- Email: [email protected]
Carcinoembryonic Antigen Peptide-Pulsed Dendritic Cells in Patients with Metastatic Cancer
c urrent trials Carcinoembryonic Antigen Peptide-Pulsed Dendritic Cells in Patients with Metastatic Cancer H. Kim Lyerly and Michael A. Morse Study Coordinator Doris Coleman
Rationale Because of high mortality rate of metastatic cancers and significant toxicity of chemotherapy and radiotherapy, new treatment approaches such as immunotherapy warrant evaluation. The basis for modern approaches to immunotherapy is the observation that many tumors possess antigens that can be recognized by antigen-specific T cells leading to tumor rejection. The recognition of tumor-associated antigens is generally MHC class I-restricted1 and has been localized to eight to ten amino acid peptide fragments. These fragments bind in the groove of the MHC complex where they are presented to T lymphocytes that express receptors specific for the particular peptide. Among the described tumor antigens is carcinoembryonic antigen (CEA), expressed on most gastrointestinal adenocarcinomas, 50% of breast cancers, and 70% of non–small-cell lung cancers.2 The immunogenicity of CEA in man has been established in studies where patients have been immunized with recombinant, human CEA along with GM-CSF3, vaccinia vectors containing the gene for CEA4, and anti-idiotype specific for CEA.5 Peptide fragments of CEA have been found to be epitopes for T-cell lines generated from patients preSubmitted: February 15, 1999
Accepted: May 30, 1999
Clinical Lung Cancer, Vol. 1, No. 1, 70-72, 1999.
70
August, 1999
viously vaccinated with vaccinia-CEA. The most potent peptide in this study, designated CAP-1, is an HLA-A2 restricted CTL epitope.4 Because naive precursors of cytotoxic T cells specific for tumor antigens are present in very low numbers, methods for stimulating the T cells are necessary. Approaches have included viral vectors, administration of antigen with adjuvants and gene-transduced tumor cells. A more recent development has been the emerging understanding of the critical role that antigen-presenting cells, such as dendritic cells (DC), play in inducing T and B-cell responses in vivo. Numerous studies have demonstrated that DC loaded with tumor antigen can induce T-cell responses in vitro and in vivo murine models without significant toxicity. We and others have developed methods for generating a sufficient number of DC in vitro for use in immunotherapy studies.6-9 Furthermore, we have demonstrated that these DC loaded with CEA and other peptides are capable of inducing CEA specific T-cell responses in vitro.10 Therefore, we hypothesize that DC loaded with tumor-associated peptides such as the CEA peptide CAP-1 will be safe and induce CAP-1 specific Tcell responses in vivo in humans with metastatic cancers. Our objectives are: a)
Dendritic Cell Photomicrograph: Courtesy of Dr. Joseph Fay, Baylor University Medical Center, Dallas, TX
to determine the safety and dose-limiting toxicity of intravenous and intradermal injections of autologous, DC pulsed with an HLA-A2 restricted CEA peptide; b) to evaluate cellular immune response to the CEA peptide.
Treatment Plan All components of the treatment plan must be performed at Duke University Medical Center (DUMC). a) Active Immunotherapy Dose Level Assignment: Patients will be entered into a dose escalating study at the next available dosage slot.
Address for correspondence: H. Kim Lyerly, Department of Surgery, Box 2606, Duke University Medical Center, Durham, NC 27710, Phone: 919-681-3480, FAX: 919-681-7970, email: [email protected]
Table 1:
Eligibility
numbers of cells for immunologic tests.
Inclusion Criteria
Exclusion Criteria
Age ≥ 18 years
Chemotherapy, radiation therapy, or immunotherapy within the past 4 weeks
histologically confirmed Metastatic adenocarcinoma expressing CEA as defined by immunohistochemistry
History of CNS metastases. (Pre-enrollment head CT no required.)
Measurable or evaluable disease (includes CEA level elevation)
History of anto-immune disease
KPS > 80%
Serious intercurrent chronic or acute illness considered by the P.I. to constitute an uwarranted high risk for investigational drug treatment
HLA=A2
Second malignancy (within the past 10 years) other than non-melanoma skin cancer
WBC ≥ 3000/mm3 and absolute lymphocyte count ≥ 1000/mm3; Hgb ≥ mg 9 /dl; platelets ≥ 100,000/mm3; PT < 1.25 x nl, PTT < 1.66 x nl; fibrinogen > 0.75 ng; g)
Presence of HIV infection or viral hepatitis
Creatinine < 2.5 mg/dl; bilirubin < 2.0 mg/dl
b) Leukapheresis: Leukapheresis will consist of up to a 41/2 hour collection of 10-14 liters. Each collection should contain a minimum of 1 x 108 nucleated cells/recipient kg. c) Generation, CEA-Peptide Pulsing and Cryopreservation of DC: Peripheral blood mononuclear cells will be separated from the leukapheresis product on a cell separator, resuspended in AIM V media, and plated onto tissue culture flasks. The adherent cells will be cultured for 7 days in GM-CSF and IL-4 containing AIM V media to generate dendritic cells. Harvested DC will be pulsed with CAP-1 peptide for 4 hours, sterility tested, and then cryopreserved. d) Administration of Peptide-Pulsed DC: Doses will be administered intravenously and intradermally on weeks 0, 2, 4, and 6 for 4 complete treatments at DUMC in an outpatient unit. Premedication will not be routinely given. The cell preparation will be delivered over 2-3 minutes into an intravenous line (via arm, vein, or central line) in which 0.9% saline is running at 250 mL/hr. The intradermal dose will be administered in a volume of 100 μL.
Immunosuppressive (including, oral or IV steroids) in the prior 6 wks.
Following administration, blood pressure and pulse monitoring will be performed over a 1-hour observational period. e) Repeat Leukapheresis: Will be performed during week 8 to obtain large
Long-Term Evaluation and FollowUp (Weeks 12+) Patients who have disease progression will be removed from the study. Those who remain on study will be asked to maintain scheduled long-term follow-up at DUMC. The following parameters listed in Table 4 will be evaluated every month for six months, every three months for the first year following the study and then every year.
Anticipated Toxicities Based on previous studies of DC immunizations, the anticipated toxicities are minimal. Theoretical risks include: allergic reactions such as fever, hives, or rash, pulmonary compromise, and accelerated procoagulant activity, although these have not been reported. Induction of auto-immunity especially to granulocytes (which express an antigen with 40% homology to CEA) and the colon which has small amounts of CEA are theoretically possible.
August, 1999
71
Carcinoembryonic Antigen Peptide-Pulsed Dendritic Cells Table 3:
Short Term Treatment Evaluation (Weeks 0-12)
Interval history and complete physical examination Blood chemistry and hematology CBC with differential and platelet count. BUN, Na, Ka+, Cl, CO2, Gluc, Crea, TP, Alb, Ca, Phos, uric acid, AST, AlkP, T Bili, ALT; Pt, PTT and fibrinogen Serum CEA (week 8) Immunologic assays as above (week 8) To be performed at DUMC on weeks 0, 2, 4, 6, 8, 12
Table 4:
Long-Term Evaluation and Follow-Up (Weeks 12+)
Interval history and complete physical examination Blood chemistry and hematology CBC with differential and platelet count. BUN, Na, Ka+, Cl, CO2, Gluc, Crea, TP, Alb, Ca, Phos, uric acid, AST, AlkP, T Bili, ALT; Pt, PTT and fibrinogen Serum CEA Available radiographs 8. Romani N, Reider D, Heuer M, et al. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods 1996;196:137-151. 9. Morse MA, Zhou LJ, Tedder TF, et al. Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha for use in cancer immunotherapy. Ann Surg 1997;226:6-16. 10.Wong C, Morse M, Nair SK. Induction of primary, human antigen-specific cytotoxic T lymphocytes in vitro using dendritic cells pulsed with peptides. J Immunother 1998;21:32-40.
72
August, 1999
References
1. Darrow T, Slingluff C, Jr., Seigler H. The role of HLA class I antigens in recognition of melanoma cells by tumor-specific cytotoxic T lymphocytes. Evidence for shared tumor antigens. J Immunol 1989;142: 33293335. 2. Thompson JA, Grunert F, Zimmermann W. Carcinoembryonic antigen gene family: molecular biology and clinical perspectives. J Clin Lab Anal 1991; 5:344366. 3. Samanci A, Yi Q, Fagerberg J, et al. Pharmacological administration of granulocyte/macrophage-colony stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen. Cancer Immunol Immunother 1998; 47:131-142. 4. Tsang KY, Zaremba S, Nieroda CA, et al. Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst 1995;7:982-990. 5. Pervin S, Chakraborty M, Bhattacharya-Chatterjee M, et al. Induction of antitumor immunity by an antiidiotype antibody mimicking carcinoembryonic antigen. Cancer Res 1997;57:728-734. 6. Romani N, Gruner S, Brang D, et al. Proliferating dendritic cell progenitors in human blood. J Exp Med 1994; 180:83-93. 7. Bender A, Sapp M, Schuler G, et al. Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. J Immunol Methods 1996;196:121-135.
Rationale Because of high mortality rate of metastatic cancers and significant toxicity of chemotherapy and radiotherapy, new treatment approaches such as immunotherapy warrant evaluation. The basis for modern approaches to immunotherapy is the observation that many tumors possess antigens that can be recognized by antigen-specific T cells leading to tumor rejection. The recognition of tumor-associated antigens is generally MHC class I-restricted1 and has been localized to eight to ten amino acid peptide fragments. These fragments bind in the groove of the MHC complex where they are presented to T lymphocytes that express receptors specific for the particular peptide. Among the described tumor antigens is carcinoembryonic antigen (CEA), expressed on most gastrointestinal adenocarcinomas, 50% of breast cancers, and 70% of non–small-cell lung cancers.2 The immunogenicity of CEA in man has been established in studies where patients have been immunized with recombinant, human CEA along with GM-CSF3, vaccinia vectors containing the gene for CEA4, and anti-idiotype specific for CEA.5 Peptide fragments of CEA have been found to be epitopes for T-cell lines generated from patients preSubmitted: February 15, 1999
Accepted: May 30, 1999
Clinical Lung Cancer, Vol. 1, No. 1, 70-72, 1999.
70
August, 1999
viously vaccinated with vaccinia-CEA. The most potent peptide in this study, designated CAP-1, is an HLA-A2 restricted CTL epitope.4 Because naive precursors of cytotoxic T cells specific for tumor antigens are present in very low numbers, methods for stimulating the T cells are necessary. Approaches have included viral vectors, administration of antigen with adjuvants and gene-transduced tumor cells. A more recent development has been the emerging understanding of the critical role that antigen-presenting cells, such as dendritic cells (DC), play in inducing T and B-cell responses in vivo. Numerous studies have demonstrated that DC loaded with tumor antigen can induce T-cell responses in vitro and in vivo murine models without significant toxicity. We and others have developed methods for generating a sufficient number of DC in vitro for use in immunotherapy studies.6-9 Furthermore, we have demonstrated that these DC loaded with CEA and other peptides are capable of inducing CEA specific T-cell responses in vitro.10 Therefore, we hypothesize that DC loaded with tumor-associated peptides such as the CEA peptide CAP-1 will be safe and induce CAP-1 specific Tcell responses in vivo in humans with metastatic cancers. Our objectives are: a)
Dendritic Cell Photomicrograph: Courtesy of Dr. Joseph Fay, Baylor University Medical Center, Dallas, TX
to determine the safety and dose-limiting toxicity of intravenous and intradermal injections of autologous, DC pulsed with an HLA-A2 restricted CEA peptide; b) to evaluate cellular immune response to the CEA peptide.
Treatment Plan All components of the treatment plan must be performed at Duke University Medical Center (DUMC). a) Active Immunotherapy Dose Level Assignment: Patients will be entered into a dose escalating study at the next available dosage slot.
Address for correspondence: H. Kim Lyerly, Department of Surgery, Box 2606, Duke University Medical Center, Durham, NC 27710, Phone: 919-681-3480, FAX: 919-681-7970, email: [email protected]
Table 1:
Eligibility
numbers of cells for immunologic tests.
Inclusion Criteria
Exclusion Criteria
Age ≥ 18 years
Chemotherapy, radiation therapy, or immunotherapy within the past 4 weeks
histologically confirmed Metastatic adenocarcinoma expressing CEA as defined by immunohistochemistry
History of CNS metastases. (Pre-enrollment head CT no required.)
Measurable or evaluable disease (includes CEA level elevation)
History of anto-immune disease
KPS > 80%
Serious intercurrent chronic or acute illness considered by the P.I. to constitute an uwarranted high risk for investigational drug treatment
HLA=A2
Second malignancy (within the past 10 years) other than non-melanoma skin cancer
WBC ≥ 3000/mm3 and absolute lymphocyte count ≥ 1000/mm3; Hgb ≥ mg 9 /dl; platelets ≥ 100,000/mm3; PT < 1.25 x nl, PTT < 1.66 x nl; fibrinogen > 0.75 ng; g)
Presence of HIV infection or viral hepatitis
Creatinine < 2.5 mg/dl; bilirubin < 2.0 mg/dl
b) Leukapheresis: Leukapheresis will consist of up to a 41/2 hour collection of 10-14 liters. Each collection should contain a minimum of 1 x 108 nucleated cells/recipient kg. c) Generation, CEA-Peptide Pulsing and Cryopreservation of DC: Peripheral blood mononuclear cells will be separated from the leukapheresis product on a cell separator, resuspended in AIM V media, and plated onto tissue culture flasks. The adherent cells will be cultured for 7 days in GM-CSF and IL-4 containing AIM V media to generate dendritic cells. Harvested DC will be pulsed with CAP-1 peptide for 4 hours, sterility tested, and then cryopreserved. d) Administration of Peptide-Pulsed DC: Doses will be administered intravenously and intradermally on weeks 0, 2, 4, and 6 for 4 complete treatments at DUMC in an outpatient unit. Premedication will not be routinely given. The cell preparation will be delivered over 2-3 minutes into an intravenous line (via arm, vein, or central line) in which 0.9% saline is running at 250 mL/hr. The intradermal dose will be administered in a volume of 100 μL.
Immunosuppressive (including, oral or IV steroids) in the prior 6 wks.
Following administration, blood pressure and pulse monitoring will be performed over a 1-hour observational period. e) Repeat Leukapheresis: Will be performed during week 8 to obtain large
Long-Term Evaluation and FollowUp (Weeks 12+) Patients who have disease progression will be removed from the study. Those who remain on study will be asked to maintain scheduled long-term follow-up at DUMC. The following parameters listed in Table 4 will be evaluated every month for six months, every three months for the first year following the study and then every year.
Anticipated Toxicities Based on previous studies of DC immunizations, the anticipated toxicities are minimal. Theoretical risks include: allergic reactions such as fever, hives, or rash, pulmonary compromise, and accelerated procoagulant activity, although these have not been reported. Induction of auto-immunity especially to granulocytes (which express an antigen with 40% homology to CEA) and the colon which has small amounts of CEA are theoretically possible.
August, 1999
71
Carcinoembryonic Antigen Peptide-Pulsed Dendritic Cells Table 3:
Short Term Treatment Evaluation (Weeks 0-12)
Interval history and complete physical examination Blood chemistry and hematology CBC with differential and platelet count. BUN, Na, Ka+, Cl, CO2, Gluc, Crea, TP, Alb, Ca, Phos, uric acid, AST, AlkP, T Bili, ALT; Pt, PTT and fibrinogen Serum CEA (week 8) Immunologic assays as above (week 8) To be performed at DUMC on weeks 0, 2, 4, 6, 8, 12
Table 4:
Long-Term Evaluation and Follow-Up (Weeks 12+)
Interval history and complete physical examination Blood chemistry and hematology CBC with differential and platelet count. BUN, Na, Ka+, Cl, CO2, Gluc, Crea, TP, Alb, Ca, Phos, uric acid, AST, AlkP, T Bili, ALT; Pt, PTT and fibrinogen Serum CEA Available radiographs 8. Romani N, Reider D, Heuer M, et al. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods 1996;196:137-151. 9. Morse MA, Zhou LJ, Tedder TF, et al. Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha for use in cancer immunotherapy. Ann Surg 1997;226:6-16. 10.Wong C, Morse M, Nair SK. Induction of primary, human antigen-specific cytotoxic T lymphocytes in vitro using dendritic cells pulsed with peptides. J Immunother 1998;21:32-40.
72
August, 1999
References
1. Darrow T, Slingluff C, Jr., Seigler H. The role of HLA class I antigens in recognition of melanoma cells by tumor-specific cytotoxic T lymphocytes. Evidence for shared tumor antigens. J Immunol 1989;142: 33293335. 2. Thompson JA, Grunert F, Zimmermann W. Carcinoembryonic antigen gene family: molecular biology and clinical perspectives. J Clin Lab Anal 1991; 5:344366. 3. Samanci A, Yi Q, Fagerberg J, et al. Pharmacological administration of granulocyte/macrophage-colony stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen. Cancer Immunol Immunother 1998; 47:131-142. 4. Tsang KY, Zaremba S, Nieroda CA, et al. Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst 1995;7:982-990. 5. Pervin S, Chakraborty M, Bhattacharya-Chatterjee M, et al. Induction of antitumor immunity by an antiidiotype antibody mimicking carcinoembryonic antigen. Cancer Res 1997;57:728-734. 6. Romani N, Gruner S, Brang D, et al. Proliferating dendritic cell progenitors in human blood. J Exp Med 1994; 180:83-93. 7. Bender A, Sapp M, Schuler G, et al. Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. J Immunol Methods 1996;196:121-135.