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Abstract

The main objective of this study was the characterization of preclinical tumor models based on their expression of alpha-fetoprotein receptor (RECAF) for targeting cancer cells with a new non-covalent complex (AIMPILA) containing alpha-fetoprotein as the carrier and Atractyloside as an apoptosis-inducing agent. For that purpose, we measured the amount of RECAF in the homogenates of the grafted tumors T47D and SW620 and in HepG2 cell extracts. We also determined the alpha-fetoprotein binding specificity of the targeting drug AIMPILA using a solid-phase chemiluminescent assay with AIMPILA-Acrdidinium. We found that RECAF is practically absent from healthy mice tissues (100 Units/mg) where in malignant cells, the amount of alpha-fetoprotein receptors follows this order: T47D (9152 Units/mg) > HepG2 (4865 Units/mg) > SW620 (2839 Units/mg). This agrees with our findings regarding AIMPILA-induced tumor growth inhibition (T47D (T/C = 22%) > HepG2 (T/C = 51%) > SW620 (T/C = 70%), where T/C is the ratio of tumor volume in treated vs control animals). Our results demonstrate that the therapeutic response to the targeting drug AIMPILA strongly depends on the RECAF expression by human tumors and confirms the choice of the tumor models used for an AIMPILA preclinical study.

Keywords

Alpha-fetoprotein receptor RECAF AIMPILA nude mice human tumors homogenate chemiluminescent assay

Introduction

Alpha-fetoprotein (AFP), an oncofetal antigen described by Abelev,¹ was the first cancer marker of clinical use. AFP receptors (RECAF) in tumor cells have been studied for decades. Following the first proposal for such a receptor in 1981,² the presence of AFP-RECAF complexes in tumor cells was described.^3–5 The complex structure of the AFP transmembrane receptor still requires further characterization of the structure/function correlation of the molecules involved.⁶ While the mechanism by which AFP affects cancer cell proliferation is not quite understood, there is a great deal of evidence demonstrating that AFP receptors are expressed on the membrane surface of cancer cells.^7–10 In vitro studies confirmed that the activation of AFP receptors can promote proliferation of the BEL-7402 cell line through the protein tyrosine kinase (PTK)-Ras-mitogen-activated protein kinase (MAPK) pathway.¹¹ Alternatively, the inhibition of caspase-3 caused by AFP binding to its receptors leads to the blocking of apoptosis signal transduction in human hepatocellular carcinoma (HCC) cells.¹² AFP active peptides can induce specific cytotoxic T-lymphocytes to eliminate AFP-positive tumor cells and can cause an immune effect to kill cancer cells.⁶

After the discovery that AFP penetrates rat’s rhabdomyosarcoma tumor cells and mouse YAC-1 cells via receptor-mediated endocytosis, a series of publications described a transmembrane pathway of AFP receptor’s penetration on the cell surface of other mammals and humans.^13,14 Using the MCF-7 cell line, it was demonstrated that AFP reacts with a soluble form of RECAF present in cytosol fractions.¹⁵ This is consistent with a previous report by Uriel et al.¹⁶ showing binding between labeled estrogens complexed to AFP and an 8s ultracentrifugation fraction in immature rat uterine cytosols. Later, it was demonstrated that the soluble AFP receptor behaves like an oncofetal antigen with clinical potential for diagnostics, screening, and monitoring of cancer patients.^8,9

Since normal adult cells express minimal quantities of RECAF, in the last two decades, AFP has been considered as a prospective anti-cancer drug carrier molecule that could be used for specific tumor targeting. In this regard, three features of AFP are of importance: (1) the transport and delivery of polyunsaturated fatty acids and other small molecules into fetal cells until a certain level of differentiation is reached;^2–4 (2) the immunosuppressive action that might help prevent maternal rejection of the fetus; and (3) the binding of estrogens.^16–18 One of the most important regulatory mechanisms of AFP is its estrogen-binding activity localized in its third Domain, which prevents the degradation of estrogens and facilitates their transport into different organs and tissues and also binds to both the free and immobilized hormones. The inhibition mechanism of cell proliferation or tumor growth stimulation is unclear.

Both native and recombinant AFP have been used as vectors for delivering various cytotoxic agents and plant toxins to tumor cells.¹⁹ The presence of AFP receptors in malignant tumors allows targeting them with a variety of complexes of AFP and different toxic substances. An example is AIMPILA, an oral drug formulation comprising a composition of a non-covalent complex of porcine AFP (PAFP) and the apoptosis-inducing agent Atractyloside. The drug is intended for the specific delivery of Atractyloside to the cancer cells via the AFP receptors on their surface.²⁰

In this report, we provide data regarding the targeting specificity of AIMPILA using a specific immunoassay based on RECAF and specific anti-RECAF monoclonal antibodies. Our objective was to select suitable tumor models for a preclinical study on AIMPILA efficacy based on their RECAF expression. Therefore, we (1) quantitatively evaluated the AFP receptors in serum of immunodeficient mice bearing human tumor xenografts and in the homogenates of the grafted tumors, (2) evaluated the binding specificity of AIMPILA to RECAF in human tumors, and (3) determined the correlation between the receptor expression in different tumors and tumor growth inhibition by AIMPILA.

Materials and methods

Immunodeficient murine models

For the experiments involving subcutaneous human tumor xenografts, we used 8-week-old female and male Balb/c nude mice weighing 20–22 g from the virus-free vivarium at the Federal State Budgetary Institution, N.N. Blokhin Medical Research Center of Oncology of the Ministry of Health of the Russian Federation. Moscow, Russia. Animals were divided into three groups with 10 specimens each.

Tumor models

Transplantable human tumor models were from the collection of human tumor strains of the Federal State Budgetary Institution, N.N. Blokhin Medical Research Center of Oncology of the Ministry of Health of the Russian Federation. The following original cell lines were obtained from ATCC^®: estrogen-dependent breast carcinoma T47D (Cat. No. HTB-133^™), colorectal adenocarcinoma SW620 (CCL-227^™), and HCC HepG2 (Cat. No. HB-8065^™). The MCF-7 human breast cancer carcinoma (ATCC Cat. No. HTB 22^™) and K-562 myelogenous leukemia (ATCC Cat. No. CCL-243^™) were used as positive controls of RECAF expression. As negative controls, homogenates of healthy mice tissues were used.

In accordance to the guidelines of the Russian Federation, we subcutaneously injected 40 mg of the tumor tissue in 0.5 mL of 199 media into Balb/c nude mice. Cell suspensions were inoculated subcutaneously into the side of each mouse, using a volume of 0.2 mL containing 12 × 10⁶ tumor cells.

Cell culturing methods

HepG2 and K-562 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) cell culture media containing 10% fetal bovine serum (Life Technologies, Inc., USA) at 37°С in 5% СО₂. After 4 days of growth, the cells were harvested and centrifuged at 580 r/min for 10 min at room temperature. The supernatant was discarded and the cells were washed twice with 0.1 M Tris-buffered saline (TBS) buffer pH 7.4 and then re-suspended to 5 mL total volume with 0.1 M TBS buffer. Following the addition of 90 µL Trypan Blue Solution (T-8154, 0.4%; Sigma, USA) to 10 µL of cell suspension, the cells were counted and adjusted to 150 million cells per/mL. After that, the suspension of HepG2 cells was sonicated as described below.

Targeting drug studied

AIMPILA is a non-covalent complex of PAFP with an inductor of apoptosis Atractyloside. PAFP was prepared from blood and amniotic fluid from porcine embryos of about 3–14 weeks gestation by butanol extraction. Atractyloside (an azulenon glycoside) is a natural extremely toxic glycoside from a family of sesquiterpenoids. It is extracted from the roots of a plant named Atractylis Lancea, which grows in South-East Asia and China.²¹ The chemical formula of Atractyloside is (2β, 4α, 15α)-15-hydroxy-2-[[2-O-(e-methyl-1-oxobutyl)-3, 4-di-O-sulfo-β-D-glucopyranosyl]oxy]-19-norkaur-16-en-18-oic acid dipotassium salt. The primary mechanism of Atractyloside poisoning is known to be inhibition of the mitochondrial dinitrophenol (DNP) transporter. Atractyloside in large amounts gives rise to massive necrosis, but in vitro studies have shown that at lower doses, cells progress to apoptosis.²¹ The AFP-Atractyloside conjugation was performed as described elsewhere.²⁰

A stock solution of AIMPILA at 50 mg/mL was introduced into the stomach of each animal using a metal probe, as a single dose of 20 mg/kg (a total therapeutic dose). The drug dosage was adjusted for each mouse according to its weight. The drug was administered 48 h after the tumor transplantation.

Characterization of the specific monoclonal antibodies 1.4G11 against RECAF

Murine monoclonal antibody (Mab) 1.4G11, isotype IgM, was purified by ammonium sulfate precipitation at 40% saturation followed by dialysis and chromatography on AcA34 (Ultragel). The IgM was recovered from the void volume of the column. Binding specificity of the purified Mab for the RECAF glycoprotein was confirmed by polyacrylamide gel electrophoresis (PAGE), western blot with labeled RECAF, and with chemiluminescence immunoassay (CLIA) such as direct binding of the Mab to RECAF or a competitive assay with native RECAF antigen. All these tests showed high binding specificity between the 1.4G11 Mab and the RECAF antigen (Figures 1 and 2).

Figure 1.

Western blot analysis with anti-RECAF Mab and AFP. Lane 1: protein markers. Lanes 2 and 3: whole MCF-7 extract stained with 1.4G11 Mab and AFP, respectively. Lanes 4 and 5: pure RECAF antigen stained with 1.4G11 Mab and AFP, respectively. Both AFP and 1.4G11 recognize the same bands (for visualization purposes, image was enhanced with photoshop).

Figure 2.

(a) Different concentrations of AFP were added to microtiter wells containing 100 µL each of 10 µg/mL of 1.4G11 Mab. Wells were coated with MCF-7 extract. AFP competes with the antibody. (b) Different concentrations of 1.4G11 were added to microtiter wells containing 0.5 µg/mL of AFP biotin. Wells were coated with MCF-7 extract. The Mab competes with AFP.

Detection of AFP receptors in human tumors

The AIMPILA strategy relies on the target cells producing RECAF. To verify the presence of RECAF in these tumors, we measured the RECAF amount in homogenates of the grafted tumors, in the serum of the mice with tumor xenografts and in the serum of healthy mice. We were also interested in determining whether there is a correlation between the RECAF expression in the tumor and its serum concentration in the grafted animals. To measure RECAF in the serum of Balb/c nude mice or in the human tumor homogenates, we used a previously developed CLIA Serum RECAF test.²² We hypothesized that the tumor growth inhibition resulting from the administration of AIMPILA would correlate with the expression of RECAF in the studied tumor models.

CLIA for AFP receptors in tumor homogenates

The assay was based on a previously developed CLIA human serum RECAF test.^8,9,22 Briefly, 96-well black Greiner plates were coated with 1.4G11 anti-RECAF Mab at 5 µg/mL in phosphate-buffered saline (PBS) buffer pH 7.2. Acridinium-labeled RECAF (100 ng/mL) was then added to the wells together with a series of homogenate dilutions. Thus, the native RECAF in the samples and the Acridinium-labeled RECAF competed for binding to the monoclonal antibody attached to the plastic. After incubating the plates for 2 h at room temperature, the wells were washed to remove unbound material and the luminescence was then measured with a flash-chemiluminometer. The amount of labeled RECAF bound to the Mab on the solid phase is inversely proportional to the amount of RECAF in the sample. RECAF standards calibrated in RECAF Units were tested simultaneously. The standards were calibrated in arbitrary units by assigning a value of 4700 Units to the readings corresponding to the 95% percentile of a population (N > 300) of normal human serum. The standard curve follows a negative slope linear-log curve onto which the RECAF Units in the unknown samples are interpolated. A typical calibration curve is shown in Figure 3.

Figure 3.

The calibration curve of RECAF CLIA follows a linear-log function with negative slope.

Detection of AFP receptors in the serum of Balb/c nude mice bearing human tumors

To a 96-well plate coated with anti-RECAF antibody prepared as described in section “Detection of AFP receptors in human tumors,” standard calibrators and two-fold dilutions of the murine serum samples were added. The reagents were mixed thoroughly and incubated for 2 h at room temperature. After that, the content of the wells was discarded and the plate was thoroughly washed three times with distilled water using a W3000 Micro Plate Washer (Guangzhou Maya, China), followed by the addition of 50 µL/well of a pre-trigger solution of 1 mM nitric acid containing 0.1% H₂O₂ in distilled water The plate was then immediately read with a flash-chemiluminometer (GLOMAX^TM 96 Microplate Luminometer ТМ278; Promega, USA). Measurements were recorded in relative lights units (RLU). Specific reading parameters used for the experiments were as follows: pre-trigger volume: 50 µL/well, 1 s delay between readings, 1 s integration time, and 1 s lag time.

Determination of RECAF in human tumors sensitive to AIMPILA

For this experiment, we chose human tumor models with different affinity for AIMPILA and with anticipated diverse levels of RECAF elevation such as T47D, SW620, or HepG2 cell lines. The concentration of RECAF was measured in the homogenates of the abovementioned subcutaneous xenografts. As previously known positive controls, we used a homogenate of the human myelogenous leukemia K-562 suspension cell line and pure RECAF extracted from K-562 cells.^8,9 As a negative control, we used a homogenate of normal muscle and spleen tissue from healthy Balb/c nude mice.

Preparation of the human tumor homogenates and HepG2 cell extract

Fragments of T47D subcutaneous tumors were cut into small pieces with a scalpel and transferred to a cryovial with 1 mL of TBS buffer containing 10 mM of each СаCl₂, MgCl₂, and MnCl₂. The material was sonicated using an ultrasound homogenizer (BILON-150Y; Shangai Bilon Instrument Co., Ltd., China) at 60 W (40%). Sonication was carried out six times, 30 s each, in an ice bath, followed by centrifugation for 10 min at 14,000 r/min in an Eppendorf microfuge. The supernatant (denoted henceforth as “homogenate(s)”) was collected for analysis. The total protein concentration was 13.5 mg/mL as measured by the Bradford method using a commercial protein assay (“Sileks”, Moscow, Russia).

Homogenates of SW620 tumor tissue, HepG2 cell line extract, and negative control (a mix of muscle and spleen fragments from healthy mice) were obtained using the same procedure. The total protein concentration in the SW620 and HepG2 homogenates was 12.4 and 21 mg/mL, respectively. For the negative control (a homogenate of the muscle and a spleen tissue), the total amount of protein was 40 mg/mL.

PAGE and western blots

A volume of 10% polyacrylamide gels containing 10% sodium dodecyl sulfate (SDS) were used in a BioRad (USA) PAGE apparatus following the standard Laemmle’s procedure. The sample buffer consisted of 4 mL of distilled water, 1 mL of 0.5 M Tris-HCl, 0.8 mL of glycerol, 1.6 mL of 10% SDS, 0.4 mL of 2-mercaptoethanol, and 0.2 mL of 0.05% bromophenol blue. Gels were run for 2 h at a constant current of 0.2 A (70–100 V) in Tris-glycine running buffer using a BioRad system. Western blots were carried out according to standard procedures, using a BioRad Mini Trans-Blot apparatus and 0.45 µm pore nitrocellulose membranes (BioRad). After blocking the membranes with 1% bovine serum albumin (BSA) in TBS, the RECAF bands were evidenced by incubating the membranes with the specific monoclonal antibody at 5 µg/mL followed by washing and incubation with a commercial goat-anti-mouse IgM antibody conjugated to horseradish peroxidase (Sigma) at a 1:2000 dilution. Colored bands were developed with diaminobenzidine (DMB) and hydrogen peroxide following standard procedures.

AIMPILA labeling with Acridinium

AIMPILA was labeled at a 40:1 Acridium/AIMPILA molar ratio as follows: 1 mL of dH₂O containing 500 µg of AIMPILA was mixed with 100 µL of dimethyl sulfoxide (DMSO) to maintain the Acridinium–NHS Ester (Toronto Research Chemicals, Inc., Canada) in solution. Then, 33 µL of 5 mg/mL Acridinium solution in DMSO (152 µg, 2.2 × 10⁻⁴ M) was added to the reaction vial, which was mixed by inversion and incubated overnight protected from the light. The AIMPILA–Acridinium conjugate was dialyzed overnight at 4°C–6°C using Spectra/Por dialyzing tubing (cutoff = 12–14 kDa; Spectrum Labs, USA) against two changes, 5 L each, of 100 mM PBS, pH 7.2. Protein concentration was determined using a standard Lowry protein assay and found to be 200 µg/mL. After the addition of 0.02% Thimerosal, the AIMPILA–Acridinium conjugate was stored protected from the light at 4°C until use.

Binding specificity of AIMPILA to RECAF in tumor homogenates

The targeting ability of an anti-cancer drug such as AIMPILA largely depends on the specific binding of the drug to cancer cells. In order to determine the AIMPILA “targeting action,” we developed an immune assay based on the 1.4G11 monoclonal anti-RECAF Mab and Acridinium-labeled AIMPILA, where the T47D tumor homogenate was reacted with the specific anti-RECAF antibodies. We expected that the RECAF molecules in the homogenate would attach to the anti-RECAF 1.4G11 Mab, and therefore, they could be detected with an AIMPILA–Acridinium conjugate. While RECAF could be detected using labeled AFP, such approach has a few substantial problems: the receptor must be functionally intact and RECAF molecules must not be already in a complex with AFP. Thus, in order to study the AFP receptors binding specificity for AFP in the non-covalent AIMPILA complex, we developed a two-step “Sandwich”-like CLIA immunoassay on immobilized anti-RECAF Mab using an AIMPILA–Acridinium conjugate as shown in Figure 4.

Figure 4.

“Sandwich”-like CLIA immunoassay on immobilized anti-RECAF Mab.

Anti-RECAF 1.4G11 Mab was coated at 5 µg/mL in PBS (pH 7.2) and 100 µL/well onto black 96-well plates (Greiner, Germany) for 16 h at 4°C. After washing the plates three times with distilled water using an automatic plate washer, non-specific binding was blocked by adding, to each well, 200 µL of 0.25 M malonate buffer pH 4.0, containing 6% Tween-80. That was followed by three washes as described above.

Different dilutions of T47D homogenate (from 1/10 to 1/10240 with protein concentration from 1.3 to 0.001 mg/mL) were added to the immobilized anti-RECAF Mab. A 0.25 M Malonate buffer with 6% Tween-80, pH 4.0, was used as a solvent for the homogenate dilutions. As negative controls, we used wells without the Mab and wells with no homogenate. After the first incubation, the plate was thoroughly washed to remove unbound reagents. During this 2 h incubation at room temperature, the RECAF present in the homogenate specifically binds to the immobilized anti-RECAF 1.4G11 Mab.

To determine the optimal reagent concentrations, we designed a matrix on a 96-well microplate, in which two-fold dilutions of AIMPILA–Acridinium conjugate ranging from 6 to 800 ng/mL in 100 mM PBS buffer, pH 7.2, were placed in the plate rows and the homogenate dilutions were carried out in the plate columns. The bound conjugate was measured by chemiluminescence using RLU as described above. In this format, the light intensity is directly proportional to the concentration of AIMPILA bound to the immobilized AFP receptors of the homogenate.

The formation of the “sandwich” happens during the second incubation with the addition of AIMPILA–Acridinium conjugate, which binds to the RECAF molecules attached to the anti-RECAF Mab during the first incubation. Specific binding was confirmed by the functional curve of the luminescent signal intensity from AIMPILA–Acridinium conjugate bound on specific anti-RECAF Mab, as compared to a non-specific binding to the cell surface or to the antibody.

Results

Determination of AFP receptors present in human tumors and their binding specificity for AIMPILA

To confirm the presence of RECAF molecules in human tumors transplanted into Balb/c nude mice, we used a solid-phase competitive CLIA with the anti-RECAF Mab 1.4G11 coating the plate and Acridinium-labeled RECAF extracted from K-562 (a human myelogenous leukemia), as described elsewhere.²² To measure RECAF in serum, a constant amount of RECAF–Acridinium was set to compete with the RECAF in the sera of the grafted mice as described below.

RECAF in serum of mice bearing human tumors

The results obtained using 1/100 mouse sera for the CLIA are presented, in RECAF Units, in Table 1. The mice bearing T47D and SW620 human xenografts had significantly more circulating RECAF (10,654 and 11,124 Units, respectively) than the healthy mice controls (3096 Units). Thus, mice bearing human T47D and SW620 tumors had ~3.5 times more serum RECAF than the controls (p < 0.05; Figure 5).

Table 1.

RECAF in serum of healthy mice and mice bearing xenografts of T47D and SW620 human tumors.

Serum origin^a	Animal number	RECAF Units	Mean	SD	t value from independent t-test^b	p value
Healthy Balb/c nude mice	1	3802	3096	1485.1	0.173	–
	2	1143
	3	4593
	4	2845
Mice grafted with T47D	1	10,344	10,654	786.4	0.173	0.00071
	2	11,694
	3	10,850
	4	11,337
	5	10,935
	6	9990
	7	9426
Mice grafted with SW620	1	11,824	11,124	2237.8	0.532	0.000137
	2	12,277
	3	14,051
	4	8523
	5	11,694
	6	8374

SD: standard deviation; AFP: alpha-fetoprotein.

It should be noted that AFP from one species recognizes RECAF from other species and that the 1.4G11 Mab also detects RECAF across species. Thus, in our assays, we are actually measuring normal mouse circulating RECAF in addition to human RECAF produced by the grafted tumors.

t-test and p values were obtained from comparing serum from mice with xenografts to serum from normal mice.

Figure 5.

RECAF in serum of nude mice bearing T47D and SW620 subcutaneous human xenografts and in the serum of healthy mice.

Quantitative evaluation of AFP receptors in human tumor homogenates

The results obtained for the homogenates of the tumors studied using SERUM RECAF test with RECAF–Acridinium conjugate and 1.4G11 Mab are presented in arbitrary RECAF Units in Figure 6 and Table 2.

Figure 6.

RECAF in the homogenates of T47D and SW620 human subcutaneous xenografts, in healthy mice, and in HepG2 culture medium.

Table 2.

RECAF Units in T47D and SW620 homogenates, HepG2 culture medium, and normal mouse tissue homogenates.

Homogenate or cell culture	RECAF Units	Total protein by Bradford (mg/mL)	RECAF Units per milligram of tumor
T47D^a	123,552	13.5	9152
HepG2	60,326	12.4	4865
SW620^a	59,619	21.0	2839
Control normal tissue^a	4000	40.0	100

Volume of the tumor fragments was not less than 200 mm³.

There is no correlation between the amounts of RECAF in the tumors and in the blood of the mice with the same tumor models.

SDS gel electrophoresis and western blot

PAGE showed AFP receptor bands with molecular weight (MW) ~62 kDa (Figure 7), which is consistent with the literature.^23–25 The presence of RECAF was confirmed immunologically by western blot using the 1.4G11 Mab, which was in turn evidenced by a commercial anti-mouse IgM-HRP conjugate, as shown in Figure 8.

Figure 7.

SDS-gel electrophoresis of T47D homogenate and control samples.

Figure 8.

Western blot of human subcutaneous xenograft homogenates and cell line HepG2 as detected with anti-RECAF 1.4G11 Mab followed by anti-mouse IgM-HRP.

Binding specificity of AIMPILA for AFP receptors in T47D homogenate

The results obtained in this study provide confirmation that the AIMPILA–Acridinium conjugate exhibits highest RECAF binding at 800 ng/mL, independent of the tumor homogenate dilution. In our experiments, the highest binding was achieved at 1/10 dilution of the T47D homogenate, without reaching saturation of the binding sites since the difference between 1/10, 1/20, and 1/40 dilutions is significant. This difference in the specific binding offers the possibility to saturate the system by increasing the concentration of AIMPILA–Acridinium and the density of the homogenate. The sensitivity of such a system is limited only by the dilution of the tumor homogenate, in this particular study > 1000 times (Figure 9). Figure 9 shows a significant binding increase in the presence of the homogenate.

Figure 9.

Specific binding of AIMPILA–Acridinium conjugate to AFP receptors in the homogenate of the human breast cancer T47D xenograft.

To confirm that the binding of AIMPILA–Acridinium conjugate is specific, we used the following negative and positive controls: (1) non-specific binding (<1530 RLU) of AIMPILA–Acridinium conjugate to T47D homogenate without the immobilized 1.4G11 Mab, (2) non-specific binding of AIMPILA–Acridinium conjugate to immobilized 1.4G11 Mab (<1530 RLU), (3) non-specific binding of AIMPILA–Acridinium conjugate to the well plastic (<1530 RLU), and (4) specific binding of AIMPILA–Acridinium to the RECAF molecules present in T47D homogenate bound to 1.4G11 Mab (7645 RLU). The specific binding of the AIMPILA Conjugate to the RECAF molecules present in T47D tumor homogenate is confirmed by a hyperbolic reaction curve, where the maximum of binding is five times higher than that of the non-specific binding.

Conclusion and discussion

The overall goal of an AIMPILA preclinical study is the evaluation of the antitumor efficacy on the human tumor xenografts on immunodeficient Balb/c nude mice. The main objective of this study was the characterization of some selected tumor models with regard to their expression of RECAF, which is the AIMPILA’s entry vehicle to cancer cells. The results obtained for the expression of RECAF were compared to the efficacy level of AIMPILA in order to confirm the targeting mechanism responsible for its antitumor action. For that purpose, we measured the amount of RECAF in the homogenates of the grafted tumors T47D and SW620 and in HepG2 cell extract as well as the binding specificity of the AFP in AIMPILA for RECAF. Preliminary results show a significant correlation between RECAF expression in different tumors and their therapeutic response to the targeting drug AIMPILA (in preparation, see Figure 10).

Figure 10.

(Preview of an article in preparation): Correlation between RECAF expression in selected tumors and the efficacy of AIMPILA.

The results presented herein were obtained using a solid-phase CLIA with a RECAF–Acridinium conjugate and a “Sandwich”-like CLIA using an AIMPILA–Acridinium conjugate. The presence of AFP receptors with a molecular weight of 62 kDa in the homogenates of human tumors T47D, as well as SW620, and in the HepG2 cell line was confirmed by SDS-PAGE and western blots using a specific monoclonal anti-RECAF Mab, as well as with the specific CLIA SERUM RECAF test.

In this study, we show that RECAF is practically absent in healthy mice tissues (100 Units/mg) and that the amount of AFP receptor in the examined homogenate of tumor models follows this order: T47D > HepG2 > SW620. This order coincides with our findings (publication in preparation), for the therapeutic efficacy of AIMPILA on these tumor models. This therapeutic efficacy is expressed as tumor growth inhibition, which is denoted as the T/C ratio, where T = tumor volume in the treated mice and C = tumor volume in the control (untreated) mice. The therapeutic effect of AIMPILA follows the same order: T47D (T/C = 22%) > HepG2 (T/C = 51%) > SW620 (T/C = 70%), as will be presented in a separate communication. A preview of the results to be presented in that report is shown in Figure 10, depicting a high correlation between RECAF expression and antitumor activity. In addition, the level of RECAF in the serum of the immunodeficient mice with T47D or SW620 xenografts reached 3.5 times the mean level of RECAF in the serum of the control mice. This is relevant because the size of the xenografts producing RECAF is rather small compared to the mass of healthy tissue.

AIMPILA’s specific binding to T47D homogenate using the developed “Sandwich” assay with an immobilized anti-RECAF Mab revealed that the highest binding of AIMPILA conjugate was achieved at 1.3 mg/mL (1/10 dilution) of T47D homogenate. At this concentration, the binding sites did not appear to be saturated, which means that the concentration of AIMPILA–Acridinium could be further increased. Overall, we found that 800 ng/mL of “AIMPILA–Acridinium” on T47D homogenate yielded good specific binding.

The results obtained in this study have confirmed previous reports showing an elevated level of RECAF in serum samples of cancer patients bearing various malignant tumors.^8,9,25 Since the antitumor activity of AIMPILA seems to be proportional to the RECAF concentration in the malignant tumor tissue, it follows that RECAF concentration could be used to predict the efficiency of AIMPILA for treating human cancers.

Both native and recombinant AFP have been used for some time as vectors for delivering various cytotoxic agents and plant toxins to tumor cells expressing surface AFP receptors.^19,20 The first direct evidence of a receptor for AFP was described on MCF-7 cells by Villacampa and colleagues^13,14 who found results consistent with the presence of a two-site receptor model exhibiting positive binding. The higher affinity site showed a K_d of 1.5 × 10⁻⁹ M and a number of 2000 sites per cell. The lower affinity site, with a number of 320,000 sites per cell, exhibited a K_d of 2.2 × 10⁻⁷ M. Similar AFP receptor systems were subsequently detected on the surface of the YAC-1 mouse T-lymphoma¹³ (but not on normal adult mouse T-cells) and on the human U937 and THP-1 cell lines.⁷ The number of sites per cell as well as the binding affinity differs from one cell line to another. Upon binding to its specific receptor, AFP is internalized by tumor cells via a receptor-mediated mechanism of endocytosis. The binding assays were not restricted to intact cells but also included MCF-7 and primary breast cancer cytosols, thus evidencing a soluble RECAF fraction.¹⁵ The presence of AFP receptors on the surface of malignant tumor cells allows targeting them with a variety of complexes of AFP and different toxic substances. As early as 1983, Deutsch et al. conjugated daunomycin to arachidonic and docosahexaenoic acids and showed, both in vitro and in vivo, that the conjugates were effective against AFP producing hepatomas but ineffective against hepatomas that did not synthesize that protein as mentioned in details.²⁶ A more direct approach was taken by Feldman et al., who conjugated Doxorubicin directly to AFP. This resulted in a five-fold increase in the conjugate toxicity compared to the drug alone.²⁷

A “delivery system” designed as (protein vector AFP)–linker–(drug) conjugates was employed by another group^28–31 that demonstrated the advantage of using AFP as a vector both in its uptake by tumor cells and its cytotoxic effect. The conjugates were constructed using 3-maleimidobenzoic acid hydrazide as the linker and a number of cytotoxic agents such as doxorubicin, daunomycin, calicheamicin, bleomycin, esperamicin, cisplatin, carboxy phosphamide, vinblastine, methotrexate, phthalocyanines, and chlorine. However, such conjugates have demonstrated high toxicity in vivo.

The conjugation chemistry is important because AFP is known to enter and exit cancer cells intact,¹³ whereas the toxic payload must be retained and reach its cellular target. To address this issue, Yabbarov et al.²⁶ used an acid-labile aconitum link between rAFP3D (a recombinant third domain fragment of AFP) and Doxorubicin. They found that while Doxorubicin alone was slightly more cytotoxic to SCOV3 cells as compared to the conjugate, the latter was significantly less toxic to normal lymphocytes than Doxorubicin alone, thus allowing the use of a higher concentration of the drug. Interestingly, the conjugate was far more effective on SKVLB ovarian cancer cells, which are resistant to Doxorubicin, than the drug alone. This suggests that the resistance mechanism might be related to poor cell internalization of the drug, which would be enhanced by the delivery mechanism provided by the AFP-RECAF system. Similar results were obtained with paclitaxel-loaded nanoparticles attached to rAFP3D.²⁷

In summary, we conclude that the breast adenocarcinoma T47D with highest expression of RECAF within the studied tumors³² tumors on immunodeficient mice Balb/c nude is the most adequate model to pursue preclinical studies on targeting cancer with AIMPILA.

Footnotes

Acknowledgements

This research was done in accordance to the contract with the Ministry of Industry and Trade of the Russian Federation # 12411.1008799.13.148, according to the Federal Program of the Development of the Pharmaceutical and Medical Industry known as “PharmaMed2020” (). The authors thank Angela Gerber (BSc) for her help in the corrections to this manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Guarantor

Helen Treshalina.

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Binding characterization of the targeting drug AIMPILA to AFP receptors in human tumor xenografts

Abstract

Keywords

Introduction

Materials and methods

Immunodeficient murine models

Tumor models

Cell culturing methods

Targeting drug studied

Characterization of the specific monoclonal antibodies 1.4G11 against RECAF

Detection of AFP receptors in human tumors

CLIA for AFP receptors in tumor homogenates

Detection of AFP receptors in the serum of Balb/c nude mice bearing human tumors

Determination of RECAF in human tumors sensitive to AIMPILA

Preparation of the human tumor homogenates and HepG2 cell extract

PAGE and western blots

AIMPILA labeling with Acridinium

Binding specificity of AIMPILA to RECAF in tumor homogenates

Results

Determination of AFP receptors present in human tumors and their binding specificity for AIMPILA

RECAF in serum of mice bearing human tumors

Quantitative evaluation of AFP receptors in human tumor homogenates

SDS gel electrophoresis and western blot

Binding specificity of AIMPILA for AFP receptors in T47D homogenate

Conclusion and discussion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

Guarantor

References