PL234874B1 - Protein sequence, preferably a fragment of antibody, constituting VEGF-C inhibitor and its application - Google Patents

Description

Description of the invention

The invention relates to a novel scFv format antibody that is an inhibitor of the VEGF-C protein. In particular, the antibody of the invention is single chain human antibody fragments (scFv) which bind to and neutralize VEGF-C. The invention also relates to the use of this inhibitor.

State of the art

Cancer is one of the leading causes of death in the world. Effective therapy related to the surgical removal or irradiation of primary tumors is often impossible due to the migration of cancer cells from the primary environment to other organs and tissues in the body. Malignant neoplasms use both blood vessels and lymph vessels to form metastases (Bzowska M et al., Acta Biochim Pol 2013, 60 (3): 263-75). Until now, researchers focused on the phenomenon of angiogenesis, i.e. the formation of new blood vessels. It has become the main target of cancer metastasis inhibiting therapies, although their efficacy has been lower than expected. One of the many possible reasons for the failure of this type of therapy is the neglect of the role of lymphatic vessels as an alternative route for cancer cells to escape from the primary tumor (Potente M et al. Cell 2011; 146 (6): 873-87). The most promising target for inhibiting lymphangiogenesis is the blockade of the signal axis between the VEGF-C protein (vascular endothelial growth factor C) and its receptors VEGFR-2 and VEGFR-3 (vascular endothelial growth factor receptor; vascular endothelial growth factor receptor) (Stacker SA et al. Nat Rev Cancer 2014; 14 (3): 159-72). VEGF-C belongs to the family of vascular endothelial growth factors, consisting of seven proteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF (placental growth factor). growth factor). Discovered in 1996, the VEGF-C protein was described as a non-covalent homodimer ligand for VEGFR-2 and VEGFR-3 (Joukov V et al. EMBO J. 1996; 15 (7): 290298), although some reports also report the occurrence of covalently linked VEGF-C dimers (Leppanen VM et al. Proc. Natl Acad Sci USA 2009; 107 (6): 2425-2430). Its affinity for receptors is regulated by a multistage proteolytic processing, during which the strength of the interaction of successively emerging forms of VEGF-C with VEGFR-3 increases. In turn, VEGFR-2 can only recognize the mature form of VEGF-C (Joukov V et al. EMBO J 1997; 16 (13): 3898-3911). The sequences of the mature forms of human and murine VEGF-C proteins differ by only two amino acid residues (UniProtKB / Swiss-Prot database records P97953 and P49767 for murine and human VEGF-C, respectively). VEGFR-2 and VEGFR-3 are receptors with tyrosine kinase activity, which, as a result of ligand binding, become activated - they undergo dimerization and mutual phosphorylation of cytoplasmic domains (Joukov 1996, loc. Cit.). Under the influence of VEGF-C interaction with its receptors, only VEGFR-3 homodimers or VEGFR-2/3 heterodimers may be formed (Nilsson I et al. EMBO J 2010; 29 (8): 1377-88). Probably an important role in the signaling axis between VEGF-C and its receptors is played by neuropilins that do not have enzymatic activity, which act as co-receptors for VEGFR-2 and VEGFR-3 (Karpanen T et al. FASEB J 2006; 20 (9): 1462-72 ).

VEGF-C plays an important role in lymphangiogenesis, and therefore takes part in many different physiological and pathological processes, such as: development of the lymphatic system during embryogenesis, wound healing, chronic inflammation, autoimmune diseases or tumor metastasis (Chachaj A and Szuba A [in:] Angiogenesis and Vascularisation Dulak J et al. (eds.); Springer Vienna 2013, pp. 27-66). VEGF-C, by activating VEGFR-3, stimulates the proliferation and migration of lymphatic endothelial cells and provides them with a survival signal (Makinen T et al. EMBO J 2001; 20 (17): 4762-4773). Moreover, while VEGF-A remains the major pro-angiogenic factor, VEGF-C also can stimulate angiogenesis by interacting with VEGFR-2/3 heterodimers (Nilsson 2010, loc. Cit.).

The main pro-lymphangiogenic factors of key importance in tumor metastasis are the VEGF-C and VEGF-D proteins, which induce the formation of new lymphatic vessels around the tumor by activating VEGFR-3 and the neuropilin-2 coreceptor (Karpanen 2006; op. Cit .; Mandriota) SJ et al. EMBO J 2001; 20 (4): 672-82; Chen Z et al. Cancer Res 2005; 65 (19): 9004-9011; Xu Y et al. J Cell Biol 2010; 188 (1): 115-30). In this way, cancer cells, secreting VEGF-C, provide an escape route to a nearby lymph node, from which they can spread further both through the lymphatic and bloodstream (Stacker SA et al. Nat Rev Cancer 2002; 2 (8): 573-583 ). The essential role of VEGF-C in tumor-related lymphangiogenesis was confirmed by in vivo experiments in which blocking this factor inhibited metastasis of neoplastic cells (Chen 2005; op. Cit .; Wang F et al. Exp

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Ther Med 2010; 1 (5): 899-904). Therefore, it has become an interesting target for potential anti-lymphangiogenic therapy.

One of the more extensively studied strategies aimed at inhibiting the formation of lymphatic vessels during tumor progression is the use of monoclonal antibodies specific to individual biological factors involved in lymphangiogenesis. Blocking the signal axis between VEGF-C and VEGF-D and VEGFR-2 and VEGFR-3 receptors is considered the most promising target of this type of therapy. Blocking the signaling pathway for VEGF-C / VEGF-D and VEGFR-3 is a theoretically safe therapeutic approach as it should not affect the already existing lymphatic vessels (Chachaj and Szuba 2013, op. Cit.). In vivo studies seem to confirm the above assumption, as blocking VEGFR-3 did not cause any side effects on previously developed lymphatic vessels in adult mice (Karpanen T et al. Am J Pathol 2006; 169 (2): 708-718). However, it is worth bearing in mind the impact of inhibition of lymphangiogenesis on the processes of wound healing or tissue regeneration (after Chachaj and Szuba 2013, op. Cit.).

Inhibitors of the process of biological signal transmission through the VEGF-C axis - its receptors are known in the art. PCT application published as WO2005112971 discloses a method of inhibiting angiogenesis and lymphangiogenesis by disrupting the maturation process of the VEGF-C and VEGF-D proteins. On the other hand, US20020164667 discloses sequences of peptides capable of binding to the VEGFR3 receptor and inhibiting its biological activity, including the ability to bind the VEGF-C protein. The prior art also includes molecules that interact with the VEGF-C protein. In particular, these molecules may be other proteins, e.g. the soluble sVEGFR receptors disclosed in US20140057851. The VEGF-C-interacting proteins may also include antibodies, particularly monoclonal antibodies. A VEGF-C specific IgG antibody is disclosed in US2011143428. Table 1 shows monoclonal antibodies known from the state of the art with the ability to inhibit the lymphangiogenesis process. The therapeutic potential of two of them is being assessed in clinical trials. The IMC-35C antibody, preventing the binding of VEGF-C and VEGF-D to VEGFR-3, in preclinical studies in combination with chemotherapy allowed to reduce the rate of progression of neck and head cancers. The VGX-100 antibody, which is an inhibitor of VEGF-C, was also directed to clinical trials and, in combination with the anti-VEGF-A antibody, inhibited the growth of tumors in animal models of glioblastoma as well as prostate and pancreatic cancers (after: Bzowska 2013, op. Cit.) . The use of a strategy involving the simultaneous use of therapeutics with anti-angiogenic and anti-lymphangiogenic properties is a very promising approach that may improve the effectiveness of currently used anti-cancer therapies.

Table 1. Monoclonal antibodies developed so far as potential inhibitors of lymphangiogenesis (Bzowska 2013, op. Cit).

Objective Name Description Research phase YEGFR-3 IMC- 3C5 human IgG 1 antibody; prevents the binding of YEGF-C and YEGF-D to VEGFR-3 phase 1 VEGF-C VGX- 100 human antibody; blocks the binding YEGF-C to VEGFR-2 and YEGFR-3 phase 1 VEGF-D cVE199 chimeric IgG1 antibody; blocks the binding of VEGF-D to YEGFR-3 research preclinical prevents the formation of homodimers YEGFR-3 2Ε11 YEGFR-3 and YEGFR-2/3 hctcrodiniers, but does not inhibit the binding of VEGF-C to research preclinical VEGFR-3 neuropilin- prevents YEGF-C from binding to neuropilin research 2 2 preclinical YEGF-C VC2 human scFv, inhibits the binding of YEGF-C to YEGFR-2 and YEGFR-3 in vitro research human scFv bispecific dimer ( YEGFR-2 and YEGFR-3 - diabody) directed against VEGFR-2 and VEGFR-3; inhibits the activation of the above-mentioned in vitro research

receptors

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Antibodies are molecules of relatively large size, which may limit their therapeutic use (e.g. due to low penetration of cancerous tissue). Moreover, the production of monoclonal antibodies is expensive (e.g. requires the use of eukaryotic cell culture). Single-chain variable fragment (scFv) antibodies, which are about 6 times smaller than IgG class antibodies and can be produced with the use of prokaryotic expression systems, do not have these drawbacks.

ScFv antibodies that specifically interact with the VEGF-C protein are known in the art (Rinderknecht M et al. PLoS One 2010; 5 (8): e11941). However, the antibodies described have a number of limitations. The protein used to select the scFv antibodies came from the yeast organism, as it was produced in a system using Pichia pastoris cells.

Therefore, this protein was characterized, among others, by a glycosylation pattern different from that of the mammalian VEGF-C protein. Moreover, the biological activity of the protein used to select scFv antibodies has not been confirmed. Furthermore, the biological activity of the described antibodies has not been disclosed, so their influence on VEGF-C signaling in biological systems is unknown.

The object of the invention is to provide molecules that can be an efficient and specific inhibitor of human and murine VEGF-C proteins. An additional object of the invention is to provide a specific inhibitor of the lymphangiogenesis process in the form of a monoclonal antibody of the scFv format that inhibits the biological activity of the VEGF-C protein. A further object of the invention is to provide a method for the production of a drug substance containing proteins according to the invention for the treatment of diseases associated with abnormal biological activity of VEGF-C.

The essence of the invention

The essence of the invention is an antibody of the scFv format, which is an inhibitor of the VEGF-C protein, characterized in that it contains the amino acid sequence of Seq. No. 3 or Seq. no 4.

Another aspect of the invention is an antibody of the scFv format as defined above for use as a medicament in the prevention and / or treatment of diseases and / or conditions associated with an abnormal amount of VEGF-C protein, including lymphangiogenesis dysfunction.

Preferably the diseases and / or conditions are selected from the following group: lymphatic metastatic tumors, acute myeloid leukemia, acute lymphoblastic leukemia, Kaposi's sarcoma, anti-VEGF-A resistant tumors, VEGF-C induced bone loss , age-related macular degeneration, diabetic retinopathy, inflammatory diseases associated with excessive VEGF-C levels, including rheumatoid arthritis and psoriasis.

The subject of the invention is presented in non-limiting examples of implementation and in the drawing, in which:

Figure 1 illustrates that the selected monoclonal antibodies are specific for both murine VEGF-C (A) and human VEGF-C (B);

Figure 2 illustrates antigen binding by selected soluble anti-VEGF-C scFv antibodies;

Figure 3 illustrates that the 1F2, 1F5 and 1F6 antibodies are specific for only

VEGF-C;

Fig. 4 illustrates the biological activity of the resulting anti-VEGF-C antibodies analyzed by a VEGF-C-induced tubule formation assay on a matrigel-containing medium;

Figure 5 illustrates an analysis of the effect of 1F2 and 1F5 antibodies on the formation induced by

VEGF-C spikes in immortalized human Lymphatic Endothelial Cells (ihFEC) A. For cells incubated with 1F2. B. For cells incubated with 1F5. C. For cells incubated without scFv. D. Analysis of the effect of 1F2 and 1F5 antibodies on the number of spikes formed by ihLEC cells after VEGF-C stimulation. E. Analysis of the Effect of 1F2 and 1F5 Antibodies on the Length of the Spikes Formed by ihLEC Cells After VEGF-C Stimulation.

Fig. 6 illustrates the amino acid sequences of the antibodies isolated from the phagemid library, converged to MSA (Multi-Sequence Alignment).

Execution example

Isolation of phages showing the expression of recombinant monoclonal antibodies of the scFv format specific for the murine and human VEGF-C protein

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A phage display method based on the use of a phagemid library of human recombinant antibodies of the Tomlinson I + J scFv format was chosen for the production of antibodies. Using the polyclonal phage of the library, three selection rounds were performed on the immobilized murine VEGF-C protein produced by mammalian cells (the antigen was therefore the mature recombinant VEGF protein-Canac). The biological activity of the protein used for selection was previously confirmed by the methods described below using in vitro cell culture. As a result of selection on the antigen, phages were obtained which expressed on their surface antibodies capable of binding the murine recombinant VEGF-C protein (Fig. 1A). Although the phage selection of the library was performed using the murine VEGF-C protein as the antigen, it was confirmed that antibodies expressed on the surface of the isolated phages also bind human VEGF-C (Fig. 1B). Thus, the selected antibodies are specific for both murine and human VEGF-C proteins.

Obtaining soluble antibodies of the scFv format binding murine and human VEGF-C

An antibody expressed on the surface of a filamentous phage is not a soluble protein - it is produced as a fusion protein together with the pili protein, a phage's structural coat protein. To express the soluble scFv antibodies, the coding sequence of the selected antibody was cloned into the pETscFv expression vector. The expression of the soluble anti-VEGF-C antibodies was obtained in E. coli Rosetta Blue bacteria. The antibodies were isolated from the periplasmic region of the bacteria and purified using protein A affinity chromatography. Figure 2 shows the binding of selected scFv monoclonal antibodies to the antigen.

Confirmation of the specificity for the VEGF-C protein

Since the proteins belonging to the VEGF family show great similarity in the amino acid sequence, it was confirmed using ELISA that the obtained antibodies 1F5, 1F6 and 1F2 recognize and bind VEGF-C while having no affinity for VEGF-A and VEGF-D (Fig. 3).

Study of the biological activity of anti-VEGF-C antibodies

The obtained scFv antibodies 1F2, 1F5 and 1F6 were also tested for biological activity, i.e. it was checked whether they were able to inhibit the induction of effects resulting from activation of its receptors by VEGF-C. For this purpose, the Immortalized Human Lymphatic Endothelial Cells (ihLEC) assay was used for matrix formation on matrigel-containing medium. IhLEC cells form such tubes only under the influence of VEGF-C, so if we do not observe VEGF-C-induced tubules formation in the presence of anti-VEGF-C antibody or if this process is impaired, it can be concluded that the ability of VEGF-C to activate its receptor (s).

It was observed that in the presence of scFv 1F2 or 1F5 antibodies this process is disturbed (Fig. 4), which may indicate that in the presence of this antibody there is no activation of VEGF-C receptors.

The biological activity of the isolated antibodies of the VEGF-C-specific scFv format was also tested using the spheroidal spike culture assay. The test of spike formation from cultures of nodular endothelial cells of blood or lymph vessels suspended in collagen is one of the most widely used methods to assess endothelial differentiation and capillary formation. Under the influence of substances that stimulate vascular growth, projections are formed from cell aggregates, which resemble angiogenesis or lymphangiogenesis in vivo. The results of the conducted studies showed that the 1F2 and 1F5 antibodies reduce both the number and the length of spikes formed by ihLEC cells in the presence of VEGF-C, which indicates the blocking of the biological activity of VEGF-C by these antibodies.

Obtaining the sequence of antibodies of the scFv format binding murine and human VEGF-C

The pIT2 phagemid vectors encoding the antibodies displayed on the surface of the extracted phages were sequenced to obtain the amino acid sequence of the encoded antibodies. The sequences obtained were then aligned in the form of MSA (Multi-Sequence Alignment) and presented in Fig. 6. Within them, CDR regions can be distinguished, which are responsible for binding the protein sequence to the antigen.

Individual CDRs in the antibody sequence can be thought of as interchangeable fragments. In addition, the above sequences containing heavy chain fragments and light chain fragments can be subjected to further optimization; in particular, they can be converted to a single heavy chain antibodies (camelid heavy chain antibodies) which characterize naturally occurring immunoproteins (Hamers-Casterman et al. Nature 1993; 363: 446-448),

PL 234 874 B1 (Muyldermans S and Lauwereys M. J Mol Recognit 1999; 12: 131-140). In this case, the entire protein sequence constituting the scFv antibody is not required for antigen binding, only a fragment (single chain) thereof. Thus, the use of such a fragment will be sufficient to preserve the antigen binding ability of the protein sequence. The use of such an optimization strategy for antibodies of the scFv format is known from the literature (Rinderknecht M et al., Loc. Cit.).

The selected monoclonal antibodies are specific for both murine and human VEGF-C as shown in Figures 1A and 1B, respectively. The analysis was performed using an ELISA test. Plates coated with antigens (mouse or human VEGF-C) were incubated with the phage extracted and the non-binding VEGF-C phage, kn negative control. The obtained result confirms that 1) the antibodies are capable of recognizing and specifically binding the murine VEGF-C protein and the human VEGF-C protein, and 2) the antibodies can be used to detect VEGF-C in both murine samples and human material.

Antigen binding by selected soluble anti-VEGF-C scFv antibodies is illustrated in Figure 2. The analysis was performed using an ELISA assay. Expression of anti-VEGF-C antibodies was obtained in E. coli Rosetta blue bacteria. The antibodies were isolated from the periplasmic area of the bacteria. The antibodies were bound to the antigen immobilized on the ELISA plate. Detection of antigen-bound scFvs was made using a solution of L-HRP (horseradish peroxidase-labeled L protein) as it binds to the scFv light chain. kn denotes an antibody not specific for VEGF-C. The obtained result confirms that not only the antibodies expressed on the surface of the selected phages are able to bind the VEGF-C protein, but also the corresponding soluble antibodies that were produced using the bacterial expression system based on the pETscFv vector.

The selected 1F2, 1F5 and 1F6 antibodies were tested for the specificity of their interaction with VEGF-C (Figure 3). The analysis was performed using an ELISA test. The plate was coated with VEGF-C, VEGF-A and VEGF-D proteins at a concentration of 2 µg / ml. All antibodies were diluted to a concentration of 5 µg / ml in PBS buffer containing 3% bovine albumin. Detection of antigen bound scFvs was accomplished with the L-HRP protein. K - control scFv antibodies specific for a protein other than VEGF-C. The obtained result confirms that the obtained scFv antibodies are specific only for the VEGF-C protein. The VEGF-C protein is very similar to other proteins of the VEGF family (for example, VEGF-A or VEGF-D), so these antibodies are a very valuable tool for the specific detection and selective binding of VEGF-C.

The analysis of the biological activity of the anti-VEGF-C antibodies of the invention, performed by the VEGF-C-induced tube formation assay on matrigel-containing medium, is illustrated in Fig. 4. Human vascular endothelial cells and hLEC were plated on matrigel plates and cultured. : in EGM2 medium, in 100 ng / ml VEGF-C medium or in VEGF-C medium with 1F2, 1F5 or 1F6 antibodies (8 µg / ml), respectively. After 48 hours the amount of tubules present on the matrix surface was assessed.

The results of the test are shown in Fig. 4. The plates cultivated with antibodies 1F5 (Fig. 4C) and 1F2 (Fig. 4E) showed results similar to the negative control - without VEGF-C (Fig. 4A). On the other hand, on the plate cultivated with the 1F6 antibodies (Fig. 4D), the results were similar to the positive control - containing VEGF-C (Fig. 4B).

The obtained results indicate that the VEGF-C specific antibodies 1F2 and 1F5 are able to inhibit the biological activity of the VEGF-C protein and thus act as its inhibitor. In turn, the 1F6 antibody used at the same concentration, which also efficiently binds VEGF-C, is not able to inhibit the biological activity of this protein.

VEGF-C is a protein that stimulates the processes of cell migration and proliferation necessary for the formation of new lymphatic vessels. Such action of VEGF-C is possible after the binding of this protein to the VEGFR2 or VEGFR3 receptor present on lymphatic endothelial cells. If the interaction of VEGF-C with its receptor is disturbed, for example as a result of binding VEGF-C by a specific scFv antibody, the biochemical processes leading to the stimulation of the lymphangiogenesis process will not be stimulated and new lymphatic vessels will not arise. Antibodies 1F2 and 1F5, due to their strong interaction with VEGF-C, are able to block its prolymphangiogenic activity and act as inhibitors of VEGF-C, as shown in the tube forming assay on matrigel-containing medium.

The analysis of the effect of 1F2 and 1F5 antibodies on VEGF-C-induced spike formation in nodular culture of ihLEC cells is illustrated in Figure 5. For culturing hLEC spheroidal cells

Anti-VEGF-C antibodies (1F2 or 1F5) were added at a concentration of 8 µg / ml or the buffer alone in which the antibodies were suspended (no scFv control) was added followed by VEGF-C at 100 ng / ml. After 24 hours pictures of the spheroid cultures were taken and analyzed for the number (Fig. 5D) and the length of the spikes per sphere (Fig. 5E).

This test was used to additionally confirm that the obtained 1F2 and 1F5 antibodies block the interaction of VEGF-C with its receptors, and thus block the prolymphogenic properties of VEGF-C. In the presence of these scFv antibodies, less spike formation was observed by VEGF-C-stimulated hLEC cells in spheroidal culture.

Antibody sequences (Figure 6) were determined by phagemid vector sequencing. As sequencing templates, pIT2 phagemids isolated from TOP10 bacteria, containing the sequences of individual selected antibodies, were used. LMB3 (5 'CAG GAA ACA GCT ATG AC 3') and pHEN (5 'CTA TGC GGC CCC ATT CA 3') primers were used in the sequencing process. The obtained sequences were analyzed with the use of programs available on the Mobyle @ Pasteur internet platform (revseq, transeq, needle), ClustalX and Jalview.

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Sequences:

Sequence No. 1a:

> 1F2_CDRH2

SIQAAGPTTD

Sequence No. Ib:

> 1F2_CDRH3

ADWEF

Sequence No. Ic:

> 1F2_CDRL2

CASV

Sequence No.ID:

> 1F2_CDRL3

ALRVPS

Sequence No. 2a:

> 1F5_CDRH2

SILDHGQGTQ

Sequence No. 2b:

> 1F5_CDRH3

EGTML

Sequence No. 2c:

> 1F5_CDRL2

RASR

Sequence No. 2d:

> 1F5_CDRL3

LRASPR

Sequence number 3:

> 1F2

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTF3SYAMSWVR QAPGKGLEWVSSIQAAGPTTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK ADWEFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS QSISSYLNWYOOKPGKAPKLLIYGASVLQSGVPTRFSGSGSGTDFTLTTSSLQPEDFATY YCQQALRVPSTFGQGTKVEIKRAAAHHHHHH

Sequence number 4:

> 1F5

MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSSILDHGQGTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK EGTMLDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRAS QSISSYLNWYQQKPGKAPKLLIYRASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY

YCQQLRAS PRT FGQGT KVEIKRAAAHHHHHH

Claims (3)

Patent claims 1. An antibody of the scFv format, which is an inhibitor of the VEGF-C protein, characterized in that it comprises the amino acid sequence of Seq. No. 3 or Seq. no 4. 2. An antibody of the scFv format which is an inhibitor of the VEGF-C protein as defined in claim 1. 1 for use as a medicament in the prevention and / or treatment of diseases and / or conditions associated with an abnormal amount of VEGF-C protein, including dysfunction of the lymphangiogenesis process. 3. An antibody of the scFv format which is an inhibitor of the VEGF-C protein for use according to claim 1. The method of claim 2, characterized in that the diseases and / or conditions are selected from the following group: lymphatic metastatic tumors, acute myeloid leukemia, acute lymphoblastic leukemia, Kaposi's sarcoma, anti-VEGF-A resistant tumors, bone loss caused by VEGF-C, age-related macular degeneration, diabetic retinopathy, inflammatory diseases associated with excessive VEGF-C levels, including rheumatoid arthritis and psoriasis.