KR101968246B1 - Anti-CD20 Monoclonal Antibody and Tacrolimus Combination Immunosuppressive Therapy in Xenotransplantation - Google Patents

Abstract

The present invention relates to a protocol for inhibiting an immune rejection reaction that may occur upon corneal xenotransplantation. According to the immune response suppression method of the present invention, it is possible not only to effectively suppress the immune rejection by the cornea transplanted after xenotransplantation, but also to maintain the engraftment of the cornea stably for a long period of time, And the like, can be effectively used, and thus can be usefully used for xenotransplantation.

Description

(Anti-CD20 Monoclonal Antibody and Tacrolimus Combination Immunosuppressive Therapy in Xenotransplantation)

The present invention relates to a protocol for inhibiting immune rejection reactions that may occur upon xenograft transplantation.

Cornea is a translucent membrane located in front of the eyeball wall, which occupies about 1/6 of the anterior aspect of the fiber wall. The cornea has a circular shape when viewed from the back, and a flat oval shape when viewed from the front. The cornea is the foremost transparent part of the eyeball, covering the iris, pupil, and anterior chamber and allowing the correct focus of the light to form on the retina. Corneal blindness due to loss of corneal transparency is known to be a major cause of visual loss. Also, eye wound and corneal ulcers lead to blindness of 1.5 to 2 million patients each year. The only effective treatment for this type of blindness is corneal transplantation (also known as keratoplasty).

However, despite the unique treatment, there is a significant difference between the supply and demand of corneal implant in clinical practice. In an environment where allogenic corneas are so scarce and difficult to change in a short period of time, researchers are making a multifaceted effort to develop alternative implant materials to address the problem of corneal scarcity. Newly reported or proven new implants are biological implants and xenografts. According to conventional reports, biological implants have unsatisfactory tissue compatibility and can not be used under large-scale conditions. In comparison, the heterotopic keratoplasty has a broader applicability.

Xenotransplantation is one of several ways to replace human organs. Until recently, xenotransplantation has developed around organ transplantation. As a heterogeneous organs or source of cells, primates such as Baboons were widely used around 1963 when a heterotopic transplant was first attempted. In particular, Reemtsma, who is called the father of xenotransplantation, maintained chimpanzee kidneys transplanted into kidney failure patients for up to 9 months (Reemtsma K, et al. Ann Surg 1964; 160: 384-410) We have survived for 20 days by transplanting the baboon's heart to Baby Fae with congenital heart disease (Bailey LL, et al., JAMA 1985; 254: 3321-9). However, primates are not only endangered, they are difficult to breed, have a high social rejection to utilize them as heterogeneous organs, and have a high risk of infection. Therefore, pigs with the advantage that the size of organs other than primates are very similar to those of humans, the gestation period is as short as 127 days and breeding is easy, and a large number of pups (6 to 12) . However, there has been a problem that, when transplanting organs of pigs into the human body, xenograft rejection, which is much more severe than allograft, occurs.

 That is, in carrying out xenotransplantation, modulating the immune response referred to as a rejection reaction is a problem faced in the art, and it is an object of the present invention to provide an effective method for preventing the systemic immune reaction and death caused by the corneal xenotransplantation There is a need for an immunosuppressive response.

The present inventors have studied a variety of drug combinations and their administration protocols that can effectively suppress the immune response that may occur in the corneal xenotransplantation, and found that when administering the drug according to the appropriate combination of drugs and administration time, It was confirmed that the engraftment of the transplanted cornea can be effectively maintained without inducing an immune response, and the present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for inhibiting immune responses by mammalian corneal xenotransplantation.

In order to accomplish the above object, the present invention provides a method of treating corneal xenotransplantation, comprising: intravenously administering an anti-CD20 antibody; Intravenously administering an anti-IL2R antibody; Intravenously administering intravenous immunoglobulin; Intramuscular injection of tacrolimus; And steroids; A method for inhibiting an immune response by a corneal xenotransplantation of a mammal other than a human,

The present invention also provides a method of treating corneal xenotransplantation, comprising administering rituximab in an amount of 10 to 30 mg / kg on the day of corneal xenotransplantation and in the first week after surgery, and intravenously every 8 to 24 weeks of operation; Intravenous administration of basiliximab at 0.1 to 0.5 mg / kg on the day of surgery and on day 4 after surgery; Intravenous administration of Intravenous Immunoglobulin at 0.1 to 5 g / kg on days 1 and 14 after surgery; Intramuscular injection of tacrolimus 0.01 to 0.5 mg / kg twice daily for 12 months after surgery; Intramuscular daily administration of methylfrednisolone at a dose of 2 mg / kg to 0.25 mg / kg for 5 weeks after surgery; Peddling prenosolone acetate daily for up to 3 months postoperatively; And dexamethasone at a dose of 1.0 mg / 0.3 ml to 1.5 mg / 0.45 ml for 6 months after surgery; The present invention also provides a method for inhibiting immune responses caused by xenogeneic xenobiotics in mammals other than humans.

According to the immune response suppression method of the present invention, it is possible not only to effectively suppress the immune rejection by the cornea transplanted after xenotransplantation, but also to maintain the engraftment of the cornea stably for a long period of time, Therefore, it can be effectively used for intercellular keratoplasty including porcine-primate and pig-human liver.

1 is a diagram showing a drug administration protocol for inhibiting the immune response by xenogeneic corneal xenotransplantation.
2 is a schematic diagram of a method for performing a whole-layer keratoplasty.
FIG. 3 is a graph showing clinical results of corneal transplant recipients according to the drug administration protocol of the present invention. FIG.
FIG. 4 shows the results of comparing the changes of B cells and T cells according to the drug administration protocol of the present invention with an immunosuppression method using an anti-CD40 antibody.

The present invention relates to a method for inhibiting an immune response by xenograft transplantation.

According to the method of the present invention, the immune rejection reaction that may occur in the recipient after xenotransplantation can be effectively inhibited, so that the engraftment of the cornea can be stably maintained for a long period of time and the central corneal thickness and intraocular pressure can be kept very stable have. In the present invention, the immune response can be used interchangeably in the sense of including immune rejection.

More specifically, the method of the present invention comprises intravenously administering an anti-CD20 antibody in a corneal xenotransplantation step; Intravenously administering an anti-IL2R antibody; Intravenously administering intravenous immunoglobulin; Intramuscular injection of tacrolimus; And steroids; To a method for inhibiting an immune response by a corneal xenotransplantation of a mammal other than a human.

In the present invention, it is preferable that the anti-CD20 antibody is rituximab, and when the anti-CD20 antibody is rituximab, it is administered in an amount of 10 to 30 mg / kg, preferably 20 mg / kg, and it is possible to repeatedly administer the same dose every 8 weeks after 8 weeks of operation. In the subsequent repeated administration, the presence or absence of an increase in the IgG antibody is confirmed, and the dose is appropriately determined And the like.

In the present invention, the anti-IL2R antibody may be basiliximab, and the above-mentioned basiliximab is preferably administered on the day of surgery and on the fourth day after the surgery. In addition, the bacillicum may be administered at 0.1 to 0.5 mg / kg, more preferably 0.3 mg / kg, once a day.

In the present invention, the intravenous immunoglobulin (IVIG) can be used interchangeably with intravenous immunoglobulin, and is an IgG antibody extracted from a blood plasma and used for the treatment of immune deficiency, autoimmune diseases, .

The intravenous immunoglobulin may be administered at a dose of 0.1 to 5 g / kg on days 1 and 14 after surgery, preferably 1 g / kg on days 1 and 14 after surgery And the like. The intravenous immunoglobulin may be administered further depending on the immune response of the individual.

In addition, the present invention can be characterized in that tacrolimus is administered by intramuscular injection at a dose of 0.01 to 0.5 mg / kg twice a day, pre-operatively to 12 months after surgery. The tacrolimus may be administered to the recipient 3 to 5 days prior to the xenograft transplantation, preferably 2 days before the surgery, and is preferably administered twice a day for 12 months after the operation. The administration of the above-mentioned tacrolimus can be appropriately controlled depending on the periodic blood concentration test.

The present invention is characterized in that taclomerus is administered through intramuscular injection at 0.01 to 0.5 mg / kg. However, in the case of an adverse reaction to tacrolimus, the dose of tacrolimus is adjusted to 0.035 mg / kg, It is most preferable to administer it once.

The present invention also provides the administration of a steroid preparation, which may be used without limitation in any of the approved steroid preparations known in the art, but is preferably a steroid preparation, such as methylprednisolon, prednisolone acetate, And at least one selected from the group consisting of meta-zones. More preferably, methylprednisolone, prednisolone acetate, and dexamethasone may be administered as a steroid preparation, wherein the methylprednisolone is administered at a dose of 2 mg / kg to 0.25 mg / kg daily for 5 weeks after surgery It can be administered intramuscularly with a gradual decrease in dosage.

In addition, prednisolone acetate may be administered in ophthalmic formulations, preferably prednisolone acetate 1% ophthalmic formulation, until 3 months after surgery. After 3 months after surgery, After months, the interval can be gradually increased by one week.

The dexamethasone may be injected subconjunctivally with 1.0 mg / 0.3 ml to 1.5 mg / 0.45 ml, preferably 1.5 mg / 0.3 ml, for 6 months after the operation. On the day of surgery, Day, 10 days, 15 days, and 21 days after administration, and 7 days after 21 days. The deck samata zone may be administered after 6 months of operation according to the common knowledge of the ordinary artisan in the art.

The method of the present invention can effectively suppress the immunological rejection that may occur in all corneal xenotransplantation, and the corneal xenotransplantation may include partial corneal xenotransplantation and whole-layer corneal xenotransplantation, Xenotransplantation, and more preferably, full thickness corneal xenotransplantation of the visual membrane.

In the present invention, the term " suspected film " refers to a film that is prepared by dissolving a pig's eyeball, immersing it in a 5% betadine solution for 1 minute, and sterilizing and washing with 20 cc of 10% It means an organization that has become available.

The present invention relates to a method for inhibiting an immune response by intra-mammalian corneal xenotransplantation. In the xenotransplantation of the present invention, the pig may be used as a donor of the cornea, and the primate may be a recipient of the cornea. The primate may include human, monkey, chimpanzee, gorilla, orangutan, and raccoon monkey. Therefore, the present invention can be useful for xenotransplantation of a porcine cornea into a primate, preferably a monkey or a human.

More particularly, the present invention relates to a method for treating rheumatoid arthritis, comprising administering rituximab in an amount of 10 to 30 mg / kg on the day of corneal xenotransplantation and 1 week after surgery, and intravenously every 8 to 24 weeks of operation; Intravenous administration of basiliximab at 0.1 to 0.5 mg / kg on the day of surgery and on day 4 after surgery; Intravenous administration of Intravenous Immunoglobulin at 0.1 to 5 g / kg on days 1 and 14 after surgery; Intramuscular injection of tacrolimus 0.01 to 0.5 mg / kg twice daily for 12 months after surgery; Intramuscular daily administration of methylfrednisolone at a dose of 2 mg / kg to 0.25 mg / kg for 5 weeks after surgery; Peddling prenosolone acetate daily for up to 3 months postoperatively; And dexamethasone at a dose of 1.0 mg / 0.3 ml to 1.5 mg / 0.45 ml for 6 months after surgery; The present invention is directed to a method for inhibiting the immune response caused by xenogeneic xenogeneic transplantation of mammals other than humans, and further explanation of each step is omitted in order to avoid redundant description with the above-described contents.

Example  1. Pig - Primate  Xenotransplantation anti-CD20 combination systemic immunosuppression protocol

1.1 Establishment of a systemic immunosuppression protocol

In order to establish a protocol for inhibiting the immune response in the manufacture of xenotransplantation cornea, the following anti-CD20 combination systemic immunosuppression protocol was devised. In this protocol, anti-CD20 Ab, anti IL2R Ab, intravenous immunoglobulin (IVIG), tacholimus, and methylprednisolone were administered at fixed intervals. Rituximab was used as an anti-CD20 mAb and bacillic was used as an anti-IL2R Ab. The protocol according to the present invention is shown in FIG. 1, and is specifically as follows.

Anti-CD20 Ab Was injected via slow intravenous infusion and was administered at 20 mg / kg on the day of xenotransplantation and 1 week after surgery. After 8 weeks of operation, , And more frequently when necessary.

Basiliximab, an anti IL2R antibody, was administered once daily on a day of xenotransplantation at the dose of 0.3 mg / kg and on day 4 after surgery with slow intravenous infusion.

Intravenous Immunoglobulin (IVIG) was administered at a dose of 1 g / kg, and was administered at 1 and 2 weeks after surgery and was further administered according to the immune response.

Tacrolimus was administered at a dose of 0.05 mg / kg via intramuscular injection twice daily for 12 months starting from 2 days prior to surgery, and the dose was adjusted by periodic blood tachylylmus concentration test. The dose was reduced to 0.035 mg / kg for individuals exhibiting adverse reactions to tacrolimus.

Methylprednisolone, prednisolone acetate eye drops and dexamethasone were administered together with the other steroids.

In the case of methylprednisolone, the dose was gradually decreased from 2 mg / kg to 0.25 mg / kg daily for 5 weeks after the operation, and intramuscular administration was carried out. More specifically, the dose was gradually decreased 2 mg / kg from the day of surgery to 7 days after surgery, 1.5 mg / kg from day 8 to day 14, 1.0 mg / kg from day 15 to day 21, and 0.5 mg / / kg, 0.25 mg / kg from 29 days after surgery to 35 days.

 Prednisolone acetate 1% ophthalmic solution was administered dropwise daily until 3 months after surgery, and gradually increased from one week to two or three times a week from three months to six months after surgery and six months after surgery Medication. In the case of dexamethasone, 1.5 mg / 0.3 ml was injected through subconjunctival injection at 0 day, 1 day, 4 day, 7 day, 10 day, 15 day, and 21 day after surgery. For six months.

1.2 Identification of immunosuppressive effect

1.2.1 Different keratoplasty methods

Penetrating keratoplasty was used for transplantation of heterogeneous corneas, with genetically unmodified adult SNU miniature pigs as donors and Chinese rhesus macaques as recipients (n = 5). The method of whole-layer keratoplasty is shown in Fig.

As shown in FIG. 2, the cornea of the recipient was cut to 7.0 mm in diameter through a trephination, and the donor's 7.5-mm-sized cornea was placed and then sealed with nylon.

1.2.2 Identification of xenotransplantation corneal immunosuppressive effect

In accordance with the administration protocol of the present invention, the xenografts were transplanted into primates and immunosuppressive therapy was performed. The results of the postoperative clinical trials were confirmed by immunoregulation, corneal thickness, and intraocular pressure. Specifically, primate corneas with differentiated keratocytes were examined with slit-lamp microscopy for transparency, edema, and neovascularization. Regular confirmation was made of whether the rejection occurred or not. The central corneal thickness and intraocular pressure were measured with an ultrasonic corneal thickness gauge (Quantel Medical, Clermont-Ferrand, France) and Tono-Pen (Medtronic Solan, Jacksonville, FL). The mean corneal thickness was measured five times, and the mean value of the intraocular pressure was measured twice. The results are shown in FIG.

As shown in FIG. 3, the subjects receiving the immunosuppressive treatment according to the protocol of the present invention after the corneal transplantation did not show any immunity rejection after 135 to 373 days after the surgery. In addition, the thickness of the central cornea was stable at a constant thickness even after the postoperative period, and the intraocular pressure (IOP) could be kept constant over the range of 20 mmHg.

In addition, the T cells and B cell concentrations in the blood were confirmed by flow cytometry in the individuals immunosuppressed according to the protocol of the present invention. The immunocompromised protocol using the already established anti-CD40 antibody (Kim J, et al (Xenotransplantation. 2017 Apr 10. doi: 10.1111 / xen.12298). The results are shown in FIG.

As shown in FIG. 4, it was confirmed that in the subjects treated according to the protocol of the present invention, the concentration of T and B cells in the blood was kept lower than the protocol using the existing anti-CD40 antibody to achieve effective immunosuppression.

According to the above results, when the immunosuppression protocol according to the present invention is used, it is possible to effectively suppress the immunity rejection reaction that may occur when the xenograft transplantation is carried out, thereby enhancing the safety of the xenobiotic keratoplasty and significantly improving the success rate Respectively.

Claims (15)

(a) intravenously administering the anti-CD20 antibody intravenously at 10-30 mg / kg on the day of corneal xenotransplantation and 1 week after surgery, and intravenously every 2 months after 8 weeks of operation;
(b) administering an anti-IL2R antibody intravenously;
(c) intravenously administering Intravenous Immunoglobulin;
(d) intramuscular injection of tacrolimus at a dose of 0.01 to 0.5 mg / kg twice a day before the corneal xerotransplantation operation and 12 months after the operation; And
(e) administering a steroid; Wherein the immune response is inhibited by a corneal xenotransplantation of a mammal other than a human.
2. The method according to claim 1, wherein the anti-CD20 antibody is rituximab. 2. A method for inhibiting immune response by a corneal xenotransplantation in a mammal other than a human.
delete The method according to claim 1, wherein the anti-IL2R antibody is bacillicum. A method for inhibiting an immune response by corneal xenotransplantation of a mammal other than a human.
5. The method according to claim 4, wherein the basiliximab is administered at a dose of 0.1 to 0.5 mg / kg on the day of surgery and on the fourth day after the operation, wherein the inhibition of the immune response by the corneal xenotransplantation of mammals other than humans Way.
The method according to claim 1, wherein the intravenous immunoglobulin is administered at 0.1 to 5 g / kg on days 1 and 14 after the surgery. .
delete The method according to claim 1, wherein the steroid is at least one selected from the group consisting of methylprednisolone, prednisolone acetate, and dexamethasone.
9. The method according to claim 8, wherein the methylprednisolone is administered intramuscularly daily at a dose of 2 mg / kg to 0.25 mg / kg for 5 weeks after surgery. Lt; / RTI >
9. The method according to claim 8, wherein the prednisolone acetate is not administered every day for up to 3 months after surgery.
[Claim 10] The method according to claim 8, wherein the dexamethasone is injected subconjunctivally with 1.0 mg / 0.3 ml to 1.5 mg / 0.45 ml for 6 months after the surgery. How to suppress.
The method according to any one of claims 1, 2, 4, 6, and 8 to 11, wherein the corneal xenotransplantation is a full-thickness corneal xenotransplantation or a partial corneal xenotransplantation. A method for inhibiting an immune response by a corneal xenotransplantation in a mammal other than a human.
The corneal xenotransplantation method according to any one of claims 1, 2, 4, 6, and 8 to 11, wherein the porcine xenotransplantation is performed by using pig as a donor and primate as an acceptor A method for inhibiting an immune response by a corneal xenotransplantation of a mammal other than a human.
The method according to any one of claims 1, 2, 4, 6, and 8 to 11, wherein the corneal xenotransplantation is a xenotransplantation of a visceral membrane. Methods for inhibiting immune response by xenograft transplantation.
a) intravenously administering rituximab intravenously at 10-30 mg / kg on the day of corneal xenotransplantation and postoperative 1 week and intravenously every 2 months for 8- 24 weeks of surgery;
b) intravenously administering basiliximab 0.1 to 0.5 mg / kg on the day of surgery and on day 4 after surgery;
c) intravenously administering Intravenous Immunoglobulin at 0.1 to 5 g / kg on days 1 and 14 after surgery;
d) Intramuscular injection of tacrolimus at a dose of 0.01 to 0.5 mg / kg twice a day before the corneal xerotransplantation operation and 12 months after the operation;
e) Intramuscular administration of methylprednisolone daily from 2 mg / kg to 0.25 mg / kg for 5 weeks post-surgery with reduced doses;
f) not administering prednisolone acetate daily for up to 3 months after surgery; And
g) subconjunctival injection of dexamethasone with 1.0 mg / 0.3 ml to 1.5 mg / 0.45 ml for 6 months after surgery; A method for inhibiting an immune response caused by xenobiotic embryo transfer of a mammal other than a human.

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