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Clinical Cancer Research Vol. 11, 4460-4468, June 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Clinical

Combination of Imatinib Mesylate with Autologous Leukocyte-Derived Heat Shock Protein and Chronic Myelogenous Leukemia

Zihai Li1,3, Yi Qiao1, Bei Liu1, Elizabeth J. Laska2, Priyamvadha Chakravarthi3, Judith M. Kulko3, Robert D. Bona3, Min Fang3, Upendra Hegde3, Victor Moyo3, Susan H. Tannenbaum3, Antoine Ménoret1, Judy Gaffney3, Laura Glynn2, Carolyn D. Runowicz3 and Pramod K. Srivastava1,3

Authors' Affiliations: 1 Center for Immunotherapy of Cancer and Infectious Disease, 2 General Clinical Research Center, and 3 University of Connecticut Cancer Center, University of Connecticut School of Medicine, Farmington, Connecticut

Requests for reprints: Zihai Li, Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, 263 Farmington Avenue, Farmington, CT 06030-1601. Phone: 860-679-7979; Fax: 860-679-1265; E-mail: zli{at}up.uchc.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: To test the feasibility, safety, immunogenicity, and clinical efficacy of an autologous vaccine of leukocyte-derived heat shock protein 70-peptide complexes (Hsp70PC), in conjunction with imatinib mesylate, in patients with chronic myeloid leukemia (CML) in chronic phase.

Experimental Design: Patients had cytogenetic or molecular evidence of disease, despite treatment with imatinib mesylate for all except one patient, at the beginning of study. Hsp70PCs were purified from the leukopheresed peripheral blood mononuclear cells and were administered in eight weekly intradermal injections at 50 µg/dose without adjuvant. Clinical responses were assessed by bone marrow analysis before and after vaccinations. An IFN-{gamma} enzyme-linked immunospot assay was used to estimate the effect of treatment on natural killer cells and T cells against CML.

Results: Twenty patients were treated. The manufacturing of Hsp70PCs was successful and the administration was safe for all patients. Minimal or no side effects were reported. Clinical responses were seen in 13 of 20 patients as measured by cytogenetic analysis of bone marrow Philadelphia chromosome–positive cells in metaphases and/or, when possible, the level of Bcr/Abl transcript by PCR. Immunologic responses were observed in 9 of 16 patients analyzed, characterized by an increase in the frequency of CML-specific IFN-{gamma}-producing cells and IFN-{gamma}-secreting natural killer cells in the blood. A significant correlation between clinical responses and immunologic responses was observed.

Conclusions: Autologous Hsp70PC vaccination is feasible and safe. When combined with imatinib mesylate, it is associated with immunologic and possible clinical responses against CML in chronic phase.

Key Words: Tumor vaccine • chronic myeloid leukemia • heat shock protein 70 • NK cell • Dendritic cell

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of the hematopoietic stem cells with a balanced translocation between the long arms of chromosome 9 and 22, the Philadelphia (Ph+) chromosome (1, 2). This abnormality generates a Bcr/Abl chimeric gene that expresses an abnormal fusion protein with altered tyrosine kinase activity. Although allogeneic hematopoietic stem cell transplant remains the only proven curative therapy for CML (3, 4), Bcr/Abl tyrosine kinase inhibitor imatinib mesylate has emerged as the frontline therapy for CML in chronic phase (CML-CP) due to its remarkable tolerability and efficacy in inducing complete cytogenetic remission (CCR) in >75% of patients (5–7). However, imatinib mesylate rarely induces molecular remission of CML (8, 9). In addition, chronic treatment with imatinib mesylate might induce resistance to imatinib mesylate (10–12) and suppress cell-mediated immune responses (13–15), highlighting the need for developing other effective therapies for this disease that may be used in combination with imatinib mesylate.

There has been a significant interest in immunologic interventions in CML, in part because translocations of chromosome 9 and 22 generate a true tumor-specific antigen. Studies have shown that peptides spanning the junctional region of both Bcr/Abl and Abl/Bcr fusion proteins bind to human leukocyte antigen (HLA; refs. 16, 17). Vaccination of patients with Bcr/Abl breakpoint fusion peptides could generate specific immune responses (18). In addition, for patients who relapsed after transplant, donor lymphocyte infusion was effective in inducing remission, indicating a positive role for immune interventions (19). In patients treated with either IFN or transplant (20), the presence of CD8+ T cells against a myeloid antigen correlated significantly with cytogenetic remission of CML (20).

As molecular chaperones, heat shock proteins (HSP) play important roles in maintaining intracellular protein homeostasis (21, 22). Recent interest in using tumor-derived HSPs as tumor vaccines stem from the observation that HSPs, including Hsp70, chaperone antigenic peptides (23) and activate dendritic cells (24–26). The efficacy of the HSP-based vaccine was shown in several tumor models (27–29) and evidence consistent with clinical efficacy has been seen in several clinical trials (30, 31). However, no human studies have been done to address the roles of HSP vaccines against leukemias.

We have determined the feasibility and safety of autologous Hsp70-peptide complex (Hsp70PC) in conjunction with imatinib mesylate for 20 patients with CML-CP. Clinical and immunologic responses were measured. We show here that vaccination with autologous Hsp70PC is feasible and safe for CML patients. In addition, we show that most immunized patients developed a robust immunologic response against CML, despite the recent appreciation of immunosuppressive effects of imatinib mesylate (13–15).


   Materials and Methods
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Patients. Eligible patients were ages >18 years and had been diagnosed with Ph+ CML-CP. Chronic phase was defined by the presence of <15% blasts, <20% basophils, and <30% blasts plus promyelocytes in the peripheral blood or bone marrow. All patients had persistent diseases, as shown by the lack of CCR (n = 16) or the lack of molecular remission for patients with CCR (n = 4), despite imatinib mesylate. Other eligibility criteria include Eastern Cooperative Oncology Group performance score of <2, absence of other serious illnesses, including inadequate renal or hepatic function, and lack of active therapy with immunosuppressants, IFN, or other cytotoxic agents.

The study protocol was reviewed and approved by the U.S. Food and Drug Administration and by the University of Connecticut Institutional Review Board. A informed consent form was obtained from all patients.

Study design and treatments. This was a single-institution, prospective trial that was conducted in an outpatient setting. Leukocytes were collected by pheresis; Hsp70PCs were isolated from them. The vaccine was injected weekly without premedication at 50 µg/dose intradermally for 8 weeks. Clinical responses were evaluated 6 to 18 weeks before the first vaccine ("pre"), 1 week after the fourth vaccine ("mid"), and 2 weeks after the eighth vaccine ("post"). The immunologic responses were evaluated at the similar period, except that the pre-vaccination refers to 1 week before the first vaccine. In some cases, the collection of clinical information continued after completion of the study, with permission from patients.

End points. The primary end points were the feasibility of Hsp70PC production and vaccine-related toxicity. All patients had complete physical examinations, blood counts and chemistries, electrocardiogram, and chest X-ray pre-, mid-, and post-vaccinations. Toxicities were assessed and graded per National Cancer Institute Common Toxicity Criteria version 2.0. Secondary end points were the rate of immunologic and clinical responses. Clinical response was defined by the cytogenetic study of bone marrow cells and/or, when possible, for the Bcr/Abl translocation by fluorescence in situ hybridizations and for the level of Bcr/Abl transcript by the Bcr/Abl-specific PCR of cDNA after reverse transcription of mRNA using oligo(dT) primers [reverse transcription-PCR (RT-PCR)]. The quantitative PCR (Q-PCR) was done by the Cytogenetics Laboratory at the Oregon Health and Science University (Portland, OR).

Preparation of heat shock protein 70-peptide complex. Leukocytes were washed with sterile PBS and Dounce homogenized as described (32). Briefly, Hsp70PC was purified using ADP-affinity chromatography followed sequentially by Blue-Sepharose column to remove serum albumin and by DEAE anion exchange chromatography. Purified Hsp70PCs were resuspended in PBS, tested by SDS-PAGE, and immunoblotted using antibodies specific for various HSPs as described (32). The vaccine was then filtered through 0.2 µm membrane, tested for sterility and endotoxin levels, and frozen at –80°C in 50 µg aliquots for future use.

Evaluation of natural killer and T cells. To minimize interexperimental variations, all blood samples were initially cryopreserved and later analyzed by enzyme-linked immunospot (ELISPOT) assay at the same time on the same plate, with the same batch of reagents, by the same researcher (B.L. or Y.Q.). Briefly, ELISPOT plates (Millipore, Billerica, MA) were coated with IFN-{gamma}-specific capture antibody (clone 1-DK, Mabtech, Nacka, Sweden) and blocked with 10% FCS (Life Technologies, Carlsbad, CA) followed by incubation with peripheral blood mononuclear cells (PBMC; 1 x 106 per well) and stimulators, in triplicates, for 2 days. In some experiments, PBMCs were further separated into CD56+ and CD56– populations using MACS column conjugated with antibody against CD56 (Miltenyi Biotech, Auburn, CA). Simulators included irradiated autologous leukocytes (2 x 105 per well), Hsp70PC with and without ATP treatment (25 µg/mL; refs. 33, 34), intact K562 cells (2 x 105 per well), K562 lysate prepared by rapid freeze and thaw for four times (5 x 105 cells equivalent per well), or phytohemagglutinin (10 µg/mL, Sigma-Aldrich, St. Louis, MO). The biotinylated anti-human IFN-{gamma} antibody (clone 7-B6-1, Mabtech) and avidin-peroxidase complex (Vector Laboratories, Burlingame, CA) were then added sequentially followed by washing and development using 3-amino-9-ethylcarbazole and H2O2 (Sigma-Aldrich). The spots were counted using a computer-assisted ELISPOT image analyzer (Immunospot, Cleveland, OH) and the numbers of spots are expressed as the mean of triplicates, with the error bar indicating the SEs. Flow cytometry (35) and 51Cr release assay (36) were done as described.

Definition of responses. Based on the percentage of bone marrow Ph+ cells, the cytogenetic responses are categorized as no (100% Ph+), complete (0% Ph+), major (1-34% Ph+), or minor (35-99% Ph+) response. Responsiveness to imatinib mesylate was assessed only if patients were on imatinib mesylate at ≥400 mg/d for >5 months. In some cases, quantitations of Bcr/Abl+ cells by fluorescence in situ hybridization were done to assess residual disease. For patients with CCR at time of vaccinations, clinical responses were determined by PCR. Response by qualitative PCR was determined if there was a conversion from positive value (RT-PCR+) to negative value (RT-PCR–). Q-PCR value is based on the ratio of Bcr/Abl transcript over the transcript of glucose-6-phosphate dehydrogenase. A response is determined if there was a reduction of the ratio by 3.5-fold at mid- or post-vaccination study. This was based on the consideration that anything less than 3.5-fold changes could simply be a result of laboratory variations.4

Statistical analysis. Student's t test was used to compare the difference between pre- and post-vaccination immune responses. The correlation between immunologic and clinical responses was analyzed by the {chi}2 test. Ps < 0.05 were considered to represent statistically significant differences.


   Results
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 Materials and Methods
 Results
Discussion
 References
 
Patient characteristics. A total of 20 patients were treated on study, and their pre-vaccination characteristics are presented in Table 1. At the time of entry into the study, the patients had a median age of 47.5 years (range, 31-61 years). There were 8 male and 12 female patients. The median duration of disease was 18 months (range, 2-148 months). Fourteen were low risk by Hasford score estimation (37) and 2 were intermediate risk. We were unable to determine the score in four patients. All but one patient were on ≥400 mg/d imatinib mesylate. The median duration of imatinib mesylate before vaccination was 15 months. Nine (45%) patients had no or minor cytogenetic response to imatinib mesylate before study entry, including four patients on >400 mg/d imatinib mesylate. All 20 patients had persistence of Bcr/Abl translocation by either cytogenetics or PCR analysis while on imatinib mesylate.


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  		Table 1. Baseline characteristics of the 20 patients in the study

 
Twenty-five patients signed consent to be screened for the study. Four were ineligible due to additional new chromosomal changes in the bone marrow that were not present at the diagnosis. Twenty-one patients enrolled in the study; however, one patient did not complete the vaccinations due to her decision to be treated by an allogeneic stem cell transplant. The 20 treated patients completed the planned number of vaccinations, and the schedule of treatments was not interrupted. Imatinib mesylate dose was maintained for all patients, except patient 391018 whose imatinib mesylate dosage was changed from 400 to 600 mg/d before the third vaccinations. One additional patient was intolerant to imatinib mesylate; therefore, the patient was treated with Hsp70PC alone.

Vaccine characterization. Leukapheresis was done for 21 patients, with a collection of a median number of cells per patient of 16.7 x 1010 cells (range, 5 x 1010-39.5 x 1010). The total amount of Hsp70PC prepared for each patient ranged from 300 to 1,900 µg (median, 950 µg) or from 20.8 to 270 µg/1010 cells (median, 47 µg/1010 cells). The purified Hsp70 from each patient was subjected to SDS-PAGE followed by a silver staining, which showed a predominant single 70-kDa band (Fig. 1). Immunoblot analysis using non-cross-reactive antibodies showed that Hsp70PC contains about a similar amount of Hsp70, the inducible member of the Hsp70 family, and Hsc70, the constitutive member. Grp78, the endoplasmic reticulum member of Hsp70 or immunoglobulin heavy chain binding protein (Bip) was not present. No serum albumin, a major concern of contamination, was detected by immunoblot. The endotoxin content in all cases was <0.2 EU/mL as measured by the Limulus Amebocyte Lysate assay. Hsp70PC was resuspended in PBS for intradermal injection in a volume of 0.1 to 2.2 mL without the addition of any exogenous adjuvant.



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  		Fig. 1. Biochemical characterization of Hsp70PC vaccine. A, a total of 1 µg Hsp70PC preparations at the last step of purifications were resolved on 10% SDS-PAGE followed by a silver staining of the gel. The molecular weight marker was not shown. B, proteins from (A) were transferred onto nylon membrane and immunoblotted for Hsp70, Hsc70, Grp78, and albumin using respective monoclonal antibodies.

 
Toxicity. A total of 160 injections of Hsp70PC were administered. No patients had any adverse reactions during the 30-minute post-injection monitoring period. No cardiac or pulmonary toxicities were observed. Grade 1 toxicities included four patients with injection site soreness, erythema, and pruritus; four patients with pruritic rash; and one patient with chills, myalgia, and other flu-like symptoms. All of these symptoms resolved spontaneously within 24 hours. No signs of autoimmune diseases were reported nor any changes in laboratory measures of renal or hepatic function. No patients had any significant changes of serum immunoglobulin levels (data not shown). Extensive immunophenotypic analysis by flow cytometry revealed no major alterations of the main subset of WBC, including T cells (CD4 and CD8), B cells (CD19), natural killer (NK) cells (CD56), and antigen-presenting cells (CD11c), after four or eight consecutive injections of Hsp70PCs. In addition, costaining with CD62L, CD69, and CD45RB for T cells revealed no significant changes of memory T-cell populations in PBMCs (data not shown).

T-cell responses to individually specific as well as shared chronic myeloid leukemia antigens. To determine if Hsp70PC vaccine stimulated antigen-specific T-cell responses, we did ELISPOT analysis to enumerate IFN-{gamma}-producing cells in the PBMCs of the immunized patients. We aimed to determine T-cell responses against any antigens specific to the individual patient's CML as well as any antigens common to all CML cells. For the former, we tested the ELISPOT reactivity of pre-, mid-, and post-vaccination PBMCs against pre-vaccination autologous leukocytes, which contain the Bcr/Abl-expressing cells. This would allow measurement of T-cell response against Bcr/Abl-derived epitopes as well as epitopes that may be uniquely expressed by individual patient's CML cells. All data points were available for 16 patients; of these, 9 patients showed significantly increased numbers of IFN-{gamma}-producing cells (P < 0.05) at mid- or post-vaccinations or at both time points compared with the corresponding pre-vaccination numbers (Fig. 2A). For four patients, data were available at only two time points; of these, two patients showed significant post-vaccination responses (data not shown).



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  		Fig. 2. Vaccination with Hsp70PC leads to increased T-cell activity in the peripheral blood. A, pre-vaccination leukocytes were irradiated, washed, and incubated with PBMCs collected 1 week before the first vaccine (pre), 1 week after the fourth vaccine (mid), and 2 weeks after the last vaccine (post) for the enumeration of the frequency of IFN-{gamma}-producing cells. N/A, data points not available due to insufficient cells. *, P < 0.05; **, P < 0.005. B, cellular antigens from Bcr/Abl+ K562 cells were prepared by lysing the cells with four cycles of freeze and thaw followed by removal of nuclei. The 5 x 105 K562 cell equivalent lysates were then incubated with 1 x 106 PBMCs collected pre-, mid-, and post-vaccinations for the enumeration of the frequency of IFN-{gamma}-producing cells by ELISPOT. N1, N2, and N3, results from three normal individuals. Nine patients with significant post-vaccination increases of IFN-{gamma}-producing cells were shown. *, P < 0.05; **, P < 0.005. C, same as in (B), except that W6/32 or control antibody was added. Representative wells are shown at the bottom. D, IFN-{gamma} spots in response to Hsp70PC or peptide-free Hsp70 in vitro. E, immune response in a patient who received Hsp70PC vaccination alone.

 
To detect immunologic responses to antigens shared between all CMLs, we stimulated PBMCs with K562 cell lysates. Derived from a patient with CML, K562 cells do not express MHC class I or II molecules (38, 39) and hence are not expected to trigger major alloreactive T-cell responses. Indeed, when PBMCs from normal subjects were incubated with K562 lysates, an insignificant number of IFN-{gamma} spots were detected (Fig. 2B). We analyzed PBMCs from 16 of 20 patients in the K562 assay due to insufficient number of PBMCs from the remaining 4 patients. Eleven patients showed post-vaccination increases in the frequency of IFN-{gamma}-producing cells against antigens in K562 lysates (Fig. 2B). Further, the reactivity was largely mediated by CD8+ T cells, as incubation with a blocking antibody against HLA class I W6/32 abrogated IFN-{gamma} production (Fig. 2C).

We used a third assay to detect peptide-specific T-cell response. In this re-presentation assay, which were used by several murine and human systems before (30, 31, 40, 41), a HSP binds antigen-presenting cells and is internalized followed by re-presentation of the HSP-chaperoned peptides by the MHC I of the antigen-presenting cell. The MHC I-peptide complex stimulates cognate CD8+ T cells whose stimulation can be read by IFN-{gamma} production. In addition, we took advantage of the fact that ATP treatment of Hsp70PC leads to dissociation of peptides from Hsp70 (42, 43). We observed that peptide-bound Hsp70 stimulated IFN-{gamma} production (Fig. 2D). Removal of peptides resulted in a significant reduction but not complete elimination of IFN-{gamma} production, indicating that Hsp70 is immunostimulatory for PBMCs in both peptide-dependent and peptide-independent manners.

The data thus far derive from patients who were treated with imatinib mesylate plus Hsp70PC. We were in a fortuitous position to determine, anecdotally, the T-cell response to Hsp70PC alone in a patient who was intolerant to imatinib mesylate due to prior grade 3 hepatotoxicity. As shown in Fig. 2E, this patient (391016) showed statistically significant augmentation of ELISPOT response to autologous CML cells, to common CML antigens (as determined by the K562 lysate assay), and to Hsp70PC itself in a manner that is temporally associated with vaccination.

Enhancement of natural killer activity but not numbers in response to vaccination. NK cell activity is critical in the effector phase for tumor rejection after HSP vaccinations (27). Furthermore, Hsp70 has been shown to directly bind to NK cells and induce cytolytic activity of NK cells (44). Phenotypical analysis of the PBMCs, however, did not indicate an increased number of CD56high+ NK cells after the Hsp70PC vaccinations (data not shown). To understand if Hsp70PC vaccines have resulted in the functional activation of NK cells, we developed an ELISPOT assay to measure IFN-{gamma}-secreting capacity of NK cells by incubating unfractionated PBMCs or CD56+ cells with K562 whole cells. K562 cells have been widely used for measuring cytolytic activity of NK cells (36, 45). We found that K562 cells can trigger IFN-{gamma} release from PBMCs that are independent of class I molecule of HLA (Fig. 3A, left). Such an activity is mainly due to CD56+ cells as IFN-{gamma}-secreting cells resides mainly in magnetically purified CD56+ cells but not CD56– cells (Fig. 3A, right). As a further control for specificity, we incubated CD56+ cells with a HLA class I–expressing and NK-resistant human colon cancer cell line HCT116. No IFN-{gamma} release was elicited (data not shown). Using this assay, we found that normal subjects have 340 (range, 154-580; n = 11) functional NK cells in the 1 x 106 PBMCs. In contrast, 7 of 16 CML patients had significantly decreased NK activity, with pre-vaccination NK cells of only 16 to 107 per 1 x 106 PBMCs. After the vaccinations, the majority of patients (10 of 16) had an increase in NK activity as shown by the greater frequency of IFN-{gamma}-producing cells against K562 (Fig. 3B). The increased NK activity was apparent even after four vaccines. In most patients (8 of 10), the increased activity of NK cells persisted after eight vaccinations.



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  		Fig. 3. Vaccination with Hsp70PC leads to increased NK cell activity in the peripheral blood. A, PBMCs, CD56+ cells, or CD56– cells from one patient (391021, mid-vaccination) were incubated with K562 whole cells, in the presence or absence of HLA class I blocking antibody, for 40 hours. The frequency of IFN-{gamma}-producing cells was measured by ELISPOT. B, intact K562 cells were incubated with 1 x 106 PBMCs collected pre- and post-vaccinations for the enumeration of the frequency of IFN-{gamma}-producing cells by ELISPOT. *, P < 0.05; **, P < 0.005.

 
Clinical analyses. The clinical focus of our study is on vaccine-related toxicity. However, cytogenetic analyses of bone marrow cells were done on all patients, as appropriate and as indicated, before, during, and at the end of the treatment. All but one patient remained in chronic phase at the end of the study (Table 2). Of the 16 patients with cytogenetic evidence of disease pretreatment, 9 had reductions of Ph+ cells by cytogenetic studies, including 7 with CCR, 1 with major response, and 1 with minor response (Tables 2 and 3). Four patients had only molecular evidence of disease at the beginning of the study. Of these, one patient had a negative Bcr/Abl level by RT-PCR and three patients had a reduction of Bcr/Abl transcript level by 3.6-, 7.5-, and 11-fold, respectively, by Q-PCR. Altogether, 13 of 20 patients derived clinical benefit after treatment with Hsp70PC plus imatinib mesylate; 7 of 20 patients did not derive clinical benefit, including 1 patient with emergence of t(3,21) (Table 3).


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  		Table 2. Disease status of the study patients before and after therapy

 

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  		Table 3. Summary of clinical responses to imatinib mesylate and the Hsp70PC vaccine

 
We looked for possible correlations between immunologic and clinical responses. Although multiple immunologic variables were measured, only the frequency of IFN-{gamma}-producing cells in response to pre-vaccination leukocytes was done for all patients for at least two time points. Sixteen of 20 patients had data at three time points (Fig. 2). These immunologic variables were compared with the clinical responses (Table 4). It was observed that 8 of 11 clinical responders had a significant increase of IFN-{gamma}-producing cells, whereas only 1 of 5 clinical nonresponders showed such immunologic activity. The correlation between clinical and cellular immune responses was statistically significant (P = 0.048; Table 4).


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  		Table 4. Correlation between immunologic responses and clinical responses

 

   Discussion
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 Materials and Methods
 Results
 Discussion
References
 
There is extensive murine data on the use of tumor-derived Hsp70PC as a prophylactic and therapeutic cancer vaccine (27, 46, 47). We have now completed the first human clinical trial on the use of autologous Hsp70PC vaccines against CML. We show that Hsp70PC can be prepared for all patients with CML-CP and the vaccine is associated with minimal toxicity. As seen in studies with the murine systems (24, 26, 46, 48), we have now observed that Hsp70PC vaccination associates with both innate immunity (NK cells) and adaptive immunity (CD8+ T cells) against CML in the human setting. In addition, treatment of CML patients with the combination of Hsp70PC and Bcr/Abl tyrosine kinase inhibitor imatinib mesylate has resulted in significant clinical responses. Our data on the correlation of immunologic responses with clinical responses, and the fact that a large proportion of patients who showed clinical responses had been on imatinib mesylate alone previously, suggest but by no means prove that vaccination with Hsp70PC vaccine has contributed to the overall therapeutic values against CML-CP. This premise is examined more closely and critically in the following paragraphs.

This study was initiated in 2001, when the clinical experience with imatinib mesylate was rather limited. The optimal dosage of imatinib mesylate and the kinetics of responsiveness of patients to imatinib mesylate became only clearer later (49). Therefore, the dosage and duration of imatinib mesylate treatment were not defined stringently when our study was designed. This was also due to the fact that the primary end point of the study was to assess feasibility and safety of Hsp70PC and not to determine its clinical efficacy. Nevertheless, several observations are of note. First, the combination of Hsp70PC and imatinib mesylate seems to be most effective early in the treatment of CML. Three patients fell into this category; all enjoyed a rapid CCR, including one patient with complete molecular responses by Q-PCR several months later (Table 2). Second, although clinical response at the end of the study is an excellent predictor for the future clinical course of CMLs, delayed clinical responses may be interesting to follow, in light of the immunologic activity. Two patients, who did not have significant clinical responses immediately after Hsp70PC vaccinations, showed responses at later time points. Third, the inclusion of Hsp70PC into the treatment was most beneficial to patients who had obtained partial clinical benefit from imatinib mesylate alone. No clinical responses were seen in three patients who were refractory to imatinib mesylate evidenced by lack of any cytogenetic responses after prolonged (>2 years) therapy with imatinib mesylate at ≥400 mg/d. Collectively, these data suggest that a more controlled study with Hsp70PC and imatinib mesylate in CML-CP patients is worth pursuing.

Three types of immune response were seen in this study: individual patient–specific immune responses, CML-specific immune responses, and NK responses. The individual patient–specific immune responses were assessed by incubating post-vaccination PBMCs with autologous leukocyte targets harvested before vaccinations (Fig. 2). It is to be noted that, although the proportion of Ph+ cells in this material varied significantly, all except four patients had at least 5% Ph+ cells in pre-vaccination leukocytes, which apparently provided enough antigens in stimulating IFN-{gamma} release from post-vaccination PBMCs. We further showed that in 9 of 16 vaccinated patients but not in any normal subjects there were HLA class I–dependent recognitions of antigens expressed by a CML cell line K562. The difference in the magnitude of IFN-{gamma} release in response to two different targets (pre-vaccination leukocytes versus K562 lysates) by some patients could be due to the difference in the amount/compositions of CML-specific antigens present in the two sources of antigens. Collectively, our data suggest that Hsp70PC vaccine was able to elicit CML-specific immune responses.

Our study also pointed out that the peptide-independent activity of Hsp70 might be due to its ability to activate NK cells. Indeed, NK activity was induced in most patients. Although Hsp70 has been shown to activate NK cells directly in some systems (44), we are not able to observe any direct effect of Hsp70PC on NK cells from CML patients in vitro (data not shown). Although the underlying mechanism is unclear, it is plausible that the NK responses, as a result of combined therapy of imatinib mesylate and Hsp70PC vaccine, may have protective values against CML. This thought, derived from a small sample of clinical data, required further experimental and clinical exploration.

Immunologic effects of imatinib mesylate should also be considered. Understanding of the immunomodulating properties of imatinib mesylate is just beginning. It was shown recently that imatinib mesylate was able to restore the normal differentiation of plasmacytoid dendritic cells in patients with CML (50). However, imatinib mesylate also has a potent in vivo inhibitory effect on the roles of Flt3 ligand in inducing dendritic cell expansion in mice (13). Consequently, Flt3 ligand-mediated antitumor effects were severely hampered by imatinib mesylate. Furthermore, imatinib mesylate inhibits differentiation and maturation of dendritic cells from human CD34+ progenitors (15). Additionally, exposure to imatinib mesylate inhibited the induction of primary T-cell responses (14). The data suggest that imatinib mesylate has previously unappreciated properties to impair immune response in vivo. These considerations raise a possibility that the immunologic and clinical responses seen in this present study may have underestimated the contribution of Hsp70PC. Of note, one patient was treated with Hsp70PC alone because the patient was off imatinib mesylate for ~2 years due to prior severe liver toxicities. Both NK activity and CML-specific cellular activity were shown (Fig. 2F). However, we wish to emphasize that more non–imatinib mesylate–treated patients are necessary to understand the effect of Hsp70PC alone on the immune response. Additionally, the observed clinical and immunologic responses in our study cannot be definitely attributed to the combination of imatinib mesylate and Hsp70PC vaccine. The contribution of Hsp70PC vaccine can only be addressed by future controlled clinical trials when imatinib mesylate alone is directly compared with imatinib mesylate in conjunction with Hsp70PC.

The availability of imatinib mesylate has clearly revolutionized the treatment of CML-CP. However, the cure of CML as defined by molecular remission is elusive (8, 9). In addition, primary imatinib mesylate resistance and relapse after an initial response to imatinib mesylate have been reported (10–12). These considerations indicate a need for therapies complementary to imatinib mesylate in the treatment of CML. Our current data on the ability of Hsp70PC to elicit CML-specific immunity, coupled with its extensive scientific and preclinical pedigree, indicate that Hsp70PC is a reasonable candidate for further clinical development in the treatment of CML.


   Acknowledgments
 
We thank Tracy Urso for isolating PBMCs, Chris Abreu and Jie Dai for performing immunophenotypic analyses of PBMCs, Diahann Wilcox and Judy Fox for bone marrow biopsy, Janet Li for secretarial assistance, and Susan Wasik and Priscilla Adler for administrative support.

This study is dedicated to all the participating patients.


   Footnotes
 
Grant support: NIH grants CA90337 (Z. Li) and MO1 RR06192 (General Clinical Research Center, University of Connecticut). P.K. Srivastava is supported by NIH grant CA84479 and a sponsored research agreement with Antigenics, Inc., in which he has a significant financial interest.

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.

4 R. Press, personal communication. Back

Received 2/ 2/05; revised 3/11/05; accepted 3/24/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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