**3. A clinical development paradigm shift is needed for cancer vaccines: Why?**

Therapeutic cancer vaccines are active immunotherapy approaches that provoke an immune response against antigens relevant for tumor cell survival or growth (Talucka, 2011). As their action is very specific towards the cancer associated antigen, cancer vaccines has very low toxicity profiles. That makes possible long lasting use as well as combination with other drugs. In general, not tumor shrinkage is expected, but a prolonged patient´s survival as a consequence of disease control with chronic vaccinations compatible with good quality of life (Lage & Crombet, 2010).

In this very different scenario, the development paradigm used for chemotherapeutics is not working well (Table 1):


The first trials with cancer vaccines are mainly devoted to demonstrate the proof of the therapeutic principle, i.e. immunogenicity of the vaccine preparation, eliciting an immune response to the antigen they are intended to target; and they are also used for testing different formulations, doses, schemes and, by sure, safety. The novel paradigm of clinical development of cancer vaccines makes then Phase I trials better defined as Proof of Principle trials (PPT).

Clinical Development Paradigms for Cancer Vaccines: The Case of CIMAvax EGF® 149

Such an emerging paradigm for the development of cancer vaccines should also translate

The need of a new paradigm for cancer vaccine development can be illustrated by the story of CIMAvax EGF®, a therapeutic vaccine recently registered in Cuba for advanced lung cancer (Gonzalez et al, 1998, 2003, 2007, 2011; Lage et al, 2003; Crombet et al, 2006; Gonzalez

Endocrine therapy is an old and well validated approach for cancer treatment. Some tumors are considered as hormone-dependent because they require hormone stimulation for the continued growth of the tumor cells. In such cases, cancer bearing patients may benefit from hormone therapy, mainly based in deprivation of hormonal stimulus by different

But in addition to sex steroid hormones, tumor can be regulated by growth factors, such as EGF, through the binding to their receptors and activation of phosphorilation cascades leading to tumor cell proliferation. Currently, research in new anticancer drugs is increasingly focused in growth factors, their receptors and signal transduction mechanisms. Active and passive immunotherapies can be instrumental in the aim of interfering growth-

The Epidermal Growth Factor(EGF) based cancer vaccine (CIMAvax EGF®) is a conjugate of human recombinant EGF with the P64K protein of *Neisseria meningitides* (acting as a carrier protein) (Rodríguez et al, 2008). It was designed to induce specific anti-EGF antibodies (Abs) which, in turn, recognize and bind circulating EGF, avoiding further binding to the cell

These ligand /Ab unions, forms immune-complexes that are eliminated from the circulation through the liver, as other immune-complexes do (González et al, 1996). As can be realized, it is like an immune-castration effect, more similar to some hormone-therapies approaches aiming to deprive the cells of the hormone, but with the difference that it is the self immune

This vaccine is not meant to destroy the tumor by inducing immune effectors mechanisms, but to inhibit further tumor cell growth. This completely different mechanism of action could not only stop tumor growth, but could shift the balance between uncontrolled

Anti-EGF Abs are then a main marker of response to vaccination. This has been verified in the clinical setting by the observation that patients with higher anti-EGF Ab titers survived

The clinical development of CIMAvax EGF began in 1995. From then up to date, more than

Our very first objective was to demonstrate that the vaccine elicited anti-EGF Abs as it was designed to do. Clinical trials (Table 2) where then designed to demonstrate that the vaccine was immunogenic and provoked a depletion of circulating EGF. All trials have also safety as

 **moved through clinical development: Applying the** 

& Lage, 2007; García et al, 2008; Neninger et al, 2008a, 2008b; Rodriguez et al, 2010).

**: Immune response translates into clinical impact** 

into the attitude of Regulatory Authorities for granting Registration.

factor stimulation of cancer cell proliferation. (Gonzalez & Lage, 2007).

**4. CIMAvax EGF®**

procedures.

membrane receptor (EGF R).

proliferation and cell death.

**5. How CIMAvax EGF®**

**paradigm shift** 

primary objective.

system which deprives the growth factor (EGF).

significantly more than patients with low anti-EGF Ab titers.

1000 advanced cancer patients have received this vaccine.

For cytotoxic drugs, tumor reductions are expected and objective responses predict clinical benefit. In cancer vaccines it has been demonstrated that objective response is not always a good SV predictor; and therefore SV remains the appropriate end point. For SV analysis, comparison between randomized vaccine and control groups is required. Then after PPT, cancer vaccines moves directly towards randomized trials to test the efficacy of the product.


Table 1. Why general principles that direct oncologic drugs development does not apply to cancer vaccines?

As can be realized, these trials do not fulfill the characteristics of classic Phase II trials defined for cytotoxic drugs, firstly because the end point is different, and secondly because are already randomized trials.

In the novel paradigm for cancer vaccines, these trials are called ¨Efficacy trials¨ (ET).

The number of patients in these trials is adjusted to show statistical significance of a therapeutic advantage which is considered medically meaningful. With a wise medical judgment of what is medically relevant these ET could be often smaller to the current Phase III trials of the classic paradigm.

For cytotoxic drugs, tumor reductions are expected and objective responses predict clinical benefit. In cancer vaccines it has been demonstrated that objective response is not always a good SV predictor; and therefore SV remains the appropriate end point. For SV analysis, comparison between randomized vaccine and control groups is required. Then after PPT, cancer vaccines moves directly towards randomized trials to test the efficacy of the product.

The increase in dose should increase efficacy. Maximal tolerated doses are not optimal

cancer vaccines

patient.

effect.

therapies.

Table 1. Why general principles that direct oncologic drugs development does not apply to

As can be realized, these trials do not fulfill the characteristics of classic Phase II trials defined for cytotoxic drugs, firstly because the end point is different, and secondly because

The number of patients in these trials is adjusted to show statistical significance of a therapeutic advantage which is considered medically meaningful. With a wise medical judgment of what is medically relevant these ET could be often smaller to the current Phase

In the novel paradigm for cancer vaccines, these trials are called ¨Efficacy trials¨ (ET).

Why these principles are not well adapted to

doses. Due the low toxicity of cancer vaccines, it is not evident that a maximal tolerable dose will be coincident with the optimal dose to reach the vaccine effect,

Objective response is not a good SV predictor. Not necessarily, a patient with objective response has increase SV, which is the most important factor for the cancer

Therapeutic benefit could be delayed on time. Cancer vaccines require some time to exert their effect, unlike cytotoxic drugs that provokes an immediate cell destruction

Cancer vaccines could be actives even beyond disease progression. Progression doesn´t means less effectiveness of the vaccine, measured by increase in SV, which could be reached by slower progression related with a good quality of life.

Therapeutic vaccines can be effectives in combinations with other onco-specific drugs, even when not individually active. They can be used additionally and not instead of other

which could be much lower.

General principles that have ruled oncology

Pharmacokinetics is relevant for finding

Objective responses predict clinical benefit.

A fast tumor reduction is expected.

An objective progression is considered a treatment failure. Drugs are NOT active if the

Drugs assayed in combination must be

drug development.

optimal dose.

tumor is growing.

individually actives.

are already randomized trials.

III trials of the classic paradigm.

cancer vaccines?

Such an emerging paradigm for the development of cancer vaccines should also translate into the attitude of Regulatory Authorities for granting Registration.

#### **4. CIMAvax EGF® : Immune response translates into clinical impact**

The need of a new paradigm for cancer vaccine development can be illustrated by the story of CIMAvax EGF®, a therapeutic vaccine recently registered in Cuba for advanced lung cancer (Gonzalez et al, 1998, 2003, 2007, 2011; Lage et al, 2003; Crombet et al, 2006; Gonzalez & Lage, 2007; García et al, 2008; Neninger et al, 2008a, 2008b; Rodriguez et al, 2010).

Endocrine therapy is an old and well validated approach for cancer treatment. Some tumors are considered as hormone-dependent because they require hormone stimulation for the continued growth of the tumor cells. In such cases, cancer bearing patients may benefit from hormone therapy, mainly based in deprivation of hormonal stimulus by different procedures.

But in addition to sex steroid hormones, tumor can be regulated by growth factors, such as EGF, through the binding to their receptors and activation of phosphorilation cascades leading to tumor cell proliferation. Currently, research in new anticancer drugs is increasingly focused in growth factors, their receptors and signal transduction mechanisms. Active and passive immunotherapies can be instrumental in the aim of interfering growthfactor stimulation of cancer cell proliferation. (Gonzalez & Lage, 2007).

The Epidermal Growth Factor(EGF) based cancer vaccine (CIMAvax EGF®) is a conjugate of human recombinant EGF with the P64K protein of *Neisseria meningitides* (acting as a carrier protein) (Rodríguez et al, 2008). It was designed to induce specific anti-EGF antibodies (Abs) which, in turn, recognize and bind circulating EGF, avoiding further binding to the cell membrane receptor (EGF R).

These ligand /Ab unions, forms immune-complexes that are eliminated from the circulation through the liver, as other immune-complexes do (González et al, 1996). As can be realized, it is like an immune-castration effect, more similar to some hormone-therapies approaches aiming to deprive the cells of the hormone, but with the difference that it is the self immune system which deprives the growth factor (EGF).

This vaccine is not meant to destroy the tumor by inducing immune effectors mechanisms, but to inhibit further tumor cell growth. This completely different mechanism of action could not only stop tumor growth, but could shift the balance between uncontrolled proliferation and cell death.

Anti-EGF Abs are then a main marker of response to vaccination. This has been verified in the clinical setting by the observation that patients with higher anti-EGF Ab titers survived significantly more than patients with low anti-EGF Ab titers.

#### **5. How CIMAvax EGF® moved through clinical development: Applying the paradigm shift**

The clinical development of CIMAvax EGF began in 1995. From then up to date, more than 1000 advanced cancer patients have received this vaccine.

Our very first objective was to demonstrate that the vaccine elicited anti-EGF Abs as it was designed to do. Clinical trials (Table 2) where then designed to demonstrate that the vaccine was immunogenic and provoked a depletion of circulating EGF. All trials have also safety as primary objective.

Clinical Development Paradigms for Cancer Vaccines: The Case of CIMAvax EGF® 151

Both Tetanus Toxoid and P64K protein from *Neisseria meningitides* showed carrier effect. In each of both treatment groups 60% of patients developed anti-EGF Ab responses after immunization. However, the Ab response against the TT in the group of patients vaccinated with hu-r-EGF-TT was very high, but not the Ab response against P64K in the group of patients vaccinated with hu-r-EGF-P64K. Trying to avoid any phenomenon related with epitopic suppression, P64K was selected as carrier protein for further trials (Gonzalez et al,

The second clinical trial was designed to demonstrate the safety and immunogenicity of the EGF based cancer vaccine using two different adjuvants: aluminum hydroxide (alum) or Montanide ISA 51 (Seppic, France). In this case, 20 stages IIIb-IV NSCLC patients were enrolled in the trial one month after concluding their first line chemotherapy, and then randomized for the adjuvant to be used. Ten patients received the conjugated vaccine in

A secondary aim of this study was to evaluate the effect of 5 single doses of the vaccine in the induction period (days 0, 7, 14, 21 and 51) so as of re-immunization when antibody titers decreased to, at least, 50% of their peak titer reached at the induction phase. Also the relation between anti-EGF antibody titers and patient's SV was evaluated (Gonzalez et al,

The third clinical trial had the very same design that the second one, but all patients in both treatment groups, received a low dose CPM as an immune-enhancer, 100 mg/m2 body

In both, second and third trials, sera were collected on days 0, 14, 28, 60 and then monthly for anti-EGF Ab titers determination. Tumor response was evaluated on the first month after inclusion and then, every 3 months, by chest X-ray, abdominal ultrasound, and thoracic and abdominal computerized tomography (CT) scan. Objective responses were classified

Pooled results from both trials were analyzed looking for immunogenicity, safety and effect

For immunogenicity analysis, two variables were analyzed: % of patients able to develop an

According to this classification, the higher % of either, seroconversion and GAR, were obtained in the groups of patients using Montanide ISA 51 as adjuvant. Cyclophosphamide pre-treatment did not show any improvement regarding % of seroconversion neither % GAR. It was concluded that the use of Montanide ISA 51 as adjuvant with the EGF vaccine

When analyzing Ab titer levels in responder patients, measured as geometric means of sera dilutions, an improvement was observed when Montanide ISA 51 was used as adjuvant.

Taken together, the data from both trials indicates that, regarding immunogenicity (% of responders and Ab titer levels), the best results were obtained when using Montanide ISA51

surface area 3 days before starting vaccination schedule. (Gonzalez et al, 2003).

of EGF vaccination on SV in the different treatment groups. (Gonzalez et al, 2007).

Patients able to develop an Ab response after vaccination were grouped as:

Pre-treatment with cyclophosphamide also improved the Ab titer levels.

alum and 10 patients in Montanide ISA 51 as adjuvant.

1998).

2003).

according to the WHO criteria.

1. Patient that seroconverts.

Ab response as well as Ab titer levels.

2. Good Antibody Responders (GAR). 3. Poor Antibody Responders (PAR).

improves % of anti-EGF Ab responder's patients.

as adjuvant and cyclophosphamide pre-treatment.

Different formulations, schemes and doses of the vaccine were tested and related with immunogenicity and safety.

As can be realized, these trials were not designed with the main goals of Phase I trials defined for cytotoxic drugs (pharmacokinetics and definition of maximal tolerable dose). Trials designed for this vaccine can be better called PPT.

Five PPT were performed allowing the selection of the optimal formulation, scheme and doses for moving forward to the ET.

In the first clinical trial, 10 patients with epithelial tumors were enrolled. Patients received two single doses of the vaccine composed by human recombinant EGF coupled to either Tetanus Toxoid (hu-r-EGF-TT) (5 patients) or the recombinant P64K protein from *Neisseria meningitidis* (hu-r-EGF-P6K) (5 patients), both groups used aluminum hydroxide (alum) as adjuvant. Each single dose of the vaccine was given on days 0 and 14.


Table 2. Clinical trials of CIMAvax EGF

The main objectives of the trial were to look at safety and immunogenicity of vaccination with self EGF in humans as well as to compare between the 2 carrier proteins: TT and P64K. Following vaccination there were no significant adverse events. Seroconversion (defined in this study as a doubling of Ab titer above baseline) was reported in 6 of the 10 patients included in the trial.

Both Tetanus Toxoid and P64K protein from *Neisseria meningitides* showed carrier effect. In each of both treatment groups 60% of patients developed anti-EGF Ab responses after immunization. However, the Ab response against the TT in the group of patients vaccinated with hu-r-EGF-TT was very high, but not the Ab response against P64K in the group of patients vaccinated with hu-r-EGF-P64K. Trying to avoid any phenomenon related with epitopic suppression, P64K was selected as carrier protein for further trials (Gonzalez et al, 1998).

The second clinical trial was designed to demonstrate the safety and immunogenicity of the EGF based cancer vaccine using two different adjuvants: aluminum hydroxide (alum) or Montanide ISA 51 (Seppic, France). In this case, 20 stages IIIb-IV NSCLC patients were enrolled in the trial one month after concluding their first line chemotherapy, and then randomized for the adjuvant to be used. Ten patients received the conjugated vaccine in alum and 10 patients in Montanide ISA 51 as adjuvant.

A secondary aim of this study was to evaluate the effect of 5 single doses of the vaccine in the induction period (days 0, 7, 14, 21 and 51) so as of re-immunization when antibody titers decreased to, at least, 50% of their peak titer reached at the induction phase. Also the relation between anti-EGF antibody titers and patient's SV was evaluated (Gonzalez et al, 2003).

The third clinical trial had the very same design that the second one, but all patients in both treatment groups, received a low dose CPM as an immune-enhancer, 100 mg/m2 body surface area 3 days before starting vaccination schedule. (Gonzalez et al, 2003).

In both, second and third trials, sera were collected on days 0, 14, 28, 60 and then monthly for anti-EGF Ab titers determination. Tumor response was evaluated on the first month after inclusion and then, every 3 months, by chest X-ray, abdominal ultrasound, and thoracic and abdominal computerized tomography (CT) scan. Objective responses were classified according to the WHO criteria.

Pooled results from both trials were analyzed looking for immunogenicity, safety and effect of EGF vaccination on SV in the different treatment groups. (Gonzalez et al, 2007).

For immunogenicity analysis, two variables were analyzed: % of patients able to develop an Ab response as well as Ab titer levels.

Patients able to develop an Ab response after vaccination were grouped as:

1. Patient that seroconverts.

150 Advances in Cancer Therapy

Different formulations, schemes and doses of the vaccine were tested and related with

As can be realized, these trials were not designed with the main goals of Phase I trials defined for cytotoxic drugs (pharmacokinetics and definition of maximal tolerable dose).

Five PPT were performed allowing the selection of the optimal formulation, scheme and

In the first clinical trial, 10 patients with epithelial tumors were enrolled. Patients received two single doses of the vaccine composed by human recombinant EGF coupled to either Tetanus Toxoid (hu-r-EGF-TT) (5 patients) or the recombinant P64K protein from *Neisseria meningitidis* (hu-r-EGF-P6K) (5 patients), both groups used aluminum hydroxide (alum) as

Clinical trial Target population End point Reference

Carrier selection (TT vs. P64)

Adjuvant selection (Alum vs. Montanide)

Cyclphosphamide (CPM) pre-treatment (CPM or not)

> Dose escalation (2 doses)

Schedule evaluation Vaccine-Chemotherapy-Vaccine

SV benefit Vaccine vs. BSC (2nd line therapy)

SV benefit Vaccine vs. BSC (2nd line therapy)

Ann Oncol 1998;9(4):431

Ann Oncol 2003; 4(3):461

Ann Oncol 2003; 4(3):461

Cancer Biol Ther. 2006; 5, 15.

> J Immunother 2009;32:92–99

J Clin Oncol. 2008 6(9):1452.

> Ongoing, not published.

immunogenicity and safety.

PPT Exploratory trial (1)

PPT Exploratory trial (2)

PPT Exploratory trial (3)

PPT Exploratory trial (4)

PPT Exploratory trial (5)

included in the trial.

doses for moving forward to the ET.

Trials designed for this vaccine can be better called PPT.

adjuvant. Each single dose of the vaccine was given on days 0 and 14.

Solid tumor patients (n=10)

> NSCLC patients (n=20)

> NSCLC patients (n=20)

> NSCLC patients (n=20)

> NSCLC patients (n=20)

> > (n=80 s)

(n=579)

The main objectives of the trial were to look at safety and immunogenicity of vaccination with self EGF in humans as well as to compare between the 2 carrier proteins: TT and P64K. Following vaccination there were no significant adverse events. Seroconversion (defined in this study as a doubling of Ab titer above baseline) was reported in 6 of the 10 patients

ET (6) NSCLC patients

ET (7) NSCLC patients

Table 2. Clinical trials of CIMAvax EGF


According to this classification, the higher % of either, seroconversion and GAR, were obtained in the groups of patients using Montanide ISA 51 as adjuvant. Cyclophosphamide pre-treatment did not show any improvement regarding % of seroconversion neither % GAR. It was concluded that the use of Montanide ISA 51 as adjuvant with the EGF vaccine improves % of anti-EGF Ab responder's patients.

When analyzing Ab titer levels in responder patients, measured as geometric means of sera dilutions, an improvement was observed when Montanide ISA 51 was used as adjuvant. Pre-treatment with cyclophosphamide also improved the Ab titer levels.

Taken together, the data from both trials indicates that, regarding immunogenicity (% of responders and Ab titer levels), the best results were obtained when using Montanide ISA51 as adjuvant and cyclophosphamide pre-treatment.

Clinical Development Paradigms for Cancer Vaccines: The Case of CIMAvax EGF® 153

Additionally, in these trials EGF sera levels were measured. A significant increase in SV was directly correlated with decreased EGF blood levels. Vaccinated patients with an EGF sera concentration after vaccination ≤168 pg/mL showed a significant increase in SV as compared with patients with levels >168 pg/mL ( (Table 4). This is also an important result, and could means that EGF levels can be considered as an adequate surrogated marker of

There was a trend to increased SV in patients in the double dose group as compared with

According to the results obtained from the previous trials, it was decided to move forward with the vaccine formulation composed by EGF-P64k in Montanide ISA 51 as adjuvant (CIMAVAX EGF) in a scheme including pre-treatment with low dose of cyclophosphamide. A trial was designed where 80 patients, 1 month after concluding their 1rst line chemotherapy, were randomized in 2 groups, one receiving the vaccine CIMAvax EGF and the other only Best Supportive Care. According to the statistical design, with this small size randomized trial we expected to find a statistical improve in survival of 4 month for vaccinated patients as compared with the non vaccinated controls. An induction step of five single doses were given on days 0, 7, 14, 21 and 51, followed by monthly re-immunizations

(months)

15,28 11,3 <0,05

p (log rank test)

SV Mean (months) Median

PAR 7,84 7,07

for testing chemotherapeutics. It was called our first ET.

GAR 17,1 11,87 <0,05

5,79 5

generates anti-EGF antibody titers and decreases the EGF sera concentration.

concentration in the vaccine group, but not in the control group.

control patients inside the GAR group but not inside the PAR group

Table 4. Relation between Ab titers, sera EGF concentration and SV in the 4th clinical trial

It is evident that this trial design does not correspond with the Phase II trials currently used

Results from this trial showed that, the% of patients that seroconverted (2X original anti-EGF antibody levels) or GAR, was significantly greater in vaccinated patients than in controls. The 50% of vaccinated patients were GAR. Serum EGF concentration was significantly lower in vaccinated patients than those in controls. That means, the vaccination

It was also demonstrated that anti-EGF Ab titers correlates with decreasing EGF serum

Again it was corroborated that SV in GAR patients was significantly longer than in PAR patients. Additionally, there was a statistical difference in SV between vaccinated and

Survival in patients with lower EGF sera concentration was longer than in patients with higher sera EGF concentration. There was a significant increase in SV between patients that

anti-tumor response.

(Neninger et al, 2008a)

EGF sera concentration ≤168 pcg/ml

EGF sera concentration >168 pcg/ml

with the EGF based vaccine.

patients in the single dose group.

The kinetics of anti-EGF Ab response was also studied. Re-immunizations when Ab titers decreased did not provoked a characteristic booster effect (stronger and maintained Ab responses). Ab titers increased after re-immunization but only to the same levels reached as previous maximal values and decreased again in a short period of time. It is likely to confirm that continuous re-immunizations were needed to maintain anti-EGF Ab titer levels.

Results showed a significant increase in SV for GAR as compared with PAR and with an historical control group (Table 3). In pre-clinical studies, we demonstrated a direct relation between anti-EGF Ab titers and SV in tumor challenged mice (González et al, 1996). The results obtained from clinical trials indicate that a similar association occurs in vaccinated cancer patients.


Table 3. Relation between Ab titers provoked by vaccination and patient´s survival in 2nd and 3rd clinical trials with the EGF based vaccine.

One of the patients that reached a maintained high anti-EGF Ab response showed a tumor regression on month 12 after receiving the first vaccination. This is a single case and should be looked carefully, but it should be noted that this was one of the patients that developed higher and maintained anti-EGF Ab titers after vaccination.

No evidence of severe clinical toxicity was observed. Secondary reactions were mild or moderated, limited to 14 of 40 patients. Main reactions consisted on chills, fever, vomits, nausea, hypertension, headache, dizziness, flushing, and pain at the site of injection, bone pain, mouth dryness or hot flashes that, in all cases, disappeared after medication. Hematological data and blood chemistry remained within normal ranges during the immunization and follow-up period.

A fourth trial was then designed, in this case an scale up dose Phase I trial, in which patients were randomized to receive a single dose of the conjugate vaccine hu-r-EGF-P64K in alum as adjuvant (10 patients) or a double dose of the same vaccine (in alum also) in 2 injection sites (10 patients). The immunization schedule consisted in 5 immunizations with the vaccine (single or double doses), on days 0, 7, 14, 21 and 28, and then monthly reimmunizations. This trial was further extended to include 0 more patients (10 in each group) (Crombet et al, 2006).

Regarding immunogenicity, improved results were obtained in patients receiving double doses of the vaccine, considering Ab titer levels (sera dilution geometric means).

In this trial, EGF levels in sera were also tested. An inverse correlation was observed between EGF levels in patient sera and anti-EGF Ab titers. This result agrees with the vaccine working hypothesis. Anti-EGF Ab should bind to circulating EGF provoking a decrease in the EGF concentration. Less (or none) EGF is available to bind the EGF receptor, avoiding then the proliferation mechanisms derived from such binding.

Patients in the double dose group reduced more their EGF sera concentration that patients in the single dose group.

Again a significant increase in SV was observed for GAR as compared with PAR (Table 4).

The kinetics of anti-EGF Ab response was also studied. Re-immunizations when Ab titers decreased did not provoked a characteristic booster effect (stronger and maintained Ab responses). Ab titers increased after re-immunization but only to the same levels reached as previous maximal values and decreased again in a short period of time. It is likely to confirm that continuous re-immunizations were needed to maintain anti-EGF Ab titer

Results showed a significant increase in SV for GAR as compared with PAR and with an historical control group (Table 3). In pre-clinical studies, we demonstrated a direct relation between anti-EGF Ab titers and SV in tumor challenged mice (González et al, 1996). The results obtained from clinical trials indicate that a similar association occurs in vaccinated

GAR 12,41 9,41

and 3rd clinical trials with the EGF based vaccine.

immunization and follow-up period.

(Crombet et al, 2006).

in the single dose group.

higher and maintained anti-EGF Ab titers after vaccination.

SV Mean (months) Median (months) p (log rank test)

P<0,05 PAR 5,47 4,5

7,41 5,67

One of the patients that reached a maintained high anti-EGF Ab response showed a tumor regression on month 12 after receiving the first vaccination. This is a single case and should be looked carefully, but it should be noted that this was one of the patients that developed

No evidence of severe clinical toxicity was observed. Secondary reactions were mild or moderated, limited to 14 of 40 patients. Main reactions consisted on chills, fever, vomits, nausea, hypertension, headache, dizziness, flushing, and pain at the site of injection, bone pain, mouth dryness or hot flashes that, in all cases, disappeared after medication. Hematological data and blood chemistry remained within normal ranges during the

A fourth trial was then designed, in this case an scale up dose Phase I trial, in which patients were randomized to receive a single dose of the conjugate vaccine hu-r-EGF-P64K in alum as adjuvant (10 patients) or a double dose of the same vaccine (in alum also) in 2 injection sites (10 patients). The immunization schedule consisted in 5 immunizations with the vaccine (single or double doses), on days 0, 7, 14, 21 and 28, and then monthly reimmunizations. This trial was further extended to include 0 more patients (10 in each group)

Regarding immunogenicity, improved results were obtained in patients receiving double

In this trial, EGF levels in sera were also tested. An inverse correlation was observed between EGF levels in patient sera and anti-EGF Ab titers. This result agrees with the vaccine working hypothesis. Anti-EGF Ab should bind to circulating EGF provoking a decrease in the EGF concentration. Less (or none) EGF is available to bind the EGF receptor,

Patients in the double dose group reduced more their EGF sera concentration that patients

Again a significant increase in SV was observed for GAR as compared with PAR (Table 4).

doses of the vaccine, considering Ab titer levels (sera dilution geometric means).

avoiding then the proliferation mechanisms derived from such binding.

Table 3. Relation between Ab titers provoked by vaccination and patient´s survival in 2nd

levels.

cancer patients.

Historical control group

Additionally, in these trials EGF sera levels were measured. A significant increase in SV was directly correlated with decreased EGF blood levels. Vaccinated patients with an EGF sera concentration after vaccination ≤168 pg/mL showed a significant increase in SV as compared with patients with levels >168 pg/mL ( (Table 4). This is also an important result, and could means that EGF levels can be considered as an adequate surrogated marker of anti-tumor response.

There was a trend to increased SV in patients in the double dose group as compared with patients in the single dose group.

According to the results obtained from the previous trials, it was decided to move forward with the vaccine formulation composed by EGF-P64k in Montanide ISA 51 as adjuvant (CIMAVAX EGF) in a scheme including pre-treatment with low dose of cyclophosphamide.

A trial was designed where 80 patients, 1 month after concluding their 1rst line chemotherapy, were randomized in 2 groups, one receiving the vaccine CIMAvax EGF and the other only Best Supportive Care. According to the statistical design, with this small size randomized trial we expected to find a statistical improve in survival of 4 month for vaccinated patients as compared with the non vaccinated controls. An induction step of five single doses were given on days 0, 7, 14, 21 and 51, followed by monthly re-immunizations (Neninger et al, 2008a)


Table 4. Relation between Ab titers, sera EGF concentration and SV in the 4th clinical trial with the EGF based vaccine.

It is evident that this trial design does not correspond with the Phase II trials currently used for testing chemotherapeutics. It was called our first ET.

Results from this trial showed that, the% of patients that seroconverted (2X original anti-EGF antibody levels) or GAR, was significantly greater in vaccinated patients than in controls. The 50% of vaccinated patients were GAR. Serum EGF concentration was significantly lower in vaccinated patients than those in controls. That means, the vaccination generates anti-EGF antibody titers and decreases the EGF sera concentration.

It was also demonstrated that anti-EGF Ab titers correlates with decreasing EGF serum concentration in the vaccine group, but not in the control group.

Again it was corroborated that SV in GAR patients was significantly longer than in PAR patients. Additionally, there was a statistical difference in SV between vaccinated and control patients inside the GAR group but not inside the PAR group

Survival in patients with lower EGF sera concentration was longer than in patients with higher sera EGF concentration. There was a significant increase in SV between patients that

Clinical Development Paradigms for Cancer Vaccines: The Case of CIMAvax EGF® 155

stratification, patients were randomized for receiving vaccination or only Best Supportive

The sample size was calculated taking into consideration previous results and considering a 3 month of increase in SV for vaccinated patients in the strata 1 and 2 months of increase in

As may be noticed, this trial design corresponds to a second ET that aimed to corroborate

This trial is currently ongoing and a partial cut on the results according to the statistical

As well as the classical Phase I-II-III paradigm, the new sequence of cancer vaccine clinical trials also approaches the goals of understanding safety and efficacy. In such a way, the commonly called Phase I and Phase II clinical trials matches with PPT, while Phase III trials

**6. The relationship between clinical trials and "levels of Evidence" CIMAvax** 

In the last decades, as the emergence of new medical technology accelerates, there has been a growing claim to assess and to classify emerging knowledge according to the strengths of

Evidence level Ia: The evidence comes from a meta-analysis of randomized, controlled, well

Evidence Level IIb: The evidence comes from, at least, one study not completely experimental, well designed, as cohort studies. Is referred to the situation when the application of an

Evidence Level III: The evidence comes from studies of well-designed non-experimental descriptive, such as comparative studies, correlation studies or case-control studies . Evidence level IV: The evidence comes from documents or opinions of expert committees or

Levels of evidence then translate into the strength of recommendation that can be assigned

During the clinical development of CIMAvax EGF®, the vaccine has transited through

We can conclude that CIMAvax EGF has 5 PPT, 2 ET (one ongoing) and has reached a level

In summary, the historical development of CIMAvax EGF, illustrates the new development

Firstly, the vaccine dose and schedule was selected across 5 different PPT, considering the specific immune response and its duration, and not the MTD or pharmacokinetics. The

Evidence Level Ib: The evidence comes from, at least, one randomized, controlled trial. Evidence level IIa: The evidence comes from, at least, one controlled, well designed, not

intervention is out of the investigators control, but whose effect can be assessed.

clinical experience of prestigious authorities or series of cases.

C: Evidence level III : Recommendation favorable but not conclusive

of evidence Ib that makes it highly recommended for its use in patients.

D: Evidence level IV: Expert consensus without adequate research evidence.

different stages of clinical trials and through different Evidence levels (Table 5).

Care. All patients received a 4X vaccine dose administered in 4 injection sites.

survival for vaccinated patients in the strata 2.

design is very encouraging.

matches with ET.

designed trials.

randomized trial.

 **example** 

the evidence (Elstein, 2004)

to a given new technology.

paradigm for cancer vaccines.

A: Evidence level I: Highly recommended B: Evidence level II: Favorable recommendation

**EGF®**

the results of our first ET results optimized conditions.

A "Level of Evidence" ladder has emerged as follows:

reached EGF sera concentration levels below168 pcg/ml as compared with patients didn´t reached these levels.

There was a trend toward better SV in vaccinated patients as compared with controls, which becomes significant when considered only those patients younger or equal 60 years old. Vaccination efficacy was well demonstrated in this patient cohort.

Differences in SV were much more advantageous for vaccinated patients when considered, for both vaccinated and controls, only those patients' responders to the first line of chemotherapy, but the trial has not a design strong enough to make the difference significant. Also in those patients with performance status (PS) 0 or 1 the effect of vaccination in SV was stronger. Again, the small sample number didn´t allow finding a statistical significance.

Summarizing we were looking an advantage in SV for vaccinated patients over non vaccinated controls that become increased in some patient niches, such as age under 60 years old, response to chemotherapy and PS.

At our experience, once safety of the vaccine is established, PPT needs not to be consecutive. After concluding the patient recruitment in our first ET, we decided to design and perform a new PPT to test the optimal conditions that, up to this date, given the best results regarding immunogenicity (Neninger et al, 2008).

This trial design included:


With this immunization schedule, a huge improvement in immunogenicity was observed with 95 % of patients reaching the GAR condition and up to 20 fold increase in anti-EGF titers as compared with the levels previously obtained.

Because of the high anti-EGF antibody levels, an additional classification of immune response was considered. Patients were considered super Gar (sGAR) when reached anti-EGF Ab titers of 1:64,000 or more. The 55% of patients in the trial reached this condition while only 2,8% did in the previous Efficacy trial..

The association between Ab response and survival was corroborated in this series, where sGAR patients survived significantly more than GAR patients.

In all patients vaccinated under this V-Ch-V scheme, the EGF sera concentration decreased to 78 pcg/mL (baseline of the detection kit).

All vaccinated patients survived significantly more than the control group of the phase II trial.

From this trial, it was demonstrated the possibility of given 4X the vaccine dose used in the first efficacy trial, and the significant improvement in immunogenicity and EGF depletion that such scheme produced.

It was then designed a new trial, where, taking into account the results from both, the first ET and the fifth PPT, Five hundred and seventy nine patients were selected after being responsive to the first line of chemotherapy and subsequently stratified per age. One hundred ninety eight patients were included in the group of age equal or minor to 60 years old (strata 1) and 381 patients in the group of ages older than 60 years old (strata 2). After

reached EGF sera concentration levels below168 pcg/ml as compared with patients didn´t

There was a trend toward better SV in vaccinated patients as compared with controls, which becomes significant when considered only those patients younger or equal 60 years old.

Differences in SV were much more advantageous for vaccinated patients when considered, for both vaccinated and controls, only those patients' responders to the first line of chemotherapy, but the trial has not a design strong enough to make the difference significant. Also in those patients with performance status (PS) 0 or 1 the effect of vaccination in SV was stronger. Again, the small sample number didn´t allow finding a

Summarizing we were looking an advantage in SV for vaccinated patients over non vaccinated controls that become increased in some patient niches, such as age under 60

At our experience, once safety of the vaccine is established, PPT needs not to be consecutive. After concluding the patient recruitment in our first ET, we decided to design and perform a new PPT to test the optimal conditions that, up to this date, given the best results regarding

4. Vaccine-Chemotherapy-Vaccine (V-Ch-V) approach: That means, a previous vaccination induction step on days 0 and 14 (4 single doses in 4 injection sites) is given before the first line of chemotherapy, then, after chemotherapy, vaccinations continue, 4

With this immunization schedule, a huge improvement in immunogenicity was observed with 95 % of patients reaching the GAR condition and up to 20 fold increase in anti-EGF

Because of the high anti-EGF antibody levels, an additional classification of immune response was considered. Patients were considered super Gar (sGAR) when reached anti-EGF Ab titers of 1:64,000 or more. The 55% of patients in the trial reached this condition

The association between Ab response and survival was corroborated in this series, where

In all patients vaccinated under this V-Ch-V scheme, the EGF sera concentration decreased

All vaccinated patients survived significantly more than the control group of the phase II

From this trial, it was demonstrated the possibility of given 4X the vaccine dose used in the first efficacy trial, and the significant improvement in immunogenicity and EGF depletion

It was then designed a new trial, where, taking into account the results from both, the first ET and the fifth PPT, Five hundred and seventy nine patients were selected after being responsive to the first line of chemotherapy and subsequently stratified per age. One hundred ninety eight patients were included in the group of age equal or minor to 60 years old (strata 1) and 381 patients in the group of ages older than 60 years old (strata 2). After

1. Use of Montanide ISA51 as adjuvant and cyclophosphamide pre-treatment. 2. Four single doses of the vaccine given in 4 different anatomical sites.

single doses (in 4 injection sites) each 14 days and then monthly.

Vaccination efficacy was well demonstrated in this patient cohort.

reached these levels.

statistical significance.

This trial design included:

years old, response to chemotherapy and PS.

3. Time intervals between vaccinations of 14 days.

titers as compared with the levels previously obtained.

sGAR patients survived significantly more than GAR patients.

while only 2,8% did in the previous Efficacy trial..

to 78 pcg/mL (baseline of the detection kit).

that such scheme produced.

trial.

immunogenicity (Neninger et al, 2008).

stratification, patients were randomized for receiving vaccination or only Best Supportive Care. All patients received a 4X vaccine dose administered in 4 injection sites.

The sample size was calculated taking into consideration previous results and considering a 3 month of increase in SV for vaccinated patients in the strata 1 and 2 months of increase in survival for vaccinated patients in the strata 2.

As may be noticed, this trial design corresponds to a second ET that aimed to corroborate the results of our first ET results optimized conditions.

This trial is currently ongoing and a partial cut on the results according to the statistical design is very encouraging.

As well as the classical Phase I-II-III paradigm, the new sequence of cancer vaccine clinical trials also approaches the goals of understanding safety and efficacy. In such a way, the commonly called Phase I and Phase II clinical trials matches with PPT, while Phase III trials matches with ET.

#### **6. The relationship between clinical trials and "levels of Evidence" CIMAvax EGF® example**

In the last decades, as the emergence of new medical technology accelerates, there has been a growing claim to assess and to classify emerging knowledge according to the strengths of the evidence (Elstein, 2004)

A "Level of Evidence" ladder has emerged as follows:

Evidence level Ia: The evidence comes from a meta-analysis of randomized, controlled, well designed trials.

Evidence Level Ib: The evidence comes from, at least, one randomized, controlled trial.

Evidence level IIa: The evidence comes from, at least, one controlled, well designed, not randomized trial.

Evidence Level IIb: The evidence comes from, at least, one study not completely experimental, well designed, as cohort studies. Is referred to the situation when the application of an intervention is out of the investigators control, but whose effect can be assessed.

Evidence Level III: The evidence comes from studies of well-designed non-experimental descriptive, such as comparative studies, correlation studies or case-control studies . Evidence level IV: The evidence comes from documents or opinions of expert committees or clinical experience of prestigious authorities or series of cases.

Levels of evidence then translate into the strength of recommendation that can be assigned to a given new technology.

A: Evidence level I: Highly recommended

B: Evidence level II: Favorable recommendation

C: Evidence level III : Recommendation favorable but not conclusive

D: Evidence level IV: Expert consensus without adequate research evidence.

During the clinical development of CIMAvax EGF®, the vaccine has transited through different stages of clinical trials and through different Evidence levels (Table 5).

We can conclude that CIMAvax EGF has 5 PPT, 2 ET (one ongoing) and has reached a level of evidence Ib that makes it highly recommended for its use in patients.

In summary, the historical development of CIMAvax EGF, illustrates the new development paradigm for cancer vaccines.

Firstly, the vaccine dose and schedule was selected across 5 different PPT, considering the specific immune response and its duration, and not the MTD or pharmacokinetics. The

Clinical Development Paradigms for Cancer Vaccines: The Case of CIMAvax EGF® 157

Many people working in cancer vaccines development are facing the need of a change in clinical development paradigm as described by Axel Hoos et al and currently been being borne out in practice. As usual in the history of science, crises arise in different places at once, in this case, caused by the emergence of new therapies (cancer vaccines) that do not agree with the mechanism of action of these previously used (cytotoxic antitumor therapies). These people that faced firstly the needs of changes are the pioneers of the new paradigm and have the challenge of ¨converting¨ the rest of the scientific community to the new ideas. How to induce the conversion and how it resists? Probably the single most common argument put forward by proponents of a new paradigm is that they can solve problems that have led to the old paradigm to crisis. As it has been extensively explained in this chapter, new paradigms proposed for cancer vaccines development solve the problems for

The challenge now is to persuade the whole medical community as well as the Regulatory bodies on the new conceptions, which will result in a more direct development of new

As Thomas Kuhn said in his book The Structure of Scientific Revolutions: ¨In science occurs as in manufacturing: a change of tools is an extravagance that is reserved for occasions that demand it. The significance of crises is that they provide an indication that it is time to

Cancer vaccines are, as other biological molecules, product of the development of Biotechnology, a tool of science which opened a new wave of opportunity for cancer immunotherapy. It created the possibility of finding and manufacturing biological molecules with the same purity, reproducibility and scalability of classic chemical pharmaceuticals. These novel therapeutical tools have the attribute of specificity, which means possibility of therapeutic effect with lack of toxicity. That property makes them very

Because of that, biotechnology drugs claimed for a change in the paradigm through which antitumor drugs has been up to date investigated, developed, and finally approved. This is

We are facing times of changes. As soon as new paradigms are implemented for new drugs

This is just the beginning. Cancer therapy is a field of Science in transition. The changes in the paradigm of product development come together with other changes also intrinsic to

There are other important differences between cancer vaccines and cytotoxic antitumor drugs: one is that they are intended to restrain tumor growth, not necessarily to reduce

These features connect with another paradigm shift which is going on in medical oncology, which is the transition of advanced cancer from a rapidly fatal disease into a chronic

Such a transition is not new in the history of medicine. Diabetes Mellitus Type I was also a rapidly fatal disease until the introduction of Insulin in 1923. Now it is a chronic condition,

drugs and a more rapid availability of the same for patients with cancer.

change tools¨ .This is precisely what is going on in the field of cancer vaccines.

different to the cytotoxic tumor drugs approach for cancer treatment.

development, the faster the profits of these new drugs will be available.

especially pertinent in the field of therapeutic cancer vaccines.

tumor mass, and the second is that they can be used long term.

novel biotechnology drugs attributes.

condition, compatible with years of quality life.

which cannot be cured, but can be controlled long term.

which the old paradigms no longer work.

**8. Conclusion: A field of science in transition** 

vaccine was very well tolerated, provoking only grade 1 or 2 of adverse events; the MTD was never achieved.

Secondly, after optimizing dose and schedule, a randomized trial was conducted to assess the preliminary efficacy in terms of survival and not response rate. Tumor shrinkage was rarely observed while all vaccinated patients had a trend toward SV benefit that was significant in those patients with 60 years old or younger. The Kaplan Meier survival curve showed a non-proportional hazard ratio, illustrating the delayed (not immediate) effect of the drug. CIMAVax EGF needed at least 3 months to induce a mature, neutralizing immune response and a consequent survival curve separation.

Finally, vaccination was not stopped at the moment of clinically irrelevant, radiologic progression. Patients received chronic vaccination, which increased their probability of becoming good responders and long survivors (González et al, 2011)


Table 5. Relation between clinical trials stages and Evidence Levels
