Immunosuppression Profile of CFZ533 (Iscalimab),a Non-Depleting Anti-CD40 Antibody,and the Presence of Opportunistic Infections in a Rhesus Monkey Toxicology Study

Abstract

CFZ533 (iscalimab) is a nondepleting anti-CD40 antibody intended for inhibition of transplant organ rejection and treatment of autoimmune diseases. In a safety assessment in rhesus monkeys, CFZ533 was administered for 13 weeks up to 150 mg/kg/week subcutaneously. CFZ533 was shown previously to completely inhibit primary and secondary T-cell-dependent antibody responses. CD40 is expressed on B cells, antigen-presenting cells, and endothelial and epithelial cells, but is not expressed on T cells. Here, we demonstrate the complete suppression of germinal center formation in lymphoid organs. CFZ533 was well tolerated and did not cause any dose-limiting toxicity. However, the histological evaluation revealed increased numbers of CD4⁺ and CD8⁺ T cells in the T-cell-rich areas of lymph nodes enlarged in response to observed adenovirus and Cryptosporidium infections which suggest that T-cell immune function was unaffected. Background infections appear as the condition leading to unraveling the differential immunosuppressive effects by CFZ533. The presence of T cells at lymph nodes draining sites of infections corroborates the immunosuppressive mechanism, which is different from calcineurin-inhibiting drugs. Furthermore, CFZ533 did not show any hematological or microscopic evidence of thromboembolic events in rhesus monkeys, which were previously shown to respond with thromboembolism to treatment with anti-CD154 antibodies.

Keywords

biotherapeutics immunomodulator lymphoid system infection CD40 CD154

Introduction

CFZ533 (iscalimab) is in clinical development for the therapeutic prevention of organ rejection after transplantation and for the treatment of autoimmune disorders. CFZ533 is a fully human, IgG1 anti-CD40 antibody that blocks recombinant CD154-induced CD40 activation of human and nonhuman primate leukocytes.^1,2 CFZ533 is Fc-silent and therefore does not deplete CD40-expressing cells via antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), while it blocks the CD40-CD154 co-stimulatory pathway and inhibits cellular proliferation and other cellular effector functions.² In vivo, CFZ533 completely blocks the generation of T-cell-dependent antibody responses (TDAR).^2,3

CD40, a member of the tumor necrosis factor (TNF) super-family of proteins, is constitutively expressed on B lymphocytes as well as monocytes, macrophages, antigen-presenting cells (APC), platelets, and eosinophils. CD40 is also expressed at low levels and upregulated under inflammatory conditions through interferon-gamma activation on endothelium, kidney epithelial cells, synovial membranes, keratinocytes, and dermal fibroblasts.^4-8 Expression patterns of CD40 in human and nonhuman primates (i.e., rhesus and cynomolgus monkeys) were shown to be similar in a tissue cross-reactivity study.³

CD154 (CD40L), the endogenous ligand for CD40, is expressed not only on activated T cells⁹ but also in an inducible fashion on B lymphocytes, mast cells, monocytes, basophils, eosinophils, natural killer (NK) cells, activated platelets, and vascular endothelial cells and is upregulated in areas of local inflammation.¹⁰ Upon cellular activation of lymphocytes and platelets, membrane CD154 is cleaved to form soluble CD154. The engagement of membrane or soluble forms of CD154 by CD40 recruits TNF receptor-associated factors which trigger downstream activation of multiple signaling pathways (e.g., JNK, ERK1/2, p38) leading to nuclear translocation of a variety of transcription factors such as NF-kB and AP1 and inducing the expression of numerous genes involved in cell survival, activation, and differentiation.^11,12

CD40 activation by CD154 is essential not only for primary TDAR, including germinal center (GC) formation, immunoglobulin (Ig) isotype switching, somatic mutation, and differentiation of memory B and plasma cells, but also for activation of APC inducing cytokine secretion and expression of surface activation molecules involved in the regulation of CD4⁺ T-helper cells and CD8⁺ T-cell cross-priming and activation.^13,14

Previous nonclinical and clinical experience with anti-CD154 antibodies indicated a potential for thromboembolic complications.^15,16 These thromboembolic complications were consistently reproduced in naive rhesus monkeys in a 8-week investigative study,¹⁷ whereas the cynomolgus monkey appeared to be less sensitive to this effect.¹⁸ Consequently, the rhesus monkey was selected to investigate the potential for CFZ533 to produce thromboembolic events in a 13-week repeat dose toxicology study. This study was part of a general toxicological assessment of CFZ533, which also included a 26-week toxicology study in the cynomolgus monkey.³

In this article, we describe the nonclinical toxicological and toxicokinetic evaluation of CFZ533 in the rhesus macaque. In addition to the potential thromboembolic liability, the rhesus monkey was considered a relevant species for toxicological assessment because CFZ533 binds to rhesus CD40 and inhibits proliferation of rhesus B cells.^2,15,19 Moreover, CFZ533 binds to nonhuman primate and human B cells with similar EC₅₀ values (less than 2-fold difference).

Materials and Methods

Animals and Husbandry

Toxicology studies were conducted at Labcorp Early Development Services GmbH, Muenster, Germany, using experimentally naïve, purpose-bred, group-housed rhesus monkeys (Macaca mulatta) originating from China. At the initiation of dosing, the animals were approximately 3 to 5 years old and males weighed 3.2 to 6.3 kg and females weighed 3.2 to 5.5 kg. The study was conducted according to an approved study protocol and local standard operating procedures in strict compliance with national legal regulations on animal welfare law and accepted animal welfare standards. The animals were kept in quarantine for at least four weeks and housed in standard ETS 123-compliant (ETS 123, Appendix A: Guidelines for Accommodation and Care of Animals, June 18, 2007) housing systems. Climate conditions with a temperature of 19°C to 25°C and a relative humidity of 40% to 70% were continuously maintained and animals were kept on a stable light/dark cycle. The animals were fed a pellet diet and offered filtered tap water ad libitum. The animals were supplied with environmental enrichment (wood chips, movable stainless-steel mirrors, colored plastic tools, and colored plastic balls). All animals underwent a comprehensive health evaluation. Male and female monkeys were assigned to one of the treatment groups by a stratified randomization scheme designed to achieve similar group mean body weights. All animals were acclimated to routine handling procedures prior to study initiation and were not sedated for dose administration.

Study Design

CFZ533 was administered once weekly subcutaneously in a dose volume of 2 ml/kg to 4 groups (3 or 5/sex/group) of rhesus monkeys at doses of 0 (vehicle only), 10, 50, or 150 mg/kg (control, low-dose, mid-dose, and high-dose, respectively) for 13 weeks (14 doses in total). An additional group received weekly intravenous doses of 150 mg/kg to assess bioavailability. Subcutaneous injection is the intended clinical route of administration. In a previous 5-week exploratory toxicity study in cynomolgus monkeys, CFZ533 at 100 mg/kg/week inhibited antigen-specific immune responses to KLH (keyhole limpet hemocyanin) and was well tolerated without any adverse findings.³ Therefore, the selected doses were expected to produce clear pharmacodynamic (PD) effects (inhibition of primary and secondary TDAR and absence of GC) as it was shown later in the 26-week cynomolgus monkey toxicity study.³ Effects arising from binding targets outside the pharmacological context—T and B cell as well APC and lymphocyte interactions—were theoretically possible; however, they were not expected in young, healthy animals and deemed unlikely for a Fc-silent, purely antagonistic antibody. Weekly dosing was selected based on CFZ533 half-life of 12 days which, due to target-mediated disposition, is shorter compared to typical IgG1 antibodies.² At the end of the 13-week dosing phase, two males and two females from the control and high-dose groups continued on study for an additional 30 weeks without administration of CFZ533 or placebo. This recovery phase was included to assess the reversibility of any test article-related changes observed at the end of the dosing phase. Cardiovascular safety pharmacology evaluation, on non-anesthetized temporarily restrained animals, was assessed 2 hours post-dose on weeks 6 and 13 of dosing phase. Electrocardiography parameters (i.e. heart, rate, RR, PR, QRS, QT, and QTC interval as well as voltage measurements) and blood pressure by high-definition oscillometric method (diastolic, systolic, and mean arterial) were measured.

Materials

CFZ533 is a human Fc-silent IgG1 antibody containing a N297A mutation, which abolishes Fc-dependent antibody effector functions.²⁰ CFZ533 is produced in CHO (Chinese hamster ovary) cell lines. CFZ533 was shown to bind to CD40 molecule in an enzyme-linked immunosorbent assay (ELISA). After reconstitution of the lyophilized powder with 1.0 ml water for injection, the resulting solution contained 150 mg/ml CFZ533 and the excipients l-histidine, sucrose, and polysorbate 20, pH 6.0 ± 0.5 and was a hyper-osmolar solution. A 20% overfill was added to permit complete removal of the intended dose. The drug was stored at 2°C to 8°C. The vehicle used in control group was composed of 0.06% Tween 20, 30 mM l-histidine, and 270 mM sucrose at pH 6.0 ± 0.5 and was kept at −20°C ± 4°C for 6 months after formulation.

Toxicokinetic Assessment and Immunogenicity Analysis

Serum samples for toxicokinetics were collected prior to dosing and 6, 24, 48, 72, 96, 120, and 168 hours post-dose on days 1 and 85 from all animals and on day 92 from recovery animals. In addition, samples were collected before dosing from all animals on days 29 and 64 and from recovery animals on days 106, 120, 134, 148, 162, 176, 190, 204, 218, 232, 246, 260, 274, 288, and 302. Serum CFZ533 was determined by a validated sandwich ELISA in which CFZ533 was captured by recombinant CD40 and detected with a mouse anti-human kappa light chain antibody followed by a horseradish peroxidase (HRP) goat anti-mouse IgG antibody conjugate. This ELISA produced a LLOQ (lower limit of quantification) of 0.15 μg/ml and an upper limit of quantification (ULOQ) of 5.00 μg/ml on rhesus monkey serum samples.

For immunogenicity analysis, serum samples from all animals were collected before dosing on days 1, 29, 64, 85, and 92 and on days 232, 246, 260, and 302 from recovery animals. Anti-CFZ533 antibodies (anti-drug antibodies or ADAs) were measured using a validated bridging ELISA in which ADAs were captured by immobilized CFZ533 and detected by biotinylated CFZ533 followed by binding of a streptavidin-HRP conjugate. Goat anti-human IgG was used as positive control, and pooled rhesus monkey serum as negative control.

Immunophenotyping

Whole blood samples, collected at predose on day 1 and in weeks 1, 7, and 13, were analyzed by flow cytometry for the evaluation of peripheral blood lymphocyte subpopulations. The blood was collected into EDTA tubes. At necropsy, samples of spleen and lymph nodes (axillary for subcutaneous [SC] and inguinal for intravenous [IV] groups) were obtained, mashed, and processed to single-cell suspensions and suspended in buffered saline for flow cytometric analyses. Flow cytometric analyses for immunophenotyping of naive B cells (CD20⁺CD21⁺CD27⁻), memory B cells (CD20⁺CD21⁺CD27⁺), CD20^highCD21⁻ and CD20^lowCD21⁺ B cells, T cells (CD4⁺ helper and CD8⁺ cytotoxic), and NK cells (CD16⁺) were performed on the FacsCalibur Flow Cytometer (Beckton Dickinson, USA) (see Supplement Table 4).

Clinical Pathology

Blood samples were collected for clinical pathologic evaluations (hematology, serum chemistry, and coagulation) from the vena cephalica antebrachii or vena femoralis, from animals prestudy and on days 48 and 86 in dosing phase, and on day 204 in recovery phase. Blood samples for hematology were also collected predose on each day of dosing (i.e., days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, and 92 of the dosing phase). Standard hematology evaluation of whole blood in EDTA was performed in Sysmex XT2000iV multiparameter automated hematology analyzer (Sysmex, Germany) (Supplement Table 1). Standard clinical chemistry evaluation of serum samples was analyzed on a Konelab 60i automated chemistry analyzer (Beckman-Coulter Instruments, Palo Alto, California) (Supplement Table 2). For assessment of coagulation parameters, plasma from whole blood collected into tubes containing trisodium citrate anticoagulant was analyzed on a Destiny Max (TCOAG Inc, Ireland) (Supplement Table 1).

Urine for urinalysis was collected from metabolic cage from animals prestudy and during weeks 7 and 13 of dosing phase and on last week of recovery phase. Standard urinalysis was performed on Urisys 1800 (Roche, Switzerland) (Supplement Table 3).

Based on clinical and anatomic pathology data indicating inflammation in two mid-dose animals (animals M8 and M9), predose cytokine levels were measured in immunogenicity backup samples from days 1, 64, 85, and 92. The sampling time points were selected based on the onset of hematological and clinical chemistry changes in the two mid-dose animals with inflammation. In addition, samples from two male and two female recovery control animals were included to increase the concurrent control animal dataset. A Multiplex Luminex assay was used for the quantification of interferon (IFN)-γ, TNF-α, interleukin (IL)-1β, IL-4, IL-6, and IL-8 in the serum.

Anatomic Pathology and Immunohistochemistry

A complete necropsy (macroscopic examination, organ weights, collection of tissues) was performed on all control and dosed animals on day 93 at the end of the dosing phase or day 302 at the end of the recovery phase. Tissues were fixed in formalin, embedded in paraffin, sectioned at a nominal 5 µm and stained with hematoxylin and eosin (H&E). Histopathological examination was performed on a full panel of tissues (following Committee for Proprietary Medicinal Products, Food and Drug Administration, and Environmental Protection Agency guidelines) of all animals from the dosing and recovery phases. Because CFZ533 is an immunomodulatory compound, an enhanced assessment of the lymphoid tissues was performed with evaluation of the different compartments of lymphoid organs using semiquantitative descriptive terminology to identify and define any changes in cellularity and/or architecture.^21,22 Lesions were graded by a pathologist on a severity scale of 1 to 5 (1 = minimal, 2 = mild, 3 = moderate, 4 = marked, and 5 = severe).

In addition to H&E staining, cells in lymphoid organs (mesenteric, inguinal and axillary lymph nodes, thymus, and spleen) were characterized using the following IHC markers: CD20, CD40 (not competing with CFZ533 epitope), CD3, CD4, CD8, CD56, CD68, and Ki67. Tissues were immunostained using an indirect immunoperoxidase method (HRP+DAB from Dako) and scored by a pathologist using a semiquantitative approach based on percentage of stained cells for each respective lymphoid compartment with control as baseline (1 = 0%-10%, 2 = 11%-20%, 3 = 21%-30%, 4 = 31%-40%, 5 = 41%-50%, 6 = 51%-60%, 7 = 61%-70%, 8 = 71%-80, and 9 = >80%). Additional IHC markers (anti-SV40, adenovirus, cytomegalovirus, and lymphocryptovirus antibodies) and special stains (Weber’s trichrome and Ziehl-Neelson for encephalitozoon, Jones’ methenamine silver and Gomori’s silver for fungi, and Gram’s stain for bacteria) were also performed to detect opportunistic pathogens.

Results

Treatment with CFZ533 was generally well tolerated, and there were no CFZ533-related clinical observations, body weight changes, or early deaths. In addition, there were no CFZ533-related changes in cardiovascular safety parameters with neither electrocardiographic evidence of cardiotoxicity or arrhythmogenesis nor effects on blood pressure measurements.

Toxicokinetic Analyses and Immunogenicity

Exposure (Table 1) increased proportionally to dose, indicating linear pharmacokinetic (PK) and that absorption or routes of elimination of CFZ533 were not saturated by doses of 10 to 150 mg/kg given weekly. The subcutaneous bioavailability of CFZ533 in animals was high as the systemic exposure (AUC_tau) of CFZ533 on week 13 was similar between subcutaneous and intravenous dose routes at 150 mg/kg. In addition, exposure in recovery animals at 150 mg/kg SC and IV was quantifiable up to days 221 and 225, respectively, indicative of similar duration of exposure for the 2 administration routes.

Two animals, both in the recovery phase, had measurable ADA. In these two animals (one female at 150 mg/kg SC and one female at 150 mg/kg IV), ADAs were detected from day 232 when CFZ533 serum levels dropped to undetectable levels during the recovery phase. Because CFZ533 blocks the generation of antigen-specific antibodies, the generation of ADA is inhibited as long as exposure to CFZ533 is sufficient as demonstrated in previous studies.³

Table 1.

Mean toxicokinetic parameters of CFZ533 on day 1 and at steady state on week 13 following weekly administration for 13 weeks (SC or IV).

Mean (M + F)Weekly dose	Cmax^a	Dose normalized Cmax^a	AUC_tau^a,b	Dose normalized exposure^a	Cav,ss^a
10 mg/kg (SC ) – Low-dose
Day 1	147	14.7	792	79.2	—
Week 13	602	60.2	3495	350	499
50 mg/kg (SC) – Mid-dose
Day 1	631	12.7	3605	72.1	—
Week 13	2265	45.3	13,800	276	1971
150 mg/kg (SC) – High-dose SC
Day 1	1985	13.3	10,900	72.5	—
Week 13	8735	58.3	52,900	353	7557
150 mg/kg (IV) – High-dose IV
Day 1	4060	27.1	13,950	93.2	—
Week 13	11,650	77.6	55,300	369	7900

Abbreviation: IV, intravenous; SC, subcutaneous.

Units: Cmax in µg/ml, Dose normalized Cmax in (µg/ml)/(mg/kg), AUC_tau in µg day/ml, Dose normalized exposure in (µg day/ml)/(mg/kg), and Cav,ss (calculated as AUC_tau/Tau) in µg/ml.

Subscript tau refers to the dosing interval (weekly).

Clinical Pathology

Standard hematology parameters (such as red blood cell [RBC] count and RBC indices, platelet count, and white blood cell [WBC] counts, including WBC differentials), coagulation parameters (prothrombin time, activated partial prothrombin time, and serum fibrinogen), and chemistry parameters (indicators of hepatobiliary and renal functions, serum proteins, enzymes of muscle and pancreatic origins, lipids, glucose, and electrolytes) were normal throughout the dosing phase with the exception of the two mid-dose males described below (Supplement Tables S5-S7, respectively). To note, a reversible increased IgG concentrations (32%-52% above baseline) in animals dosed with 150 mg/kg was noted and considered likely the result of CFZ533 administration, a recombinant IgG, being detected by the assay and not due to increases in the endogenous IgG concentrations. Neither P-selectin nor sCD154 concentrations in plasma indicated platelet activation (data not shown).

Compared with their respective baseline values, two animals at mid-dose (animals M8 and M9 at 50 mg/kg) had regenerative anemia starting on day 7 (e.g., mildly decreased RBC mass including lowest hematocrits of −8% and −14%), which was associated with mildly to moderately increased absolute reticulocyte count (2.9-fold and 3.6-fold), and mildly shortened prothrombin times (−10% and −29%, at day 86 of the dosing phase). One animal had moderately increased fibrinogen concentration at day 86 of the dosing phase (2.2-fold), and the other animal had mildly increased absolute eosinophil count throughout most of the dosing phase with two peak episodes on day 15 (ca. 4.9-fold) and on days 57 to 64 (ca. 4.2-fold) during the dosing phase (Supplement Tables S5 and S6). Compared with their respective baseline values, these two animals also exhibited moderately decreased total protein concentration (−24% and −25%, respectively) and albumin-to-globulin ratio (up to −50% and 42%), due to moderately decreased albumin concentration (−44% and −39%), and moderately increased triglyceride concentration (5.1-fold and 7.1-fold) (Supplement Table S7). Animal M8 had, compared to baseline and control animals, increased serum concentrations of IL-6 on days 85 and 92 in line with the fact that this animal was more affected in terms of inflammatory signs than animal M9 (data not shown).

Blood Immunophenotyping

Starting at seven weeks of treatment and persisting to the end of the dosing phase, a non-dose-related reduction (−47% in females and −64% in males) of blood CD20⁺ B cells, more precisely a reduction in CD21⁻ B cells, was detected in animals at all CFZ533 dose levels (Supplement Table S8). CD21⁺ B cells and other lymphocytes (NK cells, T-helper cells, and cytotoxic T cells) were within the normal limits throughout the dosing phase (Supplement Table S8). B-cell numbers returned to prestudy levels after 30 weeks of recovery.

Anatomic Pathology and Immunohistochemistry

There were no changes in organ weights considered to be related to treatment with CFZ533. The only macroscopic observation attributed to CFZ533 was enlarged lymph nodes seen at necropsy (Table 2) which correlated with increased cellularity in cortex and medulla at microscopy (see below).

Table 2.

Summary of lymph node changes (macroscopic, microscopic, and CD8 staining) in relation to presence of opportunistic infection following subcutaneous administration of vehicle or CFZ533.

Sex	Treatment weekly dose	Animal no	Enlarged LN	Inc. cellularity (inflam.)^a	Axillary T-cell area CD8 staining^b		Opportunistic infection
Sex	Treatment weekly dose	Animal no	Enlarged LN	Inc. cellularity (inflam.)^a	Axillary	Mesenteric	Opportunistic infection
Male	Vehicle	M1			5	7
		M2			7	7
		M3		Mes: Slight	6	7
	CFZ53310 mg/kg(low-dose)	M4			5	7
		M5			6	6	Crypto
		M6	Ax: Moderate		6	8
	CFZ53350 mg/kg(mid-dose)	M7	Mes: Moderate	Mand: Slight	8	8	Crypto
		M8	Ax: Marked Ing: Marked	Ax: Moderate Ing: Moderate(LG, KD, TR, and EY)	8	7	Crypto
		M9	Ax: Marked Ing: Marked	Ax: Marked Ing: Marked Mand: Marked(LG and KD)	6	6	Crypto
	CFZ533150 mg/kg(high-dose SC)	M10	Mes: Moderate	Mes: Slight	6	7
		M11	Mes: Slight		7	8
		M12			5	8
Female	Vehicle	F1			4
		F2	Mes: Moderate		5
		F3			6
	CFZ53310 mg/kg(low-dose)	F4			4		Adeno
		F5			5
		F6	Mes: Marked	Mes: Moderate	5
	CFZ53350 mg/kg(mid-dose)	F7			7
		F8			6
		F9			5
	CFZ533150 mg/kg(high-dose SC)	F10F11F12	Mes: Moderate	Mes: Moderate	446		Crypto

Abbreviations: LN, lymph nodes; Mes, mesenteric lymph node; Ax, axillary; Crypto, Cryptosporidium; Mand, mandibular; Ing, inguinal; LG, lung; KD, kidney; TR, trachea; EY, eyes; Adeno, adenovirus.

Inc. cellularity (mono cell infilt): Lymph node(s) observed with increased cellularity (tissues seen with inflammatory process: mononuclear cell infiltration, degenerative process).

Percent of positive cells are represented by grading: 1 = 0%-10%, 2 = 11%-20%, 3 = 21%-30%, 4 = 31%-40%, 5 = 41%-50%, 6 = 51%-60%, 7 = 61%-70%, 8 = 71%-80%, 9 = >80%. No grading for CD8 staining of female mesenteric lymph node was reported.

In H&E-stained tissues, the most prominent effect of CFZ533 treatment was the complete absence of GCs, the site of B lymphocyte clonal expansion (Supplement Table S9). This effect was observed in the axillary, inguinal, mandibular, and mesenteric lymph nodes and spleen at all dose levels, but was not dose-dependent. The use of the IHC B-cell marker CD20 and the cell cycle and proliferation IHC marker Ki67 confirmed the absence of GCs in the axillary lymph nodes, which are the draining lymph node for the subcutaneous injection sites (Figure 1). In lymph nodes draining mucosal surfaces, such as the mesenteric and mandibular lymph nodes, GC suppression was incomplete in few animals at all dose levels (Figure 2). This reduced effect was likely due to inflammatory signals resulting from opportunistic infections in the intestinal tract as described below.

Figure 1.

Representative photomicrographs of germinal center (GC) development in mesenteric lymph node using Ki67 immunohistochemistry with marked developed GC in a vehicle-dosed animal (A), mild developed GC in an animal dosed with CFZ533 at 10 mg/kg subcutaneous (B), minimal developed GC in an animal dosed with CFZ533 at 10 mg/kg subcutaneous (C), and absence of GC in an animal dosed with CFZ533 at 150 mg/kg subcutaneous (D). (Original objective 8X).

Figure 2.

Summary of germinal center development in axillary and mesenteric LN at the end of the 13-week treatment phase (Main) or at the end of the 30-week treatment free phase (Recovery). Grading of germinal center (GC) development was based on Ki67 immunohistochemistry staining with gray: GC absent, yellow: minimal, orange: mild, red: moderate, and violet: marked GC development. IV indicates intravenous; LN, lymph nodes; and SC, subcutaneous.

Ki67 and CD20 staining were not reduced in T-cell-rich areas of the lymph nodes from animals administered CFZ533. Ki67-positive cells were present in the paracortex, lymphoid tissue around high endothelial venules (HEVs), and other regions of the medulla. The paracortex contained very few CD20⁺ B cells in any group, including controls, and CD20⁺ B cells numbers associated with the HEVs and the medulla were not reduced by CFZ533 (see Supplement Figure S1B). Of note, in a few animals administered CFZ533, there was an increased number of B cells in the HEVs.

CD40⁺ cells were seen as expected in B-cell populations in the cortex of lymph nodes and follicles in splenic white pulp and also in dendritic cells of paracortex and medulla of lymph nodes and periarteriolar lymphoid sheaths (PALS) T-cell-rich areas of splenic white pulp. The CD40 staining appeared not affected by CFZ533 treatment except in GC (absence of staining) due to their absence (see Supplement Figure S1D).

Eight animals administered CFZ533 (animals M6 and F6 at 10 mg/kg, M7, M8, and M9 at 50 mg/kg, and M10, M11, and F10 at 150 mg/kg) and one control female (F2) had enlarged mesenteric, tracheobronchial, axillary, and/or inguinal lymph nodes noted at necropsy (Table 2). In six animals administered CFZ533, the enlarged lymph nodes could be attributed to increased numbers of lymphocytes in the paracortex, HEVs, and medullary T-cell-rich areas in histologic sections of the lymph nodes (Figure 3B and Supplement Table S9). The increased lymphocytes were identified as T cells using immunohistochemical assays against CD3 (Figure 3D), CD4 (Figure 3F), and CD8 (Figure 3H). In contrast, few CD56⁺ NK cells were observed in lymph nodes and spleen, and there was no difference between control and dosed animals.

Figure 3.

Representative photomicrographs of a normal mesenteric lymph node from control animal M2 (A: hematoxylin and eosin, C: CD3, E: CD4, and G: CD8) and an enlarged mesenteric lymph node from animal M7 (CFZ533 at 50 mg/kg subcutaneous). The lymph node from M7 has increased number of lymphocytes in the paracortical, high endothelial venules, and medullary T-cell-rich areas in hematoxylin and eosin-stained tissue (B), which are identified as T cells using CD3 (D), CD4 (F), and CD8 (H) immunohistochemistry. (Original objective 2X).

The increase in T cells in the context of the intended pharmacologic immunomodulation effect on B-cell numbers and GC formation was most likely due to immune stimulation triggered by asymptomatic/subclinical opportunistic infections. Adenovirus intranuclear inclusions were identified by IHC in the intestine in one low-dose female (Figure 4) and Cryptosporidium organisms, focally infecting the enterocytes of the intestinal mucosa, were identified in one low-dose animal, three mid-dose animals, and one high-dose animal (Figure 5). The presence of adenovirus or Cryptosporidium was not associated with clinical signs or intestinal histopathological changes. A partial correlation between these infections and the lymph node enlargements with increased cellularity in paracortical, medullary, and HEVs regions was established. The presence or absence of the infection was likely related to the different stages of elimination of infectious agents with microscopic examination capturing a picture at the time of sacrifice on limited tissue sampling. The true incidence of infections was likely higher than what was detected. We could assume that animals with dramatic T-cell expansion are or were infected with adenovirus and/or Cryptosporidium.

Figure 4.

Adenovirus-positive immunohistochemistry staining in enterocytes of jejunum of female F4 dosed with CFZ533 at 10 mg/kg subcutaneous. (Original objective 20X).

Figure 5.

Cryptosporidium (arrow) infecting enterocytes of the colon mucosa of animal M4 dosed with CFZ533 at 50 mg/kg subcutaneous. Section stained with hematoxylin and eosin. (Original objective 40X).

Although no clinical findings were noted, the two mid-dose animals (animals M8 and M9 at 50 mg/kg) that had changes in clinical pathology parameters described above (i.e., regenerative anemia, decreased albumin levels, and increased coagulation parameters) had mild to moderate inflammatory cell infiltrates in several organs including lung and kidney (Supplement Table S9). Minimal to mild inflammation in the trachea and eyes (ciliary body and choroid) of one animal (animal M8) was also noted in addition to elevated liver and kidney weights (see Supplement Figure S2), moderate multifocal renal tubular atrophy associated with moderate degeneration/regeneration in cortex, and mild proteinuria in this animal (see Supplement Figure S3A). The mild to moderate multifocal, mostly perivascular, mononuclear inflammatory cell infiltrations in the lung (see Supplement Figure S3B) and kidney of these mid-dose males were comprised mainly of CD8⁺ cytotoxic T cells. Some CD40 expression was also seen in the mononuclear cell infiltrations in the kidney and lung and in injured tubular epithelial cells in the kidney of the two mid-dose (50 mg/kg) males. As CFZ533 is an Fc-silent antibody, direct ADCC was not considered to be a reason for the observed pathology. The observed inflammatory responses in these two mid-dose males suggested opportunistic infections secondary to pharmacological B-cell depletion as a likely reason for the increase in lymph node size and increased cellularity of the predominantly T-cell-rich areas of the lymph nodes, and as presence of opportunistic pathogens was established for five animals with enlarged lymph nodes. Nevertheless, no infectious lesions were detected in the lung or kidney of these two mid-dose males using different investigative methods. These two animals also showed minimal to moderate thymic cortical atrophy plus mild increased thymic medullary cellularity with CD20⁺ lymphoid follicles, which was also observed in two low-dose animals. Worthy of note is that EBNA-2 staining of lymph nodes did not reveal a Lymphocryptovirus (LCV) reactivation in the two affected mid-dose males. In addition, there was no histological evidence, in these two animals and other CFZ533-dosed animals, of LCV reactivation as this would involve the proliferation of infected B cells.

Following CFZ533 treatment, no thrombi were observed in any of the examined tissues including the most sensitive tissues for thrombus detection (e.g., lungs and brain).^17,23

In the recovery animals, CD20⁺ regions such as cortical follicles including GCs, medullary follicles, and HEVs surrounding regions returned to normal or slightly increased values and correlated with increased Ki67 staining in normal sized and enlarged GCs. This was considered indicative of compensatory B-cell proliferation after CFZ533-mediated inhibition of B cells was reversed. This rebound was mostly noted in mesenteric lymph nodes exposed to gastrointestinal antigens, even if no signs of active Cryptosporidium, adenovirus, or any other active opportunistic infection were found in the recovery animals. CD4 and CD8 staining of lymph node sections was similar between control and dosed groups, indicating that the increased T-cell cellularity was a reversible change.

Discussion

Toxicological evaluation of CFZ533 in animals demonstrated a good safety profile and tolerability of weekly subcutaneous and intravenous doses up to 150 mg/kg for 13 weeks. Plasma concentrations of CFZ533 at all doses were sufficient to cause full receptor occupancy on blood B cells. In accordance with the pharmacological mode of action, CFZ533 was immunosuppressive in vivo at ≥10 mg/kg, as indicated by ADCC/CDC-independent reduction in blood B cells after day 49 and the absence of GC in lymphatic organs. Immunohistochemical investigation confirmed that the decrease in GC was due to an absence of CD20⁺ B cells. The decrease of B-cell counts in blood and lymphatic organs was considered a consequence of the pharmacological inhibition of the T-cell and B-cell interaction and intracellular signaling by CFZ533 rather than antibody-mediated cell killing. Importantly, after clearance of CFZ533 following chronic treatment, animals regained normal lymphatic tissue architecture and immune function. Immune function was considered recovered based on the presence of ADA in two recovery animals after CFZ533 exposure dropped below full CD40 occupancy on B cells. Recovery of immune function in this study is consistent with the described recovery of the suppression of primary and secondary TDAR following single dose and multiple doses of CFZ533 in rhesus or cynomolgus monkey toxicology studies.^2,3 The recovery animals had a few lymph nodes with increased cellularity in B-cell areas, which was attributed to compensatory B-cell proliferation and reconstitution of GC after drug withdrawal.

Macroscopic enlargement and/or increased lymph node cellularity, affecting the paracortex, HEVs, and medulla, was associated with increased T-cell populations (increased Ki67, CD3, CD4, and CD8 positivity) and was attributed to an infectious background activating innate immunity and cell-mediated responses.^24,25 Active adenovirus and Cryptosporidium infections were identified in the intestine of some animals with this lymph node change. Of note, CD154-CD40 signaling is involved in intestinal immunity to Cryptosporidium infection in man.^24,26 While Cryptosporidium is a common protozoan parasite with more than 95% of nonhuman primates being seropositive, detection of many Cryptosporidium organisms in histological sections is associated with immunosuppressed or immunodeficient status of nonhuman primates.^27,28 Because opportunistic infections may have stimulated host defenses and caused inflammation prior to necropsy dates, absence of demonstrable pathogens at this time in all the affected animals does not exclude their likely role in inducing pathology. In addition, the range of microscopic findings associated with opportunistic infections, including the composition and severity of inflammatory infiltrates, is likely dependent on the degree of immune modulation and timing of samples during the disease course and findings at necropsy may not be fully representative of the full spectrum of morphologic alterations. The higher incidence of infection noted in dosed males was likely related to the group-housing of animals as Cryptosporidium was mostly seen in the three mid-dose (50 mg/kg) males.

The lymph node changes observed in this study, with loss of B cells and increases in T cells, differ from the effects associated with lymphocryptovirus (LCV)-infected primates in post-transplantation lymphoproliferative disorder (PTLD) where B-cell proliferation is predominant.^29-31 In this study, there was no histological evidence of focal, uncontrolled lymphoproliferation and there were no signs of LCV reactivation, as confirmed by the absence of EBNA-2 staining in the two mid-dose animals with inflammation in several organs. While CD8⁺ cytotoxic T cells were reported to respond to LCV-infected cells in rhesus monkeys,³² our results point to a CD8⁺ T-cell activation due to opportunistic infection in the intestine with the mesenteric lymph node being more affected than other lymph nodes (Table 2).

The infiltration of CD8⁺ T cells in the lung and kidney of the mid-dose males, as well as the finding of increased T cells in the lymphoid organs in other animals, suggests that T-cell surveillance was not compromised by CFZ533. However, the impact of CD40 blockade on the interaction of these T cells with epithelial cells in an inflammatory setting is not entirely clear. CD40 expression in epithelial cells, especially airway and tubular epithelial cells, can stimulate the expression of inflammatory mediators upon engagement by its ligand.^5,33 Activation of CD40 on renal proximal tubular epithelial cells stimulates expression of IL-6, IL-8, RANTES, MCP-1, and IL-15, which in turn contribute to tubular injury.¹⁰ Since CFZ533 is an antagonistic anti-CD40 monoclonal antibody, it was not expected to exert any further stimulus to epithelial/endothelial cell inflammatory responses as confirmed by histological evaluation of the present toxicology study, which did not reveal findings in line with a direct stimulation of epithelial/endothelial cells neither in kidneys nor in lungs. This absence of CFZ533 agonistic activity on epithelial/endothelial cells was confirmed by in vitro studies using HUVECs (human umbilical vein endothelial cells) demonstrating a complete CFZ533-mediated suppression of rCD154-mediated upregulation of MCP-1 and ICAM-1. The first being a potent attractant for monocytes, memory T cells, and dendritic cells to sites of tissue injury and inflammation, whereas the latter (ICAM-1) is a central interaction molecule with immune cells to initiate migration to injured and inflamed tissues (data not shown). Following engagement by CD154, CD40 activation is a major mediator of endothelial cell activation by production of adhesion molecules, cytokines, chemokines, and inflammatory mediators important for wound healing upregulation of genes involved in virus defense (i.e., TLR3 and IFIH1) and downregulation of the vasodilator apelin.^34,35 Based on the non-agonistic pharmacology of CFZ533, effects on wound healing, cardiovascular function, and fluid homeostasis were not expected and have not been observed in any of the nonhuman renal transplant studies.¹ The expression of CD154 on T cells likely determines which kind of interaction with epithelial cells takes place, CD154-dependent or CD154-independent. Of note, Durlanik et al.³⁶ reported that CD154 expression by CD4⁺ but not CD8⁺ T cells regulates antiviral immune responses in acute lymphocytic choriomeningitis virus (LCMV) infection in mice demonstrated by virus-specific CD8⁺ T cells in conditional knockout mice. Furthermore, the exact consequences of CD40 blockade on the fate of CD154 expression and activity on T cells remain subject of further investigations.

Compared to standard immunosuppressant drugs to prevent graft rejection, in particular calcineurin inhibitors, CFZ533 has shown less undesirable effects. Pimecrolimus, tacrolimus, and cyclosporin A were associated with pancreatic toxicity (Langerhans islet eosinophilic staining with β-cell depletion and secondary hyperglycemia) and nephrotoxicity (basophilic tubule with inflammatory cells infiltration) in toxicology studies in nonhuman primates, which were further confirmed in patients in clinical studies.^37-42 In addition, immunosuppressant mTOR inhibitor drugs like rapamycin (also called sirolimus) are associated with hyperlipidemia, pancreatic toxicity (Islet cell vacuolation with secondary hyperglycemia and cataract), cardiotoxicity (myocardial degeneration), pulmonary toxicity (increased alveolar macrophages), and gastrointestinal toxicities (gastritis, colitis, and typhlitis with secondary diarrhea) in rat, dog, and/or cynomolgus monkey toxicology studies; some of these toxicities have also been observed in the clinic.^43-45 In addition, infections (e.g., herpes simplex and zoster, mycobacterial, cytomegalovirus) were observed in the clinic with calcineurin and mTOR inhibitors. None of the toxicological changes associated with calcineurin inhibitors or rapamycin were observed with CFZ533 in 13-week or a 26-week chronic toxicology studies in the cynomolgus monkey.³ In addition, calcineurin inhibitors and rapamycin exert their pharmacological activity on T cells, whereas CFZ533 targets CD40, which is not expressed on human T cells. Calcineurin inhibitors indirectly inhibit the production of KLH-specific IgG antibodies without full abrogation of GC development due to their directly induced reduction of CD4+ T cells (i.e., PALS in spleen).^46,47 In contrast, CFZ533-related suppression of GC and the presence of T cells at sites of infection suggest that key surveillance capabilities were preserved following CFZ533 administration.

No hematological (platelet counts, P-selectin) and microscopic (thrombi) evidence of thromboembolic events were observed in this toxicology study or in other toxicology studies with CFZ533.³ It is important to note that targeting CD154 has been associated with an increase in thromboembolic events both preclinically^15-18,48-50 and clinically.^51,52 However, no clinical instances of thromboembolic events have been observed after treatment with a number of different anti-CD40 antibodies,^53-56 including CFZ533.⁵⁷ Thus, it is considered that inhibition of CD40-CD154 interactions does not increase the risk of thromboembolic events per se; rather these data suggest a target-specific effect of anti-CD154 antibodies. The potential of anti-CD154 to induce thromboembolic effects was shown to be linked to the active Fc part of the antibody binding to FcγRIIa and leading to platelet activation. Both the corresponding Fc-silent anti-CD154 and the PEGylated Fab version did not induce thromboembolism in vivo in rhesus monkey toxicology studies and platelet activation in vitro in a platelet aggregation assay using human and rhesus monkey blood.¹⁷ In addition, investigation in human FcγR transgenic mice revealed the absence of thromboembolic activity of a blocking, non-depleting anti-CD40 antibody, whereas anti-CD154 antibody caused thrombocytopenia and thrombi formation, further confirmed in an in vitro whole blood aggregation assay with human blood (data not shown, in preparation). These data collectively suggest that the thromboembolic risk is very low, if existent at all for CFZ533.

In summary, iscalimab (CFZ533) was well tolerated and did not show thromboembolic events. Findings observed were closely related to expected pharmacology of an immunomodulating therapeutic antibody. The result of infection by microorganisms was therefore considered secondary to immunosuppression mediated by iscalimab. The increased numbers of CD4 and CD8 T cells in response to the infections suggest that T-cell immune function was unaffected, revealing the differential immunosuppression effects of iscalimab compared to calcineurin-inhibiting drug. This conclusion is supported by the antagonistic and Fc-silent nature of CFZ533, as well as the biology of CD40 expressed on B cells, antigen-presenting cells, and endothelial and epithelial cells.

Supplemental Material

sj-docx-2-tpx-10.1177_01926233221100168 – Supplemental material for Immunosuppression Profile of CFZ533 (Iscalimab), a Non-Depleting Anti-CD40 Antibody, and the Presence of Opportunistic Infections in a Rhesus Monkey Toxicology Study

Supplemental material, sj-docx-2-tpx-10.1177_01926233221100168 for Immunosuppression Profile of CFZ533 (Iscalimab), a Non-Depleting Anti-CD40 Antibody, and the Presence of Opportunistic Infections in a Rhesus Monkey Toxicology Study by Thierry D. Flandre, Keith G. Mansfield, Pascal J. Espié, Tina Rubic-Schneider and Peter Ulrich in Toxicologic Pathology

Supplemental Material

sj-tif-1-tpx-10.1177_01926233221100168 – Supplemental material for Immunosuppression Profile of CFZ533 (Iscalimab), a Non-Depleting Anti-CD40 Antibody, and the Presence of Opportunistic Infections in a Rhesus Monkey Toxicology Study

Supplemental material, sj-tif-1-tpx-10.1177_01926233221100168 for Immunosuppression Profile of CFZ533 (Iscalimab), a Non-Depleting Anti-CD40 Antibody, and the Presence of Opportunistic Infections in a Rhesus Monkey Toxicology Study by Thierry D. Flandre, Keith G. Mansfield, Pascal J. Espié, Tina Rubic-Schneider and Peter Ulrich in Toxicologic Pathology

Footnotes

Acknowledgements

The authors thank G. Betton, A. Kiessling, A. Piequet, D. Shaw, D. Sickert, Labcorp Muenster, and all other associates for their expertise and support provided across these studies.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Thierry D. Flandre

Keith G. Mansfield

Supplemental Material

Supplemental material for this article is available online.

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