Granulocyte Macrophage Colony-Stimulating Factor
`Secreting Allogeneic Cellular Immunotherapy for
`Hormone-Refractory Prostate Cancer
`(cid:160)
`Eric J. Small, Natalie Sacks, John Nemunaitis, et al.
`Clin Cancer Res(cid:160)(cid:160)
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`2007;13:3883-3891.
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`Research. Research.
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`002005002034
`
`AVENTIS EXHIBIT 2034
`Mylan v. Aventis, IPR2016-00712
`
`-
`

`
`Cancer Therapy: Clinical
`
`Granulocyte Macrophage Colony-Stimulating Factor ^ Secreting
`Allogeneic Cellular Immunotherapy for Hormone-Refractory
`Prostate Cancer
`Eric J. Small,1Natalie Sacks,2 John Nemunaitis,3 Walter J. Urba,4 Eugene Dula,5 Arthur S. Centeno,6
`William G. Nelson,7 Dale Ando,2 Catherine Howard,2 Flavia Borellini,2 Minh Nguyen,2
`Kristen Hege,2 and Jonathan W. Simons8
`
`Abstract Purpose: This trial evaluated the safety, clinical activity, and immunogenicity of an allogeneic
`cellular immunotherapy in 55 chemotherapy-naI« ve patients with hormone-refractory prostate
`cancer (HRPC). The immunotherapy, based on the GVAX platform, is a combination of two pros-
`tate carcinoma cell lines modified with the granulocyte macrophage colony-stimulating factor
`(GM-CSF) gene.
`Experimental Design: HRPC patients with radiologic metastases (n = 34) or rising prostate-
`specific antigen (PSA) only (n = 21) received a prime dose of 500 million cells and 12 boost
`doses of either100 million cells (low dose) or 300 million cells (high dose) biweekly for 6 months.
`End points were changes in PSA, time to progression, and survival.
`Results: Median survival was 26.2 months (95% confidence interval, 17, 36) in the radiologic
`group: 34.9 months (8, 57) after treatment with the high dose (n = 10) of immunotherapy and
`24.0 months (11, 35) with the low dose (n = 24).The median time to bone scan progression in the
`radiologic group was 5.0 months (2.6,11.6) with the high dose and 2.8 months (2.8, 5.7) with the
`low dose. In the rising-PSA group (n = 21) receiving the low dose, the median time to bone scan
`progression was 5.9 months (5.6, not reached), and median survival was 37.5 months (29, 56).
`No dose-limiting or autoimmune toxicities were seen; the most common adverse events were
`injection site reaction and fatigue.
`Conclusions: These results suggest that this GM-CSF ^ secreting, allogeneic cellular immuno-
`therapy is well tolerated and may have clinical activity in patients with metastatic HRPC. Phase 3
`trials to confirm these results are under way.
`
`Approximately 27,050 men die annually from metastatic
`hormone-refractory prostate cancer (HRPC; ref. 1). Although
`chemotherapy with docetaxel has been shown to prolong
`survival in HRPC (2, 3), alternatives to chemotherapy remain
`of considerable interest
`to many patients and physicians.
`Recent advances in the understanding of cancer immunology
`have led to the development of new cancer treatments
`specifically designed to stimulate the patient’s immune system.
`Although prostate cancer has traditionally been thought of as
`poorly immunogenic, numerous studies have shown that
`
`tumor tolerance can be reversed (4 – 6). Prostate cancer is a
`good target for immunotherapy due to the typically slow
`growth rate of most prostate tumor cells, which in turn permits
`an appropriately stimulated immune system time to mount
`antitumor responses (4, 5).
`Immunotherapy typically involves presenting one or more
`tumor antigens to the patient’s immune system in vivo or to
`harvested immune cells in vitro (4, 6). An immune system
`stimulant may be included in the treatment to enhance the
`immune response to the antigens. Whole tumor cells have been
`
`Authors’Affiliations: 1UniversityofCalifornia,SanFrancisco,ComprehensiveCancer
`Center, San Francisco, California; 2Cell Genesys, Inc., South San Francisco, California;
`3Mary Crowley Medical Research Center, Dallas,Texas; 4Earle A. Chiles Research
`Institute, Providence Portland Medical Center, Portland, Oregon; 5West Coast Clinical
`Research,Tarzana, California; 6Urology San Antonio, San Antonio,Texas; 7Sidney Kimmel
`Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, Maryland;
`and 8Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia
`Received 12/11/06; revised 4/3/07; accepted 4/10/07.
`Grant support: Cell Genesys, Inc., South San Francisco, CA, to each investigator
`(E.J. Small, J. Nemunaitis, W.J. Urba, E. Dula, A.S. Centeno, W.G. Nelson, and J.W.
`Simons). N. Sacks, M. Nguyen and K. Hege are employees of Cell Genesys, Inc. D.
`Ando, C. Howard, and F. Borellini were employees of Cell Genesys, Inc., at the time
`of the study.W.G. Nelson is a paid consultant to Cell Genesys and a member of the
`company’s Medical Advisory Board. The terms of this arrangement are being
`managed by The Johns Hopkins University in accordance with its conflict of
`
`interest policies. W.G. Nelson and J.W. Simons are coinventors in a patent
`application (USPTO 20060078544) with Cell Genesys, Inc. J.W. Simons received
`honoraria from Cell Genesys for speaking engagements.
`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.
`Statement of significance: This translational study showed that the GM-CSF ^
`secreting, allogeneic cellular immunotherapy was well tolerated, clinically active, and
`broke immunologic tolerance to prostate cancer in chemotherapy-naI« ve patients
`with metastatic HRPC.
`Requests for reprints: Eric J. Small, University of California, San Francisco, 1600
`Divisadero Street, 7th Floor Box 1711, San Francisco, CA 94115. Phone: 415-353-
`7095; Fax: 415-353-7779; E-mail: smalle@medicine.ucsf.edu.
`F 2007 American Association for Cancer Research.
`doi:10.1158/1078-0432.CCR-06-2937
`
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`Cancer Therapy: Clinical
`
`proposed as an antigen source in immunotherapy because
`relevant prostate cancer tumor-rejection antigens have not been
`convincingly identified, and a polyvalent source of antigens can
`better address ‘‘antigen escape’’ resulting from the modulation
`and down-regulation of antigens during tumor growth (7). The
`rationale for employing a granulocyte macrophage colony-
`stimulating factor (GM-CSF) – transduced whole cell immuno-
`therapy is to use whole tumor cells as the source of multiple
`tumor-associated antigens and to use GM-CSF to induce
`growth, maturation, and recruitment of dendritic cells, which
`process and present antigens, to the immunotherapy injection
`sites (8). Preclinical studies in several poorly immunogenic
`rodent HRPC models have shown prolonged survival
`in
`animals treated with GM-CSF – transduced whole cell immu-
`notherapy (4, 9 – 11).
`The first clinical trial of GM-CSF – secreting, cellular immu-
`notherapy for prostate cancer was conducted with autologous
`cells derived from resected tumor material in patients with
`hormone-naBve prostate cancer following prostatectomy (12).
`Treated patients exhibited tumor-associated humoral immune
`responses, delayed-type hypersensitivity reactions to autolo-
`gous prostate cancer cells, and new T-cell and B-cell responses
`against prostate cancer-associated antigens. However, the small
`number of cells that can be obtained from surgically removed
`tumors limits the practicality of this approach (12). Therefore,
`two cell lines, derived from a lymph node metastasis (LNCaP)
`and a bone metastasis (PC-3), were selected for an allogeneic
`cellular
`immunotherapy with the expectation that
`their
`combined antigenic profile would broadly represent
`the
`spectrum of metastatic prostate cancer (13). These cell lines
`were modified with a human GM-CSF gene to secrete high
`levels of bioactive GM-CSF. An initial phase 1/2 trial
`in
`hormone-naBve patients with prostate cancer showed a favor-
`able safety profile, statistically significant changes in the slope
`of prostate-specific antigen (PSA) velocity, and a PSA decline of
`>50% in one patient, suggesting an antitumor effect (13).
`An open-label, phase 1/2, multicenter trial was therefore
`conducted to evaluate the safety, clinical activity, and immu-
`nogenicity of the GM-CSF – secreting, allogeneic cellular immu-
`notherapy in chemotherapy-naBve patients with metastatic
`HRPC. The protocol was amended to allow administration of
`a higher dose level after an interim analysis showed that the
`initial dosage tested was well tolerated.
`
`Materials and Methods
`
`This study was conducted according to the precepts established by
`the Helsinki Declaration and the NIH Guidelines for Research Involving
`Recombinant DNA. The protocol was approved by each site’s Human
`Investigations Committee. Each patient provided signed informed
`consent. The study was initiated on May 19, 1999, and completed on
`January 16, 2001.
`Materials. This immunotherapy is based on the GVAX platform
`(Cell Genesys, Inc.) and consists of two prostate cancer cell lines, PC-3
`and LNCaP, modified to express the human GM-CSF gene. The cell lines
`are propagated, frozen, and irradiated to arrest further cell division
`(13). The product is stored and shipped on dry ice and thawed before
`administration. All manufacturing is conducted according to good
`manufacturing practice and NIH containment guidelines for recombi-
`nant DNA.
`Patients. Men with histologically confirmed adenocarcinoma of the
`prostate and disease progression despite androgen deprivation were
`
`eligible. All patients had metastatic disease, had two or more successive
`increases in serum PSA (z2 ng/mL) taken at least 2 weeks apart, and
`were asymptomatic (without bone pain due to HRPC). Patients in the
`radiologic group had overt metastatic disease (positive bone scan,
`bidimensionally measurable disease, or both). Patients in the rising-
`PSA group had biochemical metastases with increasing PSA levels but
`negative bone scan, computed tomography (CT) scan (abdomen and
`pelvis) and chest X-ray. Patients were excluded for primary HRPC, brain
`metastases, uncontrolled medical problems, or previous chemotherapy,
`bisphosphonate therapy, biological therapy, immunotherapy, or gene
`therapy for cancer.
`Treatment. All patients received a priming dose of 500 million cells
`(250 million cells of each cell line). This was deemed a maximum
`feasible dose due to the number of injections required. Patients in the
`rising-PSA group and the first 24 patients in the radiologic group
`received the low dose boost of 100 million cells (50 million of each cell
`line). Because no dose-limiting toxicities were seen at this dose level, a
`high boost dose of 300 million cells (150 million of each cell line) was
`given to 10 additional patients in the radiologic group. Although the
`500 million cell priming dose was well tolerated, a boost dose higher
`than 300 million cells was avoided due to the number of injections
`required. Dose levels were selected based on an earlier trial of a similar
`GVAX platform – based immunotherapy in pancreatic cancer, which
`showed that 100 – 500 million cell dose levels were immunologically
`and clinically active (14, 15). The increase in boost dose level allowed
`further exploration of tolerability and a potential dose response in
`patients with radiologically detectable metastases, presumably with a
`heavier disease burden than patients in the rising-PSA group. Each cell
`type was injected intradermally in opposite limbs every 2 weeks for
`6 months.
`Evaluation. The prospectively defined primary study end points
`were PSA decline of at
`least 50%,
`time to PSA and bone scan
`progression (16), change in PSA over time (slope), local or systemic
`immune response, and safety. PSA was tested at a central laboratory
`(Abbott AxSYM) at 2-week intervals during treatment and monthly
`during the 6-month follow-up period. Bone scans, CT scans (abdomen
`and pelvis), and chest X-rays were done at screening and months 3, 6, 9,
`and 12 in the radiologic group or at screening and when clinically
`indicated for the rising-PSA group. Serum levels of carboxyterminal
`telopeptide of type I collagen (ref. 17; ICTP) were measured in the
`radiologic group. B-cell
`immune responses were measured in the
`radiologic group pre- and posttreatment by immunoblot analyses (two-
`dimensional electrophoresis) using lysates of the LNCaP and PC-3 cell
`lines against patient sera as in the earlier studies (12, 13). The two-
`dimensional electrophoresis was done according to the method of
`O’Farrell (18) by Kendrick Labs, Inc. A posttreatment 250-kDa band
`from a PC-3 immunoblot was of interest, and the protein spot was
`excised from a Coomassie blue – stained 10% acrylamide slab gel. Mass
`spectrometry (MS) fingerprinting of the protein spot was done by
`subsequent digestion with endoproteinase Lys-C and analysis by
`matrix-assisted laser desorption ionization MS (Protein Chemistry Core
`Facility, Howard Hughes Medical
`Institute/Columbia University).
`Serum samples were tested for antibodies against PSA by ELISA using
`donkey antihuman immunoglobulin G (IgG) IgM horseradish perox-
`idase (HRP) and compared with a negative control (normal serum) and
`two positive controls (rabbit anti-PSA with donkey anti-rabbit IgG HRP;
`human IgG). A greater than 2-fold induction in titer posttreatment was
`considered positive. Patients were assessed for human leukocyte antigen
`(HLA) type on enrollment, but HLA type was not an exclusion criteria.
`Safety assessments included physical examinations, laboratory evalua-
`tions, and recording of adverse events, which were graded by the
`National Cancer Institute (NCI) Common Toxicity Criteria, version 2.
`All patients were followed for survival. Data collection was monitored
`according to Good Clinical Practice Guidelines, and data were double
`entered into a database before analysis.
`Statistical analysis. A sample size of 30 patients with radiologic
`metastases and 20 patients with biochemical metastases (rising PSA)
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`

`
`Cellular Prostate Cancer Immunotherapy
`
`Table 1. Patient characteristics at baseline
`
`Patients enrolled
`Age (y), median (range)
`PSA* (ng/mL), median (range)
`Alkaline phosphatase
`(units/L), median (range)
`Hemoglobin (g/dL),
`median (range)
`
`Ethnic group
`Caucasian
`African-American
`Asian
`Extent of disease
`Bone disease only
`Soft tissue only
`Bone and soft tissue
`PSA-only disease
`ECOG performance status
`0
`1
`Gleason score z7
`
`*PSA: prostate-specific antigen.
`
`All patients
`
`55
`71 (58-88)
`35.9 (1.3-1,207)
`77.0 (48-659)
`
`Radiologic group:
`low dose
`
`Radiologic group:
`high dose
`
`Rising-PSA group:
`low dose
`
`24
`73 (58-85)
`49.5 (3.8-1,207)
`97.5 (63-659)
`
`10
`70 (58-76)
`79.6 (3.7-846.7)
`78.5 (48-263)
`
`21
`70 (62-88)
`16.5 (1.3-92.5)
`67.0 (49-99)
`
`13.2 (9.3-16.1)
`
`12.6 (9.3-15.1)
`
`13.7 (10.3-16.0)
`
`13.4 (11.7-16.1)
`
`n (%)
`
`52 (95)
`2 (4)
`1 (2)
`
`20 (36)
`8 (15)
`6 (11)
`21 (38)
`
`45 (81.8)
`10 (18.2)
`34 (62)
`
`n (%)
`
`23 (96)
`1 (4)
`0 (0)
`
`15 (63)
`6 (25)
`3 (13)
`0 (0)
`
`18 (75.0)
`6 (25.0)
`15 (63)
`
`n (%)
`
`9 (90)
`1 (10)
`0 (0)
`
`5 (50)
`2 (20)
`3 (30)
`0 (0)
`
`10 (100.0)
`0 (0.0)
`7 (70)
`
`n (%)
`
`20 (95)
`0 (0)
`1 (5)
`
`0 (0)
`0 (0)
`0 (0)
`21 (100)
`
`17 (81.0)
`4 (19.0)
`12 (57)
`
`was calculated to allow detection of adverse events that occur at an
`underlying rate of >5% and a single PSA response if the underlying rate
`was 10%. Analyses were conducted on these two populations
`separately. Variables measured on a continuous scale were characterized
`by summary statistics (mean and SD). Variables that were dichotomous
`in nature or categorical in outcome were summarized using counts and
`proportions with exact binomial confidence limits. The time to
`progression was measured from the first day of treatment to the day
`progression was documented. Log-transformed PSA values were plotted
`against time, and a linear regression model was used to calculate the
`pretreatment slope based on at least three successive PSA values taken at
`least 2 weeks apart and the posttreatment slope based on all PSA values
`collected during the treatment and follow-up period. Survival time and
`time to progression (PSA and bone scan) were estimated according to
`the Kaplan-Meier method (19). Patients who had not reached an end
`point by the date of analysis were censored. In a post hoc analysis, a
`predicted median survival time was calculated based on baseline patient
`characteristics [including PSA, alkaline phosphatase, hemoglobin,
`lactate dehydrogenase (LDH), Gleason score, performance status, and
`visceral disease] following a validated pretreatment prognostic model
`developed by Halabi et al. (20) and compared with the observed
`survival time. Because LDH was not collected during this trial, the
`median LDH collected from a similar population of HRPC patients in a
`subsequent immunotherapy trial was used (21). Exploration of factors
`influencing survival time and the primary clinical end points (PSA
`decrease, time to progression, change in PSA velocity) was assessed by
`categorizing patients by HLA type and separately by posttreatment
`immunoreactivity to the tumor cell lines (immunoblot) regardless of
`dose group.
`
`Results
`
`Patients. All 55 patients had metastatic HRPC. The radio-
`logic group consisted of 34 men: 24 received the low dose, and
`10 received the high dose. The rising-PSA group consisted of 21
`men: all received the low dose. Patient characteristics are
`
`summarized in Table 1. Of the 55 patients enrolled, 29 (53%)
`completed the 6-month treatment period. The primary reasons
`for the discontinuation of treatment were progressive disease
`(17), initiation of alternative treatment (4), unrelated adverse
`events (4), and other (nonspecified) reasons (1). Twelve
`patients completed the 1-year study, and 17 discontinued
`during the 6-month follow-up phase due to initiation of
`alternative treatment (9), progressive disease (5), and other
`reasons (3).
`the 55 patients (11%) had a
`Clinical response. Six of
`decrease of more than 25% in PSA, including a decrease of
`more than 50% in one patient in the radiologic group (high
`dose). This patient had a baseline PSA value of 10 ng/mL,
`which began to drop 2 weeks after the first dose, reached
`0.1 ng/mL at 10 weeks, and subsequently began to increase at
`24 weeks. The duration of response was 267 days (Fig. 1). The
`
`Fig. 1. Serum PSA over time in a patient in the radiologic group on the high dose of
`immunotherapy (patient G03-018-804SS).
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`
`Cancer Therapy: Clinical
`
`Fig. 2. Kaplan-Meier estimate of (A) time
`to bone scan progression and (B) overall
`survival time. Hash marks, patients who
`have not reached end point at the time of
`data analysis.
`
`patient had resolution of a bone lesion on bone scan at week
`12 and developed no new lesions during the trial. This patient
`was not an HLA class I match to either cell line comprising the
`immunotherapy and had no evidence of antibodies against
`PSA. A posttreatment reduction in PSA slope was observed in
`25 of 34 (73.5%) patients in the radiologic group, including
`16 of 24 (66.6%) receiving the low dose and 8 of 10 (80%)
`receiving the high dose, and 11 of 21 (52.4%) patients in the
`rising-PSA group.
`Time to progression. The median time to PSA progression
`was 2.6 months in the radiologic group, including 2.3 months
`[95% confidence interval (95% CI), 1.8, 3.2] with the low dose
`and 3.7 months (95% CI, 3.2, 5.5) with the high dose. In the
`rising-PSA group, the median time to PSA progression was
`3.9 months (95% CI, 3.2, 7.8). The median time to bone
`scan progression was 3.0 months in the radiologic group,
`including 2.8 months (95% CI, 2.8, 5.7) with the low dose and
`5.0 months (95% CI, 2.6, 11.6) with the high dose of
`immunotherapy (Fig. 2A). The median time to a positive bone
`scan in the rising-PSA group was 5.9 months (95% CI, 5.6, not
`reached).
`ICTP, a biological marker of
`ICTP. Serum levels of
`metastatic bone turnover, were analyzed in the radiologic
`group. At 12 weeks, levels of ICTP were decreasing or stable
`(<25% change) in 20/29 (69%) patients in the radiologic
`group: 13/20 (65%) on the low dose and 7/9 (78%) on the
`high dose of immunotherapy (5 patients did not have data).
`Immunoblot analysis of patient
`B-cell immune responses.
`serum against lysates of the two immunotherapy cell lines, PC-
`3 and LNCaP, was done in the radiologic group to assess the
`induction of antibody responses reactive against the prostate
`
`cancer cells. New or enhanced immunoreactive bands appeared
`posttreatment in 19/28 (67.8%) patients in the radiologic
`group, including 13/19 (68.4%) on the low dose and 6/9
`(66.7%) on the high dose (6 patients did not have data). A
`larger percentage of patients showed immunoreactivity to the
`PC-3 cell lysate (18/28; 64.3%) compared with the LNCaP
`lysate (12/28; 42.8%). The immune response to prostate
`antigens was oligoclonal; some bands were shared between
`multiple patients, and others were unique to individual patients
`(Fig. 3). A median of 2 new or enhanced bands (range, 1-6)
`were induced against PC-3 and a median of 1 (range 1-3)
`against LNCaP. Induction of serum antibodies against PSA was
`evaluated by ELISA in 52 patients, and no evidence of induced
`anti-PSA antibodies was observed (Fig. 4). A more than 250-
`kDa band was present on immunoblot for 11/19 immunore-
`active patients, including 8 on the low dose and 3 on the high
`dose (all in the radiologic group). In the patient whose PSA
`dropped to 0.1 ng/mL, the band was excised and identified by
`mass spectrometry as filamin B (h), a cytoskeletal protein that
`has been linked to cancer and is involved in cell shape,
`division, adhesion, motility, signal transduction, and protein
`sorting (22 – 24).
`the overall median
`In the radiologic group,
`Survival.
`survival time after initiation of treatment was 26.2 months
`(95% CI, 17, 36), including 24.0 months (95% CI, 11, 35) with
`the low dose and 34.9 months (95% CI, 8, 57) with the high
`dose (Fig. 2B). Based on a pretreatment prognostic model
`developed by Halabi et al. (20), an expected median survival
`time of 19.5 months (95% CI, 17, 22) was estimated for the
`34 patients in the radiologic group. At the end of the study,
`13/34 patients in the radiologic group received subsequent
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`chemotherapy (taxane in 9/13); their median survival was more
`than 35.2 months (95% CI, 29, 44). The median survival of the
`nonchemotherapy-treated patients (21/34) was 17.2 months
`(95% CI, 9, 32). The differences in survival times were not
`statistically significant (P = 0.1). Median survival in the rising-
`PSA group was 37.5 months (95% CI, 29, 56). In the radiologic
`group, patients with reduced posttreatment PSA slopes had a
`longer survival time (26.2 months) compared with those with a
`stable or increasing PSA slope (11.5 months; P = 0.12). By
`contrast, in the rising-PSA group, median survival times were
`similar regardless of the direction of change in PSA slope.
`Immunoreactivity was not a
`Factors influencing outcome.
`significant predictor of survival
`time or clinical response.
`Immunoblot data were available on 3/6 patients who had a
`>25% decrease in PSA,
`including the patient whose PSA
`dropped to 0.1 ng/mL. All three patients showed induction of
`immunoreactivity to one or both cell
`lines. There was no
`difference in treatment-associated changes in PSA slope, ICTP
`levels,
`time to PSA or bone scan progression, or overall
`survival based on induction of immunoreactivity to one or
`both cell lines. The improved median survival time observed
`in patients who received subsequent chemotherapy was
`evident in patients who were immunoreactive on immuno-
`blot (29.8 months with chemotherapy versus 23.9 months
`without chemotherapy), but not
`in nonreactive patients
`(24.6 months with chemotherapy versus 24.8 months
`without chemotherapy). Although presence of a 250-kDa
`band on immunoblot was not a significant predictor of
`survival time, the median survival time in the 11 patients
`who showed this band was 31.2 months compared with
`24.3 months in the 17 patients with immunoblot data that
`did not show the 250-kDa band.
`The HLA class 1 type in 29 of 48 patients matched that of one
`or both immunotherapy cell lines (A2 and A24), including
`20/31 (64.5%) in the radiologic group and 9/17 (52.9%) in the
`rising-PSA group. HLA type was not available in seven patients.
`HLA type did not significantly impact survival time or the
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`Cellular Prostate Cancer Immunotherapy
`
`primary end points; however, the patient group whose HLA
`class 1 type was not a match to either cell line showed a greater
`response for every clinical end point (PSA, ICTP, time to
`progression, and survival) in comparison to the HLA-matched
`group. Immunoreactivity was noted in 7/9 (78%) mismatched
`and 11/18 (61%) HLA-matched patients.
`Adverse events. Most adverse events (57%) were judged by
`the investigator to be not related to treatment. Of the 760
`reported adverse events, 45% were grade 1, 49% were grade 2,
`5% were grade 3, and <1% were grade 4 (five events in two
`patients). There was a higher incidence of a flu-like syndrome
`in the high dose (50%) than in the low-dose (8%) group, but
`the incidence of fatigue was higher in the low-dose (37%) than
`in the high-dose (20%) group; these adverse events resolved
`without sequelae. There were no other notable differences in
`the overall rate or NCI toxicity grade of adverse events between
`dose levels.
`Injection site reaction was the most common treatment-
`related adverse event (Table 2). Fifty-three patients had a grade
`2 injection site reaction consisting of pruritus, pain, and/or
`swelling, and one patient had a grade 3 reaction that included
`skin ulceration, all of which resolved without sequelae. Serious
`adverse events (n = 21) occurred in 16 patients, and none were
`related to treatment. One patient died due to disease
`progression within 30 days of the last treatment.
`All posttreatment antinuclear antibody (ANA) titers
`(available for 53 patients) were V1:40. One patient had a titer
`of 1:360 at screen and <1:40 at week 24. For 17 of 24 patients,
`the posttreatment ANA lab report stated ‘‘unidentified autoanti-
`bodies present,’’ signifying the presence of IgG antibodies
`specific for non-nuclear antigens in the Hep2 cell line, which
`were not present at screening. For two patients, unidentified
`autoantibodies were noted at screen only. A review of the
`adverse events for these patients did not reveal symptoms of
`autoimmune disease. Five of the 17 patients with unidentified
`autoantibodies had a repeat ANA test at month 12, and no
`autoantibodies were observed.
`
`Fig. 3. Immunoblot of pre- and
`posttreatment patient sera against lysates
`of the PC-3 and LNCaP cell lines. Red
`arrows, examples of posttreatment
`induction of new or enhanced tumor-
`reactive antibodies.
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`Cancer Therapy: Clinical
`
`Discussion
`
`This open-label multicenter study was undertaken to evaluate
`the safety, immunogenicity, and clinical activity of a GM-CSF –
`secreting, allogeneic cellular immunotherapy for prostate
`cancer. The immunotherapy was designed to stimulate an
`immune response through presentation of multiple tumor-
`associated antigens and targeted secretion of GM-CSF. This
`study showed that the immunotherapy was well tolerated and
`immunogenic in the majority of metastatic HRPC patients.
`There seemed to be an advantage with the higher dose in
`patients with radiologically detectable metastases with regard to
`time to progression, PSA changes, and overall survival. The
`median survival time in the radiologic group was longer with
`the high dose compared with the low dose of immunotherapy
`(34.9 versus 24.0 months, respectively). The median time
`to bone scan progression was longer with the high dose
`(5.0 months versus 2.8 months). Similarly, the median time to
`PSA progression was longer with the high dose (3.7 months
`versus 2.3 months), and a higher frequency of patients in the
`high-dose group had a reduced PSA slope following treatment.
`One patient on the high dose had a complete response
`(a decline in PSA from 10 to 0.1 ng/mL that lasted 267 days
`and resolution of a bone lesion at week 12). The clinical
`relevance of posttreatment changes in PSA continues to be
`debated; however, the group of patients with reduced post-
`treatment PSA slopes had a longer survival time (26.2 months)
`compared with those with a stable or increasing PSA slope
`(11.5 months; P = 0.12), suggesting that these PSA changes are
`not random fluctuations. This study was not designed or
`powered for statistical comparison between dose groups or
`other subgroups of patients; nevertheless, these findings are
`
`provocative and have helped to generate the hypothesis that the
`higher dose of immunotherapy may provide a clinical benefit.
`Patients with metastatic HRPC are a heterogeneous popu-
`lation, and survival times are influenced by patient and tumor
`characteristics (20, 25 – 28). Because this study did not include
`a control arm, the observed survival times are difficult to
`interpret. Therefore, we calculated a predicted median survival
`time based on baseline patient characteristics following a
`validated pretreatment prognostic model, the Halabi Nomo-
`gram, to provide a context for the observed survival time (20).
`The Halabi Nomogram is based on the relationship between
`patient characteristics (PSA, alkaline phosphatase, hemoglo-
`bin, lactate dehydrogenase, Gleason sum, Eastern Cooperative
`Oncology Group (ECOG) performance status, and visceral
`disease) and overall survival observed during six chemother-
`apy trials of 1,101 patients with metastatic HRPC whose range
`of characteristics encompassed the immunotherapy study
`population. The median survival predicted by the Halabi
`nomogram for the 34 patients in the radiologic group in this
`trial was 19.5 months. The observed median Kaplan-Meier
`survival time for those 34 patients (26.2 months) exceeded
`that predicted by the Halabi Nomogram. These results should
`be interpreted with caution because the utility of
`this
`nomogram to evaluate survival data from immunotherapy
`trials has not been validated. Nevertheless, as a hypothesis-
`generating exercise, this result raises the possibility that this
`immunotherapy may improve survival time for patients with
`metastatic HRPC.
`During the follow-up phase of this study, patients in the
`radiologic group who received subsequent chemotherapy had a
`longer survival time than those who did not receive chemo-
`therapy (35.2 versus 17.2 months). Although this result could
`
`Fig. 4. PSA ELISA. Pre- to posttreatment
`fold induction of serum antibodies to PSA.
`Responses above 2-fold (dotted line) are
`considered induced.
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`Cellular Prostate Cancer Immunotherapy
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`Table 2. Adverse events judged by the investigator to be possibly or probably related to treatment