Methods for Treating or Reducing the Risk of Transplant Rejection - Patent application

Abstract

The present disclosure relates to methods of treating or reducing the risk of transplant rejection, or extending the period before transplant rejection occurs, in a subject (e.g., a human) in need thereof by administering an antagonist that targets CD40 or CD154, such as an anti-CD40 antibody or antigen-binding fragment thereof (e.g., a humanized anti-CD40 antibody or antigen-binding fragment thereof). The transplant may be an allograft or a xenograft (e.g., cells, tissues, or organs, or portions thereof).

Description

[Technical field]

[0001] (Sequence Listing) This application contains a Sequence Listing that has been filed electronically in XML format, which is incorporated herein by reference in its entirety. Said XML copy, created on December 29, 2022, is named 51383-008WO4_Sequence_Listing_12_29_22.XML and is 10,397 bytes in size. [Background technology]

[0002] Tissue and organ transplantation is a powerful therapeutic tool for replacing damaged or defective organs or tissues in subjects who need it.However, serious problems after transplantation occur when the recipient's immune system attacks the graft, resulting in transplant rejection and failure.There is a severe shortage of human tissues and organs for tissue and organ transplantation.

[0003] Xenotransplantation, the transplantation of organs or tissues from a non-human species to a human recipient, may help address the shortage of human donor material. Xenotransplantation presents significant obstacles because certain antigens on the non-human donor organ can elicit an immune response by the human recipient, leading to acute rejection of the transplanted material.

[0004] Thus, there is a need for new methods of suppressing the immune system in human subjects undergoing transplants (e.g., allogeneic or xenogeneic transplants) to treat or reduce the risk or likelihood of transplant rejection, or to prolong the time until transplant rejection occurs. Summary of the Invention

[0005] A first aspect features a method of treating or reducing the risk of transplant rejection or extending the period before the onset of transplant rejection in a human subject undergoing or having undergone a transplant (e.g., a xenotransplant). The method includes administering an anti-CD40 antibody or antigen-binding fragment thereof about 1 hour to about 24 hours (e.g., about 3 hours to about 24 hours, or about 10 hours to about 24 hours) prior to transplantation, and administering the anti-CD40 antibody or antigen-binding fragment thereof within about 1 hour to about 24 hours after transplantation, e.g., once the subject has achieved hemostasis. In certain embodiments, the method treats or reduces the risk of, or extends the period before the onset of, hyperacute, acute, or chronic rejection of the graft. The antibody or antigen-binding fragment thereof may be administered subcutaneously or intravenously.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, respectively, as set forth in SEQ ID NOs: 1-3, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3, respectively, as set forth in SEQ ID NOs: 4-6. In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 8. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof is administered at a dosage of about 1 mg / kg to about 20 mg / kg (eg, a dosage of about 5 mg / kg or about 10 mg / kg).

[0008] A second aspect features a method of treating or reducing the risk of transplant rejection or extending the time before the onset of transplant rejection in a human subject undergoing or having undergone a transplant (e.g., a xenotransplant). The method includes administering an anti-CD40 antibody or antigen-binding fragment (e.g., a humanized anti-CD40 antibody or antigen-binding fragment). In certain embodiments, the method treats, reduces the risk of, or extends the time before the onset of hyperacute, acute, and / or chronic rejection of a graft. The antibody or antigen-binding fragment thereof includes a heavy chain variable region having CDRH1, CDRH2, and CDRH3, respectively, as set forth in SEQ ID NOs: 1-3, and a light chain variable region having CDRL1, CDRL2, and CDRL3, respectively, as set forth in SEQ ID NOs: 4-6. The antibody or antigen-binding fragment thereof may be administered subcutaneously or intravenously, for example, at a dosage of about 1 mg / kg to about 20 mg / kg (e.g., at a dosage of about 10 mg / kg).

[0009] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:8. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:9, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:10.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof is administered prior to transplant (e.g., about 1 hour to about 24 hours prior to transplant, e.g., about 10 hours to about 24 hours prior to transplant, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplant).

[0011] In some embodiments, the antibody or antigen-binding fragment thereof is administered on the same day as the transplant.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof is administered after transplantation.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof is administered within about 1 hour to about 24 hours after transplantation (e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or within 24 hours), e.g., once the subject has achieved hemostasis.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and / or 1 year after transplantation.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof is administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the lifetime of the subject.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof is administered at least once per week, once per two weeks, once per three weeks, or once per month for a treatment period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0017] In some embodiments, about 1 mg / kg to about 20 mg / kg (e.g., about 5 mg / kg or about 10 mg / kg) of the antibody or antigen-binding fragment thereof is administered at least once per week, once per two weeks, once per three weeks, or once per month for a treatment period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the subject's lifetime.

[0018] In some embodiments, the method includes administering a first dose of about 1 mg / kg to about 20 mg / kg (e.g., about 1 hour to about 24 hours before transplantation, e.g., about 10 hours to about 24 hours before transplantation, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours before transplantation). The present invention includes administering an induction dose of an antibody or antigen-binding fragment thereof comprising two doses: a first dose of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 10 mg / kg) administered within about 1 to about 24 hours (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours) thereafter (e.g., once the subject has achieved hemostasis). In one embodiment, the second dose is administered once the subject has achieved hemostasis. In certain aspects of the invention, the induction dose administered prior to transplantation is a "conditioning dose" that functions to prophylactically suppress or modulate the subject's immune system with the anti-CD40 antibody or antigen-binding fragment thereof prior to transplantation. The method further includes treating the subject with subsequent maintenance dose administration (e.g., a dose of the antibody or antigen-binding fragment thereof following the induction dose) of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 10 mg / kg) of the antibody or antigen-binding fragment thereof administered once a week, once every two weeks, once every three weeks, or once a month (e.g., once every two or three weeks). In certain embodiments, administration of the maintenance dose is initiated once the anti-CD40 antibody or antigen-binding fragment thereof has reproducible (e.g., within the target therapeutic window) or predictable pharmacokinetics (PK).The maintenance dose may be administered to the subject for a treatment period of at least 1 month (e.g., a treatment period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the subject's lifetime).

[0019] In some embodiments, the induction dose comprises a single undivided dose of the antibody or antigen-binding fragment thereof. Alternatively, the induction dose may comprise a divided dose of the antibody or antigen-binding fragment thereof, comprising at least two doses. For example, in some embodiments, the induction dose is a divided dose comprising a first dose and a second dose of the antibody or antigen-binding fragment thereof administered at intervals of about 1 hour to about 24 hours (e.g., at intervals of about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, e.g., at intervals of about 1 hour to about 12 hours). For example, a first fraction of the induction dose may require administration of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 10 mg / kg) of the antibody or antigen-binding fragment thereof, and a second fraction of the induction dose may require administration of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 10 mg / kg) of the antibody or antigen-binding fragment thereof. The second fraction of the induction dose may be administered within about 1 hour to about 24 hours (e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, e.g., within about 1 to about 12 hours) after the subject receives the first fraction of the induction dose. For example, the method may entail administration of 20 mg / kg of an anti-CD40 antibody or antigen-binding fragment thereof in split doses of an induction dose, with a first split dose providing about 10 mg / kg of the antibody or antigen-binding fragment thereof and a second split dose providing about 10 mg / kg of the antibody or antigen-binding fragment thereof.

[0020] The method may include administration of multiple induction doses (e.g., either undivided or divided doses). For example, in some embodiments, the method includes administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more induction doses (e.g., about 1 to about 5 induction doses, e.g., about 3 or about 4 induction doses). In some embodiments, each induction dose (e.g., a first induction dose, a second induction dose, a third induction dose, a fourth induction dose, or more) is a divided dose, including, for example, a first and a second dose of an antibody or antigen-binding fragment thereof (e.g., each first or second dose in an amount of about 1 mg / kg to about 20 mg / kg per dose) administered at intervals of 1 to 24 hours (e.g., at intervals of 1 to 12 hours). Alternatively, the first induction dose is a split dose, and any additional induction doses (e.g., the first, second, third, fourth, or further subsequent induction doses) are administered either as split doses of the antibody or antigen-binding fragment thereof (e.g., at least two split doses administered in an amount of about 1 mg / kg to about 20 mg / kg per dose, each dose administered, for example, 1 to 24 hours apart (e.g., 1 to 12 hours apart)) or as a full dose of the antibody or antigen-binding fragment thereof (e.g., a single dose in an amount of about 1 mg / kg to about 20 mg / kg). In another embodiment, the first induction dose is a split dose (e.g., at least two split doses administered in an amount of about 1 mg / kg to about 20 mg / kg per dose, each dose administered, for example, 1 to 24 hours apart (e.g., 1 to 12 hours apart)), and any additional induction doses (e.g., the first, second, third, fourth, or further subsequent induction doses) are administered as split doses (e.g., comprising at least two doses in an amount of about 1 mg / kg to about 20 mg / kg per dose). In some embodiments, the second induction dose is administered about 1 to about 14 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days) after administration of the first induction dose.In other embodiments, the second, third, fourth, and / or fifth induction doses are administered at intervals of about 1 day to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), optionally within 1 to 30 days after administration of the first induction dose.

[0021] In some embodiments, an induction dose is provided to achieve or maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. For example, in some embodiments, an induction dose is provided to achieve or maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. For example, in some embodiments, an induction dose is provided to achieve or maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. For example, in some embodiments, an induction dose is provided to achieve or maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. a minimum blood concentration, plasma concentration, or serum concentration (e.g., in the range of about 20 ug / mL to about 150 ug / mL, about 150 μg / mL to about 300 μg / mL, about 150 μg / mL to about 200 μg / mL, or about 20 μg / mL to about 300 μg / mL) of an antibody or antigen-binding fragment thereof It is desirable to maintain a trough concentration of the antibody or antigen-binding fragment thereof over time. Thus, the method may include administering an induction dose and monitoring the blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof over time. When the concentration approaches a minimum threshold (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL, 310 μg / mL, 320 μg / mL, 330 μg / mL, 340 μg / mL, 350 μg / mL, 360 μg / mL, 370 μg / mL, 380 μg / mL, 390 μg / mL, 400 μg / mL, 400 μg / mL, 450 μg / mL, 460 μg / mL, 470 μg / mL, 480 μg / mL, 490 μg / mL, 500 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, mL, 130 μg / mL, 140 μg / mL, or about 150 μg / mL (or a concentration in the range of about 20 μg / mL to about 150 μg / mL), e.g., within about 10%, 20%, 30%, 40%, or 50% of the minimum threshold), the method may include administering an additional induction dose (e.g., prior to establishing administration of the maintenance dose, e.g., at a dosage of about 1 mg / kg to about 20 mg / kg, e.g., about 5 mg / kg or about 10 mg / kg) of the antibody or antigen-binding fragment thereof.

[0022] In some embodiments, the subject is monitored until the PK (e.g., peak and trough concentrations) of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable (e.g., C トラフ and / or C. max One or more induction doses are administered until the desired therapeutic window, such as when levels appear consistent between doses, is reached. For example, in some embodiments, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are reached. トラフ is expected or predictable to be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0023] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. トラフ is predicted or predictable to within about 20%, for example, 15%, 10%, 5% or less.

[0024] In another embodiment, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are max is expected or predictable to be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0025] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. max is predicted or predictable to be within about 20%, e.g., 15%, 10%, 5% or less. Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum is max may be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week).

[0026] Once the PK of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum becomes replicable or predictable in the desired therapeutic range, the subject may be transitioned from the induction dose to a maintenance dose. In certain embodiments, (i) two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are トラフ However, the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, L, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL; or within about 20%, e.g., about 15%, 10%, 5% or less interval), and / or (ii) two or more C of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses. max but within the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL; or within about 20%, e.g., about a 15%, 10%, 5% or less interval), the subject is transitioned from the induction dose to the maintenance dose.

[0027] Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum. トラフand / or C. max may be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week).

[0028] In some embodiments, a maintenance dose is provided to maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. For example, in some embodiments, a maintenance dose is provided to maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum. For example, in some embodiments, a maintenance dose is provided to maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum. It is desirable to maintain a minimum concentration (e.g., trough concentration) of 00 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 μg / mL (e.g., in the range of about 20 ug / mL to about 150 ug / mL, about 150 μg / mL to about 300 μg / mL, about 150 μg / mL to about 200 μg / mL, or about 20 μg / mL to about 300 μg / mL). Thus, the method may include administering an induction or maintenance dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once a week). When the concentration approaches a minimum threshold (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL, 15 ... In some embodiments, the antibody or antigen-binding fragment thereof may be administered at a concentration of about 1 mg / kg to about 20 mg / kg, e.g., at a dose of about 5 mg / kg or about 10 mg / kg, e.g., at a dose of about 20 mg / kg to about 20 mg / kg, e.g., at a dose of about 5 mg / kg or about 10 mg / kg, or at a dose of about 20 mg / kg to about 150 mg / mL (or at a concentration in the range of about 20 μg / mL to about 150 μg / mL), e.g., within about 10%, 20%, 30%, 40%, or 50% of a minimum threshold value), and the method may include administering a maintenance dose (e.g., a dose of about 1 mg / kg to about 20 mg / kg, e.g., a dose of about 5 mg / kg or about 10 mg / kg) of the antibody or antigen-binding fragment thereof.These maintenance doses can be administered, for example, once a week, once every two weeks, once every three weeks, or once a month (e.g., once every two or three weeks), or repeatedly after transplantation as needed, to maintain the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum at greater than 150 μg / mL. Maintenance doses can be administered to the subject for a treatment period of at least one month (e.g., for a treatment period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the subject's lifetime). The timing of administration of the maintenance dose and / or the timing between maintenance doses can be selected, for example, based on the PK profile of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum. The method further includes treating the subject with a maintenance dose of the antibody or antigen-binding fragment thereof of about 1 mg / kg to about 20 mg / kg, or about 5 mg / kg to about 10 mg / kg (e.g., a dose of about 5 mg / kg or about 10 mg / kg), administered once a week, once every two weeks, once every three weeks, or once a month (e.g., once every two or three weeks) (e.g., after a final induction dose). The maintenance dose may be administered to the subject for a treatment period of at least one month (e.g., for a treatment period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the life of the subject).

[0029] In some embodiments, the method further includes administering a first therapeutic agent that depletes or reduces B cells in the subject (e.g., an anti-CD20 antibody such as rituximab), and / or a second therapeutic agent that depletes or reduces T cells in the subject (e.g., an anti-thymocyte globulin, e.g., THYMOGLOBULIN® or ATGAM®, or an anti-IL-2Rα receptor antibody).

[0030] The first therapeutic agent may be administered prior to transplantation (e.g., about 10 hours to about 24 hours prior to transplantation, e.g., about 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplantation). The first therapeutic agent may be administered after transplantation (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 4 weeks after transplantation).

[0031] In some embodiments, the first therapeutic agent is administered for a treatment period of 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the first therapeutic agent is administered daily, weekly, or once every other week for a treatment period of 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0032] In some embodiments, the first therapeutic agent (e.g., an anti-CD20 antibody such as rituximab) is administered at a dosage of about 1 mg / kg to about 40 mg / kg (e.g., about 10 mg / kg). In some embodiments, the first therapeutic agent is administered intravenously.

[0033] In some embodiments, the anti-CD20 antibody (e.g., rituximab) is administered at a dose of about 250 mg / m 2 ~about 500mg / m 2 For example, a dosage of about 375 mg / m 2 is administered at a dosage of

[0034] In some embodiments, the first therapeutic agent is rituximab.

[0035] In some embodiments, the second therapeutic agent may be administered prior to transplantation (e.g., about 10 hours to about 24 hours prior to transplantation, e.g., about 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplantation).

[0036] In some embodiments, the second therapeutic agent is administered on the same day as transplantation.

[0037] The second therapeutic agent may be administered after transplantation (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 4 weeks after transplantation). In some embodiments, the second therapeutic agent is administered for a 1-day, 2-day, 3-day, 4-day, 5-day, 6-day, 1-week, 2-week, 3-week, or 4-week treatment period. In some embodiments, the second therapeutic agent is administered daily, weekly, or once every other week for a 1-week, 2-week, 3-week, or 4-week treatment period.

[0038] In some embodiments, the second therapeutic agent is administered within about 1 hour to about 24 hours after transplantation and once the subject has achieved hemostasis.

[0039] In some embodiments, the second therapeutic agent (e.g., an anti-thymocyte globulin, e.g., THYMOGLOBULIN® or ATGAM®, or an anti-IL-2Rα receptor antibody) is administered at a dosage of about 1 mg / kg to about 10 mg / kg, e.g., about 2 mg / kg or about 5 mg / kg. In some embodiments, the second therapeutic agent is THYMOGLOBULIN® and is administered at a dosage of about 2 mg / kg. In some embodiments, the second therapeutic agent is ATGAM® and is administered at a dosage of about 5 mg / kg. The second therapeutic agent may be administered intravenously.

[0040] In some embodiments, the second therapeutic agent is antithymocyte globulin.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof is administered at a dosage of about 10 mg / kg.

[0042] In some embodiments of any of the aspects described herein, the antibody or antigen-binding fragment thereof is administered intravenously.

[0043] In some embodiments of any of the aspects described herein, the antibody or antigen-binding fragment thereof is administered subcutaneously.

[0044] In some embodiments of any of the aspects described herein, the transplant is an allogeneic transplant. In other embodiments of any of the aspects described herein, the transplant is a xenogeneic transplant. The transplant may be a cell, tissue, or organ transplant. The transplant may be or include, for example, a heart, kidney, lung, liver, pancreas, intestine, thymus, skin, eye, uterus, stem cell, bone, tendon, cornea, heart valve, nerve, vein, or a portion thereof. In some embodiments, the xenogeneic transplant includes an organ or a portion thereof from a pig, cow, horse, dog, cat, sheep, goat, non-human primate (e.g., macaque (e.g., rhesus or cynomolgus), baboon, marmoset, monkey, and chimpanzee), or gorilla. The xenogeneic transplant may include a pig heart. The xenogeneic transplant may include a pig kidney. In some embodiments, the xenogeneic transplant is genetically engineered, for example, to add, modify, or remove one or more xenoreactive antigens. In some embodiments, the xenograft comprises an organ or portion thereof from a host animal that has been genetically engineered, for example, to add, modify, or remove one or more xenoreactive antigens.

[0045] In some embodiments of any of the above aspects, the method further includes administering one or more of a steroid (e.g., methylprednisolone), an antihistamine (e.g., diphenhydramine), an H2 receptor blocker (e.g., famotidine), an antiviral agent (e.g., ganciclovir), a complement inhibitor (e.g., C1 esterase inhibitor), an immunosuppressant (e.g., tocilizumab and / or mycophenolate mofetil), an anti-inflammatory agent (e.g., etanercept, or a nonsteroidal anti-inflammatory agent (NSAID, such as aspirin and naproxen), an anticoagulant (e.g., heparin, aspirin, or other known agent), an antibiotic (e.g., ceftriaxone, or other known agent).

[0046] In some embodiments, the method further comprises administering a steroid such as methylprednisolone. In some embodiments, the methylprednisolone is administered once a day or twice a day. The methylprednisolone may be administered at a dosage of about 2 mg / kg. The methylprednisolone may be administered before transplantation. The methylprednisolone may be administered one day before transplantation. The methylprednisolone may be administered the same day as transplantation. The methylprednisolone may be administered after transplantation. The methylprednisolone may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, and / or 6 months after transplantation. Methylprednisolone may be administered for a treatment period of 2 months, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

[0047] In some embodiments, the method further comprises administering an antihistamine such as diphenhydramine. Diphenhydramine may be administered at a dosage of about 50 mg. Diphenhydramine may be administered intravenously. Diphenhydramine may be administered before transplantation. Diphenhydramine may be administered one day before transplantation. Diphenhydramine may be administered the same day as transplantation. Diphenhydramine may be administered after transplantation. Diphenhydramine may be administered one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, and / or four weeks after transplantation. Diphenhydramine may be administered for a treatment period of two months, seven weeks, six weeks, five weeks, four weeks, three weeks, two weeks, one week, six days, five days, four days, three days, two days, or one day.

[0048] In some embodiments, the method further comprises administering an H2 receptor blocker, such as famotidine. Famotidine may be administered at a dosage of about 20 mg. Famotidine may be administered once a day or twice a day. Famotidine may be administered before transplantation. Famotidine may be administered one day before transplantation. Famotidine may be administered on the same day as transplantation. Famotidine may be administered after transplantation. Famotidine may be administered one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, and / or eight weeks after transplantation. Famotidine may be administered for a treatment period of two months, seven weeks, six weeks, five weeks, four weeks, three weeks, two weeks, one week, six days, five days, four days, three days, two days, or one day.

[0049] In some embodiments, the method further comprises administering an antiviral agent such as ganciclovir. Ganciclovir may be administered at a dosage of about 5 mg / kg. Ganciclovir may be administered prior to transplantation. Ganciclovir may be administered 1 day prior to transplantation. Ganciclovir may be administered on the same day as transplantation. Ganciclovir may be administered after transplantation. Ganciclovir may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, and / or 10 years after transplantation. Ganciclovir may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Ganciclovir may be administered daily for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0050] In some embodiments, the method further comprises administering a complement inhibitor, such as a C1 esterase inhibitor. The C1 esterase inhibitor may be administered at a dosage of about 20 U / kg. The C1 esterase inhibitor may be administered prior to transplantation. The C1 esterase inhibitor may be administered one day prior to transplantation. The C1 esterase inhibitor may be administered the same day as transplantation. The C1 esterase inhibitor may be administered after transplantation. The C1 esterase inhibitor may be administered one day, two days, three days, four days, five days, six days, and / or one week after transplantation.

[0051] In some embodiments, the method further comprises administering an immunosuppressant, such as tocilizumab. Tocilizumab may be administered at a dosage of about 8 mg / kg. Tocilizumab may be administered intravenously. Tocilizumab may be administered on the same day as transplantation. Tocilizumab may be administered after transplantation. Tocilizumab may be administered 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and / or 12 months after transplantation. Tocilizumab may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Tocilizumab may be administered monthly for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0052] In some embodiments, the method further comprises administering an anti-inflammatory agent such as etanercept. Etanercept may be administered at a dosage of about 0.7 IU / kg. Etanercept may be administered subcutaneously. Etanercept may be administered on the same day as transplantation. Etanercept may be administered after transplantation. Etanercept may be administered 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and / or 12 months after transplantation. Etanercept may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Etanercept may be administered weekly for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0053] In some embodiments, the method further comprises administering an immunosuppressant, such as mycophenolate mofetil. Mycophenolate mofetil may be administered at a dosage of about 20 mg / kg. Mycophenolate mofetil may be administered intravenously. Mycophenolate mofetil may be administered once a day or twice a day. Mycophenolate mofetil may be administered on the same day as transplantation. Mycophenolate mofetil may be administered after transplantation. Mycophenolate mofetil may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, and / or 10 years after transplant. Mycophenolate mofetil may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Mycophenolate mofetil may be administered daily for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0054] In some embodiments, the method further comprises administering an anticoagulant such as heparin. The heparin may be administered on the same day as the transplant. The heparin may be administered after the transplant. The heparin may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, and / or 10 years after the transplant. Heparin may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Heparin may be administered daily for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0055] In some embodiments, the method further comprises administering a nonsteroidal anti-inflammatory drug (NSAID), such as aspirin. The aspirin may be administered at a dosage of about 81 mg. The aspirin may be administered after transplantation. The aspirin may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, and / or 10 years after transplantation. Aspirin may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Aspirin may be administered daily for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0056] In some embodiments, the method further comprises administering an antibiotic such as ceftriaxone. Ceftriaxone may be administered at a dosage of about 50 mg / kg. Ceftriaxone may be administered post-transplant. Ceftriaxone may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, and / or 2 weeks after transplant. Ceftriaxone may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2 weeks. Ceftriaxone may be administered daily for a treatment period of 1 week.

[0057] A third aspect features a method of treating or reducing the risk of transplant rejection or extending the time before transplant rejection occurs in a human subject undergoing or having undergone a porcine heart xenotransplant. The method includes administering a humanized anti-CD40 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, as set forth in SEQ ID NOs:1-3, respectively, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3, as set forth in SEQ ID NOs:4-6, respectively. In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:8. In some embodiments, the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10. The method includes administering an anti-CD40 antibody or antigen-binding fragment thereof about 1 hour to about 24 hours (e.g., about 10 hours to about 24 hours) prior to transplantation and administering the anti-CD40 antibody or antigen-binding fragment thereof within about 1 hour to about 24 hours after transplantation, e.g., once the subject has achieved hemostasis. The antibody or antigen-binding fragment thereof may be administered subcutaneously or intravenously.

[0058] In some embodiments, the antibody or antigen-binding fragment thereof is administered within about 1 hour to about 24 hours after transplantation (e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or within 24 hours), e.g., once the subject has achieved hemostasis.

[0059] In some embodiments, the antibody or antigen-binding fragment thereof is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and / or 1 year after transplantation.

[0060] In some embodiments, the antibody or antigen-binding fragment thereof is administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the lifetime of the subject.

[0061] In some embodiments, about 1 mg / kg to about 20 mg / kg (e.g., about 5 mg / kg or about 10 mg / kg) of the antibody or antigen-binding fragment thereof is administered at least once per week, once per two weeks, once per three weeks, or once per month for a treatment period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the subject's lifetime.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof is administered at least once per week, once per two weeks, once per three weeks, or once per month for a treatment period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0063] In some embodiments, the method includes administering as an induction dose, e.g., a first dose of about 1 mg / kg to about 20 mg / kg administered pre-transplant (e.g., about 1 hour to about 24 hours prior to transplant, e.g., about 10 hours to about 24 hours prior to transplant, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplant). and a second dose of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 10 mg / kg) administered within about 1 to about 24 hours (e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours) thereafter (e.g., once the subject has achieved hemostasis).

[0064] In certain embodiments, the induction dose administered prior to transplantation is a "conditioning dose" that functions to prophylactically suppress the subject's immune system using an anti-CD40 antibody or antigen-binding fragment thereof prior to transplantation. Thus, the first induction dose is a conditioning dose of about 1 mg / kg to about 20 mg / kg (e.g., about 10 mg / kg) administered prior to transplantation (e.g., about 1 hour to about 24 hours prior to transplantation, e.g., about 10 hours to about 24 hours prior to transplantation, about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplantation). g dose) and an induction dose of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 10 mg / kg) administered within about 1 hour to about 24 hours (e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or within 24 hours) after transplantation (e.g., once the subject has achieved hemostasis).

[0065] Alternatively, the two divided induction doses administered pre- and post-transplant can be administered in a single undivided dose of about 1 mg / kg to about 20 mg / kg (e.g., either pre-transplant or post-transplant (e.g., after the patient has achieved hemostasis)). In one embodiment, the subject is administered a conditioning dose of about 1 mg / kg to about 20 mg / kg prior to transplant (e.g., about 1 hour to about 24 hours prior to transplant, e.g., about 10 hours to about 24 hours prior to transplant, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplant).

[0066] The method may optionally include administering a second induction dose of the antibody or antigen-binding fragment thereof (e.g., in split doses where two doses are administered at least 1-24 hours apart (e.g., 1-12 hours apart), or in a single dose) in an amount of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 10 mg / kg) about 1 day to about 10 days (e.g., about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days) after transplantation (e.g., after administration of the first induction dose). The method may optionally include administering a third induction dose (e.g., in a split dose, where at least two separate doses are administered, e.g., 1-24 hours apart (e.g., 1-12 hours apart), or in a single administration) in an amount of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 5 mg / kg or about 10 mg / kg). The second and / or third induction dose may be administered, for example, on days 2, 3, 4, 5, 6, 7, 8, 9, and / or 10 after transplantation (e.g., a second induction dose after administration of the first induction dose and a third induction dose after administration of the second induction dose). Additional induction doses (e.g., a fourth, fifth, etc. induction dose) may also be administered in split doses or in a single administration (e.g., in an amount of about 1 mg / kg to about 20 mg / kg per dose (e.g., a dose of about 5 mg / kg or about 10 mg / kg)). If administered in a single dose, the second, third, fourth, and / or fifth induction dose, etc., may be administered in an amount of about 1 mg / kg to about 20 mg / kg per dose (e.g., a dose of about 5 mg / kg or about 10 mg / kg).

[0067] In some embodiments, a subject is administered one or more induction doses until the PK (e.g., peak and trough concentrations) of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable (e.g., in a desired therapeutic window). For example, in some embodiments, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are トラフ is expected or predictable to be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0068] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. トラフ is predicted or predictable to within about 20%, for example, 15%, 10%, 5% or less.

[0069] In another embodiment, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are max is expected or predictable to be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0070] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. max is predicted or predictable to be within about 20%, e.g., 15%, 10%, 5% or less. Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum is max may be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week).

[0071] Once the PK of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum becomes replicable or predictable in the desired therapeutic range, the subject may be transitioned from the induction dose to a maintenance dose. In certain embodiments, (i) two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are トラフ However, the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, L, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL; or within about 20%, e.g., about 15%, 10%, 5% or less interval), and / or (ii) two or more C of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses. max but within the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL; or within about 20%, e.g., about a 15%, 10%, 5% or less interval), the subject is transitioned from the induction dose to the maintenance dose.

[0072] Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum. トラフand / or C. max may be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week). The method can further include treating the subject with subsequent maintenance doses (e.g., after administration of the final induction dose) of about 1 mg / kg to about 20 mg / kg of the antibody or antigen-binding fragment thereof (e.g., a dose of about 5 mg / kg or about 10 mg / kg), administered, for example, once a week, once every two weeks, once every three weeks, or once a month (e.g., once every two weeks or once every three weeks). The maintenance dose may be administered to the subject for a treatment period of at least 1 month (e.g., a treatment period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the subject's lifetime).

[0073] In some embodiments, a maintenance dose is provided to maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. For example, in some embodiments, a minimum concentration (e.g., trough concentration) of the antibody or antigen-binding fragment thereof in blood, plasma, or serum ... (e.g., trough concentration) of at least 20 μg / mL (e.g., at least 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, or 150 μg / mL (e.g., at least 1 It is desirable to maintain the concentration within the range of about 20 μg / mL to about 200 μg / mL, such as 60 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 μg / mL, e.g., about 150 μg / mL to about 300 μg / mL). Thus, the method can include administering an induction dose or a maintenance dose and monitoring the concentration of the antibody or antigen-binding fragment thereof in the blood, plasma, or serum. Once the concentration approaches a minimum threshold (e.g., about 150 μg / mL or about 20 μg / mL, or, e.g., within about 10%, 20%, 30%, 40%, or 50% of the minimum threshold), the method can include administering a maintenance dose (e.g., a dosage of about 1 mg / kg to about 20 mg / kg, e.g., about 10 mg / kg) of the antibody or antigen-binding fragment thereof. These maintenance doses can be administered repeatedly after transplantation, e.g., once per week, once per two weeks, once per three weeks, or once per month (e.g., once per two weeks or once per three weeks). The maintenance dose may be administered to the subject for a treatment period of at least 1 month (e.g., a treatment period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the subject's lifetime).The timing of administration of the maintenance dose and / or the timing between maintenance doses can be selected based on, for example, the PK profile of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum.

[0074] The subject may have been administered a therapeutic agent that depletes or reduces B cells in the subject (e.g., an anti-CD20 antibody such as rituximab). The subject may have been administered a therapeutic agent that depletes or reduces T cells in the subject (e.g., an anti-thymocyte globulin or an anti-IL-2Rα receptor antibody).

[0075] In some embodiments, the method further comprises administering to the subject one or more of a steroid (e.g., methylprednisolone), an antihistamine (e.g., diphenhydramine), an H2 receptor blocker (e.g., famotidine), an antiviral agent (e.g., ganciclovir), a complement inhibitor (e.g., C1 esterase inhibitor), an immunosuppressant (e.g., tocilizumab and / or mycophenolate mofetil), an anti-inflammatory agent (e.g., etanercept, or a nonsteroidal anti-inflammatory agent (NSAID, such as aspirin and naproxen), an anticoagulant (e.g., heparin and aspirin, or other known agents), and an antibiotic (e.g., ceftriaxone or other known agents) before and / or after transplantation.

[0076] A fourth aspect features a method of treating or reducing the risk of transplant rejection or extending the time period before transplant rejection occurs in a human subject undergoing or having undergone a xenogeneic transplant. The method includes administering a humanized anti-CD40 antibody or antigen-binding fragment thereof having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:8. The antibody or antigen-binding fragment thereof may be administered subcutaneously or intravenously, for example, at a dose of about 1 mg / kg to about 20 mg / kg (e.g., about 10 mg / kg) about 10 hours to about 24 hours prior to transplantation. The method may further include administering the antibody or antigen-binding fragment thereof on the same day as transplantation. The method may further include administering the antibody or antigen-binding fragment thereof after transplantation. The antibody or antigen-binding fragment thereof may be administered about 1 hour to about 24 hours after the subject has achieved hemostasis after transplantation. The method may further comprise administering the antibody or antigen-binding fragment thereof once a week, once every two weeks, once every three weeks, or once a month after transplantation.

[0077] The method may further include administering an anti-CD20 antibody, such as rituximab, intravenously, e.g., at a dosage of about 1 mg / kg to about 40 mg / kg, e.g., about 10 hours to about 24 hours prior to transplantation. The anti-CD20 antibody (e.g., rituximab) may be administered post-transplant, e.g., 3 days, 7 days, and / or 14 days after transplantation. The method may further include administering an anti-thymocyte globulin or an anti-IL-2Rα receptor antibody intravenously, on the same day as transplantation, at a dosage of about 1 mg / kg to about 10 mg / kg.

[0078] In some embodiments, the method further comprises administering to the subject one or more of a steroid (e.g., methylprednisolone), an antihistamine (e.g., diphenhydramine), an H2 receptor blocker (e.g., famotidine), an antiviral agent (e.g., ganciclovir), a complement inhibitor (e.g., C1 esterase inhibitor), an immunosuppressant (e.g., tocilizumab and / or mycophenolate mofetil), an anti-inflammatory agent (e.g., etanercept, or a nonsteroidal anti-inflammatory agent (NSAID, such as aspirin and naproxen), an anticoagulant (e.g., heparin and aspirin, or other known agents), and an antibiotic (e.g., ceftriaxone or other known agents) before and / or after transplantation.

[0079] In some embodiments, the xenograft comprises cells, tissues, or organs, or portions thereof, from pigs, cows, horses, dogs, cats, sheep, goats, non-human primates (e.g., macaques (e.g., rhesus or cynomolgus), baboons, marmosets, monkeys, and chimpanzees), or gorillas. The xenograft may be or comprise a heart, kidney, lung, liver, pancreas, intestine, thymus, skin, eye, uterus, stem cells, bone, tendon, cornea, heart valve, nerve, vein, or portions thereof. In some embodiments, the xenograft comprises a pig heart. In some embodiments, the xenograft is genetically engineered to add, remove, or modify one or more xenoreactive antigens.

[0080] In some embodiments, the xenograft comprises an organ or portion thereof from a host animal that has been genetically engineered to add, remove, or modify one or more xenoreactive antigens.

[0081] A fifth aspect features a method of treating or reducing the risk of transplant rejection or extending the period before transplant rejection occurs in a human subject undergoing or who has undergone a transplant (e.g., allogeneic or xenogeneic transplant). The method includes: a) administering an induction dose of a humanized anti-CD40 antibody or antigen-binding fragment thereof, in divided doses, intravenously or subcutaneously, comprising a first dose of about 1 mg / kg to about 20 mg / kg (about 10 hours to about 24 hours prior to transplant) and a second dose of about 1 mg / kg to about 20 mg / kg (after transplant, once the subject has achieved hemostasis); and b) administering a maintenance dose of a humanized anti-CD40 antibody or antigen-binding fragment thereof, in a dose of about 1 mg / kg to about 20 mg / kg, subcutaneously or intravenously, for a treatment period of at least one month.

[0082] The induction dose (e.g., the first induction dose) may alternatively be administered as a single undivided dose (e.g., about 1 mg / kg to about 20 mg / kg, e.g., about 10 mg / kg or 20 mg / kg). Alternatively, the induction dose (e.g., the first induction dose) may be administered as at least two divided doses (e.g., each divided dose contains about 1 mg / kg to about 20 mg / kg, e.g., about 10 mg / kg), with each dose administered at about 1 hour to about 24 hours apart, e.g., 1 hour to 12 hours apart.

[0083] In some embodiments, the subject is administered one or more additional induction doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional induction doses, either in split doses or in a single administration) until the PK (e.g., peak and trough concentrations) of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable (e.g., in a desired therapeutic window). For example, in some embodiments, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, wherein two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more administrations are トラフis expected or predictable to be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0084] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. トラフ is predicted or predictable to within about 20%, for example, 15%, 10%, 5% or less.

[0085] In another embodiment, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are maxis expected or predictable to be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0086] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. max is predicted or predictable to be within about 20%, e.g., 15%, 10%, 5% or less. Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum is max may be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week).

[0087] Once the PK of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum becomes replicable or predictable in the desired therapeutic range, the subject may be transitioned from the induction dose to a maintenance dose. In certain embodiments, (i) two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are トラフHowever, the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, L, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL; or within about 20%, e.g., about 15%, 10%, 5% or less interval), and / or (ii) two or more C of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses. max but within the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL; or within about 20%, e.g., about a 15%, 10%, 5% or less interval), the subject is transitioned from the induction dose to the maintenance dose.

[0088] Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum. トラフ and / or C. maxmay be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week).

[0089] The method further includes treating the subject with a maintenance dose of the antibody or antigen-binding fragment thereof of about 1 mg / kg to about 20 mg / kg, or about 5 mg / kg to about 10 mg / kg (e.g., a dose of about 5 mg / kg or about 10 mg / kg), administered once a week, once every two weeks, once every three weeks, or once a month (e.g., once every two or three weeks) (e.g., after a final induction dose). The maintenance dose may be administered to the subject for a treatment period of at least one month (e.g., for a treatment period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the life of the subject).

[0090] In some embodiments, a maintenance dose is provided to maintain a minimum blood, plasma, or serum concentration of the humanized anti-CD40 antibody or antigen-binding fragment thereof at least 150 μg / mL. In some embodiments, a maintenance dose is provided to maintain a minimum blood, plasma, or serum concentration of the humanized anti-CD40 antibody or antigen-binding fragment thereof within a range of at least about 20 μg / mL to about 200 μg / mL, e.g., at least 20 μg / mL.

[0091] In some embodiments, blood, plasma, or serum concentrations of the humanized anti-CD40 antibody or antigen-binding fragment thereof are used to guide re-administration during maintenance dosing. For example, if the serum concentration falls below a desired or predetermined threshold or range (e.g., about 20 μg / mL to about 300 μg / mL, e.g., about 50 μg / mL to about 200 μg / mL, about 50 μg / mL to about 150 μg / mL, or about 80 μg / mL to about 150 μg / mL), the subject may be administered a maintenance dose. If the serum concentration falls, for example, by more than 10%, e.g., more than 20%, more than 30%, more than 40%, or more below a predetermined threshold or range, the subject may be administered one or more "reloading doses" to raise the serum concentration to a desired therapeutic level (e.g., a concentration of about 20 μg / mL to about 300 μg / mL, e.g., about 50 μg / mL to about 200 μg / mL, about 50 μg / mL to about 150 μg / mL, or about 80 μg / mL to about 150 μg / mL). In some embodiments, a maintenance dose, when administered after a fall below a desired minimum blood, plasma, or serum concentration (e.g., to less than about 150 μg / mL or less than about 20 μg / mL), may be administered at a dosage greater than the previous maintenance dose (e.g., a dosage of more than 10 mg / kg or 20 mg / kg). In some embodiments, one or more maintenance doses, when administered after a drop below a desired minimum blood, plasma, or serum concentration (e.g., to less than about 150 μg / mL or less than about 20 μg / mL), may be administered at a shorter dosing interval than the previous maintenance dose.

[0092] In certain embodiments, if during induction or maintenance dosing, the subject has signs or symptoms of transplant rejection (e.g., elevated troponin levels or abnormal echocardiogram), the subject may be administered (i) one or more rescue doses of an anti-CD40 antibody or antigen-binding fragment thereof, (ii) an increased dose of one or more anti-CD40 antibodies or antigen-binding fragments thereof relative to the dose level administered to the subject prior to signs or symptoms of transplant rejection, or (iii) an increased dosing frequency of an anti-CD40 antibody or antigen-binding fragment thereof relative to the dosing frequency administered to the subject prior to signs or symptoms of transplant rejection, or a combination of (i)-(iii). For example, a subject receiving a dose of about 10 mg / kg once weekly can receive an increased dose of, for example, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg once weekly. Alternatively, or additionally, the dosing frequency may be increased, for example, from 10 mg / kg once per week or once every two weeks to 10 mg / kg twice per week, three times per week, four times per week, five times per week, six times per week, or seven times per week (e.g., once daily).

[0093] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 1-3, respectively, and a light chain variable region having CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NOs: 4-6, respectively.

[0094] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:8. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:9, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:10.

[0095] In some embodiments, the method further includes administering a first therapeutic agent that depletes or reduces B cells in the subject (e.g., an anti-CD20 antibody such as rituximab), and / or a second therapeutic agent that depletes or reduces T cells in the subject (e.g., an anti-thymocyte globulin, e.g., THYMOGLOBULIN® or ATGAM®, or an anti-IL-2Rα receptor antibody).

[0096] The first therapeutic agent may be administered before transplantation (e.g., about 1 hour to about 24 hours before transplantation, e.g., about 10 hours to about 24 hours before transplantation, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours before transplantation). The first therapeutic agent may be administered after transplantation (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 4 weeks after transplantation).

[0097] In some embodiments, the first therapeutic agent is administered for a treatment period of 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the first therapeutic agent is administered daily, weekly, or once every other week for a treatment period of 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0098] In some embodiments, the first therapeutic agent (e.g., an anti-CD20 antibody such as rituximab) is administered at a dosage of about 1 mg / kg to about 40 mg / kg (e.g., about 10 mg / kg). In some embodiments, the first therapeutic agent is administered intravenously.

[0099] In some embodiments, the first therapeutic agent is rituximab.

[0100] In some embodiments, the second therapeutic agent may be administered prior to transplantation (e.g., about 10 hours to about 24 hours prior to transplantation, e.g., about 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplantation).

[0101] In some embodiments, the second therapeutic agent is administered on the same day as transplantation.

[0102] The second therapeutic agent may be administered after transplantation (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 4 weeks after transplantation). In some embodiments, the second therapeutic agent is administered for a 1-day, 2-day, 3-day, 4-day, 5-day, 6-day, 1-week, 2-week, 3-week, or 4-week treatment period. In some embodiments, the second therapeutic agent is administered daily, weekly, or once every other week for a 1-week, 2-week, 3-week, or 4-week treatment period.

[0103] In some embodiments, the second therapeutic agent is administered after the subject achieves hemostasis (eg, once the subject achieves hemostasis), within about 1 hour to about 24 hours after transplantation.

[0104] In some embodiments, the second therapeutic agent (e.g., an anti-thymocyte globulin, e.g., THYMOGLOBULIN® or ATGAM®, or an anti-IL-2Rα receptor antibody) is administered at a dosage of about 1 mg / kg to about 10 mg / kg, e.g., about 2 mg / kg or about 5 mg / kg. In some embodiments, the second therapeutic agent is THYMOGLOBULIN® and is administered at a dosage of about 2 mg / kg. In some embodiments, the second therapeutic agent is ATGAM® and is administered at a dosage of about 5 mg / kg. The second therapeutic agent may be administered intravenously. In some embodiments, the second therapeutic agent is an anti-thymocyte globulin.

[0105] A sixth aspect features a method of treating or reducing the risk of transplant rejection or extending the time before transplant rejection occurs in a human subject undergoing or having undergone a pig kidney xenograft. The method includes administering (e.g., as an induction dose (e.g., as a conditioning dose) or a maintenance dose) a humanized anti-CD40 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, as set forth in SEQ ID NOs:1-3, respectively, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3, as set forth in SEQ ID NOs:4-6, respectively. In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:8. In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0106] The method includes administering an anti-CD40 antibody or antigen-binding fragment thereof (e.g., as an induction dose, which is a conditioning dose) about 1 hour to about 24 hours (e.g., about 10 hours to about 24 hours) prior to transplantation, and administering the anti-CD40 antibody or antigen-binding fragment thereof about 1 hour to about 24 hours after transplantation, e.g., once the subject has achieved hemostasis. The antibody or antigen-binding fragment thereof may be administered subcutaneously or intravenously. The induction dose of the anti-CD40 antibody or antigen-binding fragment thereof may be administered in split doses (e.g., at least two doses, each dose being about 1 mg / kg to about 20 mg / kg (e.g., about 5 mg / kg or about 10 mg / kg)) or in an undivided dose (e.g., a dose of about 1 mg / kg to about 20 mg / kg (e.g., about 5 mg / kg or about 10 mg / kg)).

[0107] In some embodiments, the antibody or antigen-binding fragment thereof is administered (e.g., as an induction dose) within about 1 hour to about 24 hours (e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours) after transplantation, e.g., once the subject has achieved hemostasis.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and / or 1 year after transplantation.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof is administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the lifetime of the subject.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof is administered at least once per week, once per two weeks, once per three weeks, or once per month for a treatment period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0111] In some embodiments, about 1 mg / kg to about 20 mg / kg (e.g., about 5 mg / kg or about 10 mg / kg) of the antibody or antigen-binding fragment thereof is administered at least once per week, once per two weeks, once per three weeks, or once per month for a treatment period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the subject's lifetime.

[0112] In some embodiments, the subject is administered one or more additional induction doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional induction doses in divided or undivided doses) until the pharmacokinetics (PK) of the antibody or antigen-binding fragment thereof is replicable or predictable. In some embodiments, the subject is administered one or more induction doses until the PK (e.g., peak and trough concentrations) of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable (within a desired therapeutic window). For example, in some embodiments, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. トラフis expected or predictable to be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0113] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. トラフ is predicted or predictable to within about 20%, for example, 15%, 10%, 5% or less.

[0114] In another embodiment, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are maxis expected or predictable to be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0115] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. max is predicted or predictable to be within about 20%, e.g., 15%, 10%, 5% or less. Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum is max may be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week).

[0116] Once the PK of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum becomes replicable or predictable in the desired therapeutic range, the subject may be transitioned from the induction dose to a maintenance dose. In certain embodiments, (i) two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are トラフHowever, the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, L, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL; or within about 20%, e.g., about 15%, 10%, 5% or less interval), and / or (ii) two or more C of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses. max but within the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL; or within about 20%, e.g., about a 15%, 10%, 5% or less interval), the subject is transitioned from the induction dose to the maintenance dose.

[0117] Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum. トラフ and / or C. maxmay be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week).

[0118] In some embodiments, a maintenance dose is provided to maintain a minimum blood, plasma, or serum concentration of the humanized anti-CD40 antibody or antigen-binding fragment thereof of at least 150 μg / mL. In some embodiments, a maintenance dose is provided to maintain a minimum blood, plasma, or serum concentration of the humanized anti-CD40 antibody or antigen-binding fragment thereof in the range of at least about 20 μg / mL to about 200 μg / mL, e.g., at least 20 μg / mL.

[0119] In some embodiments, the serum concentration of the humanized anti-CD40 antibody or antigen-binding fragment thereof is used to guide re-administration during maintenance dosing. For example, if the serum concentration falls below a desired or predetermined threshold or range (e.g., about 20 μg / mL to about 300 μg / mL, e.g., about 50 μg / mL to about 200 μg / mL, about 50 μg / mL to about 150 μg / mL, or about 80 μg / mL to about 150 μg / mL), the subject may be administered a maintenance dose. If the serum concentration falls, for example, by more than 10%, e.g., more than 20%, more than 30%, more than 40%, or more below a predetermined threshold or range, the subject may be administered one or more "reloading doses" to raise the serum concentration to a desired therapeutic level (e.g., a concentration of about 20 μg / mL to about 300 μg / mL, e.g., about 50 μg / mL to about 200 μg / mL, about 50 μg / mL to about 150 μg / mL, or about 80 μg / mL to about 150 μg / mL). In some embodiments, a maintenance dose, when administered after a fall below a desired minimum blood, plasma, or serum concentration (e.g., to less than about 150 μg / mL or less than about 20 μg / mL), may be administered at a dosage greater than the previous maintenance dose (e.g., a dosage of more than 10 mg / kg or 20 mg / kg). In some embodiments, one or more maintenance doses, when administered after a drop below a desired minimum blood, plasma, or serum concentration (e.g., to less than about 150 μg / mL or less than about 20 μg / mL), may be administered at a shorter dosing interval than the previous maintenance dose.

[0120] In certain embodiments, if during induction or maintenance dosing, the subject has signs or symptoms of transplant rejection (e.g., elevated troponin levels or abnormal echocardiogram), the subject may be administered (i) one or more rescue doses of an anti-CD40 antibody or antigen-binding fragment thereof, (ii) an increased dose of one or more anti-CD40 antibodies or antigen-binding fragments thereof relative to the dose level administered to the subject prior to signs or symptoms of transplant rejection, or (iii) an increased dosing frequency of an anti-CD40 antibody or antigen-binding fragment thereof relative to the dosing frequency administered to the subject prior to signs or symptoms of transplant rejection, or a combination of (i)-(iii). For example, a subject receiving a dose of about 10 mg / kg once weekly can receive an increased dose of, for example, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg once weekly. Alternatively, or additionally, the dosing frequency may be increased, for example, from 10 mg / kg once per week or once every two weeks to 10 mg / kg twice per week, three times per week, four times per week, five times per week, six times per week, or seven times per week (e.g., once daily).

[0121] In any of the above-described embodiments, the method may include administering, instead of the anti-CD40 antibody or antigen-binding fragment thereof, an antibody or antigen-binding fragment thereof that targets CD40 ligand (CD40L, i.e., CD154), or an antagonist that targets CD154. Suitable antibodies or antigen-binding fragments thereof and antagonists that target CD154 include, for example, dapirolizumab, VIB4920, AT-1501, and SAR441344 (INX021).

[0122] In any of the above aspects, the method may include monitoring and / or adjusting the dosage of the anti-CD40 antibody or antigen-binding fragment thereof in response to its loss or dilution as a result of a procedure during or after a transplant procedure, such as during the perioperative or postoperative care of the subject. In certain embodiments, the subject may experience a decrease in blood, plasma, or serum concentration of the anti-CD40 antibody or antigen-binding fragment thereof as a result of blood or other fluid loss or volume shift due to infusion. In such embodiments, the method may include re-administering the anti-CD40 antibody or antigen-binding fragment thereof to increase the blood, plasma, or serum concentration of the anti-CD40 antibody or antigen-binding fragment thereof in the subject. For example, the subject may be treated with additional therapeutic interventions such as dialysis, extracorporeal membrane oxygenation (ECMO), continuous veno-venous hemofiltration (CVVHW), renal replacement therapy (e.g., continuous hemofiltration and hemodialysis, intermittent hemodialysis and peritoneal dialysis), or infusion of albumin or crystalloids, which may reduce the level of anti-CD40 antibodies or antigen-binding fragments thereof in the fluid, e.g., blood, plasma, or serum of the subject, or dilute the blood, plasma, or serum concentration of anti-CD40 antibodies or antigen-binding fragments thereof. In another embodiment, the subject may experience a pleural effusion and may undergo a procedure, e.g., thoracentesis, to remove fluid from the lungs. The anti-CD40 antibodies or antigen-binding fragments thereof may be present in the pleural effusion, and thus the anti-CD40 antibodies or antigen-binding fragments thereof are removed from the circulation. In another example, the subject may require plasma exchange to remove plasma (and the antibodies contained therein) separated from the subject's blood. These therapeutic interventions, and / or the use of external devices, may reduce (directly or indirectly) the cumulative amount or concentration of the anti-CD40 antibody or antigen-binding fragment thereof in the blood, plasma, or serum of a subject, which may require re-administration or adjustment of the dose level or frequency of administration of the anti-CD40 antibody or antigen-binding fragment thereof or other therapeutic agent.To restore or increase blood, plasma, or serum concentrations of the anti-CD40 antibody or antigen-binding fragment thereof in a subject due to loss or dilution of blood or other fluids caused by these procedures and / or the use of external devices, the anti-CD40 antibody or antigen-binding fragment thereof may be re-administered to the subject, or the dosage and / or frequency of administration of the anti-CD40 antibody or antigen-binding fragment thereof may be increased, if necessary.

[0123] definition As used herein, the term "about" means + / - 10% of the recited value.

[0124] As used herein, "administration" refers to a method of giving a dosage of a pharmaceutical composition (e.g., an antibody or antigen-binding fragment thereof described herein, or any of the other agents described herein). The compositions utilized in the methods described herein can be administered according to methods known in the art for the particular agent, for example, intravenously and subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bath of direct target cells, by catheter, by lavage, by gavage, in a cream, or in a lipid composition. The method of administration can vary depending on various factors (e.g., the components of the composition administered and the severity of the condition being treated) and by using known guidance for the agent being administered.

[0125] The terms "antibody" and "immunoglobulin (Ig)" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies) and polyclonal antibodies, and may also include certain antibody fragments. Antibodies typically include both a "light chain" and a "heavy chain." The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0126] By "hemostasis" is meant the state of a subject following a surgical procedure (e.g., following a transplant operation (e.g., after the chest cavity has been sutured by a surgeon)) in which blood flow has substantially or completely stopped (e.g., no bleeding is evident).

[0127] By "pharmaceutical composition" is meant any composition containing a therapeutically or biologically active agent, such as an antibody or antigen-binding fragment thereof described herein, or any other agent described herein. For purposes of this disclosure, a pharmaceutical composition includes the active agent and one or more pharma- ceutically acceptable carriers, excipients, or diluents known in the art to be suitable for delivery of the therapeutic or bioactive agent.

[0128] "Sequence identity" or "sequence similarity" means that the identity or similarity between two or more amino acid sequences or two or more nucleotide sequences is expressed by the identity or similarity between the sequences, respectively. Sequence identity can be measured by "percentage of identity (%)", where a higher percentage indicates a higher identity shared between the sequences. Sequence similarity can be measured by percentage of similarity (taking into account conservative amino acid substitutions), where the higher the percentage, the higher the similarity shared between the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) with default settings. Such software can match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Sequence identity / similarity can be determined over all or a defined portion of two or more sequences compared.

[0129] As used herein, the phrase "specifically binds" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, for example, that is specifically recognized by an antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof that specifically binds to an antigen binds to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen binds to the antigen with a KD of up to 100 nM (e.g., 1 pM to 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or its epitope exhibits a KD of more than 100 nM (e.g., more than 500 nm, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or its epitope. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive against a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive against a protein or carbohydrate. For a description of immunoassay formats and conditions that can be used to determine specific immune reactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999).

[0130] The subject is a vertebrate such as a mammal (e.g., a primate and a human, particularly a human having an underlying disease (e.g., cardiovascular disease) that requires cell, tissue, or organ transplantation). The subject (e.g., a human) treated according to the methods described herein is a subject in need of a cell, tissue, or organ transplant. Diagnosis may be performed by any suitable means. Those skilled in the art will understand that the subject treated according to the present disclosure may have undergone standard testing or may have been identified without testing as a subject suspected of tissue or organ failure or in need of repair of cell, tissue, or organ function. The method of treating a human subject with the compositions described herein (e.g., KPL-404) is particularly useful in treating, reducing the risk of cell, tissue, or organ transplant rejection, and / or mitigating or inhibiting cell, tissue, or organ transplant rejection.

[0131] As used herein, and as is well understood in the art, a treatment is an approach to obtain a beneficial or desired result, such as a clinical result. A beneficial or desired result may include, but is not limited to, alleviation or amelioration of one or more symptoms or onset of transplant rejection (e.g., rejection of cell, tissue, or organ transplant), whether detectable or undetectable, attenuation of the degree of transplant rejection or prolongation of the period before its onset, stabilization (e.g., not worsening) of transplanted cells, tissue, or organ, or delay or slowing down the progression of cell, tissue, or organ transplant rejection. A treatment may include one or more therapeutic agents, such as one or more compositions described herein, and / or one or more additional therapeutic agents. An additional therapeutic agent may include an agent that inhibits immune response (e.g., anti-inflammatory agents, steroids, H2 receptor blockers, etc.). A treatment may include one or more therapeutic interventions, such as surgery. [Brief description of the drawings]

[0132] [Figure 1]1 is a set of graphs showing flow cytometry analysis of peripheral blood monocytes (PBMCs) performed on days -1, 0, 1, and 3 compared to xenogeneic heart transplantation. The following doses indicate the cumulative dose of each drug administered to the subject at the indicated time points of blood sample collection for flow cytometry: Day -1: Rituximab 375 mg / m2, Day 0: Post-state (s / p): Rituximab 375 mg / m2, Day 1: s / p: Rituximab 375 mg / m2 and s / p 1 mg / kg ATG, Day 3 post-dose: s / p Rituximab 375 mg / m2, s / p 2 mg / kg ATG. [Diagram 2] Flow cytometry analysis of lymph nodes performed prior to chest closure after cardiac xenotransplantation (day 2). At chest closure: s / p cumulative dose: Rituximab 375 mg / m2, s / p 2 mg / kg ATG. [Diagram 3] FIG. 1 is a graph showing daily troponin levels (ng / mL) after xenogeneic heart transplantation. [Figure 4] 1 is a graph showing serum concentrations of KPL-404 as a function of time. The top curve provides data from a Phase I study in normal healthy subjects. The bottom curve provides data in human subjects after xenogeneic heart transplantation. [Diagram 5] 1 is a graph showing the time course of serum concentrations of KPL-404 as a function of time in relation to heart transplantation (horizontal line corresponding to xenotransplantation). The time courses of abdominal surgery, continuous veno-venous hemofiltration (CVVHW) (hemofiltration), and extracorporeal membrane oxygenation (ECMO) are also shown as horizontal lines. [Figure 6A] 6A-6C are graphs showing peripheral blood B and T cell counts after heart transplantation in baboons. FIG 6A shows the percentage of peripheral blood T and B cells relative to total blood lymphocyte count. [Figure 6B] FIG. 6B shows the percentage of CD4 and CD8 T cells in peripheral blood relative to total blood CD4 and CD8 T cell counts. [Figure 6C] FIG. 6C shows the percentage of Treg cells in peripheral blood relative to total blood Treg cell count. [Figure 6D] FIG. 6D shows the absolute numbers of T and B cells in the peripheral blood. [Figure 6E] FIG. 6E shows the absolute numbers of CD4 and CD8 T cells in the peripheral blood. [Figure 6F] Figure 6F shows the absolute number of Treg cells in peripheral blood. These graphs show that T cells were still present in the animals at the time of transplant and that immediately after transplant, T cell numbers steadily increased over time, whereas B cell numbers remained low after transplant. [Figure 7] 1 is a graph showing the extended time course (compared to FIG. 5) of serum concentrations of KPL-404 as a function of time in relation to heart transplantation (horizontal line corresponding to xenotransplantation). The time courses of abdominal surgery, continuous veno-venous hemofiltration (CVVHW) (hemofiltration), and extracorporeal membrane oxygenation (ECMO) are also shown as horizontal lines. [Figure 8A] 8A-8C are graphs showing peripheral blood B and T cell counts after heart transplantation in human subjects. FIG. 8A shows the percentage of peripheral blood T cells (CD3+) and B cells (CD20+) relative to total blood lymphocyte count. [Figure 8B] FIG. 8B shows the percentage of CD4+ and CD8+ T cells in peripheral blood relative to total blood CD4+ and CD8+ T cell counts. [Figure 9] Clinical details during the postoperative course for donor-specific endothelial IgM, IgG, and troponin I levels are shown labeled on the upper x-axis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0133] The present disclosure features a method of treating or reducing the risk of transplant rejection in a human subject or extending the time period before transplant rejection occurs. The subject may be or has been receiving a transplant, such as a cell, organ, or tissue transplant. The transplant may be an allograft or a xenograft transplant. In general, the method includes administration of an antagonist that targets CD40 or CD154. For example, the method may include administering an anti-CD40 antibody or an antigen-binding fragment thereof, for example, before transplantation, on the day of transplantation, and / or after transplantation. The antagonist may be an anti-CD40 antibody or an antigen-binding fragment thereof, or the antagonist may be, for example, an antibody or an antigen-binding fragment thereof that binds to CD154. The antagonist may block the ability of CD40 to bind to CD154 without activating cells that express CD40 (e.g., B cells), thus suppressing the immune system to treat or inhibit transplant rejection. The methods described herein may further include administration of an agent that depletes or reduces B cells in a subject (e.g., rituximab) and / or an agent that depletes or reduces T cells in a subject (e.g., antithymocyte globulin). The antibody or antigen-binding fragment thereof may be derived from the murine 2C10 antibody and its humanized variants, for example, as described in PCT Publications WO2012 / 125569 and WO2017 / 040932, which are incorporated herein by reference in their entirety. The methods and pharmaceutical compositions used in the aforementioned patent documents are described in more detail below.

[0134] Antibodies and antigen-binding fragments thereof targeting CD40 or CD154, and antagonists The methods described herein include administration of an antagonist (e.g., an antibody or antigen-binding fragment thereof) that disrupts the CD40-CD154 interaction. The antagonist can target either CD40 or CD154. In some embodiments, the methods described herein include administration of an antibody or antigen-binding fragment thereof, for example, derived from the murine 2C10 antibody. The heavy chain variable region, light chain variable region, and CDRs of certain anti-CD40 antibodies described herein are shown in Table 1. [Table 1]

[0135] In certain embodiments, the antibody or antigen-binding fragment thereof comprises (e.g., consists of) a heavy chain variable region comprising an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7.

[0136] In certain embodiments, the antibody or antigen-binding fragment thereof comprises (e.g., consists of) a light chain variable region comprising an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the amino acid sequence of a light chain variable region set forth in SEQ ID NO:8.

[0137] In certain embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:7. and a light chain variable region comprising an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the amino acid sequence of the variable light chain set forth in SEQ ID NO:8.

[0138] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to a heavy chain having the amino acid sequence set forth in SEQ ID NO:9. and a light chain region comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the light chain amino acid sequence set forth in SEQ ID NO:10.

[0139] In certain embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises complementarity determining regions (CDRs) that are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the CDRs of the heavy chain variable region of the KPL-404 antibody (CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 1, 2, 3, respectively).

[0140] In certain embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof comprises CDRs that are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the CDRs of the light chain variable region of the KPL-404 antibody (CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 4, 5, 6, respectively).

[0141] In a specific embodiment, the heavy chain comprises the CDRs set forth in SEQ ID NOs: 1-3, respectively, and the light chain comprises the CDRs set forth in SEQ ID NOs: 4-6, respectively.

[0142] In certain embodiments, the heavy chain has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:7 and the CDRs set forth in SEQ ID NOs:1-3, respectively, and the light chain has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:8 and the CDRs set forth in SEQ ID NOs:4-6, respectively.

[0143] Other exemplary anti-CD40 antibodies or antibody fragments thereof include, for example, BI 55064, described in U.S. Pat. No. 8,591,900, isocalimab, described in U.S. Pat. No. 8,828,396, bleselumab, described in U.S. Pat. No. 9,125,893 and PCT Publication WO2017100305, and BMS-986325, described in U.S. Patent Application Publication No. US20210054090 and PCT Publication WO2021236546, each of the foregoing being incorporated by reference in their entirety, including the amino acid and nucleic acid sequences described herein.

[0144] In some aspects, antibodies or antigen-binding fragments thereof that target CD40 ligand (CD40L, i.e., CD154) may be contemplated within the scope of the present disclosure. Antagonists that target CD154 are also contemplated. Suitable antibodies or antigen-binding fragments and antagonists that target CD154 include, for example, dapirolizumab described in U.S. Pat. No. 8,293,237, VIB4920 described in U.S. Pat. No. 10,000,553, AT-1501 described in U.S. Pat. No. 10,106,618, and SAR441344 (INX021) described in U.S. Patent Publication No. 20210145966, including the amino acid sequences and nucleic acid sequences described herein (each of the foregoing is incorporated herein by reference in its entirety). Also included within the scope of the present disclosure are antibodies or antigen-binding fragments thereof in which specific amino acids have been substituted, deleted, or added. These modifications do not have a substantial effect on the biological properties of the peptide, such as binding activity. For example, antibodies may have amino acid substitutions in the framework regions, such as to improve binding to an antigen. In another example, a selected small number of acceptor framework residues may be replaced by the corresponding donor amino acids. The donor framework may be a mature or germline human antibody framework sequence or a consensus sequence.Guidance on how to make phenotypically silent amino acid substitutions can be found, for example, in Bowie et al. (Science, 247: 1306-1310, 1990), Cunningham et al. (Science, 244: 1081-1085, 1989), Ausubel (ed.) (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., 1994), T. Maniatis, E. F. Fritsch and J. Sambrook (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY, 1989), Pearson (Methods Mol. Biol. 243:307-31, 1994), and Gonnet et al. (Science 256:1443-45, 1992), each of which is incorporated herein by reference.

[0145] The polypeptides described herein may be functionally active variants of the antibodies or antigen-binding fragments thereof disclosed herein, e.g., having less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 1% of the amino acid residues substituted or deleted, but having essentially the same immunological properties (including, but not limited to, binding to CD40).

[0146] In some embodiments, the dissociation constant (K D ) is approximately 1 × 10 -8 Less than M, e.g., about 1 x 10 -9 Less than M.

[0147] An antibody or antigen-binding fragment thereof may also contain one or more analogs of amino acids (including, for example, unnatural amino acids, amino acids that occur naturally only in unrelated biological systems, modified amino acids from mammalian systems, etc.), polypeptides with substituted bonds, as well as other modifications known in the art.

[0148] method The methods described herein include administration of a pharmaceutical composition containing an antibody or antigen-binding fragment described herein (e.g., the antibody or antigen-binding fragment can block the ability of CD40 to bind to CD154) to a subject undergoing or having undergone a transplant, such as a cell, organ, or tissue transplant (e.g., xenotransplant). The methods can treat or inhibit transplant rejection in a subject, or extend the period before transplant rejection occurs. For example, the methods can extend that period (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more). The method may inhibit transplant rejection for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the lifetime of the subject.

[0149] The methods described herein may include administering to a subject an induction therapy during which the subject is administered one or more doses (e.g., induction doses) of an anti-CD40 antibody or antigen-binding fragment thereof during the perioperative period covering the immediate post-transplant phase as the period of highest risk for hyperacute and acute rejection of the transplant. In some embodiments, the induction therapy comprises a fixed dosing regimen in which the subject is administered a fixed or weight-based dose level of the anti-CD40 antibody or antigen-binding fragment thereof at a fixed or predetermined dosing interval. In other embodiments, the induction therapy comprises a pharmacokinetic (PK) target-based dosing regimen in which the subject is administered the anti-CD40 antibody or antigen-binding fragment thereof to achieve a desired blood, plasma, or serum concentration (e.g., peak and / or trough concentration) of the anti-CD40 antibody or antigen-binding fragment thereof sufficient to inhibit or suppress CD40 activity in the subject. In some embodiments, the induction therapy is continued until the anti-CD40 antibody or antigen-binding fragment thereof achieves replicable or predictable PK in the subject.

[0150] "Replicable" PK is when, for a given dose and dosing interval, there is consistency between doses. トラフ is observed in or near a desired range (e.g., therapeutic window), and C max is observed in or near the desired range, meaning that in either case the antibody or antigen-binding fragment thereof is administered two or more consecutive times. Once the anti-CD40 antibody or antigen-binding fragment thereof has replicable or predictable PK in the subject, maintenance therapy is administered to the subject.

[0151] Methods for determining whether PK is predictable are known in the art. Pharmacokinetic researchers routinely employ methods to generate PK analyses to predict the PK behavior of drugs in subjects, such as non-compartmental analysis (NCA), which is a standard, efficient, and effective method for estimating PK parameters, and develop models that describe the PK of drugs in subjects. In performing PK analyses, pharmacokinetic researchers identify data that should be excluded because they are insufficient to generate a PK model. Reasons for excluding data include, for example, abnormal concentration values, lack of exposure when exposure is expected, and administration failure.

[0152] The method can include administering an anti-CD40 antibody or antigen-binding fragment thereof about 1 hour to about 24 hours prior to transplantation, e.g., about 10 hours to about 24 hours prior, and administering the anti-CD40 antibody or antigen-binding fragment thereof within 1 hour to 24 hours following administration, e.g., once the subject has achieved hemostasis. The antibody or antigen-binding fragment thereof can be administered subcutaneously or intravenously at a dosage of about 1 mg / kg to about 20 mg / kg, e.g., about 5 mg / kg or about 10 mg / kg.

[0153] The antibody or antigen-binding fragment thereof may be administered prior to transplantation, for example, about 1 hour to about 24 hours prior to transplantation, for example, about 10 hours to about 24 hours prior to transplantation, for example, about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplantation. The antibody or antigen-binding fragment thereof may be administered on the same day as transplantation. The antibody or antigen-binding fragment thereof may be administered after transplantation. For example, the antibody or antigen-binding fragment thereof may be administered post-transplant within about 1 hour to about 24 hours (e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours) after transplant, e.g., once the subject has achieved hemostasis. In certain embodiments, the antibody or antigen-binding fragment thereof is administered post-transplant once the subject has achieved hemostasis.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and / or 1 year after transplantation. In some embodiments, the antibody or antigen-binding fragment thereof is administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the lifetime of the subject.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof is administered at least once weekly, once every two weeks, once every three weeks, or once a month for a treatment period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the life of the subject.

[0156] The methods described herein include administering the antibody or antigen-binding fragment thereof in two doses: a first dose (e.g., about 1 hour to about 24 hours before transplantation, e.g., about 10 hours to about 24 hours before transplantation, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours before transplantation) of about 1 mg / kg to about 20 mg / kg. The subject may be administered a first dose of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 5 mg / kg or about 10 mg / kg) within about 1 hour to about 24 hours (e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours) thereafter (e.g., once the subject has achieved hemostasis). The dose administered to the subject prior to transplantation is also referred to herein as a "conditioning dose." The conditioning dose is part of an induction therapy that functions to prophylactically suppress the subject's immune system using an anti-CD40 antibody or antigen-binding fragment thereof prior to transplantation.

[0157] Alternatively, the induction dose may be administered as a single, undivided dose (e.g., a dose of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 5 mg / kg or about 10 mg / kg) of the antibody or antigen-binding fragment thereof. As described above, the induction dose may also be administered as a divided dose comprising at least two doses of the anti-CD40 antibody or antigen-binding fragment thereof. Whether the induction dose is administered as a single, undivided dose or divided doses will depend on the administration restrictions of the anti-CD40 antibody or antigen-binding fragment thereof, e.g., restrictions set by the prescribing information of the agent or restrictions imposed by relevant clinical use experience or restrictions imposed by applicable regulatory authorities. For example, if a target blood, plasma, or serum concentration of the anti-CD40 antibody or antigen-binding fragment thereof is desired but the dosage level required to achieve this target concentration as a single dose is not recommended due to restrictions set by the prescribing information of the agent or lack of clinical experience administering a single dose, the agent may be administered in divided doses to achieve the target concentration. In certain embodiments, the split induction dose may be administered as a first and second dose of the antibody or antigen-binding fragment thereof administered at intervals of about 1 hour to about 24 hours, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, e.g., about 1 to about 12 hours. For example, the first split of the induction dose may require administration of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 10 mg / kg) of the antibody or antigen-binding fragment thereof, and the second split of the induction dose may require administration of about 1 mg / kg to about 20 mg / kg (e.g., a dose of about 10 mg / kg) of the antibody or antigen-binding fragment thereof.The second fraction of the induction dose may be administered within about 1 hour to about 24 hours (e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, e.g., within about 1 to about 12 hours) after the subject receives the first fraction of the induction dose. For example, the method may entail administration of the induction dose of an antibody or antigen-binding fragment thereof in a 20 mg / kg fractional dose, the first fractional dose providing about 10 mg / kg of the antibody or antigen-binding fragment thereof, and the second fractional dose providing about 10 mg / kg of the antibody or antigen-binding fragment thereof.

[0158] The method may include administration of multiple induction doses (e.g., either undivided or divided doses). As mentioned above, induction therapy is administered during the perioperative period to cover the immediate post-transplant period, which is the period with the highest risk of hyperacute or acute rejection of the transplant, and according to the present invention, is performed before the anti-CD40 antibody or antigen-binding fragment thereof achieves a replicable or predictable PK profile. Without wishing to be bound by any particular theory, starting from the day of transplantation, and days, weeks, or even months thereafter, the agent may not achieve a replicable or predictable PK profile in the subject, for example, as a result of target-mediated pharmacokinetics (TMDD) in the subject undergoing transplantation. Perioperative care may also contribute to the variable and unpredictable PK profile of drugs in a subject, including, but not limited to, fluid loss as a result of surgical intervention (both during the transplant procedure and any surgery or procedures required after transplantation), medical devices (e.g., extracorporeal membrane oxygenation, continuous veno-venous hemofiltration, dialysis, therapeutic plasma exchange, plasmapheresis, hemodilution, etc.) that circulate fluids in the subject and affect the PK of the drug in the subject by, for example, non-specific binding of the drug to the tubing and / or filters of the device, and / or by increasing the volume of the subject's fluids (e.g., by administering crystalloids or albumin, thereby decreasing the blood, serum, or plasma concentration of the drug). Thoracentesis may also be used to reduce pleural fluid, which may result in a decrease in the blood, serum, or plasma concentration of the anti-CD40 antibody or antigen-binding fragment thereof.

[0159] For example, the method can include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more induction doses (e.g., about 1 to about 5 induction doses, such as about 3 or about 4 induction doses). In some embodiments, each induction dose (e.g., the first induction dose, the second induction dose, the third induction dose, and / or the fourth induction dose, or subsequent induction doses) is a split dose that includes a first and a second dose of the antibody or antigen-binding fragment thereof split (e.g., in an amount of about 1 mg / kg to about 20 mg / kg per dose) administered, for example, at intervals of 1 to 24 hours (e.g., at intervals of 1 to 12 hours). Alternatively, the first induction dose is a split dose and any additional induction doses (e.g., the first, second, third, and / or fourth, or further subsequent induction doses) are administered either as split doses of antibody or antigen-binding fragment thereof (e.g., each induction dose includes at least two doses of antibody or antigen-binding fragment thereof in an amount of about 1 mg / kg to about 20 mg / kg (e.g., 5 mg / kg or 10 mg / kg) per dose), or as a single administration of antibody or antigen-binding fragment thereof (e.g., each induction dose is a single dose of antibody or antigen-binding fragment thereof in an amount of about 1 mg / kg to about 20 mg / kg (e.g., 5 mg / kg or 10 mg / kg)). In another embodiment, the first induction dose is a split dose and any additional induction doses (e.g., the first, second, third, and / or fourth, or further subsequent induction doses) are administered in split doses (e.g., each induction dose comprises at least two doses of antibody or antigen-binding fragment thereof in an amount of about 1 mg / kg to about 20 mg / kg per dose). In another embodiment, the first induction dose is a split dose and any additional induction doses (e.g., the first, second, third, and / or fourth, or further subsequent induction doses) are administered in a single administration (e.g., each induction dose is a single dose of antibody or antigen-binding fragment thereof in an amount of about 1 mg / kg to about 20 mg / kg (e.g., 5 mg / kg or 10 mg / kg)).In some embodiments, the second induction dose is administered about 1 to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) after administration of the first induction dose. In other embodiments, the second, third, fourth, and / or fifth induction doses are administered at intervals of about 1 to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days).

[0160] In some embodiments, a subject is administered one or more induction doses until the pharmacokinetics (PK) (e.g., peak and trough concentrations) of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum are replicable or predictable (e.g., a desired therapeutic window). For example, in some embodiments, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody in the subject's blood, plasma, or serum is replicable or predictable, and two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are obtained. トラフ may be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0161] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. トラフ is predicted or predictable to within about 20%, for example, 15%, 10%, 5% or less.

[0162] In another embodiment, multiple induction doses are administered (as described herein) until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are max is expected or predictable to be the same as or in a similar range of about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, or 300 ug / mL).

[0163] In other embodiments, multiple induction doses (as described herein) are administered until the PK of the anti-CD40 antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum is replicable or predictable, where two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are administered. max is predicted or predictable to be within about 20%, e.g., 15%, 10%, 5% or less. Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum ismax may be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week).

[0164] Once the PK of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum becomes replicable or predictable in the desired therapeutic range, the subject may be transitioned from the induction dose to a maintenance dose. In certain embodiments, (i) two or more Cs of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum determined after two or more doses are トラフ However, the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, L, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL, or within about 20%, e.g., about 15%, 10%, 5% or less interval), and / or (ii) two or more C of the antibody or antigen-binding fragment thereof in the blood, plasma, or serum of the subject determined after two or more doses. maxbut within the same or similar range (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL, or within about 20%, e.g., about a 15%, 10%, 5% or less interval), the subject is transitioned from the induction dose to the maintenance dose.

[0165] Optionally, the C of the antibody or antigen-binding fragment thereof in blood, plasma, or serum. トラフ and / or C. max may be determined over a period of, for example, 1-7 days, 1-14 days, 1-21 days, or 1-28 days. Thus, the method can include administering an induction dose and determining the concentration of the antibody or antigen-binding fragment thereof in the subject's blood, plasma, or serum (e.g., the concentration is determined every 6 hours, every 12 hours, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once a week).

[0166] In some embodiments, the induction dose is provided to maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. For example, in some embodiments, the induction dose is at least about 20 μg / mL (e.g., at least 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, or more. It is desirable to maintain a minimum concentration (e.g., trough concentration) of the antibody or antigen-binding fragment thereof in blood, plasma, or serum of 200 μg / mL, 240 μg / mL, or 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, 300 μg / mL (e.g., about 20 ug / mL to about 150 ug / mL, about 150 μg / mL to about 300 μg / mL, about 150 μg / mL to about 200 μg / mL, or about 20 μg / mL to about 300 μg / mL). Thus, the method may include administering an induction dose and monitoring the blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. Once the concentration approaches a minimum threshold (e.g., about 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, or about 150 μg / mL (or a concentration in the range of about 20 μg / mL to about 150 μg / mL), e.g., within about 10%, 20%, 30%, 40%, or 50% of the minimum threshold), the method may include administering an additional induction dose, e.g., about 1 mg / kg to about 20 mg / kg (e.g., about 5 mg / kg or about 10 mg / kg), of the antibody or antigen-binding fragment thereof, e.g., prior to establishment of a maintenance dose.

[0167] The method further includes treating the subject with one or more subsequent maintenance doses (e.g., after the induction dose) of the antibody or antigen-binding fragment thereof of about 1 mg / kg to about 20 mg / kg (e.g., about 5 mg / kg or about 10 mg / kg), administered, for example, once weekly, once every two weeks, once every three weeks, or once monthly (e.g., once every two weeks or once every three weeks). The maintenance doses may be administered to the subject for a treatment period of at least one month (e.g., for a treatment period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the subject's lifetime).

[0168] In some embodiments, a maintenance dose is provided to maintain a desired minimum blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. For example, in some embodiments, it is desirable to maintain a minimum concentration (e.g., trough concentration) of at least 150 μg / mL (e.g., at least 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, or 200 μg / mL, e.g., about 150 μg / mL to about 200 μg / mL) of the antibody or antigen-binding fragment thereof in the blood, plasma, or serum. Thus, the method may include administering an induction dose and monitoring the blood, plasma, or serum concentration of the antibody or antigen-binding fragment thereof. The method may include administering a maintenance dose and monitoring the concentration of the antibody or antigen-binding fragment thereof in the blood, plasma, or serum. Once the concentration approaches a minimum threshold (e.g., about 150 μg / mL, or optionally 20 μg / mL, e.g., within about 10%, 20%, 30%, 40%, or 50% of the minimum threshold), the method can include administering a maintenance dose (e.g., a dosage of about 1 mg / kg to about 20 mg / kg, e.g., about 5 mg / kg or about 10 mg / kg) of the antibody or antigen-binding fragment thereof. The maintenance dose can be administered repeatedly after transplantation, e.g., once per week, once per two weeks, once per three weeks, or once per month (e.g., once per two weeks or once per three weeks). The maintenance dose may be administered to the subject for a treatment period of at least 1 month (e.g., for a treatment period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the life of the subject). The timing of the maintenance dose and / or the time between maintenance doses may be selected based on the PK profile of the anti-CD40 antibody or antigen-binding fragment thereof in the transplant patient or the PK profile in the particular subject being treated. Additionally, the dose and / or timing may be titrated up as necessary or desired.

[0169] One of skill in the art will appreciate that in some embodiments, the minimum concentration (e.g., trough concentration) of an anti-CD40 antibody or antigen-binding fragment thereof in blood, plasma, or serum may fluctuate and / or temporarily decrease below a desired threshold concentration, e.g., at least 10 μg / mL (e.g., from about 10 μg / mL to about 200 μg / mL, e.g., from about 20 μg / mL to about 150 μg / mL, e.g., at least 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, or more).

[0170] The blood, plasma, or serum concentration of the humanized anti-CD40 antibody or antigen-binding fragment thereof may be used to guide the maintenance dose. For example, when the serum concentration falls below a desired or predetermined threshold or range (e.g., about 20 μg / mL to about 300 μg / mL, e.g., about 50 μg / mL to about 200 μg / mL, about 50 μg / mL to about 150 μg / mL, or about 80 μg / mL to about 150 μg / mL), the subject may be administered a maintenance dose. If the serum concentration falls, for example, by more than 10%, e.g., more than 20%, more than 30%, more than 40%, or more below a predetermined threshold or range, the subject may be administered one or more "reloading doses" to raise the serum concentration to a desired therapeutic level (e.g., a concentration of about 20 μg / mL to about 300 μg / mL, e.g., about 50 μg / mL to about 200 μg / mL, about 50 μg / mL to about 150 μg / mL, or about 80 μg / mL to about 150 μg / mL). In some embodiments, a maintenance dose, when administered after a fall below a desired minimum blood, plasma, or serum concentration (e.g., to less than about 150 μg / mL or less than about 20 μg / mL), may be administered at a dosage greater than the previous maintenance dose (e.g., a dosage of more than 10 mg / kg or 20 mg / kg). In some embodiments, one or more maintenance doses, when administered after a drop below a desired minimum blood, plasma, or serum concentration (e.g., to less than about 150 μg / mL or less than about 20 μg / mL), may be administered at a shorter dosing interval than the previous maintenance dose.

[0171] In certain embodiments, if during induction or maintenance dosing, the subject has signs or symptoms of transplant rejection (e.g., elevated troponin levels or abnormal echocardiogram), the subject may be administered (i) one or more rescue doses of an anti-CD40 antibody or antigen-binding fragment thereof, (ii) increased doses of one or more anti-CD40 antibodies or antigen-binding fragments thereof relative to the dose level administered to the subject prior to signs or symptoms of transplant rejection, or (iii) an increased dosing frequency of an anti-CD40 antibody or antigen-binding fragment thereof relative to the dosing frequency administered to the subject prior to signs or symptoms of transplant rejection, or a combination of (i)-(iii).

[0172] The blood, plasma, or serum concentration of an antibody or its antigen-binding fragment may be measured after administration of the antibody using an immunoassay. The immunoassay may use a plate coated with an anti-ID antibody as the capture antigen. Calibration standards and quality control (QC) samples may be prepared by diluting a stock solution of the antibody or its antigen-binding fragment into undiluted human serum, followed by serial dilutions with undiluted human serum to the desired antibody concentration. Prepared calibrators, quality control samples, and test human serum samples may be diluted to a minimum dilution factor (MRD) of 1:200 using assay diluent before being placed on the pre-coated plate. The plate is incubated to allow antibodies present in the sample to bind to the target, and then washed to remove unbound material. Mouse anti-Hu IgG4 pFc HRP may then be added to the plate to bind to the antibody. The plate may be further incubated and then washed to remove all unbound material. TMB substrate may then be added to the plate, and the reaction may then be stopped by adding 1N H2SO4. Plates can be read immediately using, for example, a SPECTRAMAX® Plus 384 (detection at 450 nm, background at 650 nm) microplate reader. OD values ​​obtained from calibration standards (detection OD at 450 nm minus background OD at 650 nm) can be fitted using the 4-PL logistic equation with 1 / y2 to calculate antibody concentrations in quality control and test samples.

[0173] The calibration curve range for this method may be, for example, 8000 ng / mL to 80.0 ng / mL in 100% human serum. Calibrators outside the evaluation range of the assay at 10000 ng / mL and 40.0 ng / mL in 100% human serum may be included as anchor points to facilitate curve fitting.

[0174] The methods described herein may include adjusting the dosage of the anti-CD40 antibody or antigen-binding fragment thereof in response to losses after administration. For example, during a transplant procedure or another procedure after transplantation, e.g., during the post-operative care of the subject, the subject may experience significant blood loss (e.g., as a result of the transplant operation or another surgical procedure) that may require re-administration to alleviate the decreased concentration of the anti-CD40 antibody or antigen-binding fragment thereof in the subject. In addition, the subject may be treated with additional therapeutic interventions such as dialysis, extracorporeal membrane oxygenation (ECMO), continuous veno-venous hemofiltration (CVVHW), mechanical ventilation, or infusion of a fluid, e.g., albumin or crystalloid, that may dilute the blood, plasma, or serum concentration of the anti-CD40 antibody or antigen-binding fragment thereof. In some cases, the subject may experience pleural effusion and may undergo thoracentesis, e.g., to remove fluid from the lungs. The anti-CD40 antibody or antigen-binding fragment thereof may be present in the pleural effusion. In other cases, the subject may require plasma exchange to remove the plasma (and the antibodies contained therein) that is separated from the subject's blood. These therapeutic interventions and / or the use of external devices may result in a reduction (directly or indirectly) in the blood, plasma, or serum concentration of the anti-CD40 antibody or antigen-binding fragment thereof in the subject, which may require re-administration of the anti-CD40 antibody or antigen-binding fragment thereof or adjustment of its dose level or frequency of administration. Thus, the serum level of the anti-CD40 antibody or antigen-binding fragment thereof can be dramatically reduced during or as a result of one or more of these procedures. Thus, the blood, plasma, or serum concentration of the anti-CD40 antibody or antigen-binding fragment thereof may be monitored before, during, or after one or more of these procedures. If necessary, the subject may be re-administered with the anti-CD40 antibody or antigen-binding fragment thereof, or the dosage and / or frequency of administration of the anti-CD40 antibody or antigen-binding fragment thereof may be increased to restore or increase the blood, plasma, or serum concentration of the anti-CD40 antibody or antigen-binding fragment thereof, and account for blood or other fluid loss, hemodilution, or loss due to one or more of these procedures and / or the use of external devices.Levels of other therapeutic agents can also be monitored for loss and re-administered as necessary.

[0175] In certain methods described herein, subjects may be treated perioperatively with the standard of care as described, for example, in Yin et al. Chin. Med. J. 124:1928-1932, 2011, or Amer. J. Transplantation, 9: Suppl 3, 2009, S1-155, each of which is incorporated by reference in its entirety.

[0176] transplant The methods described herein include performing a transplant (e.g., cell, organ, or tissue transplant) in a subject. Transplantation methods are well known and can be performed by those skilled in the art. The transplant may be an allograft. In other embodiments, the transplant may be a xenograft, e.g., cells, organs, or tissues from a different species. The transplant may include heart, kidney, lung, liver, pancreas, intestine, thymus, skin, eye, uterus, stem cells, bone, tendon, cornea, heart valve, nerve, vein, or parts thereof. The xenograft may be, for example, a cell, organ, or tissue that has been genetically engineered to add, modify, or remove one or more xenoreactive antigens. In some embodiments, the transplant is a xenograft that includes cells, organs (or parts thereof), or tissues from pigs, cows, horses, dogs, cats, sheep, goats, non-human primates (e.g., macaques (e.g., rhesus or cynomolgus monkeys), baboons, marmosets, monkeys, and chimpanzees), or gorillas. The xenograft may be a pig heart. The xenograft may be a pig kidney. In some embodiments, the xenograft comprises a host animal cell, organ or part thereof, or tissue that has been genetically engineered to add, modify, or remove one or more xenoreactive antigens. Such genetically engineered animals are described, for example, in Lu, et al. Front. Immunol. 10, 3060, 2020, which is incorporated herein by reference in its entirety (see also Table 2). See, for example, U.S. Patent Nos. 10,912,863 and 11,179,496, and US 2018 / 0249688, each of which is incorporated herein by reference in its entirety.

[0177] The cell, tissue, or organ transplant can be prepared according to methods known in the art. For example, in some embodiments, the organ transplant is a xenograft, such as an organ (e.g., heart) from a pig, and the host animal is genetically engineered. The host may be engineered to add, remove, or modify one or more xenoreactive antigens, for example, to prevent transplant rejection. Suitable systems and methods are known in the art and described, for example, in U.S. Pat. Nos. 11,179,496 and 10,912,863, and U.S. Patent Publication No. 2018 / 0249688, the disclosures of which are incorporated herein by reference in their entirety.

[0178] When a subject undergoes a transplant, significant trauma and / or bleeding of the subject's tissue or organ may occur. Thus, in some embodiments, a particular pharmaceutical composition (e.g., an anti-CD40 antibody or antigen-binding fragment thereof, or any other therapeutic agent described herein, e.g., an anti-CD20 antibody such as rituximab, and / or an anti-thymocyte globulin and / or an anti-IL-2Rα receptor antibody) may be administered, for example, on the same day as transplantation or after transplantation, once the subject achieves hemostasis. The surgeon will end the operation when no obvious bleeding is observed, and then close the skin incision when no obvious blood loss is detected. The subject may continue to be medicated with vasopressor drugs to support blood pressure for other hemodynamic reasons. Thus, hemostasis may include a visual or qualitative assessment by the surgeon. After hemostasis, the subject may be allowed to leave the operating room and move to a recovery room.

[0179] The methods described herein can be used to treat or reduce the risk of transplant rejection. In general, transplant rejection can be characterized as hyperacute rejection, acute rejection, or chronic rejection. Hyperacute rejection occurs immediately after transplantation to about 48 hours after transplantation (e.g., when the graft contains antigens that are incompatible with the recipient). Acute rejection occurs from about 2 days to about 3 months after transplantation. Chronic rejection occurs when the immune system continues to attack the graft over time, eventually reducing or permanently damaging the function of the graft. The methods described herein can be used to treat or reduce the risk of hyperacute transplant rejection. The methods described herein can also be used to treat or reduce the risk of acute transplant rejection and / or chronic transplant rejection.

[0180] Transplant rejection can be measured using methods known in the art, such as echocardiography and / or measuring or detecting troponin levels.Troponin is a biomarker of transplant rejection, and is released by cardiac muscle cells in response to injury.Therefore, troponin levels can be used as a marker of cardiac function.An increase in troponin serum levels indicates an increased risk of heart transplant rejection.

[0181] If a subject shows signs or symptoms of transplant rejection, it may be necessary to change the scheduled or expected administration of the anti-CD40 antibody or antigen-binding fragment thereof. For example, it may be beneficial to reintroduce the drug and / or increase the induction or maintenance dose or increase the administration frequency to reach a higher plasma concentration, thereby increasing the amount of drug present in the transplanted tissue. For example, in certain embodiments, if troponin levels increase during induction or maintenance administration, the subject may be administered (i) one or more rescue induction or maintenance doses of the antibody or antigen-binding fragment, respectively, in addition to the scheduled or expected dose, (ii) one or more doses of the antibody or antigen-binding fragment that are increased relative to the dose level administered to the subject before the increase in troponin levels, or (iii) an increased administration frequency of the antibody or antigen-binding fragment relative to the administration frequency administered to the subject before the increase in troponin levels. Similarly, in other embodiments, if during induction or maintenance dosing, the subject has signs or symptoms of transplant rejection, the subject may be administered (i) a rescue induction or maintenance dose, respectively, of the antibody or antigen-binding fragment in addition to the scheduled or expected dose, (ii) one or more doses of the antibody or antigen-binding fragment increased relative to the dose level administered to the subject prior to signs or symptoms of transplant rejection, (iii) an increased dosing frequency of the antibody or antigen-binding fragment relative to the dosing frequency administered to the subject prior to signs or symptoms of transplant rejection, or a combination of (i)-(iii). Other biomarkers evidencing rejection of transplanted tissue (e.g., echocardiograms) are known in the art.

[0182] B cell decline The methods described herein may further include administering a therapeutic agent that depletes or reduces B cells in the subject. The therapeutic agent may be, for example, rituximab. The therapeutic agent may be administered prior to transplantation (e.g., about 1 hour to about 24 hours prior to transplantation, e.g., about 3 hours to about 24 hours prior to transplantation, e.g., about 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplantation). The therapeutic agent may be administered post-transplantation (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 4 weeks after transplantation). In some embodiments, the therapeutic agent is administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the therapeutic agent is administered daily, weekly, or once every other week for a treatment period of 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the therapeutic agent (e.g., rituximab) is administered at a dosage of about 1 mg / kg to about 40 mg / kg (e.g., about 10 mg / kg). In some embodiments, the therapeutic agent is administered intravenously. In some embodiments, rituximab is administered at a dosage of about 250 mg / m 2 ~about 500mg / m 2 For example, a dosage of about 375 mg / m 2 is administered at a dosage of

[0183] Decrease in T cells The methods described herein may further include administering a therapeutic agent that depletes or reduces T cells in the subject. The therapeutic agent may be, for example, an antithymocyte globulin, such as THYMOGLOBULIN® or ATGAM®. The therapeutic agent may be administered prior to transplant (e.g., about 1 hour to about 24 hours prior to transplant, e.g., about 3 hours to about 24 hours prior to transplant, e.g., about 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours prior to transplant). In some embodiments, the therapeutic agent is administered on the same day as transplant. The therapeutic agent may be administered post-transplant (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 4 weeks after transplant). In some embodiments, the therapeutic agent is administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the therapeutic agent is administered daily, weekly, or once every other week for a treatment period of 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the therapeutic agent is administered once the subject has achieved hemostasis, within about 1 hour to about 24 hours after transplantation.

[0184] In some embodiments, the therapeutic agent (e.g., an anti-thymocyte globulin, e.g., THYMOGLOBULIN® or ATGAM®, or an anti-IL-2Rα receptor antibody) is administered at a dosage of about 1 mg / kg to about 10 mg / kg, e.g., about 2 mg / kg or about 5 mg / kg. In some embodiments, the therapeutic agent is THYMOGLOBULIN® and is administered at a dosage of about 2 mg / kg. In some embodiments, the therapeutic agent is ATGAM® and is administered at a dosage of about 5 mg / kg. The therapeutic agent may be administered intravenously.

[0185] Additional Therapeutic Agents The methods described herein may further include administration of additional therapeutic agents, for example, prior to transplantation, on the same day as transplantation, or after transplantation. The methods may include administration of any drugs necessary to maintain suppression of the immune system or promote post-operative recovery. Methods may include administration of steroids (e.g., methylprednisolone), antihistamines (e.g., diphenhydramine), H2 receptor blockers (e.g., famotidine), antivirals (e.g., ganciclovir), complement inhibitors (e.g., C1 esterase inhibitors), immunosuppressants (e.g., anti-IL-6R antibodies such as tocilizumab and / or mycophenolate mofetil), anti-inflammatory agents (e.g., TNFα proteins, e.g., etanercept, or nonsteroidal anti-inflammatory agents (NSAIDs, e.g., aspirin and naproxen). The method may further include administering one or more of the following: anticoagulants (e.g., heparin and aspirin, or other known agents), anticoagulants (e.g., ceftriaxone or other known agents). For example, the method may include administering one or more of methylprednisolone, diphenhydramine, famotidine, ganciclovir, C1 esterase inhibitors, tocilizumab, etanercept, mycophenolate mofetil, heparin, aspirin, and ceftriaxone.

[0186] The methods described herein may further include administering a steroid, such as methylprednisolone, or other steroids known in the art. In some embodiments, methylprednisolone is administered once a day or twice a day. Methylprednisolone may be administered at a dosage of about 2 mg / kg. Methylprednisolone may be administered before transplantation. Methylprednisolone may be administered one day before transplantation. Methylprednisolone may be administered on the same day as transplantation. Methylprednisolone may be administered after transplantation. Methylprednisolone may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, and / or 6 months after transplantation. Methylprednisolone may be administered for a treatment period of 2 months, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

[0187] The methods described herein may further comprise administering an antihistamine such as diphenhydramine, or other antihistamines known in the art. Diphenhydramine may be administered at a dosage of about 50 mg. Diphenhydramine may be administered intravenously. Diphenhydramine may be administered before transplantation. Diphenhydramine may be administered 1 day before transplantation. Diphenhydramine may be administered on the same day as transplantation. Diphenhydramine may be administered after transplantation. Diphenhydramine may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 4 weeks after transplantation. Diphenhydramine may be administered for a treatment period of 2 months, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

[0188] The methods described herein may further comprise administering an H2 receptor blocker, such as famotidine, or other H2 receptor blockers known in the art. Famotidine may be administered at a dosage of about 20 mg. Famotidine may be administered once a day or twice a day. Famotidine may be administered before transplantation. Famotidine may be administered one day before transplantation. Famotidine may be administered on the same day as transplantation. Famotidine may be administered after transplantation. Famotidine may be administered one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, and / or eight weeks after transplantation. Famotidine may be administered for a treatment period of 2 months, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

[0189] The methods described herein may further include administering an antiviral agent such as ganciclovir, or another antiviral agent known in the art. Ganciclovir may be administered at a dosage of about 5 mg / kg. Ganciclovir may be administered before transplantation. Ganciclovir may be administered 1 day before transplantation. Ganciclovir may be administered on the same day as transplantation. Ganciclovir may be administered after transplantation. Ganciclovir may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, and / or 10 years after transplantation. Ganciclovir may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Ganciclovir may be administered daily for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0190] The methods described herein may further include administering a complement inhibitor, such as a C1 esterase inhibitor, or other complement inhibitors known in the art. The C1 esterase inhibitor may be administered at a dosage of about 20 U / kg. The C1 esterase inhibitor may be administered before transplantation. The C1 esterase inhibitor may be administered one day before transplantation. The C1 esterase inhibitor may be administered the same day as transplantation. The C1 esterase inhibitor may be administered after transplantation. The C1 esterase inhibitor may be administered one day, two days, three days, four days, five days, six days, and / or one week after transplantation.

[0191] The methods described herein may further include administering an immunosuppressant, such as tocilizumab, or another immunosuppressant known in the art. Tocilizumab may be administered at a dosage of about 8 mg / kg. Tocilizumab may be administered intravenously. Tocilizumab may be administered on the same day as transplantation. Tocilizumab may be administered after transplantation. Tocilizumab may be administered 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and / or 12 months after transplantation. Tocilizumab may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Tocilizumab may be administered monthly for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0192] The methods described herein may further include administering an anti-inflammatory agent, such as etanercept, or another anti-inflammatory agent known in the art. Etanercept may be administered at a dosage of about 0.7 IU / kg. Etanercept may be administered subcutaneously. Etanercept may be administered on the same day as transplantation. Etanercept may be administered after transplantation. Etanercept may be administered 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and / or 12 months after transplantation. Etanercept may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Etanercept may be administered weekly for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0193] The methods described herein may further comprise administering an immunosuppressant such as mycophenolate mofetil, or another immunosuppressant known in the art. Mycophenolate mofetil may be administered at a dosage of about 20 mg / kg. Mycophenolate mofetil may be administered intravenously. Mycophenolate mofetil may be administered once a day or twice a day. Mycophenolate mofetil may be administered on the same day as transplantation. Mycophenolate mofetil may be administered after transplantation. Mycophenolate mofetil may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, and / or 10 years after transplant. Mycophenolate mofetil may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Mycophenolate mofetil may be administered daily for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0194] The methods described herein may further include administering an anticoagulant such as heparin, or another anticoagulant known in the art. Heparin may be administered on the same day as transplantation. Heparin may be administered after transplantation. Heparin may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, and / or 10 years after transplantation. Heparin may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Heparin may be administered daily for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0195] The methods described herein may further include administering a nonsteroidal anti-inflammatory drug (NSAID), such as aspirin. The aspirin may be administered at a dosage of about 81 mg. The aspirin may be administered after transplantation. The aspirin may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, and / or 10 years after transplantation. Aspirin may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime. Aspirin may be administered daily for a treatment period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime.

[0196] The methods described herein may further include administering an antibiotic, such as ceftriaxone, or another antibiotic known in the art. Ceftriaxone may be administered at a dosage of about 50 mg / kg. Ceftriaxone may be administered after transplantation. Ceftriaxone may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, and / or 2 weeks after transplantation. Ceftriaxone may be administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2 weeks. Ceftriaxone may be administered daily for a treatment period of 1 week.

[0197] Pharmaceutical Compositions The present invention features a method of treating or reducing the risk of transplant rejection or extending the time before transplant rejection occurs by administering a pharmaceutical composition containing an anti-CD40 antibody or antigen-binding fragment thereof, such as those described herein (e.g., an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 as set forth in SEQ ID NOs: 1-3, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NOs: 4-6, respectively, e.g., an antibody or antigen-binding fragment thereof having a heavy chain comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 7 or 9, and a light chain comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 8 or 10). The antibody or antigen-binding fragment thereof can be formulated in the pharmaceutical composition at a concentration of about 50 to about 300 mg / mL, and optionally in a volume of about 0.5 mL to about 2.0 mL (eg, about 1.0 mL).

[0198] The antibody or antigen-binding fragment thereof (e.g., KPL-404) may be incorporated into a pharmaceutical composition (e.g., at a concentration of about 50 mg / mL to about 300 mg / mL, such as about 100 mg / mL or about 200 mg / mL). The composition may include a polar excipient, such as a sugar, a polyol, or an amino acid. In some embodiments, the sugar is, for example, sucrose, trehalose, fructose, lactose, dextrose, or mannitol. In some embodiments, the polyol is, for example, polyethylene glycol or sorbitol. In some embodiments, the amino acid is one or more of alanine, arginine, aspartic acid, asparagine, carnitine, citrulline, ornithine, glycine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. The composition may also include, for example, sodium acetate (e.g., in salt form) or its ionic form acetate. In some embodiments, the composition is, for example, sucrose, arginine, glutamate, sorbitol, or a combination thereof. In some embodiments, the excipient is sucrose. In some embodiments, the polar excipient is arginine. In some embodiments, the excipient is glutamate. In some embodiments, the excipient is a mixture of arginine and glutamate. In some embodiments, the excipient is sorbitol.

[0199] A pharmaceutical composition can include, for example, an amount of an anti-CD40 antibody or antigen-binding fragment thereof described herein (e.g., KPL-404), such as, for example, about 50 mg / mL to about 300 mg / mL.

[0200] The antibody or antigen-binding fragment thereof may be provided in a container that contains the pharmaceutical composition described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container may hold a pharmaceutical composition, such as those described herein, effective for treating a condition, and may have a sterile access port. For example, the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle. A label on or associated with the container indicates that the composition is used for treating the selected condition. The kit may further include a second container with a pharma- ceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. The kit may further include other items desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0201] The antibody or antigen-binding fragment thereof may be present in the container at a concentration of about 50 mg / mL to about 300 mg / mL (e.g., 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, or 300 mg / mL), such as a concentration of about 100 mg / mL or about 200 mg / mL.

[0202] The composition may be formulated in a single-use vial with an extractable volume of about 1.0 mL or 2.0 mL. The composition can be formulated in a volume of about 0.1 mL to about 2.0 mL (e.g., 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, or 2.0 mL), such as a volume of about 1.0 mL. EXAMPLES

[0203] The following examples of specific modes for carrying out the present disclosure are offered for illustrative purposes only and are not intended to limit the scope of the disclosure in any way. EXAMPLES

[0204] The anti-CD40 antibody KPL-404 can be used to treat or reduce (e.g., inhibit or prevent) the risk of xenograft rejection, and treatment can be performed in conjunction with additional immunosuppressive therapy. The method was performed in a pig-to-baboon orthotopic xenograft heart transplantation model, using baboons that received transplants of hearts derived from pigs genetically engineered to contain 10 genetic modifications that eliminate expression of antigens known to elicit humoral and cellular immune response reactions.

[0205] Animal models Conventional Papio aubis baboons weighing 15-30 kg were screened to remove specific pathogens of interest and used as recipients. Weight-matched German Landrace pigs with the genetic properties described in Table 2 were used as heart donors (supplied by Revivicor Inc., Blacksburg, VA). Orthotopic porcine heart transplants were performed in baboons. [Table 2-1] [Table 2-2]

[0206] Immunosuppressive regimens Immunosuppressive regimens for the recipient baboons included induction and maintenance therapy, as shown in Table 3. Induction immunotherapy included antithymocyte globulin (THYMOGLOBULIN®), anti-CD20 antibody (RITUXAN®), and humanized anti-CD40 antibody (KPL-404) to block the CD40 / CD154 costimulatory pathway, as well as the anti-inflammatory drugs tocilizumab and etanercept. A C1 esterase inhibitor (Verinart) was used to inhibit complement activation. Maintenance immunotherapy included administration of mycophenolate mofetil (MMF) and anti-CD40 antibody (KPL-404), as well as the anti-inflammatory drugs tocilizumab and etanercept. The baboons received continuous heparin infusions to maintain activated clotting time (ACT) levels at twice baseline. Ganciclovir was administered daily to prevent cytomegalovirus (CMV) infection. Other medications include Epogen and Cefazolin. [Table 3]

[0207] Transplantation technique A preoperative transthoracic echocardiogram (TTE) was performed to ensure adequate cardiac function without anatomical abnormalities that would be deemed unsuitable for transplantation. Procurement of donor porcine hearts was performed using 30cc / kg blood cardioplegia (XVIVO© perfusion solution (Gothenburg, Sweden)) with XVIVO© cardioplegia solution (XHS) for induction preservation.

[0208] Heart preservation was performed using an XVIVO (Copyright) perfusion system with XHS cardioplegia solution at 8 °C, maintaining a perfusion pressure of 20 mmHg and physiological pH (7.2-7.6) at the aortic root. Life-sustaining xenocardial transplantation was performed after subjecting baboon recipients to aorto-caval cardiopulmonary bypass (CPB). Donor porcine xenografts were placed in the orthotopic position after explantation into the native heart using a bicaval anastomosis technique.

[0209] result Graft function was monitored via telemetry and regular echocardiography. Blood tests were performed weekly to measure troponin, ACT, chemistry, and CBC. Endomyocardial biopsies were performed periodically. Rejection was determined by an increase in troponin levels, a drop in pressure on telemetry, and loss of graft function on echocardiography. Blood tests were also performed whenever the baboon was anesthetized or for any reason. Rejection, if present, was assessed by histology and immunohistochemistry.

[0210] Each time blood was drawn for the blood tests described above, another blood sample was drawn according to routine procedures and stored for later evaluation using pharmacokinetic and anti-drug antibody (ADA) assays. Blood samples were also evaluated for PK and ADA analysis.

[0211] Thirty-three days after surgery, the baboon had recovered without signs of transplant rejection, as measured by echocardiograms and troponin levels, a marker of cardiac function: echocardiograms showed normal function and troponin plasma levels were normal.

[0212] The treatment did not completely eradicate T cells in the recipient baboons. ATGAM® reduces the levels of T cells to approximately 10% of pre-treatment levels. Thus, anti-CD40 treatment and mycophenolate mofetil (MMF) treatment antiproliferative agents were used to attenuate T cell functions such as T cell cytotoxicity (e.g., via CD40 blockade on antigen presenting cells). Similarly, THYMOGLOBULIN® is expected to shrink T cells to approximately 1-10% in an equivalent procedure performed in humans. Thus, it is expected that anti-CD40 treatment in human subjects undergoing transplants (whether allogeneic or xenogeneic) would mitigate transplant rejection caused by any remaining B or T cells, given that the treatment does not completely ablate these cells in the subject. Anti-CD40 treatment is also expected to reduce or inhibit transplant rejection as B and T cells begin to regenerate after B and T cell reductant treatment is stopped or tapered.

[0213] The baboons received high doses of KPL-404 antibody (e.g., 25-50 mg / kg), which was done to ensure an excess of antibody over the amount that would be necessary to treat or reduce the risk of transplant rejection. Thus, it will be understood that a reduction in the risk of transplant rejection can be achieved in a subject (e.g., a human subject receiving a dose of KPL-404 of less than 25-50 mg / kg, such as a dose of about 1 mg / kg to about 20 mg / kg (e.g., 5 mg / kg or 10 mg / kg).

[0214] B and T cell counts were assessed post-transplant. Figures 6A-6C show the percentage of B and T cell counts in the peripheral blood relative to the total blood lymphocyte count, and Figures 6D-6F show the absolute numbers of B and T cells in the peripheral blood. These graphs show that T cells were still present in the animals at the time of transplant, and T cell counts increased steadily over time immediately after transplant, while B cell counts remained low post-transplant, possibly due to the action of the B cell-depleting antibody, rituximab. EXAMPLES

[0215] We describe a 57-year-old man with nonischemic cardiomyopathy dependent on venoarterial extracorporeal membrane oxygenation without standard therapeutic candidates, including conventional allografts, who was transplanted with a heart from a genetically modified porcine source animal with 10 individual gene edits. Immunosuppression was based on CD40 blockade. The patient was able to be weaned from ECMO, and the xenograft functioned normally for 48 days, when he developed acute biventricular hypertrophy and diastolic dysfunction. The support was removed on postoperative day (POD) 60. At necropsy, the xenograft was edematous and nearly doubled in weight. Histological examination revealed scattered myocyte necrosis, interstitial edema, red blood cell extravasation, and prominent endothelial nuclei, without evidence of microvascular thrombosis, inconsistent with typical transplant rejection.

[0216] The patient presented with chronic mild thrombocytopenia, hypertension, nonischemic cardiomyopathy (NICM), and previous mitral valve repair. The patient required hospitalization for severe heart failure with a left ventricular ejection fraction (LVEF) of 10%. The patient's care was escalated to include multiple intravenous inotropes, and placement of an intra-aortic balloon pump was added on hospital day of death (HOD) 11. Despite this, the patient suffered multiple ventricular arrhythmias with arrests requiring resuscitation and was urgently placed on peripheral venoarterial (VA) extracorporeal membrane oxygenation (ECMO) on HOD day 23.

[0217] Due to severe sarcopenia and non-ambulatory status for 3 weeks, the patient did not meet the criteria for allograft or ventricular assist device (VAD). There were also concerns about the efficacy of a VAD due to severe biventricular failure with ventricular arrhythmias. Given single organ failure, the patient was considered for experimental xenotransplantation. Despite biventricular failure, the patient demonstrated preserved renal function and required only intermittent nasal cannulae for mild hypoxia. The pretransplant ICU course was significant for adrenal insufficiency, gastrointestinal bleeding, bacteremia cleared with antimicrobial therapy, and drug-induced leukopenia. The patient received a xenotransplant from a genetically modified porcine source animal. Human subjects generally received transplantation of xenotransplant porcine heart grafts according to the following protocol: [Table 4] [Table 5] [Table 6]

[0218] method Genetically engineered porcine source animals The pigs are supplied by Revivicor, Inc. and are clonally derived from fibroblasts, a cell line that contains 10 gene edits (see, e.g., U.S. Pat. Nos. 10,912,863 and 11,179,496 and US 2018 / 0249688, each of which is incorporated by reference in its entirety) to make the xenogeneic heart graft more compatible for transplantation into humans.

[0219] Preservation of xenografts Pig hearts were procured from source animals. Nonischemic perfusion of a 328 gm porcine heart using the XVIVO system continued for 114 minutes. The perfusion fluid was cooled to 8 degrees Celsius. It consisted of 4 parts Steen Cardiac Solution mixed with 1 part recipient type-matched human blood. Perfusion was fixed at 20 mmHg at the aortic root. Flow increased from 148 cc / min to 194 cc / min, suggesting coronary relaxation. Total cold ischemia time was 150 minutes. The implant required 63 minutes and was interrupted three times for intermittent cardiac arrest with XVIVO perfusion fluid taken from the circuit. An additional 13 minutes of circulatory arrest time was required after initial resuscitation of the heart for repair of a type A aortic dissection caused by aortic clamping. After both the first and second circulatory arrest and rewarming, the heart began to beat spontaneously with only temporary need for epicardial pacing.

[0220] surgical technique

[0221] The XVIVO system was transferred from the laboratory operating room to the hospital operating room. Redo sternotomy of the anesthetized patient was delayed until the donor heart was delivered. Transplantation was performed using a bitrial anastomosis, followed by isolation from cardiopulmonary bypass after xenotransplantation.

[0222] Immunosuppression and monitoring Rituximab and antithymocyte globulin (ATG) were used for B- and T-cell depletion, respectively, and a complement C1 esterase inhibitor (Verinert, King of Prussia, PA) was used for complement inhibition. A humanized monoclonal antibody (KPL-404, Kiniksa Pharmaceuticals, Hamilton, Bermuda) was administered to block CD40 costimulation. Pulsed doses of methylprednisolone (1,000 mg, day of xenotransplantation) were also administered. Maintenance immunosuppression included mycophenolate mofetil (MMF), KPL-404, and short-term tapered methylprednisolone (tapered from 125 mg to 30 mg, daily). PBMCs were monitored by flow cytometry for B (CD20+) and T (CD3+) lymphocytes. KPL-404 levels were monitored over time at peak, trough, and longitudinally. Before transplantation, donor-specific IgM and IgG antibody levels were acceptably low.

[0223] The xenografts were closely monitored by serial transthoracic echocardiography (TTE) using a Phillips EPIQ CVx Ultrasound system and X5-1 transducer at least twice weekly. Right ventricular endomyocardial biopsy (embx) and right heart catheterization for compression were planned for regular monitoring and, if necessary, permitted by clinical status. Histological examination was performed by hematoxylin and eosin (H&E) staining, and immunohistochemistry (IHC) was performed for CD3, CD20, CD68, CD3d, and CD4d markers on embx specimens.

[0224] Serum levels of troponin I were followed longitudinally. Xenograft-derived cell-free DNA (xd-cfDNA) was collected weekly and determined off-site by CareDx (Brisbane, CA).

[0225] Donor-specific antibody (DSA) assay Heat-inactivated patient serum was incubated with aortic endothelial cells (pAEC) from 10GE source animals for 2 hours at 4°C. Untreated pAEC were used as a negative assay control. Heat-inactivated serum samples from healthy subjects known to have high (high human) and low (low human) levels of anti-non-gal antibodies served as controls. After incubation, pAEC were washed twice with PBS and nonspecific protein binding sites were blocked with 10% normal goat serum (Abcam, MA, USA) for 20 minutes at 4°C. Fluorescently conjugated (FITC or Alexa-Fluor 488) goat anti-human IgM and IgG (Jackson ImmunoResearch Laboratories, PA, USA) were then added (final concentration of 1:100 in PBS) and allowed to incubate for 60 minutes at 4°C. After incubation, pAEC were washed twice with PBS and resuspended for flow cytometry (BD Accuri C6 Plus, CA, USA). Ten thousand events were counted per sample, and samples were analyzed using FlowJo software (FlowJo LLC, OR, USA).

[0226] result The organ transplant was successful and the subject recovered from surgery with no signs of xenograft hyperacute or acute rejection observed 3 days after surgery.

[0227] Various analyses were performed after surgery. As a result of changes in kinetics in subjects after surgery, the administration protocols for Tables 4-6 above were modified before, during, and after transplantation.

[0228] During the transplant procedure, blood loss was estimated to be >3 liters (KPL-404 was administered after hemostasis). Intraoperatively, 1800 cc of Cell Saver (washed packed red blood cells) was administered. Blood products infused included 8 units PRBCs, 4 units platelets, 6 units FFP (with additional C1 esterase inhibitor (Verinert) to account for infused complement), 341 cc Cryo, 24 mcg Desmopressin (DDAVP®), and 2226 mg Fibrinogen (Tables 7 and 8). [Table 7] [Table 8]

[0229] The net output was 3,767 mL (total input 13,997 mL - total output 10,230 mL).

[0230] Approximately 4-5 liters of crystalloids were used to resuspend and reinfuse lost recaptured / red blood cells during the intraoperative / perioperative period. An additional unit of exogenous PRBCs, including 1 unit of cryoprecipitate (spare FFP after state (s / p) of extra C1 esterase inhibitor (Verinert)), 1 unit of pRBCs, and 5 units of platelets, were given as intraoperative and postoperative infusions. Both preoperative and postoperative B-cell and T-cell counts (and / or lymphocyte counts) were measured from day -1 to day 7 (Figure 1). The following dosages indicate the cumulative dose of each drug administered to the subject at the indicated time points of blood sample collection for flow cytometry: Day -1: pre-induction; Day 0: s / p Total: Rituximab 375 mg / m 2 Day 1: s / p total: rituximab 375 mg / m 2 and s / p 1 mg / kg antithymocyte globulin; day 3: s / p rituximab 375 mg / m 2, s / p 2 mg / kg antithymocyte globulin. Flow cytometry analysis of peripheral blood flow showed depletion of B cells by day 0 (see quadrant 3) and T cells by day 3 (see quadrant 1). Notably, in this case, ATG was not administered until after surgery (day 1), so T cells were still present immediately after surgery, as shown in quadrant 1 of Figure 1, on days 0 and 1. Although B cells were largely ablated in the periphery, there are other sources of CD40 that may contribute to transplant rejection. CD40 is present on several other cell types, including antigen-presenting cells (APCs), which may contribute to T cell activation and transplant rejection. Furthermore, platelet-derived CD154 (CD40L) release as a result of surgery may be sufficient to initiate transplant rejection independent of any cellular source of this molecule (Xu et al., J. Clin. Invest. 2006;116(3):769-774). CD154 is a cell surface molecule expressed on activated T cells that binds to CD40, an activation molecule on APCs that is thought to promote graft rejection. Collectively, this supports the role of CD40 blockade in reducing the risk of transplant rejection, even in subjects that have undergone peripheral B cell and / or T cell ablation. The absence of transplant rejection, especially hyperacute and acute rejection, in treated subjects indicates the effectiveness of KPL-404 in mediating CD40 blockade and reducing transplant rejection.

[0231] Flow cytometry analysis was performed on lymph nodes removed from subjects on the day their chests were closed after heart transplant (day 2). At the time of chest closure, the cumulative dose of each drug administered to the subjects was as follows: s / p total: Rituximab 375 mg / m 2 , s / p 2 mg / kg antithymocyte globulin (Figure 2). Notably, flow cytometry analysis showed the presence of B cells (quartile 3) and T cells (quartile 1) in lymph nodes just prior to chest closure on day 2, suggesting (as expected) that rituximab and antithymocyte globulin did not completely deplete B or T cells in solid tissues, even though the cells were depleted in the bloodstream (Figure 1).

[0232] Rituximab and antithymocyte globulin were administered in the following regimens as a comparison to KPL-404: Day 1 WBC 3, Plt 90-100 CD3: Not collected Day 0 WBC 3.2, Plt 65; discharged from operating room; s / p Rituximab and KPL-404 Day -1 Day 1 WBC 5.8, Plt 78; 1mg / kg antithymocyte globulin, started MMF 500mg twice daily Day 2 WBC 12.6, Plt 74; after chest closure, 1mg / kg antithymocyte globulin, continued MMF and steroid withdrawal as scheduled, CD3 23 (absolute), CD4 17 (absolute), CD8 <10 Day 3 WBC 16.9, Plt 93; after hemostasis, 2mg / kg antithymocyte globulin, s / p chest closure and reassuring platelets, absolute CD3 <10, absolute CD4 <10, CD8 <10

[0233] For the KPL-404 preparation, 9 vials were used for each dose, 850 mg (10 mg / kg) drawn up to 8.5 ml.

[0234] Heparin was administered (bolus of 30,000 units) on day 0, and 250 mg of protamine was administered before leaving the operating room. The partial thromboplastin time (PTT) performed was 45-55 seconds. 1.5 × ULN was administered along with bivalverudin 0.06 mcg / kg / min for the past 3 days. Extracorporeal membrane oxygenation (ECMO) was removed on day 4.

[0235] Troponin levels were measured in subjects after transplantation (Figure 3). Troponin is a biomarker of transplant rejection and is released by cardiac myocytes in response to injury. Subjects showed increased serum troponin I levels postoperatively, but these levels decreased to baseline by POD24, suggesting that the heart had recovered from the surgical procedure and that transplant rejection had not occurred after the transplant procedure. Troponin I levels began to increase by POD35, but this was later determined by endomyocardial biopsy (POD50) to be unlikely to be the result of rejection, as there was no sign of antibodies or signs of acute cellular rejection (International Society for Heart and Lung Transplantation (ISHLT) grade 0).

[0236] Left ventricular ejection fraction (LVEF) remained normal or hyperdynamic throughout the postoperative course. Left ventricular (LV) and right ventricular (RV) thickness, LV cavity size (LV end-diastolic diameter), and longitudinal global strain (GLS) were altered. LV thickness measured 1.2-1.4 cm along the basal interventricular septum and posterior wall, whereas RV measured 1.0-1.1 cm on short-axis views. GLS was noted to be very compatible at baseline, with GLS scores ranging from -15 to -22 without contrast.

[0237] On POD34, embx showed no evidence of rejection and the heart revealed a right ventricular pressure of 5mmHg, a pulmonary artery pressure of 25 / 15mmHg, a cardiac index (L / min / BSA) of 2.7, and a mixed venous saturation of 65% (Hgb 8.1gm / dL). The patient was able to be rehabilitated without any cardiovascular support and the xenograft functioned normally without evidence of rejection.

[0238] On POD 43, the patient became more somnolent, was intubated, and developed hypotension, which responded to fluids and vasopressin administration. Chest x-ray suggested left lower lobe collapse and worsening right lung infiltrate. Bronchoscopy revealed diffuse shallow ulcerations throughout the right primary and secondary airways, suggestive of viral or fungal etiology, despite continued prophylaxis. Given the bronchoscopic findings, clinical decline suggestive of sepsis, and severe hypogammaglobulinemia (total IgG 185 mg / dL), antibacterial coverage was expanded and the patient was placed on 1 gm / kg IVIg (80 gm). Antiviral therapy was changed from ganciclovir to cidofovir. Biopsies of these airway lesions were performed but did not show any viral cytopathic effect or viral inclusion, and GMS staining was negative. Repeat bronchoscopy performed approximately 5 days later showed improvement in the diffuse airway ulceration. The patient was eventually extubated and resumed indoor rehabilitation.

[0239] On POD49, the patient's serum lactate increased from 4 mg / dL to 11.2 mg / dL. The patient became hypotensive, required vasopressors, and was intubated. The patient developed apical cyanosis, suggesting a decline in cardiac output for the first time since transplant. The patient underwent a negative test laparotomy. A newly placed pulmonary artery catheter revealed a mixed venous saturation of 33%. An echocardiogram revealed an EF of 65-70%, but a dramatic increase in LV wall thickness (1.7 cm) was observed, confirming RV thickness (1.4 cm). Both the LV and RV walls remained persistently thickened, independent of LV end-diastolic volume and loading conditions. The GLS became dramatically more positive. The patient was recannulated while on life support with VA-ECMO.

[0240] Endomyocardial biopsy (POD50) did not demonstrate antibody or acute cellular rejection (International Society for Heart and Lung Transplantation (ISHLT) grade 0). There was focal capillary damage with red blood cell extravasation and edema. There was more antibody staining IgG than IgM in the capillaries, but was negative for C3d or C4d. There was a single ischemic myocyte and no cellular infiltration on either H&E or IHC with CD3+ or CD68+ staining. Troponin I was elevated (Figure 9), and it was also determined that xd-cfDNA and serum levels of xenograft-specific IgG, and to a lesser extent IgM, were peaking. As an atypical case of antibody-mediated rejection was suspected, treatment was initiated with plasma exchange (initial 1.5 plasma volume exchange followed by 3 additional sessions with 1.0 plasma volume exchange over 5 days), IVIg 1gm / kg (60gm), complement inhibition (Verinert x2, Eculizumab x1), and B-cell depletion (Rituximab 375mg / m2). The patient continued to receive ECMO support.

[0241] On day 56, a repeat embx revealed ISHLT pAMR1 (improved capillary staining for IgG and IgM, but now with C4d staining present). There was interstitial red blood cell extravasation and interval reduction of edema, but embx revealed 40% myocyte necrosis. Again, there was no evidence of cellular rejection, but C4d, IgG, and IgM were weakly positive, more prominent in increased areas of necrotic myocytes (40%) that may have indicated nonspecific binding. There was focal capillary damage with red blood cell and interstitial cell extravasation within prominent intermyocyte edema. A repeat echocardiogram showed an EF of >70%, normal RV function, and improved longitudinal strain (-19.2). Biventricular wall thickening improved slightly. The patient was able to be slowly weaned from a VA-ECMO flow of 4.5 L / min to 3 L / min without the need for catecholamine support. Furthermore, echocardiography showed no improvement in wall thickness or GLS, and ECMO could not be weaned below 2 L / min.

[0242] Irreversible damage to the xenograft was observed and the support was compassionately extracted. A preliminary necropsy of the heart revealed an increase in cardiac weight from 328 gm at the time of implantation up to 600 gm. Cardiomyocytes were widely spaced with central nuclei separated by thin bands of fibrosis. Scattered myocyte necrosis was observed along with loss of myocyte integrity. Small caliber blood vessels showed prominent endothelial nuclei. Erythrocytes were observed scattered throughout the interstitial spaces between myocytes in a pattern consistent with extravasation. Additional studies are ongoing to characterize the pathophysiological mechanisms resulting in this damage.

[0243] Measurement of serum concentrations of KPL-404 Serum concentrations of KPL-404 were measured following administration of the antibody (Figure 4). After dose 1 (first part of the first induction dose), serum concentrations reached a maximum (peak) concentration of approximately 100 μg / mL and a minimum (trough) concentration of approximately 20 μg / mL before the subsequent second dose (second part of the first induction dose) was administered. This is in contrast to previous Phase I studies where normalized serum concentrations were greater than 250 μg / mL at similar time points.

[0244] An extended time course is shown in FIG. 5 and indicates that a minimum KPL-404 concentration of 20 μg / mL was sufficient to prevent transplant rejection.

[0245] The serum concentration of KPL-404 was determined using an immunoassay. The KPL-404 immunoassay used plates coated with anti-ID antibody as the capture antigen. Calibration standards and quality control (QC) samples were prepared by diluting a stock solution of KPL-404 in undiluted human serum, followed by serial dilutions with undiluted human serum to the desired KPL-404 concentration. The prepared calibrators, quality control samples, and test human serum samples were diluted to a minimum dilution factor (MRD) of 1:200 using assay diluent before being placed on the pre-coated plates. The plates were incubated to allow KPL-404 present in the samples to bind to the target, followed by washing to remove unbound material. Mouse anti-Hu IgG4 pFc HRP was then added to the plates to bind to KPL-404. The plates were further incubated and then washed to remove unbound material. TMB substrate was then added to the plate, and the reaction was then stopped by adding 1N H2SO4. The plate was immediately read using a SPECTRAMAX®Plus 384 (detection at 450 nm, background at 650 nm) microplate reader. The OD values ​​obtained from the calibration standards (detection OD at 450 nm minus background OD at 650 nm) were fitted using the 4-PL logistic equation with 1 / y2 to calculate the KPL-404 concentrations in the quality control and test samples.

[0246] The calibration curve range for this method was 8000 ng / mL to 80.0 ng / mL in 100% human serum. Calibrators outside the evaluation range of the assay at 10000 ng / mL and 40.0 ng / mL in 100% human serum may be included as anchor points to facilitate curve fitting.

[0247] An updated extended time course over 60 days is shown in Figure 7, at which point no signs of transplant rejection were observed in the subject. The time course of therapeutic dosing corresponding to the times shown in Figure 7 is provided in Table 9. [Table 9-1] [Table 9-2] [Table 9-3]

[0248] The dotted line in Figure 7 corresponds to the original calculation of the concentration of KPL-404 (e.g., as shown in Figure 5). Due to slight discrepancies in the assay, these samples were re-run using the same protocol, but on a different machine. The results of the re-analysis are shown in the solid line. The overall trends are similar in the dotted and solid lines, but there are slight changes in the peaks. One hypothesis for the discrepancies could be that the original assay was less sensitive and therefore provided a higher readout than was actually present.

[0249] As shown in FIG. 7, on day 44, the subject was treated with thoracentesis to remove pleural fluid, which reduced the KPL-404 antibody level to approximately 100 μg / mL. 1.6 L of pleural fluid was collected from the subject's lungs. The concentration of KPL-404 present in the pleural fluid was, unexpectedly, nearly identical to that present in the subject's plasma. On days 51, 53, and 54, the subject underwent plasma exchange, in which the plasma was separated from the remaining blood components of the subject. The plasma was removed, thus resulting in a reduction in the KPL-404 antibody level to approximately 50-75 μg / mL.

[0250] B and T cell counts were assessed post-transplant. Figure 8A and Figure 8B show the percentage of B and T cell counts in peripheral blood relative to total blood lymphocyte counts. These graphs show that T cells were still present in the subjects at the time of transplantation, and that immediately after transplantation, T cell counts steadily increased over time. In contrast, B cell counts remained low post-transplant, possibly due to the action of rituximab, a B cell-depleting antibody. The use of CD40-based immunomodulatory regimens and genetically modified xenografts avoided hyperacute rejection in the immediate post-operative period. Furthermore, endomyocardial biopsies of xenografts showed no acute cellular rejection. Antibody-mediated rejection was not detected by ISHLT criteria, except for mild C4d staining on POD 56 (including IgM / IgG staining and C4d staining of endotheliitis with intact myocardium, single cell infiltration, and myocardial thickening with fibrosis). In nonhuman primate studies, microthrombi, massive intracellular hemorrhage, myocyte necrosis with pyknotic nuclei, and occasional cellular infiltrates are commonly present in xenografts with antibody-mediated rejection, which were absent in this case. At preliminary necropsy, no evidence of extracardiac pathology related to the xenograft and associated treatment was observed.

[0251] IVIg was administered twice during the patient's postoperative course (Figure 7 and Table 9). The first dose on POD 43 was for infectious concerns. The second dose on POD 50 was for treatment of presumed antibody-mediated rejection after the first therapeutic plasma exchange. Notably, both doses were consistent with an increase in recipient donor-specific IgG and, to a lesser extent, IgM. At this point, it is unclear whether IVIg contributed to xenograft capillary damage. In a single report, IVIg has not been shown to have complement-dependent cytotoxicity against porcine xenograft tissue in vitro or in vivo (Yamamoto et al., Sci Rep 2020;10(1):11747). However, binding to donor-specific cells was observed in two laboratories, including ours. In the future, if IVIg is deemed necessary, lots should be tested to select those that show minimal xenograft-specific antibody-mediated cytotoxicity.

[0252] Postoperative monitoring of both xenografts and animal disease was performed using traditional clinical monitoring and included a novel highly sensitive cfDNA assay. A longitudinal xenograft-specific antibody assay was also used to aid in the detection of induced donor-specific antibody responses. These results were generally delayed by 1 week as they relied on the growth of cell cultures of source animal aortic endothelial cells, incubation of these cells with recipient serum, and analysis of antibody binding by flow cytometry. Point-of-care troponin I testing was most useful for detecting major early xenograft injury as the data were instantaneous and had been previously validated by NHP studies (Figure 9).

[0253] The donor spleen was positive for pCMV, indicating that the donor was likely latently infected with pCMV. Recipient PBMCs were also positive. It is unclear whether detection of pCMV via plasma mcf-DNA and / or PCR testing represents 1) viral replication in the xenograft, or 2) viral replication in the recipient, or 3) shedding of genetic material from the xenograft. The presence of pCMV in xenografts explanted from NHP recipients correlates with poorer outcomes than those without pCMV, although the reasons for this are currently unclear (Denner et al., Sci Rep 2020;10(1):17531). Human herpesvirus 6 (HHV6) was also detected in this patient's lung lavage, so further viral testing is warranted. HHV6 has been shown to cross-react with pCMV, and HHV6 is a significant cause of allograft rejection (Gu et al., Virology 2014;460-461:165-72; and Fiebig et al., Viruses 2017;9(11):317). EXAMPLES

[0254] A human subject is selected for allogeneic liver transplantation. The subject may be administered a regimen of therapeutic agents (e.g., induction therapy) prior to transplantation (e.g., within 1-24 hours prior to transplantation) including 125 mg methylprednisolone, 10 mg / kg rituximab, 20 U / kg C1 esterase inhibitor, and 10 mg / kg KPL-404.

[0255] The subject may undergo transplantation and receive 1000 mg of methylprednisolone during surgery. Once the subject achieves hemostasis after transplantation, the subject may also receive a therapeutic regimen (e.g., maintenance therapy) comprising 10 mg / kg KPL-404, 20 U / kg C1 esterase inhibitor, and 2 mg / kg antithymocyte globulin.

[0256] The next day (24 hours after surgery), subjects may receive a regimen of additional therapeutic agents including 20 U / kg of C1 esterase inhibitor and 2 mg / kg of antithymocyte globulin.

[0257] Beginning one week after transplantation, the subject may receive regular intravenous infusions of KPL-404 at a dose of 10 mg / kg (e.g., at least one dose every two or three weeks). The subject's blood is analyzed periodically to measure the subject's serum concentration of KPL-404. When the subject's serum concentration of KPL-404 is within 10% of 150 μg / mL (i.e., falls below 165 μg / mL), a maintenance dose of KPL-404 may be administered intravenously to boost the subject's serum concentration of KPL-404. The time between maintenance doses may be empirically determined based on the subject's PK profile and peak and trough levels of KPL-404. EXAMPLES

[0258] A human subject is selected for xenogeneic kidney transplantation. The kidney transplantation may include induction therapy with a biological lymphocyte depleting agent initiated before, at, or immediately after transplantation. For example, the subject may be treated with standard of care immunosuppression, including rituximab and antithymocyte globulin therapy, as described, for example, in Yin et al. Chin. Med. J. 124:1928-1932, 2011, or Amer. J. Transplantation, 9: Suppl 3, 2009, S1-155, each of which is incorporated herein by reference in its entirety. The subject may be administered a regimen of therapeutic agents (e.g., induction therapy) including B cell and / or T cell depleting agents prior to transplantation (e.g., within 1-24 hours prior to transplantation). Following administration of the B cell depleting agent or T cell depleting agent, the subject may be administered a first induction dose (i.e., conditioning dose) of 1-20 mg / kg (e.g., 5 mg / kg or 10 mg / kg) of KPL-404 subcutaneously or intravenously. The subject may also be administered one or more additional agents during the intraoperative transplantation, such as methylprednisolone (e.g., about 1000 mg).

[0259] After transplantation, once the subject has achieved hemostasis, the subject may also be administered a second dose (e.g., an induction dose) of 1-20 mg / kg (e.g., 5 mg / kg or 10 mg / kg) of KPL-404 subcutaneously or intravenously, and optionally 20 U / kg of a C1 esterase inhibitor and / or a T cell depleting agent.

[0260] The next day (24 hours after surgery), the subject may optionally receive an additional regimen of therapeutic agents including a T cell depleting agent.

[0261] The subject may also receive a second induction dose of KPL-404 administered intravenously or subcutaneously 2-10 days after surgery, either as split doses of at least two doses of 1-20 mg / kg (e.g., 5 mg / kg or 10 mg / kg per dose) administered 1-24 hours apart, or as a single undivided dose of 1-20 mg / kg (e.g., 5 mg / kg or 10 mg / kg per dose). The C of KPL-404 is then monitored until the PK of KPL-404 is replicable or predictable in the subject (as determined by periodic measurements of KPL-404 concentrations in the subject's blood, plasma, or serum). トラフ Additional induction doses of KPL-404 are delivered to the subject as needed to maintain the level at or above therapeutic levels.

[0262] Once the PK of KPL-404 in the subject's blood, plasma, or serum is replicable or predictable, the subject can receive regular intravenous infusions of KPL-404 at a dose of 10 mg / kg (i.e., maintenance doses, e.g., at least one dose every 1, 2, or 3 weeks). Optionally, the subject's blood is analyzed periodically to measure the blood, plasma, or serum concentration of KPL-404. When the subject's blood, plasma, or serum concentration of KPL-404 is within 10% of 10 μg / mL (i.e., falls below 11 μg / mL), a maintenance dose of KPL-404 may be administered intravenously to boost the subject's blood, plasma, or serum concentration of KPL-404. The optimal time between maintenance doses can be empirically determined based on the subject's PK profile and peak and trough levels of KPL-404.

[0263] The subject's troponin levels can be measured periodically to determine the level of transplant rejection. If the troponin levels increase, the subject may be administered an infusion of KPL-404 or an increased dose of KPL-404, as further described herein.

[0264] Other embodiments

[0265] Although particular embodiments of the present invention have been described and illustrated, such embodiments are merely illustrative of the invention and should not be construed as limiting the invention as interpreted according to the appended claims.

[0266] All publications and patent applications cited in this specification are incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference for all purposes. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

Claims

1. A pharmaceutical composition comprising an anti-CD40 antibody or antigen-binding fragment thereof for use in treating or reducing the risk of transplant rejection or prolonging the period before the onset of transplant rejection in a human subject undergoing or who has received a transplant, wherein the anti-CD40 antibody or antigen-binding fragment thereof is formulated to be administered subcutaneously or intravenously about 10 hours to about 24 hours before the transplant and within about 1 hour to about 24 hours after the transplant, and when the subject has achieved hemostasis.

2. 2. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 having the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

3. A pharmaceutical composition comprising a humanized anti-CD40 antibody or antigen-binding fragment thereof for use in treating or reducing the risk of transplant rejection or prolonging the period before the onset of transplant rejection in a human subject undergoing or who has received a transplant, wherein the humanized anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 having the amino acid sequences set forth in SEQ ID NOs: 1-3, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences set forth in SEQ ID NOs: 4-6, respectively, wherein the antibody or antigen-binding fragment thereof is formulated for subcutaneous or intravenous administration at a dose of about 1 mg / kg to about 20 mg / kg.

4. 2. The pharmaceutical composition of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:7 and the light chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

8.

5. 5. The pharmaceutical composition of claim 4, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, or wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:9 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

10.

6. 4. The pharmaceutical composition of claim 3, wherein the antibody or antigen-binding fragment thereof is formulated to be administered before the transplant, on the same day as the transplant, and / or after the transplant.

7. the antibody or antigen-binding fragment thereof (a) formulated for administration from about 10 hours to about 24 hours prior to said implantation; and / or (b) formulated to be administered within about 1 hour to about 24 hours after said transplantation and once said subject has achieved hemostasis; The pharmaceutical composition of claim 6, which is formulated as follows: (a) the antibody or antigen-binding fragment thereof is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and / or 1 year after the transplant; (b) the antibody or antigen-binding fragment thereof is administered for a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or the subject's lifetime; and / or (c) the antibody or antigen-binding fragment thereof is administered at least once a week, once every two weeks, once every three weeks, or monthly for a treatment period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or for the life of the subject; 8. The pharmaceutical composition of claim 7, formulated for administration to

9. 10. The pharmaceutical composition of claim 1, further comprising a first therapeutic agent that depletes or reduces B cells in the subject.

10. the first therapeutic agent is (a) prior to said transplantation; (b) after said transplantation; (c) During a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks; (d) daily, weekly, or once every other week for a treatment period of 1, 2, 3, or 4 weeks; (e) at a dose of about 1 mg / kg to about 40 mg / kg; and / or (f) Intravenously 10. The pharmaceutical composition of claim 9, formulated for administration.

11. 11. The pharmaceutical composition of claim 10, wherein the first therapeutic agent is formulated to be administered from about 10 hours to about 24 hours before the transplantation, and / or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 4 weeks after the transplantation.

12. 10. The pharmaceutical composition of claim 9, wherein the first therapeutic agent is rituximab.

13. The rituximab is administered at a concentration of about 250 mg / m ２ ~Approx. 500mg / m ２ 13. The pharmaceutical composition of claim 12, formulated to be administered at a dose of

14. 10. The pharmaceutical composition of claim 1, further comprising a second therapeutic agent that depletes or reduces T cells in the subject.

15. the second therapeutic agent is (a) prior to said transplantation; (b) on the same day as said transplantation; (c) after said transplantation; (d) During a treatment period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks; (e) daily, weekly, or once every other week for a treatment period of 1, 2, 3, or 4 weeks; (f) at a dose of about 1 mg / kg to about 10 mg / kg; and / or (g) Intravenously, 15. The pharmaceutical composition of claim 14, formulated for administration.

16. the second therapeutic agent is (a) from about 10 hours to about 24 hours before said implantation; (b) within about 1 hour to about 24 hours after said transplantation and once said subject has achieved hemostasis; and / or (c) 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and / or 4 weeks after said transplantation; 16. The pharmaceutical composition of claim 15, formulated for administration.

17. 15. The pharmaceutical composition of claim 14, wherein the second therapeutic agent is antithymocyte globulin.

18. The pharmaceutical composition of claim 1 , wherein the antibody or antigen-binding fragment thereof is formulated to be administered at a dosage of about 10 mg / kg.

19. The implant comprises: (a) includes the heart, kidney, lung, liver, pancreas, intestine, thymus, skin, eye, uterus, stem cells, bone, tendon, cornea, heart valve, nerve, vein, or part thereof; (b) is an allograft; (c) is a xenograft; The pharmaceutical composition of claim 1.

20. 20. The pharmaceutical composition of claim 19, wherein the xenograft comprises cells, tissues, or organs, or portions thereof, from a pig, cow, horse, dog, cat, sheep, goat, or non-human primate.

21. 21. The pharmaceutical composition of claim 20, wherein the xenograft comprises a pig heart and / or comprises cells, tissues, or organs, or portions thereof, derived from a host animal that has been genetically engineered to add, remove, or modify one or more xenoreactive antigens.

22. 10. The pharmaceutical composition of claim 1, further comprising one or more of a steroid, an antihistamine, an H2 receptor blocker, an antiviral agent, a complement inhibitor, an immunosuppressant, an anti-inflammatory agent, an anticoagulant, and an antibiotic.

23. A pharmaceutical composition containing an anti-CD40 antibody or antigen-binding fragment thereof for use in treating or reducing the risk of transplant rejection or extending the period before the onset of transplant rejection in a human subject undergoing or who has undergone a transplant, comprising: (a) an induction dose of an anti-CD40 antibody or antigen-binding fragment thereof comprising a first dose of about 1 mg / kg to about 20 mg / kg formulated to be administered subcutaneously or intravenously about 10 hours to about 24 hours prior to transplantation, and a second dose of about 1 mg / kg to about 20 mg / kg formulated to be administered subcutaneously or intravenously after transplantation once the subject has achieved hemostasis; and (b) a maintenance dose of an anti-CD40 antibody or antigen-binding fragment thereof formulated to be administered subcutaneously or intravenously at a dose of about 1 mg / kg to about 20 mg / kg for a treatment period of at least one month; A pharmaceutical composition comprising:

24. (a) The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, respectively set forth in SEQ ID NOs:1-3, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3, respectively set forth in SEQ ID NOs:4-6; (b) the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:7 and the light chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:8, optionally wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8; and / or (c) the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 10; 24. The pharmaceutical composition of claim 23.

25. 24. The pharmaceutical composition of claim 23, wherein the maintenance dose is provided to maintain a minimum blood, plasma, or serum concentration of the anti-CD40 antibody or antigen-binding fragment thereof of at least 150 μg / ml.

26. A pharmaceutical composition containing an anti-CD40 antibody or antigen-binding fragment thereof for use in treating or reducing the risk of transplant rejection or extending the period before the onset of transplant rejection in a human subject undergoing or who has undergone a transplant, comprising: (a) a first induction dose comprising a first fractional dose of about 1 mg / kg to about 20 mg / kg formulated to be administered subcutaneously or intravenously from about 1 hour to about 24 hours prior to transplantation and a second fractional dose of about 1 mg / kg to about 20 mg / kg formulated to be administered subcutaneously or intravenously once the subject has achieved hemostasis within about 24 hours after transplantation, and a second induction dose comprising a first fractional dose of about 1 mg / kg to about 20 mg / kg formulated to be administered subcutaneously or intravenously once the subject has achieved hemostasis within about 1 day to about 14 days after transplantation and a second fractional dose of about 1 mg / kg to about 20 mg / kg formulated to be administered subcutaneously or intravenously within about 1 hour to about 12 hours after administration of the first fractional dose of the second induction dose; and (b) A pharmaceutical composition comprising a maintenance dose of an anti-CD40 antibody or antigen-binding fragment thereof, formulated to be administered subcutaneously or intravenously at a dosage of about 1 mg / kg to about 20 mg / kg for a treatment period of at least one month.

27. 27. The pharmaceutical composition of claim 26, wherein the maintenance dose is provided to maintain a minimum blood, plasma, or serum concentration of the anti-CD40 antibody or antigen-binding fragment thereof of at least 10 μg / ml.

28. The pharmaceutical composition of claim 26, further comprising a third induction dose of about 1 mg / kg to about 20 mg / kg of the antibody or antigen-binding fragment thereof, formulated to be administered within about 1 day to about 14 days of the second induction dose.

29. A pharmaceutical composition containing an anti-CD40 antibody or antigen-binding fragment thereof for use in treating or reducing the risk of transplant rejection or extending the period before the onset of transplant rejection in a human subject undergoing or who has undergone a transplant, comprising: (a) induction doses of an anti-CD40 antibody or antigen-binding fragment thereof, comprising a conditioning dose of about 1 mg / kg to about 20 mg / kg formulated to be administered subcutaneously or intravenously about 10 hours to about 24 hours prior to transplantation and a first induction dose of about 1 mg / kg to about 20 mg / kg formulated to be administered subcutaneously or intravenously at a time point after transplantation and after the subject has achieved hemostasis; and (b) A pharmaceutical composition comprising a maintenance dose of an anti-CD40 antibody or antigen-binding fragment thereof, formulated to be administered subcutaneously or intravenously at a dosage of about 1 mg / kg to about 20 mg / kg for a treatment period of at least one month.

30. The pharmaceutical composition of claim 29, further comprising at least one or more additional induction doses of the antibody or antigen-binding fragment thereof of about 1 mg / kg to about 20 mg / kg, formulated to be administered within about 1 day to about 14 days of the first induction dose.

31. 31. The pharmaceutical composition of claim 30, wherein step (b) is initiated once the PK of the anti-CD40 antibody or antigen-binding fragment thereof becomes replicable or predictable.

Images