TW201533059A - Anti-dengue virus nonstructural protein 1 monoclonal antibody and use thereof - Google Patents

Abstract

The invention relates to an anti-dengue virus nonstructural protein 1 (NS1) monoclonal antibody and a use thereof. The monoclonal antibody comprises (a) a heavy chain variable region CDR having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and (b) a light chain variable region CDR having the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. The monoclonal antibody can be used alone or in combination with one or more acceptable carriers in medicine for manufacturing drugs to treat dengue hemorrhagic fever.

Description

Monoclonal antibody against dengue virus non-structural protein 1 and use thereof

The present invention relates to a monoclonal antibody against dengue virus non-structural protein 1 and use thereof, in particular to a monoclonal antibody or antigen-binding fragment thereof, which can specifically recognize a specific amino acid in non-structural protein 1. Sequence to treat hemorrhagic dengue caused by different serotypes of dengue virus.

According to Dengue virus (DENV), there are four different serotypes, the first type of dengue virus (DENV-1), the second type of dengue virus (DENV-2), and the third type. Dengue virus (DENV-3) and type 4 dengue virus (DENV-4). The infection of dengue virus mainly occurs in tropical and subtropical regions, and the clinical symptoms of patients include mild dengue fever and more severe hemorrhagic fever (DHF) and dengue shock syndrome (dengue shock syndrome). ). When the human body is infected with one of the above-mentioned serotypes of dengue virus, the body will have a long-term immune effect on the infection of the same serotype of dengue virus, but the protection of different serotypes of dengue virus in vivo has only a short-term effect. Once the human body re-infects different serotypes of dengue virus, the likelihood of causing severe dengue fever in patients will increase significantly, leading to higher mortality. However, good antiviral drugs and vaccines have not yet been developed for use.

Many dengue vaccine development strategies are directed to dengue membrane proteins (envelope) Protein) or dengue virus non-structural protein. Although antiviral envelope protein antibodies are the major neutralizing antibodies during dengue virus infection, this antibody also causes an effect of antibody-dependent enhancement (ADE). In the development of dengue vaccines, the antibody-dependent potentiating effect causes many problems, and the existing antibodies may cause more serious diseases. In contrast, the anti-dengue virus non-structural protein 1 (NS1) antibody does not cause an antibody-dependent potentiation effect because it destroys infected cells by activating complement. However, antibodies induced by dengue virus non-structural protein 1 may cross-react with endothelial cells and platelets, affecting coagulation, leading to bleeding (blood bleeding). The side effects of autoimmune effects caused by such vaccines remain unresolved.

According to previous studies by the present inventors, anti-dengue virus non-structural protein 1 (NS1) antibodies cross-react to human endothelial cells as well as platelets, leading to endothelial cell apoptosis and abnormal platelet aggregation mechanisms. From the proteomic sequence analysis, the C-terminal sequence of the NS1 protein (amino acid 271-352) is highly similar to the autoantigen. Considering the safety of NS1 as a vaccine, the inventors removed this fragment and produced it. A protein named ΔC NS1 was added, and the fragment removed was also replaced with a protein called DJ NS1 by the C-terminus of the Japanese encephalitis virus (JEV) NS1 protein. Please refer to the Republic of China Invention Patent Publication No. 201210615, which discloses a "dengue vaccine, a pharmaceutical composition containing the dengue vaccine, a nucleotide sequence, and an antibody composition", and the dengue vaccine of the present invention can achieve reduction and endothelium The effect of cross-reaction between cells and platelets shortened the bleeding time; from the above results, both anti-ΔC NS1 and anti-DJ NS1 antibodies can provide protection in cells infected with dengue virus and in mice. However, the treatment of patients infected with dengue virus Treatment, there is still no effective way.

Therefore, how to develop an effective anti-dengue virus treatment method without causing an antibody-dependent enhancement effect and autoimmunity has become a direction that the inventors have thought.

Now, the inventor has made many improvements in the actual implementation of the above-mentioned anti-dengue virus method. Therefore, it has been improved by its rich professional knowledge and years of practical experience. The present invention has been made.

The main object of the present invention is to provide a monoclonal antibody against dengue virus non-structural protein 1 and the use thereof, which can specifically recognize the specific amino acid sequence of non-structural protein 1; Treatment of hemorrhagic dengue caused by different serotypes of dengue virus.

In order to achieve the above-mentioned object, the present invention provides an anti-dengue virus non-structural protein 1 monoclonal antibody or antigen-binding fragment thereof, and the monoclonal antibody system comprises (a) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID. a heavy chain variable region of the sequence of the complementarity determining region of SEQ ID NO: 4, SEQ ID NO: 5, The heavy chain variable region comprises the sequence of SEQ ID NO: 7, and the light chain variable region comprises the sequence of SEQ ID NO: 8.

The present invention also provides a method for preparing a pharmaceutical composition for treating hemorrhagic dengue using an anti-dengue virus non-structural protein 1 monoclonal antibody, comprising: providing a monoclonal antibody and one or more pharmaceutically acceptable carriers; and a mixing single The antibody and the pharmaceutically acceptable carrier are used to form a pharmaceutical composition useful for treating hemorrhagic dengue.

In one embodiment of the present invention, the dengue virus non-structural protein 1 may be derived from a serotype first type Deng virus, a second type dengue virus, a third type dengue virus or a fourth type dengue disease. One of the poisons. Furthermore, the monoclonal antibody can specifically recognize the 190-200 amino acid sequence of the dengue virus non-structural protein 1, and the sequence thereof has, for example, at least 85% sequence identity with SEQ ID NO: 9, and even more, The 90-270 amino acid sequence of the virion non-structural protein 1 has, for example, at least 85% sequence identity to SEQ ID NO: 10. Preferably, the monoclonal antibody system specifically recognizes the dengue virus non-structural protein 1 derived from the second type of dengue virus.

Thereby, the monoclonal antibody of the present invention can recognize four different serotypes of dengue virus, but does not recognize the C-terminal sequence of the dengue virus NS1 and has low cross-reactivity to uninfected human endothelial cells; Since the anti-system destroys the infected cells by activating complement, it is possible to avoid the absence of an antibody-dependent potentiating effect in the individual.

First Figure: Single antibody specificity test chart of a preferred embodiment of the present invention

Second Figure: Cell lysis test chart of a preferred embodiment of the present invention

Third figure: a mouse bleeding time test chart according to a preferred embodiment of the present invention

Figure 4: Test diagram of mouse bleeding time in the second preferred embodiment of the present invention

Fifth: NS3 immunohistochemical staining map of a preferred embodiment of the present invention

Figure 6: Quantification map of NS3 performance in accordance with a preferred embodiment of the present invention

The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

The invention relates to a monoclonal antibody against dengue virus non-structural protein 1 (NS1) and the use thereof, wherein the monoclonal antibody specifically recognizes the 90-270 amino acid sequence of the non-structural protein 1 It is used to treat hemorrhagic dengue caused by four serotypes of dengue virus; and this monoclonal anti-system destroys infected cells by activating complement, and thus does not cause an antibody-dependent enhancing effect.

Anti-dengue virus non-structural protein 1 monoclonal antibody or antigen-binding sheet thereof In the paragraph, the monoclonal antibody system comprises (a) a heavy chain variable region comprising the complementarity determining region (CDR) sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and (b) comprising SEQ ID NO: 4, the light chain variable region of the complementarity determining region (CDR) sequence of SEQ ID NO: 5 and SEQ ID NO: 6; the heavy chain variable region comprising the sequence of SEQ ID NO: 7, and the light chain variable The lineage includes the sequence of SEQ ID NO: 8; the dengue virus non-structural protein 1 is derived from serotype type 1 dengue virus (DENV-1), type 2 dengue virus (DENV-2), and third One of dengue virus (DENV-3) or type 4 dengue virus (DENV-4); the above-mentioned individual strain system specifically recognizes the 190-200 amino acid sequence of dengue virus non-structural protein 1, Its sequence has, for example, at least 85% sequence identity to SEQ ID NO: 9, and even more, the 90-270 amino acid sequence of dengue virus non-structural protein 1 has, for example, at least 85% of SEQ ID NO: 10. Sequence identity. Preferably, the monoclonal antibody system specifically recognizes the dengue virus non-structural protein 1 derived from the second type of dengue virus.

Preparation of treatment hemorrhage using anti-dengue virus non-structural protein 1 monoclonal antibody A method for the medical composition of dengue fever comprising: providing a monoclonal antibody and one or more pharmaceutically acceptable carriers; and mixing the monoclonal antibody and the pharmaceutically acceptable carrier to form a medicament for treating hemorrhagic dengue Composition. The dengue virus non-structural protein 1 is derived from serotype type 1 dengue virus (DENV-1), type 2 dengue virus (DENV-2), type III dengue virus (DENV-3) or One of the four types of dengue virus (DENV-4); and, the above-mentioned individual strain system specifically recognizes the 190-200 amino acid sequence of dengue virus non-structural protein 1, and it is worth noting that dengue virus is not The 190-200 amino acid sequence of structural protein 1 is a highly conserved region, The sequence has, for example, at least 85% sequence identity to SEQ ID NO: 9, and more particularly, the 90-270 amino acid sequence of dengue virus non-structural protein 1 has, for example, at least 85% of the sequence of SEQ ID NO: Identity. Preferably, the monoclonal antibody system specifically recognizes the dengue virus non-structural protein 1 derived from the second type of dengue virus.

The inventors have invented a single document that recognizes four different serotypes of dengue virus. Strain 2E8, this strain antibody does not recognize the C-terminal sequence of dengue virus NS1 (the 271-352 amino acid sequence) and the ability to cross-react with human endothelial cells that are not infected with dengue virus is lower than that of anti-DENV NS1 antibody. This monoclonal antibody binds to the NS1 exhibited by the infected cells and activates complement to lyse the infected cells. The present inventors further tested whether the monoclonal antibody can provide a protective effect to an infected individual, and the experimental results show that the monoclonal antibody can effectively reduce the prolonged bleeding time of the mouse caused by the infection of dengue virus and the dengue virus antigen NS3. The performance of locally infected tissues.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

Experiment 1: Preparation of anti-dengue virus NS1 monoclonal antibody

First, the present inventors isolated a spleen cell and a myeloma cell line from a mouse immunized with a dengue virus non-structural protein 1 recombinant protein (containing a 90-352 amino acid sequence), and collected the obtained result. Hybridoma cells, and antibodies that are specific for dengue virus non-structural protein 1 are detected and screened by an immunological method such as enzyme-linked immunosorbent assay (ELISA) to select corresponding antibodies. The fusion tumor cells are used as a source of monoclonal antibodies. The preparation of the fusion tumor cells and the screening techniques of the subsequent antibodies are those of ordinary skill in the art to which the present invention pertains, so there are not many Make a statement. Among the obtained monoclonal antibodies, a single antibody of dengue virus non-structural protein 1 capable of simultaneously discriminating four different serotypes was named 2E8.

Experiment 2: Evaluation of the protective effect of anti-dengue virus NS1 monoclonal antibody 2E8 in cell experiments

<virus>

The dengue virus DENV2-16681 was isolated from a Thai patient with hemorrhagic dengue fever (DHF) and cultured in the laboratory for study.

<Cell culture>

Human microvascular endothelial cells (HMEC-1) were subcultured in endothelial cell growth medium M200. The baby hamster kidney cell BHK-21 line was cultured in Dulbecco's modified Eagles medium (DMEM) and used for virus amplification.

Enzyme linked immunosorbent assay

In order to confirm that monoclonal antibody 2E8 to recognize NS1 region, the entire length of 2 μ g / ml of dengue virus NS1 protein (NS1), △ C NS1 (NS1 removal of the C-terminal amino acid fragment 271-352), full-length JEV NS1 protein (JEV NS1) and JD NS1 (N-terminal amino acid 1-270 is JEV NS1 and C-terminal amino acid 271-352 is a protein of DENV NS1) dissolved in 0.0151 M sodium bicarbonate (pH 9.6). In the coating buffer, each well of the ELISA 96-well plate was covered with 100 μl of NS1, ΔC NS1, JEV NS1, and JD NS1 as antigens, and placed at 4 ° C overnight. . Thereafter, 200 μl of a buffer containing 5% bovine serum albumin (BSA) was added to each well for blocking at 4 ° C; the next day, with 0.05% Tween-20 in PBS (PBST) rinsing each well three times, and then each was added 2 μ g / ml monoclonal antibody 2E8 to each well, is placed on the role of 4 ℃. The next day, after rinsing each well three times with PBST, 100 μl of peroxidase-conjugated anti-mouse IgG was added and allowed to stand at room temperature for 2 hours; then washed three times with PBST. 100 μl of 2,2'azinobis 3-ethylbenzthiaoline sulfonic acid (ABTS) was added to each well, and the _absorbance of OD _{405 nm} was judged by a microplate analyzer.

Please refer to the first figure for the specificity test of individual antibodies according to a preferred embodiment of the present invention. In the figure, the X axis represents the antibody titer of the monoclonal antibody 2E8, and the Y axis represents the absorbance value; the experimental results show that the monoclonal antibody 2E8 only recognizes DENV NS1 and ΔC NS1, but the JD NS1 cannot be recognized. And JEV NS1, indicating that the monoclonal antibody 2E8 specifically recognizes the 90-270 amino acid sequence of dengue virus NS1.

Furthermore, this monoclonal antibody not only binds to the infected cells but also shows NS1, and activates complement to lyse infected cells. As shown in the second figure, human endothelial cells are plated on the bottom of a 96-well plate and infected with MOI=10 virus. After 72 hours, add isotype IgG1 (negative control), monoclonal antibody 2E8, control IgG (control IgG), anti-DJ NS1 antibody or anti-DENV NS1 antibody and let stand at 4 ° C for one hour, then add complement or The cell culture solution (PRF-M200) was allowed to stand at 37 ° C for 4 hours. Finally, the supernatant was taken out and the substrate of lactate dehydrogenase was added, and the absorbance of OD490nm was judged by a microplate analyzer to indicate the release amount of lactate dehydrogenase; if the cells were lysed, a higher amount would be released. Lactate dehydrogenase thus increases the absorbance. The results showed that the monoclonal antibody 2E8 disrupted the infected cells by activating complement and lysed the infected cells.

Experiment 3: Evaluation of the therapeutic effect of monoclonal antibody 2E8 in mice

<Animal Mode>

The 1 × 10 ⁷ PFU DENV2-16681 viral amount intradermal (subcutaneous, sc) injecting infected STAT1 ^{- / -} mice on the first day after intraperitoneal infection (intraperitoneally, ip) injection of 150 μ g of the isotype IgG1 (negative control) or different doses (50,100,150 μ g) monoclonal antibody 2E8 (mAb 2E8), and is measured in tail bleeding time in mice on the third day after infection; or 4 × 10 ⁸ PFU DENV2-16681 virus The amount of wild type C57BL/6 mice was infected by intradermal injection. The first day of intraperitoneal injection of 200 μ g, respectively after infection isotype IgG1 (negative control), different doses (50,100,200 μ g) monoclonal antibody 2E8 (mAb 2E8), 200 μ g of control IgG (control IgG), or 200 μ g anti DJ NS1 antibody (anti-DJ NS1) (positive control group), measured on the third day after infection mice were sacrificed tail bleeding time in mice.

<Mouse tail bleeding time test>

The bleeding time test was performed under the condition of cross-cutting of 3-mm tail end, and blood drops were collected by filter paper every 30 seconds. When the diameter of the blood drop was less than 0.1 mm, the time was recorded as the bleeding time.

As shown in the third and fourth figures, dengue virus (DENV2-16681) caused a significant increase in bleeding time in mice, whereas administration of monoclonal antibody 2E8 significantly reduced the prolongation of mice caused by infection with dengue virus. Bleeding time.

<Immunohistochemical staining>

NS3 of dengue virus can be used as an indicator of dengue virus replication, and the amount of dengue virus antigen NS3 in locally infected skin tissue is detected by immunohistochemical staining. The mouse skin tissue was embedded in paraffin and the sections were placed on a glass slide, which was then dewaxed with xylene and gradient alcohol (100%, 95%, 85%, 70%, 50%). First the slides placed in a slice containing a 2N HCl solution for 20 minutes, add containing 20 μ g / ml proteinase K of TE buffer (50mM Tris Base, 1mM EDTA, and 0.5% Triton X-100, pH 8.0 In the case, it was allowed to act at room temperature for 20 minutes. Thereafter, the slide was placed in PBS containing 3.5% H ₂ O ₂ for 15 minutes to inhibit endogenous peroxidase activity, and blocked by PBST containing 5% BSA for 30 minutes. Primary antibody and secondary antibody were diluted with Ab diluent, and the slide was first added with primary antibody (rabbit anti-DENV NS3, 1:500) at 4 °C; then biotin-labeled donkey anti-rabbit was added. The secondary antibody was allowed to act for two hours at room temperature. After rinsing the slides three times with PBST, the slides were treated with HRP-conjugated streptavidin for 15 minutes at room temperature, then treated with an AEC substrate kit, and the nuclei were stained with hematoxylin for 10 seconds at room temperature. Finally, the staining results were observed using an optical microscope. The local skin tissue of each mouse was selected from six microscopic fields (400X) to quantify the number of cells expressing NS3. Among them, mice infected with medium were used as a negative control group.

Results, please refer to the fifth and sixth figures, local infection of skin tissue utilization The DENV NS3 antibody was stained (red) and the nuclei were stained with hematoxyline (blue), and the red arrow indicated cells expressing NS3 (magnification: 40-fold). In the group given the isotype IgG1 of dengue virus-infected mice, the endothelial cells of the blood vessels and the cells of the dermis showed NS3 antigen; and the anti-DJ NS1 (anti-DJ NS1) antibody or the monoclonal antibody 2E8 (mAb 2E8) was administered. Group, NS3 showed less performance on local skin tissue. It was confirmed that the monoclonal antibody 2E8 can reduce the expression of the dengue virus antigen NS3 in locally infected tissues.

It is worth noting that an effective dose 50,100,150, 200 μ g of mouse-based calculation of the average body weight 25g used in the present invention, it can be equal in terms of the effective dose is 2,4,6,8 μ g / g or mg / kg; wherein preferred effective dose is based 4 ~ 8 μ g / g or mg / kg.

In summary, the monoclonal antibody 2E8 of the present invention can specifically recognize dengue virus The 90-270 amino acid sequence of NS1 can reduce the bleeding time caused by dengue virus and reduce the replication of dengue virus in infected mice (reduced NS3 expression); therefore, the experimental results show The monoclonal antibody 2E8 can provide a certain therapeutic effect on the infection of dengue virus, indicating that this single anti-system can be a good choice for antiviral treatment. Further, the monoclonal antibody alone or in combination with one or more pharmaceutically acceptable carriers can form a pharmaceutical composition for treating hemorrhagic dengue fever for administration to an individual infected with any serotype of dengue virus; Among them, the pharmaceutical composition for treating hemorrhagic dengue can be administered, for example, orally or by injection.

It can be seen from the above description that the present invention has the following advantages compared with the prior art:

1. This technology is a single antibody 2E8, which can recognize the 90-270 amino acid sequence of four different serotypes of dengue virus NS1, but does not recognize the N-terminus or C of dengue virus NS1. The terminal sequence (amino acid sequence other than 90-270) can reduce the cross-reaction of endothelial cells infected by dengue virus, and effectively reduce the prolonged bleeding time of dengue virus-induced mice and reduce the dengue virus antigen NS3. The performance of locally infected tissues.

2. The present invention, a monoclonal antibody 2E8 specifically binds to a specific amino acid fragment of the anti-dengue virus non-structural protein 1 (NS1), further destroys the infected cells by activating complement, and thus does not cause antibodies. The dependence-enhancing effect can greatly increase the efficacy of treating dengue.

In summary, the monoclonal antibody against dengue virus non-structural protein 1 of the present invention and its use can indeed achieve the intended efficacy by the above-disclosed examples, and the present invention has not been disclosed in Before applying, Cheng has fully complied with the requirements and requirements of the Patent Law.爰Issuing an application for a patent for invention in accordance with the law, and asking for a review, and granting a patent, is truly sensible.

The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.

<110> National Cheng Kung University

<120> Anti-dengue virus non-structural protein 1 monoclonal antibody and use thereof

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<170> PatentIn version 3.5

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Claims (15)

An anti-dengue virus non-structural protein 1 monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody resistance system comprises (a) a complementational decision comprising SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: a heavy chain variable region of a region (CDR) sequence; and (b) a light chain variable region comprising the complementarity determining region sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7, and the light chain variable region comprises SEQ ID NO: 8. The sequence. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the dengue virus non-structural protein 1 is derived from serotype type 1 dengue virus (DENV-1), type 2 One of the leather virus (DENV-2), the third type of dengue virus (DENV-3) or the fourth type of dengue virus (DENV-4). The monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the monoclonal antibody specifically recognizes the 190-200 amino acid sequence of the dengue virus non-structural protein 1 and has a sequence SEQ ID NO: 9 is at least 85% sequence identity. The monoclonal antibody or the antigen-binding fragment thereof according to the fourth aspect of the patent application, wherein the monoclonal antibody specifically recognizes the 90-270 amino acid sequence of the dengue virus non-structural protein 1 and has a sequence SEQ ID NO: 10 is at least 85% sequence identity. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the monoclonal antibody specifically recognizes a dengue virus non-structural protein derived from the second type of dengue virus (DENV-2) 1. A method for preparing a pharmaceutical composition for treating hemorrhagic dengue using an anti-dengue virus non-structural protein 1 monoclonal antibody, comprising: providing a monoclonal antibody and one or more pharmaceutically acceptable carriers; and mixing the monoclonal antibodies and The pharmaceutically acceptable carrier is used to form a pharmaceutical composition for treating hemorrhagic dengue. The method of claim 7, wherein the dengue virus non-structural protein 1 is derived from serotype first type Dengue virus (DENV-1) and second type dengue virus (DENV-2). One of the third type of dengue virus (DENV-3) or the fourth type of dengue virus (DENV-4). The method of claim 8, wherein the monoclonal antibody specifically recognizes the 190-200 amino acid sequence of the dengue virus non-structural protein 1 and has a sequence of at least 85 with SEQ ID NO: 9. % sequence identity. The method of claim 9, wherein the single anti-system specificity identifies the 90-270 amino acid sequence of the dengue virus non-structural protein 1 having a sequence of at least 85 with SEQ ID NO: 10. % sequence identity. The method of claim 9, wherein the monoclonal antibody system specifically recognizes the dengue virus non-structural protein 1 derived from the second type of dengue virus (DENV-2). The method of claim 8, wherein the monoclonal antibody system comprises (a) a weight comprising a complementarity determining region (CDR) sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: a chain variable region; and (b) comprising SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. The method of claim 12, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7, and the light chain variable region comprises the sequence of SEQ ID NO: 8. The method of claim 8, wherein the effective amount of the monoclonal antibody is between 2 mg/kg of the therapeutic subject and 8 mg/kg of the therapeutic subject. The method of claim 14, wherein the effective amount of the monoclonal antibody is between 4 mg/kg of the therapeutic subject and 8 mg/kg of the therapeutic subject.