In this chapter, we sought to develop a new class of molecular therapeutics which enable to induce antibody-mediated anti-cancer immune responses. Firstly, I introduced immunogenicity and low tissue permeability as two major drawbacks of therapeutic antibodies. Secondly, I introduced previous researches trying to address these issues. Then, main contents of our project were explained.
Therapeutic antibodies have been revolutionizing clinical outcomes of refractory diseases such as cancers and autoimmune diseases. For example, anti-CD20 monoclonal antibody (mAb) in combination with CHOP chemotherapy (Cyclophosphamide, Hydroxydaunorubicin, Oncovin, and Prednisone) significantly improved the complete response rate and survival times of B-cell lymphoma patients1. As explained in chapter 1, mAbs can provide therapeutic effects through immune responses (see Fig. 1.5), which are mechanistically different from that of conventional small molecules. We can also recognize the prosperity of mAb therapeutics in terms of market sales. 7 drugs out of top 10 drugs by sales in 2018 were therapeutic antibodies (Table 3.1)2. Although there are multiple factors contributing to the importance of mAbs in the clinic, including their high affinity and specificity to targets, potential immunogenicity of therapeutic mAbs as well as their low tissue permeability are two major factors limiting mAbs’ therapeutic potential.
Table. 3.1. Top drugs by sales globally in 2018 2. Drugs in red are antibody drugs.
Potential Immunogenicity of mAbs may induce life-threatening adverse events such as IgE-mediated anaphylaxis and cytokine storms3. In addition, production of anti-drug antibodies (ADAs) can reduce the serum concentration of mAbs and deteriorate the efficacy4. Immunogenicity of mAbs are thought to be attributed to the large molecular weight of mAbs (about 150 kDa) and the presence of non-self amino acid sequences3. Large molecular weight of mAbs may also hamper deep penetration of mAbs into solid tumors5, limiting their therapeutic effects.
To overcome these issues, small molecules which can emulate or induce effector functions of antibodies have been extensively developed. Small molecules are thought to be less immunogenic, and penetrate into tissues more efficiently than macromolecules. In 2014, novel synthetic molecules called Synthetic antibody mimics targeting prostate cancer (SyAM-Ps), which possess both targeting and effector functions of therapeutic antibodies have been reported (Fig. 3.1)6. SyAM-Ps are bispecific molecules which can bind to Fcγ receptor I (FcγRI) expressed on immune cells and prostate specific membrane antigen (PSMA) (Fig. 3.1, B to D). SyAM-Ps work as immune cell engagers to prostate cancer cells, resulting in induction of immune responses reminiscent of antibody-dependent cell-mediated phagocytosis (ADCP) (Fig. 3.2). Considering
rank Product name Sales
US$ billions) Target Company
1 Humira 19.9 TNF-α AbbVie
2 Revlimid 9.7 Cereblon (E3 ubiquitin ligase) Celgene
3 Keytruda 7.2 PD-1 Merck
4 Herceptin 7.1 HER2 Roche
5 Avastin 7.0 VEGF Roche
6 Rituxan 6.9 CD20 Roche
7 Opdivo 6.7 PD-1 BMS & Ono
8 Eliquis 6.4 Factor X BMS
9 Prevnar 13 5.8 Vaccine (Streptococcus
pneumoniae) Pfizer
10 Stelara 5.2 lL-12, IL-23 Johnson & Johnson
still technically difficult to emulate multi-functionality of therapeutic antibodies (e.g. long blood half-life and effector functions) using small synthetic molecules.
Fig. 3.1. Design and structures of synthetic antibody mimics targeting prostate cancer (SyAM-Ps).
(A) Schematic depiction of SyAM-P’s proposed mechanism of action. (B) Docking of SyAM-P into PSMA binding pocket and FcγRI binding surface to determine the linker length needed to template a ternary complex. (C) Schematic illustration of the evolution of SyAM-P’s design from a monoclonal antibody template. (D) Molecules discussed herein. CP33 is an FcγRI-targeting motif. The first-generation construct (SyAM-P1, 1) displays single FcγRI- and PSMA-binding
(SyAM-P3, 3) displays a pair of PSMA-targeting motifs tethered to a pair of CP33 motifs.
Reprinted with permission from ref6.
Fig. 3.2. Amnis flow cytometry imaging of phagocytic events induced by SyAM.
Depicted are representative images of completed phagocytosis. Channels shown are brightfield, target (stained with DiO), nuclei (stained with Hoechst), effector cell (stained with DiD, anti-CD14- APC, and anti-CD11b-APC), and merged image. Modified and reprinted with permission from ref 6.
Antibody-recruiting small molecule (ARM) is another candidate of synthetic alternatives of therapeutic antibodies 7. ARM is composed of a target-binding terminus (TBT) and an antibody-binding terminus (ABT) (Fig. 3.3). ABT plays a role to catch endogenous antibodies existing in the blood stream, and TBT plays a role in redirection of them to the target, such as tumor cells.
Subsequently, recruited antibodies induce immune responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) and eliminate the target (Fig. 3.4). While ARMs are also very small relative to antibodies, yet they potentially enable to utilize multiple functions of antibodies for therapeutic applications7. This is because, unlike SyAMs, ARMs cooperate with endogenous antibodies in the blood circulation. It would be possible for ARMs to substantially extend their blood circulation time, which is crucial for efficient drug delivery to tumor, through hitchhiking on endogenous antibodies8. Further, endogenous antibodies exclusively play roles in interaction with host immune system upon the antibody redirection. Overall, the ARM strategy can exploit multi-functionality of endogenous antibodies for therapeutic purposes while circumventing some limitations of therapeutic antibodies such as immunogenicity and low tissue penetration, thereby
Fig. 3.3. Schematic representation of antibody-recruiting small molecule (ARM).
Fig. 3.4. ARM-mediated induction of antibody-dependent cell-mediated cytotoxicity.
Immune cells such as natural kille (NK) cells recognize antibodies on cancer cells via Fc-gamma
Target-binding terminus (TBT)
Antibody-binding terminus (ABT) Fab
Fc
α-Gal trisaccharide 2,4-dinitrophenyl (DNP) α-Rhamnose
O HO OH HOMe
O
Cancer cell
Antigen CD16a
ADCC
Natural Killer
(NK) cell
Regarding the molecular structures of ARMs, various types of TBTs have been produced to evaluate the applicability of ARMs to a broad range of targets9-24, whereas only three ABTs against pre-existing endogenous antibodies have been investigated: galactosyl-(1-3)-galactose (a-Gal)
14-15, rhamnose23, and nitroarenes (Fig. 3.3)16-18, 22, 24. The limited choice of ABTs decreases the potential of ARMs because pre-existing endogenous antibodies have intra/inter-patient variabilities in characteristics such as concentration and affinities. For instance, their amounts are limited and vary among individuals; up to 2% for anti-a-Gal25-27 and 1% for anti-2,4-dinitrophenyl (DNP)28-29. In the case of anti-rhamnose antibodies, a literature suggested that its potential availability in the blood stream might be higher than anti-a-Gal and anti-DNP antibodies23. However, the literature also mentioned that anti-rhamnose antibodies in serum may have much larger individual variability23. Thus, finding new ABTs to overcome the intra/inter-patient differences observed for endogenous antibodies is a key challenge.
The Fc region of IgG is characterized by its conserved molecular structure and a variety of biological functions. The Fc region serves as a module for effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC)30-31. A recent study revealed that the Fc fragment of IgG binds to herpes simplex virus 1 (HSV1) glycoprotein E and Fc-gamma receptor IIIa (FcgRIIIa)/CD16a expressed on immune cells simultaneously, resulting in ADCC against HSV1-infected cells32. This report indicates that ADCC could be induced without antigen-Fab interactions.
Here, we report a new class of ARMs, namely Fc-ARMs, in which a Fc-binding cyclic peptide is used as an ABT and folic acid (FA) is used as a TBT to target tumor cells that overexpress folate receptor-a (FR-a). Using Fc-ARM, we sought to exploit the majority of endogenous antibodies through constant affinity to eliminate malignant cells (Fig.3.5). The binding sites of Fc-binding peptides used in this study and CD16a to the Fc region of the antibody do not overlap (Fig. 3.6), supporting the feasibility of Fc-ARM-mediated anti-tumor immune responses. We conducted quantitative evaluations of antibody recruitment as well as anti-tumor efficacy of the Fc-ARM
Fig. 3.5. Fc-binding antibody-recruiting small molecules (Fc-ARMs).
(A) Schematic illustration of Fc-ARM mediated induction of ADCC. (B) Comparison between conventional ARM and Fc-ARM regarding availability of endogenous IgG in the circulation as well as the character of affinity between ABT and antibodies.
Targeting ligand
Fc-binding peptide Fc-ARM
Blood vessel
ADCC
ARM Fc-ARM
ABT antigen Fc-binder
%Available IgG ~ 1% > 80%
Affinity between
ABT and Abs variable constant
B A
NK cell
Cancer cells
Fig. 3.6. Molecular model of the ternary complex (FR-a/Fc-ARM/IgG) bound with CD16a.
Fc-ARM is described without oligoethyleneglycol linker. IgG is only shown as Fc region (CH2 and CH3 domains). FR-a and CD16a are shown as extracellular region. This model is generated from four crystal structures (PDB ID: 1DN233, 3RJD34, 3AY435, and 4LRH36) using UCSF Chimera. Modified and reprinted with permission from ref37.