Anti-CTLA-4 antibody Ipilimumab (Ipi) or human IgGFc (hIgGFc) control were conjugated to a well-known drug payload DM1, emtansine, to furnish the corresponding ADCs: Ipilimumab-DM1 (Ipi-DM1) and hIgGFc-DM1 with corresponding size shifts in the SDS-gel (Figure 1A). The drug to antibody ratio (DAR) was calculated based on experimentally determined extinction coefficient A280 & A252 for each antibody and reported values for DM1 (Table S1). The DAR from various conjugation is ∼3.2 for Ipi-DM1 and ∼ 1.7 for hIgGFc-DM1 control (Table S1), as expected based on the sizes of the proteins. The binding for Ipi-DM1 ADC was evaluated by ELISA binding to immobilized His-hCTLA-4 and by flow cytometry using hCTLA-4 expressing CHO cells (CHO-hCTLA-4). Ipi-DM1 binding was found comparable to the parent antibody Ipi (Figures 1B, 1C). Specific killing of CTLA-4-expressing cells by Ipi-DM1 was assessed in human CTLA-4 expressing CHO (CHO-hCTLA-4) and wild type CHO (CHO-WT) cells in vitro (Figure 1D), which showed Ipi-DM1 reduced viability of CLTA-4 expressing CHO but not wild type cells. As expected, there was no change in viability for either CHO cell lines when treated with Ipi (Figure 1D).

In order to evaluate the in vivo effects of CTLA-4 ADC on Tregs, we treated human CTLA-4 knock-in (Ctla4^h/h) mice with control hIgGFc or Ipi-DM1 ADC (Figure 1E). Peripheral blood was stained for flow cytometry and gated on CD45 and T-cell subset, including CD8, CD4, CD4 Foxp3⁺ and CD4 Foxp3^- subsets (Figure 1F). We found that Ipi-DM1 ADC significantly depleted Foxp3⁺ Tregs as indicated by the reduction in percentage and cell number of Foxp3⁺ CD4 T cells when compared to hIgGFc control (Figure 1G). In addition, total CTLA-4 levels and Ki67 staining in Tregs were decreased compared to control group (Figures 1H, 1I). In contrast, Ipi-DM1 ADC did not alter either % of CD4 Foxp3^- cells among CD45⁺ leukocytes or CTLA-4 levels on the subset, but did slightly decrease the cell number of CD4 Foxp3^- cells when compared hIgGFc control (Figure 1-figure supplement 1). Unexpectedly, Ipi-DM1 ADC caused a dramatic decline of B cells as percentages of CD45 and cell number in the periphery (Figure 1J). B cells from mice treated with Ipi-DM1 exhibited higher proliferation than those from hIgGFc treated control mice (Figure 1K).

Since a dramatic loss of B cells and a slight decrease in CD4-nonTregs in Ipi-DM1 treated mice were observed, we sought ensure that this phenomenon was not a result of downstream release of payload drug DM1. As shown in Figure 1-figure supplement 2A, hIgGFc-DM1 ADC (payload control) did not alter Treg percentage and cell number. A slightly higher level of CTLA-4 was noted in CD4 subset, while Treg proliferation did not change significantly compared to control (Figure 1-figure supplement 2B, 2C). No other effects on T cell subsets were noted (Figure 1-figure supplement 2D, 2E). Importantly, B cell number and proliferation were unaffected by hIgGFc-DM1 ADC treatment (Figure 1-figure supplement 2F, 2G). Additionally, to ensure B cell depletion was not directly caused by Ipi-DM1 ADC, we stained B220⁺ B cells and Foxp3⁺ Tregs for human CTLA-4 in both human CTLA-4 knock-in (Ctla4^h/h) and WT mice to evaluate expression of CTLA-4 in B cells. As expected, CTLA-4 was detected in Foxp3⁺ Tregs of Ctla4^h/h but not B cells (Figure 1-figure supplement 3).

We then asked how Ipi-DM1 ADC induced Treg impairment impacted B cell lymphopoiesis. FACS analysis of bone marrow B cells revealed the loss of mature B cells but not immature transitional type1 or Pre-pro/Pro/Pre B cells from Ipi-DM1 treated mice, however the Pre-Pro/Pro/Pre B cell subtype percentage in B220 are enriched as a result of mature B cell loss (Figure 1L-1P, Figure 1-figure supplement 4). Additionally, CD21/35 expression level in the remaining mature B cells in Ipi-DM1 treated mice was lower than those from hIgGFc treated control mice (Figure 1Q). Taken together, data in Figure 1 showed that Ipi-DM1 can impair Treg function by depleting Tregs, preferentially the proliferating Tregs with higher CTLA-4 levels, and loss of B cells in the blood and bone marrow while B cell progenitors are not impacted.

To investigate the kinetic relationship between the decline of B cells and Treg impairment, human CTLA-4 knock-in (Ctla4^h/h) mice were treated with control hIgGFc or Ipi-DM1 and bled according to schedule diagram in Figure 2A. We observed that peripheral blood samples from mice treated with Ipi-DM1 had a gradual decrease in total leukocytes compared hIgGFc control group followed by a full recovery by Day 25 (Figure 2B). The large decrease of CD45 cells in Ipi-DM1 group is predominantly attributed to the decline of B cells, which plateaus on days 9 and 13 and recovers by day 25 (Figures 2C, 2D) in correlation with the kinetics of Tregs (Figure 2E, 2F). Similar to the bone marrow data in Figure 1, peripheral blood mature B cells decrease and transitional type1 B cells become enriched in Ipi-DM1 group compared hIgGFc control (Figure 2G-2I).

We then evaluated how Ipi-DM1-mediated impairment of Treg function affects total CD4 and CD8 T cells. Mice treated with Ipi-DM1 ADC did not affect the total percentage of CD4 or CD8 T cells among CD45⁺ leukocytes, but the cell number of both cell types decreased (Figure 3A, 3B). Staining of Ki67 in CD4 and CD8 revealed a greater percentage of T cells in hyper-proliferative state (Figure 3C). Alternatively, mice treated with hIgGFc-DM1 did not change CD4 or CD8 percentage of CD45, cell numbers and Ki67 staining compared to hIgGFc control (Figure 3-figure supplement 1). We then analyzed the functional subsets of T cells in mice that received control hIgGFc or Ipi-DM1 at day 9. Using CD44 and CD62L markers, we observed an expansion of effector memory T cells (CD44^hiCD62L^low) in Ipi-DM1 group for both CD4 and CD8 T cells (Figures 3D, 3E). Correspondingly, the frequency of naive T cells was reduced for Ipi-DM1 while central memory T cells were unchanged (Figures 3D, 3E). These results show that CTLA-4 ADC can impair Treg function thereby resulting an increase in effector memory T cells and a hyper-proliferative state similar to animals that lack the expression of CTLA-4 or Foxp3.

Since B cells are devoid of CTLA-4, Ipi-DM1 must have induced other effector cells that are directly responsible for B cell depletion. To understand which cell types are responsible for the destruction of B cells, we depleted either T cells or macrophages using either anti-Thy1.2 mAb or chlondrosome. Ctla4^h/h mice were treated with control hIgGFc, Ipi-DM1, Ipi-DM1 in combination with anti-Thy1.2, or Ipi-DM1 in combination with chlondrosome and bled on day 9 (Figure 4A). As shown in the Figure 4B, top panel, anti-Thy1.2 mAb efficiently depleted total T cells while macrophage depletion by chlondrosome had no effect (Figure 4B top panel, 4C, 4D). Additionally, combination of Ipi-DM1 with T cell depleting antibody resulted in a remarkable rescue of B cells as indicated by percentage B220 in CD45 and cell number while combination with macrophage depletion did not rescue the B cells (Figures 4B bottom panel, 4E).

In order to understand which subsets of T cells played a critical role in the T cell mediated destruction of B cells, we treated Ctla4^h/h mice with hIgGFc control, Ipi-DM1, Ipi-DM1 with CD4 depleting antibody, or Ipi-DM1 with CD8 depleting antibody (Figure 4A). Both CD4 and CD8 depleting antibodies provide efficient depletion (Figure 4F, top panel) of their respective targeted T cell. Depletion of either CD4 or CD8 T cells results in B cell increase (Figures 4F bottom panel, 4I). Interestingly, the cell number of CD4 and CD8 T cells increase reciprocally by depletion of the other subsets (Figures 4G, 4H).

Taken together our CTLA-4 antibody-drug conjugate Ipi-DM1 can impair Treg function, which results in a T cell mediated destruction of B cells. Whole T cell depletion with anti-Thy1.2, depletion of CD4 or CD8 T cells rescued B cells from abrogation by Ipi-DM1.

The process for T cell activation requires two signals. The first signal is the binding of T-cell receptor (TCR) to antigen-bound major histocompatibility complex (MHC) and the second signal is a costimulatory molecule CD28 binding with B7-1/2 (CD80/86) on the antigen-presenting cell. Having shown that the destruction of B cell can be prevented by total depletion of T cell, we sought to rescue the B cells by breaking the CD28-B7 signal with a soluble CTLA-4 that can bind to B7 but not Ipilimumab or Ipi-DM1 ADC. As shown in Figure 5-figure supplement 1, Abatacept can neutralize Ipilimumab/ADC while Belatacept does not. Therefore, Belatacept can be used to test if activation of T cells is required for B cell depletion.

We treated mice with Ipi-DM1 to impair Treg function and used Belatacept to break CD28/B7 signal to block T cell activation. Briefly, Ctla4^h/h mice were treated with hIgGFc or Ipi-DM1 with/out Belatacept and bled on day 9 for flow analysis. Foxp3⁺ Tregs percentage in CD4 and cell number decreased similarly for Ipi-DM1 with/out Belatacept compared hIgGFc control (Figures 5A, 5B). Additionally, Belatacept did not impact Ipi-DM1 to target CTLA-4 expressing Tregs, as Ipi-DM1 with/out Belactacept had similar total CTLA-4 level reduction compared to control group (Figure 5C). However, Belatacept rescued B cells from Ipi-DM1-mediated depletion (Figures 5D, 5E). Correspondingly, Belatacept reduced effector memory T cells (Figures 5F, 5G) as well as granzyme B and IFN-γ expression in CD4 and CD8 T cells compared to Ipi-DM1 treated group (Figures 5H-5K, Figure 5-figure supplement 2).

Collectively, the data presented here show that Ipi-DM1 can impair Treg function resulting in T cell activation as indicated by increase in effector memory T cell markers, GranzymB, and cytokine production thereby resulting in the loss of B cells. However, under Ipi-DM1 induced Treg impairment the addition of mutant soluble CTLA-4-Ig, Belatacept, can reduce T cell activation and thereby preserving B cells.

Since T cells are the effector cells responsible for B cell abrogation, it is of great interest to evaluate the molecular mechanism by which B cells are eliminated by T cells. To address this issue, we tested if either FasL or TNF-alpha, which are produced by activated T cells are responsible. To answer this question, Ctla4^h/h mice were treated with hIgGFc control or Ipi-DM1 with/out Adalimumab a human anti-TNF-alpha that also binds to mouse TNF-alpha (Figure 6A, 6B). Flow analysis showed Tregs were decreased similarly between Ipi-DM1 with/out Adalimumab compared hIgGFc control group (Figures 6C top panel, 6D). Remarkably, the anti-TNF-alpha partially rescued B cells as shown by percentage difference of B cell marker B220 between Ipi-DM1 and in combination with Adalimumab as well as cell number (Figures 6C bottom panel, 6E). Peripheral blood samples stimulated with Iononmycin/PMA increased intracellular cytokines TNF-alpha and IFN-gamma for T cells from Ipi-DM1 treated mice compared to hIgGFc control, while that with Ipi-DM1 in combination with Adalimumab had slight increase but the change was insignificant except for IFN-gamma in CD8 T cells (Figure 6F, 6G). In contrast, blocking FAS-L with antibody did not result in B cell rescue (Figure 6-figure supplement 1).

C57BL/6 mice that express the CTLA-4 protein with 100% identity to human CTLA-4 protein under the control of endogenous mouse Ctla4 locus, Ctla4^h/h, have been previously described.²⁶ All animals used in experiments were 7-9 weeks age (female mice were used unless male mice are indicated). No blinding or randomization was used and mice were fairly distributed into different treatment groups so that initial average weight of each group were similar. All mice were maintained at the Research Animal Facility of the Institute of Human Virology at the University of Maryland Baltimore School of Medicine. All animal studies were approved by the Institutional Animal Care and Use Committee.

CHO cells that were stably transfected with human CTLA-4 have been reported.²⁷ CHO cells were grown in DMEM (Dulbecco’s Modified Eagle Medium, Gibco) supplemented with 10% FBS (Hyclone), 100 units/mL of penicillin and 100 μg/mL of streptomycin (Gibco). Mice Peripheral blood leukocytes were cultured in RPMI-1640 medium (containing 10% FBS and 2% penicillin/streptomycin). All cell lines were incubated at 37 °C and were maintained in an atmosphere containing 5% CO2.

Wild type CHO (CHO-WT) or human CTLA-4 expressing CHO (CHO-hCTLA-4) cells were seeded at 1,000 cell/well in a flat 96-well plate at 37 °C for 24 hrs in cell culture incubator. The medium was then replaced with fresh medium containing 1/4 serially diluted vehicle (PBS), Ipilimumab or Ipilimumab-DM1 ADC and cell were incubated at 37 °C for additional 72 hrs. Each treatment group was in duplicate or triplicates. CCK-8 viability dye was then added to each well according to manufacturer’s protcol and incubated for another (2-2.5 hrs) at 37 °C. Wells were subsequently read for absorbance at 450nM on Spectramax ID3 Molecular Devices plate reader. In case of testing whether soluble human CTLA-4-Ig or mutant can neutralize Ipilimumab or Ipilimumab-DM1, the same conditions above were used except that Abetacept or Belatacept concentrations were kept constant at 6 μg/mL. Data is normalized according to the following equation (treatmentOD450 - backgroundaverageOD450 /vehicleaverageOD450 - backgroundaverage) x 100.

Freshly trypsinized human CTLA-4 expressing CHO (CHO-hCTLA-4) cells were stained with 1/4 serially dilute anti-CTLA-4 Iplimiumab or Iplimiumab-DM1 ADC in FACS buffer (2% FBS with 2 mM EDTA) for 30 minutes on ice. Cells were then washed twice with FACS buffer and incubated with anti-human IgG AF488 secondary antibody for 20 minutes on ice. Cells were washed twice with FACS buffer and processed on BD Canto II flow cytometer. In the case of whether soluble human CTLA-4-Ig or mutant can neutralize Ipilimumab or Ipilimumab-DM1 and prevent them from binding cell to surface CTLA-4 the same conditions above were used except that Abetacept or Belatacept concentrations were kept constant at 6 μg/mL.

Peripheral blood samples 50 μL were treated with ACK Lysis buffer and washed with RPMI-1640 medium. Leukocytes were then stimulated with 1 µg/ml each of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO), ionomycin (Sigma Aldrich, St. Louis, MO) and BD GolgiStop™ (BD Biosciences, cat. 51-2092KZ) and cultured in RPMI-1640 medium (containing 10% FBS and 2% penicillin/streptomycin) for 4 hours at 37°C in 96-well plate. Medium was removed and cells were washed twice with FACS buffer (2% FBS with 2 mM EDTA) followed by surface staining and fix/perm and intracellular staining.

Leukocytes from blood or bone marrow were FACS stained directly or after red blood cell lysis with ACK buffer. Fc Receptor was blocked with anti-FCR clone 2.4G2 at 10 μg/mL in FACS buffer for 10 minutes at room temperature and respective surface staining antibodies cocktails were added to each sample and incubated on ice for additional 20 minutes. Cells were then washed twice with 1xPBS and stained with 1x live/Dead Fixable dye Aqua in 1x PBS for 7 minutes at room temperature. Cells were then washed twice with FACS buffer and fixed with eBioscience™ Foxp3/Transcription Factor Staining Buffer Set for 40 minutes. Samples were either washed twice and resuspended in FACS buffer and processed for flow acquisition or further permeabilized for intracellular staining using perm buffer from same kit (eBioscience™ Foxp3/Transcription Factor Staining Buffer Set). In General, all intracellular staining of Foxp3, Ki67, hCTLA-4, GranzymeB, or cytokines were done overnight at 4 degree. In the case of detecting intracellular cytokines, samples were cultured and simulated according to the above protocol followed by surface then intracellular cytokine staining. Samples were acquired by the BD Canto II Flow cytometer and data were analyzed by Flowjo software.

96-well high-binding polystyrene plates were pre-coated with 50 μL of 1 μg/mL of His-hCTLA-4, or mouse TNF-alpha in coating buffer (0.1M bicarbonate) at 4°C overnight. After washing away the unbound protein/antibody thrice with 0.05% PBST, the plates were blocked with blocking buffer (1% BSA in PBST) for 1hr at room temperature. All primary antibody incubation was done in blocking buffer at 4°C overnight or room temperature for 1 hour. For coated His-hCTLA-4 a given concentration of either Ipilimumab/DM1, or hIgGFc/DM1; For coated mouse TNF-alpha a given concentration of Adalimumab or Ipilimumab negative control were used. Following primary incubation plates were then washed with PBST for four times and incubated with a goat anti-human IgGFc HRP conjugate secondary antibody at 1/20,000 dilution for detection in blocking buffer for 1 hr at room temperature. Plates were then washed 4 times with PBST followed by development with 1-Step™ Ultra TMB-ELISA Substrate Solution for 10 minutes and stopped with 2N sulfuric acid. Wells were read at 450nM on Spectramax ID3 Molecular Devices plate reader.

Ipilimumab or hIgGFc were buffer exchanged using prepacked column PD-10 into 1X PBS. Ipilimumab or hIgGFc 5 mg each at concentration 1.5 mg/mL were conjugated with SMCC-DM1(15 eq) in 1x PBS in the presence of 10% DMSO at 37°C under mild shaking conditions for 50 minutes to furnish Ipilimumab-DM1 or hIgGFc-DM1 ADC respectively. Reaction mixture was cleaned up from excess SMCC-DM1 by buffer exchange with a in house ADC buffer at pH 6.5 containing (20 mM Histidine, 8 % sucrose, and Polysorbate 80 0.02%) by Econo-Pac 10DG prepacked column according to manufacturer protocol. Confirmed fraction containing ADC by nano drop were combined and concentrated down to 1 mL using Pierce™ Protein Concentrator PES, (30K for hIgGFc-DM1) or (50K for Ipilimumab-DM1) MWCO according manufacture protocol followed by sterile filtration. ADC concentration was determined using BCA assay according to manufacturer protocol. ADC were diluted to 0.4 – 0.5 mg/mL in 1xPBS and A280 &A252 were recorded on Nanodrop One. The drug to antibody ratio (DAR) was calculated following previous literature.²⁸

CTLA-4-Ig fusion proteins were synthesized by Sydlabs, Inc (Boston, MA). Recombinant Ipilimumab with amino acid sequence disclosed in WC500109302 was produced by Sydlabs Inc. (Boston, MA). Azide-free human IgG-Fc was purchased from Athens Research and Technology (Athens, GA, USA). Antibody-drug conjugates Ipilimumab-DM1 and hIgGFc-DM1 were prepared from parent antibodies in lab. Depletion antibodies anti-mouse Thy1.2, clone 30H12(BE0066); anti-mouse CD4, clone GK1.5 (BE0003-1); and anti-mouse CD8α, clone 2.43 (BE00-61) were purchased by Bioxcell Inc. (West Lebanon, NH, USA). Anti-mouse FASL, clone MFL3 (BE00319) was purchased by Bioxcell Inc. (West Lebanon, NH, USA). Anti-TNF-α, Adalimumab, clinical grade Humira^TM was purchased from Premium Health Services (Columbia, MD, USA).

SMCC-DM1 drug payload with cross linker (Cederlanelabs/Cayman Chemical Co, 23926-10) Anti-mouse FcγR, clone 2.4G2 (Bioxcell Inc, BE0307). Clodrosome® (Encapsula Nano Sciences, SKU# CLD-8909). Mouse anti-human IgGFc secondary antibody (Invitrogen/Thermo Fisher Scientific, 05-42-00). Goat anti-human IgGFc (HRP) preadsorbed (Abcam, ab98624). Polyhistidine-tagged human CTLA-4 (HIS-hCTLA-4) (Sino Biological Inc, 11159-H08H). Mouse TNF-alpha (Sino Biological Inc, 50349-MNAE). 123 eBeads^TM counting beads flow (Thermo Fisher Scientific/Invitrogen, 01-1234-42). NuPage^TM BIS-TRIS gels 4-12% (Thermo Fisher Scientific/Invitrogen, NP0335BOX). NuPAGE™ MOPS SDS Running Buffer (20X) (Thermo Fisher Scientific/Invitrogen, NP0001). NuPage^TM LDS sample buffer (4x) (Thermo Fisher Scientific/Invitrogen, NP0007). Protein Ladder (Fisher Scientific, BP3603500). Sucrose (Sigma Aldrich, S0389-1KG). L-Histidine (Sigma Aldrich, H8000-10G). Dimethyl sulfoxide (DMSO) (Santa Cruz Biotechnology, Sc-258801). Polysorbate 80 (Fisher Scientific, L13315). Buffer exchange prepacked column PD-10 (GE health care/ Cytiva Life Sciences, 17085101). Buffer exchange prepacked column Econo-Pac 10DG (Bio-Rad, 7322010). Cell Counting Kit-8 (CCK-8) (Bimake, B34304). 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, 34028). Gibco™ ACK Lysing buffer (Thermo Fisher scientific/Gibco, A1049201). Live/Dead™ Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific/Life Technologies, L34966). eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific/Invitrogen, 00-5523-00). Pierce™ Protein Concentrator PES, 30K MWCO, 2-6 mL (Thermo Fisher Scientific/ Pierce, 88521) Pierce™ Protein Concentrators PES, 50K MWCO, 2– 6 mL (Thermo Fisher Scientific/ Pierce, 88538). Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific/Pierce, 23227).

eBioscience/ Thermo Fisher Scientific (San Diego, CA): APC-eFlour780 anti-mouse CD45, clone 30-F11 (47-0451-82);eFlour450 anti-mouse CD4, clone GK1.5 (48-0041-82); Pacific Blue anti-mouse CD4, clone RM-4-4 (116008); PE-Cyanine7 anti-mouse CD8α, clone 53-6.7 (25-0081-82); PE-Cyanine7 anti-mouse CD8β, clone H35-17.2 (25-0083-82);PerCP-Cy5.5 anti-mouse B220, clone RA3-6B2 (45-0452-82); APC anti-mouse Foxp3, clone FJK-16s (17-5773-82);FITC anti-mouse Ki67, clone SolA15 (11-5698-82);FITC Isotype rat IgG2ak, clone eBR2a (11-4321-82);FITC anti-mouse CD44, clone IM7 (11-0441-85);PerCP-Cy5.5 anti-mouse CD62L, clone MEL-14 (45-0621-82);PE-Cyanine7 anti-mouse B220, clone RA3-6B2 (25-0452-82); APC anti-mouse IgM, clone II/41 (17-5790-82); eFlour 450 anti-mouse CD21/CD35, clone 4E3 (48-0212-82); AF488 anti-mouse TNF-α, clone MP6-XT22 (53-7321-82);AF488 Isotype rat IgG1k, clone eBRG1 (53-4301-80); APC anti-mouse IFNγ, clone XMG1.2 (17-7311-82);APC Isotype rat IgG1k, clone eBRG1 (17-4301-82);FITC anti-mouse GranzymeB, clone NGZB (11-8898-82); Alexa Fluor 488-conjugated goat anti-human IgG (H+L) cross-adsorbed secondary antibody (A-11013). BioLegend (San Diego, CA): PE anti-human CTLA-4, clone BNI3 (369604);PE Isotype mIgG2ak, clone MPC-173 (400212); PerCP-Cy5.5 anti-mouse IgD, clone 11-26c.21 (405710); BV421 anti-mouse CD21/CD35, clone 7E9 (12342);

The specific tests used to analyze each set of experiments are indicated in the figure legends. Data were analyzed using a two-tailed t-test to compare between two groups or by one-way ANOVA (analysis of variance) for multiple comparisons. In the graphs, y-axis error bars represent S.E.M. or S.D. as indicated. Statistical calculations were performed using GraphPad Prism software (GraphPad Software, San Diego, California). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Reported CTLA-4 level and absolute cell numbers are normalized by the average value of hIgGFc control group.