacksquare}) or modified CD33 1Y2L-CTLs ({square}) were evaluated for their cytotoxic activity against autologous EBV-BLCL pulsed with native CD33 peptide in a standard 51Cr-release assay. The 1Y2L-CTLs displays the higher cytotoxic activity against native CD33 peptide presenting EBV-BLCL compared with native CD33-CTLs. The percentage of specific lysis is expressed as the mean ± SE from three independent experiments.

 
The 1Y2L-CTLs Display a Distinct Phenotype.
CTLs can be classified phenotypically by the expression of distinct cell surface antigens into naïve, memory, effector, or activated memory cells. The percentage of CD8+ T cells expressing CD45RA+/CCR7+ (naïve), CD45RA–/CCR7+ (central memory), CD45RA–/CCR7– (effector memory), CD45RA+/CCR7– (effector) or CD69+/CD45RO+ (activated memory) phenotypes was analyzed in the control T cell or the 1Y2L-CTL cultures. The CTLs contained a higher percentage of CD8+ cells (92%) compared with the control T-cell cultures (35%). Gating on the CD8+ population in the control T-cell cultures revealed a population of CD45RA+/CCR7+ (80%) cells, whereas only 4% of the CD8+ cells in the 1Y2L-CTL cultures showed this naïve cell subset. The CD8+ cells in the 1Y2L-CTLs were mainly CD45RA+/CCR7–, an effector cell phenotype. By contrast, the CD8+ T-cell subset expressing CD45RA–/CCR7– (effector memory) and CD45RA–/CCR7+ (central memory) was similar between 1Y2L-CTLs and the control T cells. In addition, CD8+ T cells expressing the CD69+/CD45RO+ (activated memory) phenotype were significantly higher in the 1Y2L-CTLs (45%) compared with the control T cells (2%; Fig. 4Citation ).



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  		Fig. 4. Phenotype analysis of CD33 1Y2L-CTLs. The 1Y2L-CTLs were gated on the CD8+ cells and evaluated for the CD45RA+/CCR7+ (naïve), CD45RA+/CCR7– (effector) and CD69+/CD45RO+ (activated memory) cell subsets. The percentage of cells in (A) unstimulated control T cell or (B) 1Y2L-CTLs displaying the CD8+/CD45RA+/CCR7+ naïve cell phenotype was 80% or 4%, respectively. In contrast, the CD8+/CD45RO+/CD69+ activated memory cell subset was much lower in (C) unstimulated control T cells (2%) compared with (D) 1Y2L-CTLs (45%).

 
The 1Y2L-CD8+ CTL Clone Displays HLA-A2.1–Restricted AML Cell-Specific Cytotoxicity and Exhibits a Distinct Phenotype.
CD8+-T–cell clones were isolated by limiting dilution from polyclonal CTLs generated with the 1Y2L CD33 peptide. The isolated 1Y2L-CTL clones were expanded ex vivo and evaluated for their cytotoxic activity (Fig. 5A and B)Citation and phenotype (Table 2)Citation . The CTL clones showed the cytotoxicity of HLA-A2.1+/CD33+ AML-PBMCs but not HLA-A2.1–/CD33+ AML-PBMCs or HLA-A2.1+/CD33– CLL-PBMCs (Fig. 5A)Citation . CD33 antigen is expressed at a low level during early stem cell differentiation, making the CD34+/CD33+ progenitor cells a potential target of the CD33-CTLs. To investigate this issue, CD34+ stem cells from three G-CSF–mobilized HLA-A2.1+ normal donors were isolated and assessed for their potential targeting by the CD33 1Y2L-CTL clone. No significant lysis of CD34+ cells by the clone was observed in the cytotoxicity assays (Fig. 5A)Citation . These results show that the cytotoxic activity of the 1Y2L-CD8+ CTL clone was HLA-A2.1–restricted and AML cell-specific but not directed against HLA-A2.1+/CD34+ stem cells from G-CSF–mobilized normal donors.



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  		Fig. 5. Characteristics of CD33 1Y2L-CTL clones. The peptide-specific clones were analyzed for cytotoxicity (Fig. 5A and B)Citation and phenotype (Table 2)Citation . Figure 5ACitation shows the data (mean ± SE) displaying the51Cr-release assay results for 1Y2L-CD8+ CTL clone. The CTL clone displayed a significant level of cytotoxicity against the HLA-A2.1+ AML-PBMCs ({diamondsuit}) but not against HLA-A2.1– AML-PBMCs ({blacksquare}), HLA-A2.1+ CLL-PBMCs ({blacktriangleup}), or normal HLA-A2.1+ CD34+ stem cells (•). Fig. 5BCitation shows a cold target inhibition assay used to evaluate the antigen-specific cytotoxicity of the 1Y2L-CTL clone. The clone was incubated for 1 hour with unlabeled cold HLA-A2.1+ AML-PBMCs ({blacksquare}) or cold NK-sensitive cell line K562 (•) or no cold inhibitor ({blacktriangleup}) before the addition of 51Cr-labeled hot HLA-A2.1+ AML-PBMCs. The cold HLA-A2.1+ AML-PBMCs decreased the cytotoxicity of the CTL clone against the 51Cr-labeled HLA-A2.1+ AML-PBMCs. In contrast, the cold K562 inhibitor cells did not affect the cytotoxicity of the 1Y2L-CTL clone against the target cells. Results of the standard 51Cr-release assay prove that the CTL clone is an AML cell-specific effector cell. The percentage of specific lysis is expressed as the mean ± SE from three independent experiments. Table 2Citation shows the distinct phenotype of CD33 1Y2L-CTL clones for expression of CD69, CD95, CD45RA, and CD45RO compared from unstimulated T cells.

 

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  		Table 2 Phenotypic analysis of unstimulated T cells and CD33 1Y2L-CTL

 
A cold target inhibition assay confirmed the AML cell-specific cytotoxicity of the 1Y2L-CTL clone (Fig. 5B)Citation . Preincubation with cold HLA-A2.1+ AML-PBMCs completely inhibited the cytotoxic activity of the 1Y2L clone against hot 51Cr-labeled HLA-A2.1+ AML-PBMCs. However, preincubation with cold NK-sensitive K562 cells (HLA-A2.1–/CD33–) did not reduce the clone cytotoxicity against HLA-A2.1+ AML-PBMCs. On the basis of these results, the cytotoxicity of the 1Y2L-CTL clone was determined to be a HLA-A2.1+/CD33+ cell-specific T-cell response and not mediated by NK activity.

Three individual CD3+/CD8+/CD56– clones were compared with control T cells for expression of activation (CD69, CD95), naïve cell (CD45RA), or memory cell (CD45RO) surface markers. The clones showed a higher percentage of activation (CD69+, CD95+) and memory (CD45RO+) cells and a lower percentage of naïve (CD45RA+) cell markers compared with the control T cells (Table 2)Citation .

The 1Y2L-CD8+ CTL Clone Does Not Inhibit Normal CD34+ Progenitor Cell Proliferation.
The low level of CD33 expression on the CD34+ progenitor cells may have contributed to the low cytotoxicity directed against these cells by the 1Y2L-CTL clone (Fig. 5A)Citation . Therefore, we assessed the ability of the 1Y2L clone to inhibit the proliferation of differentiated CD34+ cells. The CD34+ progenitor cells were induced to undergo proliferation and myeloid cell differentiation by incubating them for 1 week in media containing the hematopoietic growth and differentiation cytokines including G-CSF, GM-CSF, IL-3, and stem cell factor. The CD34+ cells cultured with hematopoietic growth and differentiation factors were analyzed by flow cytometry revealing a population of CD34–/CD33+ cells (>50%), providing evidence that the differentiating stem cells were a potential target cell population for the CTLs. Cultures containing irradiated (10 Gy) 1Y2L-CTL clone, HLA-A2.1+-differentiated cells from CD34+ progenitor cells or HLA-A2.1+ AML-PBMCs were used to assess cell proliferation inhibition by the 1Y2L-CTL clone. The cultures were pulsed with [3H]thymidine on day 6 and harvested on day 7 for assessment of proliferation. Figure 6Citation displays the ability of the 1Y2L-CTL clone to inhibit the proliferation of HLA-A2.1+ primary AML cells (>80%) but not HLA-A2.1+–differentiated cells from CD34+ progenitor cells (<20%). In summary, the 1Y2L-CD8+ CTL clone did not have a substantial impact on normal progenitor cell proliferation.



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  		Fig. 6. Proliferation inhibition of HLA-A2.1+ AML-PBMCs or normal HLA-A2.1+ CD34+ progenitor cells by CD33 1Y2L-CTL clone. The proliferation of AML-PBMCs ({blacksquare}) or normal HLA-A2.1+ CD34+ progenitor cells ({blacktriangleup}) after coincubation with irradiated 1Y2L-CTL clone was measured by [3H]thymidine incorporation on day 7 of culture. The CD34+ cell cultures contained G-CSF, GM-CSF, IL-3, and stem cell factor to induce proliferation and differentiation of the progenitor cells into the myeloid lineage. The results show that the 1Y2L-CTL clone inhibited the proliferation of AML-PBMCs but not the differentiated cells from the CD34+ progenitors. The CTLs, AML-PBMCs, and CD34+ progenitor cells cultured alone were used to determine the background proliferation. The percentage of proliferation inhibition is expressed as the mean ± SE from three independent experiments.

 
The Cytotoxic Activity of 1Y2L-CD8+ CTL Clone Is Granule Exocytosis-Mediated.
The contribution of either Fas/Fas ligand-mediated or the granule exocytosis pathway of cytotoxicity was examined with either Fas antibody or CMA that is an inhibitor of perforin-mediated cytotoxicity (20 , 21) . Viability staining with trypan blue exclusion showed that Fas antibody or CMA was not toxic to the effector cells. Figure 7Citation shows the cytotoxic activity of Fas antibody–treated, CMA-treated, or untreated CD8+ CTL clone against HLA-A2.1+ AML-PBMCs. The cytotoxic activity of the CTL clone was completely abrogated by the addition of CMA. In contrast, Fas antibody treatment did not reduce the activity of the clone against the AML-PBMCs. Thus, these results show that the main pathway of cytotoxicity for the 1Y2L-CD8+ CTL clone was mediated by granule exocytosis and not Fas/Fas ligand-dependent.



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  		Fig. 7. Mechanism of cytotoxic activity of CD33 1Y2L-CTL clone. CD8+ CTL clone treated with Fas antibody ({blacksquare}), CMA ({blacktriangleup}), or untreated (•) was evaluated for cytotoxicity against HLA-A2.1+ AML-PBMCs in a standard 51Cr-release assay. Treatment with CMA (500 nmol/L per well) but not with Fas antibody (1 µg/ml per well) inhibited the cytotoxic activity of the 1Y2L-CTL clone, indicating that the cytotoxicity is mediated through a granule-dependent mechanism. The percentage of specific lysis is expressed as the mean ± SE from three independent experiments.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CD33, a self-antigen that is highly overexpressed on AML blasts but not on pluripotent stem cells, offers a unique opportunity for developing an antigen-specific immunotherapy for AML. In previous studies, we identified an immunogenic HLA-A2.1–specific CD3365–73 peptide, AIISGDSPV, contained within the full-length of CD33 protein (364 amino acids) and have shown that this peptide is capable of generating CTLs against AML cells ex vivo (15) . We also developed a heteroclitic CD33 peptide, YIISGDSPV, from the native CD3365–73 peptide that displayed increased HLA-A2.1 affinity/stability and enhanced immunogenicity for generating CTLs against AML cells (15) . Therefore, newly engineered heteroclitic CD33 peptide with higher biological potency may prove more efficacious for evoking an immune response against AML.

The objective of our current study was to develop more effective analogs of the originally identified heteroclitic YIISGDSPV peptide (15) by conserving the tyrosine (Y) substitution at position 1 (P1) and examining the effects of additional AA substitutions on the immunogenicity against AML. First, peptides were designed to improve HLA-A2.1 affinity/stability by replacing key AA residues that affect MHC-peptide binding. On the basis of our understanding of distinct allele-specific peptide motif in MHC class I molecules and information on unique anchor residues specific to HLA-A2 (22, 23, 24) , we designed two peptides (YLISGDSPV, YIISGDSPL) by replacing residues at primary anchor position, P2 or P9 with leucine (L). Alternatively, six modified peptides with AA substitution at P3, P4, P5, P6, P7, or P8 were designed to enhance their interactions with the TCR on T cells. The AA residues at positions 3–8 within a peptide are predicted to contact the TCR (25, 26, 27) , and the substitutions of AA at those positions have been shown to mediate TCR affinity, alter T-cell differentiation, and induce secretion of cytokines not detected with cognate antigen recognition (28, 29, 30) . Thus, TCR can be triggered by heteroclitic peptide analogs that enhance T-cell stimulation by inducing immunologic functions not detected with the cognate ligand (28 , 31, 32, 33) . As an example, a heteroclitic peptide of carcinoembryonic antigen605–613 has been identified with aspartate substituting for asparagine at P6 (34) . Although the substitution did not increase the MHC class I binding of the carcinoembryonic antigen peptide, it did increase its immunogenic potency through altered interactions at the level of TCR, leading to enhanced ZAP-70 phosphorylation (35) . In our study, we developed peptides with modified residues at P3 (YIGSGDSPV), P4 (YIIIGDSPV, YIILGDSPV), P6 (YIISGISPV), P7 (YIISGDLPV), or P8 (YIISGDSWV) for the enhancement of TCR interaction. We avoided AA residues which cause reduced binding to HLA-A2.1 molecules such as aspartate (D) and glutamate (E) at P3; arginine (R), lysine (K), histidine (H) and alanine (A) at P4; proline (P) at P5; arginine (R), lysine (K) and histidine (H) at P7; aspartate (D), glutamate (E), arginine (R), lysine (K) and histidine (H) at P8; and arginine (R), lysine (K) and histidine (H) at P9 (36) . These modified CD33 peptide analogs were analyzed with GenBank and were shown not too overlapped with other peptide sequences derived from other normal proteins.

Among the eight variants of CD33 peptide examined, the modification of leucine (L) for isoleucine (I) at P2 or isoleucine (I) for aspartate (D) at P6 along with tyrosine (Y) at P1 resulted in the greatest increase in peptide HLA-A2.1 affinity from native CD33 peptide (Table 1)Citation . The improvement of HLA-A2.1 affinity for the 1Y6I peptide was unexpected because of P6 being a residue for interacting with the TCR (26 , 35) . This positive effect on HLA-A2.1 affinity results from conformational changes, repulsive electrostatic interactions, or reduced steric hindrance between P6 and its neighboring AA residues (37 , 38) . Additionally, X-ray crystallography studies of five viral peptides by Madden et al. (39) showed that the main and side chain conformations of each peptide are very different in the center of the binding sites, and these differences are accessible to direct TCR recognition, whereas the peptide termini and their second and COOH-terminal anchor side chains are bound in a similar manner.

Modifications that enhance peptide affinity to MHC or TCR are important; however, the critical contribution of modified peptide to the antitumor effect is reflected by the extent of the induced CTL-specific response. In these studies, increased peptide/HLA-A2.1 affinity did not always correlate with peptide immunogenicity or their ability to induce CTLs targeting AML cells. All six modified CD33 peptides (1Y3G, 1Y4I, 1Y4L, 1Y6I, 1Y7L, 1Y8W) designed for improved interaction with the TCR displayed increased HLA-A2.1 affinity but failed to induce augmented CTL cytotoxicity against AML. Conformational changes that affect a peptide’s affinity with TCR are the most likely cause of the poor immunogenicity of these peptides despite their increased HLA-A2.1 affinities. A seventh peptide, 1Y9L, modified for increased MHC binding also did not increase the immunogenicity of the peptide. Only the 1Y2L peptide, which showed the highest HLA-A2.1 affinity/stability, showed increased immunogenicity that is consistent with the observations of other investigators showing enhanced immunogenicity of heteroclitic CTL peptides through improved MHC binding (40 , 41) .

Of particular interest was the ability of the 1Y2L-CTLs to recognize native CD33 peptide, which is possibly presented on tumor cells. In cytotoxicity assays, the cross-reactivity of the 1Y2L-CTLs was shown against native CD33 peptide presented by autologous EBV-BLCL (Fig. 2)Citation . The high cytotoxicity of the 1Y2L-CTLs against EBV-BLCL–presenting native CD33 peptide (Fig. 3)Citation correlated with their enhanced killing of primary AML or an AML cell line, which would present the naturally occurring CD33 peptide (Fig. 1)Citation . The enhanced immunogenicity of the 1Y2L peptide was shown through its ability to stimulate HLA-A2.1–restricted- and AML–specific CTLs capable of killing of primary HLA-A2.1+/CD33+ AML PBMCs (Fig. 1)Citation .

A critical aspect in developing a CD33-based cellular immunotherapy is the potential for targeting normal CD34+/CD33+ progenitor cells, thereby having an affect on normal hematopoiesis. Jilani et al. (42) reported that the level of CD33 expression on CD34+ cells from AML patients was similar to levels observed on CD34+ cells from normal donors. The CD34+ cells used as target cells in our cytotoxicity and proliferation assays were isolated from G-CSF–mobilized normal donors and were therefore primed for differentiation into the myeloid lineage (43 , 44) . We did not observe a substantial lysis of the CD34+ cells by CD33 1Y2L-CTL clone (Fig. 5A)Citation . However, the results may be reflective of a low starting percentage (<15%) of CD33+ cells in the target population. To address this issue, the isolated CD34+ cells were induced to undergo proliferation and differentiation into the myeloid lineage by incubating them for 1 week with G-CSF, GM-CSF, IL-3, and stem cell factor (45 , 46) . The cultured cells contained >50% CD33+ cells (data not shown) and were therefore potential target cells for the CD33 1Y2L-CTL clone in the proliferation inhibition assay. The results showed that the CD33 1Y2L-CTL clones did not significantly inhibit (<20%) normal CD34+ cell proliferation, but they did significantly inhibit (>90%) the proliferation of the HLA-A2.1+/CD33+ AML-PBMCs (Fig. 5B)Citation . These preliminary results of limited targeting of normal CD34+ progenitor cells by the CD33-CTL clones are encouraging but will need to be further evaluated using CD34+/CD33+ cells from AML patients in future studies.

Donor lymphocyte infusions are currently used to reinduce remission of relapse after allogeneic stem cell transplantation. Although this strategy can be successful in some hematologic disorders, it is generally involved with graft-versus-host disease, which can cause morbidity and mortality in transplant recipients (47, 48, 49) . The availability of tumor-specific CTL clones could minimize graft-versus-host disease and mainly mediate the graft-versus-leukemia (GVL) effects. In the future, we plan to use the CD33 peptide-specific T-cell clones as a consolidation therapy to enhance a GVL effect in AML patients. Therefore, CD33 peptide-specific T-cell clones were obtained by limiting dilution and characterized in these studies. We found it interesting that we were able to isolate both CD8+ and CD4+ clones after stimulating T cells with HLA-A2.1–specific peptide. The CD4+ clones also showed cytotoxic activity against primary AML-PBMCs, but their activity was lower than that of the CD8+ clones (data not shown). The CD4+ clones may represent an important helper T-cell subset that is required to elicit long-lived immunity of CD8+ CTLs as shown by Knutson et al. (50) , but we have no evidence to support this conclusion in these studies. Unlike the CD4+ clone, the CD33 peptide-specific CD8+ clones expanded readily in our culture system providing sufficient cell numbers to characterize their specific cytotoxicity and the phenotype. The CD8+ clones displayed an activated/effector cell phenotype (CD69+/CD45RO+/CD45RA–) and also showed HLA-A2.1–restricted and antigen-specific cytotoxicity against AML cells.

Because nonmalignant CD33+ myeloid-lineage cells will be subject to recognition by redirected CTLs, the persistence of the adoptively transferred CD33-specific CTLs has the potential to result in prolonged immunity against myeloid-lineage cells. However, the in vivo persistence of those specific T cells may be short-lived or can be limited using CD33-CTLs by coexpression of a suicide gene, such as thymidine kinase of herpes simplex virus that would allow the elimination of the antigen-specific CTLs with ganciclovir treatment if necessary. The clinical squeal of temporary neutropenia and thrombocytopenia may be an acceptable side effect of CD33-directed immunotherapy, especially because prolonged ablation of normal CD33+ cells in patients receiving Mylotarg therapy does not seem to result in clinically substantial complications attributable to depleted numbers of normal CD33+ cells. We are currently developing animal models to evaluate the CTL clones on CD34+ cell engraftment and leukemia-ablative activity after adoptive transfer into SCID mice bearing AML xenografts.

In conclusion, we report here the YLISGDSPV heteroclitic CD33 peptide with immunotherapeutic potential that would allow the efficient targeting of AML cells. This novel engineered heteroclitic peptide has shown enhanced HLA-A2.1 affinity/stability along with increased immunogenicity for evoking CD33-specific CTLs against AML cells compared with the native AIISGDSPV or heteroclitic YIISGDSPV CD33 peptides (15) . On the basis of these studies, we believe that the heteroclitic YLISGDSPV peptide may be an attractive strategy for developing a vaccination or adoptive T-cell immunotherapy against AML.


   ACKNOWLEDGMENTS
 
We appreciate Drs. David Peace (University of Illinois, Chicago, IL), Ann LeFever (St. Luke’s Medical Center, Milwaukee, WI), Janet Plate (Rush University Medical Center, Chicago, IL), Kevin Colon (Rush University Medical Center, Chicago, IL), and Keith Knutson (University of Washington, Seattle, WA) for their helpful discussions. We also thank Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Okayama, Japan) and Dr. Jeffrey Molldrem (University of Texas M. D. Anderson Cancer Center, Houston, TX) for their kind gifts of cell lines.


   FOOTNOTES
 
Grant support: Coleman Foundation Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Jooeun Bae, 1400 VFW Parkway, Building 3, Room 2A111, Department of Medical Oncology, Dana-Farber Cancer Institute/VA Hospital, Harvard Medical Center, West Roxbury, MA 02132. Phone: 617-323-7700, ext. 6171; Fax: 617-363-5592; E-mail: jooeun_bae{at}dfci.harvard.edu

Received 2/19/04; revised 6/26/04; accepted 7/12/04.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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