Despite advances with therapies targeting the programmed cell death protein 1 (PD-1) or its ligand (PD-L1), many cancer patients are refractory to or relapse following treatment. Resistance to anti-PD-1 treatment is associated with upregulation of other checkpoint inhibitor receptors such as LAG-3 (Lymphocyte Activation Gene 3). FS118, currently being evaluated in a Phase 1 clinical trial in patients with advanced malignancies (NCT03440437), is a tetravalent bispecific antibody targeting LAG-3 and PD-L1, two immune checkpoint molecules that promote tumour escape from immune surveillance. We have characterised both in vitro and in vivo the functional activity of FS118 and find that this bispecific antibody can overcome PD-L1 and LAG-3 immune suppressive signals. We report a potential novel mechanism of action not observed with the combination of single PD-L1 and LAG-3 antibodies. Our results indicate that FS118 represents a possible novel approach to overcome some of the mechanism of resistance to PD-(L)1 blockade.

SUMMARYPrevious studies have identi®ed a 210 000-molecular weight molecule expressed at a high level on the surface of dendritic cells (DCs) in afferent lymph of cattle and evident on cells with the morphology of DCs in lymphoid tissues. Expression is either absent from other immune cells or is present at a lower level. The molecular weight and cellular distribution suggested that the molecule, called bovine WC6 antigen (workshop cluster), might be an orthologue of human DEC-205 (CD205). To establish whether this was the case, the open reading frame of bovine DEC-205 was ampli®ed, by polymerase chain reaction, from thymic cDNA (accession no. AY264845). The cDNA sequence of bovine DEC-205 had 86% and 78% nucleic acid identity with human and mouse molecules, respectively. COS-7 cells transfected with a plasmid containing the cattle DEC-205 coding region expressed a molecule that stained with WC6-speci®c monoclonal antibody, showing that ruminant WC6 is an orthologue of DEC-205. Twocolour¯ow cytometry of mononuclear cells from afferent lymph draining cattle skin, and from blood, con®rmed the high level of expression on large cells in lymph that were uniformly DC-LAMP positive and major histocompatibility complex class II positive. Within this DC-LAMP population were subpopulations of cells that expressed the mannose receptor or SIRPa. The observations imply that DCs in afferent lymph are all high , but not a uniform population of homogeneous mature DCs.

Two major sub‐populations of dendritic cells (DC) are present in afferent lymph draining the skin of cattle distinguished by expression of signal regulator protein alpha (SIRPα). The SIRPα– population expresses the uncharacterized bovine WC10 antigen (Ag). Initial N‐terminal sequencing of the WC10 protein purified by affinity chromatography showed significant homology with human CD26. A cDNA encoding bovine CD26 was cloned and the recombinant molecule expressed in COS‐7 cells. Transfectants abrogated the ability of macrophage‐derived chemokine (MDC) to cause a calcium flux in bovine PBMC indicating enzymatic activity characteristic of CD26. They also stained with WC10 monoclonal antibody confirming that the Ag is CD26. This is the first description of CD26 expression by DC in vivo or in vitro. It is expressed on a sub‐population of ex vivo DC in afferent lymph draining the skin and on sub‐populations of DC isolated from prescapular and mesenteric lymph nodes draining the skin or intestine, respectively. CD26 is an exopeptidase with specificity for motifs within the receptor‐binding domain of several chemokines including MDC. CD26 mediated truncation of MDC affects the Th cell response effected by the chemokine and may produce a Th1 bias. Transcripts for MDC were present in both CD26+ and CD26– DC, thus CD26mediated modification of MDC may bias the immune response induced in naive T cells by DC.

Purpose: With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic. Experimental Design: A bispecific antibody (FS222) was developed by engineering CD137 antigen-binding sites into the Fc region of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated using multiple in vitro assays. The antitumor efficacy, survival benefit, pharmacodynamics, and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumor models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 were investigated in a non-human primate dose-range finding study. Results: We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T-cell activation across CD8 þ T-cell activation assays in a PD-L1-dependent manner with a CD137/PD-L1 bispecific antibody, FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumor growth inhibition and survival benefit, concomitant with CD8 þ T-cell activation, in CT26 and MC38 syngeneic mouse tumor models. Conclusions: By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumor microenvironment, FS222 has the potential to leverage a focused, potent, and safe immune response augmenting the PD-(L)1 axis blockade.

In October 2013, the International Life Sciences Institute - Health and Environmental Sciences Institute Immunotoxicology Technical Committee (ILSI-HESI ITC) held a one-day workshop entitled, "Workshop on Cytokine Release: State-of-the-Science, Current Challenges and Future Directions". The workshop brought together scientists from pharmaceutical, academic, health authority, and contract research organizations to discuss novel approaches and current challenges for the use of in vitro cytokine release assays (CRAs) for the identification of cytokine release syndrome (CRS) potential of novel monoclonal antibody (mAb) therapeutics. Topics presented encompassed a regulatory perspective on cytokine release and assessment, case studies regarding the translatability of preclinical cytokine data to the clinic, and the latest state of the science of CRAs, including comparisons between mAb therapeutics within one platform and across several assay platforms, a novel physiological assay platform, and assay optimization approaches such as determination of FcR expression profiles and use of statistical tests. The data and approaches presented confirmed that multiple CRA platforms are in use for identification of CRS potential and that the choice of a particular CRA platform is highly dependent on the availability of resources for individual laboratories (e.g. positive and negative controls, number of human blood donors), the assay through-put required, and the mechanism-of-action of the therapeutic candidate to be tested. Workshop participants agreed that more data on the predictive performance of CRA platforms is needed, and current efforts to compare in vitro assay results with clinical cytokine assessments were discussed. In summary, many laboratories continue to focus research efforts on the improvement of the translatability of current CRA platforms as well explore novel approaches which may lead to more accurate, and potentially patient-specific, CRS prediction in the future.