From Preclinics to Proof-of-Concept Clinical Trials A Development Pathway in Biotechnology

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1 Arzneimittelwesen Gesundheitspolitik Industrie und Gesellschaft Fachthemen From Preclinics to Proof-of-Concept Clinical Trials A Development Pathway in Biotechnology Sascha Tillmanns SuppreMol GmbH, Martinsried, Germany For biotech companies, it is essential to generate first clinical efficacy data to attract investors. Preclinical efficacy models are needed to get a first idea on efficacy and target dose range and can already provide useful data on toxicity. Toxicological evaluations may include standard toxicological studies with the human investigational medicinal product (IMP) format, the development of an IMP surrogate, or humanised animal models. The determination of the no observed adverse effect level (NOAEL) or minimum anticipated biological effect level (MABEL) are important toxicological milestones prior to the start of first in human (FiH) clinical trials and proof-of-concept (PoC) studies. The FiH clinical trial may include a dose escalation part, starting with the maximum recommended starting dose (MRSD), with dosing steps that are reflecting a moderate increase in activity and a hybrid design with healthy volunteers and patients. An extension arm of the anticipated optimum dose in patients does support first PoC data as first efficacy milestone. 1. Introduction For biotech companies, one of the major challenges is the limited funding until an important milestone, such as the first clinical efficacy results in proof-of-concept (PoC) studies, is reached. Biotech companies, therefore, must often find a fast and pragmatic way to bridge the development period from preclinics to clinical efficacy studies before a larger pharma partner steps in and continues the investigational medicinal product (IMP) development until marketing approval. The intention of this essay is to give a summary overview of biotech product development in small companies from preclinics to first in human (FiH) or PoC studies; a summarising Fig. 1 is placed at the end of the article. 2. Assay Development and Preclinical Efficacy Models After IMP optimisation, the first step is to establish a variety of suitable and usually non-glp efficacy models in-house or in collaboration with academic institutions to estimate an efficacious dose in humans, to determine the minimum anticipated biological effect level (MABEL) for specific products and to establish a valid potency assay for IMP release to trial centres in clinical studies later on. This can be achieved by different animal disease models, ex-vivo cellbased assays or in-vitro binding studies In-vitro binding studies, cell-based assays, potency assays and immunogenicity In-vitro binding studies are usually the first step to investigate binding characteristics of the IMP to the desired target. These studies will provide the first insight into the efficacy range of the IMP and efficacy quantification, e. g. expressed as a degree of target binding. It also supports the development of a qualified cell-based assay for IMP release and MABEL AUTHOR Sascha Tillmanns is the Medical Director for SuppreMol GmbH in Martinsried in the field of autoimmune diseases. He has worked for several pharmaceutical companies in clinical development and toxicological research. The main focus of his work includes the conception of toxicological studies and early phase clinical trials to establish first proof-of-concept data. Sascha Tillmanns is a biologist by training and holds an MSc degree in pharmacology and pharmaceutical medicine. 344 Tillmanns From Preclinics to PoC Clinical Trials ECV Editio Cantor Verlag, Aulendorf (Germany)

2 determination. Other important invitro investigations include:. A qualified IMP bioassay for IMP quantification and pharmacokinetic (PK) analysis for human and animal samples. An anti-drug antibody (ADA) assay to assess the potential of immunogenicity and associated side effects in animals and humans. A neutralising anti-drug antibody (NADA) assay to identify a potentially limited IMP treatment duration and potential cross-reactivity to endogenous targets in humans Cell-based ex-vivo assays with human samples can serve as an important tool to investigate the IMP activity on several targets such as activation markers, mediator release, cell depletion, etc. These assays may serve later on as potency assays for IMP release in clinical trials Animal Disease Models for Efficacy If the IMP expresses at least some cross-reactivity to the equivalent animal target, standard animal disease studies are usually the next step to establish the pharmacodynamic (PD) effect of the IMP. The optimal study design would include different IMP dosages, the determination of the IMP level with PK analysis on satellite animals, the inclusion of a competitor with well-known activity in this model and first toxicological evaluations (clinical signs, histopathology, immunogenicity, etc.). Although such studies are usually performed in collaboration with an academic institution under non-glp, the models are also commercially available. A positive efficacy outcome of these studies is an important development milestone and will raise first interest of potential investors. For the extrapolation to the anticipated efficacy dose range in humans, a factor to account for a lesser binding activity of the human IMP format to the animal equivalent target must normally be considered. If the cross-reactivity of the human IMP format is negligible or the human target is not expressed at all in animal species, standard animal models appear to be not useful. Instead, the use of a surrogate IMP or humanised animals with additional disease models should be considered. These animal models are able to mimic human binding characteristics, immunological pathways and on-target effects in a more relevant way. However, humanised animal models have the disadvantage that they are normally conducted under non-glp, the available animal number is usually low and the degree of the transferred human immune systems varies quite substantially from animal to animal. Surrogate IMP does not represent the actual human IMP format and the use in animal models could mask some additional pharmacological properties. Usually, the PD animal models do not support the determination of the no observed adverse effect level (NOAEL) but they are useful for the establishment of the MABEL. 3. No Observed Adverse Effect Level (NOAEL) The NOAEL determination is one of the most important outcomes in standard GLP toxicology studies. It determines the dose level where no adverse effects can be observed. With the appropriate conversion into the human dose and an additional safety margin, the maximum recommended starting dose (MRSD) [1] for the FiH clinical trial can be determined (discussed below). The MRSD determination, based on the NOAEL, is valid for all products that don t fall under the EMA risk mitigation guideline [2] and that require the determination of the MABEL for dose selection. 4. Minimum Anticipated Biological Effect Level (MABEL) The MABEL approach for potentially risky IMPs was first established after a phase I clinical trial in 2006, where the administration of a T-cell (CD28) activating IMP caused a cytokine storm in six healthy volunteers, leading to multiple organ dysfunctions [3]. These effects were not anticipated from the classical NOAEL level based on toxicology studies in cynomolgus monkeys. This event had major implications on the design of FiH clinical trials for some products, including the MRSD based on the MABEL rather than on the NOAEL, sequential randomisation of study subjects, direct access to an intensive care unit and the implementation of a data safety monitoring board (DSMB). The MABEL and subsequently the MRSD calculation should be based on a series of relevant in-vitro, ex-vivo and animal disease models to investigate the anticipated minimum human dose which causes the first biological (on-target) effects (e. g. 10 % of the maximum effect) in the most appropriate/sensitive model. Usually, a safety margin for the MRSD needs to be applied in addition for the use in FiH clinical trials. 5. Nonclinical Toxicology and Safety Pharmacology If the IMP shows favourable efficacy results in animals, the next step would be to enter the toxicological evaluation of the product. This is normally an expensive step and usually requires toxicology and safety pharmacology studies in two species performed under GLP to fulfil regulatory requirements [4]. However, due to a lack of cross-reactivity of biotech-derived IMPs to an equivalent animal target, the standard toxicological approach will not always be feasible [5]. Basically, there are three possibilities to generate relevant preclinical toxicology data prior to the start of FiH clinical trials:. Standard GLP toxicology studies in up to two species with the IMP in the intended human format performed by a commercial available CRO ECV Editio Cantor Verlag, Aulendorf (Germany) Tillmanns From Preclinics to PoC Clinical Trials 345

3 Arzneimittelwesen Gesundheitspolitik Industrie und Gesellschaft Fachthemen. GLP toxicology studies in up to two species with an IMP surrogate performed by a commercially available CRO. Toxicology evaluations in humanised animals with the human IMP usually performed in collaboration with academic institutions under non-glp 5.1. Standard GLP toxicology If the human IMP format demonstrates at least a reasonable cross-reactivity to an equivalent target in one or more animal species, it is often useful to perform a standard GLP toxicology evaluation first. For small molecules, rats and dogs are normally chosen for this purpose, however, for biotech IMPs mice and (still) cynomolgus monkeys are quite often used for the toxicological evaluation. Mice have the advantage that they are quite often also used in animal efficacy studies and, therefore, a direct comparison to the GLP toxicology studies in terms of IMP exposure is possible. Due to the higher IMP turnover in mice, it is quite often difficult to achieve a reasonable IMP exposure which is important for the determination of an adequate exposure safety margin for the clinical development later on. The dose frequency and duration in the toxicology studies should at least cover 1:1 of the intended human application in clinical trials. Before the GLP main part of a toxicology study is performed, a non-glp dose range finder (DRF) is usually initiated before to evaluate a safe dose range for the IMP in a limited number of animals, e.g. the minimum and maximum intended toxicological dose over 2 weeks. For the main GLP part, a common way is to start with a 28- day weekly dosing toxicology study in male and female mice with three different IMP doses and placebos, including a toxicokinetic (TK) evaluation, clinical signs/symptoms, immunological evaluation and histopathology. An additional recovery period of at least 2 weeks with satellite animals is recommended. The dosages used in the DRF and main study should be based on the preclinical experience with the IMP, resulting in an anticipated human dose range with an adequate safety margin. If the human target dose is xmg/kg (or a fixed dose), appropriate dosages in the toxicology evaluation could be 0 mg/kg (placebo), 5 times xmg/kg, 30 times xmg/kg and 100 times xmg/kg. In order to achieve the same safety margin for the toxicological on-target effects, the dosages in the toxicology studies should be increased by a certain factor. Normally, single dose toxicity and safety pharmacology evaluations are integrated into the multiple toxicity dosing scheme. The appearance of (neutralising) anti-drug antibodies should be evaluated as well; their presence could induce irrelevant side effects and might limit the duration of IMP administration in animals. One major outcome of these toxicological evaluations is to determine a safe IMP dosage, where no relevant side effects occur, resulting in a NOAEL. For quite a number of biotech IMPs, however, even the highest dose may not lead to any relevant side effects in these classical GLP toxicology studies Toxicology evaluation with IMP surrogate Despite the use of an IMP surrogate for animal efficacy studies in a disease model, it can be also used in a GLP environment as toxicological evaluation in healthy animals if the human IMP format does indicate no or a very low cross-reactivity in any animal species. The model sometimes provides useful information on the expected on-target side effects in humans. However, since a surrogate does not represent the actual IMP format, additional GLP toxicology with studies to investigate at least toxicological off-target effects with the human IMP, as discussed above, are most likely to be conducted in addition Humanised Animals for Toxicology In some cases, the human IMP format does not show cross-reactivity to any animal species and it is not possible to generate an IMP surrogate due to the non-existence of the target in animals. Here, despite supporting cell based assays, it is possible to also use humanised animals expressing the human target and at least a relevant part of the downstream immune activity for toxicological evaluations. Although there are now some CROs in the field, who are offering such humanised animals (normally mice), the sole toxicological evaluation remains very difficult and is usually performed in collaboration with academic institutions under non-glp. One additional concern is the huge variety in the individual degree of humanised immune setting, making it difficult to obtain consistent data over a larger number of animals. However, these models might be the only reasonable way to obtain relevant toxicity data for non-cross-reactive IMPs with an otherwise promising anticipated mode of action in patients. Sometimes, a standard GLP toxicity study in mice will be done in addition to humanised models to obtain off-target toxicological effects in a controlled GLP environment. 6. First-in-Human (FiH)/ Proof-of-Concept (PoC) Clinical Trial Once the toxicological package revealed a NOAEL and/or a MABEL for the human IMP format based on a reasonable number of experiments, the next step would include the planning of a dose-escalation FiH clinical trial in healthy volunteers or patients. For cytotoxic drugs (e. g. in oncology), it is obvious that the IMP must be tested directly in patients with the desired target indication. For other drugs/indications, however, the inclusion of healthy volunteers would guarantee a more robust 346 Tillmanns From Preclinics to PoC Clinical Trials ECV Editio Cantor Verlag, Aulendorf (Germany)

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5 Arzneimittelwesen Gesundheitspolitik Industrie und Gesellschaft Fachthemen and controlled study environment for a first human testing. Therefore, it might be worth to consider a volunteer/patient hybrid study design, where the first part consists of the inclusion of healthy volunteers covering the lower dosing cohorts, followed by patient cohorts receiving IMP dosages within the anticipated therapeutic range. This design would avoid treating patients on low, nonefficacious IMP dosages in the first cohorts. Each dosing cohort should consist of a placebo control where possible, and the trial should be conducted in a randomised, doubleblind manner. The healthy volunteer part of the study will be most likely conducted at a single investigator site. The patient part should be already conducted in a multicentre approach with inclusion of EU and US centres. Although the main focus is on safety including the determination of dose limiting toxicities (DLTs), optimal biological dose (OBD) or recommended dose (RD), the patient part would allow to see first efficacy results in terms of biomarkers or even short term clinical endpoints Maximum recommended starting dose (MRSD) The MRSD is the first dose level in a FiH clinical trial and can be determined based on:. Direct conversion from NOAEL. Human equivalent dose (HED) from NOAEL. MABEL In general, the most appropriate or sensitive model, including a reasonable safety factor, should be used for the MRSD MRSD based on direct conversion from NOAEL The NOAEL from toxicological evaluation in a relevant/most sensitive species is directly transferred into the MRSD via a safety factor of at least 10x. This can be a total dose amount of xmg or based on body weight in xmg/kg, based on the intended human application scheme MRSD based on NOAEL via HED This approach is based on the HED in mg/kg based on the relationship of the animal s and human s average body surface. The conversion factors for different species are listed in the FDA guidance on MRSD and are 0.08 and 0.32 for mice and cynomolgus monkeys, respectively. The animal dose linked to the NOAEL will be then multiplied with the appropriate species factor to obtain the HED. A safety factor of at least 10-fold will be applied to obtain the MRSD. This approach is not necessary if the human dose will be administered directly on a body surface approach (xmg/m 2 ). Figure 1 Assay Development IMP Bioassay ADA/NADA Assay Toxicological Evalua on GLP tox study human format GLP tox study surrogate Tox in humanized animals NOAEL First in Human Clinical Trial Dose escala on part Volunteer/pa ent cohorts DLT determina on Biomarker evalua on MRSD and Dosing Steps MRSD based on MABEL The MRSD of those IMPs falling under the EMA guideline risk mitigation, e. g. non cross-reactive biotech products causing immune system activation, will be calculated based on the MABEL approach. Here, on-target effects from animal efficacy studies or other preclinical efficacy evaluations will be investigated with different IMP dosages. The level of IMP dosage will generate different degrees of on-target effects, e.g. more or less target binding or more or less efficacy in animal efficacy studies (refer also to section 5). Preclinical Efficacy Binding studies Cell-based assays Animal disease models - Human IMP format - IMP surrogate - Humanised animals MABEL First in Human Clinical Trial Extension part Safety confirma on Clinical efficacy Proof-of-concept Possible development pathway until proof-of-concept (source: figure made by the author/ SuppreMol GmbH). 348 Tillmanns From Preclinics to PoC Clinical Trials ECV Editio Cantor Verlag, Aulendorf (Germany)

6 Final choice of MRSD The final choice of the MRSD should be based on the most sensitive/appropriate preclinical efficacy (MABEL) or toxicological (NOAEL) model as discussed above. Since the final choice of the MRSD is probably the most important part of the clinical trial protocol, it should be discussed with the involved regulatory agencies prior to the submission of the clinical trial protocol Dosing Steps and Maximum Dose The FiH clinical trial will most likely include an escalating dosing scheme with the MRSD in the first dosing cohort, followed by cohorts with an increasing dose up to the maximum feasible dosing cohort. For IMPs falling into the risk mitigation category, dose steps should be determined carefully to avoid inadequate dose jumps causing intolerable side effects. If the preclinical efficacy models for determination of the MABEL did include several IMP dose levels, a preliminary dose-response curve might be established and IMP dosages can be estimated causing a moderate increase in treatment response, avoiding on-target side effects. If a FiH hybrid clinical trial with volunteers and patients is used, one appropriate way could be to start the volunteer part with 1/10 of the MABEL dose, followed by larger dosing steps and then continue the study in patients with lower dosing steps according to the dose-response curves from preclinical evaluations up to the maximum dose. Each dosing step will be controlled by the DSMB based on safety data. If placebo patients are included, the absolute minimum for a dosing cohort would be 1+2, e. g., one volunteer/patient on placebo and two volunteers/patients on active drug. The number of cohorts is often in the range of eight; with the hybrid clinical trial, three healthy volunteer cohorts and five patient cohorts would require 24 subjects in total. This might be enough for the first safety evaluation. However, for a reasonable analysis on an efficacy parameter, a higher number of subjects is usually required. This can already be achieved by the implementation of an extension cohort into the design of the FiH clinical trial. Here, the RD or OBD cohort will be extended with an additional number of patients to reach important PoC data which can be used as initiation for the important partnering processes. REFERENCES [1] FDA Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, July [2] EMA guideline: Strategies to Identify and Mitigate Risks for First-in-human Medicinal Products, July [3] Suntharalingam G et al., Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412. N Engl J Med 2006;355: [4] ICH guideline M3 (R2) on non-clinical safety studies for the conduct of human clinical trials and marketing authorisation for pharmaceuticals, December [5] ICH Guideline: Preclinical Safety Evaluation of Biotechnology Derived Pharmaceuticals S6 (R1), June Correspondence: Sascha Tillmanns Medical Director SuppreMol GmbH Am Klopferspitz 19a Martinsried/München (Germany) tillmanns@suppremol.com Wir sind Ohne Prüfpräparate keine klinische Prüfung Der Grundsatz ist einfach, die Realisierung komplex. Wir wissen wie es geht seit mehr als 20 Jahren. Von logistischer und regulatorischer Planung (GMP und IMPD), über Contract QP Service, Import aus Drittländern, Shifting von Medikation wegen unterschiedlicher Geschwindigkeiten bei der Patientenrekrutierung bis hin zu Begutachtung und Reorganisation von Abläufen in der Bereitstellung der Medikation stellen wir uns jeder Herausforderung in diesem komplexen Umfeld. Sie kennen Ihre Probleme, wir kennen die Lösung. Fordern Sie uns heraus! PharmDev InnovationsGmbH Bleicherstr. Dr. R. Dietrich Im Tiergarten Konstanz, 16Germany Konstanz info@pharmdev.de +49 (0) info@pharmdev.de