OncoVAX® immunotherapy is a patient-specific (personalized) vaccine composed of irradiated, but metabolically-active, autologous tumor cells compounded with TICE® BCG, a live, attenuated mycobacteria which serves as a potent adjuvant. Using a proprietary method for dissociating and purifying cancer cells from a resected tumor, this autologous vaccine induces a robust and functional immune response. By using the entire tumor and relying on the immune system to determine which epitopes are unique, the vaccine provides a treatment in which no preconception of "known" or shared tumor antigens is needed. However, a series of steps were required to bring this treatment from proof of concept to therapeutic reality.
The first randomized, multicenter clinical trial (16) for OncoVAX® was attempted in stage I/II/III colon cancer patients under the auspices of the Eastern Cooperative Oncology Group (ECOG). While the final results showed no significant clinical benefit, this study was instructive for a number of reasons. First, vaccine preparation was accomplished in a decentralized fashion, with each clinical site manufacturing the autologous vaccine in their respective pathology departments. Due to the logistical realities of OncoVAX® preparation, this study clearly demonstrated the requirement for a central manufacturing facility to assure adequate quality control (QC) and quality assurance (QA), providing a more standardized approach to vaccine production. Additionally, this oversight needed to extend from the primary facility to the clinical sites where the final vaccine was compounded with TICE® BCG. Secondly, the treatment protocol for this study only involved three intradermal vaccine injections, delivered each week beginning 28 to 35 days after tumor resection. The first two injections were compounded with TICE® BCG while the third vaccination was comprised autologous tumor cells alone. The final injection without adjuvant is critical for monitoring whether the immune system has been trained to react to cells previously defined as “self”. Active and potent immune responses toward these cells manifest as a delayed-type hypersensitivity (DTH) reaction visible at the site of injection (Figure 7). This visible response is still the best in vivo indication of T-cell specificity and activity. Indurations greater than 5mm are considered a significant indication of a specific T-cell response. Additionally, this reaction serves as proof of concept that with prior adjuvant stimulation the immune system has been trained to recognize these cells, and hopefully any MRD remaining after surgery. Not surprisingly, induration size correlates well with patient outcome (Figure 8).
Lessons learned from the previous study were incorporated into the next phase III clinical trial (8701). This study (17) utilized a centralized manufacturing facility to address the QC and QA issues encountered in the previous trial. This required processing to occur within a reasonable geographical area, consequently production was centralized at the Free University in the Netherlands, a reasonable distance from the 12 Dutch hospitals participating in the trial. Additionally, pathologists participating in the study needed to modify their standard sampling procedures to provide maximum tumor material for vaccine production while allowing for adequate diagnosing and staging. Following resection and staging, tumor samples were sent to the production facility for dissociation, cryopreservation, irradiation, and administration. The treatment protocol was also augmented to include a four vaccine regimen: three initial weekly treatments (two with TICE® BCG, one without) and a six-month follow-up booster inoculation. The follow-up booster was added based on the results of a side phase II trial (18) that suggested initial immune responses begin to wane 6 months after the induction vaccinations (Figure 7). However, due to the addition of a fourth inoculation, larger tumors were required for sufficient vaccine production. With a minimum requirement of 3-3.5 grams of tumor, this trial was logistically limited to stage II/III patients. An additional study change involved stratifying patient randomization by tumor stage to power for prospective analysis.
Subjects randomized to the control group (n = 126) received no further treatment after surgical resection and were followed according to scheduled assessments. For subjects randomized to OncoVAX® (n = 128), patients received the four vaccine program outlined above. OncoVAX® was well-tolerated, with 102 of 128 patients receiving all four vaccinations. To determine the extent of DTH reactivity, injection sites were measured for indurations 48 hours after the third and fourth immunizations. Subjects were defined has having achieved cellular immunity if the average of both measurements were greater than 5 mm. By this criterion, 97% of patients achieved effective cellular immunity after the fourth inoculation.
When patient response in the OncoVAX® cohort was determined during follow-up, in an Intent-to-treat analysis, no statistically significant differences in RFS, overall survival, or recurrence-free interval (RFI) were observed. However, when a prospective analysis of patients were analyzed by stage, subjects with stage II disease had clinically meaningful and statistically significant outcomes in both RFI and RFS. Both five-year event-free rates and log rank rates were improved with OncoVAX® treatment in stage II patients (Figure 9). The favorable 16.4% difference between control and OncoVAX® patients represents a 41.4% relative risk reduction of disease progression (5-year survival p=0.008; log-rank analysis p=0.018). Overall survival showed a statistically significant improvement in stage II OncoVAX® treated patients (17.5%) over those patients in the control group (27.3%) (Figure 10). The favorable 9.8% difference represents a 33.3% relative risk reduction (5-year survival p=0.014; log rank analysis p=0.074).
In the intent-to-treat (ITT) population of all randomized stage II patients, there were 43 recurrences (Figure 11). The five-year recurrence free interval p-value (0.01) and the log rank analysis p-value (0.004) was highly significant, it was discovered in referee pathology diagnosis that this included a proportion of B1 patients (9 control and 4 treated patients). These were excluded in the separate Stage II (B2, B3) analysis, the control and OncoVAX® treatment groups, respectively. When compared to the control group, the favorable 16% difference represents a 57.1% relative risk reduction in the recurrence of colon cancer in the OncoVAX® group (five year survival p = 0.026; log-rank analysis p = 0.008).
Since this study was completed, surgical techniques associated with colon cancer treatment have greatly improved. Minimally invasive laparoscopic surgery has become more feasible than open colectomy, especially for patients without locally advanced disease. However, a recent multi-institutional study of 872 patients compared these surgical techniques and determined that while patients preferred the minimally invasive option, time to tumor recurrence was still equivalent after a median follow-up of 4.4 years (19). These results have also been confirmed in T3 and T4A&B colon adenocarcinoma patients (20). Thus, the recurrence-free interval curve in the control group (Figure 11) is probably still valid.
A more recent study by de Weger, et al., (21) updated 8701 patient results with 15-year follow-up data. The event-free survival data are presented as a Kaplan-Meier plot in (Figure 12) for the original study (all 254 patients). OncoVAX® patients still demonstrated improved survival compared to surgical patients alone [HR=0.62 (95% CI: 0.40-0.96), p=0.033]. Using formalin-fixed paraffin embedded blocks from 196 of these patients, the authors also determined OncoVAX® treatment was particularly effective for patients with microsatellite instability and microsatellite stable Dukes B tumors. The long-term, stable results observed with OncoVAX® treatment can only be achieved with a robust immune response employing long-term immunological memory and surveillance. All of these aspects are essential prerequisites for successful and impactful cancer treatment.
Safety was actually better in the OncoVAX® treatment cohort compared to surgery alone. One patient treated with OncoVAX® was hospitalized for treatment of a flu-like syndrome and the event resolved nine days later. Another patient required discontinuation of OncoVAX® treatment due to a 21 x 32 mm ulceration which developed after the second inoculation (BCG had been omitted due to adverse events after the first inoculation). However, as a group, control patients more commonly experienced non-fatal serious adverse events. Thirty-three patients in the OncoVAX® group (25.8%) and 46 patients in the control group (36.5%) experienced at least one non-fatal serious adverse event. Taken together, stage II colon cancer patients had fewer non-fatal serious events and improved recurrence-free and overall survival.
In the adjuvant setting, effective treatments are lacking for Stage II colon cancer patients. To address this need, the FDA has requested a second, confirmatory, randomized controlled phase III trial of OncoVAX® in stage II colon cancer patients. Based on a protocol approved by the FDA, this study will be carried out under a Special Protocol Assessment (SPA). An SPA granted by the FDA provides a mechanism for the sponsors and the FDA to reach agreement on size, execution, and analysis of a clinical trial that is intended to form the primary basis for regulatory approval.
The primary endpoint of this pivotal phase III trial is RFS with an interim and final primary analysis with one and three years follow-up, respectively. The study is powered to detect a 50% improvement in RFS with 90% certainty. If a robust statistical significance is achieved during the interim analysis (median follow up of 1.5 years or 70% of the expected events), the Biologic License Application (BLA) can be filed. Past clinical trials using the optimum four immunization regimen (8701) will be accepted as supportive studies during the FDA review of the BLA. This critical and careful approach to the clinical development of OncoVAX® should allow for approval in stage II colon cancer patients, which remains a population of true “unmet medical need.”
Human Monoclonal Antibodies
Human monoclonal antibody (HuMab) development for cancer treatment, monitoring, and diagnosis is a rapidly evolving field and new sources of cancer-specific HuMabs are in high demand. An ancillary benefit to evaluating OncoVAX® in human patients was the isolation of circulating, diploid B-cells which produced an array of cancer-specific HuMabs (22,23). In fact, we were able to isolate 36 HuMabs which positively recognized colorectal adenocarcinoma cells and tissues. Furthermore, roughly half of these antibodies appear to recognize cell surface antigens and have immediate potential for cancer diagnosis and treatment.
There are three important factors in the development and production of HuMabs for tumor antigen recognition: strategy, specificity, and stability. Tumor-specific B-cell production initiated by OncoVAX® inoculation addresses all three of these requirements. A number of strategies for producing tumor-specific antibodies have been developed over the last few years, including hybridoma fusion of lymphocytes from tumor-draining lymph nodes to EBV transformation of peripheral blood lymphocytes and splenocytes from cancer patients. The apparent instability of antibody production by EBV-transformed lymphocytes has thus far made them an impractical means of producing HuMabs. Additionally, neither approach has succeeded to reproducibly generate HuMabs reactive with cell surface antigens, presumably due to a lack of immunocompetence in the cancer patient at the time. An inability to recognize cell surface antigens limits the in vivo utility of these agents for diagnosis and therapy.
By comparison, approximately 20% of the cultures tested from our tumor immune patients produced HuMabs, with 15.6% binding colon tumor cell antigens. HuMabs generally reactive with tumors were isolated from 7 of 10 immunized patients. Our results demonstrate that while these tumors may vary widely on a genomic level, a surprising degree of immunogenicity may be shared across patients. Our preliminary HuMab results suggest many colon cancers appear to express multiple TAAs. However, none of the HuMabs isolated thus far detect a single antigen common to all of the tumors (Figure 13). Therefore, it is quite possible a convenient number of complementary antibodies could be combined to achieve the broad reactivity necessary for cancer diagnosis or therapy in the clinic (Figure 14).
The HuMabs that we have developed exhibit marked differences in reactivity with normal colonic mucosa. Quantitative differences rather than strict qualitative differences in reactivity were seen in many tests with matched tumor and noninvolved mucosa cells obtained from the same patients. In general antibodies were tested against various normal tissues such as breast, lung, liver, and skin and were found to be negative.
Generating these antibodies from isolated human sources obviates a number of problems associated with developing immunogenic agents for clinical use. First, the expensive process of “humanizing” antibodies from murine sources is not required, although antigenic responses toward these agents still need to be fully explored. Additionally, mouse monoclonal antibodies (MuMabs) very often react with well-known tissue components, particularly carcinoembryonic agent (CEA). To date, none of the HuMabs we have isolated have any reactivity towards CEA, blood group determinants, or histocompatibility antigens, suggesting that HuMab specificity is restricted to those structures recognized as immunogenic in the autologous host. It is very possible this is a reflection of the highly targeted nature of antibody production by autologous vaccination. More specifically, inoculation of mice with human tumor cells is much more likely to identify tissue-specific antigens as foreign rather than autologous vaccination which, in theory, should ignore the majority of self-antigens and focus on unique, cancer-specific epitopes. In fact, many studies comparing MuMab to HuMab cancer recognition has noted a heterogenous staining pattern. In contrast, HuMabs generated following autologous vaccination demonstrate a homogenous recognition pattern. Thus, it is quite likely MuMabs recognize many more phase- or cell cycle-specific antigens rather than true TAAs, greatly limiting their clinical potential.
Other questions concerning the ultimate in vivo application of HuMabs include their ability to enter the extravascular environment of a tumor, recognize tumor-specific structures or epitopes, and bind with sufficient avidity to be effective couriers for antitumor drugs, radionuclides, or diagnostic reporter molecules. These questions are currently being addressed and preliminary findings in colon, breast, and head and neck cancer indicate that HuMabs generated by autologous vaccination are able to access and bind these tumor cells in vivo. These and additional investigations identifying broadly applicable TAAs will be an exciting complement to the upcoming phase III trial of OncoVAX® which will generate a new repertoire of HuMabs for study.
While OncoVAX® was originally designed with tumor heterogeneity in mind, it is thrilling this process may ultimately yield a suite of tools which will allow us to standardize this disease. The degree to which these colon cancer-specific tools will be broadly applicable to other cancers remains to be seen; however, autologous cancer vaccines utilizing renal, breast, and lung tumors should be able to produce similar tumor-specific antibodies for their respective cancer subtypes. In the future, it is very possible immunomodulatory agents such as ipilimumab or nivolumab may serve to enhance the efficacy of ASI therapeutic regimens. In the meantime, novel strategies for ASI and immunomodulation need to be developed in parallel as it is clear these modalities are far from mutually exclusive.
16. Harris JE, Ryan L, Hoover Jr HC, Stuart RK, Oken MM, Benson AB, Mansour EG, Haller DG, Manola J, Hanna Jr MG (2000) Adjuvant active specific immunotherapy of stage II and III colon cancer with an autologous tumor cell vaccine: ECOG Study E5283. Journal of Clinical Oncology, 18 148-157.
17. Vermorken JB, Claessen AME, van Tinteren H, Gall HE, Ezinga R, Meijer S, Scheper RJ, Meijer CJLM, Bloemena E, Ransom JH, Hanna Jr MG, Pinedo HM (1999) Active specific immunotherapy for stage II and III human colon cancer: A randomized trial. The Lancet 353: 345-350.
18. Hoover HC, Surdyke, M, Dangle, RB, Peters, LC, Hanna, Jr. MG. (1984) Delayed Cutaneous Hypersensitivity to Autologous Tumor Cells in Colorectcal Cancer Patients immunized with an Autologous Tumor Cell: Bacillus Calmette-GuerinVaccine. Cancer Res. 44, 1671-1676.
19. Nelson H, Sargent DJ, Wieand HS, Fleshman J, Anvari M, et al (2004) A comparison of laparoscopically assised and open colectomy for colon cancer. N Engl J Med 350;20 2050-2059.
20. Benson III AB, Schrag, D, Somerfield MR, Cohen AM, Figueredo AT, et al (2006) American society of clinical oncology recommendations on adjuvant chemotherapy for stage II colon cancer. Journal of Clinical Oncology Vol 22 No. 16 3408-3419.
21. de Wegner, V A, et al., (2012) Clinical effects of Adjuvant Active Specific Immunotherapy differ between Patients with Microsatellite Stable and Microsatellite Instable Colon Cancer, Clin Cancer Res 18(3):882-9.
22. Haspel MV, McCabe RP, Pomato N, Janesch NJ, Knowlton JV, Peters LC, Hoover Jr HC, Hanna Jr MG (1985) Generation of tumor cell reactive human monoclonal antibodies using peripheral blood lymphocytes from actively immunized colorectal carcinoma patients. Cancer Res 45: 3951-3961.
23. Haspel MV, McCabe RP, Pomato N, Hoover Jr HC, Hanna Jr MG (1989) Coming full circle in the immunotherapy of colon cancer: Vaccination with autologous tumor cells … human monoclonal antibodies … active specific immunotherapy with generic tumor associated antigens. In: Human Tumor Antigens and Specific Tumor Therapy, Metzger R, Mitchell M, eds Alan R. Liss, New York 335-344.
Induction of a DTH response. Hoover HC, Surdyke, M, Dangle, RB, Peters, LC, Hanna, Jr. MG. (1984) Delayed Cutaneous Hypersensitivity to Autologous Tumor Cells in Colorectal Cancer Patients immunized with an Autologous Tumor Cell:Bacillus Calmette-Guerin Vaccine. Cancer Res. 44, 1671-1676.
Survival and disease-free survival in patients grouped according to their DTH response to the third vaccine. Harris JE, Ryan L, Hoover Jr HC, Stuart RK, Oken MM, Benson AB, Mansour EG, Haller DG, Manola J, Hanna Jr MG (2000) Adjuvant active specific immunotherapy of stage II and III colon cancer with an autologous tumor cell vaccine: ECOG Study E5283. Journal of Clinical Oncology, 18 148-157. In the ECOG study 5283, there was inadequate quality control of the vaccine specifications and a percentage of the patients received inadequate vaccines, based on the potency with respect to live tumor cell count. This inadequate potency among a group of vaccines was reflected in failure to induce a significant T-cell mediated immune response as measured by DTH. This lack of vaccine potency correlated to clinical benefit as reflected in significant differences in recurrence-free- and overall-survival.
OncoVAX® – Clinical Results
8701 Study – Recurrence-Free Survival* in Stage II Patients
OncoVAX® – Clinical Results
8701 Study – Overall Survival in Stage II Patients
OncoVAX® – Clinical Results
8701 Study – Recurrence-Free Interval in Stage II Patients
OncoVAX® – Clinical Results 15 year F/U
8701 Study – Recurrence-Free Interval (RFI) in Stage II Patients
Distribution of epitopes as detected by human monoclonal antibodies (MCA)
Reactivity of two human monoclonal antibodies (MCA)