AntibodyâDrug Conjugates and Small MoleculeâDrug Conjugates

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Antibody-drug conjugates and small molecule-drug conjugates: opportunities and challenges for the development of selective anti-cancer cytotoxic agents Giulio Casi, and Dario Neri J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00457 • Publication Date (Web): 16 Jun 2015 Downloaded from http://pubs.acs.org on June 22, 2015

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Journal of Medicinal Chemistry

Antibody-drug conjugates and small molecule-drug conjugates: opportunities and challenges for the development of selective anti-cancer cytotoxic agents

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

(1) Philochem AG, Libernstrasse 3, CH8112, Otelfingen, 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; small molecule-drug conjugates; tumor targeting; antibody internalization; targeted cytotoxics

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

1 ACS Paragon Plus Environment


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Abstract Conventional cancer chemotherapy heavily relies on the use of cytotoxic agents, which typically do not preferentially localize at the tumor site and cause toxicity to normal organs, preventing dose escalation to therapeutically active regimens. In principle, antibodies and other ligands could be used for the selective pharmacodelivery of cytotoxic agents to the neoplastic mass. For many years, the availability of ligands, capable of selective internalization into tumor cells, has been considered to be an essential requirement for the development of targeted cytotoxics. This assumption, however, has recently been challenged on the basis of therapeutic data obtained with non-internalizing drug conjugates. Moreover, quantitative evaluations of the tumor targeting properties of antibodies and of small organic ligands have provided new insights for the implementation of optimal strategies for the development of targeted cytotoxics. In this article, we highlight opportunities and challenges associated with the clinical and industrial development of antibody-drug conjugates and small molecule-drug conjugates for cancer therapy.


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Conventional anti-cancer drugs and the first series of antibody-drug conjugates Conventional cancer chemotherapy typically relies on the use of small-molecule drugs (e.g., cytotoxic agents, kinase inhibitors), which inhibit the growth of cells undergoing rapid proliferation (or even kill them), thus offering a potential therapeutic benefit to cancer patients. The combination of highly toxic agents has revolutionized the treatment of many hematological malignancies, some of which are now curable. The induction of cancer cures for disseminated solid tumors by means of cytotoxic agents is more difficult, but some metastatic malignancies (e.g., metastatic testicular cancer)

1

can be eradicated by a combination of chemotherapeutic

drugs.

Unfortunately, the majority of patients with metastatic solid tumors die from the disease, after chemotherapeutic regimens have failed to induce objective response or when resistance develops. 2

One of the most serious limitations of conventional cancer chemotherapy relates to the fact that

most small molecule therapeutic agents (e.g., cytotoxic agents) do not preferentially localize at the tumor site. This pharmacokinetic limitation has long been appreciated in mouse models of cancer.

3, 4

Importantly, recent PET studies with radiolabeled drugs in cancer patients have

unambiguously shown that cytotoxic agents accumulate in certain healthy organs (e.g., those involved in the excretion process; Figure 1), while failing to efficiently target neoplastic masses in vivo. 5, 6



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

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11

C-docetaxel.

Drug uptake in the neoplastic lesions is not visible, while accumulation in the liver and in some other normal organ structures can be seen at multiple time points. The arrow to the chest indicates the pleural localization of the mesothelium. Reproduced with permission from van der Veldt, A.A.M. et al (2010) Eur. J. Nucl. Med. Mol. Imaging 37, 1950-1958.

Monoclonal antibodies have been considered as possible vehicles for the selective in vivo pharmacodelivery of drugs, under the assumption that the exquisite binding specificity of the antibody molecule would facilitate a selective accumulation at the site of disease. The history of the development of antibody-drug conjugates (ADCs) has extensively been reviewed elsewhere 79

and will not be repeated here. Nonetheless, a few aspects of antibody-based drug delivery

deserve a closer analysis:


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(i)

The real ability of antibody to preferentially localize at the site of disease, which is essential for the implementation of efficient pharmacodelivery strategies in cancer patients, remains largely unexplored in clinical trials. Few monoclonal antibodies have been studied with nuclear medicine procedures and adequate dosimetric analyses in a sufficiently large number of patients.10-19 In those studies, a large variability in tumor uptake, from patient to patient and from lesion to lesion, was observed. Antibodies which display tumor:organ and tumor:blood ratios > 5:1, 24 hours after intravenous injection, represent the exception rather than the rule.

(ii)

The antibody format (scFv, diabody, miniantibody or small immune proteins (SIP), IgG; Figure 2a) greatly impacts on pharmacokinetic properties and on tumor uptake. A number of independent studies have found that mini-antibodies or SIPs may exhibit the best compromise between efficient tumor uptake and a sufficiently rapid clearance from circulation.20-22 Nonetheless, if maximal antibody accumulation at the tumor site is desired, the IgG format may represent the preferred one, as its long circulation time in blood counterbalances the inherently slow extravasation process of proteins and of antibodies.21, 23, 24 However, in humans, typically more than 99% of the antibody administered, does not reach the tumor.

10-19

The persistent high-levels

of drug-conjugate in blood may be responsible for undesired toxicities as a consequence of both premature, and undesired drug release in off-target organs (e.g. endothelium, liver; Figure 2b). (iii)

A microscopic analysis of antibody localization within the tumor mass exhibits a preferential accumulation on perivascular tumor cells.25 This feature, which may hinder certain pharmacodelivery strategies, most probably results from the inefficient



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extravasation of antibody molecules and by their trapping by antigen on perivascular tumor cells (the so-called “antigen barrier effect”;26-28 Figure 2c). (iv)

While initially low-potency drugs (e.g., doxorubicin) were used for ADC development programs,29 the field has gradually progressed towards the use of ultrapotent payloads, often capable of killing cells at sub-nanomolar concentrations. The use of highly cytotoxic drugs is also justified by cost-of-goods considerations, as monoclonal antibodies (in IgG format) have a molecular weight of 150’000 Da, while drugs are typically smaller than 1’000 Da. It is probably not economically feasible to administer doses of more than one gram of antibody-drug conjugate to a patient. Less potent drugs may be used for ADC applications, if multivalent (e.g., dendrimeric) linkers are used, leading to high drug:antibody ratios.30

a)

b)

c)

Figure 2: (a) Schematic representation showing domain composition of engineered fragments including scFv (25 kDa), diabody (55 kDa), SIP/minibody (80 kDa) and intact antibodies (150 kDa). (b) Macroscopic distribution of

64

Cu-

DOTA-trastuzumab in breast cancer patients (1, 24 and 48 h) following intravenous administration. The majority of injected antibody molecules do not reach their target in vivo: virtually all of them accumulate (at least transiently) in excretory organs (liver for intact antibodies, kidneys for small antibody fragments)(c) microscopic images of tumor tissue after trastuzumab-FITC conjugate injection (green). Tumor blood vessels (red) and nuclei (blue) were also stained. The image depicts trapping of Trastuzumab in the vicinity of blood vessels (Adapted by permission from the


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American Association for Cancer Research: Dennis, M. S. et al. Imaging tumors with an albumin-binding Fab, a novel tumor-targeting agent, Cancer Res, January 1, 2007, 67, 254–261, 10.1158/0008-5472.can-06-2531).

Two ADC products are currently available on the market for cancer therapy applications: brentuximab vedotin and trastuzumab emtansine.

Brentuximab vedotin is an anti-CD30 antibody, coupled to the MMAE auristatin, by a cleavable linker comprising a valine-citruline moiety and a self-immolating spacer (Figure 3). The product has been approved for the treatment of last-line Hodgkin lymphoma and anaplastic large cell lymphoma, on the basis of compelling objective responses observed in phase II clinical trials in heavily pretreated patients.31 In particular, brentuximab vedotin (when dosed at 1.8 mg/Kg, every three weeks) mediated 75% overall responses in Hodgkin lymphoma patients that had failed autologous stem cell transplant, with 34% of the patients experiencing complete responses which lasted on average 29 months. In the anaplastic large cell lymphoma settings, brentuximab vedotin provided a substantial benefit to patients that failed at least one multi-agent chemotherapy regimen, with 87% overall response rates, of which 57% were complete responses, and a progression free survival of 12.6 months. Importantly the treatments were well tolerated, with the most common side effects reported as neutropenia, peripheral sensory neuropathy, fatigue, nausea, fever and thrombocytopenia. These encouraging data have recently been complemented by the results of a randomized phase III clinical trial in the consolidation setting for Hodgkin lymphoma patients who had received a bone marrow transplant. A new phase III trials that features a combination of brentuximab vedotin and chemotherapy was recently started in younger patients with newly diagnosed Hodgkin lymphoma (NCT-02166463).



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Trastuzumab emtansine consists of the anti-HER2 trastuzumab antibody, armed with the maytansinoid DM1, equipped with a non-cleavable linker (Figure 3). It has been proposed that the drug may be released after product internalization, upon intracellular proteolytic digestion of the antibody moiety.32 33 However, a certain instability of the ADC in blood has been documented, with maleimide elimination occurring slowly under physiological conditions.34 Trastuzumab emtansine has received marketing authorization for the second-line treatment of a subset of patients with metastatic breast cancer. Specifically, it has been approved for the treatment of patients with HER2-positive metastatic breast cancer that has previously been treated with Herceptin and a taxane chemotherapy. Trastuzumab emtansine showed convincing clinical efficacy in a phase III clinical study in 991 patients treated with 3.6 mg /Kg of ADC every three weeks, in comparison to established chemotherapy with lapatinib and capecitabine. Progression free survival (9.6 months versus 6.4 months) and overall survival (30.9 months versus 25.1 months) were improved in the ADC-treatment arm. Safety was significantly improved compared to chemotherapy, with substantially fewer grade 3/4 adverse events.35 In a recent front-line phase III clinical study in breast cancer patients, however, trastuzumab emtansine was not found to be superior to the corresponding naked trastuzumab antibody, used in combination with either docetaxel or paclitaxel chemotherapy.

Figure 3: Structures of the two antibody-drug conjugate products currently available on the market for cancer therapy: Brentuximab vedotin (left) and Trastuzumab emtansine (right).


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Trastuzumab emtansine is used in the clinic at 3.6 mg/Kg doses, while tumor eradications with the same product have been observed in mice at doses of 15 mg/Kg.36 Chemically defined antiHer2 ADCs, with precise control of site and stoichiometry of drug conjugation, displayed improved half-life, efficacy and safety, relative to conventional heterogeneous ADCs, in rodent xenograft models.37-40 Large clinical experience is available from gemtuzumab ozogamicin, a humanized IgG4 kappa CD33 antibody conjugated to the DNA-binder calicheamicin-γ1 via a hydrolysis sensitive hydrazone. Gemtuzumab ozogamicin received accelerated US marketing approval in 2000 for the treatment of patients of 60 years of age and older with CD33+ AML in first relapse, who were not eligible for standard chemotherapy. Subsequent trials failed to confirm therapeutic benefits at doses of 9 mg/m2 when GO was used in combination with chemotherapy. These data and the slight increase in deaths in the group receiving the ADC compared to those receiving chemotherapy, led to voluntary withdrawal of the drug in 2010 from the market. Interestingly the acid-sensitive hydrazone was required to elicit potent antitumor activity in an antigen independent manner on solid tumors in vivo.41 Gemtuzumab ozogamicin is also active in AML patients whose cells apparently do not express CD33.

42

The product remains available for compassionate use,

and recent studies have showed promising efficacy when the ADC was administered either following a fractionated schedule

43, 44

or to patients with more favorable prognosis AML during

standard induction therapy.45-48

Additionally, many promising ADC products can eradicate tumors in mice, at doses which are well tolerated. For example, an anti-CD33 antibody, coupled to a potent DNA alkylating drug of the pyrrolo-benzodiazepine family, has recently been shown to eradicate subcutaneously-grafted AML chloroma lesions in mice at doses as low as 0.1 mg/Kg.49



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It remains to be seen, however, how easily these promising preclinical therapeutic observations can be translated to the human situation. For example, if the tumor targeting performance (e.g., tumor uptake) of an ADC is much better in rodents than in patients, the corresponding therapeutic behavior may be different in the two species.

The need for antibody internalization: fiction or reality? Virtually all small molecule anti-cancer drugs exert their activity by binding to an intracellular target. For this reason, it has been widely assumed that the use of internalizing pharmacodelivery vehicles may represent an absolute requirement for ADC development. 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“.50 In principle, the ability to deliver cytotoxic agents only to target cells which express the antigen recognized by the cognate antibody, sparing all other cells in the body, would represent an extremely attractive therapeutic feature. In practice, however, several factors limit the performance of internalizing ADCs, including the moderate accumulation of antibody molecules at the tumor site (typically < 0.1 % injected dose per gram of tumor in humans)10-19 and the heterogenous antibody distribution within the tumor mass. In addition, while the antibody internalization process can easily be studied in vitro, the same process often remains a “black box” in vivo, as it is difficult to recover and analyze neoplastic masses from patients.

Recently, an increasing body of experimental evidence has shown that certain ADCs, based on non-internalizing ligands, can efficiently deliver cytotoxic agents to neoplastic masses.51-55 When suitable linker-payload combinations are used, these targeted cytotoxics can liberate their payload


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at the tumor site and display a potent anti-cancer activity in mouse models of cancer.56-58 In principle, it should be possible to use antibodies for the efficient delivery and release of drugs in the extracellular tumor space (e.g., through proteolytic cleavage or by reduction of disulfide bonds).56-60 The drugs could then internalize into tumor cells or other cellular targets (e.g., tumor endothelial cells, tumor resident leukocytes), causing localized damage. The therapeutic performance of these ADCs is likely to depend on many factors, including the antibody’s tumor targeting efficiency, the kinetics of drug release, as well as the diffusion properties of the drug payload.

Splice isoforms of fibronectin and of tenascin-C are abundantly found in the tumor subendothelial extracellular matrix making them attractive non-internalizing targets for pharmacodelivery applications.51-55 These antigens are more accessible, abundant, and genetically stable than tumorassociated antigens located on the cell membrane, thus facilitating the development of ADC products with long residence time at the tumor site. The ADCs would release their payload in the extracellular space, facilitating a by-stander effect on neighboring tumor cells. As antibody internalization would no longer be needed, biodistribution and imaging data, obtained with noninternalizing antibodies, could be more easily integrated into modeling studies for the prediction of ADC in vivo performance.

Small ligands and peptides as alternative to antibodies for pharmacodelivery applications In principle, small organic ligands and peptides could be used as alternatives to antibodies for tumor targeting applications. While high-affinity human monoclonal antibodies can be raised against virtually all protein targets (for example, by panning combinatorial phage display libraries )61 it is not always possible to isolate small organic or peptidic ligands to proteins of



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pharmaceutical interest. Nonetheless, a number of small ligands to tumor-associated antigens have been characterized in terms of their tumor targeting properties and for pharmacodelivery applications.4, 62 Some of the most promising compounds in this category are depicted in Figure 4. They include folate derivatives (binding to the folate receptor, a protein over-expressed in ovarian cancer and in other malignancies)63, glutamic acid urea derivatives (binding to the prostate specific membrane antigen, a surface marker of prostate cancer cells)64-66, somatostatin analogues (particularly indicated for neuroendocrine tumors)67 and certain aromatic sulfonamides, specific to carbonic anhydrase IX (a marker of hypoxia and of renal cell carcinoma).58, 68-71

Figure 4 Structures of relevant classes of small molecule tumor targeting moieties. For a more comprehensive list of small molecule-drug conjugates (SMDCs) in preclinical and clinical development, see Low et al. (2015) [Ref. 63].


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The opportunity of using small ligands for pharmacodelivery applications has recently been reviewed in detail.4, 62 There is a strong evidence that ligands with a dissociation constant to their target of 10 nM or better may be needed, in order to achieve an efficient tumor targeting performance.62, 70 In some cases, multivalent binding (“avidity”) may compensate for insufficient affinity,69, 72-74 but almost all products in clinical development feature a single tumor-targeting moiety, coupled to a potent cytotoxic payload.62 Traditionally, the discovery of small ligands to tumor associated antigens has been difficult and has often relied on the synthesis of analogues to naturally-occurring ligands. However, progress in DNA-encoded chemical library technologies promises to facilitate the hit discovery process.4 75-77

Indeed, the tagging of individual compounds with DNA-fragments, serving as amplifiable

identification barcodes, in split&pool synthetic strategies, allows the construction and screening of chemical libraries containing millions of molecules. Similarly, the discovery of high-affinity binding cyclic peptides has been greatly facilitated by the construction and selection of very large combinatorial libraries, featuring the incorporation of non-natural aminoacids 78 or the cyclization of cysteine-containing peptides with reactive chemical moieties serving as structural scaffolds.79

Folic acid derivatives probably represent the first small molecule ligands, which have successfully been used for the selective pharmacodelivery of cytotoxic drugs and other payloads to the tumor environment.62 The folic acid receptor is present at low levels in most normal tissues, with the exception of kidney, spleen and lung tissue.80 By contrast, the same target is strongly upregulated in many epithelial cancers, including tumors of the ovary, breast, lung, colon and kidney.62 Vintafolide (also known as EC145)81 is a conjugate consisting of folic acid as a targeting moiety, connected to desacetylvinblastine as toxic payload through a linker comprising a charged peptide linker and a disulfide bond with a self-immolative spacer for drug release



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(Figure 4). Endocyte developed Vintafolide for several indications. Initially the conjugate was studied for the treatment of patients with platinum-resistant ovarian cancer in combination with pegylated lisosomal doxorubicin (PLD) versus PLD alone. The combination extended progression-free survival for approximately 2 months in direct correlation with folate receptor expression.82 Moreover, Endocyte has recently reported promising results of an interim analysis of phase II data, comparing the use of Vintafolide plus docetaxel versus docetaxel alone for the treatment of patients with non-small cell lung cancer. The combination treatment extended progression-free survival by 1.2 months and overall survival by 5.9 months.62, 83 Vintafolide is currently developed in combination with Doxil in a phase III trial for the treatment of platinumresistant ovarian cancer; the enrolment in this trial was suspended in May 2014 following the recommendation from the data safety monitoring board (DSMB) based on futility. The clinical data are currently being reviewed.62 Endocyte and Bristol-Myers Squibb have also investigated conjugates based on other payloads, such as tubulysins or epothilones, in clinical trials.62

Prostate-specific membrane antigen (PSMA) is an accessible cell-surface bound homodimeric endopeptidase, which can be efficiently targeted using urea-based derivatives of glutamic acid (Figure 4). PSMA is usually absent in normal adult tissues, exception made for normal prostate tissue and duodenum.

84, 85

In most cases PSMA-selective imaging agents showed high uptake in

salivary and lacrimal glands, which may reflect either target expression in those tissues or ligand binding to non-cognate proteins.65, 86 By contrast, the antigen is strongly expressed in the majority of localized and metastatic prostate cancer cells.87, 88 PSMA has also been reported to be overexpressed in the neo-vasculature of many malignancies.89, 90 The tumor-targeting performance of


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various PSMA ligands has been validated in rodents and in cancer patients, using nuclear medicine procedures.65, 86 64, 66

Carbonic anhydrase IX (CAIX) represents an additional example of a tumor-associated antigen, which can be targeted both with antibodies and with small organic ligands. Several types of carbonic anhydrases can be found inside (CAI, CAII, CAIII, CAVII and CAXIII), or on the surface (CAIV, CAIX, CAXII, CAXIV and CAXV) of cells. Interestingly, CAIX is virtually undetectable in normal adult tissues, except for a strong expression in stomach, duodenum and gall bladder.68, 84, 91 By contrast, CAIX is over-expressed in the majority of clear-cell carcinomas, as a result of a von Hippel-Lindau mutation or deletion.92

93

Additionally, the antigen can be

found at sites of hypoxia.94, 95 An ADC product targeting CAIX has been brought to clinical trials by Bayer.96 Certain small aromatic sulfonamides (in particular, acetazolamide; Figure 4) can be used as delivery vehicles for tumor targeting applications.

69, 97

For example, an acetazolamide

moiety coupled to a near-infrared fluorophore has been shown to selectively localize to subcutaneously grafted human renal cell carcinomas.58 The improvement of acetazolamide affinity to its target, using DNA-encoded chemical library technology, led to a further increase in tumor uptake ( Figure 5).70



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Figure 5 Evaluation of targeting performance of IRDye 750 conjugates in near-infrared fluorescence imaging of balb/c nu/nu mice bearing SK-RC-52 xenografts. 3 nmol of ligand-IRDye 750 conjugate were injected: untargeted control, targeted based on acetazolamide 1 (b) and targeted based on pharmacophore pair 2 (c). Chemical structures of 1 and 2 are provided in Figure 4. . Reproduced with permission from Wichert, M. et al (2015) Nat. Chem. 7, 241-249.

In our experience, CAIX ligands do not efficiently internalize into cancer cells, but they can be used for the selective delivery of cytotoxic payloads (e.g., DM1, MMAE) to the tumor environment, using disulfide-based or peptide-based cleavable linkers,

58, 69

[Pretto, F. Casi, G.

unpublished data] with promising therapeutic results. A direct comparison of tumor targeting experiments based on two high-affinity CAIX antibodies or on acetazolamide derivatives revealed a superiority of the small ligands, in terms of speed of targeting, tumor uptake and selectivity

70

[N. Krall, F. Pretto, M. Mattarella, D. Neri, manuscript submitted; L. Gualandi, C.

Hutchinson, F. Pretto, D. Neri, S. Wulhfard, unpublished data].

Challenges and opportunities for the development of next-generation cytotoxic agents As mentioned previously, the high efficacy of targeted therapies often seen in rodent models of cancer rarely translate to patients in the clinic. Ideally, one would like to characterize the


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biodistribution properties of therapeutic agents in both settings, thus learning about tumor targeting performance in mouse and in man. A number of companies and investigators have realized the importance of using companion diagnostics for the development of targeted therapeutics. Nuclear medicine procedures based on radiolabeled products equipped with radionuclides suitable for SPECT or PET detection appear to be the methodology of choice for the quantitative characterization of tumor targeting results. This approach is equally valuable for antibody-based therapeutics 10, 11, 15, 18 and for small molecule-drug conjugates.98, 99 Unfortunately, logistical challenges (e.g., need to work simultaneously with oncology and nuclear medicine units), regulatory hurdles (e.g., requirement to consider the radiolabeled product as a second investigational medicinal agent, with the need to provide adequate documentation and GMP preparation) and radioprotection considerations have so far limited the routine execution of these important investigations.

Nuclear medicine procedures are also ideally suited also for studying the fate of delivery vehicle (e.g., antibody or small organic ligand) and of the therapeutic payload (e.g., cytotoxic drug) in vivo. In a pioneering study, the groups of Guus van Dongen and of Matteo Zanda have recently reported the results of biodistribution studies, featuring the simultaneous labeling of the antibody moiety and of a tubulysin payload with two different radionuclides 100. We anticipate that similar studies, if performed in patients with cancer, could provide a much more detailed understanding of the pharmacodelivery process, than what is currently available. Alternatively, the fate of a targeted cytotoxic agent could be studied in vivo, if biopsies and suitable analytical methodologies are available.



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It is becoming increasingly clear that delivery vehicle (e.g., antibody or small ligand), cleavable linker and payload may all contribute to the anticancer activity of conjugates in vivo. It has become common practice to speak about “linker-payload combinations”, as these chemical moieties both contribute to the success (or failure) of a given therapeutic approach. Cleavable linkers may release the toxic payload directly or indirectly (e.g., after subsequent cleavage of a self-immolative spacer) 101-103. In some cases, a traceless (i.e., linker-less) coupling of drug to the delivery vehicle is possible

104, 105

. Linkers may be cleaved by uncatalyzed hydrolytic processes

(which are not likely to differ between mouse and man)106 or by other procedures (e.g., reduction of disulfide bonds, proteolytic cleavage of peptides) which are greatly influenced by the site of in vivo delivery.56-60 While these “triggered” release processes may provide a highly desirable additional selectivity (e.g., if the trigger is more abundant at the tumor site), clinical translational activities become more challenging, as it is difficult to adequately characterize the drug release process in mouse and man.

Various antibody-drug coupling methodologies can be considered, as the antibody moiety contains various structures that can be used for chemical modification (e.g., primary amines, cysteines, oligosaccharidic moieties). Site-specific conjugation strategies are emerging as important features for the development of a successful ADC product,37, 39, 40, 49, 104, 105, 107 as the corresponding conjugates tend to be more homogenous and to display improved pharmacokinetic profiles. While site-specific drug coupling strategies were not implemented in first-generation ADC products, these features have always been incorporated in the design of small moleculedrug conjugates (SMDCs), as they are assembled by stepwise chemical synthesis processes.


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One of the most puzzling aspects in the field of targeted cytotoxics relates to the choice between antibodies and small ligands as preferred vehicles for pharmacodelivery applications. On one hand, antibodies can be rapidly generated against virtually all targets of interest, including proteins of exquisite selectivity (e.g., virtually undetectable in normal adult tissues). Knowledge about the blood clearance and tissue distribution properties of antibodies is largely predictable and transferable from one antigen to the other. However, the tumor targeting properties of anticancer antibodies are still largely unexplored, both at a macroscopic and microscopic level, as discussed in a previous section. In addition, for certain antigens, the tumor targeting process may be slow and/or inefficient. By contrast small organic ligands may target tumors rapidly and efficiently. However, certain “off-target” liabilities are often observed in preclinical and clinical imaging studies with SMDCs. Virtually all small ligands characterized so far show some level of kidney uptake, which may be the consequence of a renal clearance process and precipitation (or binding) in renal structures. In addition, an undesired accumulation in normal organs may be observed, for example, when the cognate antigen is expressed at that site. PSMA ligands efficiently accumulate in salivary parotid glands, while CAIX ligands may localize to stomach (where the antigen is expressed) or lung (where other accessible carbonic anhydrase isoforms can be found). Interestingly, some of these “off-target” accumulation features in normal organs can be minimized by the pre-administration of competitors

108

or by the administration of judiciously

chosen doses. Intact monoclonal antibodies in IgG format are cleared via the hepatobiliary route. By contrast, small antibody fragments and most SMDC products studied so far are cleared via the renal route. The development of small drug conjugates, which may preferably be eliminated via the liver, remains an attractive but largely unexplored possibility.

Cancer cures and combination strategies



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ADCs and SMDCs are able to cure cancer in certain animal models of the disease, which do not respond to conventional cytotoxic agents. However, cancer cures in patients treated with ADC and SMDC products are rare. It is important to investigate the reasons behind the different activity observed in rodent models of cancer and in patients. As mentioned above, a quantitative characterization of the ligand-based tumor targeting process and of drug release mechanisms in mouse and man will be important. Imaging technologies will play a crucial role for a “Precision Medicine” approach to drug development.

If ADCs and SMDCs fail to cure cancer as single agents, then combination strategies may be recommended. The striking activity observed using brentuximab vedotin and nivolumab as single agents in last-line patients with Hodgkin lymphoma has prompted the clinical investigation of combination strategies. In addition, preclinical studies performed in immunocompetent tumorbearing mice have shown that the combination of ADCs with antibody-cytokine fusion proteins (immunocytokines) or the development of trifunctional antibody products, which simultaneously carry a cytokine moiety and a cytotoxic payload (immunocytokine-drug conjugates, or IDCs) may lead to very potent in vivo activity.109, 110 The antibody-based pharmacodelivery of cytokines and cytotoxic moieties is particularly attractive, due to the orthogonal toxicity profiles of these classes of payloads.

Concluding remarks The development of targeted cytotoxics represents an area of pharmaceutical research in constant evolution. The modular nature of ADC and SMDC products facilitate an “engineering” approach to drug discovery, in which a precise knowledge and a suitable modeling of the pharmacodelivery process allows to predict therapeutic performance.111,

112

In practice, in spite of substantial


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advances over the last two decades, several aspects of ADC and SMDC development remain insufficiently studied and will require dedicated future investigations. A detailed characterization of the tumor targeting process and of the drug release process will continue to be important. Different types of linker-payload combinations may be required for different types of malignancies. However, an empirical choice determined by success (or failure) in exploratory clinical trials will continue to be problematic, both from an economical viewpoint and in consideration of the patients’ legitimate request for efficacious drugs. Sparing normal organs by the improvement of pharmacokinetic properties or by designing drugs with minimal toxicity to healthy tissue (as a consequence of their intrinsic mechanism of action or of an inactivation process in clearance-associated organs) will probably represent one of the most important challenges for the future. In addition, the marriage of targeted cytotoxics and of immunostimulatory products, which represent two of the most prominent classes of anticancer agents, is likely to bear fruits.



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CORRESPONDING AUTHOR: Prof. Dr. Dario Neri, Professor for Biomacromolecules, ETH Zurich, Institute of Pharmaceutical Sciences, HCI G396.4, Vladimir-Prelog-Weg 1-5/10, CH-8093 Zurich; Tel: +41 44 63 37401; email: [email protected].

ABBREVIATIONS: ADC: antibody-drug conjugates; SMDC: small molecule-drug conjugates; MMAE: monomethyl auristatin-E; AML: acute myeloid leukemia; CAIX; carbonic anhydrase IX; IDC: immunocytokine-drug conjugates; PSMA: prostate specific membrane protein;

BIOGRAPHIES

Giulio Casi Giulio Casi studied chemistry at Florence University (Italy) and ETH Zurich (Switzerland). After postdoctoral research at Roche Penzberg (Germany) in 2009 he returned to Switzerland to work for Philochem AG, where he is currently responsible for the research and development of targeted cytotoxics. Dario Neri Dario Neri studied Chemistry at the Scuola Normale Superiore in Pisa (Italy), obtained his PhD in Chemistry from the ETH Zürich (Switzerland) in 1992, under the supervision of Prof. Kurt Wüthrich. After postdoctoral research at the MRC Centre in Cambridge, under the supervision of Sir Gregory Winter, he became professor at the ETH Zürich in 1996. He is a co-founder of Philogen and Philochem.

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Page 34 of 39

TABLE OF CONTENTS GRAPHIC

DRUG

DRUG


Page 35 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45


ACS Paragon Plus Environment


a) 1

b)

H

H

L

SIP / Minibody VH

VL

VL

CH4 CH4

V

H

IgG antibody V

VH

VL CL

H

1 CH

C

H

1

VL

2 3scFv 4 5V V 6 7 8 Diabody 9 10 V V 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

C

L

CH2

CH2 Fc

L

interdomain disulfide bond

CH3

CH3


Page 36 of 39

c)

Page 37 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


O O

H N

N H

O

O

O N H

O

NH

N

O

O

N

H N O

O N

H N

N OMe O

OH

OMe O

MeO

N Cl

N

O

O

O

O

S O

NH 2

O O

S

N H MeO OH


O

O

N

HN O


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

FOLATE RECEPTOR

PSMA O

O N

HN H 2N

N

O

OH

N H

N H

N

Page 38 of 39

OH

O

O O

HO

N H

O

SOMATOSTATIN RECEPTOR

OH

N H

O

CAIX

O

OH

N H

N H

S O S

HO NH

O O

O

O

H N

O HN

N N N

HN HO

H N O

N

HN

O

O

NH

N S

SO2NH 2

1

CH3 O

HN

N

O N N N

NH 2 HN

OH

CO2H O HN

N NH

N S

SO2NH 2

2

CO2H O HN O


OH

Page 39 of 39

Untargeted dye mouse #1

a

1h

2h

6h

8h

11 h

23 h

1.0 0.8 0.6 0.4

x 10 9

0.2 Tumour

b

4h

1h

Kidneys 2h

4h

6h

8h

11 h

23 h

0.0 5.0

Epi-fluorescence [radiant efficiency] = p/s/cm2 /sr µW/cm 2

Colour scale Min = 0.00 Max = 1.00 x 10

9


9


9

Targeted 1 mouse #1

4.0 3.0 2.0

c

x 10 9

1.0 Tumour 1h

2h

4h

6h

8h

11 h

23 h

0.0 5.0 4.0

Targeted 2 mouse #1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.0 2.0 1.0 Tumour

0.0


x 10 9

AntibodyâDrug Conjugates and Small MoleculeâDrug Conjugates

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