key: cord-1040475-sam88wrq authors: Shin, Jong; Phelan, Paul J.; Gjoerup, Ole; Bachovchin, William; Bullock, Peter A. title: Characterization of A Single Chain variable fragment of Nivolumab that targets PD-1 and Blocks PD-L1 Binding date: 2020-09-25 journal: Protein Expr Purif DOI: 10.1016/j.pep.2020.105766 sha: 4611f7f39b6089cc4d4afb51eb484d519dfaca8b doc_id: 1040475 cord_uid: sam88wrq Activated T-cells express Programmed cell Death protein 1 (PD-1), a key immune checkpoint receptor. PD-1 functions primarily in peripheral tissues, where T cells may encounter tumor-derived immunosuppressive ligands. Monoclonal antibodies that disrupt the interaction between T-cell derived PD-1 and immunosuppressive ligands, such as PD-L1, have revolutionized approaches to cancer therapy. For instance, Nivolumab is a monoclonal Ab that targets human PD-1 and has played an important role in immune checkpoint therapy. Herein we report the purification and initial characterization of a ∼ 27 kDa single chain variable fragment (scFv) of Nivolumab that targets human PD-1 and blocks binding by PD-L1. The possibility that the anti-PD-1 scFv can serve as both an anti-tumor agent and as an anti-viral agent is discussed. IMPORTANCE: The clinical significance of anti-PD-1 antibodies for treatment of a range of solid tumors is well documented (reviewed in [1-4]). In this report, we describe the results of studies that establish that an anti-PD-1 scFv purified from E. coli binds tightly to human PD-1. Furthermore, we demonstrate that upon binding, the anti-PD-1 scFv disrupts the interaction between PD-1 and PD-L1. Thus, the properties of this scFv, including its small size, stability and affinity for human PD-1, suggest that it has the potential to be a useful reagent in subsequent immunotherapeutic, diagnostic and anti-viral applications. Activation of "immune checkpoints" is a fundamental event that limits collateral tissue 69 damage during T cell responses to infection (reviewed in [5] [6] [7] ). However, activation of 70 immune checkpoints also enables cancer cells to avoid destruction by the human immune A seminal discovery in the immunotherapy field was that monoclonal antibodies that 86 bind PD-1, and thereby disrupt PD-1's interactions with ligands such as PD-L1, promote 87 T cell proliferation and activation (e.g., [17] ; reviewed in [4, 18, 19] ). This led to the 88 observation that broad-spectrum antitumor activity can result from antibody-based 89 inhibition of the interaction between PD1 and its ligands (e.g., [3, 5, 8, 16, 20] ). Anti-PD1 90 J o u r n a l P r e -p r o o f 8.0, 300 mM NaCl, 10 % glycerol, 20 mM imidazole and 0.02 % Tween 80). The bound 160 anti-PD-1 scFv was then eluted with 5 ml of "Elution buffer" (20 mM Tris.HCl pH 8.0, 0.3 161 M NaCl, 10 % glycerol, 0.02 % Tween 80 and 300 mM imidazole) and ten 0.5 ml fractions 162 were collected. To immediately lower the pH away from the pI of the scFv (~8. 65 Na citrate dihydrate and 20 % glycerol). The purified anti-PD-1 scFv (~0.2 mg/ml) was 171 stored at -80 0 C until needed. Regarding the determination of the concentration of the 172 purified anti-PD-1 scFv; using the ExPASy portal, the extinction coefficient for the 6X His 173 containing anti-PD-1 scFv at 280 nm was determined to be 51,130 M -1 cm -1 . After 174 obtaining the spectra of the purified anti-PD-1 on an Agilent 8453 UV-visible 175 spectrophotometer, and subtracting the spectra of the Storage buffer, the concentration of 176 anti-PD-1 scFv samples was determined using Beer's Law. The accuracy of this 177 approach was confirmed by SDS-PAGE of aliquots of the purified anti-PD-1 scFv and 178 subsequent quantitation using ImageJ. The internal alignment function of PyMol was then used to align the predicted structure of 185 the anti-PD-1 scFv with the corresponding region of Nivolumab (calculation of alignment 186 was placed with a restriction to alpha carbons of the backbone of the proteins). 187 C. A molecular model demonstrating that the anti-PD-1 scFv bound to PD-1 will block 188 binding to PD-L1. The residues on PD-1 that bind to the Fab region of nivolumab [40, 41] 189 and those that bind to PD-L1 [42] , were previously reported. The anti-PD-1 scFv was 190 positioned opposite the nivolumab-binding site via standard procedures. In brief, starting 191 with the nivolumab/ PD-1 co-structure [40], the predicted structure of the anti-PD-1 scFv 192 (described herein) was aligned to nivolumab using the alignment function of PyMol [43] . 193 D. Western blotting: Human PD-1 was purchased from ACROBiosystems and the 194 indicated amounts were separated by SDS-PAGE on a 10 % acrylamide gel. The PD-1 195 protein, and BSA control, was then transferred to a PVDF membrane (Millipore). After 196 overnight blocking at 4 0 C with 5 % dry milk in TBST (20 mM Tris (pH 7.5), 137 mM NaCl 197 and 0.1 % Tween-20) the membrane was incubated for 1 hr at RT with the purified anti-198 PD-1 scFv (0.25 ug/ml) in ~ 20 ml of TBST containing 2 % dry milk. The 6xHis tag on the 199 scFv was subsequently detected using a horseradish peroxidase-conjugated anti-200 polyhistidine monoclonal antibody ((Abcam: ab49781 diluted 1:2500) and the blot was 201 developed using an enhanced chemiluminescence detection kit (Millipore). 202 E. Determining the IC 50 of the anti-PD-1 scFv for PD-1: BPS Bioscience has developed 203 a FRET-based high-throughput assay that measures the effectiveness of molecules at 204 inhibiting the PD-1/ PD-L1 interaction http://bpsbioscience.com/pd-1-pd-l1-tr-fret-72038. A. The antigen-binding site on the anti-PD-1 scFv: Figure 4A presents a space-filling 276 model of the anti-PD-1 scFv that depicts both the predicted antigen-binding region (shown 277 in cyan) and residues in nivolumab (shown in teal) that are known to interact with PD-1 278 [40, 41]. Importantly, residues derived from the (Gly 4 S) 3 linker are not predicted to be in a 279 position to obscure the residues used to bind to PD-1. However, given the previously 280 discussed uncertainties regarding the location(s) of the N-terminal 6xHis and TEV 281 protease regions, these N-terminal sequences might be situated on the surface of the 282 anti-PD-1 scFv in a manner that could interfere with binding to PD-1. Therefore, to 283 eliminate this possibility, and to confirm the hypothesis that we had identified the amino 284 acid residues needed for a functional anti-PD-1 scFv, we tested if the 6xHis and TEV 285 protease site-containing anti-PD-1 scFv binds to purified PD-1. 1 with lanes 3-4) . Therefore, it was concluded that the anti-PD-1 293 scFv is active and that the N-terminal 6xHis and TEV protease regions do not obscure 294 residues needed for binding to PD-1. Finally, it is noted that the full-length human PD1 295 from ACROBiosystems is heavily glycosylated; therefore, this molecule does not run as a 296 distinct species. In terms of the mechanism utilized by the anti-PD-1 scFv to disrupt the PD-1/ PD-L1 375 interaction, the purified anti-PD-1 scFv has an IC50 of 26 nM, while the IC50 of full-length 376 nivolumab is 2.52 nM (as determined by surface plasmon resonance [72]). Thus, the 377 IC50s, obtained using two separate methods, are not identical. Whether this difference is 378 because the anti-PD-1 scFv is actually 10-fold weaker than full-length nivolumab, or if 379 only 10% of the scFv molecules are active, remains to be determined. Nevertheless, the 380 tight binding of the anti-PD-1 scFv to PD-1 is in keeping with previous experiments that 381 demonstrated that scFv fragments that contain the complete antigen-binding site of an 382 antibody, and have not undergone reorientation of the two domains, have the same 383 monomeric binding affinity as the parental monoclonal antibody [73] . Furthermore, based 384 on the design of the BPS Bioscience FRET-based assay, it is apparent that once tightly 385 bound to PD-1, the anti-PD-1 scFv blocks the ability of PD-L1 to interact with PD-1. functions (including targeted cell lysis and phagocytosis (e.g., [79, 80] An additional concern is that checkpoint therapies utilizing full-length antibodies scFv to PD-1, using the residues needed to bind Nivolumab, it is apparent that the V L 869 region of the anti-PD-1 scFv (in red) would obscure the PD-1 residues needed for binding 870 to PD-L1. 871 Structure of the complex of human 593 programmed death 1, PD-1, and its ligand PD-L1 Protein structure prediction on the web: A 596 case study using the Phyre server His-598 tag impact on structure Structural basis of 601 checkpoint blockade by monoclonal antibodies in cancer immunotherapy Features and 604 development of Coot Structural basis for small molecule targeting of the 606 Programmed death ligand 1 (PD-L1) A molecular and 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cysteine in VH: consequences for thermodynamic stability and folding The V H sequences used to form the anti-PD-1 scFv 783 are in blue, while the V L sequences are in red. The two pairs of cysteines used to form the 784 intradomain disulfides (i.e., Cys 23 & 88 in V L and Cys 22 and 96 in V H ) that are critical for 785 the stability of scFv fragments [97] are highlighted. A2. A diagram of the V L and V H 786 containing anti-PD-1 scFv; the (Gly 4 S) 3 linker is symbolized by the yellow line. B1. A 787 schematic of the region of plasmid pLIC-His-anti-PD-1 that encodes the anti-PD-1 scFv 788 including the 6X His, TEV cleavage, V H , (Gly 4 S) 3 linker and V L sites. B2. An expanded 789 view showing the DNA sequences from the 6X His (in purple) and TEV cleavage sites Met that establishes the N-terminus of the anti-PD-1 scFv. Finally, presented in blue are 792 the first two amino acids from the V H domain of Nivolumab (and the corresponding DNA 793 sequence) Protein induction and purification of the anti-PD-1 scFv from E. coli BL21 cells Representative SDS-PAGE gel showing the distribution of the anti-PD-1 scFv during 797 stages of purification. Pre-stained "SeeBlue Plus 2" protein size markers were run in Lane 1. The whole cell lysate following induction with IPTG (20 ul) is shown in 799 Lane 2; a key feature is the prominent band running at the expected location of the anti-800 Lane 3, the lysate supernatant (20 ul) following centrifugation at 801 18,000 RPM. Lane 4, the cell pellet following solubilization in Buffer B (20 ul). Lane 5, an 802 aliquot of the loaded Ni-NTA resin (~5 ul) after in situ renaturation of the anti-PD-1 scFv 803 and washing with ten column volumes of low imidazole The peak fractions of the anti-PD-1 scFv were identified by 806 spotting aliquots onto Whatman filter paper and staining with Coomassie Brilliant Blue, as 807 well as subsequent analyses of 20 ul aliquots by SDS-Page (lanes 6-8). Finally, our yield 808 of purified anti-PD-1 scFv was lower than expected (i.e., ~ 1 mg from 2.5 grams of 809 bacterial cell pellet). It is noted that during elution with 300 mM imidazole not all of the 810 anti-PD-1 scFv was removed from the Ni-NTA column While we have yet to determine why these purification steps are sub-optimal, a likely 813 contributor is insoluble aggregation of some fraction of the scFv