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Therapeutic Delivery
The use of a-fetoprotein for the delivery of cytotoxic payloads to cancer cells
One approach to improving the activity of anticancer drugs is to bind them to the human a-fetoprotein (HAFP) that recognizes the tumor-associated cell-surface HAFP receptor. A drug can be bound to the HAFP by covalent conjugation or within a noncovalent complex. Specially designed linkers couple cytotoxins to the HAFP and ensure the stability of the HAFP–drug conjugate in the circulation and the activation of the drug in the cancer cell. On the other hand, AFP–drug non-covalent complexes can exploit the natural role of the AFP as a nutrition delivery “shuttle”. In this article we review the design of HAFP–drug conjugates and AFP–drug complexes and their potential uses.
Background The HAFP is a 70 kDa major fetal serum glycoprotein consisting of a 591 amino acid polypeptide and a carbohydrate moiety. It is synthesized mainly by the yolk sac, fetal liver and the GI tract. It is present in both fetal and maternal circulation during pregnancy [1] and is thus commonly used as a pregnancy marker. The HAFP serum levels drop rapidly after birth and only trace amounts of it can be detected in the blood of normal adults. It has been suggested that the HAFP may play an immunomodulating role and prevent the rejection of an embryo by the maternal immune system [2] . The observation that women with autoimmune diseases often experience a considerable relief from their symptoms during pregnancy has lead researchers to examine the HAFP as a possible treatment for auto-immune diseases [3,4] . The HAFP extracted from aborted human tissues was a marketed immunomodulator drug in the Russian Federation (Alfetin, 0.75 mcg/ampoule) for several years but has since been withdrawn for ethical reasons [5] . Merrimack Pharmaceuticals attempted to develop a rHAFP for treating rheumatoid arthritis, psoriasis and uveitis. Unfortunately, their rHAFP (MM-093) has failed to achieve the primary efficacy endpoint in Phase 2 clinical
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Vladimir Pak Freelancer [email protected]
trials for rheumatoid arthritis [6] . The natural HAFP is a glycoprotein with a single glycosylation site, while rHAFP manufactured by Merrimack was not glycosylated. This could be the reason for the different immunomodulating properties of Alfetin and MM-093. Nevertheless, the clinical trials demonstrated the safety of MM-093 at doses significantly higher than the HAFP serum concentrations during pregnancy [7] . The observed excellent safety profile of the rHAFP makes MM-093 a very attractive platform for targeted delivery of cytotoxins to cancer cells. The HAFP is able to bind non-covalently different low-molecular weight substances from the mother’s blood, such as polyunsaturated fatty acids (PUFAs), hydrophobic antibiotics, steroidal hormones, retinoids, bilirubin, certain dyes, and other [8] . For this ability and high amino acids sequence homology to albumin AFP is named an “embryo albumin”. Immature fetal cells internalize the HAFP through the HAFP receptor-mediated endocytosis. It has been suggested that the purpose of that internalization is to deliver nutrients into fetal cells [9] . Upon delivering its payload into cells the HAFP returns to the circulation where it picks up another payload and continues shuttling nutrients to fetal cells.
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Key terms Polyunsaturated fatty acid: Avital nutrition for growing cells. HAFP–drug conjugate: A cytotoxic drug covalently linked to a HAFP molecule that recognizes a tumor-associated HAFP receptor. HAFP–drug complex: A cytotoxic drug bound noncovalently to the HAFP that recognizes a tumor-associated HAFP receptor. Dox: Doxorubicin, a cytotoxic drug and a mutagen. Apoptosis inducer: An agent that activates the programmed cell death. Teratogen: an agent that interferes with the normal embryonic development. In this article: small molecule embryonic toxin.
HAFP and the HAFP receptor can be re-expressed by cancer cells, which facilitate the cells nutrient supply to these rapidly growing cells. The synthesis of the HAFP is a common feature of hepatocellular carcinoma, but it has occasionally been detected in tumors of other types [10] . The HAFP receptors are universally expressed on the surface of all embryonic cells but they are not detected on normal adult cells. However, most cancer cell types express HAFP receptors [11–13] . As many as four types of AFP receptors with different affinities and densities have been described [1] . The complete structure of the receptor has not yet been elucidated. In vitro and in vivo studies demonstrate that cancer cells regain the ability to take up the HAFP via this receptor [14,15] . As already mentioned, the HAFP receptor is mostly absent in normal adult cells except for a small population of activated immune cells. Therefore, the HAFP loaded with cytotoxins can target specifically the cancer cells. Either glycosylated or unglycosylated rHAFP can be chosen for this purpose [16,17] . HAFP–drug conjugates or HAFP–drug complexes can be used to deliver cytotoxic payloads to cancer cells with a high degree of selectivity. Such conjugates/complexes can target the majority of cancer cells with the exception of HAFP receptor-negative ones. HAFP–drug conjugates The above discussion provides the rationale for the use of the HAFP–drug conjugates: the high selectivity with a potential for improved efficacy and higher safety than the conventional chemotherapeutics [18] . An ideal conjugate should retain the favorable pharmacokinetic (long half-life) and functional properties of the HAFP, remain stable and nontoxic during the circulation in blood, and release a sufficient quantity of
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the active drug form to destroy the tumor cell. An optimal linker must allow for efficient and rapid release of the cytotoxin in active form upon internalization into the cancer cell. The linker used to conjugate the cytotoxin to the HAFP has to be cleaved enzymatically inside a cancer cell and release the cytotoxin leading to the cell’s death [19] . This highly selective approach minimizes the side effects of the treatment. The desirable characteristics for a cytotoxic drug include a low molecular weight, the lack of immunogenicity, and the ability to link to the HAFP under the conditions that do not affect its binding to the protein receptor. It has been reported that the HAFP conjugates with numerous anticancer drugs including Dox, daunomycin, calichemicin, carboxyphosphamide, bleomycetin, methotrexate, carminomycin and others. Most of the tested conjugates had 1 to 5 cytotoxic molecules per HAFP molecule [1] . For example, the HAFP-Esperamicin A1 conjugate (1:1 molar ratio) injections demonstrated an excellent anti-tumor activity in the mice xenograft model [20] . The multi-drug resistance (MDR) is a major obstacle to the successful cancer chemotherapy [21] . In MDR cancer cells the activity of the MDR1 gene of the P-glycoprotein pump (that removes various anti-cancer drugs from the cytoplasm) is increased, resulting in the reduced drug accumulation inside the cell. Receptor-mediated endocytosis of HAFP-toxin conjugates bypasses the membrane pumps and thus overcomes the MDR mechanism [22] . The full-size HAFP conjugates are limited by the capacity of 1–5 cytotoxic molecules per HAFP molecule that do not interfere receptor binding and by the immunogenicity of the conjugates. In order to avoid the immunogenicity and to deliver numerous toxins, one can use conjugates with HAFP-derived vector peptides. This includes strategies such as nanoparticles, micelles, liposomes, etc. [23–25] . AFP–drug complexes “If one way is better than another, than you can be sure it is nature’s way.” – Aristotle. During the fetal period the HAFP binds and transports PUFA from the mother’s blood through placenta into the embryo’s blood. The human hemochorial placenta has 3 cell layers while porcine epitheliochorial placenta has 6 cell layers that separate the mother’s blood from that of the embryo. It is possible that the HAFP receptor expressed in the normal human placenta during the fetal development at term [26] facilitates the absorption of the HAFP-PUFA complex and its transportation through placenta. The AFP has a binding affinity to PUFA docosahexaenoic acid
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The use of a-fetoprotein for the delivery of cytotoxic payloads to cancer cells
(DHA) that is 53 higher than that between albumin and DHA [27] . Due to the hydrophobic pocket the AFP binds more than 75% of DHA even in the presence of x10 albumin excess. Most nutrition substances (and drugs) do not bind the HAFP strong enough compared with albumin whereas the HAFP-payload binding affinity is crucial to cross placenta transport. In vivo studies with rats have shown that more than 70% of estrone and estradiol (which bind strongly to the rodent AFP) when injected into the maternal circulation were subsequently found in the fetus in association with the AFP. The synthetic estrogens with a lower AFP-binding affinity do not concentrate in the fetus [28] . The rat AFP can bind drugs warfarin and phenylbutazone. These agents bind to AFP at the same large hydrophobic pocket as estrogens, PUFAs, pyrrazolic compounds and proprionic drugs [29] . To be useful for anti-cancer treatment, a drug in a HAFP–drug complex should have two features: the high binding affinity to the HAFP and the high cytotoxicity. Potent drugs (IC50 in the nM-pM range) are preferable for the complex, as they can be released only inside the cancer cell avoiding the bystander effects. Being a natural product of a living system, the HAFP as a drug delivery protein reduces the drug’s toxicity. Ideally, a potent drug should substitute DHA in the HAFP hydrophobic pocket and follow its way (Figure 1) . Cytotoxins for AFP–drug complexes The traditional chemotherapy drugs (e.g. Dox) suppress the proliferation and serve as indirect apoptosis inducers (AIs) that rely on healthy apoptosis. At the same time, the tumor suppressor p53 - the key proliferation/apoptosis control protein - is not working in >50% of cancers [30] , making them insensitive to and helping them survive the traditional chemotherapy. This is an alternative MDR mechanism to the activated P-glycoprotein pump in cancer cells [31] . Thus, direct AIs that restore the apoptosis of MDR cancer cells downstream of the inactive apoptosis elements (e.g. p53) are superior to the indirect AIs [32] . The electron microscopy was used to follow the HAFP receptormediated endocytosis and the intracellular pathway of the HAFP conjugates with horseradish peroxidase. The HAFP was observed in coated vesicles, endosomes and a tubular vesicular network localized in the Golgicentrosphere region [2] . On this intracellular pathway a HAFP–drug complex can release the drug that will hit the closest target. The endoplasmic reticulum, the mitochondrion (the “point of no return” in the intrinsic apoptosis pathway), lysosomes (“suicide bags”), peroxisomes and their membranes can all be effective targets for anti-cancer drugs [33] , unlike the popular anti-cancer targets DNA or tubulin.
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The selection of appropriate drug candidates for HAFP–drug complexes can be done empirically, based on the computer 3D modeling, or a candidate may be chosen from teratogens. In our study, we empirically chose the polyene (polyunsaturated) antibiotic amphotericin B for the HAFP-amphotericin B complex since polyunsaturated fatty acids have a stronger binding affinity to the HAFP than monounsaturated ones. Alfetin (the natural HAFP) and amphotericin B, the two registered drugs in Russia were used in the complex for injections. The intracellular target of anti-fungal ion-pore antibiotic amphotericin B in unknown [34] and it is not a potent cytotoxin. Nevertheless, the complex has shown a response in 6 out of 8 cancer patients [33] . HAFP was found empirically to bind the environmental toxin dioxin (1:2 molar ratio). The HAFPdioxin complex was successfully used to destroy cancer cells [35] . However, if dioxin is released from the complex in the bloodstream or delivered in a quantity insufficient to destroy a tumor cell, it serves as a mutagen and a carcinogen [36] . Therefore, the anticancer application of the HAFP-dioxin complex is problematic. The empirical study of spectral changes in the presence of the rHAFP has demonstrated the rHAFP ability to bind curcumin and genistin. The rHAFP-curcumin and rHAFP-genistin complexes can also destroy cancer cells [17] . Nevertheless, the curcumin and genistin are poor candidates for HAFP–drug complexes, since less potent cytotoxins need more HAFP for the delivery to cancer cells and the cell destruction. The HAFP strongly binds the synthetic estrogen diethylstilbestrol (DES), unlike natural estrogens. A 3D computer model of DES docking in the HAFP hydrophobic pocket was made [37] . The model has a U-shaped structure in which a cavity may be distinguished (Figure 1) . The putative estrogen-binding site is
Toxin
Figure 1. HAFP U-shaped structure with the cavity [37] that can fit the natural ligand or toxin.
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localized in this cavity. Two types of interactions were proposed between the amino acid residues of HAFP and a DES molecule: hydrogen and hydrophobic binding. Again, DES is a carcinogen and the HAFP-DES complex anti-cancer application is questionable. An optimal anti-cancer drug can be characterized by specificity, efficacy and drug-tolerance by the normal cells. A teratogen specifically destroys embryo cells but is tolerated by the adult mother cells. Chemotherapy is fairly safe to both the child and the mother when given during the second and the third trimesters of pregnancy. Cyclophosphamide, 5-FU, Dox, bleomycin, vincristine and etoposide have been given safely during pregnancy [38] . It is assumed that some teratogens (unlike chemotherapy drugs) can bind HAFP and cross the placenta, enter thus the blood of the embryo and destroy the embryo cells. HAFP-teratogen complexes suppress embryogenesis and can also suppress cancer cells. AFP complexes with teratogens dioxin and atractyloside were successfully used to destroy cancer cells [35,39] . Unlike teratogens dioxin or DES that target the DNA, atractyloside is targeted at mitochondrion and hence is not mutagen and carcinogen. HAFP–drug complexes are different from HAFP–drug conjugates Comparison of the AFP–drug conjugate and AFP–drug complex properties are given in Table 1. Cancer cells specific targeting and receptor-mediated endocytosis of AFP–drug conjugates or complexes was reviewed in [1] . HAFP–drug conjugate stability in the circulation is provided by the covalent link. As for the complex the rigidity of the HAFP tertiary structure is controlled by the payload [17,40] . Fitting 1–2 small drug molecules into the HAFP hydrophobic pocket should naturally stabilize a HAFP–drug non-covalent complex in the bloodstream. A HAFP–drug complex alters the PK profile of drugs positively along with the stabilization of the HAFP. This profile is supposed to be similar to that of the natural HAFP-DHA complex or to the PKs of the HAFP alone (MM-093). Receptor-mediated endocytosis of HAFP-toxin conjugate/complex bypasses the membrane pumps and thus overcomes the MDR mechanism [22] . AFP recycling is possible for complex due to the non-covalent AFP–drug link [41] . The release of the payload from a HAFP complex inside a cancer cell happens with the pH change into acidic at a definite compartment within about an hour [2] . After that, the HAFP recycles back. Its approximate half-life in the blood is 5 days [7] . During this time the recycled HAFP can deliver multiple further payloads, acting as a natural shuttle. Curcumin, genistin, other supplements and drugs that feature a competitive to the blood proteins binding to HAFP can potentiate the rHAFP–drug
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complex anti-cancer action. The optimal AFP:drug conjugation ratio was shown to range from 1:3 to 1:5 [1] . The increase of this number can interfere with HAFP receptor interaction. HAFP–drug complex can fit into the HAFP hydrophobic pocket only 1–2 molecules. The drugs non-covalently bound at the HAFP surface will be shared with serum proteins and will not be targeted to cancer cells. To provide the integrity of the targeting and toxicity parts in the AFP–drug conjugate they should be bound with a linker(s), unlike AFP–drug complex that grabs the drug by the hydrophobic pocket. The AFP–drug conjugate linker(s) are designed to be cleaved enzymatically inside a cancer cell and release the drug [19] . The release of the drug from an AFP–drug complex upon internalization by cancer cell goes naturally.AFP does not elicit an immune response in the species in which the protein originated [1] . Conjugate expose 3–5 drugs on the surface of the HAFP that makes them immunogenic. On the other hand the immunogenicity of the drugs hidden inside the HAFP hydrophobic pocket is decreased. HAFP–drug complex immunogenicity is supposed to be as low as of the HAFP one. A possibly superior strategy is to supply cancer patients with small doses of AFP–drug complex on a permanent basis (maintaining the AFP concentrations that are typical during pregnancy and known to be low immunogenic) rather than to give them periodically a fraction of the maximum tolerated dose of HAFP–drug conjugate. Maximum tolerated dose should be determined for the artificial HAFP–drug conjugate, while drug delivery by the complex is effective with physiological HAFP doses. The full-size glycosylated HAFP has immunosuppressive or cancer stimulating properties [42] . To avoid the unwanted effects, HAFP–drug complex may be built with the unglycosylated rHAFP (e.g. MM-093) that has demonstrated no immunomodulating activity in clinical trials. Full-size HAFP is not important for conjugate: drugs can be targeted to cancer cells with HAFP vector peptides conjugate. HAFP–drug complex do not need linkers for drug binding that makes manufacturing less costly than HAFP–drug conjugates. Oral AFP–drug complex formulations The unenhanced bioavailability for oral routes of administration for protein/peptide pharmaceuticals is about 0 - 1% [43] . The enzymatic instability of proteins and the GI permeation are the main challenges. Moreover, HAFP–drug complexes can dissociate in the acidic stomach environment. Nevertheless, the AFP–drug complexes as oral formulations have shown promising anti-cancer results. Impila decoction containing mitochondrion toxin atractyloside [44] is an oral folk remedy of South African Zulu aborigines. In our study, an oral formulation
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Table 1. Comparison of the biochemical and anti-cancer properties of a-fetoprotein–drug conjugate versus a-fetoprotein–drug complex. Properties
AFP–drug Conjugate
Complex
Cancer cells targeting
Yes
Yes
Receptor-mediated endocytosis
Yes
Yes
Stability in circulation
Yes
Yes
Overcomes MDR
Yes
Yes
AFP recycling
No
Yes
Toxins per AFP molecule
1–5
1–2
Need a special linker
Yes
No
Need drug release design
Yes
No
Immunogenicity
High
Low
Maximum tolerated dose
Yes
No
Full-size AFP is important
No
Yes
Manufacturing cost
High
Low
AFP: a-fetoprotein; MDR: Multi-drug resistance.
of the atractyloside and the porcine AFP complex has shown response in 6 out of 12 patients with metastatic colorectal cancer [39,45] . Two of the patients who responded had indications of a possible MDR breakage during the previous chemotherapy treatment. The AFP-atractyloside complex improves the quality of life and the longevity of terminal cancer patients. Future perspective The majority of cancers displays the HAFP receptor and will therefore be sensitive to treatment with HAFP–drug conjugates/complexes; cancer stem cells, which are responsible for metastasis [46] , have a higher probability of expressing the HAFP receptor and, as such, can be treated using this approach. Taking this into account, positive testing for the HAFP recep-
tor among cancer patients can therefore allow for personalized cancer therapy. HAFP tumor-targeting peptides have now substituted full-size HAFP in HAFP–drug conjugates. These are less immunogenic and can be conjugated by optimal linkers with numerous potent cytotoxins (e.g., strategies such as nanoparticles and liposomes). It is projected that, over the next 5–10 years, drugs with high binding affinities to the HAFP will be identified using 3D modeling and the screening of chemical libraries. Among these candidates, the highly toxic drugs should be chosen for HAFP–drug complexes and the cancer treatment. In Table 2 the potential properties of AFP–drug complex are compared against registered chemotherapy, demonstrating the advantages of the proposed therapeutic approach.
Table 2. a-fetoprotein–drug non-covalent complex comparison with Abraxane. Properties
AFP–drug complex [39]
Abraxane [47]
AFP (embryo albumin)
1–10 mg
Albumin
900 mg
Toxin delivered
0.1–1 mg
100 mg Paclitaxel
Cancer cells targeted delivery
Yes
No
Receptor-mediated endocytosis
Yes
No
Maximum tolerated dose
No
260 mg/m2
Overcomes MDR
Yes
No
Side effects
No
Yes
Cancer stem cells affected
Yes
No
AFP: a-fetoprotein; MDR: Multi-drug resistance.
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The recycled AFP binding of some drugs, or endogenous molecules from the microenvironment, can potentiate the anti-cancer action of AFP–drug complexes. Teratogens or registered drugs with teratogenic properties are promising drug candidates for use simultaneously with AFP–drug complexes in anti-cancer therapy. In addition, the registered drug data package can shortcut clinical trials and accelerate the FDA approvals. The GI absorption of HAFP–drug complexes is still in need of additional research. HAFP is synthesized mainly by the yolk sac, fetal liver and GI tract cells and, since it binds the ligands of high nutritional value, it should be absorbed by AFP receptor positive cells. Assuming that the GI tract absorbs AFP-DHA or AFP–drug complexes
in a similar way to their absorption and transportation through placenta, oral AFP–drug complexes have the potential to enter the blood of the patient via this route and thereby prevent metastasis – or other explanations of the data may exist. Financial & competing interests disclosure V Pak is shareholder of Constab Pharmaceutical Inc. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Executive summary Background • The HAFP receptor-based targeting may emerge as a rewarding strategy in the treatment of cancer in the near future as it exploits the HAFP selectivity and the high cytotoxic potency of a drug. Factors that determine the anti-tumor activity of the HAFP–drug conjugates/complexes include the HAFP receptor expression, internalization and the direction of intracellular trafficking of HAFP–drug conjugates/complexes, the rate, compartment and/or the degree of the intracellular release of the potent drug. • The majority of cancers re-expresses HAFP receptors and can be sensitive to the specific HAFP-cytotoxin delivery mechanism provided by HAFP–drug conjugates or AFP–drug complexes. HAFP–drug conjugates or AFP–drug complexes by-pass membrane pumps and thus overcome the MDR mechanism of cancer cells.
HAFP–drug conjugates • The chances for a clinical success increase due to the continued improvements in the design of HAFP–drug conjugates, namely the development of more potent cytotoxic drugs and the optimization of HAFP–drug linkers. The full-size HAFP–drug conjugates are substituted today with the HAFP tumor-targeting peptide– drug conjugates. This way, the conjugate becomes less immunogenic and carries multiple potent drugs attached via optimal linker(s).
AFP–drug complexes • The AFP–drug complexes exploit the natural shuttle-like delivery potential of the HAFP. They can be obtained by fitting a potent drug into the hydrophobic pocket of the HAFP. The AFP–drug complexes for injections or oral formulations have demonstrated promising results and are well tolerated by cancer patients.
Cytotoxins for AFP–drug complexes • The AFP complexes with cytotoxins (1:1–2 molar ratios) should have both a high toxicity and a high binding affinity to the AFP. Such binding affinity can be provided by the AFP’s hydrophobic pocket that naturally transports DHA in a shuttle manner. Direct AIs targeted to organelles can be effective anti-cancer drugs. Teratogens or registered drugs with teratogenic properties should be included in the drug candidate list.
HAFP–drug complexes are different from HAFP–drug conjugates • The HAFP–drug complexes having close to conjugates drug/HAFP molecule ratio, at the same time they are not immunogenic, don’t need linkers, and hence less costly in manufacturing. The pharmacokinetic profiles of AFP–drug complexes are supposed to be similar to those of the AFP stabilized with natural ligands, e.g. DHA.
Oral AFP–drug complex formulations • The mechanism of the anti-cancer action of the oral AFP–drug complexes needs additional research. Their GI absorption can be similar to the absorption and the transportation of AFP-DHA complexes through placenta. Other plausible explanations of the data may exist.
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Pak V, Molchanov O, Vincent M. Treatment of metastatic colorectal cancer with aimpila, a glycoside/alpha-fetoprotein complex. J. Clin. Oncol. 25(Suppl. S18), S160 (2007).
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Belyaev NN, Abramova VA. Transmission of “split anergy” from tumor infiltrating to peripheral NK cells in a manner similar to “infectious tolerance”. Med. Hypothesis. 82, 129–133 (2014).
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Abraxane. http://www.rxlist.com/abraxane–drug.htm
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Therapeutic Delivery
The use of a-fetoprotein for the delivery of cytotoxic payloads to cancer cells
One approach to improving the activity of anticancer drugs is to bind them to the human a-fetoprotein (HAFP) that recognizes the tumor-associated cell-surface HAFP receptor. A drug can be bound to the HAFP by covalent conjugation or within a noncovalent complex. Specially designed linkers couple cytotoxins to the HAFP and ensure the stability of the HAFP–drug conjugate in the circulation and the activation of the drug in the cancer cell. On the other hand, AFP–drug non-covalent complexes can exploit the natural role of the AFP as a nutrition delivery “shuttle”. In this article we review the design of HAFP–drug conjugates and AFP–drug complexes and their potential uses.
Background The HAFP is a 70 kDa major fetal serum glycoprotein consisting of a 591 amino acid polypeptide and a carbohydrate moiety. It is synthesized mainly by the yolk sac, fetal liver and the GI tract. It is present in both fetal and maternal circulation during pregnancy [1] and is thus commonly used as a pregnancy marker. The HAFP serum levels drop rapidly after birth and only trace amounts of it can be detected in the blood of normal adults. It has been suggested that the HAFP may play an immunomodulating role and prevent the rejection of an embryo by the maternal immune system [2] . The observation that women with autoimmune diseases often experience a considerable relief from their symptoms during pregnancy has lead researchers to examine the HAFP as a possible treatment for auto-immune diseases [3,4] . The HAFP extracted from aborted human tissues was a marketed immunomodulator drug in the Russian Federation (Alfetin, 0.75 mcg/ampoule) for several years but has since been withdrawn for ethical reasons [5] . Merrimack Pharmaceuticals attempted to develop a rHAFP for treating rheumatoid arthritis, psoriasis and uveitis. Unfortunately, their rHAFP (MM-093) has failed to achieve the primary efficacy endpoint in Phase 2 clinical
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Vladimir Pak Freelancer [email protected]
trials for rheumatoid arthritis [6] . The natural HAFP is a glycoprotein with a single glycosylation site, while rHAFP manufactured by Merrimack was not glycosylated. This could be the reason for the different immunomodulating properties of Alfetin and MM-093. Nevertheless, the clinical trials demonstrated the safety of MM-093 at doses significantly higher than the HAFP serum concentrations during pregnancy [7] . The observed excellent safety profile of the rHAFP makes MM-093 a very attractive platform for targeted delivery of cytotoxins to cancer cells. The HAFP is able to bind non-covalently different low-molecular weight substances from the mother’s blood, such as polyunsaturated fatty acids (PUFAs), hydrophobic antibiotics, steroidal hormones, retinoids, bilirubin, certain dyes, and other [8] . For this ability and high amino acids sequence homology to albumin AFP is named an “embryo albumin”. Immature fetal cells internalize the HAFP through the HAFP receptor-mediated endocytosis. It has been suggested that the purpose of that internalization is to deliver nutrients into fetal cells [9] . Upon delivering its payload into cells the HAFP returns to the circulation where it picks up another payload and continues shuttling nutrients to fetal cells.
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Key terms Polyunsaturated fatty acid: Avital nutrition for growing cells. HAFP–drug conjugate: A cytotoxic drug covalently linked to a HAFP molecule that recognizes a tumor-associated HAFP receptor. HAFP–drug complex: A cytotoxic drug bound noncovalently to the HAFP that recognizes a tumor-associated HAFP receptor. Dox: Doxorubicin, a cytotoxic drug and a mutagen. Apoptosis inducer: An agent that activates the programmed cell death. Teratogen: an agent that interferes with the normal embryonic development. In this article: small molecule embryonic toxin.
HAFP and the HAFP receptor can be re-expressed by cancer cells, which facilitate the cells nutrient supply to these rapidly growing cells. The synthesis of the HAFP is a common feature of hepatocellular carcinoma, but it has occasionally been detected in tumors of other types [10] . The HAFP receptors are universally expressed on the surface of all embryonic cells but they are not detected on normal adult cells. However, most cancer cell types express HAFP receptors [11–13] . As many as four types of AFP receptors with different affinities and densities have been described [1] . The complete structure of the receptor has not yet been elucidated. In vitro and in vivo studies demonstrate that cancer cells regain the ability to take up the HAFP via this receptor [14,15] . As already mentioned, the HAFP receptor is mostly absent in normal adult cells except for a small population of activated immune cells. Therefore, the HAFP loaded with cytotoxins can target specifically the cancer cells. Either glycosylated or unglycosylated rHAFP can be chosen for this purpose [16,17] . HAFP–drug conjugates or HAFP–drug complexes can be used to deliver cytotoxic payloads to cancer cells with a high degree of selectivity. Such conjugates/complexes can target the majority of cancer cells with the exception of HAFP receptor-negative ones. HAFP–drug conjugates The above discussion provides the rationale for the use of the HAFP–drug conjugates: the high selectivity with a potential for improved efficacy and higher safety than the conventional chemotherapeutics [18] . An ideal conjugate should retain the favorable pharmacokinetic (long half-life) and functional properties of the HAFP, remain stable and nontoxic during the circulation in blood, and release a sufficient quantity of
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the active drug form to destroy the tumor cell. An optimal linker must allow for efficient and rapid release of the cytotoxin in active form upon internalization into the cancer cell. The linker used to conjugate the cytotoxin to the HAFP has to be cleaved enzymatically inside a cancer cell and release the cytotoxin leading to the cell’s death [19] . This highly selective approach minimizes the side effects of the treatment. The desirable characteristics for a cytotoxic drug include a low molecular weight, the lack of immunogenicity, and the ability to link to the HAFP under the conditions that do not affect its binding to the protein receptor. It has been reported that the HAFP conjugates with numerous anticancer drugs including Dox, daunomycin, calichemicin, carboxyphosphamide, bleomycetin, methotrexate, carminomycin and others. Most of the tested conjugates had 1 to 5 cytotoxic molecules per HAFP molecule [1] . For example, the HAFP-Esperamicin A1 conjugate (1:1 molar ratio) injections demonstrated an excellent anti-tumor activity in the mice xenograft model [20] . The multi-drug resistance (MDR) is a major obstacle to the successful cancer chemotherapy [21] . In MDR cancer cells the activity of the MDR1 gene of the P-glycoprotein pump (that removes various anti-cancer drugs from the cytoplasm) is increased, resulting in the reduced drug accumulation inside the cell. Receptor-mediated endocytosis of HAFP-toxin conjugates bypasses the membrane pumps and thus overcomes the MDR mechanism [22] . The full-size HAFP conjugates are limited by the capacity of 1–5 cytotoxic molecules per HAFP molecule that do not interfere receptor binding and by the immunogenicity of the conjugates. In order to avoid the immunogenicity and to deliver numerous toxins, one can use conjugates with HAFP-derived vector peptides. This includes strategies such as nanoparticles, micelles, liposomes, etc. [23–25] . AFP–drug complexes “If one way is better than another, than you can be sure it is nature’s way.” – Aristotle. During the fetal period the HAFP binds and transports PUFA from the mother’s blood through placenta into the embryo’s blood. The human hemochorial placenta has 3 cell layers while porcine epitheliochorial placenta has 6 cell layers that separate the mother’s blood from that of the embryo. It is possible that the HAFP receptor expressed in the normal human placenta during the fetal development at term [26] facilitates the absorption of the HAFP-PUFA complex and its transportation through placenta. The AFP has a binding affinity to PUFA docosahexaenoic acid
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The use of a-fetoprotein for the delivery of cytotoxic payloads to cancer cells
(DHA) that is 53 higher than that between albumin and DHA [27] . Due to the hydrophobic pocket the AFP binds more than 75% of DHA even in the presence of x10 albumin excess. Most nutrition substances (and drugs) do not bind the HAFP strong enough compared with albumin whereas the HAFP-payload binding affinity is crucial to cross placenta transport. In vivo studies with rats have shown that more than 70% of estrone and estradiol (which bind strongly to the rodent AFP) when injected into the maternal circulation were subsequently found in the fetus in association with the AFP. The synthetic estrogens with a lower AFP-binding affinity do not concentrate in the fetus [28] . The rat AFP can bind drugs warfarin and phenylbutazone. These agents bind to AFP at the same large hydrophobic pocket as estrogens, PUFAs, pyrrazolic compounds and proprionic drugs [29] . To be useful for anti-cancer treatment, a drug in a HAFP–drug complex should have two features: the high binding affinity to the HAFP and the high cytotoxicity. Potent drugs (IC50 in the nM-pM range) are preferable for the complex, as they can be released only inside the cancer cell avoiding the bystander effects. Being a natural product of a living system, the HAFP as a drug delivery protein reduces the drug’s toxicity. Ideally, a potent drug should substitute DHA in the HAFP hydrophobic pocket and follow its way (Figure 1) . Cytotoxins for AFP–drug complexes The traditional chemotherapy drugs (e.g. Dox) suppress the proliferation and serve as indirect apoptosis inducers (AIs) that rely on healthy apoptosis. At the same time, the tumor suppressor p53 - the key proliferation/apoptosis control protein - is not working in >50% of cancers [30] , making them insensitive to and helping them survive the traditional chemotherapy. This is an alternative MDR mechanism to the activated P-glycoprotein pump in cancer cells [31] . Thus, direct AIs that restore the apoptosis of MDR cancer cells downstream of the inactive apoptosis elements (e.g. p53) are superior to the indirect AIs [32] . The electron microscopy was used to follow the HAFP receptormediated endocytosis and the intracellular pathway of the HAFP conjugates with horseradish peroxidase. The HAFP was observed in coated vesicles, endosomes and a tubular vesicular network localized in the Golgicentrosphere region [2] . On this intracellular pathway a HAFP–drug complex can release the drug that will hit the closest target. The endoplasmic reticulum, the mitochondrion (the “point of no return” in the intrinsic apoptosis pathway), lysosomes (“suicide bags”), peroxisomes and their membranes can all be effective targets for anti-cancer drugs [33] , unlike the popular anti-cancer targets DNA or tubulin.
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The selection of appropriate drug candidates for HAFP–drug complexes can be done empirically, based on the computer 3D modeling, or a candidate may be chosen from teratogens. In our study, we empirically chose the polyene (polyunsaturated) antibiotic amphotericin B for the HAFP-amphotericin B complex since polyunsaturated fatty acids have a stronger binding affinity to the HAFP than monounsaturated ones. Alfetin (the natural HAFP) and amphotericin B, the two registered drugs in Russia were used in the complex for injections. The intracellular target of anti-fungal ion-pore antibiotic amphotericin B in unknown [34] and it is not a potent cytotoxin. Nevertheless, the complex has shown a response in 6 out of 8 cancer patients [33] . HAFP was found empirically to bind the environmental toxin dioxin (1:2 molar ratio). The HAFPdioxin complex was successfully used to destroy cancer cells [35] . However, if dioxin is released from the complex in the bloodstream or delivered in a quantity insufficient to destroy a tumor cell, it serves as a mutagen and a carcinogen [36] . Therefore, the anticancer application of the HAFP-dioxin complex is problematic. The empirical study of spectral changes in the presence of the rHAFP has demonstrated the rHAFP ability to bind curcumin and genistin. The rHAFP-curcumin and rHAFP-genistin complexes can also destroy cancer cells [17] . Nevertheless, the curcumin and genistin are poor candidates for HAFP–drug complexes, since less potent cytotoxins need more HAFP for the delivery to cancer cells and the cell destruction. The HAFP strongly binds the synthetic estrogen diethylstilbestrol (DES), unlike natural estrogens. A 3D computer model of DES docking in the HAFP hydrophobic pocket was made [37] . The model has a U-shaped structure in which a cavity may be distinguished (Figure 1) . The putative estrogen-binding site is
Toxin
Figure 1. HAFP U-shaped structure with the cavity [37] that can fit the natural ligand or toxin.
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localized in this cavity. Two types of interactions were proposed between the amino acid residues of HAFP and a DES molecule: hydrogen and hydrophobic binding. Again, DES is a carcinogen and the HAFP-DES complex anti-cancer application is questionable. An optimal anti-cancer drug can be characterized by specificity, efficacy and drug-tolerance by the normal cells. A teratogen specifically destroys embryo cells but is tolerated by the adult mother cells. Chemotherapy is fairly safe to both the child and the mother when given during the second and the third trimesters of pregnancy. Cyclophosphamide, 5-FU, Dox, bleomycin, vincristine and etoposide have been given safely during pregnancy [38] . It is assumed that some teratogens (unlike chemotherapy drugs) can bind HAFP and cross the placenta, enter thus the blood of the embryo and destroy the embryo cells. HAFP-teratogen complexes suppress embryogenesis and can also suppress cancer cells. AFP complexes with teratogens dioxin and atractyloside were successfully used to destroy cancer cells [35,39] . Unlike teratogens dioxin or DES that target the DNA, atractyloside is targeted at mitochondrion and hence is not mutagen and carcinogen. HAFP–drug complexes are different from HAFP–drug conjugates Comparison of the AFP–drug conjugate and AFP–drug complex properties are given in Table 1. Cancer cells specific targeting and receptor-mediated endocytosis of AFP–drug conjugates or complexes was reviewed in [1] . HAFP–drug conjugate stability in the circulation is provided by the covalent link. As for the complex the rigidity of the HAFP tertiary structure is controlled by the payload [17,40] . Fitting 1–2 small drug molecules into the HAFP hydrophobic pocket should naturally stabilize a HAFP–drug non-covalent complex in the bloodstream. A HAFP–drug complex alters the PK profile of drugs positively along with the stabilization of the HAFP. This profile is supposed to be similar to that of the natural HAFP-DHA complex or to the PKs of the HAFP alone (MM-093). Receptor-mediated endocytosis of HAFP-toxin conjugate/complex bypasses the membrane pumps and thus overcomes the MDR mechanism [22] . AFP recycling is possible for complex due to the non-covalent AFP–drug link [41] . The release of the payload from a HAFP complex inside a cancer cell happens with the pH change into acidic at a definite compartment within about an hour [2] . After that, the HAFP recycles back. Its approximate half-life in the blood is 5 days [7] . During this time the recycled HAFP can deliver multiple further payloads, acting as a natural shuttle. Curcumin, genistin, other supplements and drugs that feature a competitive to the blood proteins binding to HAFP can potentiate the rHAFP–drug
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complex anti-cancer action. The optimal AFP:drug conjugation ratio was shown to range from 1:3 to 1:5 [1] . The increase of this number can interfere with HAFP receptor interaction. HAFP–drug complex can fit into the HAFP hydrophobic pocket only 1–2 molecules. The drugs non-covalently bound at the HAFP surface will be shared with serum proteins and will not be targeted to cancer cells. To provide the integrity of the targeting and toxicity parts in the AFP–drug conjugate they should be bound with a linker(s), unlike AFP–drug complex that grabs the drug by the hydrophobic pocket. The AFP–drug conjugate linker(s) are designed to be cleaved enzymatically inside a cancer cell and release the drug [19] . The release of the drug from an AFP–drug complex upon internalization by cancer cell goes naturally.AFP does not elicit an immune response in the species in which the protein originated [1] . Conjugate expose 3–5 drugs on the surface of the HAFP that makes them immunogenic. On the other hand the immunogenicity of the drugs hidden inside the HAFP hydrophobic pocket is decreased. HAFP–drug complex immunogenicity is supposed to be as low as of the HAFP one. A possibly superior strategy is to supply cancer patients with small doses of AFP–drug complex on a permanent basis (maintaining the AFP concentrations that are typical during pregnancy and known to be low immunogenic) rather than to give them periodically a fraction of the maximum tolerated dose of HAFP–drug conjugate. Maximum tolerated dose should be determined for the artificial HAFP–drug conjugate, while drug delivery by the complex is effective with physiological HAFP doses. The full-size glycosylated HAFP has immunosuppressive or cancer stimulating properties [42] . To avoid the unwanted effects, HAFP–drug complex may be built with the unglycosylated rHAFP (e.g. MM-093) that has demonstrated no immunomodulating activity in clinical trials. Full-size HAFP is not important for conjugate: drugs can be targeted to cancer cells with HAFP vector peptides conjugate. HAFP–drug complex do not need linkers for drug binding that makes manufacturing less costly than HAFP–drug conjugates. Oral AFP–drug complex formulations The unenhanced bioavailability for oral routes of administration for protein/peptide pharmaceuticals is about 0 - 1% [43] . The enzymatic instability of proteins and the GI permeation are the main challenges. Moreover, HAFP–drug complexes can dissociate in the acidic stomach environment. Nevertheless, the AFP–drug complexes as oral formulations have shown promising anti-cancer results. Impila decoction containing mitochondrion toxin atractyloside [44] is an oral folk remedy of South African Zulu aborigines. In our study, an oral formulation
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Table 1. Comparison of the biochemical and anti-cancer properties of a-fetoprotein–drug conjugate versus a-fetoprotein–drug complex. Properties
AFP–drug Conjugate
Complex
Cancer cells targeting
Yes
Yes
Receptor-mediated endocytosis
Yes
Yes
Stability in circulation
Yes
Yes
Overcomes MDR
Yes
Yes
AFP recycling
No
Yes
Toxins per AFP molecule
1–5
1–2
Need a special linker
Yes
No
Need drug release design
Yes
No
Immunogenicity
High
Low
Maximum tolerated dose
Yes
No
Full-size AFP is important
No
Yes
Manufacturing cost
High
Low
AFP: a-fetoprotein; MDR: Multi-drug resistance.
of the atractyloside and the porcine AFP complex has shown response in 6 out of 12 patients with metastatic colorectal cancer [39,45] . Two of the patients who responded had indications of a possible MDR breakage during the previous chemotherapy treatment. The AFP-atractyloside complex improves the quality of life and the longevity of terminal cancer patients. Future perspective The majority of cancers displays the HAFP receptor and will therefore be sensitive to treatment with HAFP–drug conjugates/complexes; cancer stem cells, which are responsible for metastasis [46] , have a higher probability of expressing the HAFP receptor and, as such, can be treated using this approach. Taking this into account, positive testing for the HAFP recep-
tor among cancer patients can therefore allow for personalized cancer therapy. HAFP tumor-targeting peptides have now substituted full-size HAFP in HAFP–drug conjugates. These are less immunogenic and can be conjugated by optimal linkers with numerous potent cytotoxins (e.g., strategies such as nanoparticles and liposomes). It is projected that, over the next 5–10 years, drugs with high binding affinities to the HAFP will be identified using 3D modeling and the screening of chemical libraries. Among these candidates, the highly toxic drugs should be chosen for HAFP–drug complexes and the cancer treatment. In Table 2 the potential properties of AFP–drug complex are compared against registered chemotherapy, demonstrating the advantages of the proposed therapeutic approach.
Table 2. a-fetoprotein–drug non-covalent complex comparison with Abraxane. Properties
AFP–drug complex [39]
Abraxane [47]
AFP (embryo albumin)
1–10 mg
Albumin
900 mg
Toxin delivered
0.1–1 mg
100 mg Paclitaxel
Cancer cells targeted delivery
Yes
No
Receptor-mediated endocytosis
Yes
No
Maximum tolerated dose
No
260 mg/m2
Overcomes MDR
Yes
No
Side effects
No
Yes
Cancer stem cells affected
Yes
No
AFP: a-fetoprotein; MDR: Multi-drug resistance.
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The recycled AFP binding of some drugs, or endogenous molecules from the microenvironment, can potentiate the anti-cancer action of AFP–drug complexes. Teratogens or registered drugs with teratogenic properties are promising drug candidates for use simultaneously with AFP–drug complexes in anti-cancer therapy. In addition, the registered drug data package can shortcut clinical trials and accelerate the FDA approvals. The GI absorption of HAFP–drug complexes is still in need of additional research. HAFP is synthesized mainly by the yolk sac, fetal liver and GI tract cells and, since it binds the ligands of high nutritional value, it should be absorbed by AFP receptor positive cells. Assuming that the GI tract absorbs AFP-DHA or AFP–drug complexes
in a similar way to their absorption and transportation through placenta, oral AFP–drug complexes have the potential to enter the blood of the patient via this route and thereby prevent metastasis – or other explanations of the data may exist. Financial & competing interests disclosure V Pak is shareholder of Constab Pharmaceutical Inc. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Executive summary Background • The HAFP receptor-based targeting may emerge as a rewarding strategy in the treatment of cancer in the near future as it exploits the HAFP selectivity and the high cytotoxic potency of a drug. Factors that determine the anti-tumor activity of the HAFP–drug conjugates/complexes include the HAFP receptor expression, internalization and the direction of intracellular trafficking of HAFP–drug conjugates/complexes, the rate, compartment and/or the degree of the intracellular release of the potent drug. • The majority of cancers re-expresses HAFP receptors and can be sensitive to the specific HAFP-cytotoxin delivery mechanism provided by HAFP–drug conjugates or AFP–drug complexes. HAFP–drug conjugates or AFP–drug complexes by-pass membrane pumps and thus overcome the MDR mechanism of cancer cells.
HAFP–drug conjugates • The chances for a clinical success increase due to the continued improvements in the design of HAFP–drug conjugates, namely the development of more potent cytotoxic drugs and the optimization of HAFP–drug linkers. The full-size HAFP–drug conjugates are substituted today with the HAFP tumor-targeting peptide– drug conjugates. This way, the conjugate becomes less immunogenic and carries multiple potent drugs attached via optimal linker(s).
AFP–drug complexes • The AFP–drug complexes exploit the natural shuttle-like delivery potential of the HAFP. They can be obtained by fitting a potent drug into the hydrophobic pocket of the HAFP. The AFP–drug complexes for injections or oral formulations have demonstrated promising results and are well tolerated by cancer patients.
Cytotoxins for AFP–drug complexes • The AFP complexes with cytotoxins (1:1–2 molar ratios) should have both a high toxicity and a high binding affinity to the AFP. Such binding affinity can be provided by the AFP’s hydrophobic pocket that naturally transports DHA in a shuttle manner. Direct AIs targeted to organelles can be effective anti-cancer drugs. Teratogens or registered drugs with teratogenic properties should be included in the drug candidate list.
HAFP–drug complexes are different from HAFP–drug conjugates • The HAFP–drug complexes having close to conjugates drug/HAFP molecule ratio, at the same time they are not immunogenic, don’t need linkers, and hence less costly in manufacturing. The pharmacokinetic profiles of AFP–drug complexes are supposed to be similar to those of the AFP stabilized with natural ligands, e.g. DHA.
Oral AFP–drug complex formulations • The mechanism of the anti-cancer action of the oral AFP–drug complexes needs additional research. Their GI absorption can be similar to the absorption and the transportation of AFP-DHA complexes through placenta. Other plausible explanations of the data may exist.
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