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Increased Tumor Penetration of Single-Domain Antibody Drug Author Manuscript Published OnlineFirst on January 15, 2020; DOI: 10.1158/0008-5472.CAN-19-2295 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Increased Tumor Penetration of Single-Domain Antibody Drug Conjugates Improves In Vivo Efficacy in Prostate Cancer Models Ian Nessler1, Eshita Khera1, Steven Vance2, Anna Kopp1, Qifeng Qiu3, Thomas A. Keating3, Adnan O. Abu-Yousif4, Thomas Sandal2, James Legg2, Lorraine Thompson2, Normann Goodwin2, and Greg M. Thurber1,5 1 – Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109 2 – Crescendo Biologics, Cambridge, United Kingdom 3 – ImmunoGen, Waltham, MA, 02451 4 – Takeda Pharmaceuticals, Cambridge, MA, 02139 5 – Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109 Statement of Significance A mechanistic study of protein-drug conjugates demonstrates that a lower potency compound is more effective in vivo than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution. Conflict of Interest SV, TS, JL, LT, and NG were employed by Crescendo, QQ and TAK were employed by Immunogen, and AA was employed by Takeda during the study. GMT sits on the Scientific Advisory Board of Advanced Proteome Therapeutics. Running Title: Tumor penetration drives efficacy of protein-drug conjugates Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 15, 2020; DOI: 10.1158/0008-5472.CAN-19-2295 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA approved antibody-drug conjugates (ADCs). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of Prostate-Specific Membrane Antigen (PSMA)-binding single- domain (Humabody®) ADC constructs to demonstrate that tissue penetration of protein-drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower in vitro potency resulted in higher in vivo efficacy than other protein-drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin binding domain to the single-domain ADCs. A construct lacking an albumin binding domain was rapidly cleared leading to lower tumor uptake (%ID/g) and decreased in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro potency alone for these protein-drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing. Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 15, 2020; DOI: 10.1158/0008-5472.CAN-19-2295 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Antibody-drug conjugates (ADCs) have opened a new field of targeted therapeutics based on ‘hybrid’ drugs combining desirable targeting properties of biologics with the potency of small molecule cytotoxic payloads. For ADCs, the protein carrier is typically a monoclonal antibody that specifically binds to a target antigen expressed on cancer cells, increasing the delivery of the small molecule payload to the site of action in vivo. To date, seven ADCs have been FDA approved1, 2, most recently enfortumab vedotin and trastuzumab deruxtecan, with a large pipeline of >70 in clinical trials. However, one drawback of antibodies is slow tumor penetration. The tumor uptake of antibodies is limited by their extravasation rate, and they tend to penetrate only a few cell layers outside of blood vessels due to their rapid antigen-binding rate relative to intratumoral diffusion3, 4. In the clinic, unconjugated antibodies are often well tolerated, such that they can be delivered at very high doses that saturate receptors on cell layers closer to the blood vessel, enabling the antibody to diffuse farther through the tumor. However, the payload toxicity of ADCs limits the dose and frequency of administration, restricting tumor penetration depths and allowing regrowth between doses (typically given every 3 weeks for current therapies)5. Therefore, devising design and treatment strategies that increase tumor penetration may yield greater efficacy and improve clinical success rates for ADCs and other protein-drug conjugates. Many current ADCs in late-stage clinical trials are targeting receptors with high expression (e.g. TROP-2, HER2, folate receptor, and Nectin-4) where tissue penetration can play a significant role in efficacy. Previous work based on co-administration of trastuzumab with the ADC T-DM1 (ado-trastuzumab emtansine) demonstrated that higher tissue penetration could yield better efficacy in a mouse model of highly expressed HER2 positive cancer6. Based on a literature review of studies involving ADCs with a range of drug-to-antibody ratios (DAR)7, ADCs that delivered the same payload dose at a lower DAR (i.e. higher protein doses) generally yielded an increase in efficacy in animal models with moderate to high expression, likely as a result of better tissue penetration8. Although bystander effects can help mitigate transport challenges by allowing the payload to diffuse deeper into the tissue following release from the protein, analysis of literature data and predictive simulations indicated that higher tissue penetration is more efficient at improving efficacy even when using bystander payloads8. When the ADC is more uniformly distributed, the same total amount of cytotoxic payload delivered to the tumor is spread more homogeneously, so cells adjacent to vessels that receive an overdose of payload with heterogeneous ADC distribution now receive fewer payloads. Therefore, implicit in this approach is that the amount of payload delivered per cell exceeds the intrinsic potency of the payload, thereby maintaining a lethal cellular dose while increasing penetration to reach more cells. Similarly, lower target receptor expression can improve efficacy for extremely potent ADCs by enabling deeper tissue penetration. Lower receptor expression limits the amount of bound and internalized ADC for perivascular cells but increases tumor tissue penetration since penetration depth is inversely proportional to expression7, 9. Based on these and other results10, tumor tissue penetration is critical for the overall efficacy of ADC treatment and should be analyzed when optimizing protein-drug conjugates for maximum effectiveness. Besides increasing the dose or co-administering the ADC with unconjugated antibody, there are protein engineering approaches to develop scaffolds for improved tissue penetration11- 14. This may be particularly advantageous for highly expressed targets that would require very Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 15, 2020; DOI: 10.1158/0008-5472.CAN-19-2295 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. large antibody doses to saturate the tumor or for targeting metastases with varying expression levels. Reducing affinity is one method of increasing tissue penetration15. However, for large, bivalent IgGs, the intrinsic affinity of each Fab arm must be very low to penetrate tissue with 16 highly expressed antigens (e.g. 270 nM Kd) . Instead of modulating affinity, lower molecular weight proteins can penetrate tissue farther before being immobilized through target binding17 because of their faster diffusion coefficient (which is the dominant mode of transport in tumors due to elevated interstitial pressure18). Some smaller scaffolds are already being tested in the clinic, such as caplacizumab - a bivalent single-domain antibody (~30kDa) used to block platelet aggregation19, providing precedent for these formats. A major limitation for these smaller scaffolds is their rapid renal clearance11, 20, 21. However, fusion to albumin binding domains can extend plasma half-life and avoid glomerular filtration by increasing the effective molecular weight while still enabling better tissue penetration22-26. In this work we investigated the tumor tissue pharmacokinetics and efficacy of 3 different Prostate-Specific Membrane Antigen (PSMA)-binding single-domain antibody (Humabody) drug conjugates and an IgG drug conjugate (referred to collectively as antibody drug conjugates or simply antibodies/antibody constructs if no drug is present). Humabodies are fully human, single heavy chain variable (VH) domains generated using a proprietary transgenic mouse platform27, 28 (Crescendo Biologics). The mice lack murine heavy chain, light chain, and light
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