Antibody is a protein that binds to an antigen with high specificity and high affinity to neutralize the antigen. Furthermore, the antibody has antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity, which are the functions of the heavy-chain constant region thereof, and has a long serum half-life by binding to FcRn (neonatal Fc receptor). Due to the property of binding to an antigen with high specificity and high affinity, undesirable side effects can be reduced, and due to antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity, the antibody can induce apoptosis of disease-causing cells, and the long serum half-life of the antibody enables the long-lasting effects of the antibody. Because of such properties of the antibody, studies have been actively conducted to develop the antibody into therapeutic proteins.
Cancer therapeutic antibodies developed to date are divided into two categories: antibodies for treatment of solid tumors; and antibodies for treatment of blood cancer (leukemia/lymphoma). According to the single administration of the antibodies in each category, the two antibodies show different response rates. When referring to statistics of several commercially available antibodies, in case where a single administration of an antibody for treatment of blood cancer, the response rate reaches 30 to 51%, whereas in case where a single administration of an antibody for treatment of solid tumors, the response rate is 8 to 15%, which is relatively low. This is because the antibody for treatment of blood cancer targets cancer cells in blood, whereas the antibody for treatment of solid tumors should be subjected to the following processes in order to exhibit its therapeutic effects: 1) reaching tumor blood vessels in solid tumor tissue through blood vessels after systemic intravenous injection or subcutaneous injection (tumor homing step); 2) flowing out from tumor blood vessels toward tumor tissue (extravasation step); 3) penetrating into vessel-free tissue even in tumor tissue (tumor tissue penetration step); and 4) binding to an antigen expressed in tumor cells and acting on the antigen (targeted antigen binding & effector function step) (Scott A M et al. 2012). In such series of processes, various factors are present which interfere with tumor tissue accumulation, and penetration into tumor tissue of the solid tumor therapeutic antibody, which leads an arrival of the antibody at tumor cells in the tumor tissue. For this reason, the amount of antibody that is accumulated to the tumor tissues in human body is very small (0.01 to 0.0001% of injected dose per gram tumor tissue), and thus the antibody shows a low response rate (Thurber et al. 2008). Accordingly, the development of antibody technology that enables an antibody to be accumulated selectively in tumor tissue and to have a high ability to penetrate tumor tissue makes it possible to increase the therapeutic effect of a solid tumor therapeutic antibody, and thus is very important.
There are two major reasons that an antibody has a deficiency in penetrating tissue: 1) intrinsic properties of the antibody (size (˜150 kDa), antigen-binding barrier, etc.) (Thurber and Dane Wittrup, 2012), and 2) microstructural/physiological properties of tumor tissue (e.g., incomplete and abnormal angiogenesis, very low lymphatic gland formation, high cellular density, high extracellular density, etc.), which differ from those of normal tissue (Jain and Stylianopoulos, 2010). Thus, efforts have been made to increase the tumor tissue penetrability of antibodies by use of various methods, including an antibody engineering technique that regulates the size and antigen binding specificity of antibodies, or a method of combination administration an antibody with a molecule (i.e., promoter agent) that promotes the tumor tissue penetration of the antibody.
Antibodies in blood are hardly delivered to tumor tissue by diffusion or convection, because the antibody is a 150-kDa large molecule consisting of 12 domains (Baker et al. 2008). To overcome this difficulty, there has been an attempt to administer antibody fragments alone, such as an antigen-binding fragment (Fab) (50 kDa), a single-chain variable fragment (scFv) (30 kDa), and a heavy-chain variable domain (VH) (14 kDa), which have reduced sizes. However, the antibody fragment has no Fc fragment and is small in size, and for this reason, when it is administered in vivo, it is released in large amounts through the kidneys to reduce the half-life thereof, indicating that the efficacy of the antibody is not significantly improved (Behr et al. 1998).
Another reason why an antibody is not distributed in a large amount in tissue is an antigen-binding capability of the antibody. An antibody for treatment of solid tumors is overexpressed on a tumor-associated antigen or in a tumor, and has a high affinity for a target which is important for tumor growth. Even when the antibody may reach the tissue where a specific antigen is present, in a tumor tissue composed of cells with a great amount of antigen expression, the antibody is stayed on the surface of the tumor tissue while binding to an antigen expressed in cells on the tumor tissue surface, due to its high affinity (Lee and Tannock, 2010). After binding to the antigen, the antibody is endocytosed, i.e., penetrates into the cells along with the antigen and is degraded in the cells. In other words, the antibody may be located on the tumor tissue surface, but is degraded after binding to an overexpressed tumor antigen, and thus does not efficiently penetrate the tumor tissue. Accordingly, the antibody cannot reach a tumor cell antigen in a tissue distant from tumor blood vessels, and thus the anti-tumor activity of antibody may decrease, and antibody resistance and tumor recurrence may be induced. To overcome this shortcoming, studies have been conducted to regulate antibody affinity or increase antibody half-life (Dennis et al. 2007).
The physiological properties of tumor tissue, which interfere with the penetration and distribution of antibodies in tumor tissue, can be largely classified into four cases: endothelial barrier; high tumor interstitial fluid pressure; stromal impediment; and epithelial barrier.
As for the endothelial barrier, a tumor overexpresses and secretes a pro-angiogenic factor that promotes the growth of vascular endothelial cells located around blood vessels, in order to receive large amounts of nutrients due to its rapid growth rate. Accordingly, a large amount of new blood vessels are non-uniformly produced to reduce the overall blood flow rate. In an attempt to overcome this shortcoming, there has been proposed a method of increasing extravasation to enable a therapeutic agent to flow out from blood vessels so as to be distributed to tissue. Furthermore, a case has been reported in which TNF-α and IL-2, which are pro-inflammatory cytokines associated with extravasation, a promoter chemical drug that promotes extravasation, and a therapeutic agent were co-administered to promote drug to tumor tissue (Marcucci et al. 2013). However, these attempts are difficult to be commercialized and clinically experimented in that it is required to produce two substances such as antibody and extravasation promoter.
High tumor interstitial fluid pressure results from a situation where a pressure difference allowing a drug to be convected from a blood vessel to tissue is small, or where the fluid pressure of tissue is higher than that of blood. High tumor interstitial fluid pressure is mainly caused due to the accumulation of interstitial fluid pressure in the absence of a lymphatic duct in tumor tissue, unlike in normal tissue, and also contributes to abnormal angiogenesis. In an attempt to overcome this, there has been proposed a method of inhibiting the activity of a factor promoting the growth of vascular endothelial cell, particularly vascular endothelial cell growth factor-A (VEGF165A), to inhibit angiogenesis to normalize the blood vessel, or a method of increasing the fluid pressure of blood vessel. With regard to the method of increasing the fluid pressure of blood vessel, a case has been reported in which the plasma protein albumin was administered in combination with an antibody to increase the osmotic pressure of blood vessels, thereby enhancing delivery of the antibody to tumor tissue (Hofmann et al. 2009).
The stromal impediment is an extracellular matrix barrier that an antibody meets when being convected to tissue after flowing out from micro-vessels. The stromal impediment mainly consists of collagen and hyaluronan. The extracellular matrix greatly affects the shape of tumor. Accordingly, there is a great difference between an area where a drug is well distributed and an area where the drug is not well distributed, and thus drug distribution becomes non-uniform. Additionally, as the expression level of extracellular matrix increases, the tumor interstitial fluid pressure increases due to high cell density with solid tumor stress (solid stress). In an attempt to overcome this limitation, there has been a method of inducing apoptosis of tumor tissue cells to reduce cell density in tumor tissue. Additionally, there has been reported an example in which solid stress was reduced by treatment with collagenase degrading collagen of tumor tissue, thereby increasing drug delivery about twice compared to a control group (Eikenes et al. 2004).
In the epithelial barrier, intercellular adhesion factors of interstitial epithelial cells in tumor tissue densely fill up an intercellular space, and thus they prevent a therapeutic agent from being diffused and convected between the cells. E-cadherin is well known as a main factor of the intercellular adhesion. Since a substance that reduces E-cadherin was found in virus (adenovirus-3), a case has been reported in which only a portion (JO-1) having an activity of reducing cellular E-cadherin, among proteins constituting the virus, was co-administered with an antibody, thereby increasing the anti-cancer effect of the antibody (Beyer et al. 2011).
In addition, there have been proposed methods in which a peptide that binds to neuropilin (NRP) that is overexpressed in tumor-associated endothelial cells and tumor cells is used to enhance antibody penetration into tumor tissue. One of the methods that use the neuropilin-binding peptide is to co-administer an iRGD peptide with an antibody (Sugahara et al. 2010). However, in the case of the method of co-administering the peptide, the amount and frequency of peptide that is actually administered to a patient should be very large due to pharmacokinetics attributable to the small molecular size of the peptide. Furthermore, the industrial feasibility of the method is low, because a therapeutic agent and a tumor-penetrating peptide are separately produced due to an inevitable process of co-administration. In recent technologies to overcome this limitation, there has been an example in which an A22p peptide that binds to neuropilin is fused to the heavy-chain C-terminus of a monoclonal antibody, so that the long half-life of the antibody will be maintained and tumor tissue penetration of the antibody will be enhanced (Shin et al. 2014; Korean Patent Application No. 10-2014-0061751; and PCT Patent Application No. PCT/KR2014/004571).
Neuropilin, a transmembrane glycoprotein, is divided into two types: neuropilin-1 (NRP1) and neuropilin-1 (NRP2) (Kolodkin et al. 1997). Neuropilin-1 and -2 consist of 923 and 931 amino acids, respectively, and show an amino acid sequence homology of about 44%, and share several structural aspects and biological activities. Neuropilin-1 and 2 consist commonly of extracellular a1, a2, b1, b2 and MAM domains and an intracellular PDZ-binding domain (Appleton et al. 2007). Neuropilin is very weakly expressed in normal cells, but is overexpressed in most tumor-associated endothelial cells, solid tumor cells and blood tumor cells (Grandclement, C. and C. Borg 2011). Neuropilin acts as a co-receptor of VEGF receptors (VEGFRs) by binding to VEGF family ligands. Particularly, NRP1 acts as a co-receptor of VEGFR1, VEGFR2 and VEGFR3 to bind to various VEGF ligands, thereby contributing to angiogenesis, cell migration & adhesion and invasion. On the other hand, NRP2 acts as a co-receptor of VEGFR2 and VEGFR3, thereby contributing lymphangiogenesis and cell adhesion. Furthermore, neuropilin 1 and 2 act as a co-receptor of plexin family receptors to bind to secreted class-3 semaphorin ligands (Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, Sema3F, Sema3G). Since neuropilin has no domain in functional cells, it has no activity by itself, even if a ligand is binding thereto. It is known that neuropilin signal transduction occurs through VEGF receptor, which is a co-receptor, or through plexin co-receptor. Sema3 binds to neuropilin and plexin receptor at a ratio of 2:2:2 and acts. However, many study results show that neuropilin protein alone can perform signal transduction without its interaction with the VEGF receptor or plexin co-receptor. However, an exact molecular mechanism for this signal transduction is still unclear.
Cases have been reported in which the activities of neuropilin and co-receptor are inhibited even when only neuropilin is targeted. For example, it has been reported that anti-neuropilin-1 antibody binds to only neuropilin-1 competitively with VEGF-A known to bind to VEGFR2 and neuropilin-1, and functions to inhibit angiogenesis, cell survival, migration & adhesion and invasion, which are the actions of VEGFR2 (Pan Q et al. 2007). It has been reported that anti-neuropilin-2 antibody binds to neuropilin-2 competitively with VEGF-C known to binds to both VEGFR3 and neuropilin-2, and functions to inhibit lymphangiogenesis and cell adhesion, which are the operations of VEGFR3 (Caunt M et al. 2008).
The C-terminal region of each of the VEGF ligand family and Sema3 ligands, which bind to neuropilin 1 and 2, binds to the VEGF-binding sites (so-called arginine-binding pocket) in the b1 domain present commonly in neuropilin 1 and 2 (MW Parker et al. 2012). Herein, binding to the arginine-binding pocket occurs by a motif of R/K-x-x-R/K (R=arginine, K=lysine, and x=any amino acids), which is present commonly in the C-terminal region of neuropilin binding ligands. When mutation is induced with an amino acid sequence deviating from the motif, the ligands have a reduced binding affinity for neuropilin or do not bind to neuropilin, and thus lose their biological activity. Particularly, cationic arginine (Arg) or lysine (Lys) in the C-terminal region is essential for binding, and thus when it is substituted with another amino acid residue, the ligand loses its binding affinity for neuropilin, and loses its biological activity. Accordingly, the necessity of the R/K-x-x-R/K motif in the C-terminal region of such neuropilin binding ligands is called “C-end rule” (CendR) (Teesalu et al. 2009). A protein or peptide containing a C-end rule sequence is capable of binding to neuropilin by the C-terminal arginine (Arg) or lysine (Lys) residue (Zanuy et al, 2013).
The C-terminal regions of VEGF ligands and Sema3 ligands commonly have the R/K-x-x-R/K motif, and thus most of the ligands have the property of binding to both neuropilin 1 and 2 rather than binding selectively to any one of neuropilin 1 and 2.
In addition to ligands that bind to neuropilin 1 and 2, many peptides that bind to neuropilin have been selected or designed and reported. These peptides all have the R/K-x-x-R/K motif, and thus appear to bind to the arginine-binding pocket in the b1 domain of neuropilin 1 and 2. Furthermore, an iRGD peptide (Sugahara et al. 2010) that binds to neuropilin 1 and 2 to increase tumor tissue penetration of a co-administered drug, and an A22p peptide (Shin et al. 2014) that is fused to the heavy-chain end of an antibody to increase tumor tissue penetration of the antibody, also have amino acid sequences, following the CendR rule.
With respect to the peptides that bind to neuropilin, a peptide that binds specifically to any one of neuropilin 1 and 2 has not been reported, and these peptides have the CendR sequence motif, and thus bind to the arginine-binding pocket of both neuropilin 1 and 2.
As described above, neuropilin-1 is overexpressed only in newly formed blood vessels and plays an important role in angiogenesis, and neuropilin-2 is expressed in lymphatic vessels and contributes to lymphatic vessel production. Thus, a peptide that binds specifically to neuropilin-1 or neuropilin-2 with high affinity may have the capability to specifically regulate the biological activity of each neuropilin, but has not yet been reported. Furthermore, neuropilin 1 and 2 are activated as a homodimer or a heterodimer, and conventional peptides have been developed as monomeric peptides that have a very weak ability to regulate biological activity. Thus, a peptide that binds neuropilin-1 as a homodimer to regulate the biological activity of neuropilin-1 is preferred. Moreover, neuropilin-1 is overexpressed in endothelial cells and is stimulated by VEGF ligands, and thus plays an important role in angiogenesis. Accordingly, a peptide, which binds to only neuropilin-1 with high specificity and high affinity competitively with VEGF ligands, may have the ability to home and accumulate in tumor tissue and to inhibit angiogenesis. Furthermore, neuropilin-1 is overexpressed in tumor tissue blood vessels and tumor cells (epithelial cells) and stimulated by VEGF ligands, and thus plays an important role in tumor growth and angiogenesis. Accordingly, a peptide, which binds specifically to neuropilin-1 competitively with VEGF ligands, may have an activity of inhibiting tumor growth. In addition, when neuropilin-1 is activated, it has an activity of reducing the endothelial barrier VE-cadherin and the epithelial barrier E-cadherin. Accordingly, a peptide, which binds specifically to neuropilin to reduce the levels of VE-cadherin in vascular endothelial cells and E-cadherin in tumor cells, may increase tumor extravasation and tumor tissue penetration of a protein, an antibody, a nanoparticle or a small-molecule drug, with which the peptide is fused or co-administered, and may also increase tumor tissue penetration.
Accordingly, the present inventors have attempted to overcome the limitation of conventional peptides that bind to both neuropilin 1 and 2 and to identify a novel peptide that binds specifically to neuropilin 1 with high affinity without binding to neuropilin 2. Furthermore, the present inventors have attempted to identify a novel peptide that binds bivalently to the VEGF-binding pocket (arginine-binding pocket) of the b1 domain of neuropilin-1 to induce signaling to activate neuropilin-1 to be endocytosed into cells, thereby increasing tumor tissue distribution and accumulation of a fused or co-administered protein, antibody or the like and promoting extravasation of this protein or antibody into tumor tissue, and has the ability to penetrate tumor tissue. Therefore, the present inventors have attempted to develop a novel peptide that is always present as a homodimer and is fused to the C-terminus of the heavy-chain constant region of an antibody while maintaining its activity.
To this end, the present inventors have attempted to construct the yeast surface-displayed immunoglobulin Fc-fused peptide library, and then select a clone that binds to the b1 domain of neuropilin-1. To select peptides that bind only to neuropilin-1, neuropilin-2 was used as a competitor in the selection process. Among the selected clones, a clone which has the ability to penetrate tumor tissue and binds to the b1 domain of neuropilin-1 was identified, and this peptide was bound bivalently to the C-terminus of the heavy-chain of an antibody to construct an antibody-peptide fusion protein that retains the intrinsic function of the antibody. According to this fusion antibody technology, the antibody was accumulated selectively in tumor tissue overexpressing neuropilin-1, and had an increased ability to penetrate tumor tissue. In addition, the present inventors have developed a fusion antibody technology that interferes with binding of vascular endothelial growth factor-A (VEGF165A) to neuropilin-1 to thereby inhibit angiogenesis.
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