Specific humoral response in cancer patients treated with a VEGF-specific active immunotherapy procedure within a compassionate use program

Vascular endothelial growth factor A (hereafter denominated VEGF) has been broadly studied due to its relevant role in physiological and pathological angiogenesis [1]. During malignant tumor development and progression, cancer cells produce VEGF, among other pro-angiogenic factors, to compensate hypoxia and promote proliferation. Pathological angiogenesis is also important for cancer dissemination and metastases, and for the resistance of tumor cells to the natural immune response. The biological activity of VEGF is mediated by its binding to VEGFR receptor 2 (VEGFR2) or VEGF receptor 1 (VEGFR1) [2]. Both interactions have been implicated in tumor-induced angiogenesis and immunosuppression [3, 4].

All these properties made VEGF an attractive target for cancer immunotherapy. Passive and active immunotherapies targeting this molecule have been developed, and are currently in different stages of preclinical or clinical development. So far, the most successful passive immunotherapy directed to VEGF is bevacizumab, a recombinant monoclonal antibody that neutralizes the binding of VEGF to its receptors [5]. In combination with chemotherapy, bevacizumab has been approved in many countries for the treatment of different tumors [6‐12].

VEGF-targeted active immunotherapies are based on different approaches, from DNA to peptide or protein-based active immunotherapeutic procedures [13, 14]. Only the latter has been clinically evaluated. Our group has developed an active immunotherapy procedure (CIGB-247) that uses as antigen a recombinant mutated version of human VEGF genetically coupled to the first 47 aminoacids of the p64K protein. Discrete aminoacid mutations, in the receptor-binding domain sequence of VEGF, were made with the aim of blocking the binding between the antigen and VEGFR2, and hence avoiding any CIGB-247 potential proangiogenic activity [15]. The antigen is formulated with a bacterially-derived adjuvant VSSP [16]. CIGB-247 has shown anti-tumor and anti-metastatic effects in mice, stimulating the development of VEGF-blocking antibodies and specific T cell responses [15, 17]. CIGB-247 has been already evaluated in two phase I clinical trials known as CENTAURO and CENTAURO-2, where safety and immunogenicity were studied in patients with advanced solid tumors [18, 19].

The results obtained from these two trials demonstrated the excellent safety profile of CIGB-247, and also indicated that it is possible to induce a polyclonal antibody response against human VEGF, characterized by the presence of specific IgG, IgM and IgA antibodies. This specific polyclonal antibody response was able to inhibit the interaction between VEGF and its receptors and reduce VEGF bioavailability within platelets [18, 19].

Wentink et al. have also developed a therapeutic vaccine that uses as antigen a truncated form of human VEGF (aminoacids 26–104). This antigen sequence represents the complete bevacizumab binding site. Using RFASE as adjuvant, the vaccine (hVEGF_26–104/RFASE) induces an immune response with VEGF neutralizing activity and anti-tumor effect [20]. In rats and monkeys immunized with this vaccine candidate, the VEGF-specific polyclonal antibody response has also demonstrated its capacity to impair the binding of bevacizumab to VEGF, suggesting the presence of antibodies that target the same VEGF epitope as bevacizumab [20, 21]. This vaccine is being investigated in a phase I open-label clinical trial (NCT02237638), and preliminary results of the first included patients indicated that hVEGF_26–104/RFASE has a good safety profile. However, no VEGF-specific antibody responses were found in any of the patients evaluated [22].

As mentioned before, patients enrolled in the CENTAURO clinical trial were in frank progression and they had previously received all available therapies and were no longer responding [18]. After the end of the trial period (week 16), CENTAURO surviving patients were voluntarily enrolled to receive off-trial monthly re-immunizations with CIGB-247 and participate in regular immunological studies. In these individuals we have been able to show an excellent safety profile and sustained specific immune responses [23, 24]. As immunizations have increased over the years, anti-VEGF IgG polyclonal antibody response shifts gradually in some patients from IgG1 to IgG4 [19, 24]. Objective clinical benefits have been documented in a number of surviving CENTAURO patients [23, 24].

All the aforementioned led us to propose to the Cuban Regulatory Authority (CECMED), the initiation of a compassionate use program (CUP) for CIGB-247 in cancer patients that did not meet the strict entry criteria applied for the CENTAURO and CENTAURO-2 clinical trials, but could potentially benefit from the application of CIGB-247. It is well known that a large proportion of cancer patients with life-threatening disease are excluded from clinical trials, despite the fact that these subjects are better reflections of the real-world population of cancer patients [25].

Treating a broader spectrum of cancer patients with CIGB-247 in non-trial conditions would also allow us to continue evaluating the nature, magnitude, persistency of the specific humoral response as well as the safety profile of CIGB-247. To achieve this, cancer patients were vaccinated with 400 μg of antigen combined with 200 μg of VSSP, the highest antigen dose that at that point of the initiation of this program had been found to be safe. Vaccination was administered until death, intolerance, marked disease progression or patient’s withdrawal of consent.

This paper mainly describes and discusses the results of the study of the immune humoral response to CIGB-247 in a sample of the CUP patients treated so far. This work also presents a brief analysis of some safety and product administration aspects that were not possible to assess under the strict patient enrollment criteria applied for the CENTAURO and CENTAURO-2 clinical trials.

CIGB-247 can be considered the most clinically advanced strategy worldwide targeting VEGF via active immunotherapy. The proposed mechanism of action for this intervention is related to the induction of a long lasting, self-regulated, nontoxic response of both humoral and cellular nature [13]. The immune response elicited by CIGB-247 has been extensively investigated in two phase I clinical trials (CENTAURO and CENTAURO-2), with follow up studies in long-term surviving patients [18, 19, 24], with documented clinical benefits in the latter [23]. The CIGB-247 CUP was initiated mainly as a way of potentially benefiting cancer patients that were/are ineligible for the early phase clinical trials that evaluate this vaccine candidate. This means that cancer patients with different stages of their disease, brain primary tumors, brain metastases, additional chronic uncompensated, autoimmune or immune suppressing diseases as well as patients receiving immune modulator drugs, chemotherapy or biological therapies have been recruited for the CUP. The CIGB-247 CUP is developed in medical Cuban institutions, and headed by physicians and specialists trained in clinical trial development. This effort also opened the opportunity of gathering new information about the specific antibody response and safety profiles of CIGB-247 in a broader clinical spectrum sample of cancer patients, treated within the context of day-to-day clinical practice settings.

Some clinical studies on cancer vaccines have revealed a relationship between the high levels of elicited antibodies and an improved survival; and even have been able to elucidate the specific immunoglobulin class associated with overall survival [26‐28]. Although these types of correlations are not applicable to this CUP study of CIGB-247, there is no doubt about the importance of studying in depth the humoral response in early evaluations of cancer vaccines in humans [29]. A special emphasis was done in this paper in the characterization of the vaccine-induced humoral response in terms of quantity, quality and composition.

This report confirms some of the results obtained from the CENTAURO and CENTAURO-2 clinical trials, where CIGB-247 induced a polyclonal antibody response against human VEGF with a dual blocking activity on the interactions with VEGFR2 and VEGFR1. The polyclonal antibody response was characterized by the presence of IgM, IgA and IgG antibodies specific to VEGF, being the latter the predominant immunoglobulin [19]. So far VEGF-specific IgE antibodies have not been detected in any of the studies of CIGB-247, and the presence of this class of immunoglobulin has been reported in very few studies of specific active immunotherapy with other antigens and adjuvants [28, 30]. Although IgE has been pointed out as a mediator of anti-tumor effect [31, 32], the generation of IgE antibodies by active immunization is probably the result of the specific antigen, adjuvants or schedules, or a combination of these.

Additionally, this work shows now that the polyclonal antibody response elicited by CIGB-247 is able to block the binding of bevacizumab to VEGF, adding this property to other previously described for the interaction of VEGF with VEGFR2 and VEGFR1 [19].

This VEGF-neutralizing activity is probably caused in a greater part to a steric hindrance, and in a lesser extent to a competition for the same critical binding determinants on VEGF. This hypothesis is based on the fact that CIGB-247 uses as antigen a protein representative of human VEGF 121 isoform, in which amino acids R82, K84 and H86 have been mutated to E in order to prevent possible undesired pro-angiogenic activities [15]. These three residues have been reported as important for the bindings between VEGF with VEGFR2, VEGFR1 and bevacizumab [33‐37]. Because of the aforementioned, the blocking activity induced by CIGB-247 against these three molecules could be probably explained mainly due to steric hindrance, and could very well be also at the basis of the inhibition of the interaction of VEGF with VEGFR2, VEGFR1 and bevacizumab. The polyclonal antibody response elicited after immunization blocks the receptor binding domain on natural VEGF as well as the binding site for bevacizumab. The finding of the presence of bevacizumab-blocking antibodies within the polyclonal response elicited by CIGB-247 indicates that the specific humoral response is directed to relevant domains on VEGF. The induction of bevacizumab-blocking antibodies by active immunization has been also developed by Wentink et al. This approach is based on vaccination against the bevacizumab binding site on VEGF using a truncated protein (human VEGF _26–104) as antigen and RFASE as adjuvant [21]. This VEGF therapeutic vaccine has shown antiangiogenic and antitumor activity in pre-clinical models [20], but discrete results in terms of immunogenicity have been observed in the first cancer patients treated with the vaccine [22].

Because VEGF binding determinants for VEGFR2 and VEGFR1 overlap only partially, and their binding sites are located at opposite ends of the molecule [38] steric hindrance is a suitable explanation for the VEGF-blocking activity detected in the serum of vaccinated patients. In fact, it has been proposed that the neutralizing effect of bevacizumab for VEGF binding to VEGFR2 and VEGFR1 is also based on steric hindrance [37]. The polyclonal nature of the response to CIGB-247 and the showed evolution with immunization time in different individuals, imply that different classes and subclasses of antibodies, with different affinity, avidity and antigen-recognition sites, are present in the patients. The specific polyclonal antibodies elicited by CIGB-247 probably have a more effective VEGF neutralization than that of the monoclonal antibody. The polyclonal nature of the response to CIGB-247 as compared with monoclonal antibody bevacizumab could yield the same levels of VEGF blocking activity with a lower amount of specific antibodies. The potential benefits of a vaccine strategy over a passive monoclonal antibody immunotherapy, in terms of the efficacy of the binding antigen/antibody, have been also outlined by others. An epidermal growth factor receptor 2 (HER 2)-based vaccine has been compared to the monoclonal antibodies trastuzumab and pertuzumab [39], which have been approved for the treatment of HER 2 overexpressed breast cancers. The immunization with the extracellular domain of HER 2 (HER 2-ECD) elicited polyclonal antibodies with specificity for 14 different epitopes. These polyclonal antibodies inhibited the binding of trastuzumab and pertuzumab [39, 40], which target different subdomains of HER 2-ECD. The properties exhibited by HER 2-induced antibodies included higher growth inhibition and significant receptor internalization, not observed when HER 2 overexpressing tumor cells were treated with trastuzumab [39, 40].

To achieve a clinically relevant effect using a cancer vaccine, it is not only important to generate a robust immune response, but also to sustain this response over time with booster vaccinations. On this regard, patients recruited for the CENTAURO and CENTAURO-2 clinical trials received off-trial monthly re-immunizations until death, intolerance, marked disease progression or patient’s withdrawal of consent [18, 19]. This re-immunization phase was important to sustain the VEGF-specific immune response generated during the induction phase [23, 24]. On this basis, individuals participating in the CIGB-247 CUP were also submitted to re-immunizations. In line with our findings in the previous clinical studies, re-immunizations helped to maintain the seroconversion status and blocking activity in most of the patients. Booster vaccination has been broadly used in cancer vaccines [28, 41, 42], indicating the importance of this strategy for the maintenance of the immune response.

During repetitive vaccinations and the generation of an immune response, there is a programmed order of IgG subclass usage. Collins et al. have been proposed a sequential switch where B cells do a first switch from IgM to IgG3, then to IgG1 and to IgG2 and finally to IgG4, although the switch can also occur directly from IgM to a particular IgG subclass. For this mechanism the affinity maturation increases in the following order: IgG3 < IgG1 < IgG2 < IgG4 [43]. The humoral response against VEGF elicited by CIGB-247 has been characterized by the presence of IgG1 as predominant subclass from the induction phase to 1 year of monthly re-immunizations [19]. After long term immunization and up to 3 years, IgG4 is the predominant immunoglobulin [24]. On the other hand, IgG3 subclass has a trend to disappear after 1 year of vaccinations; however it can be detected in some patients after 3 years of long term immunizations [19, 24]. Most of these results have been confirmed in the present study. It is likely that the putatively high affinity VEGF-specific IgG4 antibodies elicited after chronic vaccinations with CIGB-247 have an active role as effective blockers of the binding between VEGF and its receptors VEGFR2 and VEGFR1. In the case of IgG3, this is considered a relatively transient immunoglobulin, commonly associated with a primary immune response after initial exposure to an antigen [43]. The detection of VEGF-specific IgG3 antibodies during the re-immunization phase could be explained by the generation of new B cell clones, ready to initiate a programed process of sequential switching. The generation of new B cell clones during different time points of booster vaccination has been described by other investigators when two healthy donors were immunized with the tetanus toxoid (TT) vaccine [44]. The TT-specific serum IgG repertoire after booster vaccination comprised new clonotypes not observed before and their frequencies varied between subjects.

Despite the fact that since 2011 a thoroughly characterization of the humoral response elicited by CIGB-247 in cancer patients has been performed [18, 19, 24], no data about a possible cross-reactivity of the VEGF-specific polyclonal response with other VEGF family members had been done. The VEGF family is integrated by several members: VEGF, VEGF-B, VEGF-C, VEGF-D, and others less studied like VEGF-E and VEGF-F [45‐47]. VEGF is a principal mediator of angiogenesis through its binding to VEGFR2; meanwhile VEGF-C and VEGF-D can also bind to the same receptor and exert their biological activity. The amino acid sequence similarity between VEGF and VEGF-C or VEGF-D is 47 and 35% respectively. Herein presented results indicate that the antibody response induced by CIGB-247 is highly specific to VEGF and does not cross-react with VEGF-C and VEGF-D [48].. The lack of antibody cross-reactivity with molecular families of the antigen has been also reported in other cancer vaccines. For example, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) belong to EGF family, and both structurally related soluble proteins (between 30 and 40% amino acid homology) exert their action after interaction with a common cell surface EGF receptor [49]. CIMAvax-EGF is a therapeutic vaccine able to induce in humans a polyclonal antibody response directed to EGF, with the ability to block the interaction between EGF and the EGF receptor. However, the presence of antibodies against TGF-α has not been detected in immunized individuals [50]. In another example, Hosseini-Ghatar and colleagues generated three polyclonal antibodies against HER 2-ECD and none were able to bind to the other members of the human HER family [39].

As mentioned before, the CIGB-247 CUP allowed us to evaluate the preparation in cancer patients, under clinical conditions not yet explored before. In our previous clinical trials [18, 19], cancer patients with brain metastases, Hodgkin lymphoma or with other concomitant chronic diseases were not eligible for enrollment. Simultaneous onco-specific treatment and surgical procedures were also exclusion criteria.

In the sample presented in this paper, it is noteworthy that immunization with CIGB-247 was safe and well tolerated in all these cases. In addition to this experimental evidence, a detectable VEGF-specific antibody response was elicited in patients concomitantly treated with different chemotherapeutic agents. These findings provide the basis to further investigate the combination of CIGB-247 with standard-of-care drugs in patients with different types of cancer. Combinations of conventional chemotherapeutics with specific active immunotherapy are known [51‐53]. Chemotherapy is generally administered in regimens allowing and sometimes enhancing the development of the immunotherapy-induced response. We have recently initiated a phase II clinical trial with CIGB-247 in advanced ovarian cancer patients, where individuals receive neoadjuvant chemotherapy followed by interval debulking surgery, and all these procedures in concomitancy with immunization (RPCEC00000246).

Finally, two individuals included in our present study were submitted to major urgent surgeries during immunization with CIGB-247 without reports of wound healing impairment. It is know that bevacizumab treatment increases the risk of bleeding and wound healing complications in cancer patients [54]. These clinical observations with CIGB-247 could be explained by the incomplete abrogation of platelet VEGF levels after immunization with CIGB-247 [55]. After vaccination, remaining active VEGF molecules within platelets are probably sufficient to maintain the recovery response in wound healing, normal adult vasculature or other VEGF-dependent normal physiological processes.

In the CENTAURO clinical trial, platelet VEGF levels were evaluated by ELISA test and antigen-specific interferon-gamma (IFN-γ)-secreting cells were also measured by enzyme-linked immunospot assay (ELISPOT) using stimulated-peripheral blood mononuclear cells (PBMC) [18]. This investigation does not show data about the effect of CIGB-247 on platelet VEGF levels and frequency of cytokine-producing T cells, the latter as indicative of the cellular immune response.

Mandatory and programmed blood sample collections were not required in the CIGB-247 CUP, and these elements were optional and subjected to the physician’s criteria, limiting the number of immunogenicity tests. Also, during the routine clinical evaluation of CUP patients, the blood sample collection, processing, handling and storage (− 20 °C) of serum and plasma were suitable for antibody tests (immunoglobulin classes, IgG subclasses and blocking activity vs VEGFR2 and VEGFR1); however these conditions are not appropriate for VEGF quantification in serum and plasma, needing a storage temperature of − 70 °C [55].

On the other hand, between 50 and 60 mL of blood are required for isolation of PBMC by Ficoll density gradient. This volume of blood is at least five times higher than the volume used for routine clinical monitoring of cancer patients. For that reason, no data about cellular immune response elicited by CIGB-247 are shown here. Despite these limitations, ELISA tests used in this CIGB-247 CUP have been of importance for the immunological evaluations performed in all patients included in the CENTAURO and CENTAURO-2 clinical trials and their off-trial follow-up studies [19, 24].

Objective clinical benefits have been previously documented in a number of surviving CENTAURO patients [23, 24]. The information regarding clinical benefits observed in some patients only vaccinated with CIGB-247, and included within this CUP, will be presented as case reports by physicians, detailing the clinical evolution of every patient as well as the associated immunological data. These preliminary evidences of objective clinical benefits indicate that CIGB-247 could be a potentially promising therapeutic option for the treatment of certain types of cancer.

The CIGB-247 CUP confirms in a variety of cancer patients, under day-to-day clinical practice conditions in several Cuban medical institutions that CIGB-247 is immunogenic and safe. The results also indicate that CIGB-247 is the only one strategy already tested in humans able to induce a VEGF-specific antibody response. Administration of CIGB-247 induces IgM, IgA and IgG antibodies highly specific to human VEGF. This polyclonal response is able to block the interaction between VEGF with VEGFR2, VEGFR1 and bevacizumab. The immunological properties elicited during the induction phase are conserved at the re-immunization phase by monthly vaccinations. In both phases, VEGF-specific IgG1, IgG2, IgG3 and IgG4 subclasses were found, being IgG4 the predominant subclass after 3 years of chronic vaccination. Immunogenicity and preliminary safety data for immunization with CIGB-247 have been extended to previously untested clinical scenarios. The information unraveled by this study provides rationale for the potential combination of CIGB-247 with other onco-specific treatments of many kinds.

Relevant information of all commercially available reagents was provided in Additional file 2.