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The use of phage display technology to develop peptide-based immune checkpoint inhibitors disrupting PD-1/PD-L1 interaction for immunotherapy of cancer


For a considerable period, immunotherapy employing cytokines such as interleukin-2 or alpha-interferon has been lagging behind other standard treatments, i.e. surgery, chemotherapy and radiotherapy for cancer.  It became less prioritized with the event of 'targeted therapy' as the latter inhibiting oncogenic signaling pathways received greater attention.  However, within the last decade, there has been a great resurgence of interest in immunotherapy due to its noticeable impact on melanoma.

Prior to the recent revival of interest, immunotherapy has been experimenting with the potential of triggering a quasi-autoimmune reaction against cancer.  Though the anticancer vaccines are intended to target tumor specific antigen generated through mutation, it is still part of one's own physiological repertoire.  Thus, there is a risk of triggering autoimmunity against other unintended (normal) targets in the process of destroying cancer.  To avoid such mishap, the immune system has installed a monitoring system called 'immune checkpoint'.  (Note: the checkpoint is distinct from 'DNA damage checkpoint' which monitors damaged DNA to allow time for repair before replicating)  (Sharma et al., 2021).

The activity of host immune system consists of both the antibody-based response and the cellular response.  For the latter, the target protein is proteolytically cleaved into short peptides within the cytosol, followed by its presentation by the major (or minor) histocompatibility complex (MHC) for the recognition by T cell receptors.  In addition to MHC molecules, the antigen presenting process requires co-stimulation by other factors, which include proteins such as B7-1 (CD80) present on dendritic cells and others.  The co-stimulatory molecule B7-1 present on antigen presenting cells is recognized by CD28 present on T cells during the MHC-to-TCR interaction.  In addition to the above, the immune system also consists of Tregs (regulatory T cells) which function to suppress the activation and proliferation of effector T cells.  CTLA-4 (expressed in Treg cells or upregulated in activated T cells) can bind to B7 to turn off the activation.  

PD-1 (programmed cell death protein-1, CD279) is a member of the immunoglobulin superfamily and is expressed on T or B cells, which was discovered by T. Honjo  of Kyoto University (Japan; Nobel prize 2018).  As an 'immune checkpoint' protein, PD-1 functions to suppress immune response via facilitating the apoptosis of antigen-recognizing T cells or inhibiting the death of Treg cells.  PD-1 (expressed in T or B cells) is a receptor for the ligand PD-L1 or PD-L2 (member of B7 family), whose interaction with PD-1 negatively regulates the immune response of cytotoxic lymphocytes (i.e. CD8+ T cells) (Robert, 2020).

Hence, disrupting the interaction between PD-1 to PD-1L (expressed by tumor cells) has become the major focus of pharmaceutical industries to enhance the immunological response to tumors.  This has led to the development of FDA-approved antibodies recognizing PD-1 (Inivolumab) or CTLA4 (Ipilimumab) [in addition to 3 PD-L1 inhibitors] following the demonstration of their therapeutic efficacy by J. Allison of Univ. of Texas M. D. Anderson Cancer Center (USA; Nobel prize 2018).  Despite the media hype, the therapeutic efficacy of 'immune checkpoint blockade' drugs is limited to ~20% of melanoma patients (plus small cell lung cancer, head and neck cancer and others potentially), which is difficult to explain based on the mechanism.  The puzzle remains a key unresolved issue along with the side effects it causes (ex. thyroiditis requiring hormone therapy). (Bardhan et al. 2016).

                         

All currently FDA approved immune checkpoint inhibitors are monoclonal antibodies.  As such, their limited ability to penetrate solid tumors represents a critical disadvantage for solid tumor therapy (Deng et al. 2016).  Another issue (not unexpected) is the reduction of T cells expressing PD-1 or PD-L1 by the anti-PD-1 antibody-based drugs, which compromises the efficacy of immunotherapy.  The decrease in T cells occurs due to the recognition of antibody-coated T cells by NK (Natural Killer) immune cells, which release perforin and proteolytic enzymes (granzymes) to kill the target cells (Brahmer et al., 2010).  Another mechanism through which antibody-coated cells can be lysed is via 'complement mediated cytotoxicity'; however, this path can be bypassed for type 4 IgG antibodies, which have been used to develop the 'immune checkpoint inhibitors'.

To remedy the situation, peptides capable of disrupting the PD-1 and PD-L1 interaction are increasingly being sought.  Unlike the antibodies, small synthetic peptides can penetrate solid tumors effectively, exhibit little immunogenicity, conjugate to tumor-targeting agents or encapsulate in nanoparticles readily, manufacture easily, and disrupt the protein-to-interaction (unlike the chemical drugs).  To this end, the investigators at the University of Missouri (USA) isolated a peptide that blocks the PD-1/PD-L1 interaction by screening a random-peptide displaying phage library (Liu et al., 2019).

Of relevance is the continuing modification of the peptides displayed by the bacteriophages to improve the phage display technology.   These include the peptides (whose termini could be bound to a scaffold) developed by the Bicycle Therapeutics (United Kingdom).  The peptide could be designed to encode multiple sequences in tandem, which could then bind to the different facets of the same scaffold.   The resultant peptide may engage multiple therapeutic targets simultaneously.

 

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References

Bardhan K, Anagnostou T, et al. The PD1:PD-L1/2 Pathway from Discovery to Clinical Implementation.  Front Immunol.  7:550 (2016).  PMID: 28018338

Brahmer JR, Drake CG, et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.  J Clin Oncol. 28:3167-75 (2010). PMID: 20516446

Deng R, Bumbaca D, et al. Preclinical pharmacokinetics, pharmacodynamics, tissue distribution, and tumor penetration of anti-PD-L1 monoclonal antibody, an immune checkpoint inhibitor.   MAbs. 8:593-603 (2016).   PMID: 26918260

Liu H, Zhao Z, et al.  Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy. J Immunother Cancer.  7:270 (2019).  PMID: 31640814

Robert C. A decade of immune-checkpoint inhibitors in cancer therapy.   Nat Commun. 11:3801 (2020)  PMID: 32732879

Sharma P, Siddiqui BA, et al. The Next Decade of Immune Checkpoint Therapy.  Cancer Discov.  11:838-857 (2021).  PMID: 33811120