Engineering Bifunctional Antibodies with Constant ... - ACS Publications

Subscriber access provided by Queen Mary, University of London

Article

Engineering Bifunctional Antibodies with Constant Region Fusion Architectures Juanjuan Du, Yu Cao, Yan Liu, Ying Wang, yong Zhang, Guangsen Fu, Yuhan Zhang, Lucy Lu, Xiaozhou Luo, Chan Hyuk Kim, Peter G Schultz, and Feng Wang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09641 • Publication Date (Web): 29 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 10

Engineering Bifunctional Antibodies with Constant Region Fusion Architectures Juanjuan Du†‡, Yu Cao‡, Yan Liu†, Ying Wang†, Yong Zhang†, Guangsen Fu†, Yuhan Zhang†, Lucy Lu†, Xiaozhou Luo‡, Chan Hyuk Kim†, Peter G. Schultz†‡, Feng Wang† † California Institute for Biomedical Research, 11119 N. Torrey Pines Road, La Jolla, CA 92037, United States ‡ Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, United States KEYWORDS Antibody fusion, protein engineering, bispecific antibodies, cancer immunotherapy ABSTRACT: We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies have excellent in-vitro activity, physiochemical properties, and stability. Among these, a Her2×CD3 bispecific antibody (BsAb) was constructed by inserting an anti-Her2 ScFv into an anti-CD3 Fab. This bispecific antibody efficiently induces targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations in vitro. Moreover, the Her2 ×CD3 BsAb shows potent in-vivo antitumor activity in mouse Her22+ and Her21+ xenograft models. These results demonstrate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multi-functional antibodies for therapeutic applications.

Previously, we developed a strategy to generate antibody agonists and antagonists by fusing biologically active proteins and peptides into antibody hypervariable loops based on the X-ray crystal structure of the bovine antibody BLV1H12 which has an ultralong heavy chain complementarity-determining region 3 (CDR3H). The hypervariable loop folds into a novel structural motif,18 consisting of a solvent-exposed antiparallel β-strand stalk which terminates in a disulfide-crosslinked knob domain to afford a unique “stalk-knob” structure. On the basis of

this novel structure, we have grafted a number of cytokines, growth factors, and conformationally restricted peptides into the CDR loops of bovine and human immunoglobulins to generate functional antibody-CDR fusions.19-27 The immunoglobulin scaffold affords long serum half-life and ease of recombinant protein production, while the fused polypeptide endows the antibody with new agonist, antagonist or inhibitory activities. Insert Protein

Insert Protein

Linker

Linker

CDR1 CDR2

CDR3

N

C

F

G

A

B

E

D

C''

C'

C

A2

F

G

A1

N

B

Monoclonal antibodies are increasingly important therapeutics due to their high affinity and specificity, long circulating half-life, and low immunogenicity.1-3 A new generation of engineered antibodies, including bispecific antibodies (BsAb) and antibody conjugates, have given rise to antibodies with dual functions.1-6 Since the pioneering work of Dr. Nisonoff,7 a plethora of methods to constructs bispecific antibodies have emerged in the past fifty years, including Ig-fusion,8 quadromas,9 diabodies,10 tandem ScFvs,11,12 DART,13 knobs-into-holes,14 DVD-Ig,15 etc.16,17 With over 30 different bifunctional antibodies in clinical trial, it has become increasingly evident that each format has its own limitation. The BsAb format is best chosen to match the specific mechanism of action. Expanding the format arsenal thus facilitates the BsAb development. Therefore, exploring more and novel BsAb formats remains on the cusp of coming years.

E

Introduction

D

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

C

C Variable Domain D F

β Strands Consisting Two β Sheets

Constant Domain Loop Linker

Insert Protein Disulfide Bond

Figure 1. The topologies of CDR3 fusion in variable region and non-CDR loop fusion in constant region. Diagrams show the protein insertion sites and the strand nomenclature for the β-sheets of IgG variable domains and constant domains.

Similar to the antibody variable domain, the constant domains also have the characteristic immunoglobulin fold

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(Figure 1).28 This fold consists of two β sheets, formed from antiparallel β strands that surround a central hydrophobic core. Based on the structural similarities between the antibody variable and constant domains, we reasoned that our CDR loop fusion strategy could be extended to the non-CDR loops of the latter domain. In contrast to a typical CDR loop fusion, constant region loop fusions are expected to retain the binding affinity and specificity of the parent scaffold, resulting in a bifunctional antibody with the additional activity of the grafted polypeptide. In this work, we show that non-CDR loops are able to accommodate a variety of biologically active protein inserts. Results Full-length erythropoietin can be grafted in the non-CDR loops of an anti-Her2 antibody. We initially chose to fuse human erythropoietin (EPO) into an antiHer2 antibody (Herceptin) to explore different non-CDR loops as fusion sites (Figure 2a). Previously, we have fused EPO into the CDR3H loop of Herceptin to generate a stable antibody fusion protein (EPO-Herceptin-CDR3H). The fusion protein retained the biological activity of EPO and gained a long serum half-life.22,27 Fusing EPO into non-CDR loops in the constant domain of Herceptin allows a direct comparison of the ability of variable and constant domains to accommodate loop fusions. To avoid interference with Fc receptor binding, we grafted EPO into loops between the β strands D and E either in the

Page 2 of 10

CH1 (replacing S182 and G183 at the splice site) or CL (replacing K169 at the splice site) regions (Figure 2b). To spatially separate the EPO insert and Herceptin backbone, an anti-parallel coiled-coil “stalk” (14 amino acids in each chain) was used as a rigid linker to connect EPO and Herceptin (Figure 2b). GGSG and GGGGS adapters were placed at each end of the coiled-coil sequences to afford flexibility (Table S1). A similar linker strategy was previously used successfully to fuse EPO, Granulocyte-colony stimulating factor (GCSF) and Exendin-4 into the CDR loops of human and bovine antibodies.1-3,22-25,29 The distance between the N- and C-termini of EPO (~15.8 Å, PDB# 1BUY) is close to the axial distance between the two coiled-coil chains in the stalk (~11 Å, Figure S1). Thus, fusion of the N- and C-termini of EPO with the coiledcoil“stalk”should not interfere with folding of either the antibody or EPO. Moreover, because the receptor-binding surface of EPO is on the opposite face to the fusion site, the fusion protein should retain EPO receptor (EpoR) binding. The two fusion constructs (hereafter referred to as EPO-Herceptin-CL and EPO-Herceptin-CH1) were expressed in Free-Style HEK293 cells by transient transfection and purified using protein G chromatography. The unoptimized expression yields of EPO-Herceptin-CL and EPO-Herceptin-CH1 after purification are 13 mg/L and 5 mg/L, respectively.

Figure 2. Design, generation, and characterization of EPO-Herceptin constant domain fusions. (a) Model of the EPO-Herceptin constant domain fusion. (b) Map of the key elements of EPO-Herceptin fusions: numbers indicate the fusion sites in the Kabat numbering scheme. (c) Dose-dependent TF-1 proliferation stimulated by EPO-Herceptin-CL, EPO-Herceptin-CH1 and EPOHerceptin-CDR3H (positive control). (d) Binding of EPO-Herceptin-CL, EPO-Herceptin-CH1 and Herceptin (positive control) against immobilized Her2-Fc antigen measured by ELISA.

The proteins were then analyzed with SDS-PAGE (Figure S2). Under non-reducing conditions, Herceptin has an apparent molecular weight of ~160 kDa, higher than the calculated mass (145 kDa) due to glycosylation. EPOHerceptin-CH1 (Lane 2) and EPO-Herceptin-CL (Lane 3) migrate at ~200 kDa, higher than the theoretical mass of 189 kDa, again due to glycosylation. Under reducing con-

ditions, the light chain of EPO-Herceptin-CL migrates at ~55 kDa, higher than the theoretical molecular weight of 45.6 kDa due to glycosylation. The heavy chain of EPOHerceptin-CL migrates at ~50 kDa, consistent with the expected molecular weight of 49.1 kDa. Similarly, EPOHerceptin-CH1 shows a heavy chain of ~80 kDa and a light chain of ~ 23 kDa, consistent with the theoretical


Page 3 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


molecular weights of 71.3 kDa plus glycans and 23.5 kDa, respectively. In addition, after treatment with peptide-Nglycosidase to remove N-glycosylation and dithiothreitol (DTT), the molecular weights of the heavy chains and light chains of Herceptin, EPO-Herceptin-CL and EPOHerceptin-CH1 were measured by mass spectrometry. As shown in Figure S3 and Figure S4, the mass spectrum of EPO-Herceptin-CH1 heavy chain shows multiple peaks with two major peaks of 71138 Da (22020 Da higher than the Herceptin chain, consistent with the expected molecular weight increase of 22016 Da) and 72086 Da (multiple peaks, due to O-glycosylation). The molecular weights of the light chains of Herceptin and EPO-Herceptin-CH1 are the same (23968 Da) and consistent with the theoretical mass (23972 Da). Similarly, compared with Herceptin (Figure S3), EPO-Herceptin-CL shows the same heavy chain molecular weight (Figure S5), and a molecular weight gain of 22185 Da (non-glycosylated form, theoretical molecular increase: 22184 Da) in light chain, indicating one EPO is fused in each light chain. Gel filtration analyses in PBS (pH 7.4) indicated that EPO-Herceptin-CL and EPO-Herceptin-CH1 both have apparent molecular weights of ~200 kDa, as expected. Additionally, aggregation was determined by size exclusion chromatography, in which EPO-Herceptin-CL (8.2 mg/mL) or EPOHerceptin-CH1 (10.5 mg/mL) was loaded. Integrated UV absorbance peaks at 280 nm indicate that less than 5% protein forms aggregation in both samples from protein G purification (Figure S6). EPO-Herceptin non-CDR loop fusions retain the activities of both the insert and the scaffold. We next examined the activity of EPO-Herceptin-CL and EPOHerceptin-CH1 using an EPO-dependent TF-1 proliferation assay. EPO-Herceptin CDR3H loop fusion (EPOHerceptin-CDR3H) is used as a positive control to compare non-CDR fusion and CDR fusion.1-6,22,27 TF-1 cells are human bone marrow erythroblasts, which have a strong growth dependency on EPO. Both EPO-Herceptin-CL and EPO-Herceptin-CH1 stimulate TF-1 cell proliferation in a dose-dependent manner (Figure 2c). The EC50 is 0.16±0.01 nM for EPO-Herceptin-CL, 0.30±0.02 nM for EPOHerceptin-CH1 and 0.21±0.02 nM for EPO-HerceptinCDR3H, similar to the EC50 of recombinant EPO (0.13±0.02 nM). These observations indicate that the fusion of EPO into non-CDR loops of constant domains does not significantly affect EPO activity, similar to fusion into CDR3H loop.18,22 To test whether non-CDR loop fusion affects the antigen binding affinity of the antibody variable domain, we carried out an ELISA against immobilized Her2 extracellular domain fused with Fc (Her2-Fc) (Figure 2d). Both EPO-Herceptin-CH1 and EPO-Herceptin-CL bind to Her2-Fc in a dose-dependent manner, with EC50 of 2.4±0.2 nM and 1.2±0.1 nM, respectively, which are comparable to that of Herceptin (1.9±0.1 nM). In addition, flow cytometry analysis shows that both EPO-HerceptinCL and EPO-Herceptin-CH1 bind to Her2+ SK-BR-3 cells with similar affinities (EC50 = 7.5±4.3 nM and 11.0±2.3 nM, respectively) to that of the parental antibody Herceptin

(EC50 = 15.3±4.0 nM) (Figure S7). These results demonstrate that the antibody binding affinity is not significantly affected by the non-CDR loop fusion. Collectively, these observations demonstrate grafting of a functional protein in non-CDR loops in constant region can generate fusion proteins retaining both the biological activity of the insert and the antigen-binding affinity of the scaffold antibody. Her2×CD3 bispecific antibodies can be generated by fusing an anti-Her2 ScFv into the non-CDR loops of an anti-CD3 antibody. Next we determined whether an antibody single-chain variable fragment (ScFv) could be grafted into the loops of constant domains. This strategy would enable us to generate BsAbs targeting cancer cells using for example, an antibody fragment specific for tumor associated antigens, and a parent antibody scaffold specific for CD3 on T cells. As shown in Figure 3a, a Herceptin ScFv (hereafter referred to as Her2ScFv) was inserted into the loop connecting β strands D and E (to replace K169) in the CL domain of a humanized anti-CD3 antibody (SP34). We opted to fuse Her2ScFv into SP34Fab instead of fusing SP34 ScFv into Herceptin-Fab, because the properties of Her2ScFv are well documented.1927,30,31 We chose the CL domain as the insertion site due to higher expression yield of CL domain fusion in the case of EPO-Herceptin fusion. In contrast to EPO, whose N- and C-termini are located in close proximity to each other, the N- and C-termini of Her2ScFv are separated by approximately 34 Å (Figure S8). To ensure a correct folding of the fusion protein, a long flexible linker (GGGGS)3 was used between the stalk and the N-terminus of the Her2ScFv (Figure 3a). Additionally, it was previously demonstrated that an extra disulfide bond at the termini or in the middle of coiled coils increases the stability of synthetic coiled-coil structures.28,32-34 Therefore, in our design, a disulfide bond was introduced at the site where the coiled-coil stalk connects to SP34 CL domain to enhance stability (Table S1). The Her2×CD3 BsAb (referred to as Her2ScFv-SP34-CL) was expressed in HEK293 FreeStyle system by co-transfection with plasmids encoding the fused light chain and the heavy chain (Fab) of SP34. After purification with protein G and size exclusion chromatography, we obtained Her2ScFv-SP34-CL with a yield of 12 mg/L. Gel filtration analysis of Her2ScFv-SP34-CL (2 mg/ml) in PBS showed that around 83% species purified from protein G chromatography corresponds to the monomer (Figure S9). The isolated fraction was then reanalyzed by analytical size-exclusion chromatography, which showed a single peak with molecular weight corresponding to the monomeric Her2ScFv-SP34-CL (Figure S9). This indicates that the purified monomer is stable in solution without formation of observable dimers or oligomers. Figure S10 shows the SDS-PAGE of SP34 Fab (Lane 1) and Her2ScFv-SP34-CL (Lane 2). Compared with SP34, which migrates at ~45 kDa under non-reducing condition, Her2ScFv-SP34-CL has an apparent molecular weight of ~75 kDa on SDS-PAGE. The 30 kDa molecular weight increase is consistent with the molecular weight of the ScFv insert (29 kDa). Under reducing condition, the heavy



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

chains of SP34 Fab and Her2ScFv-SP34-CL migrate at the same position (~23 kDa). The light chain of Her2ScFvSP34-CL migrates at ~54 kDa, approximately ~30 kDa higher than the light chain of SP34 Fab. Additionally, after reducing with DTT, two peaks with molecular weights of 24528 Da and 54252 Da were observed in the mass spectra, consistent with the theoretical masses of 24532 Da (heavy chain) and 54277 Da (light chain) (Figure S11). It is worth noting that ScFv fusion proteins, including the Bi-specific T-cell engager (BiTE) developed by Micromet/Amgen, tend to form non-specific oligomers/aggregations. A large portion of these oligomers are in equilibrium with monomers in solution and therefore difficult to separate, which cause serious manufacture and toxicity issues.22,27,35 Dynamic light scattering (DLS) was used to evaluate the aggregation in concentrated Her2ScFv-SP34-CL. As shown in Figure S12, a single fraction with hydrodynamic radius of ~10 nm was observed at 10 mg/ml protein concentration, indicating that no aggregation was formed. To determine the thermostability of Her2ScFv-SP34-CL, the melting temperature (TM) was measured using the Protein Thermal Shift™ Dye Kit (ThermoFisher Scientific). The melting curve indicates two TMs at 70 and 79 °C, likely corresponding to the unfolding of ScFv and Fab domains, respectively (Figure S13). Her2ScFv-SP34-CL fusion protein retains the binding affinities of the anti-Her2 ScFv insert and the anti-CD3 Fab fragment. The binding affinity of Her2ScFvSP34-CL to CD3 was measured by flow cytometry on CD3+ Jurkat cells (Figure 3b). SP34 Fab was used as a positive control. Similar to SP34 Fab, Her2ScFv-SP34-CL binds to Jurkat cells in a dose-dependent manner. Her2ScFv-SP34-CL has an EC50 of 8.7±1.0 nM, slightly higher than the EC50 of SP34 Fab (3.8±0.4 nM). The binding affinity of Her2ScFv-SP34-CL to Her2 was next measured by ELISA on immobilized Her2-Fc (Figure 3c). Compared with Herceptin Fab (EC50 = 0.9±0.1 nM), Her2ScFvSP34-CL binds to Her2-Fc with an EC50 of 6.3±0.7 nM. ScFv fragments reportedly have lower binding affinities than the corresponding Fab. Nevertheless, the EC50s to CD3 and Her2 still remain in the nanomolar range.

Figure 3. Design, generation, and characterization of the Her2×CD3 bispecific antibody (a) Key elements of Her2ScFvSP34-CL fusion proteins. Numbers indicate the insertion site in the CL domain (Kabat numbering scheme). (b) The bind+ ing of SP34 Fab and Her2ScFv-SP34-CL to CD3 Jurkat cells determined by flow cytometry. (c) The binding of Herceptin

Page 4 of 10

and Her2ScFv-SP34-CL to immobilized Her2-Fc antigen by ELISA.

Her2ScFv-SP34-CL can effectively recruit CD3+ cells to target Her2+ cancer cells in vitro. Next we determined whether Her2ScFv-SP34-CL could mediate the formation of an immunological synapse between Her2+ target cells and CD3+ T cells. To visualize the association of Her2+ cells and CD3+ cells mediated by Her2ScFv-SP34CL, live Her2+ SK-BR-3 cells and CD3+ Jurkat cells were stained with calcein AM and cell tracker Orange, respectively. After co-culturing with or without 100 nM Her2ScFv-SP34-CL for 12 hours, cells were extensively washed to remove the unbound Jurkat cells. The cells treated with Her2ScFv-SP34-CL showed significantly more Jurkat cells bound to the SK-BR-3 cells than that in the absence of BsAb (Figure 4a). This result demonstrates that Her2ScFv-SP34-CL mediates cell-cell interaction between CD3+ cells and Her2+ cells. To measure the activity of Her2ScFv-SP34-CL to selectively direct T cells to kill Her2 expressing cancer cells, we performed a cytotoxicity assay using cells expressing different levels of Her2. Peripheral blood mononuclear cells (PBMCs) from healthy donors were purified with Ficoll and incubated with the target cells in the presence or absence of Her2ScFv-SP34-CL. LDH release from the lysed cells was used to evaluate in-vitro cytotoxicity. As shown in Figure 4b, Her2ScFv-SP34-CL demonstrates excellent cytotoxicity against Her2-expressing cells. On Her23+ MDA-MB-435/Her2 (with stable Her2 expression) and SKBR-3 cells, the EC50s of Her2ScFv-SP34-CL are 1.0±0.2 pM and 0.32±0.04 pM, respectively (Table S2). The subpicomolar potency suggests that Her2ScFv-SP34-CL efficiently recruit T cells to lyse Her2 positive cells. On Her21+ MDA-MB-231 cells, which have 100-fold lower Her2 expression compared to Her23+ SK-BR-3 cells,36 Her2ScFvSP34-CL has an EC50 of 5.6±0.5 pM. The maximum cytotoxicity on Her21+ MDA-MB-231 (22%) is lower than that on Her23+ cells (51% on SK-BR-3 and 47% on MDA-MB435/Her2). It was previously reported that the surface antigen expression level positively correlates to maximum cytotoxicity and negatively correlates to EC50, which is consistent with our observations.37,38No significant cytotoxicity was observed on Her2 negative MDA-MB-468 cells. The correlation between EC50s and surface Her2 expression levels suggests that the T cell killing mediated by Her2ScFv-SP34-CL is highly selective to Her2 expressing cells. Non-specific T-cell activation might result in potential off-target toxicity. However, no detectable cytotoxicity on Her2- MDA-MB-468 cells in the T cell killing assay was observed. To further validate this observation, nonspecific T cell activation was evaluated. As shown in Figure 4c, T cells activated with anti-CD3 and anti-CD28 antibodies showed a significant upregulation of the early T-cell activation marker CD69 compared with the nontreated group. After cells were treated with 200 pM Her2ScFv-SP34-CL, a dose over 200-fold higher than the EC50 on Her23+ cells, no significant CD69 up-regulation was observed. These results demonstrate that Her2ScFv-


Page 5 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SP34-CL does not cause significant non-specific T cell

activation.

Figure 4. T cell recruitment, cytotoxicity and non-specific T cell activation mediated by Her2ScFv-SP34-CL (a) Fluorescence microscope images of the interaction between SK-BR-3 (green) cells and Jurkat cells (red) in the presence of PBS or Her2ScFv1+ SP34-CL. Scale bar = 100 µm. (b) Dose-dependent cytotoxicity on MDA-MB-468 (Her2 ), MDA-MB-231 (Her2 ), MDA-MB3+ 3+ 435/Her2 (Her2 ), and SK-BR-3 (Her2 ) cells in the presence of Her2ScFv-SP34-CL and human peripheral blood mononuclear cells (PBMCs) from healthy donors. (c) Flow cytometry analysis of T-cell activation marker CD69 on human peripheral blood mononuclear cells (PBMCs) after 20-hour treatment of 200 pm Her2ScFv-SP34-CL (labeled as “Her2ScFv-SP34-CL”). Untreated PBMCs purified from healthy donors were used as the negative control (labeled as “no treatment”). Human PBMCs activated with plate-bound anti-CD3 antibody (clone OKT3, eBioscience) and 2 µg/mL of soluble anti-CD28 antibody (clone CD28.2, eBioscience) were used as the positive control (labeled as “OKT3+aCD28”).

The serum stability and pharmacokinetics of Her2ScFv-SP34-CL. The serum stability of Her2ScFvSP34-CL was then assessed to determine whether Her2ScFv-SP34-CL is stable against proteolytic activities. Her2ScFv-SP34-CL was incubated in mouse serum for up to 96 hours. To quantify the concentration of the active fraction of Her2ScFv-SP34-CL, a sandwich ELISA was established with immobilized Her2-Fc as the antigen and anti-human kappa chain as the secondary antibody. The sandwich ELISA detects two separate epitopes on the parental Fab and the insert, respectively. Thus, the sandwich ELISA results can quantify the amount of the fusion protein. As shown in Figure 5a, after 96 hours, ~65% of original Her2ScFv-SP34-CL was present, demonstrating that Her2ScFv-SP34-CL is relatively stable in serum. The pharmacokinetic (PK) properties of Her2ScFvSP34-CL were assessed in mice by intravenously injection. The concentration of the fusion protein was determined by the ELISA described above. Her2ScFv-SP34-CL showed a characteristic two-phase pharmacokinetic behavior (Figure 5b). The half-lives of the distribution and elimination phases were determined with the WinNonlin® phar-

macokinetic software package. The half-life of the elimination phase is 9.8±1.9 hour for Her2ScFv-SP34-CL, significantly longer than ~1.3 hour for a typical Fab.39 In contrast, Blinatumomab, the anti-CD19/CD3 bispecific T cell engager (BiTE), has a reported short terminal phase halflife of only ~2 hours even in human.40 With a molecular weight of approximately 78 kDa, Her2ScFv-SP34-CL is above the first-pass renal clearance limit, offering a pharmacokinetic advantage over smaller BiTE format. Her2ScFv-SP34-CL recruits T cells to suppress tumor growth in vivo. To evaluate the in vivo efficacy of Her2ScFv-SP34-CL, xenograft models were established by subcutaneous implantation of 5×106 Her22+ (MDA-MB453) or 2.5×106 Her21+ (MDA-MB-435) cells in female NSG mice.41 One day later, 2×107 fresh human PBMC were injected into the intraperitoneal space. Upon formation of a palpable tumor, mice were intravenously infused with 2×107 activated human T cells. Her2ScFv-SP34-CL was then intravenously administered daily at a dose of 1 mg/kg for 10 days. Mice treated with PBMCs and PBS were used as negative controls. The tumor growth and body weight change were monitored for up to 50 days. As



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

shown in Figure 5c and 5d, shortly after treatment was initiated, tumor shrinkage was observed in the Her2ScFvSP34-CL group, whereas the vehicle group showed rapid tumor outgrowth. In the Her22+ MDA-MB-453 model, the tumor size decreased and then stabilized in the treatment group within the 50-day period of this study; and no apparent relapse was observed for at least 30 days after treatment was stopped. In the Her22+ MDA-MB-435 model, similarly, tumor growth was significantly suppressed by Her2ScFv-SP34-CL. During these studies, no overt body weight loss was observed in mice in any group (Figure S14), indicating Her2ScFv-SP34-CL has no obvious toxicity to mice in this dosing paradigm. These observations demonstrate that BsAbs generated by non-CDR loop grafting represent a potential effective treatment for Her2+ cancers with all types of Her2 overexpression levels.

Page 6 of 10

the cognate antigen. Therefore, unlike the monofunctional antibody CDR-fusions, non-CDR loop fusions yield antibody fusions for applications such as the generation of immunocytokines42 and immune cell recruiting bispecific antibodies.17 Current published Ig fusion strategies mainly include N- or C- terminal fusions and hinge insertions. As Ntermini are close to the antigen-binding site, large-size Nterminal fusion in antibodies often decreases the binding affinity of the antibody.43 C-terminal fusion, comparatively, does not affect the activity of the parental antibody. Therefore, it is often chosen for immunocytokines and immunotoxins. However, C-terminal fusion has reportedly shorter serum half-life, likely due to interference with neonatal Fc receptor (FcRn) binding and/or destabilized Fc dimerization.8 In addition to terminal fusions, insertion of peptides/proteins into the hinge region has been reported. However, the inserted proteins have reduced FcγR-binding as well as shorter half-lives.8 In comparison, non-CDR fusion site is distant from the antigen binding site or the Fc domain, minimizing the influence on antigen binding or PK properties. Additionally, as the insert protein is stemmed out from the antibody scaffold by a rigid stalk, allowing retained biological functions of the insert protein. As we demonstrated in our studies, neither EPO insert nor the anti-Her2 ScFv insert had significant decrease in its biological activity. To demonstrate the potential utility of this strategy, we generated a Her2×CD3 bispecific antibody (Her2ScFvSP34-CL) capable of recruiting T cells to Her2+ cancer cells with high in vitro and in vivo cytotoxity.

Figure 5. Serum stability, pharmacokinetics, and in vivo efficacy of Her2ScFv-SP34-CL (a) The stability of Her2ScFvSP34-CL in mouse serum. The Her2ScFv-SP34-CL concentration is determined by sandwich ELISA with Her2-Fc as capnd ture antigen and HRP-anti-human kappa 2 Ab. (b) Pharmacokinetics of Her2ScFv-SP34-CL in mice by i.v. dosing (n=3). The Her2ScFv-SP34-CL concentration is determined by sandwich ELISA with Her2-Fc as capture antigen and nd HRP-anti-human kappa 2 Ab. (c) Tumor regression medi2+ ated by 1 mg/kg Her2ScFv-SP34-CL in MDA-MB-453 (Her2 ) xenograft in NSG mice (n=5). (d) Tumor regression mediated 1+ by 1 mg/kg Her2ScFv-SP34-CL in MDA-MB-435 (Her2 ) xenograft in NSG mice (n=5).

Discussion In our previous work, we reported successful protein/peptide grafting into CDR3 and CDR2 in antibody variable regions.19-27 Herein, we extend this insertion strategy to the loops in the constant regions, which structurally also belong to the immunoglobulin scaffold. We demonstrate that these conserved loops are also capable of accepting large functional protein insertions. In general CDR loop fusions reduce or destroy the binding properties of the parent antibody. In contrast, the non-CDR loops are spatially separated from the antigen-binding site, and as a result, protein/peptide grafting into these loops does not significantly impact the binding affinity to

The use of bispecific antibodies (bsAbs) to retarget the immune system to treat cancer has been highly effective but still faces several challenges.44,45 Catumaxomab, the first clinically approved bispecific antibody, was constructed from rat-mouse antibody quadromas and has significant immunogenicity in humans.44 Bispecific T cell engager (BiTE)12 and dual-affinity re-targeting (DART)46 bispecific constructs have shown excellent efficacy. However, they suffer from short half-lives and can have poor physical properties. More recent attempts to generate heterodimeric IgG-like bispecific antibodies have afforded long circulation times and high potencies. However, complete removal of unwanted homodimeric species to generate highly purified heterodimers remains a manufacturing challenge. 47 Tumor penetration, effective immune synapse formation, and stability are critical properties of T-cell recruiting bispecific antibodies. The optimal size of a bsAb is a trade-off between serum half-life and tumor penetration.48,49 It has been suggested that proteins with molecular weight between 65 kDa and 110 kDa are most suitable for penetrating solid tumors.49 Thus the small size of BiTE and DART (~ 50 kDa) afford excellent efficacy, but suffer from fast clearance. On the other hand, IgG-like bispecific antibodies have long serum half-lives, but less efficiently penetrate through the dense extracellular matrix (ECM) of solid tumors.48 The scFv-Fab non-CDR loop


Page 7 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


fusion, with molecular weight around ~ 78 kDa, nicely fall within the optimal range for tumor penetration, and have a terminal half-life of ~ 10 hours 0Her2ScFv-SP34-CL0 in mice, significantly longer than for BiTEs. Efficient immunological synapse formational also affects the ability of immune cells to kill cancer cells. Reducing the distance between the T-cell-binding site and tumor antigen binding sites on bsAbs normally results in higher potency.50 Indeed, the excellent in vitro and in vivo efficacies of BiTE and DART are due in part to the short distance between T-cell- and cancer-cell binding sites. Similarly, the distance between the CD3-binding site on the parent SP34 antibody and the Her2-binding site on the Herceptin scFv insert is small, and the in vitro cytotoxicity of Her2ScFv-SP34-CL reaches EC50 values of 10-12 10-13 M. Stability is another important requirement for bispecific antibodies. Blinatumomab, for example, has a narrow therapeutic window, due to non-specific T cell activation likely caused by bsAb aggregations.40 Although small amounts of Her2ScFv-SP34-CL oligomers are present after protein G chromatography of the expressed fusion protein, no further aggregation is observed after the monomer is purified by size exclusion chromatography, and no aggregates can be detected by dynamic light scattering. Indeed, the physiochemical properties of Her2ScFv-SP34CL upon expression and purification are similar to those of a typical Fab. More importantly, no nonspecific in vitro cytotoxicity or T cell auto-activation is observed for this construct. The non-CDR loop fusion can be constructed on either Fab scaffolds or full-length IgG scaffolds, affording either monovalent or bivalent binding modes. It is also likely that one can generate multi-valent binding formats that can include binding to multiple tumor antigens, checkpoint proteins or serum albumins. Additionally, unlike other full-length IgG fusion strategies (such as N- and Cterminal fusion),8,43 there are multiple options for the non-CDR loop fusion sites to that allow one to retain full antigen-binding and Fc mediated functions. We are currently exploring the extension of this strategy to trispecific antibody fusions. Conclusions In summary, we demonstrate that full-length proteins and single chain variable fragments (scFv) can be inserted into the non-CDR loops of an IgG or a Fab. The antibody fusion proteins retain the antigen binding activity of the scaffold antibody as well as full activities of new functionalities introduced by the insert. The non-CDR loop fusion proteins have excellent in vitro activity, physiochemical properties, and thermal and serum stabilities. The Her2×CD3 bispecific antibodies generated by this approach can induce targeted cell lysis in the presence of effector cells at sub-picomolar concentrations in breast cancer cells. No T cell activation was observed in the absence of target cells even at high concentration of Her2ScFv-SP34-CL. Furthermore, Her2ScFv-SP34-CL

showed potent in vivo antitumor activity in mouse Her22+ and Her21+ xenograft models. Future work includes evaluation of candidate constructs for developability, as well as potential immunogenicity and toxicity in non-human primates. Methods Cloning of EPO-Herceptin-CL and EPO-Herceptin-CH1 IgG Fusion Protein. The genes encoding Herceptin Fab heavy chain and light chain were synthesized by Genscript (NJ, USA), and amplified by polymerase chain reactions (PCR). The mammalian expression vector of Herceptin full-length IgG heavy chain was generated by in-frame ligation of amplified Herceptin Fab heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Gene encoding antibody Herceptin light chain was amplified and cloned into the pFuse vector without hIgG1 Fc fragment. The gene encoding EPO was synthesized by Genscript (NJ, USA), and amplified by PCRs. Coiled-coil stalk was added to both ends of the EPO insert sequence. The sequence of the ascending stalk peptide with linkers at each end is: H2N-GGSGAKLAALKAKLAALKGGGGS-COOH; the sequence of the descending peptide with linkers at each end is: H2NGGGGSELAALEAELAALEAGGSG-COOH. The EPO-HerceptinCL light chain was created by replacing the K169 in the CL region of Herceptin light chain by EPO with a coiled-coil stalk. EPO-Herceptin-CH1 heavy chain was created by replacing the S182 and G183 in the CH1 region of Herceptin heavy chain by EPO with a coiled-coil stalk. The genes encoding the EPOHerceptin-CH1 heavy chain and EPO-Herceptin-CL light chain were obtained by overlap extention PCRs. And the vectors of the EPO-Herceptin-CH1 heavy chain and the EPO-Herceptin-CL light chain were generated by in-frame ligation of the amplified PCR products to pFuse-hIgG1-Fc backbone vector. The resulting mammalian expression vectors were confirmed by DNA sequencing. Cloning of Her2ScFv-SP34-CL Fab Fusion Protein. The genes encoding SP34 Fab heavy chain and light chain were synthesized by Genscript (NJ, USA), and amplified by PCRs. The mammalian expression vectors of SP34 Fab heavy and light chains were generated by in-frame ligation of amplified SP34 Fab heavy chain (VH and CH1) or light chain (VL and CL) to pFusehIgG1-Fc backbone vector (InvivoGen, CA) without the Fc fragment. Gene encoding Her2ScFv with the coiled-coil linkers was synthesized as gBlock gene fragment by IDT, Inc (IA, USA), and amplified by PCRs. The sequence of the ascending stalk peptide with linkers at each end is: H2NGGSGCAKLAALKAKLAALKGGGGS-COOH; the sequence of the descending peptide with linkers at each end is: H2NGGGGSELAALEAELAALEACGGSG-COOH. Subsequently, Her2ScFv-SP34-CL light chain was created by replacing the K169 (for Her2ScFv-SP34-CL) in the CL region of SP34 light chain by Her2ScFv with a coiled-coil stalk. The gene encoding the Her2ScFv-SP34-CL Fab light chain was obtained by overlap extention PCR. And the vector of Her2ScFv-SP34-CL Fab light chain was generated by in-frame ligation of the amplified PCR products to the pFuse-hIgG1-Fc backbone vector. The resulting mammalian expression vectors were confirmed by DNA sequencing. Expression and purification of fusion proteins. Fusion proteins were expressed through transient transfection of FreeStyle HEK 293 cells with expression vectors (Table S1), according to the manufacturer’s protocol. Briefly, 28 mL FreeStyle HEK 293 cells containing 3×107 cells were seeded in a 125 mL shaking flask. Defined amounts of plasmids encoding the light chain and heavy chain (Table S1) were diluted in 1 mL Opti-MEM medium and added to 1 mL Opti-MEM containing 60 µL 293fectin (Invitro-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

gen, Inc). After the plasmids were incubated with 293fectin for 30 min at room temperature, the lipoplex mixture was added to the cell suspension. Cells were then shaken at 125 rpm in a 5% CO2 environment at 37 ºC. Culture medium containing secreted proteins was harvested every 48 hours for twice after transfection. Fusion proteins were purified by Protein G and sizeexclusion chromatography. Purified proteins were analyzed by SDS-PAGE, mass spectrometry and size exclusion chromatography. In-vitro assay of EPO activity. Human TF-1 cells were cultured at 37 °C with 5% CO2 in RPMI-1640 medium containing 10% fetal bovine serum (FBS), penicillin/streptomycin (50 U/mL), and 2 ng/mL human granulocyte macrophage colony stimulating factor (GM-CSF). To test the proliferative activity of EPO fusion proteins, cells were washed three times with RPMI1640 medium plus 10% FBS, resuspended in RPMI-1640 medium with 10% FBS at a density of 1.5×105 cells/ml, plated in 96-well plates (1.5×104 cells per well) with various concentrations of EPOHerceptin-CL, EPO-Herceptin-CH1, Herceptin-hEPO-CDR3H (positive control) and then incubated for 72 h at 37 °C with 5% CO2. Cells were then incubated with Alamar Blue (Invitrogen) for 4 h at 37 °C. Fluorescence intensity measured with λex = 570 nm and λem = 595 nm is proportional to cell viability and plotted versus protein concentrations. The EC50 values were determined by fitting data into a logistic sigmoidal function: y = A2 + (A1 − A2)/(1 + (x/x0)p), where A1 is the initial value, A2 is the final value, x0 is the inflection point of the curve, and p is the power. In vitro PBMC-mediated cytotoxicity of Her2ScFv-SP34CL on breast cancer cells. For in vitro cytotoxicity assays, PBMCs were purified from fresh healthy human donor blood (from The Scripps Research Institute normal blood donor service) by conventional Ficoll-Hypaque gradient centrifugation (GE Healthcare). Purified PBMCs were washed and resuspended in RPMI with 10% (vol/vol) FBS and were incubated with target cells and different concentrations of Her2ScFv-SP34-CL for 24 h at 37 °C. Cytotoxicity of each well was measured by LDH levels in supernatant using the Cytotox-96 nonradioactive cytotoxicity assay kit (Promega). Lysis solution provided in the same kit (10 μL) was added to wells containing only target cells to achieve the maximum killing; and spontaneous killing was measured in wells with effector and target cells treated with vehicle (10 μL PBS). The absorbance at 490 nm was recorded using a SpectraMax 250 plate reader (Molecular Devices Corp.). Percent cytotoxicity was calculated by: % cytotoxicity = (absorbance experimental − absorbance spontaneous average)/ (absorbance maximum killing average − absorbance spontaneous average). Pharmacokinetics of Her2ScFv-SP34-CL in Mice. 8 mg/kg Her2ScFv-SP34-CL in PBS (pH 7.4) was administrated by intravenous (i.v.) injection into CD1 mice (6 per group). Blood was collected from 5 min, 15 min, 30 min, 1h, 2h, 4h, 6h, 8h, 24 hr, 32 hr, 48 hr after injection. 75µl of whole blood sample is collected via retro-orbital sinus into a heparinized capillary tube and stored on wet ice until processed. Each sample is spun at 12,000RPM for 3 min; the resulting plasma is placed into a uniquely identified location of a 96 well plate. The plate is stored at -80°C until analyzed. The plasma concentration of Her2ScFvSP34-CL is determined by ELISA against immobilized hErbB2-Fc (R&D Systems) with HRP anti-human Kappa (Abcam) as secondary antibody. The half-lives for elimination phase were determined by fitting the last four data points into the first-order equation, A = A0 e−kt, where A0 is the initial concentration, t is the time, and k is the first order rate constant. In vivo efficacy study of Her2ScFv-SP34-CL. All procedures were approved by The Scripps Research Institute Animal Care and Use Committee and were performed according to national

Page 8 of 10

and international guidelines for the humane treatment of animals. All efficacy studies were conducted with 6 to 8-week-old female NOD-SCID-γ(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (Jackson Laboratory). Human breast cancer cell lines Her22+ (MDA-MB-453), and Her21+ (MDA-MB-435)] were used to evaluate the in vivo efficacy of Her2ScFv-SP34-CL. For Her22+ tumor model, 5×106 MDA-MB-453 cells in 50% Matrigel (BD Bioscience) were subcutaneously implanted into the right flank of mice. One day after that, 2×107 fresh PBMC were injected into the intraperitoneal space. Meanwhile, human PBMCs were activated with plate-bound anti-CD3 antibody (clone OKT3, eBioscience) and 2 µg/mL of soluble anti-CD28 antibody (clone CD28.2, eBioscience), and maintained in RPMI1640 media supplemented with 10% FBS and 50 IU/mL of recombinant human IL-2 (R&D Systems). Eight days and eleven days after tumor implantation, mice received 2×107 activated T cells via intraperitoneal injection. Ten days after tumor inoculation, when tumors reached a volume of 200-300 mm3, mice were intravenously administered Her2ScFv-SP34-CL (1 mg/kg) or saline daily for ten days. For Her21+ tumor model, 2.5×106 MDA MB435 cells in 50% Matrigel were subcutaneously implanted to the right flank of mice. One day after that, 2×107 fresh PBMC were injected into the intraperitoneal space. Eight days and eleven days after tumor implantation, mice received 2×107 activated PBMC cells via intraperitoneal injection. Ten days after tumor inoculation, when tumors reached a volume of 200-300 mm3, mice were intravenously administered Her2ScFv-SP34-CL (1 mg/kg) or saline daily for ten days. Tumors were measured twice weekly by calipers. Tumor volume was calculated based on width × length × height.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed materials and methods, supplementary figures and tables (PDF file).

AUTHOR INFORMATION Corresponding Author [email protected] [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT We thank Prof. Liangfang Zhang (UCSD) and his student Yue Zhang in helping with the DLS measurement.

ABBREVIATIONS CDR, complementarity determining region; EPO, Erythropoietin; scFv, single-chain variable fragment; ICK, inhibitor cysteine knot; IgG, immunoglobulin G; Fab, fragment antigen-binding; Fc, fragment constant; bsAb, bispecific antibody.


Page 9 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1)

Ecker, D. M.; Jones, S. D.; Levine, H. L. mabs 2015, 7 (1),

9. (2) Chames, P.; Van Regenmortel, M.; Weiss, E.; Baty, D. Brit J Pharmacol 2009, 157 (2), 220. (3) Scott, A. M.; Wolchok, J. D.; Old, L. J. Nat Rev Cancer 2012, 12 (4), 278. (4) Evans, J. B.; Syed, B. A. Nat Rev Drug Discov 2014, 13 (6), 413. (5) Liu, T.; Du, J.; Luo, X.; Schultz, P. G.; Wang, F. Current Opinion in Chemical Biology 2015, 28, 66. (6) Beck, A.; Wurch, T.; Bailly, C.; Corvaia, N. Nat Rev Immunol 2010, 10 (5), 345. (7) Nisonoff, A.; Rivers, M. M. Arch. Biochem. Biophys. 1961, 93, 460. (8) Coloma, M. J.; Morrison, S. L. Nat. Biotechnol. 1997, 15, 159. (9) Milstein, C.; Cuello, A. C. Nature 1983, 305 (5934), 537. (10) Holliger, P.; Prospero, T.; Winter, G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90 (14), 6444. (11) Löffler, A.; Kufer, P.; Lutterbüse, R.; Zettl, F.; Daniel, P. T.; Schwenkenbecher, J. M.; Riethmüller, G.; Dörken, B.; Bargou, R. C. Blood 2000, 95 (6), 2098. (12) Dreier, T.; Lorenczewski, G.; Brandl, C.; Hoffmann, P.; Syring, U.; Hanakam, F.; Kufer, P.; Riethmüller, G.; Bargou, R.; Baeuerle, P. A. Int. J. Cancer 2002, 100 (6), 690. (13) Johnson, S.; Burke, S.; Huang, L.; Gorlatov, S.; Li, H.; Wang, W.; Zhang, W.; Tuaillon, N.; Rainey, J.; Barat, B.; Yang, Y.; Jin, L.; Ciccarone, V.; Moore, P. A.; Koenig, S.; Bonvini, E. J Mol Biol 2010, 399 (3), 436. (14) Carter, P.; Ridgway, J.; Presta, L. G. Immunotechnology 1996. (15) Wu, C.; Ying, H.; Grinnell, C.; Bryant, S.; Miller, R.; Clabbers, A.; Bose, S.; McCarthy, D.; Zhu, R.-R.; Santora, L.; Davis-Taber, R.; Kunes, Y.; Fung, E.; Schwartz, A.; Sakorafas, P.; Gu, J.; Tarcsa, E.; Murtaza, A.; Ghayur, T. Nat. Biotechnol. 2007, 25 (11), 1290. (16) Spiess, C.; Zhai, Q.; Carter, P. J. Molecular Immunology 2015, 67 (2), 95. (17) Brinkmann, U.; Kontermann, R. E. mabs 2017, 9 (2), 182. (18) Wang, F.; Ekiert, D. C.; Ahmad, I.; Yu, W.; Zhang, Y.; Bazirgan, O.; Torkamani, A.; Raudsepp, T.; Mwangi, W.; Criscitiello, M. F.; Wilson, I. A.; Schultz, P. G.; Smider, V. V. Cell 2013, 153 (6), 1379. (19) Liu, T.; Fu, G.; Luo, X.; Liu, Y.; Wang, Y.; Wang, R. E.; Schultz, P. G.; Wang, F. J. Am. Chem. Soc. 2015, 137 (12), 4042. (20) Liu, T.; Zhang, Y.; Liu, Y.; Wang, Y.; Jia, H.; Kang, M.; Luo, X.; Caballero, D.; Gonzalez, J.; Sherwood, L.; Nunez, V.; Wang, D.; Woods, A.; Schultz, P. G.; Wang, F. Proc Natl Acad Sci Usa 2015, 112 (5), 1356. (21) Luo, X.; Liu, T.; Wang, Y.; Jia, H.; Zhang, Y.; Caballero, D.; Du, J.; Wang, R. E.; Wang, D.; Schultz, P. G.; Wang, F. Angew. Chem. Int. Ed. Engl. 2015, 54 (48), 14531. (22) Zhang, Y.; Liu, Y.; Wang, Y.; Schultz, P. G.; Wang, F. J. Am. Chem. Soc. 2015, 137 (1), 38. (23) Zhang, Y.; Zou, H.; Wang, Y.; Caballero, D.; Gonzalez, J.; Chao, E.; Welzel, G.; Shen, W.; Wang, D.; Schultz, P. G.; Wang, F. Angew. Chem. Int. Ed. 2014, 54 (7), 2126. (24) Liu, T.; Liu, Y.; Wang, Y.; Hull, M.; Schultz, P. G.; Wang, F. J. Am. Chem. Soc. 2014, 136 (30), 10557. (25) Zhang, Y.; Goswami, D.; Wang, D.; Wang, T.-S. A.; Sen, S.; Magliery, T. J.; Griffin, P. R.; Wang, F.; Schultz, P. G. Angew. Chem. Int. Ed. Engl. 2014, 53 (1), 132.

(26) Zhang, Y.; Wang, D.; de Lichtervelde, L.; Sun, S. B.; Smider, V. V.; Schultz, P. G.; Wang, F. Angew. Chem. Int. Ed. 2013, 52 (32), 8295. (27) Zhang, Y.; Wang, D.; Welzel, G.; Wang, Y.; Schultz, P. G.; Wang, F. ACS Chem. Biol. 2013, 8 (10), 2117. (28) Bork, P.; Holm, L.; Sander, C. J Mol Biol 1994, 242 (4), 309. (29) Kontermann, R. E. Arch. Biochem. Biophys. 2012, 526 (2), 194. (30) Eigenbrot, C.; Randal, M.; Presta, L.; Carter, P.; Kossiakoff, A. A. J Mol Biol 1993, 229 (4), 969. (31) Zhao, Y.; Wang, Q. J.; Yang, S.; Kochenderfer, J. N.; Zheng, Z.; Zhong, X.; Sadelain, M.; Eshhar, Z.; Rosenberg, S. A.; Morgan, R. A. The Journal of Immunology 2009, 183 (9), 5563. (32) Zhou, N. E.; Zhu, B. Y.; Kay, C. M.; Hodges, R. S. Biopolymers 1992, 32, 419. (33) Pandya, M. J.; Cerasoli, E.; Joseph, A.; Stoneman, R. G.; Waite, E.; Woolfson, D. N. J. Am. Chem. Soc. 2004, 126 (51), 17016. (34) Dombkowski, A. A.; Sultana, K. Z.; Craig, D. B. FEBS Lett. 2014, 588 (2), 206. (35) Nelson, A. D.; Hoffmann, M. M.; Parks, C. A.; Dasari, S.; Schrum, A. G.; Gil, D. Journal of Biological Chemistry 2012, 287 (51), 42936. (36) Hicks, D. G.; Schiffhauer, L. Lab Med 2011, 42 (8), 459. (37) Friedrich, M.; Henn, A.; Raum, T.; Bajtus, M.; Matthes, K.; Hendrich, L.; Wahl, J.; Hoffmann, P.; Kischel, R.; Kvesic, M.; Slootstra, J. W.; Baeuerle, P. A.; Kufer, P.; Rattel, B. Molecular Cancer Therapeutics 2014, 13 (6), 1549. (38) Lopez-Albaitero, A.; Xu, H.; Guo, H.; Wang, L.; Wu, Z.; Tran, H.; Chandarlapaty, S.; Scaltriti, M.; Janjigian, Y.; de Stanchina, E.; Cheung, N.-K. V. OncoImmunology 2017, 6 (3), 1. (39) Nguyen, A.; Reyes, A. E.; Zhang, M.; McDonald, P.; Wong, W. L. T.; Damico, L. A.; Dennis, M. S. Protein Eng. Des. Sel. 2006, 19 (7), 291. (40) Portell, C. A.; Wenzell, C. M.; Advani, A. S. Clin Pharmacol 2013, 5 (Suppl 1), 5. (41) Ito, R.; Takahashi, T.; Katano, I.; Ito, M. Cell. Mol. Immunol. 2012, 9 (3), 208. (42) List, T.; Neri, D. Clin Pharmacol 2013, 5, 29. (43) Wu, X.; Sereno, A. J.; Huang, F.; Lewis, S. M.; Lieu, R. L.; Weldon, C.; Torres, C.; Fine, C.; Batt, M. A.; Fitchett, J. R.; Glasebrook, A. L.; Kuhlman, B.; Demarest, S. J. mabs 2015, 7 (3), 470. (44) Chames, P.; Baty, D. mabs 2009, 1 (6), 539. (45) Thakur, A.; Lum, L. G. Curr. Opin. Mol. Ther. 2013, 12 (3), 340. (46) Moore, P. A.; Zhang, W.; Rainey, G. J.; Burke, S.; Li, H.; Huang, L.; Gorlatov, S.; Veri, M. C.; Aggarwal, S.; Yang, Y.; Shah, K.; Jin, L.; Zhang, S.; He, L.; Zhang, T.; Ciccarone, V.; Koenig, S.; Bonvini, E.; Johnson, S. Blood 2011, 117 (17), 4542. (47) Liu, H.; Saxena, A.; Sidhu, S. S.; Wu, D. Front. Immunol. 2017, 8, 9. (48) Davies, C. de L.; Berk, D. A.; Pluen, A.; Jain, R. K. Br J Cancer 2002, 86 (10), 1639. (49) Cuesta, Ã. N. M.; Sainz-Pastor, N.; Bonet, J.; Oliva, B.; lvarez-Vallina, L. Ã. Trends Biotechnol. 2010, 28 (7), 355. (50) Li, J.; Stagg, N. J.; Johnston, J.; Harris, M. J.; Menzies, S. A.; DiCara, D.; Clark, V.; Hristopoulos, M.; Cook, R.; Slaga, D.; Nakamura, R.; McCarty, L.; Sukumaran, S.; Luis, E.; Ye, Z.; Wu, T. D.; Sumiyoshi, T.; Danilenko, D.; Lee, G. Y.; Totpal, K.; Ellerman, D.; Hötzel, I.; James, J. R.; Junttila, T. T. Cancer Cell 2017, 31 (3), 383.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 10

Insert Table of Contents artwork here


10

Engineering Bifunctional Antibodies with Constant ... - ACS Publications

Recommend Documents