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LIST OF TABLES Table 1: Abbreviations and Specialist Terms .............................................................................6 |
Table 2: Test Articles Used in Study ALPN100-PHRM-001 .....................................................9 |
Table 3: Test Materials Used in Study ALPN100-PHRM-001 ..................................................9 |
Table 4: Analysis Tools Used in Study ALPN100-PHRM-001 ...............................................11 |
Table 5: ALPN-101 Binding Affinity (EC50) to CD28, ICOS, and CTLA-4 Across Species Assessed by Flow Cytometric Analysis ........................................................16 |
Table 6: ALPN-101 Demonstrates Superior Binding to CD28 Compared to that of WT ICOSL-Fc1.1 .......................................................................................................17 |
Table 7: ALPN-101 KD, ka, kd, Errors, and Full R2 Measured by BLI Analysis.....................18 |
Table 8: ALPN-101 Flow Cytometric Raw Binding Values (MFI): Human Counter Structures ....................................................................................................................25 |
Table 9: ALPN-101 Flow Cytometric Raw Binding Values (MFI): Cynomolgus Counter Structures ....................................................................................................................26 |
Table 10: ALPN-101 Flow Cytometric Raw Binding Values (MFI): Rat Counter Structures ....................................................................................................................27 |
Table 11: ALPN-101 Flow Cytometric Raw Binding Values (MFI): Mouse Counter Structures ....................................................................................................................28 |
Table 12: ALPN-101 Flow Cytometric Raw Binding Values (MFI): Mock Transfectants ...............................................................................................................29 |
Table 13: ALPN-101 BLI Binding Responses for Each Replicate in the Titrations ..................30 |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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LIST OF FIGURES Figure 1: Flow Cytometric Analysis Gating Strategy (A) and Representative Histograms of ALPN-101 Binding to Human Counter Structures (B) .......................14 |
Figure 2: ALPN-101 Binding to Human, Cynomolgus Monkey, Rat, and Mouse CD28, ICOS, and CTLA-4 .........................................................................................15 |
Figure 3: General Description of Binding Sensorgrams ............................................................19 |
Figure 4: CD28 Binding Sensorgram .........................................................................................19 |
Figure 5: ICOS Binding Sensorgram .........................................................................................20 |
Figure 6: CTLA-4 Binding Sensorgram .....................................................................................20 |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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2. LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS |
Table 1: Abbreviations and Specialist Terms |
Abbreviation or specialist term Explanation |
AIS Alpine Immune Sciences, Inc. |
ALPN-101 ICOSL vIgD 236 IgV (DG44)-Fc1.1 or ICOSL vIgD-Fc1.1 |
ADCC Antibody-dependent cellular cytotoxicity |
BLI Bio-Layer Interferometry (e.g. using the Forté Bio™ Octet system) |
BSA Bovine serum albumin |
CD28 Cluster of Differentiation 28 |
CDC Complement-dependent cytotoxicity |
CTLA-4 Cytotoxic T lymphocyte-associated antigen 4 |
EC50 Half maximal effective concentration |
Expi239FTM Subline of HEK 293, human embryonic kidney cell line |
EDTA Ethylenediaminetetraacetic acid |
Fc Fragment crystallizable region of immunoglobulin |
Fc1.1 Fragment crystallizable region of human immunoglobulin IgG1, engineered to lack binding to Fc receptors or complement ("effectorless") |
FSC Forward scatter |
GLP Good Laboratory Practice |
ICOS Inducible T cell costimulator |
ICOSL Inducible T cell costimulator ligand |
IgV Immunoglobulin-like variable domain |
KBI KBI Biopharma (contract manufacturing site) |
KD Equilibrium dissociation constant (= kd/ka) |
kd Disassociation constant, off-rate constant |
ka Association constant, on-rate constant |
MFI Mean fluorescence intensity |
min Minute(s) |
NaN3 Sodium azide |
SSC Side scatter |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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3. ABSTRACT |
ALPN-101 [ICOSL vIgD 236 IgV (DG44)-Fc1.1] is an Fc fusion protein of a human inducible T cell costimulator ligand (ICOSL) variant immunoglobulin domain (vIgD™) designed to inhibit simultaneously the CD28 and ICOS T cell costimulatory pathways (ALPN100-CMC-002). This study determined binding by flow cytometry of ALPN-1... |
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... |
As assessed by the ForteBio Octet system, ALPN-101 bound recombinant soluble human CD28, ICOS, and CTLA-4 with KD values of 192, 707, and 241 pM, respectively. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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4. STUDY RATIONALE AND OBJECTIVES |
ALPN-101 is an Fc fusion protein containing a variable immunoglobulin domain (vIgDTM) of ICOSL engineered to have increased affinity for both ICOS and CD28, designed to provide dual inhibition costimulatory signaling (ALPN100-CMC-002). |
ALPN-101 is designed to bind counter-structures ICOSL, CD28 and CTLA4. The objective of this study was to determine binding activity of ALPN-101 to its counter-structures: |
1. By flow cytometric analysis of ALPN-101 binding to cells transduced and expressing CD28, CTLA-4, and ICOS based on sequences derived from species relevant for preclinical and clinical development: human (Homo sapiens), cynomolgus monkey (Macaca fascicularis), rat (Rattus norvegicus), and mouse (Mus musculus) (Rixon,... |
2. Via BLI analysis using the Forté Bio™ Octet system to determine affinity (KD) of ALPN-101 for human recombinant CD28, CTLA-4, and ICOS. |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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5. STUDY MATERIALS |
5.1. Test Articles |
The test articles used in Study ALPN100-PHRM-001 are listed in Table 2. |
Table 2: Test Articles Used in Study ALPN100-PHRM-001 |
Item Source Catalog/Lot No. |
ALPN-101 [ICOSL vIgD 236 IgV (DG44)-Fc1.1] or ICOSL vIgD-Fc1.1 KBI Biopharma, Inc. Lot# 622 p23A (used for flow cytometry analyses) |
ALPN-101 [ICOSL vIgD 236 IgV (DG44)-Fc1.1] or ICOSL vIgD-Fc1.1 KBI Biopharma, Inc. Lot# PDB00242 (used for BLI analyses) |
WT ICOSL-Fc1.1 [WT ICOSL ECD-Fc1.1] AIS N10229 |
Fc1.1 control (see ALPN100-CMC-001) AIS N10118.3 |
ZLZ (ICOS WT ECD) FLAG His AIS P1011 |
ZLZ (CD28 WT ECD) FLAG His AIS P1012 |
(CTLA4 WT ECD) GSS His6 AIS A145 |
5.2. Test Materials |
The test materials used in Study ALPN100-PHRM-001 are listed in Table 3. |
Table 3: Test Materials Used in Study ALPN100-PHRM-001 |
Item Source Catalog/Lot No. |
Expi293F™ Cells Gibco A14527 |
Expifectamine 293 transfection kit Gibco A14524 |
Expi293 expression media Gibco A14351-01 |
Opti-MEM Gibco 31985-010 |
PBS Gibco 14190-136 |
Poloxamer 188/Pluronic Acid Corning 13-901-Cl |
24 well plates Thomson Instrument Co. 931568 |
R-Phycoerythrin AffiniPure F(ab') Fragment Goat Anti-Human IgG, Fc Fragment Specific Jackson ImmunoResearch 109-116-098 |
pcDNA3.1 Plasmid Expression Vector ThermoFisher Sciences V79020 |
Pharmacology Study Report Alpine Immune Sciences, Inc. ALPN100-PHRM-001 |
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Table 3: Test Materials Used in Study ALPN100-PHRM-001 (Continued) |
Item Source Catalog/Lot No. |
PAIS47_CD28 (HUMAN) AIS N/A |
PAIS195_ICOS_CD4_TM (HUMAN) AIS N/A |
PAIS50_CTLA4 (HUMAN) AIS N/A |
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