Noninternalizing Targeted Cytotoxics for Cancer Therapy - Molecular

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Non-internalizing targeted cytotoxics for cancer therapy Giulio Casi, and Dario Neri Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500798y • Publication Date (Web): 04 Mar 2015 Downloaded from http://pubs.acs.org on March 11, 2015

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Molecular Pharmaceutics

Non-internalizing targeted cytotoxics for cancer therapy

Giulio Casi (1) and Dario Neri * (2)

(1) Philochem AG, Libernstrasse 3, CH8112, Otelfingen (ZH) Switzerland. (2) Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093 Zürich, Switzerland

Keywords: Antibody-drug conjugates; drug quantitative biodistribution; dosimetries; small molecule drug conjugates; non-internalizing antibody-drug conjugates; cancer

Conflict of interest disclosure: G.C. is employed at Philochem AG, D.N. is a co-founder and shareholder of Philogen SpA.

Graphical Abstract

1 ACS Paragon Plus Environment


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Abstract Conventional cancer chemotherapy is limited by the fact that small organic cytotoxic agents typically do not preferentially localize at the tumor site, causing unwanted toxicities to normal organs and limiting dose escalation to therapeutically active regimens. In principle, antibodies and other ligands could be used for the selective pharmacodelivery of cytotoxic agents to the tumor environment. While traditionally internalizing ligands have been used for such targeting applications, increasing experimental evidence suggests that the ligand-based delivery of anticancer drugs to the extracellular space in the tumor, followed by suitable release strategies, may mediate a potent anti-cancer activity. In this review, we outline the main requirements for the development of non-internalizing targeted cytotoxics.


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Internalizing vs. non-internalizing targeted cytotoxics Conventional cancer chemotherapy typically relies on the use of small-molecule drugs (e.g., cytotoxic agents), which are expected to preferentially kill cancer cells, or at least stop their growth. In most cases, drugs are used which target cells in rapid proliferation. As tumor cells are not the only class of cells in rapid division within the body, other healthy tissues (e.g., epithelial structure, cells in the bone marrow) are also frequently attacked by cancer chemotherapy, causing considerable side-effects, which may limit the dose escalation to therapeutically active regimens. One of the most important limitations of conventional cancer chemotherapy consists in the fact that most small molecule therapeutic agents (e.g., cytotoxic agents) do not preferentially localize at the tumor site. While this limitation has long been appreciated in mouse models of cancer 1, recent PET studies with radiolabeled drugs have unambiguously shown that conventional anticancer agents fail to preferentially target neoplastic structures in vivo and, indeed, accumulate in other healthy organs (e.g., those involved in the excretion process; Figure 1) 2,3

Figure 1: PET images at multiple time points of a patient with metastatic mesothelioma, injected with

11

C-docetaxel. Drug uptake in the neoplastic lesions is not visible, while the agent



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accumulates in various non-target tissues. Reproduced from van der Veldt, A.A.M. et al (2010) Eur. J. Nucl. Med. Mol. Imaging 37, 1950-1958 with permission.

Certain monoclonal antibodies have been shown to preferentially localize to tumors in vivo, even though a quantitative understanding of the targeting process in patients is often missing. For this reason, antibodies have been used as vehicles for the selective delivery of potent cytotoxic agents to neoplastic sites. The history of the development of antibody-drug conjugates (ADCs) has extensively been reviewed elsewhere 4,5 and will not be repeated here. More recently, peptides 6, small organic ligands 7-12 and polymers 13-16 have been used as vehicles for the delivery and slow release of cytotoxic agents in a novel class of targeted cytotoxics named small-targeted drug conjugates (SMDC). Typically the novel constructs are evaluated in imaging or biodistribution studies with diagnostic radionuclides. Subsequently, the most promising delivery agents are coupled on cytotoxic molecules for the treatment of cancer 17-19 and of chronic inflammation 20 in animal models. Some SMDC products have been studied in the clinic. 21,22 Virtually all small molecule anti-cancer drugs act at the intracellular level. For this reason, it has been widely assumed that the use of an internalizing ligands is an absolute requirement for the development of ADCs and similar classes of targeted cytotoxics. Indeed, it has been claimed that “targeting an ADC to a noninternalizing target antigen with the expectation that extracellulary released drug will diffuse into the target cell is not a recipe for a successful ADC “23. In principle, it would be highly desirable to deliver cytotoxic agents only to target cells which express the antigen recognized by the cognate antibody, sparing all other cells in the body. In practice, however, several factors limit the efficacy of internalizing targeted cytotoxics (e.g., internalizing ADCs). For example, IgG-based ADCs display an efficient binding to the neonatal FcRn receptor, which mediates the continuous internalization of ADC products in the endothelium and a long


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residence time for antibodies in the liver

24

. In addition, as outlined below, the real ability of

antibodies (and other ligands) to preferentially localize to solid tumors is often unknown. Even in preclinical models with lesions which strongly and homogenously express a tumor-associated target, the inefficient extravasation of antibodies and the antigen-barrier effect dramatically limit the targeting process, restricting the antibody accumulation to few perivascular tumor cells 25.

Recently, increasing experimental evidence has suggested that ADCs (and SMDCs), based on non-internalizing ligands, can efficiently deliver cytotoxic agents to neoplastic sites

26-30

. When

suitable linker-payload combinations are used, these targeted cytotoxics can mediate a potent anti-cancer activity in mouse models of cancer

31,32

In principle, it should be possible to

efficiently accumulate antibodies (or other ligands) in the tumor environment, releasing drugs in the extracellular space (e.g., through proteolytic cleavage or by reduction of disulfide bonds). These drugs could then internalize into tumor cells or other cellular targets (e.g., tumor endothelial cells), causing a localized damage. The real efficiency of this process is likely to depend on many factors, including the efficiency of antibody accumulation in the tumor, the kinetics of drug release, as well as the diffusion properties of the drug itself 33.

Non-internalizing ADCs (and SMDCs) would have a number of attractive features. First, a payload released in the extracellular space could diffuse in the immediate surroundings, thus causing a by-stander effect and targeting cells which may be antigen-negative. Second, antibody internalization would no longer represent a “black box” in the targeting process, which could be conveniently studied by imaging or biodistribution studies. Obviously, the study of drug release would remain a challenge for the quantitative description of the therapeutic process, but the differential labeling of antibodies and drugs could facilitate experimental investigations, at least



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at the microscopic level. Third, it would be possible to direct ADCs to markers expressed in the tumor neovasculature and in the subendothelial extracellular matrix. Those structures can be more accessible, abundant, and genetically stable, thus allowing an efficient tumor targeting with long residence time of the ligand at the site of disease. Alternatively, innovative technologies have been developed to promote potent anticancer activity also with poorly internalizing antibodies. 34

In addition to antibodies, non-internalizing small organic ligands (e.g., binders to carbonic anhydrase IX;

33,35,36

and certain polymeric carriers

13-16

) have been shown to be able to

preferentially localize at the tumor site and to mediate an efficient anti-cancer activity, when equipped with suitable linker-payload combinations (Figure 2)


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Figure 2: Immunofluorescence findings with ligands specific to carbonic anhydrase IX. a) Immunofluorescence studies on SKRC-52 tumor cells, revealing that acetazolamide-based ligands do not internalize; b) tumor accumulation of CAIX ligands in SKRC-52 lesions, revealed



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by immunofluorescence analysis 30 min and 1 hour after intravenous administration (Adapted from Krall, N et al (2014) Angew. Chem. Int. Ed. Engl., 53, 4231-4235 with permission).

Targeting efficiency and off-target effects In the mouse, the best monoclonal antibodies in IgG format are able to deliver anything between 10 and 100 percent of the injected dose per gram of tissue to the tumor (please consider that values > 100 % ID/g are possible, if the tumor is smaller than 1 gram)

29,37-39

. Full

immunoglobulins clear slowly from circulation, exhibiting %ID/g values in the blood of ~5-10 at 24 hours (immediately after injection, the %ID/g in the blood is 100:2.5 = 40 % ID/g, if the mouse has 2.5 ml of blood). Normal organs typically exhibit values similar to the ones for blood. However, clearance-associated organs (e.g., liver) tend to give higher values compared to blood, while some special structures (e.g., brain and muscle) exhibit lower antibody uptake values. The best monoclonal antibodies in IgG format have exhibited tumor:blood ratios of ~ 6:1 at 24 h in preclinical studies, while tumor:blood as large as ~ 20:1 and ~ 10:1 have been reported at the same time point for antibodies in diabody format and in mini-antibody format (e.g., small immune format), respectively 37.


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Figure 3: Biodistribution results in mice bearing F9 teratocarcinomas 24 hours after intravenous administration, obtained with radioiodinated preparations of the F8 antibody in diabody and in SIP format 37

In humans, the absolute uptake of antibodies decreases compared to the mouse, as a result of the different mass of the two organisms (50-100 Kg, compared to 20-25 grams). The best monoclonal antibodies in IgG format have exhibited ~0.01 % ID/g at 24 h in the tumor, with tumor:blood ratios of ~ 5:1

40-43

. Thus, antibodies are able to preferentially localize at the tumor site in patients,

but the vast majority of the antibody product does not end up in the tumor. In addition, a large variability from patient to patient and from lesion to lesion can be observed, based on dosimetric evaluation of Nuclear Medicine studies with radiolabeled antibodies

44,45

. One could say that

almost 100% of injected antibody products transit through clearance organs over time, while only a small portion of the same product ends up in the tumor.



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The tumor targeting properties of antibodies (and in particular IgGs) are limited by the slow and inefficient extravasation of these molecules. By contrast, small ligands can extravasate very rapidly (in some cases, in a matter of seconds), but would not remain at the tumor site, unless they displayed a potent binding affinity to abundant and accessible tumor-associated targets

33,46

.

The rapid diffusion properties of small molecules may contribute to exceptionally high tumor uptake values at early time points 10,12,20,35,36,47-49. At the same time, small ligands will be typically cleared very fast, in most cases via the renal route. Thus, small molecule drug conjugates (SMDCs) will transiently deliver large amounts of cytotoxic agents to clearance-associated organs, even though the kinetics of the process may be different, compared to antibodies. The identification of SMDCs, which rapidly transit through the kidneys without internalization, is likely to facilitate the development of efficacious targeted cytotoxics. This task, however, is not easy and requires both sophisticated imaging procedures and unconventional clinical translational activities, as outlined in the next section.

Hurdles which hamper the development of next-generation cytotoxic agents The field of antibody engineering has made tremendous progress over the last 20 years 50 51 and, in most cases, it is possible to raise a high-affinity fully human antibody against virtually any target protein of interest. In spite of this, the number of validated antibodies for tumor targeting applications, with a proven ability to preferentially localize on neoplastic masses, remains disappointingly low. While immunohistochemical data are becoming available for more than thousands of target antigens from the Protein Atlas Project [www.proteinatlas.org], quantitative biodistribution studies or imaging analyses (e.g. PET, SPECT) are seldom performed, possibly due to the limitations which many groups experience in the use of radioactively labeled products.


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Traditionally, it has been difficult to generate high-affinity binding peptides and small organic ligands against tumor-associated antigens. However, emerging technologies allow the synthesis and screening of combinatorial libraries of chemically modified peptides and small organic molecules, containing over 1 billion members 33,52-54. These technologies, which include ribosome display, phage display and DNA-encoding of chemical libraries, are likely to revolutionize the way small tumor targeting agents are discovered. As a consequence, it is conceivable that SMDCs and ADCs may be developed against the same targets, allowing a direct comparison of the two technologies.

The possibility to radioactively label both antibody and drug moieties has provided, for the first time, a quantitative understanding of the tumor targeting properties of ADC products in preclinical models

55

. However, at the clinical level, a quantitative evaluation of the targeting

properties of ADC products is often missing, hampering pharmaceutical development and patient selection strategies. In principle, attractive regulatory provisions are in place for Phase 0 imaging studies.1 56However, the microdosing requirements associated with Phase 0 studies prevent the imaging of ADC products at the doses, which are required for therapeutic purposes. The development of surrogate companion diagnostics has been clinically implemented for folatebased tumor targeting agents

22

. It is conceivable that similar theranostic strategies will be

implemented in the future, both for SMDC and ADC products.

1

EMEA ICH guideline M3(R2) on non-clinical safety studies for the conduct of human clinical trials and marketing authorisation for pharmaceuticals: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/ WC500002720.pdf



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Finally, the design of conventional Phase I clinical trials, with dose escalation procedures based on cohorts of multiple patients (e.g., 3 patients per cohort), often results in long Phase I clinical trials. In many countries, regulatory provisions for dose escalation studies have become more strict after the TGN412 disaster 57. However, the use of conservative dose escalation studies may expose patients to suboptimal doses, denying them an active therapeutic option and increasing the time to Phase II trials (i.e., the stage in drug development at which an experimental therapeutic agent is administered at the recommended dose to all patients, thus potentially offering the maximum benefit).

Cancer cures, combination strategies and concluding remarks ADCs and SMDCs have been reported to cure cancer in animal models of the disease, which do not respond to conventional cytotoxic agents. However, complete responses in cancer patients with clinical-stage ADC and SMDC products are rare. This striking difference in pharmacological activity will continue to stimulate scientific investigations, aimed at understanding the reasons behind the difficult translation of targeted cytotoxics from rodent models to patients. A quantitative characterization of the ligand-based tumor targeting process and of drug release mechanisms in mouse and man will be necessary for the development of nextgeneration targeted cytotoxics. Indeed, molecular imaging technologies are likely to play a crucial role for a “Precision Medicine” approach to drug development. If and when ADCs and SMDCs fails to cure cancer when used as single agents, then combination strategies may be required. Promising preclinical studies indicate that the combination of ADCs with immunostimulatory agents (e.g., immunocytokines), or the simultaneous “arming” of antibodies with cytotoxic agents and with immunostimulatory moieties

58,59

may facilitate the development of potent anti-cancer


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pharmaceuticals. The combination of cytokines and cytotoxic moieties is particularly attractive, due to their orthogonal toxicity profiles.

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Noninternalizing Targeted Cytotoxics for Cancer Therapy - Molecular

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