Bispecific therapeutics for next-generation combination immunotherapy

Introduction: Immunotherapy has brought a paradigm shift in cancer treatment. However, most patients have failed to show an objective response to immunotherapy. Many factors undermine the response of immunotherapy, which include (1) activation of complementary immune checkpoints, (2) heterogeneous immune checkpoint expression, (3) lack of functional immune cells in the tumor microenvironment, (4) adaptive resistance to the therapy, etc. So, translational approaches are necessary to develop integrative technology by combining multiple therapeutic strategies.

Recently, the use of bispecific antibodies has gained tremendous attention because of their specificity and dual antigen-binding properties1-3. In a parallel paradigm, antibody-drug conjugates (ADCs) showed enhanced clinical efficacy by delivering the cytotoxic payload specifically to the cancer cells by leveraging the precise antigen-targeting capability of the antibody4-8. We bridge the gap between these two paradigms by rationally designing bi-specific therapeutics. We have explored both (1) bispecific antibody-drug conjugate (ADC) and (2) bispecific antibody-conjugated drug-loaded nanotherapeutics (Fig.1). The ADCs are superior in terms of favorable pharmacokinetics, whereas the ADNs have advantages because of their huge drug loading capacity (drug to antibody ratio, DAR ~300), higher tumor homing capability, and easily modifiable formulation. Here, we demonstrate the bi-specific antibody conjugated drug-loaded nanotherapeutics (ADNs) consisting of anti-PDL1 and anti-CD47 immune checkpoint inhibitors (ICIs) and pharmacological PI3K inhibitor PI103. We hypothesized that (1) using multiple ICIs activating adaptive and innate immunity, (2) combining with targeted therapy inhibiting PI3K, and (3) precise delivery of the drugs to the cancer cells may offer increased treatment outcomes in solid tumors.

Materials and

Methods: At first, the drugs, PI103, were conjugated with cholesterol via an EDC-NHS-based coupling reaction to convert the drug to an active supramolecule. The liposomal nanoparticles (ADN) were prepared by a thin-film hydration technique after uniform mixing of phosphatidylcholine (Soy-PC), DSPE-PEG-COOH, and drug with the molar ratio 6:3:1. The antibodies were conjugated with the liposomes via coupling reaction with the terminal COOH groups of DSPE-PEG-COOH. The COOH group has been converted to activate ester via EDC-NHS reaction followed by conjugation of the antibody using EDC-NHS chemistry. Human lung adenocarcinoma (A549), mouse Lewis-lung carcinoma (LLC), and lung squamous cell carcinoma (KLN-205) cells were used for in vitro studies, and the in vivo study has been carried out in syngeneic LLC and KLN-205mouse model using male C57BL/6 and DBA/2 mice. The LLC (0.5 million) and KLN-205 (0.75 million) cancer cells were injected subcutaneously, and the treatment was started after the tumor volume reached 70mm3. The endpoint was decided based on tumor volume>2000mm3 or body weight loss of more than 20% or death of the animal. We have used CD47 and PDL1 antigen-coated ELISA plates to understand the binding of the ADNs to the antigen. We have used a Novocyte flow cytometer and Nikon Ti2 inverted fluorescence microscope for internalization studies. The patient tissue microarray was purchased from TissueArray.com, and the images were acquired in a TissueFAXS Plus slide scanner (TissueGnostics USA) equipped with Hamamatsu Orca Flash 4.0 V2 cooled digital CMOS camera. The quantification of the images was performed by TissueQuest 7.0.1.147 software.

Results, Conclusions, and Discussions: Result and

Discussion: At first, we streamlined an ex-vivo pipeline for the estimation of the immune checkpoint expression in solid tumors by immunohistochemistry. The fluorescence image analysis revealed the heterogenous expression of both CD47 and PDL1 in non-small cell lung cancer (NSCLC) patients (Fig.2). We demonstrate the synthesis of a bispecific-ADN composed of anti-CD47 and anti-PDL1 (Fig.3A) and the characterization of diameter and surface charge (Fig.3B-D). The successful conjugation of both anti-CD47 and anti-PDL1 antibodies was evaluated by investigating the immobilization of the ADNs on antigen-coated ELISA plate (Fig.3E). It was further verified by internalization and the colocalization of the red and green fluorescence of the anti-CD47(FITC)-PDL1(APC)-ADNs in A549 cells (Fig.3F). The anti-CD47-PDL1-ADN has shown increased cellular internalization than monospecific-ADNs and non-targeted IgG-ADN because of the preferential cell surface attachment and receptor internalization (Fig.4A-C). Increased cellular internalization also contributes to the increased cytotoxicity of the anti-CD47-PDL1-ADN than monospecific-ADNs (Fig.4D). The anti-CD47-PDL1-ADN has shown significantly lower cytotoxicity in normal lung fibroblast (MRC-5), which has lower CD47 and PDL1 expression than A549 cells (Fig.4E). We have found increase in phagocytosis of LLC cells by mouse macrophage (Raw264.7) in presence of the anti-CD47-PDL1-ADN (Fig.4F). The anti-CD47-PDL1-ADN has shown significantly lower tumor growth compared to untreated control and traditional ICIs in both LLC (Fig.5A) and KLN-205 (Fig.5B) tumor models. The blood biochemistry of anti-CD47-PDL1-ADN treated mice shows no significant toxic side effect (Fig.5C). The anti-CD47-PDL1-ADN treatment mice have shown increased survival than other treatment groups (Fig.5D). The single-cell analysis of the tumor by FACS revealed an increased CD8 T-cells in the anti-CD47-PDL1-ADN treated mice than control mice (Fig.5E). The immunofluorescence images form the tumor sections show the increased T cells and monocyte presence in the anti-CD47-PDL1-ADN tumor (Fig.5F).

Conclusion: In summary, we have introduced a bispecific-ADN platform that enables targeting multiple immune checkpoints and specific delivery of drugs to the tumor tissue. The ADNs are even effective in tumors with a lower expression of either PDL1 or CD47, which makes it applicable in patients with low PDL1 expression, where traditional immunotherapy fails to respond. The studies related to bispecific ADC are in progress.

Acknowledgements (Optional):

References:
1 Labrijn, A. F., Janmaat, M. L., Reichert, J. M. & Parren, P. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov 18, 585-608 (2019). https://doi.org:10.1038/s41573-019-0028-1
2 Piccione, E. C. et al. A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells. MAbs 7, 946-956 (2015). https://doi.org:10.1080/19420862.2015.1062192
3 Rader, C. Bispecific antibodies in cancer immunotherapy. Curr Opin Biotechnol 65, 9-16 (2019). https://doi.org:10.1016/j.copbio.2019.11.020
4 Coats, S. et al. Antibody-Drug Conjugates: Future Directions in Clinical and Translational Strategies to Improve the Therapeutic Index. Clin Cancer Res 25, 5441-5448 (2019). https://doi.org:10.1158/1078-0432.Ccr-19-0272
5 Gupta, N. et al. Computationally designed antibody-drug conjugates self-assembled via affinity ligands. Nat Biomed Eng 3, 917-929 (2019). https://doi.org:10.1038/s41551-019-0470-8
6 Gupta, N. et al. Development of a facile antibody-drug conjugate platform for increased stability and homogeneity. Chem Sci 8, 2387-2395 (2017). https://doi.org:10.1039/c6sc05149a
7 Strop, P. et al. Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading. Nat Biotechnol 33, 694-696 (2015). https://doi.org:10.1038/nbt.3274
8 Sau, S., Petrovici, A., Alsaab, H. O., Bhise, K. & Iyer, A. K. PDL-1 Antibody Drug Conjugate for Selective Chemo-Guided Immune Modulation of Cancer. Cancers (Basel) 11 (2019). https://doi.org:10.3390/cancers11020232