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 preclinical 608 comparison of the PD-1-targeted T-cell checkpoint inhibitors nivolumab and 609 pembrolizumab

New checkpoint inhibitors ride the immunotherapy tsunami

PD-1 and its ligands are important immune 613 checkpoints in cancer

Nivolumab and Ipilimumab versus

Nivolumab as Programmed Death-1

Inhibitor for Targeted Immunotherapy in Tumor

Combined Nivolumab and Ipilimumab or Monotherapy in 623

Top 10 Challenges in Cancer Immunotherapy

Pharmacokinetics and biodistribution of genetically-engineered 628 antibodies

Recombinant antibody constructs in cancer therapy

Structural basis of anti-PD-633 L1 monoclonal antibody avelumab for tumor therapy

Production and 636 Evaluation of Specific Single-Chain Antibodies against CTLA-4 for Cancer-637

Targeted delivery of a PD-1-641 blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo

antibodies that function in checkpoint blockade, discovered using microfluidics 646 and molecular genomics

Restoring function in exhausted CD8 T cells during 649 chronic viral infection

PD-1/PD-L1 pathway and T-cell 651 exhaustion in chronic hepatitis virus invfection

PD-1 expression on HIV-653 specific T cells is associated withT-cell exhaustion and disease progression

The PD-1/PD-L1 Axis and Virus Infecdtions: A 656

Delicate Balance

Checkpoint inhibitors for the treatment of JC virus-659 related progressive multifocal leukoencephalopathy

Controversies about COVID-19 and anticancer treatment 662 with immune checkpoint inhibitors

PD-1 Blockade with Pembrolizumab in Advanced 664

Durable 668 Tumor Regression and Overall Survival in Patients with

Carcinoma Receiving Pembrolizumab as First-Line Therapy

Metastatic Merkel Cell Carcinoma Response to Nivolumab

Anelumab in patients with chemotherapy-refractory

metastatic Merkel cell carcinoma: a multicentre, single-group, open-label, phase 677 2 trial

Vitro 686 Characterization of the Anti-PD-1 Antibody Nivolumab, BMS-936558, and In Vivo

Toxicology in Non-Human Primates

F(ab')2 693 molecules made from Escherichia coli produced Fab' with hinge sequences 694 conferring increased serum survival in an animal model

Pharmacodynamic enhancement of the anti-platelet

antibody fab abciximab by site-specific pegylation

Dosimetric evaluation and radioimmunotherapy of anti-tumor multivalent 700

Fab fragments

Engineered antibodies

Specificity and affinity of human Fc gamma receptors and 705 their polymorphic variants for human IgG subclasses

Trastuzumab causes 709 antibody-dependent cellular cytotoxicity-mediated growth inhibition of 710 submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug 711 resistance

Antibody therapeutics: isotype and glycoform selection

Structure of full-length human anti-PD1 therapeutic 716

IgG4 antibody pembrolizumab

In vivo imaging reveals a tumor-associated macrophage-720 mediated resistance pathway in anti-PD-1 therapy

Balance and imbalance in the immune 723 system: life on the edge

Immune dysregulation in 726 human subjects with heterozygous germline mutations in CTLA4

Immune-related adverse events with 730 immune checkpoint blockade: a comprehensive review

Therapeutic uses of anti-PD-1 and anti-PD-L1

Brahmer, a. et., Safety, Activity and Immune 735 Correlates of Anti-PD-1 Antibody in Cancer

Nivolumab plus

Ipilmumab in advanced melanoma

Stability 742 engineering of scFvs for the development of bispecific and multivalent antibodies

Bifunctional PD-1 X alphaCD3 X alphaCD33 747 fusion protein reverses adaptive immune escape in acute myeloid leukemia

Targeting Human-Cytomegalovirus

Infected Cells by Redirecting T Cells Using an Anti-CD3/Anti-Glycoproteing B 752

Bispecific antibodies for viral 755 immunotherapy

Designer antibodies fight cancer by tethering immune cells to 757 tumor cells

Whole body PD-1 and PD-L1 positron emission tomography in patients 763 with non-small-cell lung cancer

A 768 HER2 bispecific antibody can be efficiently expressed in Escherichia coli with 769 potent cytotoxicity

A natural antibody missing a 771 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