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Counter-structures CD28, ICOS, or CTLA-4 were transiently transfected and expressed in Expi293FTM cells. |
Flow cytometric analysis was performed on an LSRII flow cytometer (BD Biosciences, USA). The MFIs were graphed using Prism software (GraphPad Software, USA, v7.02). Prism was also used to determine the half maximal effective concentration (EC50) through nonlinear regression analysis using the following parameters: fit ... |
BLI analysis using the Forté Bio™ Octet system was used to determine the association rate (ka) and dissociation rate (kd) to calculate the affinity (KD) (KD =kd/ka) of ALPN-101 binding to recombinant monomeric human CD28-Flag-His, ICOS-Flag-His and CTLA-4-His proteins. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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6. EXPERIMENTAL PROCEDURES |
6.1. Flow Cytometric Analysis: |
6.1.1. Cell Transfections (Day 0) |
Expi293 cells were plated at 10 x 106 cells per well in a 24 deep-well plate in a total of 3.6 mL Expi293 expression media containing 1% pluronic acid and incubated overnight at 37°C/5% CO2 with shaking at 125 rpm. A DNA tube for each expression plasmid encoding each species' CD28, ICOS, or CTLA-4 was created by suspen... |
6.1.2. FACS Staining (Day 2) |
Transfected Expi293 cells were harvested, counted, and adjusted to a concentration of 5 x 106 cells/mL in PBS. A 1:1000 dilution of NEAR-IR Live/Dead dye (stock vial reconstituted in 50 µL DMSO) was added and the cells were incubated for 15 min at RT, washed in FACS buffer (PBS, 0.5% BSA, 0.1% NaN3, 0.5 mM EDTA) and ad... |
Gating on Near IR Live/Dead dye positive cells was used to exclude any remaining dead cells from analysis. The Near IR Live/Dead fluorescent dye is reactive with amines. The dye cannot penetrate live cell membranes, so cell surface proteins are only dimly stained on live cells. The dye can easily permeate damaged membr... |
6.2. Bio-Layer Interferometry (BLI) Analysis: |
Following a 60 second baseline to confirm sensor stability, ALPN-101 was loaded onto anti-human Ig capture sensors in Forté Bio™ kinetic buffer at a concentration of 10 µg/mL. Another baseline reading was performed, followed by binding of recombinant monomeric CD28-Flag-His, ICOS-Flag-His, and CTLA-4-His in a 7 point 2... |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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required to achieve a 10% decrease in signal, corresponding to 900 seconds for ICOS and CTLA-4, and 3600 seconds for CD28. |
After the data was acquired, the blank buffer sample signal was subtracted from each titration and the triplicate curves were globally fit with a 1:1 model to determine the ka and kd of each counter structure. The error of the association and dissociation were calculated along with the KD and full R2. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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7. DATA ANALYSIS |
[THIS IS FIGURE: Figure 1 showing Flow Cytometric Analysis Gating Strategy (A) and Representative Histograms of ALPN-101 Binding to Human Counter Structures (B)] |
A) Exclusion of dead cells was accomplished by gating on healthy cells in a forward scatter (FSC) x side scatter (SSC) gate as shown in the left-hand panel. Gating out Near IR Live/Dead dye positive cells excluded any remaining dead cells from analysis, as described in Section 6.1.2. |
B) Representative flow cytometric histograms of Fc1.1 control, human WT ICOSL-Fc1.1, and ALPN-101 binding to human counter structures CD28, ICOS, and CTLA-4. Each protein is at a concentration of 4000 pM and was detected with goat anti-human IgG-PE, as described in Section 6.1.2. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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[THIS IS FIGURE: Figure 2 showing multiple graphs of ALPN-101 Binding to Human, Cynomolgus Monkey, Rat, and Mouse CD28, ICOS, and CTLA-4] |
Figure 2: ALPN-101 Binding to Human, Cynomolgus Monkey, Rat, and Mouse CD28, ICOS, and CTLA-4 |
Flow cytometric analysis of ALPN-101, human WT ICOSL-Fc1.1, and Fc control protein binding (MFI) to Expi293 cells transfected with human, cynomolgus monkey, rat, or mouse CD28, ICOS, or CTLA-4. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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Raw binding data (MFI) for all counter structures are located in the Appendix as follows: human (Table 8), cynomolgus (Table 9), rat (Table 10), mouse (Table 11), mock control (Table 12). |
Table 5: ALPN-101 Binding Affinity (EC50) to CD28, ICOS, and CTLA-4 Across Species Assessed by Flow Cytometric Analysis |
Species CD28 EC50 (pM) ICOS EC50 (pM) CTLA-4 EC50 (pM) |
Human 648 633 680 |
Cynomolgus monkey 440 579 704 |
Rat 1,064 1,028 793 |
Mouse 1,153 1,428 1,112 |
The EC50 were calculated using Prism and nonlinear regression (curve fit) analysis: fit = log(agonist) vs response- FindECanything; HillSlope = 1; F = 50 |
The EC50s of WT ICOSL-Fc1.1 binding to ICOS across all species were comparable to those of ALPN-101; the EC50 of WT ICOSL-Fc1.1 for CD28 and CTLA-4 binding could not be determined. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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Table 6: ALPN-101 Demonstrates Superior Binding to CD28 Compared to that of WT ICOSL-Fc1.1 |
Species Protein Conc. (pM) Average MFI1 Fold Increase in MFI2 ALPN-101 WT ICOSL-Fc1.1 |
Human 100000 32480 6076 5.3 |
20000 33835 1388 24.4 |
4000 35343 1254 28.2 |
800 18413 1119 16.5 |
160 4511 600 7.5 |
Cynomolgus 100000 24408 4878 5.0 |
20000 24160 1867 12.9 |
4000 24487 1475 16.6 |
800 16924 1364 12.4 |
160 5187 989 5.2 |
Rat 100000 79493 7435 10.7 |
20000 84573 5387 15.7 |
4000 93342 5354 17.4 |
800 25094 5436 4.6 |
160 6124 2826 2.2 |
Mouse 100000 87485 6197 14.1 |
20000 81393 3997 20.4 |
4000 91109 3384 26.9 |
800 25887 3751 6.9 |
160 6205 1975 3.1 |
1 Binding of ALPN-101 or WT ICOSL-Fc1.1 at various concentrations to Expi293 cells transfected with CD28 from each of the species noted was assessed by flow cytometry; average MFI is listed. |
2 The fold increase in MFI for binding of ALPN-101 compared to that of WT ICOSL-Fc1.1 is provided (i.e. MFI for ALPN-101/MFI for WT ICOSL-Fc1.1). |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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Table 7: ALPN-101 KD, ka, kd, Errors, and Full R2 Measured by BLI Analysis |
Both pM and M versions of the KD are reported for ease, along with ka, kd, and their respective errors. The error of each calculated value is reported to give confidence to the values reported and ideally should be less than 10%. Full R2 is also reported to give confidence to the global fit of a 1:1 model; ideally, thi... |
Sample ID KD (pM) KD (M) KD Error ka(1/Ms) ka Error kd(1/s) kd Error Full R2 |
ICOS 706.6 7.066E-10 9.344E-11 93940 962.2 6.638E-05 8.752E-06 0.9371 |
CD28 192 1.92E-10 1.187E-11 135400 938.3 0.000026 1.598E-06 0.8865 |
CTLA-4 240.5 2.405E-10 3.745E-11 412400 21110 9.916E-05 1.459E-05 0.9065 |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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Figure 3: General Description of Binding Sensorgrams |
ALPN-101 was loaded onto sensors at 10µg/mL via anti-human Ig capture. Flag-His or His-tagged monomeric CD28, ICOS or CTLA-4 counter structure titrations were then associated for 300 seconds. Samples were then allowed to dissociate equal to the time required to achieve a 10% decrease in signal: i.e. 900 seconds for ICO... |
Figure 4: CD28 Binding Sensorgram |
As described in Figure 3, ALPN-101 was loaded onto sensors via anti-human Ig capture and then CD28-Flag-His protein associated for 300 seconds. Samples were then allowed to dissociate equal to the time required to achieve a 10% decrease in signal: i.e. 3600 seconds for CD28. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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Figure 5: ICOS Binding Sensorgram |
As described in Figure 3, ALPN-101 was loaded onto sensors via anti-human Ig capture and then ICOS-Flag-His protein associated for 300 seconds. Samples were then allowed to dissociate equal to the time required to achieve a 10% decrease in signal: i.e. 900 seconds for ICOS. |
Figure 6: CTLA-4 Binding Sensorgram |
As described in Figure 3, ALPN-101 was loaded onto sensors via anti-human Ig capture and then CTLA-4-His protein associated for 300 seconds. Samples were then allowed to dissociate equal to the time required to achieve a 10% decrease in signal: i.e. 900 seconds for CTLA-4. |
Alpine Immune Sciences, Inc. |
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Pharmacology Study Report ALPN100-PHRM-001 |
8. STORAGE OF DATA |
Confirmation of cell surface expression of human CD28, ICOS, and CTLA-4 on transiently transfected Expi293F cells: Notebook reference # ALPINE.LSE.2 (p.15-18). |
Transfection Notebook reference #: EAR.007 Exp#TF20171204 (p. 88-89). |
Binding Notebook reference #: ALPINE.LSE.004 (p. 82-90). |
BLI KD determination Notebook reference #: JEH04-84 & 85. |
All raw data generated for this study is securely stored on the AIS server. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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9. RESULTS |
ALPN-101 bound human CD28, ICOS, and CTLA-4 in flow cytometric assays with EC50s of 648, 633, and 680 pM, respectively. ALPN-101 bound cynomolgus monkey homologs of its target molecules with affinities (440, 579, 704 pM) similar to those for the human targets, and ALPN-101 bound rat (1064, 1028, 793 pM) and mouse (1153... |
Weak WT ICOSL binding to CD28 and CTLA-4 has been demonstrated previously by flow cytometric binding, surface plasmon resonance (SPR), and mutational analysis (Yao et al., 2011). In the studies described herein, ALPN-101 exhibited improved CD28 binding compared to WT ICOSL-Fc1.1, demonstrated by a >10-fold increase in ... |
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