Immunomodulation in Established Murine Tumors: Response and Survival Rate Enhancement by Blood Leukocyte-Augmenting Substance 236 (Cl⁻), a Novel Synthetic Compound

There are many striking examples of long-lasting remission and cure of some malignancies. However, the prognosis for patients with advanced invasive or metastatic disease is not much improved over the situation of 25 years ago (1), and there is an obvious need for more effective cancer therapy regimens to improve patient survival and cure rates (2). Recent efforts in this research line have taken advantage of a renewed interest in immunotherapy based on highly purified biological products obtained through recombinant DNA engineering (3).

Experimental immunotherapy through active vaccination procedures has resulted in remarkable, although transient, tumor growth inhibition (Refs. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37; see “Discussion”). However, after 10 years of clinical trials in patients with advanced disease, the current immunotherapy approach to cancer has not lived up to its initial promise (Refs. 38, 39, 40, 41, 42, 43, 44, 45; see “Discussion”).

Immunomodulation takes on the role of the nonspecific natural immune pathway and is an alternative to immunotherapy. Some new immunomodulatory products have proved highly successful in animal therapy models (Refs. 46, 47, 48, 49, 50, 51; see “Discussion”) but again have been less effective in human clinical trials (Refs. 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62; see “Discussion”).

Immunosuppression is a major problem in cancer patients because of their inability to fully restore peripheral pool T lymphocytes and functions after profound disease and therapy-related T-lymphocyte depletion. The novel product BLAS 236 (Cl⁻) presented here is an immunomodulatory compound that has no overt side effects. In contrast with thymic hormones and extracts, BLAS 236 (Cl⁻) gives rise to a broader than normal population of peripheral T lymphocytes responding to PHA² stimulation, as shown by a significant 191% increase in the lymphoproliferative response to PHA of peripheral blood mononuclear cells from blood bank donors, over the self-PHA baseline (100%) in “in vitro” assays (63). This enhancement exceeds that induced in healthy human adults and animals by fetal tissues and extracts (64, 65, 66). The administration of BLAS 236 (Cl⁻) doses to adult White rabbits has been related to a 169% global increase in the WBC count, namely lymphocytes, over the self baseline (67). To date, the product has also significantly improved the overall survival of mice bearing s.c. tumors (68). This report describes the response and survival rates observed using suitable BLAS 236 (Cl⁻) doses (68), both in single-agent and combined therapy protocols with effective doses of CTX or G-CSF, in mice bearing established syngeneic s.c. tumors.

Adult Swiss mice were purchased from PANLAB (Barcelona, Spain) and caged and kept in a conventional environment until use at 20–27 weeks of age. Mice live weight was 35 g for males and 30 g for females on average. EAC (hypertriploid) specimens from the Institute Gustave Roussy (París, France) were kept in the ascites form by serial i.p. passage every 10–11 days in both male and female mice. Briefly, tumor free-cells were aseptically harvested from the ascitic fluid and washed twice in sterile saline solution. Appropriate final suspensions were prepared in normal saline solution to establish primary s.c. tumors [containing 150,000 (80% viable) free-cells in 0.1 ml].

BLAS 236 (Cl⁻) was kindly supplied by Totiam S.L. (Madrid, Spain). The molecular formula of this product is C₁₁H₁₅C_l2N₃O₃, H₂O and its chemical name is 4-ammonium-4 (1-pyridiniomethyl) pyroglutamic acid dichloride. Its chemical identity has been confirmed by nuclear magnetic resonance, high-resolution mass spectrometry, infrared spectroscopy, and elemental CNH analysis (63). A 0.2-mm stock solution of BLAS 236 (Cl⁻) was prepared in sterile pyrogen-free isotonic saline in aseptic vials, was sterilized using Millex-GS single use filter units (Millipore S.A.), and was stored at 4°C. To treat the animals, suitable 1:50 sterile dilutions in isotonic saline were prepared as needed. The final concentration of the active product in both stock and working dilutions was verified by UV light absorption at a wavelength of 260 nm. G-CSF, kindly supplied by AMGEN (Neupogen 30; Barcelona, Spain), was diluted to a final concentration of 15 μg/ml (1.5 mU) in sterile, pyrogen-free, 5% glucose 1–20 h before administration. Both stock and working solutions were stored at 4°C. CTX was bought at a private drug store in vials containing 200 mg/10 ml, (Genoxal; Prasfarma S.A., Barcelona, Spain) and diluted 50:50 in sterile isotonic saline immediately before administration. Sterile pyrogen-free, isotonic normal saline and 5% glucose were used as vehicle controls (placebo).

Primary tumors were established by s.c. inoculating 0.1 ml containing 150,000 of tumor free-cells in the right hind flank of adult syngeneic mice. The sample population was composed of 333 mice, 131 (39%) males and 202 (61%) females. A total of 102 (31%) were given single-agent therapy, 144 (43%) received combined therapy, and the remaining 87 (26%) were administered the control vehicle (placebo). The population subsets are provided in Table 1.

Cohorts of at least five mice were used in all of the experimental groups, which were randomly assigned to treatment with either placebo or a specific protocol. Single therapy or the first agent in combined therapy was initiated 4–14 days after tumor inoculation. All of the agents [CTX, G-CSF, BLAS 236 (Cl⁻) or vehicle solution-placebo] were administered i.p. as 0.2-ml injections. CTX and G-CSF doses were calculated according to their specifications; BLAS 236 (Cl⁻) was administered at a suitable dose defined in a previous study (68). Single therapy included doses of CTX (C1–C3 subsets) or G-CSF (GR1–GR3 subsets) or BLAS 236 (Cl⁻) (B1–B6 subsets). Combined therapy included doses of CTX + BLAS 236 (Cl⁻) (A1–A7 subsets) or G-CSF + BLAS 236 (Cl⁻) (D1–D9 subsets). The treatment strategies for each protocol are described in Table 2.

All of the studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by NIH and European Union (EU) Guidelines (European Economic Community directive no. L-358, December 18, 1986).

Follow-up for mice with tumor progression was monitored twice weekly until death and, for the contingent of tumor-free alive mice, ended over 1 year after tumor inoculation. Tumor surface (mm²) and volume (mm³) were estimated by measuring the largest (L) and smallest (W) dimensions of each tumor in millimeters using calipers, and calculating the product using the formulae: L × W (mm²) and L × W² × ½ (mm³), respectively. The mean ± SE of tumor size in mm² was plotted as a function of treatment time in days for the follow-up of tumor growth and regression. Independent Student’s t tests or ANOVA were used to compare tumor size expressed as mean ± SE in mm² or mm³. Bonferroni’s test was applied for multiple contrasts. The proportions of mice subjected to each protocol showing a curative response were compared using Pearson’s χ² or Fisher’s exact tests. Survival rates were estimated by the Kaplan-Meier method. Breslow’s exact test was used to evaluate differences in survival time. Univariate and adjusted RRs and their 95% CI were calculated using Cox’s regression model. Model assumptions were tested. The null hypothesis was rejected in each statistical test when P < 0.05. Analyses were performed using Windows “SPSS” version 7.5 software.

Primary tumors developed at the site of inoculation in all of the mice after tumor grafting at 6–9 days after implant. These tumors steadily progressed to death in control mice, after local invasion and widespread metastasis to lymph nodes, lungs, liver, and so forth. The early tumor growth rate in untreated control mice was slower in females than in males; mean relative tumor sizes were 45, 25, 38, and 51% (P < 0.001) during the first 4 weeks, respectively. However, by day 48, the tumor load in females had reached the highest load attained by males on day 30.

Complete tumor remission after treatment occurred in 30% of the mice bearing established primary tumors, compared with 0% in control mice. Best results were obtained in males in the BLAS 236 (Cl⁻) single-therapy B3 and B6 subsets (80% for both) and in the G-CSF plus BLAS 236 (Cl⁻) combined therapy D6 and D7 subsets (both 100%). In females, best results were recorded for the CTX plus BLAS 236 (Cl⁻) combined therapy A4 subset (67%); the significant difference was P < 0.001 for each group. In contrast, almost no effects were observed in the subset of male mice subjected to CTX treatment (Table 3).

Eradicated tumors were, on the whole, fairly large, especially those in female mice. The mean time to eradication was 21 ± 1.1 days (range, 11–49 days): 13 ± 0.7 days for males, and 27 ± 1.2 days for females (data not shown).

Of the treated mice showing complete remission, 98.6% remained tumor free over the year after treatment [1 (1.4%) died by accident without a tumor], whereas 100% of the control mice had died by day 48. Among the mice with established s.c. tumors, all of the therapeutic subsets significantly exceeded the mean survival time of appropriate placebo controls, with the exception of protocol GR1 subset in males (Table 4). The maximum overall survival time, 232 ± 29.0 days, was achieved in the female mouse CTX-BLAS 236 (Cl⁻) A4 subset. The male B3 subset, with a mean survival time of 215 ± 33.5 days, followed. The control placebo subset showed maximum survival times of only 45 ± 1.5 days, P > 0.001 for each group (Table 4).

Specifically, protocols B3 and A6 showed up to an 87.2-fold (95% CI, 9.4–808) and 156.7-fold (95% CI, 12.8–1925) reduction in the HR for males, and protocol A4 was related to a 56.3-fold decrease (95% CI, 17.3–183) in the HR for females.

Table 4 and Figs. 1,2,3 show the significance values for manifold comparisons of cumulative survival curves and RR survival for the protocol regimens versus placebo for single and combined therapies using the KM-method and Cox’s regression model. The cumulative survival graphs for the B3–B6, D6–D7, and D1–D3 subsets overlap because survival values were identical from a given day onwards. The HR for protocols D6 and D7 was undetermined because none of the animals had died at the experiment cutoff point.

Male and female mice responded differently to the treatment schedules, and their primary tumor development also differed in the early phase of EAC tumor growth (see “Tumor Growth versus Remission” in “Results” section). This was also observed in a pilot study designed to determine the significant optimal BLAS 236 (Cl⁻) therapeutic dose (68). There are no previous reports in the literature that offer an explanation for this difference in EAC tumor growth between males and females, although similar examples of gender-related differences in responses to hormone-dependent pathologies, not considered nowadays, have been described.

Surprisingly, male tumors that generally showed faster growth at presentation, responded less well to chemotherapy but were highly sensitive to BLAS 236 (Cl⁻) modulation either as a single agent or combined with G-CSF. In contrast, tumors in females, which were slower at becoming established, showed a better response to single-agent or combined-agent chemotherapy, yet also responded well to combined G-CSF/BLAS 236 (Cl⁻) immunomodulation (Tables 3, 4).

A neuronal-endocrine-immunological axis, normally acting on this three-systems interrelation, could support the explanation to the different therapeutic activity observed in this immunomodulation study with BLAS 236 (Cl⁻).

Today, the interpretation of cancer biotherapy effects as dictated by the killing paradigm is insufficient, because there is clear disparity between tumor remission and long-term survival rates. Relapse and the development of a secondary tumor after long periods of remission indicate that remission does not predict cure (69), and long-term survival continues being rare. The ultimate control of neoplasia depends on new hypotheses that evoke both the molecular spread of cancer and host resistance toward a minimal residual tumor burden.

Experimentally, enhancement of the host immune response to cancer has led to remarkable, yet generally transient, tumor growth inhibition. This boosting of the immune system has been achieved in many ways including: active vaccination procedures (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15), monoclonal antibodies alone or combined with chemotherapy regimens (16, 17, 18, 19, 20), recombinant immunotoxin constructs targeted toward oncogene products (21, 22, 23, 24, 25), immunocytokine-fusion proteins (26, 27, 28, 29), radioimmunotherapy alone (30, 31), or, after the administration of immunotoxins (32), adoptive immunotherapy by transferred antitumor immunological cells (33, 34) and immunogenetic therapy modalities (35, 36, 37, 70). In contrast, the overall response and survival rates recorded here through nonspecific immunomodulation with BLAS 236 (Cl⁻) remained unchanged throughout the entire follow-up of over 1 year. Phase I and Phase II clinical trials using vaccinia virus encoding carcinoembryonic antigen, or Herceptin, rituximab, or targeted immunotoxins and immunoconjugates, and so forth, have achieved variable responses, but no complete tumor remission was achieved in most cases, and associated toxicity was found (38, 39, 40, 41, 42, 43, 44, 45, 71, 72).

Nonspecific immunomodulation in mice bearing B16F10, M5076, or TSA-mammary adenocarcinoma using IL-12, rIL-2, thymosin α1 (Tα1), or IFN, either as single agents or in combined chemotherapy protocols (46, 47, 48, 49, 50, 51), led to complete tumor remission and cure in a significant proportion of mice, although toxicity problems were observed at optimal treatment doses.

In human clinical trials, thymic hormones and factors (thymosin Fv, thymosin α1, thymostimulin, and so forth) were able to prolong survival when used as adjuvant or given after surgery in select patients (52, 53, 54, 55, 56). Immunomodulation experience gained in 15 trials conducted by the National Biotherapy Study Group using high-dose IL-2 alone or combined with lymphokine-activated killer cells, CTLs, tumor-infiltrating lymphocytes, IFN, tumor necrosis factor, and so forth, gave rise to low response rates overall and high toxicity (57, 58). In metastatic renal cancer treated with high-dose rIL-2, only 8% of patients showed no disease progression (59). In the Eastern Cooperative Oncology Group (ECOG) trial EST 1684, IFN α-2b was the first agent to be related to increased survival times of about 1 year (60). Finally, in pilot trials in which patients were subjected to s.c. Rhu IL-12 treatment, neither partial nor complete remission was achieved (61, 62).

The consensus on the strong link between lymphocyte depletion and cancer outcome is unanimous (73), but there is practically no knowledge on the mechanisms responsible for regulating lymphocyte numbers (74, 75) and promoting their recovery (76, 77). Cancer patients often show severely diminished circulating T-lymphocyte populations, both in terms of numbers and functions. This situation is further impaired by classic cytotoxic therapy-related changes, which may last for years (78). In addition, the recovery of the mainly naïve CD45RA⁺ CD45RO⁻ CD4⁺ T-cell phenotype is incomplete and occurs only in about 50% of adult patients (79, 80). Thus, therapeutic strategies aimed at lymphocyte regeneration are crucial for improving the treatment of cancer and other diseases associated with a depleted immune system (75).

Experimental evidence thus far indicates that BLAS 236 (Cl⁻) achieves a sizeable peripheral pool of PHA-sensitive, naïve T lymphocytes (63) and increases lymphocyte counts in vivo (67). This potentially ensures an improved immune response to neoplasmic challenges. Administered as a single agent or in a combined-therapy protocol, the drug was able to safely achieve definitive tumor remission or prolonged the animal’s overall life span and brought down the death HR, compared with placebo controls even in mice bearing huge tumors (Tables 3 and 4; Figs. 1,2,3). These results represent a significant improvement over those derived from preclinical trials based on current immune approaches to cancer in which long-term survival has been scarce at best, and toxicity is always a major limitation. Lastly, we envisage the use of this innocuous immunomodulation based on BLAS 236 (Cl⁻) as adjuvant therapy to surgery, radiotherapy, and chemotherapy to restore or boost the available peripheral T-lymphocyte repertoire. This therapeutic approach would have the ultimate goal of overcoming the process of carcinogenesis at the residual stage.

We thank A. Alonso-Diaz de Isla, M. P. Pérez-Villalobos, and J. J. Pérez-Villalobos for technical assistance and Totiam S. L. (Madrid, Spain) and AMGEN (Barcelona, Spain) for kindly supplying BLAS-236 (Cl⁻) and G-CSF, respectively.