KR20210157688A - Antigen binding fragment platform conjugated with anticancer peptides - Google Patents

Abstract

The present invention relates to an antigen-binding fragment platform for cancer treatment combined with anticancer peptides, wherein the platform includes an antigen-binding fragment-anticancer peptide conjugate. The conjugate has excellent tumor cell targeting ability by the antigen-binding fragment, and it is possible to kill tumor cells by anticancer peptides, and thus the platform can be usefully used for cancer treatment.

Description

Antigen-binding fragment platform combined with anticancer peptide {ANTIGEN BINDING FRAGMENT PLATFORM CONJUGATED WITH ANTICANCER PEPTIDES}

The present invention relates to an antigen-binding fragment platform for cancer treatment combined with an anti-cancer peptide.

Chemotherapy for cancer treatment causes proliferation inhibition and apoptosis of normal cells as well as tumor cells, causes non-tumor specific systemic toxicity and cytotoxicity, and resistance due to long-term use of drugs can induce

On the other hand, antibody drugs have the advantage of inhibiting the proliferation and inducing death of tumor cells without damaging normal cells because they bind to specific antigens on the surface of tumor cells. have. Therefore, antibody drugs that are specific to tumor cells, have high anticancer efficacy, and have few side effects, which can reduce the number of administrations, are positioning themselves as next-generation anticancer agents and have high development value.

Recently, antibody fragments that have a good penetration rate into tissues or tumors have been widely used due to their smaller size compared to intact antibodies. The antibody fragment can be used for biodiagnosis, used for drug delivery by conjugation with a bacterial toxin, or produced as a bispecific antibody with an antibody fragment that binds to cytotoxic T cells and developed as a cancer treatment agent. In fact, various types of antibody fragments have been developed as improved therapeutic antibodies and are being used for clinical use (abciximab, ranibizumab, and certolizumab).

The present inventors completed the present invention by confirming that a conjugate of an anticancer peptide and an antibody fragment has excellent tumor targeting ability and anticancer activity while researching a new cancer therapeutic agent using an antibody fragment.

1. Republic of Korea Patent No. 10-1232454

An object of the present invention is to provide an antigen-binding fragment platform for cancer treatment combined with an anti-cancer peptide that can be used as a cancer treatment agent.

In order to achieve the above object, one aspect of the present invention is (a) an antigen-binding fragment heavy chain; (b) an anticancer peptide module linked to the C-terminus of the antigen-binding fragment heavy chain; And (c) antigen-binding fragment light chain bound to the antigen-binding fragment heavy chain; provides an antigen-binding fragment-anticancer peptide conjugate comprising a.

As used herein, the term "antibody binding fragment (Fab)" refers to an antibody fragment including an antigen binding site, and consists of one variable region of a heavy chain and one variable region of a light chain. . The heavy and light chains constituting the antigen-binding fragment are called Fab Fd and Fab Lc, respectively. Antigen-binding fragments are widely used as therapeutic agents because they are smaller in size than full-length antibodies while maintaining antigen-binding ability and thus easily penetrate into tissues or tumors.

According to one embodiment of the present invention, the Fab Fd and Fab Lc may be covalently linked, such as a disulfide bond, or covalently linked by a peptide linker sequence.

According to one embodiment of the present invention, the antigen-binding fragment heavy chain and the antigen-binding fragment light chain are cetuximab, panitumumab, bevacizumab, pertuzumab, trastuzumab (trastuzumab), rituximab, ofatumumab, obinutuzumab, ipilimumab, ramucirumab, nivolumab and pembrolizumab ( pembrolizumab) may be selected from the group consisting of, preferably cetuximab may be used.

According to one embodiment of the present invention, the antigen-binding fragment imparts targeting ability to the antigen-binding fragment-anticancer peptide conjugate, and can also kill tumor cells by its anticancer effect. For example, when the antigen-binding fragment of cetuximab is used, the conjugate can bind to EGFR1-expressing tumor cells, and can kill tumor cells by the action of the antigen-binding fragment and anticancer peptide.

According to one embodiment of the present invention, the antigen-binding fragment heavy chain and the antigen-binding fragment light chain may have a signal peptide bound to the N-terminus, and the signal peptide has the sequence of SEQ ID NO: 7 to the antigen-binding fragment heavy chain (Fd) (H4). ), the sequence (L1) of SEQ ID NO: 8 may be used for the antigen-binding fragment light chain (Lc) (see FIG. 3A). The signal peptide (SP sequence) serves to promote the extracellular secretion efficiency of the synthesized antigen-binding fragment-anti-cancer peptide conjugate, and when the synthesized antigen-binding fragment-anti-cancer peptide conjugate passes through the endoplasmic reticulum, it acts as a signal peptide peptidase. so that it can be cut accurately.

According to one embodiment of the present invention, the anticancer peptide module includes a protease cleavage sequence (FK), an endosome fusion inducing sequence (S19) and a transcription factor CP2c binding sequence (ACP), and a tumor cell penetration sequence (iRGD) It may further include (FIG. 3).

According to one embodiment of the present invention, the anti-cancer peptide module comprises a protease cleavage sequence represented by SEQ ID NO: 4, an endosome fusion inducing sequence represented by SEQ ID NO: 5, a transcription factor CP2c binding sequence represented by SEQ ID NO: 3 and SEQ ID NO: and a tumor penetrating sequence represented by 6.

In the present invention, the tumor cell penetration sequence (iRGD) is a sequence introduced to introduce ACP into a cell, and can specifically bind to an integrin/neurophilin receptor. ACP bound to the integrin/neurophilin receptor moves into the cell by sendR and induces cell death. By including the iRGD sequence, the antigen-binding fragment-anticancer peptide conjugate of the present invention primarily targets tumor cells with the antigen-binding fragment, and secondarily targets tumor cells with the iRGD sequence.

In the present invention, the protease cleavage sequence (FK) was introduced so that the antigen-binding fragment-anticancer peptide conjugate that entered tumor cells was separated into an antigen-binding fragment (Fab) and transcription factor CP2c-binding sequence (ACP) by protease. . The free ACP may flow into tumor cells and induce cell death.

In the present invention, the endosome fusion inducing sequence (S19) serves to induce the antigen-binding fragment-anticancer peptide conjugate to easily escape from the endosome. The isolated anticancer peptide inhibits the growth of cancer cells and causes cell death.

In the present invention, the transcription factor CP2c binding sequence is a peptide that binds to the transcription factor CP2c, which is a gene frequently expressed in cancer cells, to inhibit the growth of cancer cells and induce cell death. According to one embodiment of the present invention, the anticancer peptide The module may be linked only to the C terminus of the heavy chain of the antigen-binding fragment by the sequence shown in SEQ ID NO: 11.

In the present invention, the cetuximab antigen-binding fragment-anticancer peptide conjugate injected in vivo binds to a specific receptor on the surface of a tumor cell by the antigen-binding fragment, and is introduced into the cell by receptor-mediated endocytosis. At this time, the intracellular influx enhancing effect is shown by the iRGD sequence, and the antigen-binding fragment trapped in the endosome-anti-cancer peptide conjugate according to the inflow is the antigen-binding fragment by truncating the FK sequence by the proteolytic enzyme cathepsin B. and anticancer peptide module (S19-ACP-iRGD). The truncated S19-ACP-iRGD sequence easily escapes endosomes because of the S19 sequence, and the escaped S19-ACP-iRGD sequence kills cancer cells by the action of ACP.

According to one embodiment of the present invention, the antigen-binding fragment-anti-cancer peptide conjugate can be prepared by expressing the antigen-binding fragment heavy chain-anti-cancer peptide and the antigen-binding fragment light chain, respectively, and then covalently linking them. For example, when ExpiCHO-S™ cells are transformed with an antigen-binding fragment heavy chain-anticancer peptide expression vector and antigen-binding fragment light chain expression vector, a covalent bond is formed between the expressed antigen-binding fragment heavy chain-anticancer peptide and the antigen-binding fragment light chain. and can be obtained in the form of a conjugate.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the antigen-binding fragment-anticancer peptide conjugate as an active ingredient.

The present inventors confirmed that tumor growth was significantly inhibited as a result of injecting cetuximab antigen-binding fragment-anticancer peptide conjugate into tumor-forming mice.

The pharmaceutical composition of the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceuticals.

The pharmaceutical composition according to the present invention may be formulated and used in the form of a sterile injection solution according to a conventional method. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, and freeze-dried preparations. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a 'pharmaceutically effective amount' means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level includes the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The antigen-binding fragment-anti-cancer peptide conjugate according to an embodiment of the present invention has excellent tumor cell targeting ability by the antigen-binding fragment and can kill tumor cells by the anti-cancer peptide, so it can be usefully used for cancer treatment.

1 schematically shows the structure of a cetuximab antigen-binding fragment-anticancer peptide conjugate according to an embodiment of the present invention.
2 shows the results of confirming the expression of various cetuximab antigen-binding fragment-anticancer peptide conjugates.
Figure 3 shows the structure (A) of the cetuximab antigen-binding fragment-anticancer peptide conjugate according to an example of the present invention and the result of checking the expression (B).
FIG. 4 shows the mass spectrometry results of the cetuximab antigen-binding fragment-anticancer peptide conjugate shown in FIG. 3A .
5 shows the results of confirming the EGFR1 binding ability of the cetuximab antigen-binding fragment-anticancer peptide conjugate by ELISA (A) and FACS analysis (B).
6 shows the results of confirming the anticancer activity of the cetuximab antigen-binding fragment-anticancer peptide conjugate through a cell experiment.
7 shows the results of confirming the anticancer activity of the cetuximab antigen-binding fragment-anticancer peptide conjugate in mice with tumors.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Preparation Example 1: Cet Fab-ACP fusion protein expression

1-1. Cet Fab-ACP fusion protein expression vector construction

A vector having the structure shown in FIG. 1 was prepared as follows. In Figure 1, D1 is cetuximab (cetuximab) Fab (antigen binding fragment, antigen-binding fragment) Fd (SEQ ID NO: 1) to the ACP module is fused to the N and C terminus, D2 is Lc of cetuximab Fab ( In SEQ ID NO: 2), the ACP module is fused to the N and C terminals. Fd and Lc in the Fab of cetuximab were used by humanizing some sequences (SEQ ID NOs: 1 and 2). The ACP module includes CB (cathepsin B cleavage site), EED (endosomal escape domain), ACP (CP2c binding peptide; SEQ ID NO: 3) and TAT (transactivator of transcription) sequences, and as a signal sequence, Fd of D1 has an H4 sequence ( SEQ ID NO: 7), L1 sequence (SEQ ID NO: 8) was used for Lc of D1.

The D1 or D2 coding sequence was cloned into the pUC57 vector by request of Genewiz (pUC57 D1 and pUC57 D2). The synthesized D1 or D2 coding sequence was subjected to PCR with forward and reverse primers (SEQ ID NOs: 12 to 15), respectively, and the PCR product was ^{TA cloned into pcDNA™ 3.4-TOPO ®} vector (Invitrogen, CA, USA). After mixing Top10 cells and the vector, heat shock was applied at 42° C. to transform them, incubate them in SOC medium for 1 hour, and then spread them on a solid medium containing ampicillin antibiotics and grow overnight. The next day, 8 colonies were selected and colony PCR was performed with CMV forward primer and pcDNA™ 3.4 reverse primer. After electrophoresis of the PCR product on an agarose gel, colonies with the same band size were selected, and plasmid DNA was isolated. Thereafter, the plasmid DNA was sequenced to confirm that the cloning was performed properly.

Expression vectors (ΔB, ΔX) in which the ACP module was removed from the N-terminus and/or C-terminus were constructed as follows.

Each of the pcDNA ^TM 3.4-TOPO ^® D1 and pcDNA ^TM 3.4-TOPO ^® vectors prepared above was inactivated after treatment with BamHI or XhoI, and the vector was ligated again with T4 ligase. After mixing each of pcDNA ^TM 3.4-TOPO ^® D1 and pcDNA ^TM 3.4-TOPO ^® vectors in ΔBamHI and ΔXhoI state with Top10 cells, heat shock was applied at 42° C. It was spread on a solid medium and grown overnight. The next day, 8 colonies were selected and colony PCR was performed with CMV forward primer and pcDNA™ 3.4 reverse primer. The PCR product was electrophoresed on an agarose gel, and colonies with matching band sizes were selected to isolate plasmid DNA. The isolated plasmid DNA was treated with a restriction enzyme, and the product was electrophoresed to select a plasmid with the correct band size, and finally ΔB and ΔX expression vectors were prepared. The ΔX expression vector was treated with BamHI, and the above process was repeated to construct a ΔBX expression vector.

Hereinafter, expression vectors from which the ACP module has been removed from the N-terminus are referred to as D1ΔB and D2ΔB, and expression vectors from which the ACP module has been removed from the C-terminus are referred to as D1ΔX and D2ΔX. Expression vectors in which the ACP module has been removed at both the N-terminus and C-terminus are described as D1ΔBX and D2ΔBX.

1-2. Recombinant protein expression

ExpiCHO-S™ cells (Invitrogen) were prepared at a concentration of ⁶ ×10 6 cells/mL in a 125 mL non-baffled flask. ExpiFectamine™ CHO Reagent (Invitrogen) (3.2 μl/medium mL) and both D1 and D2 expression vectors were diluted in OptiPRO™ SFM (40 μl/medium mL), mixed and allowed to stand for 1 minute, and then evenly sprayed on the flask. . After culturing ExpiCHO-S™ cells for about 20 hours, ExpiCHO™ Enhancer was added to overexpress D1 and D2, and ExpiCHO™ Feed was added to further culture for 13 days.

After incubation, only the supernatant was recovered by centrifugation, and the supernatant was filtered with a 0.3 μm filter syringe, mixed with KappaSelect resin (Capacity: 15 mg Fab/ml; GE Healthcare), and the resin and antibody at 4°C for 2 to 4 hours. was induced to bind. KappaSelect was washed several times with PBS (Phosphate buffered saline) pH 7.4 (0.01 M phosphate buffer, 0.0027 M KCl, 0.14 M NaCl), and an elution buffer (0.1 M glycine buffer, pH 2.5 to 3.0) was added to elute the antibody. . Thereafter, a neutralization buffer (1 M Tris, pH 7.5 to 8.5) was immediately added to adjust the pH of the eluate to pH 7.6.

A total of 16 protein combinations comprising each of the four constructs of D1 and D2 (full length, ΔB, ΔX and ΔBX) were expressed by the above method. Thereafter, each protein combination was loaded on a 12% SDS-PAGE gel to perform electrophoresis, and the protein band was identified by staining and decolorizing the SDS-PAGE gel.

As a result, as shown in FIG. 2 , only the D1ΔB+D2ΔBX combination (a protein in which the ACP module was fused only at the C terminus of Fd) among 16 protein combinations was successful in expression. An attempt was made to use this combination as a Cet Fab-ACP fusion protein, but only a portion of the fusion protein was expressed. As a result of molecular weight measurement using MALDI-TOF/TOF, D1ΔBX and D1ΔB should have a difference of about 3988 Da, which was only 368 Da. It was confirmed to be cleaved from phosphorus CB (GFLG (392 Da)) (experimental results not shown). Therefore, the fusion protein and ACP module were modified as described in 1-3 below.

1-3. Construction of the final Cet Fab-ACP fusion protein

A new ACP module (FK+S19+ACP+iRGD) was cloned into the restriction enzyme XhoI site in the D1ΔB vector cloned into pcDNA ^{TM 3.4, which is referred to as D1' (FIG. 3A).} The new ACP module (FK+S19+ACP+iRGD) is changed to the FK sequence (SEQ ID NO: 4) because the CB sequence of FIG. 1 cannot be used, and the EED is an S19 sequence (SEQ ID NO: 5) that is likely to have a better effect , The TAT sequence was entirely changed to iRGD (SEQ ID NO: 6), which seems to have a better effect.

As a result of changing the ACP module of D1ΔB, it was confirmed that all proteins were expressed as shown in FIG. 3B, so the D1′+D2ΔBX combination was selected as the final Cet Fab-ACP fusion protein.

In Figure 3A, FK is a linker sequence that can be cleaved by an intracellular protease to release a drug (https://pubmed.ncbi.nlm.nih.gov/27089329/), and S19 is an endosome to a specific target cell. is a sequence that fuses , and iRGD is a tumor-penetrating peptide.

4 is a result of mass spectrometry of the produced Cet Fab-ACP fusion protein (D1'+D2ΔBX).

Experimental Example 1: Confirmation of EGFR binding ability of Cet Fab-ACP fusion protein

1-1. Enzyme-linked immunosorbent assay

EGFR1 dissolved in carbonate/bicarbonate buffer was aliquoted into an immunoplate, and stored overnight at 4° C. to coat each well of the plate. The next day, the buffer solution was removed and the plate was washed several times with 100 μl of PBST (0.05% tween20). 200 μl of BSA dissolved in PBS was placed in each well and left at room temperature for 1 hour to block the remaining portion to which EGFR1 was not bound. After removing the solution from each well, 100 μl of Cet Fab-ACP protein diluted to 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml and 0.625 ng/ml was added to each well and reacted at room temperature for 1 hour. made it After the reaction was completed, the solution was removed, and the plate was washed several times with 100 μl of PBST (0.05% tween20). After washing, 100 μl of a primary antibody (Anti-Human IgG in goat) diluted 1/1000 in PBS was added to each well and reacted at room temperature for 1 hour. After removing the solution, wash the plate several times with 100 μl of PBST (0.05% tween20), and then 100 μl of secondary antibody (Anti-Goat IgG+HRP) diluted 1/3000 in 1% BSA (in PBS) Each was added and reacted at room temperature for 1 hour. After the reaction was completed, the solution was removed, and the plate was washed several times with 100 μl of PBST (0.05% tween20). Add 100 μl of TMB to each well of the plate and wait for a few seconds to several minutes until the color changes to blue. When the color started to change, 100 μl of 1M HCl was added to stop the reaction, and absorbance was measured at 450 nm.

As a result of the measurement, as shown in A of FIG. 5 , it was confirmed that there was no change in the EGFR1 binding ability of cetuximab even when the ACP module was bound to the Fab of cetuximab.

1-2. Flow cytometer titration

A431 cells, a cell line overexpressing EGFR1, were cultured and 4x10 ⁶ cells were suspended in 2 ml of FACS buffer (PBS, 2% FBS), and 50 μl was dispensed into each well of a v-shaped bottom 96-well plate. A control group, cetuximab, and Cet Fab-ACP protein diluted to a range of 0 to 25 nM were dispensed in each well of the plate containing 50 μl of cells, mixed well with the cells, and reacted at 4° C. for 20 minutes. made it Thereafter, the supernatant was removed by centrifugation at 300 g at 4°C. The cells were resuspended in 200 μl of PBS, centrifuged at 4° C. at 300 g, and the washing process of removing the supernatant was repeated twice. 100 μl of anti-human IgG (Fab-specific)-FITC antibody (sigma, F5512) diluted 1/32 in PBS was aliquoted into each well and mixed, followed by reaction at 4° C. for 20 minutes. After the reaction, the supernatant was removed by centrifugation at 300 g at 4°C. The cells were resuspended in 200 μl of PBS, centrifuged at 4° C. at 300 g, and the washing process of removing the supernatant was repeated twice. Thereafter, the cells were suspended in 300 μl of FACS buffer, and the FITC level was measured with a Flow Cytometer (BD FACSCalibur) and compared with cetuximab as a control.

As a result, as shown in FIG. 5B , the Cet Fab-ACP protein exhibited a similar level of EGFR1 binding ability to cetuximab and Cetuximab Fab, indicating that there was no significant change in cancer targeting properties. The above result means that the fusion of ACP to Cet Fab can effectively deliver ACP to cancer cells without significantly affecting the targeting ability.

Experimental Example 2: Confirmation of anticancer effect of Cet Fab-ACP fusion protein

2-1. cell experiment

Merck's Erbitux was ^{mixed with Pierce TM} 's Protein A agarose (Capacity: 15 mg Fab/ml) resin and left at 4°C for 2-4 hours to induce resin binding. Thereafter, Protein A agarose was washed several times with PBS pH 7.4 (0.01 M phosphate buffer, 0.0027 M KCl, 0.14 M NaCl), and cetuximab was eluted with an elution buffer (0.1 M glycine buffer, pH 2.5-3.0). Neutralization buffer (1 M Tris, pH 7.5-8.5) was immediately added to the eluate to adjust the pH to 7.6, and concentrated to a concentration of 200 uM with a centrifugal filter. Cet Fab and Cet Fab-ACP proteins were prepared as described above, and the ACP module (FK+S19+ACP+iRGD) was synthesized in Anygen (Gwangju).

The A431 cancer cell line was cultured by dispensing 2,500 each into each well of a 96-well plate (10% FBS, RPMI-1640), and the medium was removed the next day and washed once with PBS. Thereafter, 90 μl of 0% FBS and RPMI-1640 medium was exchanged, and each sample stored at 200 uM was serially diluted by ½, and 10 μl of each well containing cells was treated. Cells were cultured in an incubator at 37° C. for 5 days, and when 10 μl of CCK-8 solution was treated in each well, and the color changed after 5 hours, absorbance was measured at 450 nm.

As a result of the measurement, as shown in FIG. 6 , it was found that ACP effectively kills cancer cells in cell experiments, but cetuximab, Cet Fab and Cet Fab-ACP proteins hardly kill cells.

2-2. mouse experiment

5-7 week old female nude BALB/c mice (nu/nu) (18 to 20 g) were purchased from Orient Bio, and bred in an aseptic breeding room with a light-dark cycle for 12 hours. Food and water were provided ad libitum. A431 tumor cells ^{were cultured at a concentration of 1x10 7} cells/animal per animal, harvested, and suspended in 100 μl of Matrigel (Corning). The suspension was injected subcutaneously into the left flank of the mice, and when the tumors settled after about 2 weeks, the mice were randomly divided into 4 groups (n=5/group). When the size of the tumor reached 150 mm 3 , the samples corresponding to each group were intravenously injected by 3 nmol every 3 days. During the experiment period, the size of the tumor was measured with a caliper, and the tumor volume was calculated as follows: (short ^{axis 2} x long axis)/2. In each experimental group, the tumor size was calculated except for mice having the minimum and maximum values.

As a result of the experiment, as shown in FIG. 7 , the tumor size grew rapidly in the order of control group (Mock) > ACP administration group > Cet Fab-ACP administration group > cetuximab administration group, and cetuximab, which was not seen in in vitro experiments The effect of Mab was clearly demonstrated in vivo. In the Cet Fab-ACP administration group, the tumor size slightly increased compared to the cetuximab administration group, but it was found that the tumor growth rate was significantly reduced compared to the ACP administration group, which had an excellent anticancer effect in the in vitro experiment. This means that the ACP module itself does not have the ability to specifically target tumors in vivo. However, it can be seen that the Cet Fab-ACP protein using Cet Fab as a messenger targets the tumor by Cet Fab, so that ACP can exert its effect.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> ANTIGEN BINDING FRAGMENT PLATFORM CONJUGATED WITH ANTICANCER PEPTIDES <130> 2020-I109_P20U10C0983 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> cetuximab Fab (antigen binding fragment) Fd <400> 1 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 <210> 2 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> cetuximab Fab (antigen binding fragment) Lc <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Ala Glu 210 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> ACP (CP2c binding peptides) <400> 3 Asn Tyr Pro Gln Arg Pro 1 5 <210> 4 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> FK sequence <400> 4 Phe Lys One <210> 5 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> S19 sequence <400> 5 Pro Phe Val Ile Gly Ala Gly Val Leu Gly Ala Leu Gly Thr Gly Ile 1 5 10 15 Gly Gly Ile Gly Ser Gly 20 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> iRGD sequence <400> 6 Cys Arg Gly Asp Lys Gly Pro Asp Cys 1 5 <210> 7 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> H4 sequence <400> 7 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser <210> 8 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> L1 sequence <400> 8 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Ser Gly Ala Arg Cys 20 <210> 9 <211> 295 <212> PRT <213> Artificial Sequence <220> <223> SP-cetuximab Fab Fd-(GSS)2-ACP module=D1' <400> 9 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Gly Ser Gly Gln Val Gln Leu Lys Gln Ser Gly Pro Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Ser Ile Thr Cys Thr Val Ser Gly 35 40 45 Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly 50 55 60 Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp 65 70 75 80 Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser 85 90 95 Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr 100 105 110 Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Gly Ser Leu Asp Gly Gly Ser Gly Gly Ser 245 250 255 Phe Lys Pro Phe Val Ile Gly Ala Gly Val Leu Gly Ala Leu Gly Thr 260 265 270 Gly Ile Gly Gly Ile Gly Ser Gly Asn Tyr Pro Gln Arg Pro Cys Arg 275 280 285 Gly Asp Lys Gly Pro Asp Cys 290 295 <210> 10 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> SP-cetuximab Fab Lc=D2ΔBX <400> 10 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Ser Gly Ala Arg Cys Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 20 25 30 Val Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg 35 40 45 Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr 50 55 60 Asn Gly Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser 65 70 75 80 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Ser Ile Asn Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys 100 105 110 Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu 115 120 125 Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Ala Glu Cys Gly 225 230 235 240 Ser Leu Glu <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> (GSS)2 linker sequence <400> 11 Gly Gly Ser Gly Gly Ser 1 5 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D1 forward primer <400> 12 ggtatggact ggacctggag gatt 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D1 reverse primer <400> 13 tcactcgagt ccctgaggcc ttcg 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D2 forward primer <400> 14 ggtatggaca tgagggtccc tgct 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D2 reverse primer <400> 15 tcactcgagt ccttggggcc gtct 24

Claims (9)

(a) an antigen-binding fragment heavy chain;
(b) an anticancer peptide module linked to the C-terminus of the antigen-binding fragment heavy chain; and
(c) an antigen-binding fragment light chain bound to the antigen-binding fragment heavy chain;
The anticancer peptide module is an antigen-binding fragment comprising a protease cleavage sequence, an exosome fusion inducing sequence and a transcription factor CP2c binding sequence - an anticancer peptide conjugate.
The antigen-binding fragment-anticancer peptide conjugate according to claim 1, wherein the antigen-binding fragment heavy chain and the antigen-binding fragment light chain have a signal peptide bound to the N-terminus.
The method of claim 1, wherein the antigen-binding fragment heavy chain and the antigen-binding fragment light chain are cetuximab, panitumumab, bevacizumab, pertuzumab, trastuzumab, rituximab, ofatumumab, obinutuzumab, ipil An antigen-binding fragment-anticancer peptide conjugate selected from the group consisting of rimumab, ramucirumab, nivolumab and pembrolizumab.
The antigen-binding fragment-anticancer peptide conjugate according to claim 1, wherein the binding of (c) is a covalent bond.
The antigen-binding fragment-anticancer peptide conjugate of claim 1, wherein the anticancer peptide module further comprises a tumor cell penetration sequence.
The method according to claim 5, wherein the anticancer peptide module is represented by a protease cleavage sequence represented by SEQ ID NO: 4, an exosome fusion inducing sequence represented by SEQ ID NO: 5, a transcription factor CP2c binding sequence represented by SEQ ID NO: 3, and SEQ ID NO: 6 The antigen-binding fragment comprising a tumor-penetrating sequence to be-anti-cancer peptide conjugate.
The antigen-binding fragment-anti-cancer peptide conjugate according to claim 1, wherein the anti-cancer peptide module is linked to the C-terminus of the antigen-binding fragment heavy chain by a linker sequence.
The antigen-binding fragment-anticancer peptide conjugate according to claim 7, wherein the linker sequence comprises the sequence shown in SEQ ID NO: 11.
A pharmaceutical composition for preventing or treating cancer comprising the antigen-binding fragment-anticancer peptide conjugate according to claim 1 as an active ingredient.

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