RU2790558C2 - Antibodies to cd40 for use in the treatment of sjogren's syndrome - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a method for treating primary Sjögren's syndrome in a human, which includes administering a therapeutically effective dose of an anti-CD40 antibody to said human.

EFFECT: invention has the ability to effectively treat Sjögren's syndrome in humans.

9 cl, 27 dwg, 5 tbl, 9 ex

Description

PREVIOUS APPLICATIONS

This application claims priority of U.S. Provisional Application No. 62/581212, filed November 3, 2017, and U.S. Provisional Application No. 62/644939, filed March 19, 2018, the contents of which are incorporated herein in their entirety. volume.

FIELD OF TECHNOLOGY

The present invention relates to methods, treatment regimens, uses, kits and therapies for the treatment of Sjogren's syndrome, such as primary Sjogren's syndrome or secondary Sjogren's syndrome, using anti-CD40 antibodies such as CFZ533.

BACKGROUND OF THE INVENTION

Sjögren's syndrome (SS), such as primary Sjögren's syndrome (pSS) or secondary Sjögren's syndrome (sSS), is a common chronic autoimmune disease of unknown etiology.

Disease symptoms in patients with both primary and secondary SS include the presence of anti-Ro and anti-La autoantibodies, as well as mononuclear cell infiltrates in the salivary and lacrimal glands (Bombardieri, et al. 2012). In some cases, these accumulations of T and B lymphocytes form well-organized structures called ectopic lymphoid structures (ELS), which share morphological and functional similarities to GCs (Voulgarelis et al. 2008). Previous work reported evidence for ELS in the salivary glands of SS patients, and in preclinical models of the disease and evidence for ongoing affinity maturation (Jacobi et al. 2002; Stott et al. 1998; Bombardieri et al. 2012), linking these structures with disease pathology (Bombardieri et al. 2017).

The impact of this disease on measures of quality of life (QoL) is significant, and comparative studies show that pSS QoL has been quantified worse than congestive heart failure or many forms of cancer (Segal et al 2009; Kuenstner et al 2002; Komaroff et al 1996) . Moreover, increased B cell activity in SS results in an increased risk for malignant transformation, with the development of lymphoma being found in 5% of patients with SS. Treatment for patients with SS is limited to symptomatic care for mucosal signs and symptoms, and there is currently no evidence-based systemic therapy available for patients with SS.

Glucorticoids and typical disease-modifying antirheumatic drugs (DMARDs) are largely ineffective, and there is no effective pharmacological intervention for severe, disabling fatigue. Although there is no conclusive evidence of efficacy based on anecdotal evidence and experience with similar autoimmune diseases such as systemic lupus erythematosus, antimalarials (Tishler et al 2008), methotrexate (Winzer and Aringer 2010) or azathioprine (Kaufman et al 1999) are sometimes used , in particular for the treatment of extraglandular symptoms, including involvement of the kidneys or joints.

Although small trials with the B-cell depleting agent rituximab have demonstrated a level of therapeutic efficacy, there have not been adequate large randomized controlled trials that have demonstrated clear efficacy for disease manifestations in and outside glandular tissue. A disease-modifying agent that prevents destruction of the secretory glands and targets disease manifestations outside the glandular tissue will take the treatment of SS, such as chronic pSS, to a whole new level.

CFZ533 is a human monoclonal antibody directed against human CD40. It belongs to the IgG1 isotype subclass and contains an Fc suppressive mutation (N297A) that abolishes FcγR binding and associated effector functions such as ADCC and CDC. CFZ533 is disclosed in US8828396 and US9221913, incorporated herein by reference.

BRIEF DESCRIPTION OF THE INVENTION

Anti-CD40 human monoclonal antibodies with suppressed ADCC activity have been found to be useful in the treatment of Sjögren's syndrome. In particular, the concept study of the CFZ533 antibody demonstrated evidence that a novel treatment for clinically active pSS has been proposed.

According to a first aspect of the present invention, an anti-CD40 antibody is provided for use in the treatment of Sjögren's syndrome. In one embodiment, Sjogren's syndrome is a primary Sjogren's syndrome.

The antibody may be selected from the group consisting of:

a. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8;

b. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the hypervariable regions shown under SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and an immunoglobulin VL domain containing the hypervariable regions shown under SEQ ID NO : 4, SEQ ID NO: 5 and SEQ ID NO: 6;

c. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 13;

d. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 14; And

e. an anti-CD40 antibody that contains an IgG1 Fc region with suppressed activity.

The antibody may comprise the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, or the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12.

In one embodiment, a pharmaceutical composition is provided comprising a therapeutically effective amount of an antibody for use according to the first aspect and one or more pharmaceutically acceptable carriers.

In one embodiment, the route of administration of the antibody according to the first aspect is subcutaneous or intravenous, or a combination with the subcutaneous or intravenous route.

The dosage may be from about 3 mg to about 30 mg of the active ingredient per kilogram of the body weight of a human subject. The dose can be administered every week, every two weeks, or every four weeks.

In one embodiment, the dose is about 10 mg of the active ingredient per kilogram of the body weight of the human subject.

In one embodiment, the dose is from about 150 mg to about 600 mg of the active ingredient. The dose can be administered every week, every two weeks, or every four weeks.

In one embodiment, the dose is about 300 mg of the active ingredient.

In one preferred embodiment, the dose is 150 mg of the active ingredient. In another preferred embodiment, the dose is 300 mg of the active ingredient. In yet another preferred embodiment, the dose is 600 mg of the active ingredient.

In one embodiment, the antibody is administered via a loading dose and a maintenance dose.

In one embodiment, the administration of the loading dose is via subcutaneous injections of the first dose, and the administration of the maintenance dose is via subcutaneous injections of the second dose. The first dose may be the same as the second dose or greater than the second dose.

In one embodiment, the first dose is from about 150 mg to about 600 mg of the active ingredient, such as about 300 mg of the active ingredient, and the second dose is from about 150 mg to about 600 mg of the active ingredient, such as about 300 mg of the active ingredient.

In one embodiment, the first dose is 150 mg, 300 mg, or 600 mg of the active ingredient and the second dose is 150 mg, 300 mg, or 600 mg of the active ingredient.

In one embodiment, the administration of the loading dose comprises at least two subcutaneous injections, and the administration of the maintenance dose consists of subcutaneous injections administered weekly (Q1W), biweekly (Q2W), or monthly (Q4W). In one embodiment, the administration of the loading dose consists of two subcutaneous injections. In another embodiment, the administration of the loading dose consists of three subcutaneous injections.

In one embodiment, the subcutaneous injections for the loading dose are different doses. In another embodiment, the subcutaneous injections at the loading dose are the same dose.

According to a second aspect, a method for treating Sjögren's syndrome in a human subject is provided, comprising administering a therapeutically effective dose of an anti-CD40 antibody to said subject. In a preferred embodiment, Sjogren's syndrome is primary Sjogren's syndrome.

In one embodiment, the antibody is selected from the group consisting of:

a. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8;

b. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the hypervariable regions shown under SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and an immunoglobulin VL domain containing the hypervariable regions shown under SEQ ID NO : 4, SEQ ID NO: 5 and SEQ ID NO: 6;

c. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 13;

d. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 14; And

e. an anti-CD40 antibody that contains an IgG1 Fc region with suppressed activity.

In one embodiment, the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10, or a heavy chain amino acid sequence of SEQ ID NO: 11 and a light chain amino acid sequence of SEQ ID NO: 12.

In one embodiment, the antibody is administered together with one or more pharmaceutically acceptable carriers.

In one embodiment, the antibody is administered subcutaneously or intravenously, or in combination with subcutaneous or intravenous administration.

In one embodiment, the antibody is administered as a dose of about 3 mg to about 30 mg of the active ingredient per kilogram of the body weight of the human subject.

In one embodiment, the dose is about 10 mg of the active ingredient per kilogram of the body weight of the human subject.

In one embodiment, the antibody is administered as a dose of about 150 mg to about 600 mg of the active ingredient, such as 300 mg of the active ingredient.

In one preferred embodiment, the dose is 150 mg of the active ingredient. In another preferred embodiment, the dose is 300 mg of the active ingredient. In yet another preferred embodiment, the dose is 600 mg of the active ingredient.

In one embodiment, the antibody is administered via a loading dose and a maintenance dose.

In one embodiment, the administration of the loading dose is via subcutaneous injections of the first dose, and the administration of the maintenance dose is via subcutaneous injections of the second dose.

The first dose may be the same as the second dose or greater than the second dose.

In one embodiment, the first dose is from about 150 mg to about 600 mg of the active ingredient, such as about 300 mg of the active ingredient, and the second dose is from about 150 mg to about 600 mg of the active ingredient, such as about 300 mg of the active ingredient.

In one embodiment, the first dose is 150 mg, 300 mg, or 600 mg of the active ingredient and the second dose is 150 mg, 300 mg, or 600 mg of the active ingredient.

In one embodiment, administration of the loading dose comprises at least two subcutaneous injections and administration of the maintenance dose consists of subcutaneous injections administered weekly (Q1W), biweekly (Q2W), or monthly (Q4W).

In one embodiment, the administration of the loading dose consists of two subcutaneous injections. In another embodiment, the administration of the loading dose consists of three subcutaneous injections.

In one embodiment, the subcutaneous injections for the loading dose are different doses. In another embodiment, the subcutaneous injections at the loading dose are the same dose.

According to a third aspect, the use of a liquid pharmaceutical composition comprising an anti-CD40 antibody, a buffer, a stabilizer and a solubilizer, and a means for subcutaneously administering an anti-CD40 antibody to a patient having Sjogren's syndrome is provided for the manufacture of a medicament for the treatment of Sjogren's syndrome, wherein the antibody to CD40

a. intended for subcutaneous administration through the introduction of the first loading dose and

b. thereafter by administering a second maintenance dose, wherein said anti-CD40 antibody is selected from the group consisting of:

i. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8;

ii. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the hypervariable regions shown under SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and an immunoglobulin VL domain containing the hypervariable regions shown under SEQ ID NO : 4, SEQ ID NO: 5 and SEQ ID NO: 6;

iii. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 13;

iv. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 14;

v. antibodies to CD40, which contains the Fc region of IgG1 with suppressed activity; And

vi. an anti-CD40 antibody that contains the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, or the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12.

In a preferred embodiment, Sjogren's syndrome is primary Sjogren's syndrome.

BRIEF DESCRIPTION OF GRAPHICS

Figure 1 is a schematic representation of the study design of the first and second cohorts of the evidence-based part of the CFZ533 conceptual study in pSS patients (CCFZ533X2203).

Figure 2 is a schematic representation of the study design for the third cohort of the evidence-based part of the CFZ533 conceptual study in pSS patients (CCFZ533X2203).

Figure 3 is a graph showing pre-simulated pharmacokinetic profiles prior to the start of the evidence-based part of the CCFZ533X2203 concept study.

Figure 4 is a graph showing pharmacokinetic profiles after intravenous administration (cohort 2 of the CCFZ533X2203 study; interim analysis).

Figure 5 is a graph showing clinical scores (ESSDAI) after IV administration (cohort 2; study CCFZ533X2203).

Figure 6 is a graph showing biomarker levels (CXCL13) after IV administration (cohort 2; study CCFZ533X2203).

Figure 7 is a schematic representation of treatment outcomes for various clinical endpoints after IV administration (cohort 2; study CCFZ533X2203).

Figure 8 is a graph showing pharmacodynamic profiles (total soluble plasma CD40 after IV administration; cohort 2; study CCFZ533X2203).

Figure 9 is a graph showing clinical scores (ESSDAI) after subcutaneous administration (cohort 1; study CCFZ533X2203).

Figure 10 is a graph showing pharmacokinetic profiles after subcutaneous administration (cohort 1; study CCFZ533X2203).

Figure 11 is a graph showing pharmacodynamic profiles (total soluble CD40 after SC administration; cohort 1; study CCFZ533X2203).

Figure 12 is a graph showing clinical scores (ESSPRI) after SC administration (cohort 1; study CCFZ533X2203).

Figure 13 is a graph showing clinical scores (ESSPRI) after IV administration (cohort 2; study CCFZ533X2203).

Figure 14 is a graph showing biomarker levels (CXCL13) after IV administration (cohort 2; study CCFZ533X2203).

Figure 15 is a graph showing clinical scores (ESSDAI) after IV administration (cohort 2; study CCFZ533X2203).

Figure 16 is a graph showing ICAM and B cell levels after IV administration (cohort 2; study CCFZ533X2203).

Figure 17 is a schematic representation of treatment outcomes for various clinical endpoints (IV and SC administrations; study CCFZ533X2203).

Figure 18 is a graph showing in vitro CFZ533-mediated inhibition of rCD154-induced pathway activation.

Figure 19 is a graph showing the minimal in vitro stimulatory activity of CFZ533.

Figure 20 is a graph demonstrating that CFZ533 does not mediate cell depletion in vitro.

Figure 21 is an exemplary image of individual RI-1 B cells showing internalization of CD40 receptors after recCD154 or CFZ533 binding.

Figure 22 is a graph showing the pharmacokinetics and pharmacodynamics (target binding; no B cell depletion) of CFZ533 properties in non-human primates.

Figure 23A shows the PK/PD experimental design and vaccination study in non-human primates. Figure 23B is a graph showing plasma levels of IgG to KLH (immune response) and CFZ533 (pharmacokinetics). Figure 23C shows the results of histological analysis of germinal centers.

Figures 24A, 24B and 24C are pictorial representations of possible weekly loading doses of the active ingredient administered subcutaneously, followed by biweekly maintenance of the active ingredient (subcutaneously).

Figure 25A is a graphical representation of possible weekly loading doses of active ingredient given subcutaneously followed by active ingredient every 4 weeks (subcutaneously) and Figure 25B is a graphical representation of possible weekly loading doses of active ingredient given subcutaneously followed by active ingredient every week (subcutaneously).

Figure 26 shows the experimental results; CD40 signature in ELS in salivary glands and reduction in tertiary lymphoid organs in the salivary glands of NOD mice after 10 weeks of MR1 treatment.

Figure 27 shows the experimental results; increased percentage of AQP-5 positive cells in the salivary glands of NOD mice after 10 weeks of anti-CD154 antibody treatment.

DETAILED DESCRIPTION OF THE INVENTION

The CD40-CD154 pathway (CD154 is CD40L) is believed to play an important role in the pathogenesis of Sjögren's syndrome (SS).

Thus, any anti-CD40 monoclonal antibody capable of blocking CD40-CD154 signaling, such as an anti-CD40 antibody with ADCC suppression, would be suitable for the treatment of SS, such as primary Sjögren's syndrome (pSS).

Without wishing to be bound by theory, the present inventors have identified that sustained plasma concentrations of at least about 40 μg/mL of CFZ533 antibodies were needed during maintenance regimen to block the CD40-CD40L pathway in target tissue in patients with SS such like patients with pSS. Also, since CFZ533 undergoes a target-mediated location (which is associated with target metabolism and expression) and SS patients are characterized by high expression of CD40 in the body, a loading regimen at the beginning of treatment was necessary to fully saturate the CD40 receptors in these patients in conditions in which levels CD40s were increased, requiring higher doses or more frequent dosing regimens at the start of treatment. Thus, with a loading dose regimen that provides rapid saturation of CD40 receptors at the beginning of treatment (2-3 weeks), followed by a maintenance dose regimen that provides stable plasma concentrations of at least 40 μg / ml or at least 100 μg/ml, in situations in which CD40 expression in exposed tissues would increase (severity of the condition), is considered a therapeutic effect. The observed maximum steady state plasma concentration was approximately 300 to 400 μg/mL (Cohort 3; study CCFZ533X2203) and was generally safe and well tolerated with no significant signal to suggest an increased risk of infection. No thromboembolic complication was observed.

The appropriate dosage will differ depending on, for example, the specific CD40 pathway antagonists to be used, e.g., an anti-CD40 antibody or antigen-binding fragment thereof (e.g., mAb1, also referred to herein as CFZ533, mAb2, ASKP1240) or an anti-CD40L antibody (e.g., BIIB063 ) or antigen-binding fragment thereof, the subject to be treated, the route of administration, as well as the nature and severity of the condition being treated, and the nature of the prior treatment the patient has received. Ultimately, the attending physician will decide on the amount of CD40 pathway antagonist with which to treat each individual patient. In some embodiments, the physician may administer low doses of the CD40 pathway antagonist and observe the patient's response. In other embodiments, the initial dose(s) of the CD40 pathway antagonist administered to the patient is(are) high(s) and then titrated(s) downward until signs of relapse appear. . Higher doses of the CD40 pathway antagonist can be administered until the optimal therapeutic effect is achieved for the patient, and then the dosage is usually not increased.

In the practice of certain treatments or uses of the present invention, a therapeutically effective amount of a CD40 pathway antagonist, e.g., an anti-CD40 antibody or antigen-binding fragment thereof (e.g., mAb1, also referred to herein as CFZ533, mAb2, ASKP1240) or an anti-CD40L antibody, or the antigen-binding fragment is administered to a patient, such as a mammal (eg, a human). While it is understood that the disclosed methods provide for the treatment of patients with SS using a CD40 pathway antagonist (e.g., mAb1/CFZ533, mAb2, ASKP1240), this does not preclude that if the patient was ultimately treated with a CD40 pathway antagonist, then such therapy using a CD40 pathway antagonist is necessarily monotherapy. Indeed, if the patient is selected for treatment with a CD40 pathway antagonist, then the CD40 pathway antagonist (e.g., mAb1/CFZ533, mAb2, ASKP1240) may be administered according to the methods of the present invention, either alone or in combination with other agents and therapies. .

It will be understood that regimen changes may be appropriate for some patients with SS, such as patients with pSS, for example, patients who show an inadequate response to treatment with CD40 pathway antagonists, such as an anti-CD40 antibody or antigen-binding fragment thereof (e.g., mAb1, also referred to as herein CFZ533, mAb2, ASKP1240) or an anti-CD40L antibody or antigen-binding fragment thereof to be used. Thus, administration (eg, mAb1/CFZ533 or mAb2) may be more frequent than monthly dosing, eg, biweekly dosing (every two weeks) or weekly dosing.

Some patients may benefit from a loading regimen (eg, weekly dosing over several weeks [eg, 1-4 weeks, eg, dosing at weeks 0, 1, 2, and/or 3, such as week 2, loading schedule at weeks 0 and 1] followed by a maintenance regimen starting, for example, from week 3 or 4, where CFZ533 can be administered weekly, every two weeks, or every 4 weeks for several weeks.

For example, a suitable regimen for mAb1/CFZ533 or mAb2 may be weekly for several weeks [eg, 1-4 weeks, eg, administered at weeks 0, 1, 2, and 3] followed by a monthly maintenance regimen.

In another example, a suitable regimen for mAb1/CFZ533 or mAb2 is weekly for several weeks (e.g., 2-8 weeks, such as 3 weeks, administered at weeks 0, 1, 2) followed by a biweekly maintenance regimen .

It will also be appreciated that administration (e.g., mAb1/CFZ533 or mAb2) may be administered at a lower frequency than monthly administration, e.g., dosing every 6 weeks, every 8 weeks (every two months), quarterly (every three months), and etc.

It should be understood that dose escalation based on disease severity may be appropriate for certain patients with SS, such as patients with pSS, e.g., patients who show an inadequate response to treatment with CD40 pathway antagonists, e.g., an anti-CD40 antibody or antigen-binding fragment thereof (e.g., mAb1, also referred to herein as CFZ533, mAb2, ASKP1240) or an anti-CD40L antibody or antigen-binding fragment thereof to be used. Thus, subcutaneous (s.c.) dosages (loading or maintenance doses) may be greater than about 150 mg to about 900 mg s.c., e.g., about 75 mg, about 100 mg, about 125 mg, about 175 mg, about 200 mg, about 250 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, etc.; similarly, intravenous (i.v.) dosages may be greater than about 10 mg/kg, e.g., about 11 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg , 35 mg/kg, etc. It will also be understood that dose reduction may also be appropriate for certain patients with SS, such as patients with pSS, for example, patients who exhibit side effects or an undesirable response to treatment with a CD40 pathway antagonist (for example , an anti-CD40 antibody or antigen-binding fragment thereof (eg, mAb1, also referred to herein as CFZ533, mAb2, ASKP1240) or an anti-CD40L antibody or antigen-binding fragment thereof). Thus, dosages of a CD40 pathway antagonist (e.g., an anti-CD40 antibody or antigen-binding fragment thereof (e.g., mAb1, also referred to herein as CFZ533, mAb2, ASKP1240) or an anti-CD40L antibody or antigen-binding fragment thereof) can be less than about 150 mg to about 900 mg s.c., e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 175 mg, about 200 mg, about 250 mg, about 350 mg, about 400 mg, about 450 mg, approximately 500 mg, approximately 600 mg, etc.

In some embodiments, a CD40 antagonist, e.g., an anti-CD40 antibody or antigen-binding fragment thereof (e.g., mAb1, also referred to herein as CFZ533, mAb2, ASKP1240) or an anti-CD40L antibody, or antigen-binding fragment thereof, may be administered to a patient at an initial dose of 600 mg administered s.c., and the dose may then be adjusted to 150 mg or 300 mg delivered by s.c., if necessary, as determined by the physician.

In some embodiments, a CD40 antagonist, such as an anti-CD40 antibody or antigen-binding fragment thereof (e.g., mAb1, also referred to herein as CFZ533, mAb2, ASKP1240) or an anti-CD40L antibody, or antigen-binding fragment thereof, may be administered to the patient at an initial dose of 10 mg/kg administered i.v. and the dose may then be adjusted to 150 mg or 300 mg s.c. if necessary, as determined by the physician.

In a specific embodiment, 3 mg/kg CFZ533 is administered s.c. on day 1 (D1), day 15 (D15), D29, D57, D85, D99, D113 and D141.

In another specific embodiment, 10 mg/kg CFZ533 is administered i.v. in D1, D15, D29, D57, D85, D99, D113 and D141.

In yet another specific embodiment, a loading dose that contains four single doses of 600 mg of CFZ533 is administered s.c. once a week (Q1W), i.e. 600 mg CFZ533 s.c. in D1, D8, D15, and D22, followed by a maintenance dose that includes single doses of 300 mg administered s.c. once a week (Q1W), i.e. 300 mg CFZ533 s.c. once a week from D29 to D85.

In another specific embodiment, a loading dose that provides for a single dose of 10 mg of CFZ533 per kg of subject weight is administered i.v. once a day 1 followed by a maintenance dose, which includes single doses of 300 mg administered by s.c. once a week (Q1W), i.e. 300 mg CFZ533 s.c. once a week from D8 to D85.

CFZ533 can be administered quarterly, monthly, weekly, or biweekly, such as subcutaneously, at a dose of about 75 mg to about 600 mg, or about 150 mg to about 300 mg administered by subcutaneous injection, in a single dose of about 75 mg, approximately 150 mg, approximately 300 mg, approximately 450 mg or approximately 600 mg.

CFZ533 can be administered by subcutaneous injection, weekly, at loading doses of about 150 mg to about 600 mg, where loading doses are administered over 1-4 weeks.

A loading dose can also be administered i.v. from about 10 mg/kg to about 30 mg/kg. Also, loading doses can be administered subcutaneously weekly or biweekly at doses of approximately 150 mg, 300 mg and/or 600 mg of the active ingredient.

A loading or dosing regimen of CFZ533 (such as 150/300/600 mg administered weekly or biweekly) is preferably followed by a maintenance regimen or dosing weekly, biweekly or monthly (every four weeks). The maintenance dose is preferably 300 mg s.c. weekly or biweekly or 600 mg biweekly or 600 mg every 4 weeks.

The anti-CD40 antibody, or antigen-binding fragment thereof, may be CFZ533, a functional derivative thereof, or a biosimilar thereof.

As defined herein, "single dose" refers to s.c. dose, which may be from about 75 mg to about 900 mg, for example, from about 150 mg to about 600 mg, for example, from about 150 mg to about 600 mg, for example, from about 300 mg to about 600 mg, or, for example, from about 150 mg to about 300 mg. For example, a single s.c. the dose is about 75 mg, about 150 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg.

Definitions

As used herein, the term CD40 refers to the 40 differentiation cluster, also referred to as member 5 of the tumor necrosis factor receptor superfamily. The term CD40 refers to human CD40, for example, as defined in SEQ ID NO: 19, unless otherwise indicated.

The term "about" in relation to the numerical value of x means, for example, +/-10%. When used before a numerical range or list of numbers, the term "approximately" is used in relation to each number in the series, for example, the phrase "approximately 1-5" should be interpreted as "approximately 1 - approximately 5", or, for example, the phrase "approximately 1, 2 , 3, 4" should be interpreted as "about 1, about 2, about 3, about 4, etc."

The word "substantially" does not exclude "completely", for example, a composition that "substantially does not contain" Y may not contain Y at all. If necessary, the word "actually" can be omitted from the definition of the present invention.

The term "comprising" encompasses "comprising" as well as "comprising", for example, a composition "comprising" X may consist solely of X, or may include something additional, such as X+Y.

AUC0-t denotes the area under the plasma concentration versus time curve from start time to time 't', where t is a specific point in time after administration [mass x time/volume].

AUCtx-ty is the area under the plasma concentration versus time curve from time 'x' to time 'y', where 'time x' and 'time y' are the specified times after administration.

C _max is the observed maximum plasma concentration following drug administration [mass/volume].

_Cmin is the observed minimum plasma concentration following drug administration.

C _trough is the observed plasma concentration before or at the end of the dosing interval.

T _max is the time to reach the maximum concentration after drug administration [time].

ss (label) indicates that the parameter is defined in steady state.

As used herein, the term "antibody" or "anti-CD40 antibody" and the like refers to whole antibodies that interact with CD40 (eg, via binding, steric hindrance, stabilization/destabilization, spatial distribution). A naturally occurring "antibody" is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains - CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single CL domain. The VH and VL regions can be further subdivided into regions of hypervariability, referred to as complementarity determining regions (CDRs), separated by regions that are more conserved, referred to as framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, or chimeric antibodies. The antibodies may be of any isotype (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass, preferably IgG and most preferably IgG1. An exemplary antibody includes CFZ533 (herein also referred to as mAb1) and mAb2 as shown in Table 1.

Both light and heavy chains are subdivided into regions of structural and functional homology. The terms "constant" and "variable" are used in a functional sense. In this regard, it is to be understood that variable domains from both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. In contrast, the light chain (CL) and heavy chain (CH1, CH2, or CH3) constant domains confer important biological properties such as secretion, placental transfer, Fc receptor binding, complement binding, etc. Domain numbers are assumed to be The constant region increases as they move away from the antigen-binding site or the amino-terminus of the antibody. The N-terminus is the variable region and the C-terminus is the constant region; the CH3 and CL domains actually contain the C-terminus of the heavy and light chains, respectively. In particular, the term "antibody" specifically includes the IgG-scFv format.

The term "antigen-binding portion" of an antibody (or simply "antigenic portion") as used herein refers to the full length or one or more fragments of an antibody, such as a protein, that retains the ability to specifically bind to an antigen or epitope (eg, a portion of CD40).

"Complementarity determining regions" ("CDRs") are amino acid sequences with boundaries defined using any of a number of well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme) and ImMunoGenTics (IMGT) numbering ( Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (numbering scheme" IMGT"). According to the IMGT, antibody CDRs can be determined using the IMGT/DomainGapAlign program.

The term "epitope" as used herein refers to any determinant capable of binding with high affinity to an immunoglobulin. An epitope is a region of an antigen bound by an antibody that specifically targets that antigen, and if the antigen is a protein, the epitope includes the specific amino acids that directly contact the antibody. Most often, epitopes are localized on proteins, but in some cases they can be localized on molecules of other types, such as nucleic acids. Epitopic determinants may include reactive surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structure characteristics and/or specific charge characteristics. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be combined using recombinant methods using a synthetic linker, which allows them to be converted into a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single chain Fvs (scFvs); see, for example, Bird et al., 1988 Science 242:423-426 and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883).

The phrase "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies having different antigen specificity (e.g., an isolated antibody that specifically binds CD40 is substantially free of other antibodies that specifically bind antigens other than CD40). An isolated antibody that specifically binds CD40 may, however, be cross-reactive with other antigens, such as CD40 molecules from other species. Moreover, the isolated antibody may be virtually free of other cellular material and/or chemicals. As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to a formulation of antibody molecules of a single molecular composition. The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human-derived sequences. A "human antibody" need not be produced by a human, human tissue, or human cell. Human antibodies of the present invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis, by adding N-nucleotides at in vivo junctions during recombination of antibody genes, or via somatic mutation in vivo ).

"Identity" with respect to a native polypeptide and a functional derivative thereof is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to those of the corresponding native polypeptide after sequence alignment and gapping, if necessary, to achieve maximum percent identity, and disregarding any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions should be considered a reduction in identity. Methods and computer programs for alignment are well known. Percent identity can be determined using standard alignment algorithms, such as the Basic Local Alignment Finder (BLAST) described by Altshul et al. ((1990) J. Mol. Biol., 215: 403 410); algorithm of Needleman et al. ((1970) J. Mol. Biol., 48: 444 453) or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci., 4: 11-17). The parameter set can be a Blossum 62 weight matrix with a gap penalty of 12, a gap extension penalty of 4, and a gap frame shift penalty of 5. The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989 ) CABIOS, 4:11-17), which was incorporated into the ALIGN program (version 2.0) using the PAM120 balance substitution weight table, a gap extension penalty of 12, and a gap penalty of 4.

The term "amino acid(s)" refers, for example, to all naturally occurring L-α-amino acids and includes D-amino acids. The phrase "variant amino acid sequence" refers to molecules with some differences in their amino acid sequences compared to the sequences in accordance with the present invention. Variants of the amino acid sequence of an antibody of the present invention, such as the specified sequence, still have the ability to bind human CD40. Amino acid sequence variants include substitution variants (those in which at least one amino acid residue has been removed and another amino acid inserted in its place at the same position in the polypeptide according to the present invention), insertion variants (variants with one or more amino acids inserted in close proximity to an amino acid at a particular position in a polypeptide of the present invention) and deletion variants (those in which one or more amino acids are deleted in the polypeptide of the present invention).

The term "Fc region" as used herein refers to a polypeptide containing CH3, CH2 and at least a portion of the hinge region of an antibody constant domain. The Fc region may optionally include a CH4 domain present in certain classes of antibodies. The Fc region may comprise the entire hinge region of an antibody constant domain. In one embodiment, the present invention provides an Fc region and a CH1 region of an antibody. In one embodiment, the present invention provides an Fc region and a CH3 region of an antibody. In another embodiment, the present invention provides an Fc region, a CH1 region, and a C _kappa/lambda region of an antibody constant domain. In one embodiment, the binding molecule of the present invention contains a constant region, for example, a heavy chain constant region. In one embodiment, such a constant region is modified from a wild-type constant region. In other words, the polypeptides of the present invention disclosed herein may contain alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and/or the light chain constant region (CL) domain. Examples of modifications include additions, deletions, or substitutions of one or more amino acids in one or more domains. Such changes can be included to optimize effector function, half-life, etc.

As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at individual antigenic sites. Within each antigenic site, the antibody "arm" variable region interacts with the antigen through weak non-covalent forces at many sites; moreover, the more interactions, the stronger the affinity. As used herein, the term "high affinity" for an IgG antibody or fragment (eg, Fab fragment) refers to an antibody that has a K _D of 10 ^-8 M or less, 10 ^-9 M or less, or 10 ^{- 10} M, or 10 ^-11 M or less, or 10 ^-12 M or less, or 10 ^-13 M or less for the target antigen. However, high affinity binding may vary 10-fold for other antibody isotypes. For example, high affinity binding for an IgM isotype refers to an antibody that has a K _D of 10 ^-7 M or less or 10 ^-8 M or less.

As used herein, the term "antibody or protein that specifically binds to a CD40 polypeptide" is intended to refer to an antibody or protein that binds to a human CD40 polypeptide with a K _D of 100 nM or less, 10 nM or less, 1 nM or less.

An antibody that "cross-reacts with an antigen other than CD40" is intended to refer to an antibody that binds that antigen to a K _D of about 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less. An antibody that "does not cross-react with a specific antigen" is intended to refer to an antibody that binds to that antigen with a K _D of 100 nM or greater, or a K _D of 1 μM or greater, or a K _D of 10 µM or more. In some embodiments, such antibodies that do not cross-react with an antigen show virtually undetectable binding to those proteins in standard binding assays.

The terms "K _assoc " or "K _a " as used herein are intended to refer to the rate of association for a particular antibody-antigen interaction, while the terms "K _dis " or "K _d " as used herein are assumed to refer to , refer to the rate of dissociation for a particular antibody-antigen interaction.

The term "K _D " as used herein is intended to refer to the dissociation constant, which is derived from the ratio of K _d and K _a (ie, K _d /K _a ) and which is expressed as a molar concentration (M). Antibody K _D values can be determined using methods well known in the art. The method for determining the K _D of an antibody is carried out using surface plasmon resonance or using a biosensor system such as the Biacore® system.

As used herein, the term "ADCC" or "antibody dependent cellular cytotoxic" activity refers to an activity that depletes cells. ADCC activity can be measured using an ADCC assay, as is well known to one of skill in the art.

As used herein, the term "suppressed" antibody refers to an antibody that exhibits no or low ADCC activity as measured by an ADCC assay.

In one embodiment, the term "no or low ADCC activity" means that the suppressed antibody exhibits ADCC activity that is below 50% specific cell lysis, e.g., below 10% specific cell lysis, as measured in a standard ADCC assay. Absent ADCC activity means that the suppressed antibody exhibits an ADCC (specific cell lysis) activity that is below 1%.

Suppressed effector functions can be obtained by mutation in the Fc region of antibodies and have been described in the prior art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691) and D265A (Baudino et al., 2008, J. lmmunol. 181:6664-69; Strohl, W., supra). Examples of antibodies with a repressed IgG1 Fc domain include the so-called LALA mutant containing mutations L234A and L235A in the amino acid sequence of the IgG1 Fc domain. Another example of a repressed IgG1 antibody contains the D265A mutation. Another repressed IgG1 antibody contains the N297A mutation, which results in aglycosylated/non-glycosylated antibodies.

The term "treatment" or "treat" is defined herein as the use or administration to a subject of an anti-CD40 antibody or protein according to the present invention, such as mAb1 or mAb2 antibodies, or the use or administration of a pharmaceutical composition containing said anti-CD40 antibody or protein of the present invention. of the invention, into an isolated tissue or cell line derived from a subject, wherein the subject has an autoimmune disease and/or inflammatory disease, a symptom associated with an autoimmune disease and/or inflammatory disease, or a predisposition to developing an autoimmune disease and/or inflammatory disease, where the aim is to reduce the severity, reduce the intensity or improve the course of an autoimmune disease and/or inflammatory disease, any associated symptoms of an autoimmune disease and/or inflammatory disease, or a predisposition to develop an autoimmune disease and/or inflammatory disease.

The term "treatment" is also intended to refer to the use or administration to a subject of a pharmaceutical composition comprising said anti-CD40 antibody or protein of the present invention, such as mAb1 or mAb2 antibodies, or the use or administration of a pharmaceutical composition comprising said anti-CD40 antibody or protein of the present invention, in isolated tissue or cell line derived from a subject, where the subject has an autoimmune disease and/or inflammatory disease, a symptom associated with an autoimmune disease and/or inflammatory disease, or a predisposition to develop an autoimmune disease and/or inflammatory disease, where the goal is to reduce severity, reduction in intensity or improvement in the course of an autoimmune disease and/or inflammatory disease, any associated symptoms of an autoimmune disease and/or inflammatory disease, or a predisposition to develop an autoimmune disease and/or inflammatory disease Evania.

The term "prevent" or "preventive" refers to prophylactic or preventive treatment; it refers to delaying the onset or preventing the onset of a disease, disorder and/or symptoms associated therewith.

As used herein, a subject "needs" treatment if such treatment would benefit that subject from a biological, medical, or quality of life point of view.

The term "pharmaceutically acceptable" means a non-toxic material that does not affect the effectiveness of the biological activity of the active(s) substance(s).

As used herein, the term “administering” or “administering” a compound in question means providing a compound of the present invention and its prodrugs to a subject in need of treatment. Administration "in combination with" one or more additional therapeutic agents includes simultaneous (competitive) and sequential administration in any order and by any route of administration. One administration may be a single injection or multiple injections delivered together with each other, depending on how much drug must be administered to achieve a therapeutic effect.

As used herein, the term "therapeutically effective amount" refers to the amount of an anti-CD40 antibody or antigen-binding fragment thereof, such as mAb1, that is effective when administered in single or multiple doses to a patient (such as a human) to treat, prevent, prevent onset, cure , delaying, reducing the severity, improving at least one symptom of the disorder or relapsing disorder, or prolonging patient survival beyond that expected in the absence of such treatment. When applied to an individual active ingredient (eg, anti-CD40 antibody, eg mAb1) administered alone, the term refers to that ingredient only. When used in relation to a combination, the term refers to the combined amounts of active ingredients that result in a therapeutic effect, whether administered in combination, sequentially or simultaneously.

The phrase "therapeutic regimen" means a regimen used to treat a disease, eg, a dosing protocol used during treatment with an SS such as pSS. The therapeutic regimen may include a loading regimen (or administration of a loading dose) followed by a maintenance regimen (or administration of a maintenance dose).

The phrase "loading regimen" or "loading period" refers to a treatment regimen (or part of a treatment regimen) that is used to initially treat a disease. In some embodiments, the disclosed methods, uses, kits, processes, and regimens (eg, methods for treating SS) use a loading regimen (or administration of a loading dose). In some cases, the loading period is a period that lasts until the maximum efficiency is reached. The overall goal of a loading regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. A loading regimen may include administering a larger dose of drug than the physician would prescribe during the maintenance regimen, administering the drug more frequently than the physician would administer the drug during the maintenance regimen, or both. Dose escalation may occur during or after the loading regimen.

The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or part of a treatment regimen) that is used to support a patient during treatment of a disease, for example, to maintain a patient in remission for an extended period of time (months or years) after load pattern or period. In some embodiments, the disclosed methods, applications, and circuits use a support circuit. A maintenance regimen may use continuous therapy (eg, administering the drug at regular intervals, such as weekly, biweekly, or monthly (every 4 weeks), yearly, etc.) or intermittent therapy (eg, interrupted treatment , intermittent therapy, relapse treatment, or treatment upon reaching a predetermined specific criterion [eg, pain, disease manifestations, etc.]). Dose escalation may occur during a maintenance regimen.

The phrase "injection means" is used to refer to any available device for systemically administering a drug to a patient, including, without limitation, pre-filled syringe, vial and syringe, pen, auto-injector, i.v. dropper and bag, pump, patch, etc. With these devices, the patient can self-administer the drug (i.e. self-administer the drug), or the doctor can administer the drug.

Example 1 Antibodies to CD40

anti-CD40 mAbs with suppressed ADCC activity are disclosed in US8828396 and US9221913, incorporated herein by reference in their entirety. It is expected that anti-CD40 mAbs with ADCC suppressed activity have an improved safety profile relative to other anti-CD40 antibodies, and in particular may be more suitable for non-oncological indications such as Sjogren's syndrome (SS) and in particular primary Sjogren's syndrome (pSS).

In accordance with the non-binding hypothesis of the present inventors, two mAbs from US8828396 and US9221913, designated mAb1 and mAb2, are considered to be suitable compounds for the treatment of SS. In particular, mAb1, also referred to as CFZ533, is preferred.

mAb1 inhibits CD154-induced activation in vitro and T-cell dependent antibody formation and germinal center formation in vivo . In pSS patients, blockade of CD40 with mAb1 demonstrated a novel treatment (Example 7).

To enable one skilled in the art to practice the present invention, the amino acid and nucleotide sequences of mAb1 and mAb2 are shown in Table 1 below.

Another anti-CD40 mAb known in the art is ASKP1240 from Astellas Pharma/Kyowa Hakko Kirin Co, as described, for example, in US8568725B2, which is incorporated herein by reference.

Another anti-CD40 mAb known in the art is BI655064 from Boehringer Ingelheim, as described, for example, in US8591900, which is incorporated herein by reference.

An additional anti-CD40 mAb known in the art is FFP104 from Fast Forward Pharmaceuticals, as described, for example, in US8669352, which is incorporated herein by reference.

Another treatment would be AstraZeneca's MEDI4920, which is a Tn3-anti-CD40L fusion protein, or Biogen's BIIB063 anti-CD40L antibody.

Antibodies with a similar mechanism of action as the above antibodies, so-called biosimilars, are also covered by the present invention, as will be understood by a person skilled in the art.

Table 1. Sequence table

SEQID NO: Sequence Description Detailed amino acid or nucleotide sequence 1 HCDR1 mAb 1 and mAb2 (Kabat) SYGMH 2 HCDR2 mAb 1 and mAb2 (Kabat) VISYEESNRYHADSVKG 3 HCDR3 mAb 1 and mAb2 (Kabat) DGGIAAPGPDY 4 LCDR1 mAb 1 and mAb2 (Kabat) RSSQSLLYSNGYNYLD 5 LCDR2 mAb 1 and mAb2 (Kabat) LGSNRAS 6 LCDR3 mAb 1 and mAb2 (Kabat) MQARQTPFT 7 mAb1 and mAb2 heavy chain variable region QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSS 8 mAb1 and mAb2 light chain variable region DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIR 9 Full-length mAb1 heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 10 Full length light chain mAb1 DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSKDSTYSLSSTLTLSKADYEKHKVYACE eleven mAb2 full length heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 12 Full length mAb2 light chain DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSKDSTYSLSSTLTLSKADYEKHKVYACE 13 Fc region mAb1 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSVMFFLYSKLTVDKSRWHEKSALPGGNFSK 14 Fc region of mAb2 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQLSGNFSKK 15 DNA encoding full-length mAb1 heavy chain CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGGTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGGCATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAGATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACGCCTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCC CCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCACAGGTGTACACACTGCCCCCCAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTCCAACGGACAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 16 DNA encoding full-length mAb1 light chain GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCGAGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCACAGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCGGTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGACCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 17 DNA encoding full-length mAb2 heavy chain CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGGTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGGCATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAGATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCC CCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCACAGGTGTACACACTGCCCCCCAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACCCCTCCGATATCGCCGTGGAGTGGGAGTCCAACGGACAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 18 DNA encoding full-length mAb2 light chain GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCGAGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAACGGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCACAGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCGGTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGACCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 19 Amino acid sequence of human CD40 MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

In one embodiment, an anti-CD40 antibody is provided, said antibody comprising an immunoglobulin VH domain comprising the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain comprising the amino acid sequence of SEQ ID NO: 8.

In one embodiment, an anti-CD40 antibody is provided, said antibody comprising an immunoglobulin VH domain comprising the hypervariable regions shown under SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and an immunoglobulin VL domain comprising hypervariable regions shown under SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

In one embodiment, an anti-CD40 antibody is provided, said antibody comprising an immunoglobulin VH domain comprising the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain comprising the amino acid sequence of SEQ ID NO: 8, and an Fc region of SEQ ID NO: 13.

In one embodiment, an anti-CD40 antibody is provided, said antibody comprising an immunoglobulin VH domain comprising the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain comprising the amino acid sequence of SEQ ID NO: 8, and an Fc region of SEQ ID NO: 14.

In one embodiment, an anti-CD40 antibody is provided, said antibody comprising a repressed IgG1 Fc region.

In a preferred embodiment, an anti-CD40 antibody referred to as mAb1 is provided. In particular, mAb1 contains the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10; and mAb2 contains the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12.

1. Expression systems

For expression of the light and heavy chains, the expression vector(s) encoding(s) for the heavy and light chains were transfected into the host cell using standard techniques. The various forms of the term "transfection" are intended to cover a wide variety of techniques commonly used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, calcium phosphate precipitation, transfection with DEAE-dextran, etc. Theoretically, it is possible to express antibodies of the present invention in either prokaryotic or eukaryotic host cells. Expression of antibodies in eukaryotic cells, such as mammalian, yeast, or filamentous fungal host cells, is contemplated, since such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

In particular, the cloning or expression vector may contain at least one of the following coding sequences (a)-(b) operably linked to a suitable promoter sequence:

(a) SEQ ID NO: 15 and SEQ ID NO: 16, encoding full-length mAb1 heavy and light chains, respectively; or

(b) SEQ ID NO: 17 and SEQ ID NO: 18 encoding full-length mAb2 heavy and light chains, respectively.

Mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO cells) cells (including dhfr-CHO cells described in Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA 77:4216-4220, used with selectable DH FR markers, for example as described in R. J. Kaufman and P. A. Sharp, 1982 Mol Biol 159:601-621), CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells. In particular for use with NSO myeloma cells, another expression system is the GS gene expression system shown in PCT publications WO 87/04462, WO 89/01036 and EP0338841.

When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, antibodies are obtained by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the cells are grown. -hosts. Antibodies can be recovered from the culture medium using standard protein purification methods (see eg Abhinav et al. 2007, Journal of Chromatography 848: 28-37).

Host cells can be cultured under suitable conditions for mAb1 or mAb2 expression and production.

2. Pharmaceutical compositions

Therapeutic antibodies are usually formulated either in an aqueous form ready for administration or as a lyophilisate for reconstitution with a suitable diluent prior to administration. The anti-CD40 antibody can be formulated either as a lyophilisate or as an aqueous formulation, eg in pre-filled syringes. The formulation is also referred to as drug product (DP).

A suitable formulation may provide an aqueous pharmaceutical composition or lyophilisate that can be reconstituted to provide a solution with a high concentration of antibody active ingredient and a low level of antibody aggregation for delivery to a patient. High concentrations of antibody are useful because they reduce the amount of material that must be delivered to the patient. Reduced dosing volumes minimize the time required to deliver a fixed dose to a patient. Aqueous compositions of the present invention with a high concentration of antibodies to CD40, in particular, are suitable for subcutaneous administration.

Thus, the present invention provides an aqueous pharmaceutical composition suitable for administration to a subject, for example for subcutaneous administration, containing an anti-CD40 antibody such as mAb1 or mAb2.

The anti-CD40 antibody can be used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. In addition to an anti-CD40 antibody such as mAb1 or mAb2, such a composition may contain carriers, various diluents, excipients, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The characteristics of the carrier will depend on the route of administration. Pharmaceutical compositions for use in the disclosed methods may also contain additional therapeutic agents for the treatment of the particular target disorder.

In one specific embodiment, the composition, also referred to as a drug product (DP), is a lyophilized formulation derived from an aqueous formulation characterized by pH 6.0 and containing:

(i) 150 mg/ml mAb1 or mAb2;

(ii) 270 mm sucrose as a stabilizer;

(iii) 30 mM L-histidine as a buffer and

(iv) 0.06% polysorbate 20 as a surfactant.

In another specific embodiment, the pharmaceutical composition, also referred to as drug product (DP), is an aqueous pharmaceutical composition characterized by pH 6.0 and containing:

(i) 150 mg/ml mAb1 or mAb2;

(ii) 270 mm sucrose as a stabilizer;

(iii) 30 mM L-histidine as a buffer and

(iv) 0.06% polysorbate 20 as a surfactant.

3. Route of administration

Typically, antibodies or proteins are administered by injection, for example, either intravenously, intraperitoneally, or subcutaneously. Methods for carrying out this administration are known to those of ordinary skill in the art. It is also possible to obtain a composition that can be administered topically or orally, or that can be penetrated through mucous membranes. As will be appreciated by one of skill in the art, any suitable means of administration suitable for the particular route of administration chosen may be used.

Examples of possible routes of administration include parenteral, (e.g., intravenous (I.V. or IV), intramuscular (IM), intradermal, subcutaneous (S.C. or SC) or infusion), oral and pulmonary (e.g., inhalation), nasal, transdermal (topical), transmucosal and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonic agents such as sodium chloride or dextrose. The pH level can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The preparation for parenteral administration can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Anti-CD40 antibody therapy can be initiated by administering a loading regimen or a loading dose of an antibody or protein of the present invention to a subject in need of anti-CD40 antibody therapy. The term "loading dose(s)" refers to the initial administration of a dose of an anti-CD40 antibody or protein of the present invention, which is administered to the subject one or more times, where the dose of the administered antibody or protein of the present invention is in the high dose range ( i.e., from about 10 mg/kg to about 50 mg/kg, such as about 30 mg/kg intravenously, or about 600 mg, or about 300 mg, or about 150 mg weekly, once every two weeks for no more than 4 weeks). "Loading dose administration" can be administered as a single administration or multiple administrations, for example, in the form of a single (s) or multiple (s) intravenous infusion (s), or as subcutaneous injections, combined in a scheme "administration of a loading dose", depending on the severity of the disease. On subsequent administrations, the "loading regimen" subject is then administered one or more additional therapeutically effective doses of an anti-CD40 antibody or protein of the present invention. Subsequent therapeutically effective doses may be administered, for example, according to a weekly dosing regimen, or once every two weeks (once every two weeks), once every three weeks, or once every four weeks. In such embodiments, subsequent therapeutically effective doses are typically in the low dose range (i.e., about 0.003 mg/kg to about 30 mg/kg, such as about 10 mg/kg, such as 10 mg/kg IV or about 150 mg, about 300 mg, or about 600 mg administered weekly, biweekly, or every 4 weeks subcutaneously).

Alternatively, in some embodiments, following a "loading regimen", subsequent therapeutically effective doses of an anti-CD40 antibody or protein of the present invention are administered according to a "maintenance regimen" wherein a therapeutically effective dose of the antibody or protein of the present invention is administered once a month, once every 6 weeks, once every two months, once every 10 weeks, once every three months, once every 14 weeks, once every four months, once every 18 weeks, once every five months, once every 22 weeks, once every six months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once every 12 months. In such embodiments, therapeutically effective doses of an anti-CD40 antibody or protein of the present invention are in the low dose range (i.e., about 0.003 mg/kg to about 30 mg/kg, such as about 10 mg/kg, such as 10 mg /kg), in particular if subsequent doses are administered at more frequent intervals, for example, weekly, from once every two weeks to once every four weeks, or in the high dose range (i.e. from 10 mg / kg to 50 mg/kg, such as 30 mg/kg), in particular if subsequent doses are administered at less frequent intervals, for example where subsequent doses are administered from once a month to once every 12 months.

The time of dosing is generally measured from the day of administration of the first dose of the active compound (eg, mAb1), which is also known as "baseline". However, different physicians use different conventions.

Notably, week zero may be referred to as week 1 by some physicians, while day zero may be referred to as day one by some physicians. Thus, it is possible for different physicians to designate, for example, the dose to be administered during week 3/day 21, during week 3/day 22, during week 4/day 21, during week 4/day 22 while referring to the same dosing regimen. For consistency, the first week of dosing will be referred to herein as week 0, with the first day of dosing being referred to as day 1. However, one of skill in the art will appreciate that this convention is used merely for consistency and should not be construed as limiting. i.e. weekly dosing is the provision of a weekly dose of an anti-CD40 antibody, eg mAb1, regardless of whether the physician refers to a particular week as "week 1" or "week 2". An example of the dosage regimens indicated herein are depicted in figures 1 and 2. It should be understood that the administration of the dose should not be provided at a precise point in time, for example, a dose to be administered around day 29 could be administered, for example, at days 24 to 34, such as day 30, as long as its administration is ensured in a suitable week.

As used herein, the phrase "a container containing a sufficient amount of anti-CD40 antibody to deliver [a given dose]" is used to mean that the container (e.g., vial, pen, syringe) contains a volume of anti-CD40 antibody (e.g., in as part of a pharmaceutical composition) that can be used to provide the desired dose. As an example, if the required dose is 500 mg, then the clinician may use 2 ml from the container that contains the 250 mg/ml anti-CD40 antibody formulation, 1 ml from the container that contains the anti-CD40 antibody formulation. 500 mg/ml, 0.5 ml from a container that contains a 1000 mg/ml anti-CD40 antibody formulation, etc. In each such case, these containers contain sufficient anti-CD40 antibody to allow deliver the required dose of 500 mg.

As used herein, the phrase "formulated at a dosage that allows the [route of administration] to deliver the [intended dose]" is used to indicate that the pharmaceutical composition can be used to provide the desired dose of an anti-CD40 antibody, such as mAb1, via the intended route of administration ( e.g. s.c. or i.v.). As an example, if the required subcutaneous dose is 500 mg, then the clinician may use 2 ml of an anti-CD40 antibody formulation having a concentration of 250 mg/mL, 1 ml of an anti-CD40 antibody formulation having a concentration of 500 mg/mL, 0.5 ml of an anti-CD40 antibody formulation having a concentration of 1000 mg/ml, etc. In each such case, these anti-CD40 antibody formulations are at a concentration high enough to allow subcutaneous delivery of the anti-CD40 antibody. Subcutaneous delivery typically requires delivery in volumes of less than about 2 ml, preferably a volume of about 1 ml or less. However, larger volumes can be delivered over time using, for example, a patch/pumping mechanism.

Disclosed herein is the use of an anti-CD40 antibody (e.g., mAb1) for the manufacture of a medicament for the treatment of Sjögren's syndrome in a patient, wherein the medicament is formulated to contain containers, where each container has sufficient anti-CD40 antibody to deliver at least about 75 mg , 150 mg, 300 mg, or 600 mg of an anti-CD40 antibody or antigen-binding fragment (eg, mAb1) per single dose.

This document discloses the use of an anti-CD40 antibody (e.g., mAb1) for the manufacture of a medicament for the treatment of Sjögren's syndrome in a patient, where the drug is formulated at a dosage that allows systemic delivery (e.g., intravenous or subcutaneous delivery) of 75 mg, 150 mg, 300 mg or 600 mg of anti-CD40 antibody or antigen-binding fragment (e.g. mAb1) per single dose.

4. Sets

The disclosure also covers kits for treating a patient with Sjögren's syndrome (as applicable) with an anti-CD40 antibody or antigen-binding fragment thereof, such as mAb1. Such kits contain an anti-CD40 antibody or an antigen-binding fragment thereof, for example, mAb1 (eg, in liquid or lyophilized form) or a pharmaceutical composition containing an anti-CD40 antibody (described above). Additionally, such kits may contain means for administering the anti-CD40 antibody (eg, syringe and vial, pre-filled syringe, pre-filled pen, patch/pump) and instructions for use. The instructions may disclose providing an anti-CD40 antibody (eg, mAb1) to a patient as part of a particular dosing regimen. Such kits may also contain additional therapeutic agents (described above) for the treatment of psoriasis, for example, to be delivered in combination with an encapsulated anti-CD40 antibody, such as mAb1.

The phrase "injection device" is used to refer to any available device for systemically administering a drug to a patient, including, without limitation, pre-filled syringe, vial and syringe, pen, auto-injector, i.v. dropper and bag, pump, patch, etc. With these items, the patient may self-administer the drug (i.e. self-administer the drug), or the drug may be administered by a caregiver or physician.

Disclosed herein are kits for the treatment of a patient who has Sjögren's syndrome, comprising: a) a pharmaceutical composition that contains a therapeutically effective amount of an anti-CD40 antibody or antigen-binding fragment thereof; b) means for administering to a patient the anti-CD40 antibody, or antigen-binding fragment thereof; and c) instructions for administering subcutaneously to a patient in need thereof, the anti-CD40 antibody, or antigen-binding fragment thereof, in the form of a dose of about 3 to about 30 mg of the active ingredient per kilogram of body weight. human subject, three times, once every two weeks, followed by monthly doses of about 3 to about 30 mg, such as 10 mg, of the active ingredient per kilogram of body weight of the human subject, or about 150 mg, about 300 mg, or about 600 mg weekly, biweekly, or monthly during maintenance regimen.

In one particular embodiment, the use of a) a liquid pharmaceutical composition comprising an anti-CD40 antibody, a buffer, a stabilizer, and a solubilizer, and b) a means for subcutaneously administering an anti-CD40 antibody to a patient who has Sjögren's syndrome, for the manufacture of a medicament for the treatment of the syndrome Sjögren, where the antibody to CD40

i) is to be administered intravenously to a patient at a dose of from about 3 to about 30 mg, such as 10 mg, of the active ingredient per kilogram of body weight of a human subject three times, once every two weeks, and

ii) thereafter to be administered intravenously to a patient in monthly doses of from about 3 to about 30 mg, such as 10 mg, of the active ingredient per kilogram of body weight of a human subject, wherein said anti-CD40 antibody is selected from the group consisting of:

a) an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8;

b) an anti-CD40 antibody that contains an immunoglobulin VH domain containing the hypervariable regions shown under SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and an immunoglobulin VL domain containing the hypervariable regions shown under SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;

c) an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 13;

d) an anti-CD40 antibody which contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 14;

e) an anti-CD40 antibody that contains a repressed IgG1 Fc region; And

f) an anti-CD40 antibody which comprises the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, or the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12.

According to a third aspect, the use of a) a liquid pharmaceutical composition comprising an anti-CD40 antibody, a buffer, a stabilizer, and a solubilizer, and b) a means for subcutaneously administering an anti-CD40 antibody to a patient who has Sjögren's syndrome, for the manufacture of a medicament for the treatment of the syndrome Sjögren, where the antibody to CD40

i) is to be administered subcutaneously to a patient as a dose of about 150 mg active, about 300 mg active, or about 600 mg active (weekly or biweekly); And

ii) thereafter, it is administered subcutaneously to the patient weekly, biweekly, or monthly (every four weeks) at doses of approximately 150 mg active, approximately 300 mg active, or approximately 600 mg active, wherein said anti-CD40 antibody selected from the group consisting of:

a) an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8;

b) an anti-CD40 antibody that contains an immunoglobulin VH domain containing the hypervariable regions shown under SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and an immunoglobulin VL domain containing the hypervariable regions shown under SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;

c) an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 13;

d) an anti-CD40 antibody which contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 14;

e) an anti-CD40 antibody that contains a repressed IgG1 Fc region; And

f) an anti-CD40 antibody which comprises the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, or the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12.

Example 2 Pharmacology

1. Primary pharmacology

mAb1 binds to human CD40 with high affinity (K _d 0.3 nM). However, it does not bind to Fcγ receptors (including CD16) or mediate antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. mAb1 inhibits human leukocyte activation induced by recombinant CD154 (rCD154), but does not induce PBMC proliferation or cytokine production by monocyte-derived dendritic cells (DCs). mAb1 binds to human and non-human primate CD40 with very similar affinity.

In vivo, mAb1 blocks primary and secondary T-cell dependent antibody responses (TDAR) and may prolong the survival of kidney allografts in non-human primates (Cordoba et al 2015). In addition, mAb1 can disrupt established germinal centers (GCs) in vivo .

The occupancy rate and functional activity of the CD40 receptor were assessed simultaneously in vitro using human whole blood cultures. Functional activity was quantified by CD154-induced expression of CD69 (an activation marker) on CD20-positive cells (B cells), and CD40 occupancy was monitored using fluorescently labeled mAb1. Nearly full occupancy of CD40 mAb1 was required to completely inhibit rCD154-induced CD69 expression.

2. Secondary pharmacology

The effects of mAb1 on platelet function and blood hemostasis were investigated, indicating that mAb1 does not induce responses to platelet aggregation, but rather exhibits some mild inhibitory effects on platelet aggregation at high concentrations.

Example 3 Non-Clinical Pharmacological Toxicology and Safety

Toxicological studies of mAb1 did not reveal any significant organ toxicity, including no thromboembolic events, as reported in clinical trials with anti-CD154 mAb (Kawai et al 2000). In a 13-week study of GLP in rhesus monkeys (weekly administration at 10, 50, and 150 mg/kg), increased lymphoid tissue cellularity was found in 5/22 animals, which was thought to be due to ongoing infection, while observing matched the pharmacology of mAb1. Inflammatory lesions were seen in the kidneys and lungs of 2 animals at 50 mg/kg, and lesions were also seen in the eyes and trachea in one of the two animals. Although a direct effect of mAb1 on the kidneys and lungs cannot be ruled out, the strength of the evidence, including evidence for the presence of opportunistic pathogens, suggests that such results are most likely mediated by factors secondary to mAb1-mediated immunosuppression and due to the source of infection. In terms of such inflammatory outcomes, the no adverse effect level (NOAEL) for the 13 week toxicity study was 10 mg/kg. In a 26-week chronic toxicity study in cynomolgus monkeys, no mAb1 related adverse outcomes were found. Based on these data, NOAEL was at 150 mg/kg (26 weeks). Mean (all animals) Cmax, ss _ss were 44, 3235, and 9690 μg/mL, respectively, at 1, 50, and 150 (NOAEL) mg/kg SC weekly. NOAEL derived from a 26-week study in cynomolgus monkeys is considered the most appropriate to support a clinical dosing regimen.

Post-mortem histological and immuno-histological evaluations revealed a decrease in GC in cortical regions of B cells in splenic and lymphatic tissues. Animals in the recovery group showed several cases of increased cellularity of the lymph nodes with normal areas of T-cells and increased areas of B-cells, which corresponded to the recovery of GC after cessation of drug administration. Animals in the recovery group developed a primary keyhole limpet hemocyanin (KLH) TDAR immediately after blood mAb1 levels fell below the level required for full receptor occupancy.

Due to the complete inhibition of T-cell dependent antibody (TDAR) responses by KLH, the formation of anti-drug antibodies (ADA) to mAb1 was not expected, and therefore ADA-related side effects were considered unlikely if mAb1 concentrations were maintained continuously at pharmacological levels.

Cross-reactivity studies with similar tissue epitopes have revealed that CD40 is present not only in immune cells, but also in various tissues. This is mainly due to its expression in endothelial and epithelial cells, where CD40 is involved in signaling, such as in response to wound healing processes, activation of virus defenses, and mediators associated with inflammation. The CD40 antagonist monoclonal antibody, like mAb1, was not expected to cause inflammatory processes, which was confirmed by an in vitro study using human umbilical vein endothelial cells (HUVEC).

So far, full-fledged, consistent with methodological recommendations, studies of toxicity in relation to the reproductive system have not been conducted. However, a dose range, embryofetal development (EFD) study was conducted in rabbits to confirm rabbits as suitable species for reproductive toxicity studies. In any of the groups, no effects on embryofetal development were observed and no external malformations associated with the treatment of the embryo were found.

In conclusion, preclinical data support the first multiple dose studies in patients with primary Sjögren's syndrome.

Example 4 Preclinical Pharmacokinetics and Pharmacodynamics

1. Pharmacokinetics (PK)

For IgG immunoglobulins, it is typical that the primary route of mAb1 removal is likely to be through proteolytic catabolism occurring at sites characterized by equilibrium binding to plasma proteins. In addition, the binding and internalization of the mAb1-CD40 complex results in fast and saturable clearance routes. This is illustrated by non-linear profiles of mAb1 serum concentration versus time showing the inflection point of the curve at approximately 10-20 μg/mL. The contribution of CD40-mediated clearance to overall clearance is dependent on mAb1 concentration, together with CD40 expression levels, internalization, and receptor metabolic rates. For serum concentrations of mAb1 > 10-20 µg/mL, linear kinetics were expected, while non-linear kinetics appeared at lower concentrations.

2. Pharmacodynamics (PD)

In a PK/PD study in cynomolgus monkeys, the inflection point (approximately 10 µg/mL) in the PK profiles was associated with a drop in CD40 saturation, as determined by an independent target lymphocyte saturation analysis. As such, this inflection point of the curve is considered as a marker for CD40 saturation levels and evidence of target binding.

The association between employment and CD40 pharmacodynamic activity was further demonstrated in rhesus monkeys immunized with KLH. Monkeys were immunized with KLH three times (the first time approximately 3 weeks prior to dosing, the second time 2 weeks after mAb1 administration and the third time after mAb1 was completely cleared). CD40 mAb1 occupancy at plasma concentrations >40 μg/mL at the second KLH vaccination completely prevented recovery of antibody responses. Once mAb1 was cleared, all animals had a complete memory for the immune response to the third KLH. These results indicate that the pre-existing memory function of B cells was not affected. After complete removal of mAb1, immunization with tetanus toxin (TTx) resulted in IgG/IgM titers to TTx, similar to untreated animals, and demonstrated that complete TDAR was restored after removal of mAb1.

3. Immunogenicity

As expected from an immunosuppressive drug, the immunogenicity data obtained in rhesus monkeys (single dose) were consistent with those obtained from the KLH-TDAR experiment and confirmed that no immune response against mAb1 was maintained at full CD40 occupancy. mAb1.

4. Therapeutic regimens

Based on the pharmacokinetic and pharmacodynamic profiles of mAb1, as well as the results from preclinical and clinical studies of mAb1, the following therapeutic regimens can be applied.

In one embodiment (see FIG. 24A, 1), the mAb1 therapeutic regimen consisted of administering a loading dose comprising two 600 mg doses of mAb1 administered with a one-week break between the two doses, followed by a maintenance dose comprising 600 mg doses of mAb1 administered every 2 weeks (Q2W). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml).

In another embodiment (see FIG. 24A, 2), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 300 mg mAb1, where the second dose was administered one week after the first dose followed by a maintenance dose. dose consisting of doses of 600 mg mAb1 administered every 2 weeks (Q2W). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml).

In another embodiment (see FIG. 24A, 3), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 150 mg mAb1, where the second dose was administered one week after the first dose followed by a maintenance dose. dose consisting of doses of 600 mg mAb1 administered every 2 weeks (Q2W). The 150 mg dose is preferably administered subcutaneously by 1 injection of 1 ml of drug product (150 mg/ml), alternatively by 1 injection of 2 ml of drug product diluted ½ times (75 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml).

In one embodiment (see FIG. 24B, 1), the mAb1 therapeutic regimen consisted of administering a loading dose comprising two 600 mg doses of mAb1 administered with a one-week break between the two doses, followed by a maintenance dose comprising 300 mg doses of mAb1 administered every 2 weeks (Q2W). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml).

In another embodiment (see FIG. 24B, 2), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 300 mg mAb1, where the second dose was administered one week after the first dose followed by a maintenance dose. dose consisting of doses of 300 mg mAb1 administered every 2 weeks (Q2W). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml).

In yet another embodiment (see FIG. 24B, 3), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 150 mg mAb1, where the second dose was administered one week after the first dose followed by maintenance dose consisting of doses of 300 mg mAb1 administered every 2 weeks (Q2W). The 150 mg dose is preferably administered subcutaneously by 1 injection of 1 ml of drug product (150 mg/ml), alternatively by 1 injection of 2 ml of drug product diluted ½ times (75 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml).

In one embodiment (see FIG. 24C, 1), the mAb1 therapeutic regimen consisted of administering a loading dose comprising two 600 mg doses of mAb1 administered with a one-week break between the two doses, followed by a maintenance dose comprising 150 mg doses of mAb1 administered every 2 weeks (Q2W). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml). The 150 mg dose is preferably administered subcutaneously by 1 injection of 1 ml of drug product (150 mg/ml), alternatively by 1 injection of 2 ml of drug product diluted ½ times (75 mg/ml).

In another embodiment (see FIG. 24C, 2), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 300 mg mAb1, where the second dose was administered one week after the first dose followed by a maintenance dose. dose consisting of doses of 150 mg mAb1 administered every 2 weeks (Q2W). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml). The 150 mg dose is preferably administered subcutaneously by 1 injection of 1 ml of drug product (150 mg/ml), alternatively by 1 injection of 2 ml of drug product diluted ½ times (75 mg/ml).

In yet another embodiment (see FIG. 24C, 3), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 150 mg mAb1, where the second dose is administered one week after the first dose followed by maintenance dose consisting of doses of 150 mg mAb1 administered every 2 weeks (Q2W). The 150 mg dose is preferably administered subcutaneously by 1 injection of 1 ml of drug product (150 mg/ml), alternatively by 1 injection of 2 ml of drug product diluted ½ times (75 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml).

In a further embodiment (see FIG. 25A, 1), the mAb1 therapeutic regimen consisted of administering a loading dose comprising two 600 mg doses of mAb1 administered with a one-week break between the two doses, followed by a maintenance dose comprising 600 mg doses of mAb1 administered every 4 weeks (Q4W). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml).

In another embodiment (see FIG. 25A, 2), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 300 mg mAb1, where the second dose is administered one week after the first dose followed by a maintenance dose. dose consisting of doses of 600 mg mAb1 administered every 4 weeks (Q4W). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml).

In another embodiment (see FIG. 25A, 3), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 150 mg mAb1, where the second dose was administered one week after the first dose followed by a maintenance dose. dose consisting of doses of 600 mg mAb1 administered every 4 weeks (Q4W). The 150 mg dose is preferably administered subcutaneously by 1 injection of 1 ml of drug product (150 mg/ml), alternatively by 1 injection of 2 ml of drug product diluted ½ times (75 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml).

In a further embodiment (see FIG. 25B, 1), the mAb1 therapeutic regimen consists of administering a loading dose comprising two 600 mg doses of mAb1 administered with a one-week interval between doses followed by a maintenance dose comprising 300 mg doses of mAb1, introduced every week (Q1W). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml).

In another embodiment (see FIG. 25B, 2), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 300 mg mAb1, where the second dose was administered one week after the first dose followed by a maintenance dose. dose consisting of doses of 300 mg mAb1 administered every week (Q1W). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml).

In yet another embodiment (see FIG. 25B, 3), the mAb1 therapeutic regimen consisted of administering a loading dose consisting of a first dose of 600 mg mAb1 and a second dose of 150 mg mAb1, where the second dose was administered one week after the first dose followed by maintenance dose consisting of doses of 300 mg of mAb1 administered every week (Q1W). The 150 mg dose is preferably administered subcutaneously by 1 injection of 1 ml of drug product (150 mg/ml), alternatively by 1 injection of 2 ml of drug product diluted ½ times (75 mg/ml). The 300 mg dose is preferably administered subcutaneously by 1 injection of 2 ml of drug product (150 mg/ml). The 600 mg dose is preferably administered subcutaneously by 2 injections of 2 ml of drug product (150 mg/ml).

The advantage of having a therapeutic regimen divided into a loading dose part and a maintenance dose part is that it allows an optimal therapeutic effect to be achieved.

For all therapeutic regimens described herein, the aim of the loading dose is to achieve the target saturation (plasma concentrations close to 40 µg/mL) and thus initiation of the therapeutic effect, and the aim of the maintenance dose is to maintain the efficacy .

Example 5 Human Safety and Tolerability Data

The safety, tolerability, PK and PD activity of mAb1 was evaluated in an ongoing randomized, double-blind, placebo-controlled, single dose incremental study of mAb1 in healthy subjects and patients with rheumatoid arthritis (RA). A total of 48 subjects were included: 36 healthy subjects who received single doses of mAb1 not exceeding 3 mg/kg IV or S.C., and 12 patients with RA, 6 of whom received single doses of mAb1 10 mg/kg IV. Overall, single doses of no more than 3 mg/kg mAb1 in healthy volunteers and single doses of 10 mg/kg mAb1 in patients with RA were safe and well tolerated, with no suspected serious side effects (SAEs). A 30 mg/kg IV dose study is ongoing in patients with RA. Because this study is still ongoing, all clinical data are preliminary and are based on interim analyzes performed at doses not exceeding 10 mg/kg in patients with RA.

Example 6 Pharmacokinetics and Pharmacodynamics in Humans (Healthy Volunteers and Patients with Rheumatoid Arthritis)

In healthy subjects, as well as in patients with rheumatoid arthritis, after a single IV or SC administration, the PK profiles of CFZ533 were consistent with a target-mediated distribution, resulting in non-linear PK profiles and faster clearance if CD40 receptor occupancy fell below about 90 %.

Despite inter-individual variability in PK profiles from Chinese subjects, the distribution of CFZ533 in Chinese subjects was generally similar to that of non-Chinese subjects, and target binding was also similar (approximately 4 weeks) after 3 mg/kg IV CFZ533. At this dose level, similar PK/PD profiles were demonstrated through profiles of free CFZ533 in plasma, CD40 occupancy on peripheral B cells, measuring free CD40, and total CD40, and total plasma concentrations of sCD40.

Following SC administration to healthy subjects, CFZ533 was rapidly absorbed and distributed in humans as expected for a typical IgG1 antibody. At 3 mg/kg SC, peak levels of CFZ533 were generally observed 3 days post-dose (7 days for 2 subjects), and 1 week post-dosing, plasma concentrations were in the same range as after IV dosing. . With SC administered at 3 mg/kg, the duration of target binding was also approximately 4 weeks.

In patients with rheumatoid arthritis at 10 mg/kg IV, as measured by free CD40 on whole blood B cells compared to pre-dose mean profiles and total plasma sCD40, full CD40 occupancy was generally maintained for 8 weeks. At 30 mg/kg IV, plasma PK and total sCD40 profiles corresponded to the duration of target binding for 16 weeks.

In healthy subjects, when monitoring CD40 occupancy on B cells, CD40 binding by CFZ533 typically resulted in a reduction in total CD40 on peripheral B cells by approximately 50%, as measured by free CD40 on B cells. This was most likely due to internalization and/or clearance of membrane-bound CD40 after binding to CFZ533. In patients with rheumatoid arthritis, a decrease in total CD40 on peripheral B cells was not confirmed.

The relationship between plasma concentration of CFZ533 and CD40 occupancy on whole blood B cells (free CD40 on B cells) was determined, and CFZ533 concentrations of 0.3-0.4 µg/mL were associated with total (defined as ≥ 90% ) CD40 occupancy on whole blood B cells.

More broadly, non-specific and specific elimination pathways have been identified for CFZ533. The non-specific and high capacity pathways mediated by FcRn receptors are usually identical to endogenous IgG. The specific target-mediated distribution of CFZ533 resulted in the formation of CFZ533-CD40 complexes, which were partially internalized (followed by lysosomal degradation) and/or cleared from the membrane. Target-mediated processes led to saturation and non-linear arrangement of CFZ533. The formation of CFZ533-CD40 complexes was dose/concentration dependent, with saturation occurring at high concentrations of CFZ533.

In general, the distribution of CFZ533 depended on the relative influence of specific (target-mediated) and non-specific elimination pathways on the overall clearance of CFZ533. A non-linear PK pattern was observed when CFZ533 concentrations were lower than target, while at higher concentrations with saturated CD40 receptors, non-specific pathways predominated and removal of CFZ533 was linear.

As expected, for a typical IgG1 antibody targeting the membrane-bound receptor and showing target-mediated distribution, the exposure to CFZ533 (AUClast) increased more than the dose increase (overproportionality). Therefore, it is expected that this is due to a decrease in the volume of distribution and elimination of CFZ533 at high doses.

One subject at an IV dose of 1 mg/kg CFZ533 (1 week full CD40 occupancy) developed specific antibodies to CFZ533 detected 6 weeks after plasma concentrations of CFZ533 were below the limit of quantitation, and definitely too low to block any relevant CD40 pathway effects in tissue. The presence of anti-drug antibodies (ADA) in this subject did not interfere with exposure and is not associated with an immune-related safety signal. In this study, this corresponded to an incidence of ADA of 2%.

A single 3 mg/kg dose (IV and SC) of CFZ533 transiently suppressed anti-KLH antibody responses at the first KLH immunization, with CFZ533 concentrations consistent with full (≥ 90%) receptor occupancy (within approximately 3-4 weeks). Primary anti-KLH antibody responses were detected in all subjects as a concentration of CFZ533 and concomitant receptor occupancy was reduced. All subjects were able to sustain recovered responses on a second KLH immunization (given after the expected loss of receptor occupancy).

The data indicate that CD40 binding by CFZ533 prevented recombinant human CD154 (rCD154) mediated B cell activation in human whole blood. rCD154-induced expression of CD69 in B cells was generally suppressed during the period corresponding to the full occupancy of CD40 on B cells. If the CD40 occupancy was incomplete, the functional activity of rCD154 was restored.

There was no evidence for the effects of CFZ533 on immunophenotyping data.

Example 7 Multicenter, Randomized, Double-Blind, Placebo-Controlled, Parallel Group Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Preliminary Efficacy of CFZ533 in Patients with Primary Sjögren's Syndrome

In order to evaluate the suitability of the use of anti-CD40 human monoclonal antibodies with suppressed ADCC activity in the treatment of Sjögren's syndrome, a clinical study was designed and conducted using the antibody CFZ533, herein also referred to as mAb1.

Sjögren's syndrome (SS) may be primary or often associated with other autoimmune diseases such as systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). In this case, SS is called secondary. Patients with secondary Sjögren's syndrome were excluded from the CFZ533 clinical trials because the primary disease (eg, RA or SLE) and its concomitant treatments would greatly interfere with the assessment of primary SS. For example, the involvement/damage of the joints in RA would not allow interpretation that the ESSDAI domain or anti-TNF antibody treatment would not be compatible with the use of CFZ533. However, the pathogenesis of secondary SS is due to the same pathobiological phenomena as in pSS, such as germinal center formation and the formation of autoantibodies mediated by abnormal activation of the CD40-CD154 pathway, while treatment with CFZ533 was expected to benefit patients with secondary SS, guaranteeing subsequent clinical trials.

Primary Sjögren's syndrome (pSS) is a systemic progressive autoimmune disease characterized by the formation of ectopic germ centers in the exocrine glands and dysfunction of the secretory glands. Some patients also develop extraglandular manifestations. CFZ533 is a novel monoclonal antibody that potently and selectively blocks CD40, a costimulatory pathway receptor required for germinal center responses and other mediated immune functions involved in the pathogenesis of pSS. A randomized, double-blind, placebo-controlled, multicenter, partially crossover (PoC) phase IIa evidence-based study was performed to calculate the safety, tolerability, and efficacy of CFZ533 in patients with pSS.

Several data sets indicate that the pathology of the disease is due to or is closely related to the CD40-CD154 pathway, which is the main one in pSS. Characteristic diagnostic features of pSS include B-cell overreactivity, such as the formation of germinal center-like structures in the salivary glands (observed in 18-59% of patients), and autoantibodies (Vossenkämper et al 2012). Activated T cells predominated in pSS foci and were able to induce B cell hyperactivity, release Ig, and facilitate glandular destruction (Manganelli and Fietta 2003). In addition, T and B cell infiltrates in these patients' salivary glands showed upregulation of CD40 and CD154. Mediated CD40-CD154 inflammation of the tissue may also determine the pathogenesis of pSS.

In addition, CD40-CD154 mediated tissue inflammation may also contribute to the pathogenesis of pSS. Epithelial cell lines derived from pSS patients constitutively activated surface CD40 (Dimitriou et al 2002). Elevated levels of platelet-derived microparticles as well as leukocytes have been reported, along with elevated serum concentrations of sCD154 in patients with pSS (Sellam et al 2009).

1. Development of the evidence-based part of the conceptual study CCFZ533X2203 in SS.

Presented here is a double-blind, open-label, randomized, placebo-controlled, non-confirmatory, parallel-group study to evaluate the safety, tolerability, pharmacokinetics, and preliminary clinical efficacy of multiple doses of CFZ533 in the following cohorts 1 and 2.

• Cohort 1: CFZ533 3mg/kg subcutaneously in pSS patients in a double-blind and placebo-controlled fashion followed by open-label treatment.

• Cohort 2: 10mg/kg CFZ533 administered by intravenous infusion to pSS patients in a double-blind and placebo-controlled fashion followed by open-label treatment.

These cohorts were followed by an open-label, randomized, parallel group that was not the confirmatory part of cohort 3.

• Cohort 3: In treatment group 1, CFZ533 was administered at a concentration of 600 mg s.c. weekly in 4 repetitions (loading scheme) followed by the introduction of 300 mg s.c. weekly in 9 repetitions (maintenance scheme based on the study of day 29).

In treatment group 2, the loading regimen was a single i.v. dose of 10 mg/kg CFZ533 (on study day 1) followed by weekly s.c. a dose of 300 mg in 12 repetitions (maintenance scheme based on day 8 of the study).

In cohorts 1 and 2, randomization will be stratified by baseline oral corticosteroid use (yes/no). There will be no stratification in cohort 3.

The study included three periods for cohort 1 and cohort 2:

1. A placebo-controlled period (Day 1 Week 1 to completion of pre-dose evaluations on Day 85 Week 13) during which 4 doses of CFZ533 or placebo were administered in addition to the standard of care (eg, low dose corticosteroid) required for pSS treatment;

2. the open-label period (from dosing day 85 week 13 to completion of evaluations on day 169 week 25) when all patients received 4 doses of CFZ533 in the open-label regimen, and

3. follow-up period (weeks 25-32) when patients were followed up without study medication.

Figure 1 is a schematic representation of the study plan for cohorts 1 and 2.

Cohort 3 included two periods:

1. the open-label period (from dosing on day 1 of week 1 to the last dose and completion of evaluations on day 85 of week 13);

2. follow-up period (from week 13 after completion of the last dosing to day 141 of week 21) when patients were followed up for 8 weeks without study medication.

During the open-label period, dosing in treatment group 1 started at 600 mg s.c. CFZ533 once weekly for 4 weeks; in treatment group 2, dosing started at 10 mg/kg i.v. CFZ533 on day 1.

Thereafter, dosing continued with 300 mg s.c. CFZ533 once weekly for 4 weeks (treatment group 1) and 9 weeks (treatment group 2), respectively.

Figure 2 is a schematic representation of the cohort 3 study design.

Cohort 1

Approximately 12 patients were randomized in this cohort. Each patient had a screening period from day -28 to day -2. Patients who met screening eligibility criteria were eligible for baseline evaluations. Baseline assessments were performed from day -6 with assessments completed on day -1 to treatment on day 1.

All results from baseline safety assessments were available prior to dosing and met the eligibility criteria. Eligible patients entered the placebo-controlled period on day 1 (week 1) and were randomized in a 2:1 ratio to receive either CFZ533 or placebo treatment. On day 1, a 3 mg/kg dose of CFZ533 or placebo was administered by subcutaneous injection (s.c.) followed by PK, pharmacodynamics (PD), and safety evaluations over a period of no more than 6 hours. Patients were discharged from the center on the same day after all evaluations were completed, with assurance that no safety issues were found.

Patients returned to the study center to receive three s.c. doses of either CFZ533 or placebo (same as they received on day 1) on day 15 (week 3), day 29 (week 5) and day 57 (week 9), respectively. During these visits, safety and efficacy assessments were performed and biomarker and PK samples were collected.

On day 85 (week 13), after safety and other evaluations that were evaluations at the end of the placebo-controlled period, all patients entered the open-label period and received s.c. dose of 3 mg/kg CFZ533 with open mode. This was followed by three s.c. doses of open-label CFZ533 (3 mg/kg each) on day 99 (week 15), day 113 (week 17), and day 141 (week 21), respectively. During these visits, safety and efficacy assessments were performed and biomarker and PK samples were collected. "Blindness" of the controlled period was maintained for the investigator and the patient until the end of the study.

On day 169 (week 25), patients received evaluations at the end of the open-label period and entered the follow-up period without study drug administration. Patients returned to the study center for safety monitoring during this period.

The end of the study visit was on day 225 (week 33), which included the completion of study evaluations followed by release from the study.

Cohort 2

When approximately 12 subjects were included in cohort 1, enrollment in cohort 2 began for randomization of approximately 30 patients to obtain approximately 24 patients undergoing 12 weeks of treatment. The study design for cohort 2 was identical to that for cohort 1, except for the dosing regimen, which included multiple intravenous doses of 10 mg/kg CFZ533 followed by PK, pharmacodynamics (PD), and safety assessments within no more than 2 hours after the end of the infusion. Additionally, body weight was measured at each dosing visit to calculate drug dosage according to actual weight (in cohort 1, baseline patient weights were used throughout the study).

Cohort 3

Approximately 24 patients were randomized in cohort 3 to obtain approximately 20 patients (10 in treatment group 1, 10 in treatment group 2) who completed 12 weeks of treatment.

Patients could remain on their standard therapies provided that the treatments were maintained at a constant level during the study.

Safety evaluations included physical examinations, ECG, vital signs, standard clinical laboratory evaluations (CBC, CBC, urinalysis, pregnancy test, blood clotting), control of adverse events and serious adverse events.

All s.c. injections and i.v. infusions took place in a control facility. Patients were closely monitored for at least 6 hours after s.c. injections in cohort 1, 2 hours after completion of i.v. infusions in cohort 2. In cohort 3, patients were monitored for at least 1 hour after completion of i.v. infusion and 30 minutes after s.c. injection or longer, at the discretion of the Investigator, to evaluate vital signs and signs or symptoms of side effects, including the development of a reaction to the injection. Pharmacokinetic, pharmacodynamic, and safety evaluations were performed no more than 6 hours after s.c. injections in cohort 1, 2 hours after completion of i.v. infusions in cohort 2 or 1 hour after completion of i.v. infusion and 30 minutes after s.c. injections in cohort 3. Subjects were released after these evaluations at the discretion of the investigator after a satisfactory analysis of the safety data.

PK assessment included measurements of free CFZ533 in plasma.

Competitive PD markers: CD40 saturation on peripheral blood B cells (free CD40 and total CD40 on B cells, cohorts 1 and 2 only), total soluble CD40, and total soluble CD154 (optionally only cohorts 1 and 2) were measured in patients' plasma to carry out PK/PD simulation. Additionally, autoantibodies were investigated, as well as some search biomarkers. Immunogenicity was assessed using a quasi-quantitative analysis of antibodies to CFZ533.

In Cohort 1 and Cohort 2, labial biopsies were optionally taken at baseline and at the end of the placebo-controlled period (Day 85, Week 13).

An interim analysis (1st IA) was performed including approximately 12 patients who completed a 12-week treatment period in cohort 1. For these patients, key data regarding safety and tolerability, as well as preliminary efficacy, were evaluated.

Additional interim safety and efficacy analyzes may have been conducted to support decisions regarding the current clinical trial, the sponsor's clinical trial projects in general, or in the event of any safety concerns.

2. Rationale for the study design

Cohorts 1 and 2

Two different doses (3 mg/kg s.c. and 10 mg/kg i.v.) of CFZ533 were evaluated in two separate cohorts.

A randomized, placebo-controlled, double-blind approach was used to eliminate potential bias in reporting safety and clinical efficacy data in this first exploratory study in patients with pSS.

Patients were randomized to CFZ533 or placebo in a 2:1 ratio in order to minimize placebo exposure and in order to collect more data on CFZ533. Stratified randomization was performed to limit the imbalance between the active and placebo groups in oral corticosteroid use at baseline.

During the open-label period, CFZ533 was administered to all patients, including those who received placebo during the placebo-controlled period. This provided a potential benefit in the treatment of these patients and the collection of additional safety and efficacy data for CFZ533 in patients with pSS. After the open-label period, patients underwent a follow-up period for which a duration of 8 weeks (12 weeks after last dosing of CFZ533) was chosen to allow sufficient time for safety monitoring and duration of response studies.

In addition to safety, efficacy, as calculated by the European League Against Rheumatism (EULAR), the Sjögren's Syndrome Disease Activity Index (ESSDAI) was chosen as the key endpoint of the study. The ESSDAI is well known to those skilled in the art (see, for example, Seror et al. 2011a). The ESSDAI demonstrated susceptibility in a retrospective analysis of a randomized controlled trial of rituximab and has been proposed as a sensitive tool to evaluate the efficacy of rituximab treatment (Moerman et al 2014). The authors reported a significantly reduced ESSDAI in the rituximab group compared to the placebo group at week 12 and week 24, demonstrating some efficacy in reducing disease activity in this small study.

Similarly, the study used an index of EULAR data from patients with Sjögren's syndrome (ESSPRI) (see, for example, Seror et al. 2011b)

Cohort 3

Because of the key goal of this cohort, placebo control was not necessary and an open-label regimen was warranted. Cohort 3 used two treatment groups to evaluate two loading regimens (multiple s.c. doses in treatment group 1 or single i.v. administration in treatment group 2) followed by a maintenance regimen (multiple s.c. doses, both groups) that were 12 weeks (from day 1 until day 85) to ensure steady state conditions for plasma concentrations of CFZ533 at a level similar to the minimum concentrations observed in cohort 2 (10 mg/kg i.v. schedule).

The duration of the follow-up period was 8 weeks (day 85 to day 141):

(i) to monitor the removal of CFZ533 under conditions where the target was fully saturated (slow removal) and when the target was not completely saturated with CD40 (fast removal), and

(ii) to characterize the possibility of a target-mediated elimination pathway for CFZ533 after 12 weeks of treatment.

3. Justification of the dose / regimen, duration of treatment

There is no previously reported experience with a CD40 blocking agent in primary human Sjögren's syndrome. However, evidence points to the involvement of CD40-dependent immune processes in the pathophysiology of the disease, including the presence of lymphoid aggregates and germinal center-like structures in the salivary glands of affected individuals, disease-specific autoantibodies, and expression of CD40 and its ligands in the glands. Preclinical and early human results indicated that full occupancy of the CD40 receptor was required to completely suppress T cell dependent B cell function as well as block CD40 related parenchymal functions and would be necessary to achieve maximum therapeutic benefit. benefit. For example, results from a 26-week toxicity study indicated that 1 mg/kg CFZ533 administered weekly measured total CD40 occupancy on peripheral B cells, but an inferior pharmacological effect was observed in some animals (3/6), manifested in partial decrease in the germinal centers.

This indicated that doses greater than 1 mg/kg CFZ533 (weekly) would be needed to completely suppress CD40-CD154 interactions and subsequent tissue signaling events.

Cohort 1 (subcutaneous, 3 mg/kg)

The highest subcutaneous dose of CFZ533 that has been tested in healthy volunteers is currently 3mg/kg and has been shown to be safe and well tolerated, as is a single dose in healthy volunteers. Doses of 1 and 3 mg/kg i.v. were associated with weeks 1 and 4 of complete (defined as >90%) CD40 occupancy on peripheral blood B cells, respectively. In addition, ex vivo CD154-induced CD69 expression was suppressed for about 4 weeks using PBMC from healthy volunteers who received 3 mg/kg CFZ533 i.v. CD40 occupancy was 90% (mean n=6; range 73-97%) 4 weeks after a single 3 mg/kg subcutaneous dose of CFZ533.

As a result, 3 mg/kg s.c. chose for the existing cohort 1 because this dose was safe and well tolerated by healthy volunteers, and also could lead to the desired pharmacological and pharmacodynamic effect.

The 2-week dosing intervals at baseline were designed to ensure complete saturation of CD40 on peripheral B cells, as well as complete suppression of CD40-CD154 interactions in tissues following subcutaneous administration. At the end of the 12-week treatment period, clinically relevant changes were expected in the primary endpoint and other key efficacy and biomarker indications. Based on published results with a single cycle of rituximab, clinically significant responses in pSS patients could be detected as early as week 5, with a maximum effect occurring at week 12 (Meijer et al 2010). During an additional 12-week open-label extension, additional data were collected regarding the long-term efficacy and safety of CFZ533.

Cohort 2 (intravenous, 10 mg/kg)

A 10 mg/kg i.v. regimen was presented. to provide a greater effect in the blood plasma during the treatment period in order to ensure complete and stable blockade of the CD40 pathway in the target tissue, in conditions where CD40 expression is likely to be higher. This scheme is supported by human safety data, relevant safety ratios from preclinical toxicology studies, relevant PD effects in non-human primates, and recently published data from ASKP1240.

Demonstrated Safety and Tolerability in Humans : Phase 1 study (CCFZ533X2101) tested single escalating doses (0.03 to 30 mg/kg) of CFZ533 iv and 3 mg/kg sc doses, showed no major safety issue upon completion up to highest dose tested (10 mg/kg iv). Based on clinical experience, the 10 mg/kg IV dosing regimen is currently expected to be safe and tolerable in patients with pSS.

Appropriate margin of safety from preclinical toxicology studies : GLP toxicology studies have so far tested CFZ533 at (i) weekly sc dosing for 13 weeks at 10, 50 and 150 mg/kg (sc and iv) in rhesus monkeys and ( ii) weekly sc dosing for 26 weeks at 1, 50 and 150 mg/kg in cynomolgus monkeys. Such studies did not reveal any major problems that prevented the use of CFZ533 on the proposed intravenous regimen for 12 weeks or 24 weeks. In a 26-week toxicity study in steady state cynomolgus monkeys, a mean concentration of 8300 µg/mL (Cav, ss) was obtained after weekly dosing at 150 mg/kg (NOAEL). The corresponding systemic exposure (AUC, steady state conditions) over a 1-month period would be 232,400 days*µg/mL, which is approximately 57 times greater than the predicted systemic exposure in plasma during the first month (AUC0-28 days; figure 3). In a 26-week NOAEL toxicology study, Cmax, ss was 9495 μg/mL, which is 24 times the expected Cmax (approximately 400 μg/mL) for the proposed intravenous regimen in patients with pSS (Figure 3).

Figure 3 shows the expected mean plasma concentration versus time profile for CFZ533 administered intravenously at 10 mg/kg (cohort 2). Mean PK profiles were simulated for 10 mg/kg iv CFZ533 was administered on study days 1, 15, 29, and 57 (placebo-controlled period) and study days 85, 99, 113, and 141 (open-label period). A Michaelis-Menten model was applied using parameters derived from a preliminary population analysis based on a cohort 5 model (3 mg/kg iv) PK data from the FIH study CCFZ533X2101 in healthy subjects. There was no previous experience with a CD40 blocking agent derived from a human with pSS, and no potential differences in CD40 biology (expression, metabolism) were known between healthy subjects and pSS patients. It is expected that the proposed iv regimen will provide stable plasma concentrations above 40 μg/ml throughout the treatment period, with the expected increased expression of CD40 in target tissues in patients with pSS. The horizontal dotted line at 40 µg/mL represents the plasma concentration above which full occupancy and blocking of the CD40 pathway in target tissues would be expected (based on PD data from a 26-week toxicology study in cynomolgus monkeys - 1 mg/day dose group). kg). The expected systemic exposure for the first month (increased dosing frequency) was 4087 days*mcg/mL (57 times less than the observed systemic exposure in plasma over one month at steady state in the 26-week Cynomolgus Monkey Toxicology Study - NOAEL at 150 mg/kg weekly), the expected C _max was approximately 400 μg/ml.

Corresponding effects of PD in non-human primate tissues : In a 26-week toxicology study (1 mg/kg dose group), animals with mean steady-state plasma concentrations ≥ 38 μg/mL had complete suppression of germline B cells. centers in the cortical regions of the lymph nodes. The 10 mg/kg iv regimen was expected to provide stable plasma concentrations above 40 µg/mL throughout the treatment period (placebo-controlled and open-label, see Figure 3) for the expected increased CD40 expression in pSS patients and the inferior effects of PD in target tissues due to loss of target saturation.

Data from ASKP1240, a CD40-blocking monoclonal antibody : A recent analysis of the disclosed PK/efficacy data of Astella ASKP1240 anti-CD40 antibody in solid organ transplantation (Harland et al 2015) demonstrated that effective target-mediated tissue clearance of the antibody can lead to loss of CD40 blockade and likely loss of effectiveness due to a significant increase in target expression in target tissues. The proposed intravenous regimen aims to saturate, throughout the treatment period, CD40 clearance pathways in conditions where CD40 overexpression is likely. Internal data suggested overexpression of CD40 in tissues derived from patients with primary Sjögren's syndrome (unpublished data), and the original subcutaneous regimen may not provide complete and stable blockade of the CD40 pathway in target tissue under these conditions.

Cohort 3

Based on the results from cohorts 1 and 2, it was considered that an effective dosing regimen in patients with pSS required a loading regimen (i.v. or s.c.) providing early full CD40 saturation and minimal target-mediated distribution (TMDD) followed by s.c. support scheme.

In cohort 3, the dose/regimen in treatment group 1 and treatment group 2 are set to assess whether a loading regimen (i.v. or s.c.) followed by s.c. maintenance regimen to deliver at stable plasma concentrations as in i.v. scheme tested in cohort 2 and have the ability to overcome the target-mediated distribution of CFZ533 through s.c. way.

Treatment group 1 : CFZ533 was administered sc at 600 mg (4 x 1 ml injections) weekly in 4 repetitions (loading) followed by 300 mg sc (2 x 1 ml injections) weekly in 9 repetitions (maintenance; starting on study day 29). During the loading phase, the saturation rate of the CD40 pool as well as the bioavailability of sc doses were unknown. However, a cumulative dose of 2700 mg for 1 month (up to day 29) was expected to have the ability to overcome TMDD (cumulative dose was 630 and 2100 mg on day 29 in cohort 1 and cohort 2, respectively). A weekly loading regimen of 600 mg followed by a maintenance regimen of 300 mg weekly was expected to provide inpatient delivery by day 85 because, if the target saturation was achieved, it was expected that the bioavailability of subcutaneous doses would be ≥ 75%, as for healthy people. volunteers. In treatment group 1, the cumulative dose up to day 85 (last dose) would have been 5100 mg compared to 1050 and 3500 mg in cohort 1 and cohort 2, respectively).

In treatment group 2, the loading regimen consisted of a single iv dose of 10 mg/kg CFZ533 (on study day 1) followed by sc 300 mg weekly in 12 repetitions (maintenance regimen based on study day 8). In cohort 3 treatment group 2, as already demonstrated iv in cohort 2, a single iv dose of 10 mg/kg (day 1) was expected to provide plasma concentrations ≥ 100 µg/mL by study day 8 (start of maintenance regimen) and conditions for full CD40 saturation. In treatment group 2, the weekly sc maintenance regimen was evaluated in the absence of significant target-mediated distribution and first pass effect (sc pathway).

In cohort 3, all subcutaneous administrations (300 mg or 600 mg weekly) were provided as fixed doses (not adjusted for body weight) to facilitate administration and improve convenience, since each drug product vial contained 150 mg/ml CFZ533.

In cohort 1 (sc 3 mg/kg regimen), at interim analysis, mean body weight was 70.7 kg (range 50.0–91.1 kg), corresponding to 212.2 mg fixed dose (range 150–273.3 mg). ). In cohort 2 (10 mg/kg schedule iv), the interim analysis had a mean body weight of 72.6 kg (range 50.0–107.2 kg), corresponding to a fixed dose of 726 mg (range 500–1072 mg). In Cohort 3, subcutaneous doses of 300 mg or 600 mg each replication were in the range of doses already used in Cohorts 1 and 2 and were unlikely to affect safety and tolerability. How body weight affected the distribution of CFZ533 has not been fully characterized, however, for both groups throughout the treatment period, it was expected that the average maximum plasma concentration (C _max ) would be below approximately 300 μg / ml (assuming that all subcutaneous doses had 100% safety bioavailability and a mean body weight of 70 kg for pSS patients) and did not show the expected excess of the mean C _max in cohort 2 (approximately 386 µg/mL, fifth dose on day 85 of the placebo-controlled period 1 cohort 2). This would be 32 times less than the C _max values (mean 9495 µg/mL) in a 26-week toxicology study in cynomolgus monkeys at NOAEL (150 mg/kg sc weekly).

PK/PD endpoints and immunogenicity were assessed during treatment and follow-up periods. Table 2 provides a synopsis of the protocol for the study.

Table 2. Protocol Synopsis

Intention and rationale • This study is designed to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary therapeutic efficacy of multiple doses of the monoclonal antibody CFZ533 in patients with primary Sjögren's syndrome (pSS), which will provide data for the further development of CFZ533 in the treatment of pSS. Primary Goals • To evaluate the safety and tolerability of multiple intravenous infusions of CFZ533 in patients with primary Sjogren's syndrome as measured by adverse events (AE).
• Compare the effect of multiple intravenous infusions of CFZ533 versus placebo on clinical disease activity in patients with primary Sjögren's syndrome as measured by change in the EULAR Sjögren's Disease Activity Index (ESSDAI) after 12 weeks of treatment. Secondary goals • To evaluate the safety and tolerability of multiple subcutaneous doses of CFZ533 in patients with primary Sjögren's syndrome as measured by adverse events (AE).
• To compare the effect of multiple subcutaneous doses of CFZ533 versus placebo on clinical disease activity in patients with primary Sjögren's syndrome, as measured by change in the EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) after 12 weeks of treatment.
• To evaluate the pharmacokinetics of multiple subcutaneous doses and multiple intravenous infusions of CFZ533 in patients with primary Sjögren's syndrome.
• To evaluate the effect of multiple subcutaneous doses and multiple intravenous infusions of CFZ533 versus placebo on self-report outcomes in patients with primary Sjogren's syndrome after 12 weeks of treatment, as measured by the EULAR Intensity Patient Report for Sjogren's Syndrome (ESSPRI), short form ( 36) health research (SF-36) and a multifactorial fatigue questionnaire (MFI).
• Assess changes in the physician's overall assessment of the patient's overall disease activity as recorded using a visual analog scale (VAS) after 12 weeks of treatment.
• Assess the change in the patient's overall assessment of their overall disease activity as recorded by the VAS after 12 weeks of treatment. Study plan • Randomized, double-blind, placebo-controlled, non-confirmatory study of approximately 12 patients in cohort 1 and 30 patients in cohort 2 and an open-label, randomized study of approximately 24 patients in cohort 3 population Male and female patients with primary Sjögren's syndrome aged 18 to 75 years (inclusive). Inclusion Criteria • Diagnosis of primary Sjögren's syndrome according to the revised EU/US Common Criteria (Vitali et al 2002).
• Moderate to severe disease activity with an ESSDAI score ≥ 6.
• The presence of autoantibodies at screening, as determined by any of the following:
• elevated serum ANA titers (≥ 1:160) and positive rheumatoid factor (RF); or
• a positive result for SSA binding.
• Stimulated saliva flow rate > 0 ml/min. for cohorts 1 and 2 and unstimulated saliva flow > 0 ml/min. for cohort 3.
• If the patient is receiving oral glucocorticoid treatment at screening, the dose should not exceed 10 mg prednisone or its equivalent per day and should be stable for at least 2 weeks prior to randomization and throughout the study period.
• If the screening patient is taking chloroquine or hydroxychloroquine, the dose should be stable for at least 4 weeks prior to randomization and throughout the study period.
• If the patient is receiving oral or parenteral methotrexate at screening, then the dose should not exceed 25 mg per week for at least 3 months prior to randomization and should be stable throughout the study.
• If the patient is being treated with oral azathioprine at screening, then the dose should not exceed 100 mg daily for at least 3 months prior to randomization and should be stable throughout the study. Exclusion Criteria • Secondary Sjögren's syndrome
• Use of other investigational medicinal products at study entry, or for five half-lives with other investigational medicinal products, or longer if required by local regulations, at study entry.
• A history of hypersensitivity to the study drug or to drugs of similar chemical classes.
• Patients received treatment:
• cyclosphosphamide for 6 months;
• IV corticosteroid bolus > 1 mg/kg for 3 months;
• rituximab for 12 months;
• belimumab for 6 months;
• any other biological product within 1 month or five times the half-life;
• any other immunosuppressive drugs such as cyclosporine A or mycophenolate for 3 months.
• Patients whose primary cause of dryness symptoms is related to medications taken regularly or intermittently, and not to primary Sjögren's syndrome.
• At significant risk of thromboembolic events.
• Pancreatic injury or pancreatitis.
• History or presence of medically significant heart disease.
• Clinically significant systemic viral, bacterial, or fungal infection within 30 days of randomization.
• A condition that can lead to a significantly increased risk of infections.
• History or event of tuberculosis.
• Significant surgical, medical, psychiatric or additional physical condition. Investigational and Reference Therapy Cohort 1:
150 mg of CFZ533 powder for injection and corresponding placebo.
• CFZ533: 3 mg/kg administered subcutaneously.
• Control: placebo CFZ533, cohort 2:
150 mg of CFZ533 powder for injection and corresponding placebo.
• CFZ533: 10 mg/kg administered by intravenous infusion.
• Control: placebo CFZ533, cohort 3:
150 mg CFZ533 liquid in vial for injection.
• 600 mg CFZ533 sc once a week for 4 weeks followed by 300 mg sc once a week for 9 weeks.
• 10mg/kg CFZ533 iv on day 1 followed by 300mg sc starting on day 8 once a week for 12 weeks. Efficacy and pharmacodynamic evaluations • EULAR Disease Activity Index for Sjögren's Syndrome (ESSDAI).
• Total soluble CD40 in plasma, total soluble CD154 in plasma, CD40 saturation in peripheral blood B cells. Safety ratings All cohorts:
• Physical examination.
• Key vital signs.
• Laboratory evaluations: hematology, clinical chemistry, urinalysis.
• Electrocardiogram (ECG).
• Pregnancy test.
• By-effect.
• Serious side effect.
• Immunogenicity (antibodies to CFZ533). Other ratings All cohorts:
• Pharmacokinetics of CFZ533.
• Soluble biomarkers (eg CXCL13).
• Patient Report on EULAR Intensity in Sjögren's Syndrome (ESSPRI).
• Physician and patient assessment of total disease activity (VAS), cohorts 1 and 2:
• Short Form (36) Health Research (SF-36);
• Multifactorial Fatigue Inventory (MFI). Data analysis Cohorts 1 and 2:
a longitudinal model describing the change in ESSDAI from baseline over time was fitted for the controlled portion of the study (up to 13 weeks) with the following covariates: baseline ESSDAI, baseline prednisone dose, treatment (placebo, 3 mg/kg CFZ533 sc or 10 mg/kg CFZ533 iv), time as a continuous factor and quadratic time effect, as well as random intercept, random slope and random quadratic effect for the subject. The change from baseline ESSDAI at week 13 was estimated based on the model for all treatments. The conclusion was made in the framework of the analysis with the frequency approach. The results of the primary analysis were evaluated against the following performance criteria to support internal decision making:
• statistically significant reduction in ESSDAI at week 13 in the CFZ533 group compared to placebo at a one-sided significance level of 10% and
• The estimated mean reduction in ESSDAI in the CFZ533 group should be 5 points or greater than placebo.
A positive sign of effectiveness was counted if both criteria were met.
Cohort 3:
descriptive statistics and graphical displays were used to summarize the PK concentrations in each treatment group.

4. Results

Cohorts 1 and 2

Forty-four patients included: 8 patients treated with 3 mg/kg CFZ533 s.c. and 4 placebos in cohort 1, and 21 treated with 10 mg/kg CFZ533 i.v. and 11 placebos in cohort 2. While PK/PD were as expected in the CFZ533 10 mg/kg i.v. cohort, based on data from the first human trial, exposure to CFZ533 was lower than expected in the 3 mg/kg cohort s.c., probably due to an effective target-mediated location (first pass effect) under conditions where CD40 expression was likely to be improved. Overall, CFZ533 was safe and well tolerated, and most AEs were mild to moderate. There was one serious AE (bacterial conjunctivitis) in the 3 mg/kg s.c. cohort that was not study drug related. Cohort 1 showed an improvement in ESSDAI of approximately 2 points compared to mean baseline scores of approximately 12 for both placebo and 3 mg/kg s.c. groups, and therefore no evidence of difference in treatment (ΔESSDAI=0.68, 95 % CI = -4.71-6.46). However, cohort 2 showed an improvement in ESSDAI from mean baseline of about 11, which was 6.35 in the 10 mg/kg i.v. compared to 1.27 in the placebo group, with a simulated difference between groups of ΔESSDAI=5.2 (95% CI=1.02-10.58), which strongly favored treatment with CFZ533 i.v. Improvements in other measures such as ESSPRI, MFI, clinician's overall score, and patient's overall score, and a decrease in the serum germinal center-associated biomarker CXCL13 were also observed in the CFZ533 i.v. 10 mg/kg group.

Figure 4 is a graph showing the pharmacokinetics of CFZ533-10 mg/kg IV. Scheme IV provided complete target saturation and complete blockade of the CD40 pathway in target tissues. Blocking of the CD40 pathway in tissues with a plasma concentration of >40 μg/mL was expected (suppression of GC development and T-dependent antigen response), dotted line in the graph. After 12/24 weeks of treatment, there were indications that CD40 expression was reduced in some patients with pSS.

Figure 5 shows the original adjusted total ESSDAI of 10 mg/kg IV. The top line represents placebo iv and the bottom line represents 10 mg/kg CFZ533 iv. As can be seen, there was a clear improvement in the CFZ533 group compared to placebo (mean delta=5.6 at week 12).

Figure 6 shows the original adjusted CXCL13-10 mg/kg IV. The top line represents placebo iv and the bottom line represents 10 mg/kg CFZ533 iv.

Figure 7 shows the difference in treatment groups at different endpoints for 10 mg/kg CFZ533 compared to placebo at week 13 (after 12 weeks of treatment - period 1).

It can be concluded that there are clear improvements in ESSDAI and Physician Overall Assessment (VAS). In addition, trends in most secondary endpoints favor CFZ533. Changes in ESSDAI were maintained during the open study period. Some improvement in the placebo group when switching to 10 mg/kg CFZ533 IV with a significant decrease in CXCL13 levels followed the same pattern.

In this proof-of-concept study testing for the first time a blocking, non-CD40-depleting antibody for primary Sjögren's syndrome, the results indicated that CFZ533 could provide a novel treatment for clinically active pSS.

In addition, figure 8 shows pharmacodynamics/target binding (10 mg/kg IV). In general, plasma soluble CD40 is stable target binding during the treatment and follow-up period. After 12/24 weeks of treatment, there were indications that CD40 expression was reduced in some patients with pSS.

Figure 9 shows a profile plot of the total ESSDAI - cohort 1: 3 mg/kg SC.

Figure 10 shows the pharmacokinetics: free CFZ533 - cohort 1: 3 mg/kg SC. As can be seen, there is an effective presystemic target-mediated clearance (first pass effect; probably due to increased expression of CD40) - blockade of the CD40 pathway in tissues was not achieved (CFZ533<40 μg/ml).

Figure 11 shows pharmacodynamics/target binding: total soluble plasma CD40 - cohort 1: 3 mg/kg SC. As can be seen, target binding was not maintained due to target-mediated deletion under conditions where CD40 expression was likely to be upregulated.

Figure 12 shows a profile plot of the total ESSPRI - cohort 1: 3 mg/kg SC.

Figure 13 shows a profile plot of the total ESSPRI - cohort 2: 10 mg/kg IV.

Figure 14 shows a profile plot of CXCL13 (pg/ml) - cohort 2: 10 mg/kg IV.

Figure 15 shows a profile plot of the total ESSDAI in the 10 mg/kg CFZ533 cohort.

Figure 16 shows a graph of the ICAM+ B cell profile (%) - cohort 2: 10 mg/kg IV.

Figure 17 shows the difference in treatment groups at different endpoints of CFZ533 compared to placebo at week 13 (after 12 weeks of treatment - period 1).

CD40 signaling has been associated with the pathogenesis of autoimmune diseases (AD), and patients with systemic AD (including pSS) typically have elevated CD40 expression and elevated serum/plasma levels of sCD40. In salivary gland biopsies from patients with primary Sjögren's syndrome, CD40 was constitutively expressed by lymphocytes, ductal epithelial cells, and endothelial cells (Dimitriou et al, 2002), consistent with elevated baseline plasma levels noted in the CCFZ533X2203 study.

CFZ533 was subject to target-mediated positioning (TMD), a process in which a significant portion of CFZ533 (relative to dose) is associated with high affinity for CD40, such that this interaction was reflected in the PK profile of CFZ533. In such circumstances, additional factors that should be considered to determine an appropriate posology for treating patients with pSS include the level of CD40 expression in the body, CD40 synthesis and degradation (target biology), and CFZ533-CD40 binding kinetics.

Previous clinical experience with CFZ533 in healthy volunteers, volunteers with rheumatoid arthritis, primary Sjögren's syndrome, kidney transplantation, Graves' disease, and myasthenia gravis showed that overexpression of CD40 is associated with a high rate of CFZ533 deletion (clearance), loss of target binding, and loss of pathway blockade. CD40 in target tissues if CD40 is not fully saturated. At full CD40 occupancy, the contribution of CD40 to the overall clearance of CFZ533 is minimal, and the location of CFZ533 was mainly due to the binding of CFZ533 to FcRn receptors (high capacity receptor responsible for IgG homeostasis via recycling/utilization).

As can be seen from the data, from a pharmacokinetic/pharmacodynamic point of view and a dose selection strategy, it is likely that the appropriate posology in patients with pSS will include a loading regimen followed by a maintenance regimen.

A loading regimen probably during the first month by IV or SC administration is warranted because CFZ533 is subject to CD40-mediated deletion. If CD40 is not completely saturated at the start of treatment, under conditions of increased CD40 expression, then the high clearance rate of CFZ533 is likely due to loss of target binding and loss of blockade of the CD40 pathway in target tissues. After a loading period, and based on preliminary modeling using PK data from the ongoing CCFZ533X2203 study, SC maintenance regimen was selected in pSS patients to achieve complete blockade of the CD40 pathway in target tissues.

Cohort 3

The reduction in ESSDAI from baseline to week 12 in Cohort 3 showed a similar pattern in both dosing groups, such as in Cohort 2, confirming the efficacy of two IV/SC or SC/SC dosing regimens of CFZ533. However, since cohort 3 was open-label and no placebo control, the efficacy data were exploratory only and should be interpreted with caution.

Example 8 Blockade of CD40-CD154 Interaction Pathways Suppressed Ectopic Germ Centers and Inhibited Pathology in a Murine Model of Sjögren's Syndrome NOD/ShiLtJ

Sjögren's syndrome (SS) is a chronic, autoimmune disease characterized by sialadenitis and exocrine gland dysfunction. It is one of the most common rheumatic systemic autoimmune diseases after RA, with an adult prevalence of 0.3-0.5%. Clinical manifestations included fatigue, keratoconjunctivitis sicca, xerostomia, nasal dryness, vaginal dryness, tracheal dryness, dry skin, arthralgia/arthritis, Raynaud's disease, lymphadenopathy, interstitial pneumonitis, vasculitis (usually cutaneous), nephritis, and lymphoma.

The EULAR Disease Activity Index for Sjögren's Syndrome (ESSDAI) was developed to measure disease activity in patients with primary SS. The ESSDAI is the HA's accepted primary outcome measure for SS and is designed to complement the results of the Patient Report (ESSPRI).

Specific manifestations of the disease in patients with both primary and secondary SS include the presence of anti-Ro and La autoantibodies, as well as mononuclear cell infiltrates in the salivary and lacrimal glands (Bombardieri, et al. 2012). In some cases, these accumulations of T and B lymphocytes form well-organized structures called ectopic lymphoid structures (ELS), which share morphological and functional similarities to GCs (Voulgarelis et al., 2008). Previous work has reported evidence of ELS in the salivary glands of patients with SS, and in preclinical models of the disease and evidence of ongoing affinity maturation (Jacobi et al., 2002; Stott et al., 1998; Bombardieri et al., 2012), linking these structures with the pathology of the disease (Bombardieri et al., 2017).

There are relatively few published data on the role of various immunostimulatory pathways in the formation and functioning of ELS, despite unequivocal data supporting the essential role of such pathways as, for example, CD40-CD154 (CD40 ligand) in GC biology (Laman et al., 1996). If such interactions played a role in ELS formation and function, then pathway blockade would be predicted to eliminate established structures in diseased tissue and potentially improve organ function. To test this hypothesis, the present inventors investigated the effect of therapeutic administration of an anti-CD154 monoclonal antibody in a non-obese diabetic mouse model with secondary SS (NOD/ShiLtJ) (Humphreys-Beher et al., 1996) and monitored salivary gland ELS antibody-secreting cells , as well as the expression of aquaporin-5 (AQP-5), a protein essential for secretory cell function (Delporte et al., 2006).

1. Materials and methods

CD40 pathway gene signature in the salivary glands

Cryosections of salivary glands from untreated NOD/ShiLtJ mice (Charles River, Germany) were used for microarray profiling of gene expression. Laser capture of the microdissection was used to enrich the ELS.

Mouse model of NOD/ShiLtJ secondary SS

The NOD/ShiLtJ strain develops type 1 diabetes-like diseases in addition to SS-like disease, and therefore could be considered as a model for secondary SS (Humphreys-Beher et al., 1994). 12-week-old NOD/ShiLtJ females were randomized into 2 treatment groups (n=15 per group, two experiments): MR1 (Armenian hamster anti-mouse CD154 IgG) and isotype control (IC, hamster anti-mouse IgG, BioXcell) at 15 mg /kg, intraperitoneally (i.p.), 2x/week for 10 weeks. All procedures were performed in accordance with Swiss animal welfare law and were approved by the cantonal veterinary office in Basel, Switzerland (Animal License No. BS-2482).

Histopathology and immunohistochemistry of the spleen and salivary glands in NOD/ShiLtJ mice

Three μm thick paraffin sections of the left salivary glands were stained with hematoxylin and eosin (HE). Automated immunohistochemical stains for CD3, CD45R, CD138, Iba-1, Ki-67, and AQP-5 were performed on a Ventana Discovery XT immunostainer (Roche Diagnostics, Switzerland). Spleens from NOD mice were stained with Ki-67.

ELISPOT Assays for Antibody Secreting Cells (ASC)

Single cell suspensions were prepared from the salivary glands and spleens (online file with additional data) and fresh CD45+ cells were added to pre-coated ELISPOT plates and incubated for 20 hours at 37° C. in the dark. ASC binding to plates was detected by adding TMB substrate solution and spot density was measured using an EliSpot Reader "AID classic".

Anti-Ro antibody, ELISA

Mouse antibody to SSA/Ro60 was evaluated in serum using the ELISA kit cat. no. 5710 (Alpha Diagnostic International, Inc. USA) according to the manufacturer's instructions.

2. Results

Activation of CD40 gene signature in salivary gland B cells

Microarray analyzes were used to identify subsequent CD40 genes in B cells following rCD154 stimulation of primary human CD19pos B cells, 43 of which can be mapped to mouse genes. It was then investigated whether the expression of these genes was modulated in salivary gland (SG) tissue in NOD mice at 12 and 24 weeks of age. Fourteen genes, including Cd40, Cd80, and Aicda, were significantly upregulated in microdissected ELS compared to whole salivary gland (WS) tissue at both time points, indicating chronic pathway activation (Figure 26A). Ingenuity Pathway Analysis also indicated that CD40 and CD154 (CD40LG) were among the upper upstream regulators at both week 12 and 24 (Figure 26B) and it was possible to demonstrate overlap between CD40 pathway genes activated in SG obtained from NOD/ShiLtJ mice and from pSS patients (Horvath et al., 2012).

CD40-CD154 interactions are required for ELS formation

NOD/ShiLtJ mice developed focal cellular infiltrates in the SG at 8-12 weeks of age with a 100% incidence that preceded the decline in SG function (Jonsson et al. 2012). The above data suggest chronic CD40 signaling in NOD SG tissues, and this was supported by evidence of CD40 expression by mononuclear cells at inflammatory sites (Roescher et al. 2012). Therefore, it was decided to test the effects of therapeutic blockade of CD40-CD154 by administering doses of anti-CD154 MR1 mAbs at 15 mg/kg once every two weeks starting at 12 weeks of age for ten weeks.

Figure 26B shows that continuous dosing of MR1 for 10 weeks in NOD/ShiLtJ mice resulted in a significant decrease in ELS in the salivary glands, as determined by clusters of Ki67-positive cells. In addition, a strong decrease in the percentage of resident SG B and T cells and macrophages was observed. Histological images showed that MR1 was able to suppress ELS ten weeks after the start of dosing (Figure 26C).

Blockade of CD40-CD154 Interactions Inhibited ASC Formation and Autoantibody Production

The introduction of MR1 also led to a decrease in total IgG (but not IgM) ASC in the spleen, SG and bone marrow (figure 26B), as well as complete suppression of spleen GC. Mice treated against CD154 also showed a significant reduction in serum anti-Ro IgG levels, although the incidence of Ro-specific ASCs in the salivary glands did not appear to be significantly affected (Figure 26C).

Blockade of CD40-CD154 interactions prevented loss of AQP-5 positive cells

Although it was possible to assess stimulated salivary flow in mice, sampling for such analyzes required fasting, anesthesia, and pilocarpine stimulation (Jonsson et al. 2006); factors that may have influenced animal welfare and interpretation of readings. Therefore, it was decided to control the expression of AQP-5, a water channel protein involved in the regulation of saliva and tear production (Delporte et al., 2006), which is thought to be involved in secretory function in SS (Yoshimura et al., 2016). The AQP-5 positivity score assessed by staining (Figure 27) showed a higher percentage of AQP-5 positive cells in treated MR1 compared to controls, suggesting that blockade of CD40-CD154 may prevent or delay loss of secretory functions of the cells of the salivary glands.

3. Discussion

Evidence for affinity maturation and the presence of autoreactive B cells in salivary ELS suggests a potential role for these structures in SS pathology (Stott et al., 1998). Given the functional similarity between ELS and GC, it has been suggested that immunological co-stimulatory pathways involved in GC biology may play a similar role in ELS. Deficiency in either CD40 or CD154 prevented GC formation, and pharmacological ligand or receptor blockade also disrupted established GCs (Ristov et al., 2018; Kim et al. 2014). CD40-CD154 interactions have also been implicated in ELS biology, as CD40 expression has been observed on infiltrating cells in SG glands derived from pSS patients as well as in NOD mice (Roescher et al. 2012; Ohlsson et al. 2002). Combined with sustained upregulation of genes below CD40 in ELS from NOD mice as well as in SG biopsies from pSS patients, these data suggested that this co-stimulatory pathway was active in salivary gland leukocyte infiltration.

In addition, it was demonstrated that therapeutic blockade of CD40-CD154 interactions resulted in the removal of ELS, a profound decrease in infiltrating leukocytes, as well as a decrease in serum levels of anti-Ro autoantibodies, blockade of CD40-CD154 also prevented the loss of cells expressing AQP-5, a protein required for secretory cell function (Delporte et al., 2006), although assessment of saliva production would be a more direct assessment of SG function. Previous data indicated that prophylactic administration of a single dose of MR1 to young NOD mice (4–5 weeks of age; 1–2 months before onset of signs of sialadenitis) could prevent the development of sialadenitis and reduce the level of anti-Ro autoantibodies (Mahmoud et al., 2016), which was consistent with the results obtained from CD40-deficient NOD mice. However, it has not been previously shown that therapeutic blockade of CD40-CD154 interactions could eliminate SG inflammation, ELS, and reduce autoantibody levels in this model.

The data contrast with earlier work where adeno-associated viral expression of a soluble CD40-Ig fusion protein in NOD mouse SG failed to reduce sialadenitis (Roescher et al., 2012). Since the authors reported no evidence of fusion protein expression in SG, it is likely that drug concentrations were insufficient to achieve complete blockade of the CD40-CD154 pathway. On the contrary, this study could clearly demonstrate the abolition of spleen GC, suggesting that there was sufficient exposure to MR1 for a full pathway-related effect of PD in tissue. Taken together, the data suggested that therapeutic blockade of the co-stimulatory CD40-CD154 pathway with biologics such as anti-CD40 mAb1 could have a beneficial therapeutic effect in patients with SS (Ristov et al., 2018).

Example 9 In vitro and in vivo characterization of CFZ533, a blocking and non-depleting anti-CD40 monoclonal antibody

1. Ways

CD40 Affinity Analysis of CFZ533 Using Surface Plasmon Resonance

Recombinant CFZ533 binding assays were performed at 25° C. with HBS-EP+ as running buffer. A typical binding assay cycle consisted of three steps: (i) capture of the antibody by ProteinA immobilized on the chip surface, (ii) binding of the CD40 antigen to the captured anti-CD40 antibody, and (iii) regeneration of the ProteinA surface. To determine the kinetic rate constants of antigen-antibody binding interactions, the binding data were processed twice referring to responses from control injections. Binding curves were fitted locally using a 1:1 interaction model from Biacore T100 Evaluation software to determine kinetic rate constants. The equilibrium dissociation constant (KD) was calculated as the ratio of the rate constants kd/ka. All binding measurements were performed in two independent experiments.

FcγRIIIA Affinity Analysis of CFZ533 Using Surface Plasmon Resonance

The extracellular domains of human FcγRIIIA labeled with a 4 amino acid purification tag (4APP; Novartis) and an Avi biotinylation tag (GLNDIFEAQKIEWHE; Avidity) were synthesized by Geneart: Human FcγRIIIA (CD16a) 158V (Uniprot: P08637, 17-199), 158F human FcγRIIIA (Uniprot : P08637, 17-199) were expressed in HEK293 cells and purified by 4APP-binding affinity chromatography. The receptors were site-directed, biotinylated BirA (Avidity) coupled to streptavidin sensor chips (General Electric) and equilibrium binding levels of various Abs were analyzed by surface plasmon resonance (T100, General Electric) as described (Warncke et al. 2012) . Equilibrium dissociation constants (K _D ) were calculated using the 1:1 model.

Cultures of human leukocytes

Whole blood buffy coats were obtained from healthy volunteers (Blutspendezentrum, Basel, Switzerland) or whole blood was collected from healthy volunteers provided with informed consent in accordance with the Swiss Human Research Law and approval of the responsible ethical committee (Ethikkommission Nordwest- und Zentralschweiz; EKNZ ). Human tonsil specimens were obtained from both Ergolz Klinik (Liestal, Switzerland) (study protocol no. 1000244 v.03; approved by Ethikkommission beider Basel; EKBB) and Kantonspital (Listal, Switzerland) (study protocol no. TRI0149 v.01; approved EKNZ). For in vitro culture experiments, please see Supplementary Materials for detailed methods. Briefly, whole blood, PBMC isolated, in vitro derived human tonsil monocytic DC or B cells were incubated with single concentrations or dose titration of CFZ533 or appropriate control antibodies. For pathway blocking experiments, these cultures also included a concentration of recombinant human CD154 EC80 (5 μg/ml) and IL-4 (75 ng/ml). The results of the in vitro assay included proliferation assessed by thymidine incorporation ( ³ H-TdR), assessment based on flow cytometry of expression of the CD69 activation molecule on B cells, and cytokine secretion assessed by ELISA. Similar analyzes were used for whole blood NHP and PBMC. In some human whole blood experiments, CD40 receptor occupancy was also assessed using fluorescently labeled CFZ533. If necessary, IC50 values were estimated using linear regression curve fitting in GraphPad Prism® software.

In vitro cell depletion assays

See Supplementary Materials for detailed methods. Briefly, the ability of CFZ533 to mediate depletion of CD20 ^pos B cells was monitored in human whole blood over a period of three days compared to depletion of the rituximab B cell antibody. For CDC, CFZ533 or rituximab was incubated with RAJI B cells in the presence or absence of rabbit complement and cell lysis was assessed by luminescence.

Internalization of CFZ533

The internalization of fluorescently labeled CFZ533 and rCD154 was assessed in vitro using the human RI-1 B cell line (Th'ng et al, 1987). The CD40 dependence of CFZ533 internalization was assessed using the CD40 knockout RI-1 cell line. Internalization was assessed using an Amnis® flow cytometer (Merck KHaA, Darnstadt) according to manufacturer's instructions and data analyzed using ImageStream® ^X software.

In vivo studies

Single-dose pharmacokinetic/pharmacodynamic (PK/PD) studies used cynomolgus macaque (Macaca fascicularis) 7.5-8.5 years old (6.5±2.6 kg) and captive-bred from the Philippines for treatment with biologicals (Siconbrec, Makati, Philippines). Animal handling, care, drug treatments and blood sampling were performed in accordance with the Swiss Federal Law for the Protection of Animals (animal licenses BS No. 1900, BS No. 1495). For immunization reconstitution experiments, the present inventors used animals from a toxicology study conducted at Covance Laboratories GmbH, Münster, Germany (original in preparation). The study was conducted in accordance with an approved study protocol and local standard operating procedures in strict accordance with national animal welfare legislation and accepted animal welfare standards.

In the PK study, CFZ533 was administered to three animals at calculated single doses of 16.2 (5532), 18.5 (5531) and 20 (5530) mg/kg. Blood samples were collected for analysis of serum CFZ533 concentrations, peripheral T and B lymphocyte counts, and CD40 occupancy on peripheral B cells by CFZ533. For TDAR recovery experiments, animals were immunized with keyhole limpet hemocyanin (KLH) in alum on study days 8 (priming) and 43 (recovery; during CFZ533 treatment), respectively. Serum was collected one day before and 7, 14 and 21 days after priming and booster immunizations. KLH-specific IgM/IgG titers were determined by sandwich ELISA using reference cynomolgus monkey KLH IgM/IgG serum as standard. PK assessment was performed as described above. For more details regarding the PK and TDAR experiments, see Supplementary Materials.

Histological analysis of germinal centers

Sections of formalin-fixed, paraffin-embedded (FFPE) spleen and lymph nodes (axillary, mandibular and mesenteric), stained with hematoxylin and eosin, and with indirect immunoperoxidase method (HRP+DAB from Dako) with the following markers: anti-CD20 antibody (M0755, Dako ), anti-CD8 (RM-9116-SO, Medac) and Ki67 (M7240, Dako). All preparations were evaluated and classified according to the intensity of staining (from negative to intense). In addition, the staining pattern and distribution of any immunohistochemically stained cells in the tissue were also described.

2. Results

CFZ533 binds human CD40 and inhibits rCD154-induced activation of many CD40-expressing cell types

Table 3 shows that the KD of CFZ533 for recombinant human CD40 was determined by surface plasmon resonance as 0.3 nM, thus it is very similar to the original HCD122 antibody (wild-type IgG1 version of CFZ533).

Table 3. Binding affinity (KD) and kinetics of HCD122 and CFZ533 with human CD40.

HCD122 CFZ533 K _D [M] 4.67±1.00×10 ^-10 3.05±0.26×10 ^-10 k _a [1/M⋅s] 2.84 ± 0.67×10 ⁵ 3.13 ± 0.73×10 ⁵ k _d [1/s] 1.26±0.03×10 ^-4 0.93 ± 0.14×10 ^-4 Chi ² [RU ² ] 0.17-0.19 0.10-0.15

Figure 18A shows the effect of CFZ533 on rCD154 and IL-4 mediated proliferation (3H-TdR) of human whole blood cultures, PBMCs, and isolated tonsil B cells from multiple donors (5, 32, and 6 donors, respectively). Data are presented as normalized counts per minute (cpm) (rCD154+IL-4=100; dotted lines). Figure 18B shows the production of CFZ533 inhibited TNF-alpha with rCD154-stimulated moDCs after overnight culture. Figure 18C shows delayed addition of CFZ533 inhibited rCD154+IL-4 mediated proliferation of human PBMCs. CFZ533 was added to human PBMC one hour before, concurrently, or two and six hours after rCD154+IL-4 stimulation, and proliferation (3H-TdR) was assessed after a further four days of culture (dashed dotted lines and dotted lines represent rCD154+IL-4 and control cells plus media). For all data, the mean and SD readings of rCD154-induced stimulation were plotted against log-transformed CFZ533 concentrations. Where appropriate, IC50 values were determined using linear regression curve fitting. Figure 18D shows the relationship between CD40 occupancy and blockade of the CFZ533 pathway. Whole human blood from 10 donors was cultured overnight with rCD154 in the presence of a dose titration of CFZ533. The extent of pathway activation (% CD69pos on B cells) and the extent of CD40 occupancy (staining with AlexaFlour 488 labeled with CFZ533) were assessed. Open and filled circles show the percentage of CD40 occupied by CFZ533 and the percentage of cells expressing CD69pos on CD20pos B cells as a function of log-transformed CFZ533 concentration, respectively (mean and SD shown). Dotted and dotted lines represent rCD154-induced CD69 expression and control cell cultures plus media normalized to all donors.

Figure 18A shows that CFZ533 completely inhibited rCD154-induced proliferation of human whole blood cultures, PBMCs, and purified tonsil B cells from multiple donors with activities (IC50 values) of 0.024 μg/mL (0.16 nM), 0.017 μg/ ml (0.12 nM) and 0.071 µg/ml (0.47 nM), respectively. In addition, the present inventors were able to demonstrate that CFZ533 completely blocked rCD154-induced TNF production by monocyte-derived primary dendritic cells (moDC) with an IC50 of 0.04 μg/mL (0.27 nM) (Figure 18B).

As previously reported, CFZ533 inhibited rCD154-induced proliferation of PBMCs from cynomolgus monkeys (Cordoba et al., 2015). CFZ533 inhibited rCD154-induced proliferation of PBMCs from humans, animal rhesus monkeys, and cynomolgus monkeys with similar activity (IC50 0.02, 0.03, and 0.01 μg/mL, respectively) and could also bind CD40 on B cells of these species with EC50 values of approximately 0.2 µg/mL, see Table 4.

Table 4. Cellular binding and functional properties of CFZ533 in humans and NHP.

Inhibition of rCD154-induced proliferation (IC50 PBMC) CD40 CFZ533 occupancy (MFI EC50 on CD20+ cells) Human 0.017+0.012 µg/l
0.12+0.08 µM
(n=32) 0.22+0.042 µg/ml
1.49+0.28 µM
(n=4) rhesus monkey 0.026+0.017 mg/ml
0.18+0.12 µM
(n=8) 0.22+0.033 µg/ml
1.49+0.22 µM
(n=6) crabeater macaque 0.010+0.003 mg/ml
0.07+0.02 µM
(n=4) 0.20+0.068 µg/ml
1.35+0.46 µM
(n=4)

The above cellular data were obtained from experiments in which CFZ533 was added before or simultaneously with rCD154, indicating that the antibody could prevent endogenous ligand binding. The present inventors were also able to demonstrate that addition of CFZ533 within no more than 6 hours after the start of culturing leukocytes containing rCD154 resulted in complete inhibition of cell activation with minimal loss of activity, indicating that CFZ533 could displace an endogenous ligand from CD40 (figure 18C).

The inventors also wanted to evaluate the relationship between the degree of CD40 CFZ533 occupancy and the degree of pathway inhibition. To this end, the inventors simultaneously evaluated the occupancy of the CD40 receptor CFZ533 and rCD154-induced CD69 in whole blood from several donors. Figure 18D shows that at least 90% CD40 receptor CFZ533 occupancy was required for complete blockade of CD40 pathway activation. A similar relationship between receptor occupancy and pathway inhibition was also observed using CD23 and CD54 as indications of CD40 pathway activation (data not shown).

CFZ533 exhibited minimal stimulatory potential in vitro

The ability of CFZ533 to stimulate human leukocyte activation was assessed using the proliferation and activation of the CD69 activation molecule on B cells in whole blood. Figure 19A shows data for i. of human whole blood from multiple donors (n=13), which was incubated with CFZ533 dose titration, and proliferation ( ³ H-TdR) was assessed after three days of culture. ii. Human PBMC from multiple donors (n=26) were incubated with CFZ533 dose titration and proliferation ( ³ H-TdR) was assessed after three days of culture. For both plots, data are presented as mean and SD normalized cpm versus log-transformed CFZ533 concentration (rCD154+IL-4=100; dotted lines, cells plus media=0; dotted lines). Figure 19B shows that CFZ533 did not induce human PBMC proliferation in the presence of additional stimuli. Human PBMCs were stimulated for 3 days by dose titration of CFZ533 in the presence of IL-4(i) or F(ab')2 IgM antibody. (ii). Mean and SD 3H-TdR (cpm) are shown as a function of log-transformed CFZ533 concentration. Figure 19C shows how human whole blood (41 donors) was cultured overnight without stimuli, CFZ533, isotype control or rCD154 and CD69 expression on B cells was assessed by FACS. Each dot represents data from a single donor with mean CD69 % values indicated by a horizontal red line.

Figure 19A shows that CFZ533 is unable to induce thymidine incorporation into human whole blood (1:10 dilution) or PBMC, unlike rCD154. The inability of CFZ533 to induce proliferation was not affected by the addition of additional costimuli such as IL-4 or IgM antibody (Figure 19B). The present inventors were also able to demonstrate that CFZ533 was unable to induce CD69 activation on B cells in whole blood from multiple donors, again in contrast to rCD154 (Figure 19C). Finally, CFZ533 was unable to induce cytokine production by CD40 expressing DCs derived from human umbilical vein monocytes or endothelial cells (HUVEC) (data not shown).

CFZ533 did not mediate cell depletion

CFZ533 was engineered to contain the N297A mutation, previously shown to abolish FcγR binding, resulting in an inability to mediate antibody-dependent cellular cytotoxicity (ADCC). CFZ533 was not able to bind FcγRIIIA compared to HCD122 (wild-type IgG1) (Table 5), and the inventors wanted to study how this lack of binding affects the ability of CFZ533 to mediate cell depletion.

Table 5. Binding affinity (k _a [1/M]) of HCD122 and CFZ533 to human FcγRIIIA

FcγR species HCD122 (IgG1 wild type) CFZ533 (N297A IgG1) Human 158V FcγRIIIA 1.72×106 n. O. 158F Human FcγRIIIA 6.99×105 n. O.

n. O. - not detected

Figure 20A shows data from human whole blood cultures incubated for 72 hours in the presence of a titration dose of CFZ533 or 50 μg/ml rituximab. The number of B cells was determined based on the CD45pos and CD19pos events entering the gates of FSC/SSC lymphocytes. Results for individual antibody concentrations were calculated as the percentage of B cells remaining relative to untreated samples and plotted against the log-transformed antibody concentration (adjusted to 100% and shown as a dotted line). Data are mean and SD of eight independent donors. Figure 20B shows results for Raji B cells incubated with various concentrations of rituximab or CFZ533 and a fixed concentration of rabbit complement. Concentration-dependent killing of Raji cells was analyzed after 2 hours, while cell viability was measured by determining the concentration of ATP in each well using luciferase. Results are presented as isotype-normalized luciferase relative units (RLU) versus log-transformed antibody concentration.

Figure 20A shows that not only was the CD20 depleting antibody rituximab able to remove approximately 80% of B cells in human whole blood, but CFZ533 was unable to mediate any cell depletion. In addition, CFZ533 was unable to mediate complement dependent cytotoxicity (CDC) of Raji B cells, unlike rituximab (Figure 20B).

CFZ533 is internalized by B cells in a CD40-dependent manner

Next, the present inventors wanted to test whether CFZ533 could be internalized by CD40 expressing the human B cell line RI-1. Figure 21A shows that rCD154 was internalized under acceptable conditions (37° C.) compared to unacceptable conditions (4° C.), where weak rCD154 staining could be observed on the plasma membrane. CFZ533 was also internalized, although there appeared to be residual membrane staining at 37°C. Figure 21B shows that the degree of internalization of rCD154 was greater than that observed for CFZ533. Using the CD40 knockout cell line RI-1, we were able to demonstrate that the binding and internalization of CFZ533 (Figure 21C) and rCD154 (data not shown) was CD40 dependent.

Figure 21A shows representative images of individual RI-1 B cells cultured with AlexaFlour 488 labeled with rCD154 or CFZ533 for 3 hours at 37°C or 4°C. Figure 21B. Relative failure of internalization of CFZ533 and rCD154 under acceptable conditions (unacceptable failure values deducted). Each point represents data from a single experiment, and the population mean is indicated by a horizontal red line. Figure 21C. Representative images of individual CD40-expressing or CD40-knockout RI-1 cells cultured with Alexa488 labeled with CFZ533 for 3 hours at 37°C. In all experiments, cells were co-stained with CD45-labeled AlexaFlour 647 to mark the cell membrane.

Pharmacokinetic properties of CFZ533 in non-human primates

Figure 22A. Serum concentrations of CFZ533 in three cynomolgus monkeys after a single dose at calculated doses of 16.2 (5532), 18.5 (5531) and 20 (5530) mg/kg IV. Figure 22B. CD40 occupancy: percentage of CD40 present (i) and percentage of total CD40 (ii) C. Peripheral B/T cells: percentage of peripheral blood B cells after a single dose. Day 0 is the time when CFZ533 was administered.

The above data indicate that CFZ533 binds NHP CD40 and can inhibit rCD154-induced NHP B cell activation with similar activities. This suggests that cynomolgus and rhesus monkeys would be suitable species for in vivo studies investigating the association between PK and PD of CFZ533. The data in Figure 22A shows the PK profiles of three cynomolgus monkeys after a single intravenous dose of CFZ533 (estimated doses of 16.2, 18.5, and 20 mg/kg). Typically, for a monoclonal antibody targeting an internalizing membrane-bound antigen (Mager et al. 2006 and Ng et al. 2006), the time course of CFZ533 concentration showed a clear target-mediated arrangement, resulting in non-linear PK profiles and half-life and clearance rates dependent on from concentration. The inflection point seen in the PK profiles was a marker of target binding and is associated with increased CD40 contribution to overall CFZ533 clearance and shorter half-life. In addition, the inflection point in the PK profiles coincided with the time when a drop in CD40 saturation was observed (Figure 22B, i). This occurred at about 10-20 μg/ml when CFZ533 was more rapidly removed. All animals showed no loss of CD40 receptor expression on cells (Figure 22B, ii). In addition, CFZ533 did not deplete peripheral blood B cells (Figure 22C) or T cells (data not shown), despite some observed changes throughout the study.

CFZ533 inhibited reductive T-cell dependent antibody production

Figure 23A shows an experimental schematic design for evaluating the effect of CFZ533 on TDAR recovery. The arrows below the x-axis represent primary and secondary KLH immunizations. The timing of a single dose of 10 mg/kg CFZ533 is shown above. Asterisks indicate time points at which anti-KLH and/or CFZ533 IgG levels were measured. Figure 23B. Each graph shows anti-KLH IgG levels (solid symbols) and plasma levels of CFZ533 (logarithmic scale; continuous line) for an individual animal. Mean anti-KLH IgG levels from control animals (open symbols) are superimposed on each plot for comparative purposes. Figure 23C. Histological analysis of germinal centers (Ki67 staining) in mLN from rhesus monkeys from a 26-week multiple subcutaneous dose study of 1 mg/kg/week using CFZ533. mLN sections from six animals are shown (i) along with a control image (ii). iii. Mean steady-state serum concentrations of CFZ533 during the dosing period from individual animals at the end of the treatment period.

The expected target effect of PD on blocked CD40 was TDAR inhibition (Kawabe et al. 1994). CFZ533 inhibited primary TDARs in NHP and humans, and the present inventors also wanted to investigate the effects of this antibody on TDAR recovery. The experimental design is summarized in Figure 23A. Briefly, four rhesus monkeys were immunized with KLH in alum on study day -28 (priming) prior to a single intravenous dose of CFZ533 at 10 mg/kg on study day 1 followed by a second KLH immunization on study day 15.

Figure 23B shows the effects of CFZ533 on recovered anti-KLH IgG responses in four individual animals compared to data from an immunized control (no CFZ533). There was inter-animal variability in the PK profiles of CFZ533 with faster clearance of CFZ533 observed in animals #1 and #3. Higher plasma concentrations were observed over a longer period of time in animals #2 and #4. Interestingly, these animals demonstrated complete suppression of the restored IgG response to KLH (and IgM; data not shown) on study day 15 (note that all animals had a primary TDAR for KLH). In contrast, IgG responses to KLH were observed (albeit somewhat delayed) in animals with faster clearance of CFZ533 (higher latency in animal #3 compared to animal #1), especially when serum levels of CFZ533 were less than about 40 µg /ml during the second KLH immunization. As observed in previous in vivo experiments with CFZ533 in transplanted (Cordoba et al. 2015) and non-transplanted animals (Figure 22B), no depletion of peripheral B cells was observed (data not shown).

The above results indicated that serum concentrations of CFZ533 higher than about 40 μg/ml were required to completely suppress TDAR recovery in NHP. The present inventors wished to further explore the relationship between exposure to CFZ533 and tissue pharmacodynamic effects dependent on the CD40 pathway. At the end of the 26 week toxicology study, at 1 mg/kg/week CFZ533 subcutaneously, the inventors performed histological and molecular analyzes of GC in the mesenteric lymph nodes (mLN). Figure 23C(i) shows that of the six dosed animals, the inventors could observe complete suppression of GC in three individuals, while GC could still be observed in mLN of the remaining animals. Figure 23C(iii) shows that serum concentrations of at least 38 μg/mL (mean steady state concentration over the dosing interval) were associated with complete inhibition of GC development in the cortical regions of lymph node B cells. , while incomplete suppression (animal 26842) or no suppression (animals 26772 and 26837) of GC was observed at serum concentrations below 20 μg/mL despite full CD40 occupancy on whole blood CD20 ^pos B cells (animals 26842 and 26772; data not shown). There was no depletion of peripheral B cells (data not shown).

Discussion

CFZ533 has been developed as a potential therapy for solid organ transplantation and autoimmune diseases associated with dysregulation of the CD40-CD154 costimulatory pathway. In this document, the authors of the present invention describe the characterization of the functional properties of CFZ533 in the CD40 pathway corresponding to the model systems in vitro and in vivo, and also explore the relationship between exposure to CFZ533 and the effects of PD.

CFZ533 was able to bind CD40 and completely prevent rCD154-induced pathway activation on various human immune cell types, including B cells and DCs. In addition, CFZ533 appears to require over 90% CD40 occupancy to completely block pathway activation in whole blood. Taken together, these data suggested that CFZ533 has the potential to block CD40 pathway-dependent effector functions regardless of cell type, provided sufficient receptor occupancy has been achieved. These data also indicate that in PBMC, CFZ533 was able to displace pre-bound rCD154 from CD40, suggesting that mAb and physiological ligand epitopes may overlap; concept under investigation in structural studies.

In vivo , concentration dependent clearance and half-life was observed for CFZ533 in single dose PK studies. This PK profile suggested that CD40 receptor expression influenced the removal of CFZ533. At low concentrations of CFZ533 (i.e. incomplete target saturation), the contribution of CD40 to the total clearance of CFZ533 was increased and the half-life was somewhat shorter than is typically observed for IgG1 type antibodies. At higher concentrations, corresponding to complete target saturation (and complete inhibition of the functional pathway), the receptor's contribution to the total elimination of CFZ533 was limited and the half-life increased. The target-mediated clearance of CFZ533 was consistent with the CD40-mediated internalization of CFZ533 observed in vitro , probably accompanied by lysosomal degradation of the complex.

Additional data from PK/PD studies confirmed the inability of CFZ533 to deplete peripheral B cells in vivo (Cordoba et al. 2015). As mentioned, the inability of CFZ533 to deplete CD40-expressing cells is due to the presence of the N297A mutation in the antibody resulting in the absence of N-linked glycosylation in the hinge region, rendering it unable to bind FcγRIIIA or mediate ADCC or CDC. Suppression of Fc CFZ533 was performed to prevent the depletion of cell types expressing CD40; Of particular concern was the wide distribution of this receptor in tissues across immune and non-immune cell types, especially in inflammatory conditions.

In addition to being effective in NHP kidney transplantation (Cordoba et al. 2015), the results in this paper indicate that CFZ533 completely inhibited TDAR recovery. This result suggests that memory B cell responses to T cell antigens were entirely dependent on CD40-CD154 interactions. The degree of inhibition of the recovered response seems to be related to the concentration of CFZ533 with serum levels in excess of 30-40 μg/ml (for at least a week after stimulation) necessary to completely suppress the antigen-specific antibody response. This relationship between serum concentration and tissue PD readings related to the CD40 pathway was also maintained when studying the effect of CFZ533 on mesenteric lymph node GC, where a minimum threshold of mean sustained serum CFZ533 concentrations was required for complete suppression of GC. These data point to the importance of establishing the relationship between peripheral drug exposures and target tissue PD effects in order to inform dosing strategies. For various autoimmune diseases, several biologics are being developed that target the CD40-CD154 costimulation pathway. In addition to anti-CD40 mAbs such as CFZ533, anti-CD154 mAbs remain in clinical practice despite the potential risk for thromboembolic complications (Boumpas et al., 2003). Recent results have shown that Fc-suppression and pegylated F(ab')2 approaches can address the thromboembolic deficiencies of CD154-targeting antibodies, however, there are reports that Fc-suppressed anti-CD154 mAbs may be less effective. To date, there are no data on thromboembolic events associated with the administration of multiple anti-CD40 antibodies in preclinical models or in clinical practice.

In conclusion, these data indicate that CFZ533 is a pathway-blocking, non-depleting anti-CD40 antibody with minimal agonist properties. With sufficient pharmacologically relevant exposures, CFZ533 is able to completely inhibit TDAR recovery and also inhibits germinal centers without depleting CD40-expressing cell types. These data, combined with preclinical efficacy in kidney transplantation, provide a solid scientific basis for the potential clinical utility of CFZ533 in certain autoimmune diseases and solid organ transplantation.

REFERENCE MATERIAL

Bombardieri M, Pitzalis C. Ectopic lymphoid neogenesis and lymphoid chemokines in Sjogren's syndrome: at the interplay between chronic inflammation, autoimmunity and lymphomagenesis. Curr Pharm Biotechnol. 2012 Aug; 13(10):1989-1996.

Bombardieri M, Barone F, Lucchesi D, Nayar S, van den Berg WB, Proctor G, et al. Inducible tertiary lymphoid structures, autoimmunity, and exocrine dysfunction in a novel model of salivary gland inflammation in C57BL/6 mice. J Immunol. 2012 Oct 1; 189(7):3767-3776.

Bombardieri M, Lewis M, Pitzalis C. Ectopic lymphoid neogenesis in rheumatic autoimmune diseases. Nat Rev Rheumatol. March 2017; 13(3):141-154.

Boumpas DT, Furie R, Manzi S, Illei GG, Wallace DJ, Balow JE et al. A short course of BG9588 (anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum 2003; 48(3):719-727.

Cordoba F, Wieczorek G, Audet M, Roth L, Schneider MA, Kunkler A et al. A novel, blocking, Fcsilent anti-CD40 monoclonal antibody prolongs nonhuman primate renal allograft survival in the absence of B cell depletion. Am J Transplant 2015; 15(11):2825

Dimitriou ID, Kapsogeorgou EK, Moutsopoulos HM, et al (2002) CD40 on salivary gland epithelial cells: high constitutive expression by cultured cells from Sjögren's syndrome patients indicating their intrinsic activation. ClinExp Immunol; 127(2):386-92.

Delporte C, Steinfeld S. Distribution and roles of aquaporins in salivary glands. Biochim Biophys Acta. 2006 Aug; 1758(8):1061-1070. Harland R, Klintmalm G, Yang H, et al. (2015) ASKP1240 in De Novo Kidney Transplant Recipients. Am J Transplant; 15(S3): Abstract #3012.

Horvath S, Nazmul-Hossain AN, Pollard RP, Kroese FG, Vissink A, Kallenberg CG, et al. Systems analysis of primary Sjogren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model. Arthritis Res Ther. 2012 Nov 1; 14(6):R238.

Jacobi AM, Hansen A, Kaufmann O, Pruss A, Burmester GR, Lipsky PE, et al. Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjogren's syndrome. Arthritis Res. 2002; 4(4):R4.

Jonsson MV, Delaleu N, Brokstad KA, Berggreen E, Skarstein K. Impaired salivary gland function in NOD mice: association with changes in cytokine profile but not with histopathologic changes in the salivary gland. Arthritis Rheum. 2006 Jun; 54(7):2300-2305.

Kaufman I, Schwartz D, Caspi D, et al (1999) Sjögren's syndrome - not just Sicca: renal involvement in Sjögren's syndrome. Ann Rheum Dis. 58:253-6.

Kawabe T, Naka T, Yoshida K, Tanaka T, Fujiwara H, Suematsu S et al. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1994; 1(3):167-178.

Kim EJ, Kwun J, Gibby AC, Hong JJ, Farris AB, 3rd, Iwakoshi NN, et al. Costimulation blockade alters germinal center responses and prevents antibody-mediated rejection. Am J Transplant. 2014 Jan; 14(1):59-69.

Komaroff AL, Fagioli LR, Doolittle TH, et al (1996) Health status in patients with chronic fatigue syndrome and in general population and disease comparison groups. Am J Med; 101:281-90.

Kuenstner S, Langelotz C, Budach V, et al (2002) The comparability of quality of life scores. A multitrait multimethod analysis of the EORTC QLQ-C30, SF-36 and FLIC questionnaires. Eur J Cancer; 38:339-48.

Laman JD, Claassen E, Noelle RJ. Functions of CD40 and its ligand, gp39 (CD40L). Crit Rev Immunol. 1996; 16(1):59-108.

Mager D.E. Target-mediated drug disposition and dynamics. Biochem Pharmacol 2006; 72(1):1-10.

Mahmoud TI, Wang J, Karnell JL, Wang Q, Wang S, Naiman B, et al. Autoimmune manifestations in aged mice arise from early-life immune dysregulation. Sci Transl Med. 2016 Oct 19; 8(361):361ra137.

Manganelli P and Fietta P (2003) Apoptosis and Sjögren syndrome. Semin Arthritis Rheum; 33(1):49-65.

Meijer JM, Meiners PM, Vissink A, et al (2010) Effectiveness of rituximab treatment in primary Sjögren's syndrome: a randomized, double-blind, placebo-controlled trial. Arthritis Rheum; 62:960-8.

Moerman RV, Arends S, Meiners PM, et al (2014) EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) is sensitive to show the efficacy of rituximab treatment in a randomized controlled trial. Ann Rheum Dis; 73:472-4.

Ng CM, Stefanich E, Anand BS, Fielder PJ, Vaickus L. Pharmacokinetics/pharmacodynamics of nondepleting anti-CD4 monoclonal antibody (TRX1) in healthy human volunteers. Pharm Res 2006; 23(1):95-103.

Ohlsson M, Szodoray P, Loro LL, Johannessen AC, Jonsson R. CD40, CD154, Bax and Bcl-2 expression in Sjogren's syndrome salivary glands: a putative anti-apoptotic role during its effector phases. Scand J Immunol. Dec 2002; 56(6):561-571.

Ristov J, Espie P, Ulrich P, Sickert D, Flandre T, Dimitrova M, et al. Characterization of the in vitro and in vivo properties of CFZ533, a blocking and non-depleting anti-CD40 monoclonal antibody. Am J Transplant. 2018 Apr 17.

Roescher N, Lodde BM, Vosters JL, Tak PP, Catalan MA, Illei GG, et al. Temporal changes in salivary glands of non-obese diabetic mice as a model for Sjogren's syndrome. OralDis. Jan 2012; 18(1):96-106.

Roescher N, Vosters JL, Lai Z, Uede T, Tak PP, Chiorini JA. Local administration of soluble CD40:Fc to the salivary glands of non-obese diabetic mice does not ameliorate autoimmune inflammation. PLOS One. 2012; 7(12):e51375.

Segal B, Bowman SJ, Fox PC, et al (2009) Primary Sjögren's Syndrome: health experiences and predictors of health quality among patients in the United States. Health Qual Life Outcomes; 7:46.

Sellam J, Proulle V, Jüngel A, et al (2009) Increased levels of circulating microparticles in primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity. Arthritis Res Ther; 11:R156.

Seror R, et al (2011a) EULAR Sjogren's syndrome disease activity index: development of a consensus systemic disease activity index for primary Sjogren's syndrome. Ann Rheum Dis.; 69(6):1103-9.

Seror R, et al (2011b) EULAR Sjogren's Syndrome Patient Reported Index (ESSPRI): development of a consensus patient index for primary Sjogren's syndrome. Ann Rheum Dis.; 70(6):968-72.

Stott DI, Hiepe F, Hummel M, Steinhauser G, Berek C. Antigen-driven clonal proliferation of B cells within the target tissue of an autoimmune disease. The salivary glands of patients with Sjogren's syndrome. J Clin Invest. 1998 Sep 1; 102(5):938-946.

Tishler M, Yaron I, Shirazi I, et al (2008) Hydroxychloroquine treatment for primary Sjögren's syndrome: its effect on salivary and serum inflammatory markers. Scand J Rheumatol. 37:213-8.

Th'ng KH, Garewal G, Kearney L, Rassool F, Melo JV, White H et al. Establishment and characterization of three new malignant lymphoid cell lines. Int J Cancer 1987; 39(1):89-93.

Vossenkämper A, Lutalo PM, Spencer J (2012) Translational mini-review series on B cell subsets in disease. Transitional B cells in systemic lupus erythematosus and Sjögren's syndrome: clinical implications and effects of B cell-targeted therapies. ClinExp Immunol; 167:7-14.

Voulgarelis M, Moutsopoulos HM. Mucosa-associated lymphoid tissue lymphoma in Sjogren's syndrome: risks, management, and prognosis. Rheum Dis Clin North Am. Nov 2008; 34(4):921-933, viii.

Warncke M, Calzascia T, Coulot M, Balke N, Touil R, Kolbinger F et al. Different adaptations of IgG effector function in human and nonhuman primates and implications for therapeutic antibody treatment. J Immunol 2012; 188(9):4405-4411.

Winzer M, Aringer M. (2010) Use of methotrexate in patients with systemic lupus erythematosus and primary Sjögren's syndrome. Clin Exp Rheumatol. 28 (5 Suppl 61): S156-9.

Yoshimura S, Nakamura H, Horai Y, Nakajima H, Shiraishi H, Hayashi T, et al. Abnormal distribution of AQP5 in labial salivary glands is associated with poor saliva secretion in patients with Sjogren's syndrome including neuromyelitis optica complicated patients. Mod Rheumatol. 2016; 26(3):384-390.

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SEQUENCE LIST

<110> Novartis AG

<120> ANTI-CD40 ANTIBODIES FOR USE IN THE TREATMENT OF SJOGREN'S SYNDROME

<130> PAT057947

<150> US 62/581212

<151> 2017-11-03

<150> US 62/644939

<151> 2018-03-19

<160> 19

<170> PatentIn version 3.5

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Ala Val Ile Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ile Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Gly Ile Ala Ala Pro Gly Pro Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 10

<211> 219

<212> PROTEIN

<213> Homo sapiens

<400> 10

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Val Leu Ile Ser Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Arg

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 11

<211> 450

<212> PROTEIN

<213> Homo sapiens

<400> 11

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ile Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Gly Ile Ala Ala Pro Gly Pro Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 12

<211> 219

<212> PROTEIN

<213> Homo sapiens

<400> 12

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Val Leu Ile Ser Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Arg

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 13

<211> 217

<212> PROTEIN

<213> Homo sapiens

<400> 13

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215

<210> 14

<211> 217

<212> PROTEIN

<213> Homo sapiens

<400> 14

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215

<210> 15

<211> 1350

<212> DNA

<213> Homo sapiens

<400> 15

caggtgcagc tggtggaatc tggcggcgga gtggtgcagc ctggccggtc cctgagactg 60

tcttgcgccg cctccggctt caccttctcc agctacggca tgcactggggt gcgacaggcc 120

cctggcaagg gactggaatg ggtggccgtg atctcctacg aggaatccaa cagataccac 180

gctgactccg tgaagggccg gttcacaatc tcccgggaca actccaagat caccctgtac 240

ctgcagatga actccctgcg gaccgaggac accgccgtgt actactgcgc cagggacgga 300

ggaatcgccg ctcctggacc tgattattgg ggccagggca ccctggtgac agtgtcctcc 360

gctagcacca agggcccctc cgtgttccct ctggccccct ccagcaagtc cacctctggc 420

ggcaccgccg ctctgggctg cctggtgaaa gactacttcc ccgagcccgt gaccgtgtcc 480

tggaactctg gcgccctgac ctccggcgtg cacacctttc cagccgtgct gcagtcctcc 540

ggcctgtact ccctgtcctc cgtggtgacc gtgccctcta gctctctggg cacccagacc 600

tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagcg ggtggaaccc 660

aagtcctgcg acaagaccca cacctgtccc ccctgccctg cccctgaact gctgggcgga 720

ccttccgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc 780

gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 840

tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacgcc 900

tccacctacc gggtggtgtc tgtgctgacc gtgctgcacc aggactggct gaacggcaaa 960

gagtacaagt gcaaggtctc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 1020

aaggccaagg gccagccccg cgagccacag gtgtacacac tgccccccag ccgggaagag 1080

atgaccaaga accaggtgtc cctgacctgt ctggtcaaag gcttctaccc ctccgatatc 1140

gccgtggagt gggagtccaa cggacagccc gagaacaact acaagaccac cccccctgtg 1200

ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 1260

cagcaggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1320

cagaagtccc tgtccctgag ccccggcaag 1350

<210> 16

<211> 657

<212> DNA

<213> Homo sapiens

<400> 16

gacatcgtga tgacccagtc ccccctgtcc ctgaccgtga cacctggcga gcctgcctct 60

atctcctgca gatcctccca gtccctgctg tactccaacg gctacaacta cctggactgg 120

tatctgcaga agcccggcca gtccccacag gtgctgatct ccctgggctc caacagagcc 180

tctggcgtgc ccgaccggtt ctccggctct ggctctggca ccgacttcac actgaagatc 240

tcacgggtgg aagccgagga cgtgggcgtg tactactgca tgcaggcccg gcagaccccc 300

ttcaccttcg gccctggcac caaggtggac atccggcgta cggtggccgc tcccagcgtg 360

ttcatcttcc cccccagcga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg 420

ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

agcggcaaca gccaggagag cgtcaccgag caggacagca aggactccac ctacagcctg 540

agcagcaccc tgaccctgag caaggccgac tacgagaagc ataaggtgta cgcctgcgag 600

gtgacccacc agggcctgtc cagccccgtg accaagagct tcaacagggg cgagtgc 657

<210> 17

<211> 1350

<212> DNA

<213> Homo sapiens

<400> 17

caggtgcagc tggtggaatc tggcggcgga gtggtgcagc ctggccggtc cctgagactg 60

tcttgcgccg cctccggctt caccttctcc agctacggca tgcactggggt gcgacaggcc 120

cctggcaagg gactggaatg ggtggccgtg atctcctacg aggaatccaa cagataccac 180

gctgactccg tgaagggccg gttcacaatc tcccgggaca actccaagat caccctgtac 240

ctgcagatga actccctgcg gaccgaggac accgccgtgt actactgcgc cagggacgga 300

ggaatcgccg ctcctggacc tgattattgg ggccagggca ccctggtgac agtgtcctcc 360

gctagcacca agggcccctc cgtgttccct ctggccccct ccagcaagtc cacctctggc 420

ggcaccgccg ctctgggctg cctggtgaaa gactacttcc ccgagcccgt gaccgtgtcc 480

tggaactctg gcgccctgac ctccggcgtg cacacctttc cagccgtgct gcagtcctcc 540

ggcctgtact ccctgtcctc cgtggtgacc gtgccctcta gctctctggg cacccagacc 600

tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagcg ggtggaaccc 660

aagtcctgcg acaagaccca cacctgtccc ccctgccctg cccctgaact gctgggcgga 720

ccttccgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc 780

gaagtgacct gcgtggtggt ggccgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 840

tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac 900

tccacctacc gggtggtgtc tgtgctgacc gtgctgcacc aggactggct gaacggcaaa 960

gagtacaagt gcaaggtctc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 1020

aaggccaagg gccagccccg cgagccacag gtgtacacac tgccccccag ccgggaagag 1080

atgaccaaga accaggtgtc cctgacctgt ctggtcaaag gcttctaccc ctccgatatc 1140

gccgtggagt gggagtccaa cggacagccc gagaacaact acaagaccac cccccctgtg 1200

ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 1260

cagcaggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1320

cagaagtccc tgtccctgag ccccggcaag 1350

<210> 18

<211> 657

<212> DNA

<213> Homo sapiens

<400> 18

gacatcgtga tgacccagtc ccccctgtcc ctgaccgtga cacctggcga gcctgcctct 60

atctcctgca gatcctccca gtccctgctg tactccaacg gctacaacta cctggactgg 120

tatctgcaga agcccggcca gtccccacag gtgctgatct ccctgggctc caacagagcc 180

tctggcgtgc ccgaccggtt ctccggctct ggctctggca ccgacttcac actgaagatc 240

tcacgggtgg aagccgagga cgtgggcgtg tactactgca tgcaggcccg gcagaccccc 300

ttcaccttcg gccctggcac caaggtggac atccggcgta cggtggccgc tcccagcgtg 360

ttcatcttcc cccccagcga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg 420

ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

agcggcaaca gccaggagag cgtcaccgag caggacagca aggactccac ctacagcctg 540

agcagcaccc tgaccctgag caaggccgac tacgagaagc ataaggtgta cgcctgcgag 600

gtgacccacc agggcctgtc cagccccgtg accaagagct tcaacagggg cgagtgc 657

<210> 19

<211> 277

<212> PROTEIN

<213> Homo sapiens

<400> 19

Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu

20 25 30

Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val

35 40 45

Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

50 55 60

Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65 70 75 80

Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr

85 90 95

Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100 105 110

Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly

115 120 125

Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys

145 150 155 160

Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln

165 170 175

Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu

180 185 190

Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile

195 200 205

Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn

210 215 220

Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp

225 230 235 240

Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His

245 250 255

Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser

260 265 270

Val Gln Glu Arg Gln

275

<---

Claims (17)

1. A method of treating primary Sjögren's syndrome in a human, comprising administering to said human a therapeutically effective dose of an anti-CD40 antibody, where the antibody is administered via a loading dose and a maintenance dose, where the administration of the loading dose is carried out by subcutaneous injections of the first dose, and the administration of the maintenance dose is carried out by subcutaneous injections of the second dose, where the first dose is from about 600 mg of the active ingredient, and the second dose is from about 150 mg to about 600 mg of the active ingredient, and where the antibody contains an IgG1 Fc region with repressed activity and is selected from the group consisting of: a. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8; b. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the hypervariable regions represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and an immunoglobulin VL domain containing the hypervariable regions represented by SEQ ID NO: 4 , SEQ ID NO: 5 and SEQ ID NO: 6; c. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence of SEQ ID NO: 8 and an Fc region of SEQ ID NO: 13; And d. an anti-CD40 antibody that contains an immunoglobulin VH domain containing the amino acid sequence of SEQ ID NO: 7 and an immunoglobulin VL domain containing the amino acid sequence under SEQ ID NO: 8 and an Fc region of SEQ ID NO: 14. 2. The method of treatment according to claim 1, wherein the antibody comprises a heavy chain with the amino acid sequence of SEQ ID NO: 9 and a light chain with the amino acid sequence of the light chain of SEQ ID NO: 10, or a heavy chain with the amino acid sequence of SEQ ID NO: 11; and a light chain with the light chain amino acid sequence of SEQ ID NO: 12. 3. The method of claim 1 or 2, wherein the antibody is administered together with one or more pharmaceutically acceptable carriers. 4. The method of claim 1, wherein the first dose is 600 mg of the active ingredient and the second dose is about 150 mg, about 300 mg, or about 600 mg of the active ingredient. 5. The method of claim 4, wherein administration of the loading dose comprises at least two subcutaneous injections and administration of the maintenance dose is subcutaneous injections administered weekly (Q1W), biweekly (Q2W) or monthly (Q4W). 6. The method according to claim 5, where at least two subcutaneous injections provide different doses when administering a loading dose. 7. The method according to claim 4, wherein the loading dose is administered with three weekly (Q1W) subcutaneous injections of 600 mg of the active ingredient at weeks 0, 1 and 2, and the maintenance dose is administered every two weeks (Q2W), starting from the 4th week in the form of subcutaneous injections of 600 mg of the active ingredient. 8. The method of claim 4, wherein the loading dose is administered as three weekly (Q1W) subcutaneous injections of 600 mg of the active ingredient at week 0, 300 mg of the active ingredient at week 1, and 300 mg of the active ingredient at week 2 th week, and a maintenance dose is administered every two weeks (Q2W) starting from the 4th week, in the form of subcutaneous injections of 300 mg of the active ingredient. 9. The method according to claim 4, wherein the loading dose is administered by three weekly (Q1W) subcutaneous injections of 600 mg of the active ingredient in the 1st week, 150 mg of the active ingredient in the 2nd week and 150 mg of the active ingredient in the 3rd week, and a maintenance dose is administered every two weeks (Q2W) starting from the 4th week, in the form of subcutaneous injections of 150 mg of the active ingredient.

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